EP3781697A1 - Method for determining chemical compounds in waste - Google Patents

Method for determining chemical compounds in waste

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Publication number
EP3781697A1
EP3781697A1 EP19716189.6A EP19716189A EP3781697A1 EP 3781697 A1 EP3781697 A1 EP 3781697A1 EP 19716189 A EP19716189 A EP 19716189A EP 3781697 A1 EP3781697 A1 EP 3781697A1
Authority
EP
European Patent Office
Prior art keywords
waste
acid
compounds
liquid
treatment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19716189.6A
Other languages
German (de)
French (fr)
Inventor
Hanne Risbjerg SØRENSEN
Peter FRISTRUP
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Renescience AS
Original Assignee
Renescience AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Renescience AS filed Critical Renescience AS
Publication of EP3781697A1 publication Critical patent/EP3781697A1/en
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H8/00—Macromolecular compounds derived from lignocellulosic materials
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the qualitative or quantitative determination of chemical compounds or classes of compounds in heterogeneous waste comprising biodegradable and non-biodegradable matter.
  • the method may especially be used for identifying, tracking and mapping geographical origin of waste, such as e.g. municipal solid waste, agricultural waste or industry waste comprising one or more specified chemical compounds and for identifying, tracking and mapping any change in occurrence of one or more specified chemical compound(s) present in the waste from a certain geographic area.
  • the current invention appears well-suited for analyzing and/or monitoring waste being subjected to waste treatment in a large-scale waste plant.
  • Incineration is one method which combines utilization of the energy stored within the organic material with disposal of waste such as household, agricultural or municipal waste. For example in Denmark, the heat generated by the incineration process is mainly used for district heating, but can also be converted into electricity. Incineration is, however, problematic if the material/waste to be incinerated includes non-organic parts like plastic, glass and metals. In order to optimally utilize the energy stored within organic material found in different types of material/waste by incineration, pre-sorting is required.
  • Pre-sorting may be provided by the consumers or by the waste station. Regardless of this, pre-sorting may not be efficient in separating all non-biodegradable material such as metal and glass from the organic waste. The waste separation process may time consuming, inefficient and expensive.
  • An alternative waste sorting method is to liquefy the organic contents while maintaining the non-organic contents in their solid phase, and afterwards separate the solid and the liquid phases.
  • the problem with pollution when e.g. incinerating the separated waste is thereby greatly diminished.
  • Recently such enviromentaly friendly waste processing methods like the Renescience technology have been developed. With the Renescience technology, ordinary unsorted household waste is mixed with water, enzymes and optionally microorganisms in order to dissolve all food waste, cardboard, paper, labels and similar types of organic waste and turns it into a liquid that can for example be used for biogas.
  • the waste that can be processed according to the present invention is heterogeneous waste, such as municipal solid waste (MSW), particularly including domestic household waste, waste from restaurants and food processing facilities, and waste from office buildings. Waste often comprises significant amounts of organic material that can be further processed to energy, fuels and other useful products. At present, only a small fraction of available waste, such as MSW, worldwide is recycled, the great majority being dumped into landfills. Due to the general heterogenity of waste, such as MSW, which comprises a mixture of diverse biodegradable and non-biodegradable matter, it has not previously been possible to sufficiently analyse the chemical composition of e.g. the soluble fraction of waste, including MSW.
  • MSW municipal solid waste
  • the degradable component of waste such as MSW can be used in“waste to energy” processing using both thermo-chemical and biological methods. Waste such as MSW can be subject to pyrolysis or other modes of thermo-chemical gasification.
  • Biological methods for conversion of degradable components of waste include fermentation to produce specific useful end-products, such as ethanol.
  • Biological conversion of waste into useful end-products can for example be achieved by anaerobic digestion to produce biomethane or biogas.
  • Pre-sorted organic component of waste, such as MSW can in some methods be converted to biomethane either directly or after a "pulping" process involving mincing in the presence of added water.
  • Pre-sorting of waste, such as MSW, to obtain the organic and bio-degradable component is typically costly, inefficient or impractical.
  • Source-sorting requires large infrastructure and operating expenses as well as the active participation and support from the community from which waste are collected - an activity which has proved difficult to achieve in modern urban societies.
  • Mechanical sorting is typically capital intensive and further associated with a large loss of organic material, on the order of at least 30% and often much higher.
  • a radically different approach to liquefaction of degradable organic components is that this may be achieved using biological process, specifically through enzymatic hydrolysis and microbial fermentation.
  • the method according to the present invention is suitable when waste has been subject to enzymatic degradation and microbial fermentation. Examples of such waste treatment processes are disclosed in W02006056838, W02007036795,
  • Enzymatic hydrolysis offers unique advantages over "autoclave” methods for liquefaction of degradable organic components. Using enzymatic liquefaction, processing of waste, such as MSW, can be conducted in a continuous manner, using comparatively cheap equipment and non-pressurized reactions run at comparatively low temperatures.
  • Enzymatic liquefaction may sometimes require thermal pre-treatment to a comparatively high temperature of at least 90- 95°C, in part to effect a "sterilization" of waste such as unsorted MSW and also so that degradable organic components can be softened and paper products "pulped.”
  • high temperature pre-treatment can be actively detrimental, since this kills ambient microorganisms which are fostering in the waste.
  • Safe enzymatic liquefaction of unsorted MSW has been described without high temperature pre-treatment. Promoting microbial fermentation concurrently with enzymatic hydrolysis at thermophillic conditions >45°C improves "organic capture,” either using "ambient” microorganisms or using selectively "inoculated” organisms.
  • thermophillic microbial fermentation safely increases the organic yield of "bioliquid," which is the term used herein for the liquefied degradable components obtained by enzymatic hydrolysis.
  • bioliquid which is the term used herein for the liquefied degradable components obtained by enzymatic hydrolysis.
  • pathogenic microogranisms typically found in waste such as MSW do not thrive.
  • typical pathogens present in waste such as MSW-borne pathogens are easily outcompeted by e.g. lactic acid bacteria and other safe organisms present in the waste to be treated, and/or in the bioreactor, where the enzymatic and microbial liquefaction takes place.
  • liquefaction and fermentation is performed at temperatures wherein ambient microoganisms survive, the specific temperature however depending on the specific kind of waste to be treated, and/or the end products to be obtained.
  • the method according to the present invention makes it possible to track the geographic origin of specific waste compounds and to optionally initiate various acts directed at the geographic origin of the waste and to follow the occurrence of specific compounds of interest over time within the same geographic area or between areas.
  • Such acts could include setting op surveillance systems; directing campaigns regarding for instance sorting of waste; identifying areas with a high contend of i.e. drug or medicinal residues and optionally directing health campaigns and/or alter social compositions;
  • the method according to the present invention makes it possible to compare the content of a certain chemical compound(s) or class(es) of compounds in waste from a given geographic area with the content of the same compound(s) or class(es) of compounds or in waste within the same geographic area or with other geographic areas.
  • Such information makes it possible to more effectively address for instance sales campaigns, surveillance of activities that leaves chemical compounds in the waste.
  • the above mentioned possibilities are suggestions of possible use of the information that may become available by use of the method according to the present invention. It is evident that it will be possible to apply the information obtained by use of the present method for other purposes also.
  • the present invention relates to a method for determining the abundance of or establishing the identity of one or more chemical compound(s) or class(es) of compounds in waste, such as a waste batch collected by a garbage truck, said method comprising the steps of:
  • a) Subjecting said waste to a combined enzymatic and microbial treatment in a large- scale plant; b) Subjecting the treated waste from step a) to one or more separation step(s), whereby a liquid fraction is provided;
  • the method according to the first aspect of the invention further comprises one or more of the following steps:
  • the present invention relates to the use of the method according to the present invention for identifying the local geographical origin of specific chemical compounds present in the waste and optionally identifying the specific leaking or additive source(s) of said chemical compound.
  • the present invention relates to the use of the method according to the present invention for identifying the local geographical origin of specific chemical compounds present in the waste and directing information campaigns, reward campaigns, sales activities, local sorting of waste, environmental or social improvement activities; to the geographic area or specific entity that has been identified as the source(s) of the waste comprising a specific chemical compound.
  • the present invention relates to the use of the method according to the present invention for decreasing the abundance of or eliminating the presence of one or more chemical compound(s) in waste such as MSW before, during or after anaerobic digestion of the waste, by adjusting one or more of the processing parameters in step a) or step b)
  • the present invention relates to the use of the method according to the present invention for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste, such as MSW; based on the presence of one or more chemical compounds measured in step c).
  • the inventors have found that it is possible to determine and quantify the presence of individual chemical compounds in very heterogeneous waste, such as municipal solid waste, using a single extraction process, comprising enzymatic and/or microbial treatment. It is thus no longer necessary to use the conventional and tideous approach of presorting waste into individual fractions, in order to extract said chemical compounds, often individually, prior to analysis.
  • the present invention appears well-suited for a large-scale environment, such as a municipal solid waste treatment plant.
  • One application of the present invention is to track the local geographical origin of one or more chemical compounds e.g. in relation to enforcement of regulations regarding storage, treatment and disposal of waste.
  • Other applications include tracking of the local
  • FIG. 1 Overview of general waste processing process.
  • FIG. 1 Overview of sample treatment.
  • Figure 3 GC-EI-MS analysis of benzenepropanoic acid.
  • Figure 4 GC-EI-MS analysis of cholestan-3-one.
  • Figure 5 GC-EI-MS analysis of d-limonene.
  • the method of the present invention allows for determining the abundance of or
  • a“Chemical compound” means an entity consisting of two or more atoms, at least two from different chemical elements, which associate via chemical bonds.
  • Class(es) of chemical compound(s) refers to one or more class(es) of chemical compound(s) classified by any kind of common classification of chemical compounds including classes based on the specific elements present in the compound, the types of bonds that the compound contains or the types of reactions that the chemical compound may undergo.
  • MSW munal solid waste
  • MSW refers to waste fractions which are typically available in a city, but that need not come from any municipality per se.
  • MSW may be any combination of one or more of cellulosic, plant, animal, plastic, metal, or glass waste including, but not limited to, any one or more of the following: Garbage collected in normal municipal collections systems, optionally processed in central sorting, shredding or pulping device, such as e.g., a Dewaster® or a reCulture®; solid waste sorted from households, including both organic fractions and paper rich fractions; waste fractions derived from industry such as restaurant industry, food processing industry, general industry; waste fractions from paper industry; waste fractions from recycling facilities; waste fractions from food or feed industry; waste fraction from the medicinal industry; waste fractions derived from agriculture or farming related sectors; waste fractions from processing of sugar or starch rich products; contaminated or in other ways spoiled agriculture products such as grain, potatoes and beets not exploitable
  • unsorted refers to a process in which waste or MSW is not substantially fractionated into separate fractions such that organic material is not substantially separated from plastic and/or other inorganic material, notwithstanding removal of some large objects or metal objects and notwithstanding some separation of plastic and/or other inorganic material may have taken place.
  • sorted refers to a process in which MSW is substantially fractionated into separate fractions such that organic material is substantially separated from plastic and/or other non-biodegradable material.
  • sorted waste refers to waste in which less than 15% by weight of the dry weight is non-biodegradable material.
  • the term “organic” refers to materials that comprises carbon and are bio-degradable and include matter derived from living organisms.
  • bio-degradable refers to organic matter that can be partly or completely degraded by into simple chemical compounds such as mono or di-saccharides by micro-organisms and/or by enzymes.
  • liquid fraction means the mainly liquid slurry obtained after the waste to be processed has been subjected to a combined enzymatic and microbial treatment and thereafter has been subjected to one or more separation step(s), separating the treated waste into a liquid, i.e. a slurry, and a solid or semi-solid fraction.
  • bioliquid is used for thehe liquefied material of the liquid fraction once separated from non-fermentable solids.
  • the term“batch” refers to any defined amount of waste that is delivered from a specified geographic area to the waste plant.
  • the amount of waste in a“batch” and the size of the geographic area will vary from plant to plant and depend of the specific renovation-collecting system and the specific size of the plant, such as a large scale plant.
  • Each batch may be treated separately in each of step a), b) and c) of the present method or several batches may be treated continuously or at least having overlapping retention times at one or more step(s).
  • a batch will be the amount of waste loaded into the waste plant by a single truck which usually comprises between 15 - 20 m 3 waste disposal per load.
  • Several batches from trucks may be collected, stored and entered into the treatment plant as one large batch.
  • the batch will usually comprise between 40 - 6000 m 3 waste.
  • “large-scale plant” is a plant wherein waste is processed at a commercial scale; wherein the plant normally operates continuously for at least 24h periods typically followed by the next continous period; wherein waste collected from different sources are processed; and wherein each batch of waste entered into the plant can be defined by the way the waste enters the plant, such a one or more truck loads of a specific size, such as a truck load of 15 - 20 m 3 waste disposal, or of a specific weight of the waste disposal, such as the weight of one or more truck loads of 15 - 20 m 3 waste disposal.
  • the large scale plant also comprises means for“waste-to-energy” transformation or is connected to means for“waste-to-energy” transformation, such as means for the production of biogas, bioethanol, syngas, heat or electricity.
  • Fermentation refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.
  • the term“fermentation medium” refers to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
  • a medium resulting from a saccharification process such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
  • SSF simultaneous saccharification and fermentation process
  • the term“fermenting microorganism” refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product.
  • the fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art.
  • Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product.
  • a“Microbial consortium” refers to a consortium comprising one or more of any bacteria and/or yeasts capable of providing microbial fermentation.
  • or“Auxiliary Activity 9 polypeptide” or“AA9 polypeptide” means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et al., 201 1 , Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips et al., 201 1 , ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20: 1051 -1061 ).
  • polypeptides were formerly classified into the glycoside hydrolase Family 61 (GH61 ) according to Henrissat, 1991 , Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.
  • GH61 glycoside hydrolase Family 61
  • the term“oversize particles” means particles above 600 mm.
  • anaerobic digestion system refers to a fermentation system comprising one or more reactors operated under controlled aeration conditions in which methane gas is produced in each of the reactors comprising the system. Methane gas is produced to the extent that the concentration of metabolically generated dissolved methane in the aqueous phase of the fermentation mixture within the “anaerobic digestion system” is saturated at the conditions used and methane gas is emitted from the system.
  • the “anaerobic digestion system” may be a fixed filter system.
  • a “fixed filter anaerobic digestion system” refers to a system in which an anaerobic digestion consortium is immobilized, optionally within a biofilm, on a physical support matrix.
  • the present invention relates to a method for determining the abundance of or establishing the identity of one or more chemical compound(s) or class(es) of compounds in waste, such as a waste batch collected by a garbage truck, said method comprising the steps of:
  • step b) Subjecting the treated waste from step a) to one or more separation step(s),
  • Relevant types of mono- and/or polysaccharide containing waste that is suitable for being processed by the combined enzymatic and microbial treatment in a large scale plant according to the present invention may include:
  • Waste fractions derived from households such as e.g.:
  • MSW processed in some central sorting, shredding or pulping device such as e.g.
  • Waste fractions derived from the industry such as e.g.:
  • Waste fraction from paper industry e.g. from recycling facilities
  • Waste fractions derived from agriculture or farming related sectors such as e.g.:
  • Waste fractions derived from municipal, county or state related or regulated activities such as e.g.:
  • the dry matter content of the mono- and/or polysaccharide containing waste fraction in the enzymatic hydrolysis and fermentation processes is in one embodiment above 20%, such as 20-80%, such as 20-50%, such as 20-45% and such as 20-40%.
  • Waste such as MSW
  • MSW is typically heterogeneous.
  • Statistics that provide firm basis for comparisons between countries concerning composition of waste materials are not widely known.
  • Standards and operating procedures for correct sampling and characterization remain unstandardized. Indeed, only a few standardised sampling methods have been reported (see e.g. Riber et al., 2007).
  • the composition exhibits seasonal and geographical variation, even over small distances of 200-300 km, see e.g., Dahlen et al., 2007; Hansen et al., 2007b; Muhle et al., 2010; Riber et al., 2009.
  • the dry weight of modern urban waste from Western Europe typically comprise on the order of 25% by weight of "vegetable and food waste” In China, in contrast, the relative proportions of "food waste” are typically increased by a factor of at least two relative to MSW from Western Europe, see e.g. Zhang et al. 2010.
  • Municipal solid waste may in particular comprise one or more of kitchen putrecibles, garden putrecibles, paper, card, plastics, miscellaneous combustible and non-combustible matters, textiles, glass, ceramics, metals, electronic devises.
  • municipal solid waste in the Western part of the world normally comprise one or more of: animal food waste, vegetable food waste, newsprints, magazines, advertisements, books and Phonebooks, office paper, other clean paper, paper and carton containers, other cardboard, milk cartons and alike, juice cartons and other carton with alu-foil, kitchen tissues, other dirty paper, other dirty cardboard, soft plastic, plastic bottles, other hard plastic, non-recyclable plastic, yard waste, flowers etc., animals and excrements, diapers and tampons, cottonsticks etc., other cotton etc., wood, textiles, shoes, leather, rubber etc., office articles, empty chemical bottles, plastic products, cigarette buts, other combustibles, vacuum cleaner bags, clear glass, green glass, brown glass, other glass, aluminium containers, alu-trays, alu-foil (including tealight candle foil), metal containers (-AI), metal foil (-AI), other sorts of metal, soil, rocks, stones and gravel, ceramics, cat litter, batteries (botton cells, alkali, thermometer
  • the waste to be processed is MSW.
  • MSW is processed in the method according to the invention as unsorted MSW, i.e. in which greater than 15% by weight of the dry weight is non- biodegradable material.
  • the dry weight of the non-biodegradable material is greater than 18% by weight, greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, or greater than 25% by weight.
  • An increase in the percentage of non-biodegradable material may indicate a decrease in degree of sorting, but also geographical and/or seasonal variations.
  • unsorted MSW may comprise organic waste, including one or more of food and kitchen waste; paper- and/or cardboard-containing materials; recyclable materials, including glass, bottles, cans, metals, and certain plastics; burnable materials; and inert materials, including ceramics, rocks, and debris.
  • waste to be processed such as MSW
  • MSW can be processed as sorted MSW.
  • waste to be processed can be source-separated organic waste comprising predominantly fruit, vegetable and/or animal waste.
  • sorting systems may be applied to MSW, for instance source sorting, where individual households dispose off different waste materials separately. Source sorting systems are currently in place in some municipalities in Austria, Germany, Luxembourg, Sweden, Belgium, the Netherlands, Spain and Denmark.
  • industrial sorting systems may be applied at the large scale plant prior to subjecting the waste to the combined enzymatic and micorbial treatment.
  • Means of mechanical sorting and separation may include any methods known in the art including but not limited to the systems described in US2012/0305688; W02004/101 183; W02004/101098; W02001/052993; W02000/0024531 ; W01997/020643; W01995/0003139; CA2563845; US5465847.
  • the combined enzymatic and microbial treatment included in the method of the invention typically produce an organic slurry, which also may be known as a bioliquid, or a liquid fraction.
  • the slurry is organic where it comprises predominantly organic material, but may also include inorganic contaminants.
  • a slurry, as used herein, is a liquid to the extent that it is pumpable, notwithstanding substantial content of undissolved solids.
  • components into a liquid slurry is increased with increased water content.
  • effective pulping of paper and cardboard which comprise a substantial fraction of MSW in some countries, is typically improved where water content is increased.
  • Water content provides a medium in which the microbial preparation can propagate and which dissolves metabolites.
  • enzyme activities may exhibit diminished activity when hydrolysis is conducted under conditions with low water content.
  • cellulases typically exhibit diminished activity in hydrolysis mixtures that have non-water content higher than about 10% by weight.
  • an effectively linear inverse relationship has been reported between substrate concentration and yield from the enzymatic reaction per gram substrate, see Kristensen et al. 2009.
  • the water content waste to be processed may preferably have a non water content of between above 10% or more and below 45%, such as 10 - 45%, or in some embodiments between 12 and 40%, or between 13 and 35%, or between 14 and 30%, or between 15 and 25%.
  • MSW may often comprise a considerable amount of water.
  • the water content may be adjusted in order to achieve appropriate non-water content.
  • the waste to be processed by the method of the present invention is suited for being processed by the method of the present invention when it comprises a mixture of biodegradable and non-biodegradable matter.
  • the method according to the invention has been applied to wet municipal solid waste, comprising above 20% biodegradable material by weight on a wet basis, such as 10 - 100%, 15 - 60%, such as 20 - 55%, such as 25 - 50%, such as 30 - 50%.
  • the content of the biodegradable matter has been determined after drying of the waste in accordance with standard methods known in the art.
  • the content of the biodegradable material in the waste processed by the method of the present invention has been found to be above 10% biodegradable material by weight on a dry basis, such as between 10-100%, such as 10 - 40%, such as 15 - 35%, such as 15 - 30% or such as 15 - 25%.
  • the waste When the method according to the present invention is to be used for approaches directed to the geographic origin of the waste, it will be necessary to register or at least to keep track of which geographic area the waste was disposed off. Normally, the waste is disposed off in the vicinity of its final use prior to becoming a“waste subject”. In the Western part of the world, renovation-collecting systems are put in place where the waste is collected by trucks or by other collecting means. Alternatively, the waste is placed at waste disposal sites usually subject to some kind of sorting principle. Regardless of whether the waste is collected by a truck at the vicinity of its final use, or collected by a truck at a waste disposal site, the waste normally enters the large scale plant in batches that correspond to the amount of waste that can be loaded on the specific truck or other transportation means. Optionally, more than one load from one truck may be collected in a collecting container such as a vessel or tank prior to subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant.
  • a collecting container such as a vessel or tank
  • step a) of the method according to the invention When the waste has been entered into the large scale plant, it is in accordance with step a) of the method according to the invention subjected to a combined enzymatic and microbial treatment.
  • Step a) The combined enzymatic and microbial treatment in step a) is performed by adding hydrolytic enzymes, supplied in either native form or in form of microbial organisms giving rise to the accumulation of such hydrolytic enzymes; and by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing
  • biochemicals ethanol, or biogas.
  • Enzymatic hydrolysis is initiated at that point in time at which isolated enzyme preparations are added.
  • isolated enzyme preparations are not added, but instead microorganisms that exhibit desired extracellular enzyme activities are used, enzymatic hydrolysis is initiated at that point which the desired microorganism is added.
  • enzymatic hydrolysis is conducted concurrently with microbial fermentation.
  • Concurrent microbial fermentation can be achieved using a variety of different methods.
  • microorganisms naturally present in the waste such as MSW
  • MSW microorganisms naturally present in the waste
  • the processed waste has not previously been heated to a temperature that is sufficient to effect a "sterilization.”
  • microorganisms present in waste such as MSW
  • the general beneficial effect of concurrent microbial fermentation is comparatively robust, meaning that a very wide variety of different organisms can, individually or collectively, contribute to organic capture through enzymatic hydrolysis of waste, such as MSW.
  • the treatment in step a) is accomplished by the use of one or more species of microorganisms present in the waste.
  • microbial fermentation can be accomplished by a direct inoculation using one or more microbial species.
  • one or more bacterial species used for inoculation so as to provide simultaneous enzymatic hydrolysis and fermentation of waste, such as MSW, can be advantageously selected where the bacterial species is able to thrive at a temperature at or near the optimum for the enzymatic activities used.
  • said combined enzymatic and microbial treatment in step a) is performed by adding hydrolytic enzymes, supplied in either native form or in form of microbial organisms giving rise to the accumulation of such enzymes; and by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing biochemicals, ethanol, or biogas.
  • Fermentable sugars obtained from the hydrolyzed waste material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a fermentation products such as one or more of volatile fatty acids (e.g. acetate, propionate, butyrate), lactate and alcohols.
  • volatile fatty acids e.g. acetate, propionate, butyrate
  • lactate e.g. acetate, propionate, butyrate
  • sugars released from the waste such as municipal solid waste material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast.
  • Hydrolysis and fermentation can be separate or simultaneous.
  • Examples of fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast.
  • Yeast include strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.
  • Examples of fermenting organisms that can ferment pentose sugars in their native state include bacterial and fungal organisms, such as some yeast.
  • Xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis ; and strains of Pichia, e.g., P. stipitis, such as P. stipitis CBS 5773.
  • Pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus. Organisms not capable of fermenting pentose sugars, such as xylose and arabinose, may be genetically modified to do so by methods known in the art.
  • Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans ⁇ , Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. scehatae ⁇ , Clostridium, such as C. acetobutylicum, C. thermocellum, and C. phytofermentans ; E. coli, especially E.
  • Geobacillus sp. Hansenula, such as Hansenula anomala ⁇ , Klebsiella, such as K. oxytoca ⁇ , Kluyveromyces, such as K. marxianus, K. lactis, K. thermotolerans, and K. fragilis ⁇ , Schizosaccharomyces, such as S. pombe ; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum and Zymomonas, such as Zymomonas mobilis.
  • the fermenting microorganism may have been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.
  • the fermenting organism may comprise one or more polynucleotides encoding one or more cellulolytic enzymes, hemicellulolytic enzymes, and accessory enzymes described herein.
  • the fermenting microorganism is typically added to the degraded waste such as municipal solid waste material or hydrolysate and the fermentation is performed for about 8 to about 96 h, e.g., about 24 to about 60 h.
  • the temperature is typically between about 26°C to about 60°C, e.g., about 32°C or 50°C, and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
  • the yeast and/or another microorganism are applied to the degraded waste, such as municipal solid waste material, and the fermentation is performed for about 12 to about 96 h, such as typically 24-60 h.
  • the temperature is above 20°C such asbetween about 20°C to about 60°C, e.g., about 25°C to about 50°C, about 32°C to about 50°C, or about 32°C to about 50°C
  • the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7 such as pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 6.5 or pH 7.
  • some fermenting organisms e.g., bacteria, have higher fermentation temperature optima.
  • Yeast or another microorganism is applied in amounts of approximately 10 5 to 10 12 , such as from approximately 10 7 to 10 10 , such as approximately 2 x 10 8 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g.,“The Alcohol Textbook” (Editors K. Jacques, T.P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.
  • a fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield.
  • A“fermentation stimulator” refers to stimulators for growth of the fermenting microorganisms, in particular, yeast.
  • Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E and any combinations thereof.
  • minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu and any combinations thereof.
  • Inoculation of the hydrolysis mixture so as to induce microbial fermentation can be accomplished by a variety of different means.
  • the treatment in step a) is accomplished by the use of one or more species of lactic acid bacteria, acetate-producing bacteria, propionate-producing bacteria, or butyrate-producing bacteria, including any combination thereof.
  • the treatment step a) comprises subjecting the waste with a microbially- derived cellulase activity of at least 30 FPU/L that is provided by one or more
  • microorganisms such as a microbial consortium, providing microbial fermentation.
  • the treatment step a) comprises addition of cellulase activity by inoculation with one or more microorganism(s) that exhibits extracellular cellulase activity.
  • the cellulase activity is preferably one or more (e.g., several) activities selected from the group comprising endoglucanases, cellobiohydrolases, and beta-glucosidases.
  • LAB inoculate using one or more species of LAB including but not limited to any one or more of the following, or genetically modified variants thereof: Lactobacillus plantarum, Streptococcus lactis, Lactobacillus casei, Lactobacillus lactis, Lactobacillus curvatus, Lactobacillus sake, Lactobacillus helveticus, Lactobacillus jugurti, Lactobacillus fermentum, Lactobacillus carnis, Lactobacillus piscicola, Lactobacillus coryniformis, Lactobacillus rhamnosus, Lactobacillus maltaromicus,
  • Lactobacillus pseudoplantarum Lactobacillus agilis, Lactobacillus bavaricus, Lactobacillus alimentarius, Lactobacillus uamanashiensis, Lactobacillus amylophilus, Lactobacillus farciminis, Lactobacillus sharpeae, Lactobacillus divergens, Lactobacillus alactosus, Lactobacillus paracasei, Lactobacillus homohiochii, Lactobacillus sanfrancisco,
  • Lactobacillus fructivorans Lactobacillus brevis, Lactobacillus ponti, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus viridescens, Lactobacillus confusus, Lactobacillus minor, Lactobacillus kandleri, Lactobacillus halotolerans, Lactobacillus hilgardi,
  • Lactobacillus kefir Lactobacillus collinoides, Lactobacillus vaccinostericus, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactobacillus leichmanni, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus salicinus, Lactobacillus gasseri, Lactobacillus suebicus, actobacillus oris, Lactobacillus brevis, Lactobacillus vaginalis, Lactobacillus pentosus, Lactobacillus panis, Lactococcus cremoris, Lactococcus dextranicum,
  • Lactococcus garvieae Lactococcus hordniae, Lactococcus raffinolactis, Streptococcus diacetylactis, Leuconostoc mesenteroides, Leuconostoc dextranicum, Leuconostoc cremoris, Leuconostoc oenos, Leuconostoc paramesenteroides, Leuconostoc
  • pseudoesenteroides Leuconostoc citreum, Leuconostoc gelidum, Leuconostoc carnosum, Pediococcus damnosus, Pediococcus acidilactici, Pediococcus cervisiae, Pediococcus parvulus, Pediococcus halophilus, Pediococcus pentosaceus, Pediococcus intermedius, Bifidobacterium longum, Streptococcus thermophilus, Oenococcus oeni , Bifidobacterium breve, and Propionibacterium freudenreichii, or with some subsequently discovered species of LAB or with other species from the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, or Carnobacterium that exhibit useful capacity for metabolic processes that produce lactic acid.
  • the treatment step a) comprises contacting the waste with a live lactic acid bacteria concentration of at least 1 .0 x 10 L 10 CFU/L.
  • a bacterial preparation used for inoculation may comprise a community of different organisms.
  • one or more naturally occurring bacteria which exist in any given geographic region and which are adapted to thrive in waste, such as MSW, from that region, can be used.
  • LAB are ubiquitous and will typically comprise a major component of any naturally occurring bacterial community within waste, such as MSW.
  • waste such as MSW
  • microbial fermentation has a pH lowering effect, especially where metabolites comprise short chain carboxylic acids/ fatty acids such as one or more of formate, acetate, butyrate, proprionate, or lactate. Accordingly, in some embodiments it can be advantageous to monitor and adjust pH of the concurrent enzymatic hydrolysis and microbial fermentation mixture.
  • inoculation is advantageously made prior to addition of enzyme activities, either as isolated enzyme preparations or as microorganisms exhibiting extra-cellular cellulase activity.
  • one or more naturally occurring bacteria adapted to thrive on waste, such as MSW from a particular region can be cultured on waste, such as MSW, or on liquefied organic component obtained by enzymatic hydrolysis of waste, such as MSW.
  • one or more cultured naturally occurring bacteria can then be added as an inoculum, either separately or supplemental to inoculation using recycled wash waters or process solutions.
  • bacterial preparations can be added before or concurrently with addition of one or more isolated enzyme preparations, or after some initial period of pre-hydrolysis.
  • one or more specific strains can be cultured for inoculation, including strains that have been specially modified or“trained” to thrive under enzymatic hydrolysis reaction conditions and/or to emphasize or de-emphasize particular metabolic processes.
  • it can be advantageous to inoculate waste, such as MSW, using one or more bacterial strains which have been identified as capable of surviving on phthalates as sole carbon source.
  • Such strains include but are not limited to any one or more of the following, or genetically modified variants thereof:
  • Tropicibacter phthalicus Gordonia JDC-2, Arthrbobacter JDC-32, Bacillus subtilis 3C3, Comamonas testosteronii, Comamonas sp E6, Delftia tsuruhatensis, Rhodoccoccus jostii, Burkholderia cepacia, Mycobacterium vanbaalenii, Arthobacter keyseri, Bacillus sb 007, Arthobacter sp. PNPX-4-2, Gordonia namibiensis, Rhodococcus phenolicus, Pseudomonas sp. PGB2, Pseudomonas sp.
  • Phthalates which are used as plasticizers in many commercial poly vinyl chloride preparations, are leachable and, in our experience, are often present in liquefied organic component at levels that are undesirable. In some
  • strains can be advantageously used which have been genetically modified by methods well known in the art, so as to emphasize metabolic processes and/or de- emphasize other metabolic processes including but not limited to processes that consume one or more of glucose, xylose or arabinose.
  • waste such as MSW
  • one or more bacterial strains which have been identified as capable of degrading lignin.
  • Such strains include but are not limited to any one or more of the following, or genetically modified variants thereof: Comamonas sp B-9, Citrobacter freundii, Citrobacter sp FJ581023, Pandorea norimbergensis, Amycolatopsis sp ATCC 39116, Streptomyces viridosporous, Rhodococcus jostii, and Sphingobium sp. SYK-6. See e.g. Bandounas et al. 201 1 ; Bugg et al. 201 1 ; Chandra et al. 201 1 ; Chen et al. 2012; Davis et al. 2012.
  • waste such as MSW typically comprises considerable lignin content, which is typically recovered as undigested residual after AD.
  • acetate-producing bacterial strain including but not limited to any one or more of the following, or genetically modified variants thereof: Acetitomaculum ruminis,
  • Acetobacterium wieringae Acetobacterium woodii, Acetogenium kivui, Acidaminococcus fermentans, Anaerovibrio lipolytica, Bacteroides coprosuis, Bacteroides propionicifaciens, Bacteroides cellulosolvens, Bacteroides xylanolyticus, Bifidobacterium catenulatum, Bifidobacterium bifidum , Bifidobacterium adolescentis, Bifidobacterium angulatum,
  • a butyrate-producing bacterial strain including but not limited to one or more of the following, or genetically modified variants thereof: Acidaminococcus fermentans, Anaerostipes caccae, Bifidobacterium adolescentis, Butyrivibrio crossotus, Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Clostridium acetobutylicum, Clostridium aurantibutyricum, Clostridium beijerinckii, Clostridium butyricium, Clostridium cellobioparum, Clostridium difficile, Clostridium innocuum, Clostridium kiuyveri, Clostridium pasteurianum, Clostridium perfringens, Clostridium proteoclasticum, Clostridium sporosphaeroides, Clostridium symbiosum
  • Coprococcus comes, Escherichia coli, Eubacterium barkeri, Eubacterium biforme,
  • Eubacterium cellulosolvens Eubacterium cylindroides, Eubacterium dolichum, Eubacterium hadrum, Eubacterium halii, Eubacterium limosum, Eubacterium moniliforme, Eubacterium oxidoreducens, Eubacterium ramulus, Eubacterium rectale, Eubacterium saburreum, Eubacterium tortuosum, Eubacterium ventriosum, Faecalibacterium prausnitzii,
  • a propionate-producing bacterial strain including but not limited to any one or more of the following, or genetically modified variants thereof: Anaerovibrio lipolytica, Bacteroides coprosuis, Bacteroides propionicifaciens, Bifidobacterium adolescentis, Clostridium acetobutylicum, Clostridium butyricium, Clostridium methylpentosum,
  • the present invention also relates to a method wherein in step a) the waste is treated with an enzyme composition comprising a cellulolytic background composition combined with one or more enzymes selected from (i) a protease; (ii) a lipase and (iii) a beta-glucanase; and optionally combined with one or more further enzymes selected from (iv) a pectate lyase; (v) a mannanase and (vi) an amylase.
  • an enzyme composition comprising a cellulolytic background composition combined with one or more enzymes selected from (i) a protease; (ii) a lipase and (iii) a beta-glucanase; and optionally combined with one or more further enzymes selected from (iv) a pectate lyase; (v) a mannanase and (vi) an amylase.
  • the treatment in step a) is accomplished by treating the waste with an enzyme composition comprising a cellulolytic background composition and one or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase.
  • the treatment in step a) is accomplished by treating the waste with an enzyme composition comprises a cellulolytic background composition and two or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase (e.g. a protease and a lipase; a protease and a beta-glucanase; or a lipase and a beta-glucanase).
  • treatment in step a) is accomplished by treating the waste with an enzyme composition comprising a cellulolytic background composition and (i) a protease,
  • the treatment in step a) is accomplished by treating the waste with an enzyme composition a cellulolytic background composition and (i) a protease, (ii) a lipase, and (iii) a beta-glucanase and further comprising one or more enzymes selected from (iv) a pectate lyase, (v) a mannanase, and (vi) an amylase.
  • an enzyme composition a cellulolytic background composition and (i) a protease, (ii) a lipase, and (iii) a beta-glucanase and further comprising one or more enzymes selected from (iv) a pectate lyase, (v) a mannanase, and (vi) an amylase.
  • the cellulolytic background composition comprises one or more enzymes selected from the group comprising: cellobiohydrolases I or variants thereof; cellobiohydrolases II or variants thereof; beta-glucosidases or variants thereof; polypeptides having cellulolytic enhancing activity; and/or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
  • the cellulolytic background composition comprises a cellobiohydrolase I or a variant thereof; a cellobiohydrolase II or a variant thereof; a beta-glucosidase or a variant thereof; and a polypeptide having cellulolytic enhancing activity; or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
  • the cellulolytic background composition comprises one or more enzymes selected from a) a cellobiohydrolase I or variant thereof;
  • cellobiohydrolase II or variant thereof (b) cellobiohydrolase II or variant thereof; (c) beta-glucosidase or variant thereof; and (d) a polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • the cellulolytic background composition may comprise one or more enzymes selected from (a) an Aspergillus fumigatus cellobiohydrolase I or variant thereof; (b) an Aspergillus fumigatus cellobiohydrolase II or variant thereof; (c) an Aspergillus fumigatus beta- glucosidase or variant thereof; and (d) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.
  • the cellulolytic background composition may comprise a commercial cellulolytic enzyme preparation.
  • Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLUCLAST® (Novozymes A/S), NOVOZYMTM 188 (Novozymes A/S), SPEZYMETM CP (Genencor Int.),
  • the cellulolytic enzyme preparation is added in an amount effective from about 0.001 to about 5.0 wt. % of solids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 to about 2.0 wt. % of solids.
  • bacterial endoglucanases examples include, but are not limited to one or more of: Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551 ; U.S. Patent No. 5,536,655; WO 00/70031 ; WO 05/093050), Erwinia carotovara
  • fungal endoglucanases examples include, but are not limited to one or more of: Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichoderma reesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reesei endoglucanase II (Saloheimo et al., 1988, Gene 63:1 1 -22),
  • Trichoderma reesei Ce ⁇ 5A endoglucanase II GenBank:M19373
  • Trichoderma reesei endoglucanase III Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563
  • GenBank:AB003694 Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228, GenBank:Z33381 ), Aspergillus aculeatus
  • cellobiohydrolases useful in the present invention include, but are not limited to one or more of: Aspergillus aculeatus cellobiohydrolase II (WO 201 1/059740), Aspergillus fumigatus cellobiohydrolase I (WO 2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO 2013/028928), Chaetomium thermophilum cellobiohydrolase I, Chaetomium
  • thermophilum cellobiohydrolase II Humicola insolens cellobiohydrolase I, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871 ), Penicillium occitan is cellobiohydrolase I (GenBank:AY690482), Talaromyces emersonii cellobiohydrolase
  • CEL6A Trichoderma reesei cellobiohydrolase II
  • Trichoderma reesei cellobiohydrolase II Trichoderma reesei cellobiohydrolase II
  • Trichophaea saccata cellobiohydrolase II WO 2010/057086
  • beta-glucosidases useful in the present invention include, but are not limited to one or more of: beta-glucosidases from Aspergillus aculeatus (Kawaguchi et al., 1996,
  • any“Auxiliary Activity 9 polypeptide” or“AA9” polypeptide can be used as a component of the enzyme composition.
  • Thermoascus crustaceous (WO 201 1/041504), Aspergillus aculeatus (WO 2012/125925), Thermomyces lanuginosus (WO 2012/1 13340, WO 2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporus alborubescens (WO 2012/122477), Trichophaea saccata (WO 2012/122477), Penicillium thomii (WO 2012/122477), Talaromyces stipitatus (WO 2012/135659), Humicola insolens (WO 2012/146171 ), Malbranchea cinnamomea (WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), and Chaetomium
  • thermophilum (WO 2012/101206), and Talaromyces thermophilus (WO 2012/129697 and WO 2012/130950).
  • a protease may be derived from the genus Bacillus, such as e.g. Bacillus
  • amyloliquefaciens such as e.g. the protease encoded by SEQ ID NO:1 as disclosed in W017076421 , or a protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 as disclosed in W017076421 .
  • a lipase is derived from the genus
  • Thermomyces sp. such as e.g. Thermomyces lanuginosus such as e.g. the lipase encoded by SEQ ID NO: 2 as disclosed in W017076421 (or a lipase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 as disclosed in W017076421 ) or wherein the (ii) a lipase is derived from the genus Humicola sp.
  • a beta-glucanase is derived from a member of the genus Aspergillus such as e.g. Aspergillus aculeatus such as e.g.
  • the beta-glucanase encoded by the sequence encoded by SEQ ID NO: 4 as disclosed in W017076421 or homologs thereof e.g., a beta- glucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 156875at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 as disclosed in W017076421 ).
  • a pectate lyase forms part of a multicomponent enzyme composition comprising pectate lyase, xylanase and cellulase activities such as e.g. Novozym 81243TM.
  • a mannanase is an endo-mannosidase derived from the genus Bacillus such as e.g. Bacillus bogoriensis such as e.g.
  • an amylase is an alpha-amylase derived from the genus
  • Rhizomucor such as e.g. Rhizomucor pusillus such as e.g. the alpha-amylase encoded by SEQ ID NO: 5 as disclosed in W017076421 or homologs thereof (e.g., an alpha-amylase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 as disclosed in W017076421 ).
  • an alpha-amylase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
  • the protease is present at a ratio between 0-20% w/w, such as e.g. 10% w/w of the total enzyme protein.
  • the beta-glucanase is present at a ratio between 0-30% w/w, such as e.g. 15% w/w of the total enzyme protein.
  • the pectate-lyase is present at a ratio between 0-10% w/w, such as e.g. 5% w/w of the total enzyme protein.
  • the mannanase or amylase is present at a ratio between 0-10% w/w, such as e.g. 5% w/w of the total enzyme protein.
  • the cellulolytic enzyme blend is present at a ratio between 40%-99% w/w, such as e.g. between 50%-90% w/w, such as e.g. 60%-80% w/w, such as e.g. 65-75% of the total enzyme protein.
  • the enzyme composition further comprises one or more enzymes selected from a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein (CIP) an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.
  • a cellulase an AA9 polypeptide
  • a hemicellulase a cellulose inducible protein (CIP) an esterase
  • an expansin a ligninolytic enzyme
  • an oxidoreductase an oxidoreductase
  • pectinase a pectinase
  • protease aswollenin
  • the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
  • the enzymatically hydrolysing of the biodegradable parts of the waste concurrently with microbial fermentation according to step a) of the present invention may be performed at a temperature above 20°C and up to 75°C resulting in liquefaction of biodegradable parts of the waste and accumulation of microbial metabolites.
  • the method according to treatment step a) is performed at a
  • enzymatic hydrolysis are conducted within the temperature range 30 to 35°C, or 35°C to 40° C, or 40°C to 45°C, or 45°C to 50°C, or 50°C to 55°C, or 55°C to 60°C, or 60°C to 65°C, or 65°C to 70°C, or 70°C to 75°C.
  • the temperature to which waste such as MSW is heated is the highest average temperature of waste such as MSW achieved within the reactor. In some embodiments, the highest average temperature may not be maintained for the entire period.
  • the heating reactor may comprise different zones such that heating occurs in stages at different temperatures. In some embodiments, heating may be achieved using the same reactor in which enzymatic hydrolysis is conducted. The object of heating is simply to render the majority of cellulosic waste and a substantial fraction of the plant waste in a condition optimal for enzymatic hydrolysis. To be in a condition optimal for enzymatic hydrolysis, waste should ideally have a temperature and water content appropriate for the enzyme activities used for enzymatic hydrolysis.
  • agitation can comprise free-fall mixing, such as mixing in a reactor having a chamber that rotates along a substantially horizontal axis or in a mixer having a rotary axis lifting the waste such as MSW or in a mixer having horizontal shafts or paddles lifting the waste such as MSW.
  • agitation can comprise one or more of shaking, stirring or conveyance through a transport screw conveyor. In some embodiments, agitation continues after waste such as MSW has been heated to the desired temperature.
  • agitation is conducted for between 1 and 5 min, or between 5 and 10 min, or between 10 and 15 min, or between 15 and 20 min, or between 20 and 25 min, or between 25 and 30 min, or between 30 and 35 min, or between 35 and 40 min, or between 40 and 45 min, or between 45 and 50 min, or between 50 and 55 min, or between 55 and 60 min, or between 60 and 120 min.
  • the enzymatic liquefaction and fermentation in step a) is performed continuously at a certain residence time, at the optimal temperature and pH for enzyme performance.
  • the treatment step a) is conducted for a period of time in the range of 1 - 10Oh, in another embodiment the treatment step a) is conducted for a period of time in the range of 2.5 - 48h, in another embodiment the treatment step a) is conducted for a period of time in the range of 4 - 36h, and in a preferred embodiment, the treatment step a) is conducted for a period of time in the range of 5 - 25h, 6 - 18h, 8 - 15h or around 12h.
  • treatment step a the microbial fermentation is conducted under pH conditions that is adjusted to the optimum pH for the enzymatic activity, which is generally a pH at 7 or below.
  • the pH may be adjusted in order to discourage methane production by methanogens, for example, by adjusting the pH to less than 6.0, or less than 5.8, or less than 5.6, or less than 5.5.
  • treatment step a) is conducted at pH conditions that is
  • pH such as at pH 6.5, or at pH 6.0, or at pH 5.5, or at pH 5.0.
  • the pH may be adjusted by any suitable means known in the art.
  • Step b) in the method of the present invention is a separation step where the bio-liquid is separated from the non-degradable fractions. Clean and efficient use of the degradable component of waste, such as MSW, combined with recycling typically requires some method of sorting to separate degradable from non-degradable material.
  • the separation in step b) may be performed by any means known in art, such as in a ballistic separator, washing drums or hydraulic presses.
  • the liquid fraction is separated from the solid fraction of the waste at one or more of the following steps:
  • the liquefied, fermentable parts of the waste are separated from non-fermentable solids.
  • at least 40% of the non-water content of this bioliquid comprises dissolved volatile solids, or at least 35%, or at least 30%, or at least 25%.
  • at least 25% by weight of the dissolved volatile solids in the bioliquid comprise any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate.
  • at least 70% by weight of the dissolved volatile solids comprises lactate, or at least 60%, or at least 50%, or at least 40%, or at least 30%, or at least 25%.
  • the liquid fraction in step b) has a pH below 7.0, such as a pH at 6.5, or a pH at 6.0, or a pH at 5.5, or a pH at 5.0, or a pH at 4.5. In other embodiments the liquid fraction in step b) has a pH below 6.5, a pH below 6.0, a pH below 5.5, a pH below 5.0, or a pH below 4.5.
  • separation of non-fermentable solids from liquefied, fermentable parts of the waste, such as MSW, so as to produce a bioliquid characterized in comprising dissolved volatile solids of which at least 25% by weight comprise any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate is conducted in less than 16 h after the initiation of enzymatic hydrolysis, or in less than 18 h, or in less than 20 h, or in less than 22 h, or in less than 24 h, or in less than 30 h, or in less than 34 h, or in less than 36 h.
  • the non-water content of the liquid fraction in step b) comprises at least 40% by weight dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate.
  • Separation of liquefied, fermentable parts of the waste from non-fermentable solids can be achieved by a variety of means. In some embodiments, this may be achieved using any combination of at least two different separation operations, including but not limited to screw press operations, ballistic separator operations, vibrating sieve operations, or other separation operations known in the art.
  • the non-fermentable solids separated from fermentable parts of the waste comprise at least about 20% of the dry weight of the waste such as MSW, or at least 25%, or at least 30%. In some embodiments, the non-fermentable solids separated from fermentable parts of the waste comprise at least 20% by dry weight of recyclable materials, or at least 25%, or at least 30%, or at least 35%.
  • separation using at least two separation operations produces a bioliquid that comprises at least 0.15 kg volatile solids per kg waste, such as MSW processed, or at least 0.10 kg volatile solids per kg waste, such as MSW, processed.
  • MSW processed at least 0.15 kg volatile solids per kg waste
  • MSW volatile solids per kg waste
  • the figure 0.15 kg volatile solids per kg waste, such as MSW, processed reflects a total capture of biogenic material in typical unsorted waste, such as MSW, of at least 80%.
  • the calculation of kg volatile solids captured in the bioliquid per kg waste, such as MSW, processed can be estimated over a time period in which total yields and total waste, such as total MSW, processed are determined.
  • the ballistic separator separates the enzymatic treated waste such as MSW into the bio liquid, a fraction of 2D non-degradable materials and a fraction of 3D non-degradable materials.
  • the 3D fraction physical 3 dimensional objects as cans and plastic bottles
  • the 2D fraction textiles and foils as examples
  • Inert material which is sand and glass is sieved from the bio-liquid.
  • the water used in all the washing drums can be recirculated, heated and then used as hot water in the first step for heating.
  • step c) of the method of the present invention the abundance of one or more chemical compound(s) or class(es) of compound(s) in the liquid fraction obtained in step b) is/are determined, or the identity of said chemical compound(s) or class(es) of compounds in the liquid fraction obtained in step b) is/are established.
  • the identity of a chemical compound can be detected if it is present in the liquid fraction obtained in step b) in an amount above 0.00001 ppm.
  • any chemical compound measured in step c) is present in the liquid fraction obtained in step b) in an amount ranging from 0.00001 ppm to 90 ppm, such as from 0.0001 ppm to 90 ppm, or such as from 0.001 ppm to 90 ppm, or such as from 0.01 ppm to 90 ppm, or such as from 0.1 ppm to 10 ppm.
  • the identity of a chemical compound can be detected if it is present in the liquid fraction obtained in step b) in an amount above 7E-02 ng/ml in 50 ml. of said liquid fraction obtained from step b).
  • step c) a sample of the liquid fraction obtained from step b) is prepared in order to separate phases by centrifugation into supernatant and precipitate prior to determining the abundance of or establishing the identity of the chemical compound(s) or class(es) of compounds.
  • step b) It may also be necessary to prepare the precipitate fraction of the separated sample of bioliquid obtained from step b) further by one or more means.
  • the separated precipitate fraction may be further prepared by one or more of the following treatments: pressurized liquid extraction of the precipitate with silica and dichloromethane; pressurized liquid extraction of the precipitate with silica and
  • the separated precipitate fraction is further prepared by pressurized liquid extraction of the precipitate with silica and dichloromethane.
  • the separated precipitate fraction may be further prepared pressurized liquid extraction of the precipitate with silica and dichloromethane followed by extraction with silica and methanol.
  • the separated supernatant fraction may further be prepared by one or more of the following treatments: liquid-liquid extraction with pentane, liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane, liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane followed by liquid- liquid extraction with dichloromethane, optionally adjusting the sample pH to 2 or 12.
  • the separated supernatant fraction is further prepared by liquid-liquid extraction with pentane. In another preferred embodiment the separated supernatant fraction is further prepared by liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane. In another preferred embodiment the separated supernatant fraction is further prepared by liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane followed by liquid-liquid extraction with dichloromethane.
  • the pH may optionally be adjusted to pH 2 or to pH 12.
  • the abundance of the chemical compound(s) or class(es) of compound(s) present in either the supernatant or the precipitate fraction may the determined, or the identity of the chemical compound(s) or class(es) of compound(s) may be established by one or more of the following methods: liquid chromatography, mass spectrometry, gas chromatography, liquid chromatography- mass spectrometry, gas chromatography-mass spectrometry, gas chromatography- electrospray ionization-mass spectrometry, liquid chromatography-electrospray ionization- mass spectrometry.
  • the liquid obtained from step b) is separated into precipitate and supernatant and the compound(s) or class(es) of compound(s) in the precipitate is determined or established in step c) by preparing with pressurized liquid extraction with silica and dichloromethane followed by gas-chromatography mass spectrometry.
  • the liquid obtained from step b) is separated into precipitate and supernatant and the compound(s) or class(es) of compound(s) in the precipitate is determined or established in step c) by preparing with pressurized liquid extraction with silica and dichloromethane followed by pressurized liquid extraction with silica and methanol followed by liquid chromatography-mass spectrometry.
  • the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by liquid chromatography-mass spectrometry.
  • the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by prepareing with liquid-liquid pentane extraction followed by gas chromatography-mass spectrometry.
  • the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by prepareing with liquid-liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry.
  • the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by preparing with liquid-liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by adjusting the aqueous phase to pH 2 or to pH12 and subjecting to liquid-liquid dichloromethane extraction followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry.
  • the identity/abundance of the chemical compound(s) or class(es) of compound(s) may be determined using one or more strategies. If the scope is to determine the
  • the results obtained from the bioliquid sample may be compared to spiked samples containing a known amount of one or more target compounds. If the scope is to screen the bioliquid for several compounds, the results obtained from the samples comprising the bioliquid may be compared with one or more pre-selected chromatography signatures of known compounds. If the scope is a broader screen for various different compounds, the results obtained from the samples comprising the bioliquid may be compared with profiles of liquid-chromatography, mass- spectrometry, and/or gas-chromatography, respectively, from databases of known compounds.
  • determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing a sample from the liquid fraction obtained in step b) with spiked samples comprising known amounts of one or more target compounds. In another preferred embodiment, determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing a sample from the liquid fraction obtained in step b) with spiked samples comprising known amounts of one or more target compounds wherein said spiked sample used for quantification comprises e.g. at or above 10 ng/mL, such at 10 ng/ml, 100 ng/mL or 1000 ng/mL of the target compound to be quantified.
  • determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing said sample from the waste batch by liquid-chromatography, and/ or gas-chromatography with one or more standards of known concentration analyzed by the same method.
  • determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by identifying one or more chemical compounds from said sample from the waste batch by one or more of liquid- chromatography, mass-spectrometry, and/or gas-chromatography and comparing the obtained data with profiles of liquid-chromatography, mass-spectrometry, and/or gas- chromatography, respectively, from databases of known compounds.
  • step c) of the present method have proven to be of very various character, and it is believed that the method will be applicable to most if not all chemical compounds that are present in the bioliquid fraction obtained from step b) according to the present invention
  • the method according to the present method has shown to be suitable for measuring ingredients and/or by-products of fat, fatty acids and derivatives, compounds from biological sources, pharmaceuticals, plasticizers, plastic additives, pesticides and anitmicrobial compounds, food additives, cosmetic additives and fluorinated compounds.
  • the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing fat and fatty acids. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to compounds from biological sources. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing pharmaceuticals. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing cosmetics. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing plastic additives.
  • the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing plasicisers. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing fluorinated compounds.
  • the method of the present invention has proven suitable for measuring chemical compound(s) or class(es) of compound(s) selected from organic acids, free fatty acids, esters, alcohols, alkanes, phthalates, amides, bisphenols, aromatic, and poly-aromatic compounds and their heterocyclic, alkyl-, hydroxyl-, and carboxylated variants.
  • the chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s). In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are free fatty acids. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are esters. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are alcohols. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are alkanes. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are phthalates.
  • the chemical compound or class(es) of compounds determined or established in step c) is/are amides. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are bisphenols. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are aromatic, and poly aromatic compounds and their heterocyclic, alkyl-, hydroxyl-, and carboxylated varieties.
  • the chemical compound(s) or class(es) of compound(s)s determined or established in step c) is/are organic acid(s) selected from one or more of: carboxylic acids, sulfonic acids, alcohols, and organic compounds comprising an acidic thiol-, enol-, or phenol-group.
  • the chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s) selected from one or more of: 1 - adamantanecarboxylic acid, benzenepropanoic acid, 4-butylbenzoic acid,
  • cyclohexanecarboxylic acid cyclohexanepentanoic acid, dicyclohexylacetic acid, diphenic acid, 1 -hydroxy-2-naphthoic acid, linolenic acid, myristic acid, 1 -naphthoic acid, 2-naphthoic acid, oleic acid, palmitic acid, palmitoleic acid, pentadecanoic acid, phthalic acid, 1 - pyrenecarboxylic acid, salicylic acid, sorbic acid.
  • the chemical compound determined or established in step c) is selected from one or more of the following groups of compounds: Linear
  • LAS alkylbenzensulfonates
  • PAH Polycyclic aromatic hydrocarbon
  • NPE Nonylphenol and ethoxylates
  • NPE comprise nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2-ethylhexyl)phthalate (DEHP); hormone-disturbing compounds e.g. bisphenol-A; drugs (amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa); pesticides; ingredients from personal care products (limonene, parabens); plasticisers other than DEHP; biocides (e.g. triclocarban, 2-phenylphenol, DEET); problematic compounds from paper and packaging industry (PFOS, PFOA).
  • PFOS PFOS
  • PFOA paper and packaging industry
  • the chemical compound determined or established in step c) is selected from one or more of: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one N,N-dimethyl-1 -dodecanamine, Benzophenone, Oleic acid, 4-hydroxy-3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, Methyl oleate, 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3- methylindole, 2-methylindole, 4-hydroxy-3,5-di-tert-butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cot
  • the method further comprises steps for linking the origin of the waste to the specific chemical compounds found in the waste.
  • steps for linking the origin of the waste to the specific chemical compounds found in the waste comprise registering the geographic origin of a waste batch; the point in time wherein a given batch is delivered to the large scale plant and the point in time for processing the batch. This may sometimes require that a marker is added to the waste.
  • the steps may also be a registration of the point in time when a batch enters and/or leaves one or more separation step(s).
  • the method according to the present invention comprises in addition to step a), b) and c) one or more of:
  • Anaerobic digestion is a series of biological processes in which microorganisms break down biodegradable material in the absence of oxygen.
  • One of the end products is biogas, which can be combusted to generate electricity and heat, or can be processed into renewable natural gas and transportation fuels.
  • a range of anaerobic digestion technologies exists in the state of the art for converting waste, such as municipal solid waste, municipal wastewater solids, food waste, high strength industrial wastewater and residuals, fats, oils and grease (FOG), and various other organic waste streams into biogas.
  • waste such as municipal solid waste, municipal wastewater solids, food waste, high strength industrial wastewater and residuals, fats, oils and grease (FOG), and various other organic waste streams into biogas.
  • Many different anaerobic digester systems are commercially available and the skilled persion will be familiar with how to apply and optimize the anaerobic digestions process.
  • the metabolic dynamics of microbial communities engaged in anaerobic digestion are complex.
  • a preferred aspect of the invention is a method comprising the following steps:
  • step c) Subjecting the liquid obtained from step b) to step c);
  • step c) if the liquid obtained in step b) is subject to further treatment and separation steps and/or to anaerobic digestion.
  • the markers used in the method can be any marker known in the art that can be tracked and the choice of specific marker may depend on the waste to be processed.
  • the marker may be selected from one or more of: a radioactive labelled marker, a specific chemical compound that is resistant to bio-degradation, or a maker that can be visualised during the various steps of the method such as a fluorescent marker.
  • the present invention relates to the use of the method according to the present invention for decreasing the abundance of or eliminating the presence of one or more chemical compound(s) in waste such as MSW before, during or after anaerobic digestion of the waste, by adjusting one or more of the processing parameters in step a) or step b).
  • the present invention relates to the use of the method according to the present invention for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste, such as MSW; based on the presence of one or more chemical compounds measured in step c).
  • said chemical compound(s) could be any organic compound(s).
  • said chemical compound(s) could be any organic compound(s)
  • compound(s) of interest preferably a compound(s) within one or more of the following groups of compounds: Linear alkylbenzensulfonates (LAS); Polycyclic aromatic
  • NPE Nonylphenol and ethoxylates
  • NPE comprise nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2-ethylhexyl)phthalate (DEHP); hormone-disturbing compounds e.g.
  • bisphenol-A bisphenol-A; drugs (amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa); pesticides; ingredients from personal care products (limonene, parabens);
  • plasticisers other than DEHP biocides (e.g. triclocarban, 2-phenylphenol, DEET);
  • said chemical compound(s) is selected from one or more of the following compound(s): butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one, Para-cresol, Skatole, N,N-dimethyl-1 - dodecanamine, Benzophenone, Oleic acid, 4-hydroxy-3,5-ditert-butylbenzaldehyde,
  • the chemical compound measured is selected from one or more of: lactic acid, acetate, propionate andbutyrate. In another embodiment, the chemical compound measured is selected from one or more of: glucose, xylose, arabinose, lactate, acetale and ethanol.
  • the processing parameters to be adjusted could be any of the parameters mentioned above in relation to step a) and/or step c) of the method of the invention i.e the ratio of biodegradable waste to non-biodegradable waste, the amount of enzyme added to the waste, the identity of the specific enzyme composition added to the waste, the addition of microorganism to the waste, the temperature applied in step a), the retention time of the waste in step a), the pH applied in step a), the non-water content of the liquid fraction in step b),
  • the processing parameter to be adjusted could be the retentiontime of the waste in bio-reactor and/or the retentiontime of the bioliqued in the subsequent AD process.
  • the method according to the present invention can be applied to any kind of waste comprising a mixture of biodegradable and non-biodegradable matter.
  • the waste is derived from or comprises any one or more of waste from household, industry, agriculture, farming, county, or state activities.
  • the method according to the invention has been applied to wet municipal solid waste, comprising above 20% biodegradable material by weight on a wet basis, such as 15 - 60%, such as 20 - 55%, such as 25 - 50%, such as 30 - 50%.
  • the content of the biodegradable matter has been determined after drying of the waste in accordance with standard methods known in the art.
  • the content of the biodegradable material in the waste processed by the method of the present invention has been found to be above 10% biodegradable material by weight on a dry basis, such as between 10 - 40%, such as 15 - 35%, such as 15 - 30% or such as 15 - 25%.
  • the waste is municipal solid waste. Such waste may be sorted or unsorted. In one embodiment, the waste is unsorted municipal solid waste. In one embodiment, the waste is centrally sorted municipal solid waste. In one embodiment, the waste is source sorted municipal solid waste from households. In one embodiment, the waste is municipal solid waste processed by shredding or pulping. In one embodiment, the waste is organic fractions and paper rich fractions. In one embodiment, the waste is Refuse-Derived-Fuel fractions. In a preferred embodiment, the biodegradable material in said municipal solid waste comprises a combination of one or more items selected from: food residues, paper, cardboard, and fines.
  • the waste is sorted municipal solid waste not comprising items selected from one or more of the following: domestic appliances, glass, ceramics, batteries, newsprints, magazines, advertisements, books, plastics, fabrics, textiles, yard waste, electrical and electronic equipment, chemicals, pharmaceuticals, metals.
  • one or more of the following groups of items are removed from the waste prior to the combined enzymatic and microbial treatment in step a): leaves, grasses, wood, fabrics, stones, plastics, metals.
  • the waste is selected from one or more of general industry waste fractions containing paper or other organic fractions, waste fractions from paper industry or recycling facilities, waste fractions from food and feed industry, waste fractions from the medicinal industry.
  • the waste is selected from one or more of agriculture or farming, waste fractions from processes of sugar or starch rich products, contaminated or spoiled agricultural products not exploitable for food or feed purposes, manure, manure derived products.
  • the waste is selected from one or more of waste fractions derived from municipal, county or state related or regulated activities, sludge from waste water treatment plants, fibre or sludge fractions from biogas processing, general waste fractions from the public sector containing paper or other organic fractions.
  • the said waste is subjected to pre-treatment prior to step a).
  • the pre-treatment may depend upon the dry-matter content of the waste to be processed.
  • the method according to the present invention is suitable for waste with high dry-matter content, such as waste having a dry-matter content above 20%.
  • the dry-matter content may be even higher if the waste to be processed is municipal solid waste.
  • the dry- matter of the waste could be up to 75%, such as waste comprising 20 - 75% dry-matter, such as 30 - 75% dry-matter, such as 40 - 75% dry matter, such as 50 - 75% dry-matter, or such as 55 - 70% dry-matter.
  • Any pre-treatment process known in the art can be used to disrupt plant cell wall components of the municipal solid waste material (Chandra et a!., 2007, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv. Biochem. Engin./Biotechnol. 108: 41 -65; Hendriks and Zeeman, 2009, Bioresource Technology 100: 10-18; Mosier et al., 2005, Bioresource Technology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci. 9: 1621 -1651 ; Yang and Wyman, 2008, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).
  • waste such as MSW
  • a mild to severe temperature pretreatment in the range 10-300°C prior to hydrolysis. Heating will normally occur together with a mixing. Heating will normally be carried out by addition of water or steam.
  • Pretreatment might also consist of a separation (manual or automatic) of waste such as MSW in different fractions.
  • the municipal solid waste material can also be subjected to particle size reduction, sieving, pre-soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art.
  • such pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
  • the MSW may be pretreated both physically (mechanically) and chemically. Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof.
  • high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi.
  • high temperature means temperature in the range of about 100 to about 300°C, e.g., about 140 to about 200°C.
  • mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.
  • the physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.
  • the municipal solid waste material may accordingly be subjected to physical (mechanical) or chemical pre-treatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose, and/or lignin.
  • Mechanical or physical pre-treatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof.
  • high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi.
  • high temperature means temperature in the range of about 100 to about 300°C, e.g., about 140 to about 200°C.
  • mechanical or physical pre-treatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.
  • the physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.
  • the waste may also be subjected to any biological pre-treatment known in the art that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the municipal solid waste material.
  • Biological pretreatment techniques can involve applying lignin- solubilizing microorganisms and/or enzymes
  • Pre-treatment in general is required if subsequent hydrolysis (e.g. enzymatic hydrolysis) of the polysaccharides requires the break down of an otherwise protecting structure (e.g. lignin) of plant materials.
  • Several pre-treatment techniques are known within e.g. the field of bioethanol production. Pre-treatment-processes may be based on e.g. acidic hydrolysis, steam explosion, oxidation, extraction with alkali or ethanol etc.
  • a common feature of the pre treatment techniques is that combined with the action of possible added reactants they take advantage of the softening and loosening of plant materials that occurs at temperatures above 100°C, i.e. a process requiring the application of pressure.
  • the waste to be treated in accordance with the present method may optionally be subjected to pre-treatment prior to step a) and the pre-treatment is selected from one or more of: acid hydrolysis, steam explosion, oxidation, extraction with alkali, extraction with ethanol, sorting, shredding, pulping, pressure, size fractionation, bag opening, free fall mixing, stirring, rotation.
  • the waste is subjected to acid hydrolysis pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to steam explosion pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to oxidation pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to extraction with alkali pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to extraction with ethanol pre-treatment prior to step a) and optionally one or more other pre treatment means.
  • the waste is subjected to sorting pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to shredding pre-treatment prior to step a) and optionally one or more other pre treatment means.
  • the waste is subjected to pulping pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to pressure pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to size fractionation pre treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to bag opening pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to free fall mixing pre-treatment prior to step a) and optionally one or more other pre-treatment means.
  • the waste is subjected to stirring pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to rotation pre-treatment prior to step a) and optionally one or more other pre treatment means.
  • Pre-treatment of municipal solid waste may be performed at atmospheric pressure which reduces the energy cost, the equipment cost and the mechanical difficulties significantly. Further more no chemical addition is needed in the pre-treatment.
  • the waste comprises fractions where the polysaccharides mainly are sugars, starch or already pre-treated cellulose as paper, cardboard or similar.
  • the costs for enzymes also are kept low, as amylases in general are cheaper than cellulases. This is a low-priced extraction of monosaccharides.
  • Unconverted lignocellulosics can possibly be sorted out after the fermentation and used for instance in a process with high pressure pre-treatment.
  • the purpose of the pre-treatment may be to minimize the amount of unwanted
  • microorganisms through biocidal activities prior to the actual enzymatic treatment.
  • Examples of such activities are radioactive radiation, UV-radiation and electroporation.
  • thermal treatment softening of processed biomass such as various paper fractions, break down of the internal structure of biomass with high water content and low lignin content such as vegetables, and opening of starch structures occurs in addition to the biocidal effect. If a proper performance of the subsequent hydrolysis of the
  • polysaccharides requires the decomposition of an otherwise protecting structure (e.g. lignin) of the original lignocellulosic material, a pressurised pre-treatment might be required.
  • an otherwise protecting structure e.g. lignin
  • the purpose of the pre-treatment in this is non-pressurised is not to break down protecting structures of non-processed biomass or the like, but rather to delimit microbial activity and to soften the processed waste fraction.
  • the non-pressurised pre-treatment process is preferably based on steam admission, but can optionally be supplemented with acidic or alkali compounds.
  • polysaccharide containing waste fractions preferably utilises steam to heat up the waste to approximately 100 5 C.
  • Pre-treatment temperature 60-1 10°C, such as 65-105°C, such as 70-105°C, such as 75- 105°C and such as 80-100°C
  • Pre-treatment time 0-120 min, such as 5-100 min, such as 10-90 min, such as 20-80 min and such as 30-60 min.
  • Pre-treatment steam admission 0-2 kg/kg dry matter, such as 0.01 -1.5 kg/kg dry matter, such as 0.02-1.0 kg/kg dry matter, such as 0.03-0.8 kg/kg dry matter and such as 0.05- 0.5 kg/kg dry matter.
  • the pre-treatment is a non-pressurised pre-treatment for up to 120 min with a temperature ranging between 60-1 10°C and a steam admission of up to 2 kg/kg dry matter.
  • the non-pressurised pre-treatment described above is particularly suitable for waste having a relatively high dry matter content, preferably above 20%, and including relatively large particles.
  • the treatment of said material can be carried out without additional water supply, detoxification or mechanical shredding.
  • Cooling and heating of the mash could optionally be performed by circulating cold or hot water (e.g. district heating water) through a vessel jacket or by injecting steam or cold water directly into the vessel.
  • cold or hot water e.g. district heating water
  • steam or cold water directly into the vessel.
  • additional water Prior to the enzymatic hydrolysis it may also be necessary to add additional water in order to reach an appropriate dry matter content, for the enzymes.
  • 0.5 - 2.5 kg water per kg waste having a DM of up to 75% is added to the waste prior to subjecting said waste to step a).
  • part or all of the water added to the waste prior to subjecting the waste to step a) is recycling wash water that is added to the incoming waste stream.
  • waste with a dry matter content above 20% is processed mechanically, e.g. by free fall mixing while subjected to pre-treatment and/or to step a).
  • Waste such as MSW
  • waste from big cities is often collected as is in plastic bags.
  • the waste, such as MSW, is typically transported to the large scale waste refinery plant where it may become stored in a silo until processing.
  • a sorting step can be installed in front of the system to take out oversize particles.
  • waste such as sorted or unsorted MSW is size fractionated into fractions, providing a fraction with a size range of e.g. 0 to 60 cm, and/or providing an oversize fraction (bulk refuse refraction), such as a fraction comprising waste with a size exceeding 60 or more cm.
  • the oversize fraction may be pre-treated mechanically by any known means in order to reduce the size of the particles.
  • the waste in the oversize fraction may be subject to further pre-treatment as described herein or directly enter into step a) of the invention.
  • An important aspect of the present invention is the use of the information that is obtained in step c) of the method of the invention.
  • step c) of the method of the invention in a given batch of waste and link this to the geographic origin of the waste. This is possible with the present invention in those embodiments where the method of the invention in addition to step a), b) and c) also comprises the following steps:
  • step b) Subjecting the treated waste from step a) to step b);
  • step c) Subjecting the liquid obtained from step b) to step c);
  • step c) if the liquid obtained in step b) is subject to further treatment and separation steps.
  • Chemical compounds that may be of interest to idenity and/or quantify for use of the method according to the invention is for identifying the local geographical origin of specific chemical compounds present in the waste and optionally identifying the specific leaking or additive source(s) of said chemical compound (s).
  • use of the method according to the invention is for identifying the local geographical origin of chemical compounds present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of fat, fatty acids or derivatives thereof present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of compounds from biological sources present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of medicinal drugs/pharmaceuticals and degradation products thereof present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of plasticizers present in the waste.
  • use of the method according to the invention is for identifying the local geographical origin of plastic additives present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of pesticides or antimicrobial compounds present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of food additives present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of cosmetic additives present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of fluorinated compounds present in the waste.
  • use of the method according to the invention is for identifying the local geographical origin of chemical compounds subject to legal restrictions regarding their manufacture, import, use or disposal present in the waste.
  • the leaking or additive source(s) of said chemical compound (s) may optionally be identified.
  • This use may be directed to approaching the residents of the geographic area where the waste originated from with the purpose of preventing materials/compositions that was found in the waste to be present in future waste or to direct information or sales campaigns to a specific geographic area.
  • the use may also be directed at a public affairs level with the purpose of urban planning, public health, detection of activities etc.
  • Use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste includes directing information campaigns, reward campaigns, fees for disposal of waste, sales activities, local sorting of waste, ; to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
  • use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing information campaigns to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
  • use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing reward campaigns to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
  • use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing sales activities to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
  • use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing local sorting of waste to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
  • use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing environmental or social improvements to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
  • the identity and/or abundance of a certain chemical compound(s) or class(es) of compounds from a certain geographic area is registered and compared over time so that the occurrence of chemical compounds of interest can be followed or surveilled.
  • a furhter embodiment of the invention is the use of a method according to the invention for decreasing the abundance of or eliminating the presence of a chemical compound measured in step c) in waste before, during or after fermentation of the waste by adjusting one or more of the processing parameters in step a) or step b) as described above.
  • the present invention relates to the use of the method according to the present invention for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste, such as MSW; based on the presence of one or more chemical compounds measured in step c).
  • a preferred embodiment of the invention is the use of a method according to the invention for degrading one or more of benzenepropanoic acid, benzyl benzoate, caffeine, ethyl oleate, eugenol, myristic acid, which are released during step a) by subsequent anaerobic digestion .
  • Another preferred embodiment of the invention is the use of a method according to the invention further comprising an anaerobic digestion step wherein cholestan-3-one is generated.
  • Another preferred embodiment of the invention is the use of a method according to the present invention wherein in step b) polar and ionic chemical compounds primarily distribute to the bioliquid and hydrophobic chemical compounds primarily partition to the solid fraction.
  • use of the method according to the invention is for identifying one or more of the following chemical compounds: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one, Para-cresol, Skatole, N,N-dimethyl-1 -dodecanamine, Benzophenone, Oleic acid, 4-hydroxy-3,5-ditert- butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, Methyl oleate, 13- Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3-methylindole, 2- methylindole, 4-hydroxy-3,5-ditert-butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine
  • this includes waste that comprises both biodegradable and non-biodegradable material and particularly when the waste comprises 10-60% biodegradable material on a wet basis.
  • the use of the method and method as such has proven to be efficient when the waste is municipal solid waste.
  • the use of the method according to the invention is for waste comprising both biodegradable and non-biodegradable material.
  • the use of the method according to the invention is for waste comprising 10-60% biodegradable material on a wet basis.
  • the use of the method according to the invention is when the waste to be processed is municipal solid waste.
  • FIG. 1 Overview of the process applied at the large scale plant for processing of the waste in the examples disclosed herein.
  • the plant was the Renescience plant situated at Amager in Denmark. Unsorted MSW was treated with enzymes in the bioreactor.
  • the generated bioliquid was suitable for biogas production e.g. in a conventional biogas plant. Solids can be recoved in 2D and 3D fraction for recycling purposes.
  • FIG. 2 Overview of the different sample pre-treatment conditions used in the examples herein.
  • the liquid-liquid extraction procedure where first a raw aqueous sample is taken out, then the same supernatant is extracted with pentane, then with DCM (dichloromethane), then the remaining aqueous phase is split into two, one part adjusted to pH 2 and the other to pH 12, and each of them was extracted.
  • the pressurized liquid extraction of the solid part includes silica clean-up and dichloromethane as first extraction solvent, methanol as second extraction solvent, final extract volumes 50 ml_.
  • # indicates samples for GC-MS analysis
  • n indicates samples for both LC-MS and GC-MS analysis. Fraction names are given in brackets.
  • FIG. 3 GC-EI-MS analysis of benzenepropanoic acid.
  • Benzenepropanoic acid is found in several fractions in the bioliquid but after anaerobic digestion it is not detected.
  • FIG. 4 GC-EI-MS analysis of cholestan-3-one.
  • FIG. 5 GC-EI-MS analysis of d-limonene.
  • the areas obtained by GC-EI-MS analysis of d-limonene of the fractions obtained during treatment of the bioliquid (A1 a) and the two solid fractions (fiber and 3mm reject) after various extraction d-limonene clearly partitions pre-dominantly to the solid fractions which is in line with the compound being hydrophobic.
  • FIG. 6 GC-EI-MS analysis of caffeine.
  • Caffeine clearly partitions pre-dominantly to the bioliquid which is in line with the compound being
  • a method for determining the abundance of or establishing the identity of one or more chemical compound(s) or class(es) of compounds in waste comprising the steps of:
  • step b) Subjecting the treated waste from step a) to one or more separation step(s), whereby a liquid fraction is provided;
  • step b) Determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in said liquid fraction obtained in step b).
  • step a) wherein said combined enzymatic and microbial treatment in step a) is performed by adding hydrolytic enzymes, supplied in either native form or in form of microbial organisms giving rise to the accumulation of such enzymes; and by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing biochemicals, ethanol, or biogas.
  • step a) is accomplished by the use of one or more species of lactic acid bacteria, acetate-producing bacteria, propionate-producing bacteria, or butyrate- producing bacteria, including any combination thereof.
  • step a) is accomplished by the use of one or more species of microorganisms present in the waste.
  • treatment step a) comprises contacting the waste with a live lactic acid bacteria concentration of at least 1 .0 x 10 L 10 CFU/L.
  • the treatment step a) comprises subjecting the waste with a microbially-derived cellulase activity of at least 30 FPU/L that is provided by one or more microorganisms, such as a microbial consortium, providing microbial fermentation. 1 1 .
  • the treatment step a) comprises addition of cellulase activity by inoculation with one or more microorganism(s) that exhibits extracellular cellulase activity.
  • step a) is accomplished by treating the waste with an enzyme composition comprising a cellulolytic background composition and one or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase.
  • composition comprises two or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase (e.g. a protease and a lipase; a protease and a beta-glucanase; or a lipase and a beta-glucanase).
  • a protease selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase
  • a beta-glucanase e.g. a protease and a lipase; a protease and a beta-glucanase; or a lipase and a beta-glucanase.
  • the enzyme composition comprises (i) a protease, (ii) a lipase, and (iii) a beta-glucanase.
  • the enzyme composition further comprises one or more enzymes selected from (iv) a pectate lyase, (v) a mannanase, and (vi) an amylase.
  • the cellulolytic background composition comprises one or more enzymes selected from the group comprising: cellobiohydrolases I or variants thereof; cellobiohydrolases II or variants thereof; beta-glucosidases or variants thereof; polypeptides having cellulolytic enhancing activity; and/or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
  • the cellulolytic background composition comprises a cellobiohydrolase I or a variant thereof; a cellobiohydrolase II or a variant thereof; a beta-glucosidase or a variant thereof; and a polypeptide having cellulolytic enhancing activity; or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
  • the treatment step a) is performed at a temperature between 20 and 75°C, 30°C and 70°C, 40°C and 60°C, 45 and 55°C, or around 50°C.
  • liquid fraction in step b) has a pH below 7.0, 6.5, 6.0, 5.5, 5.0, or 4.5.
  • non water content of the liquid fraction in step b) comprises at least 40% by weight dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate.
  • step c) The method according to any of the preceding embodiments, wherein said determination of or establishment to the identity of a chemical compound and/or class of compounds in step c) is present in an amount at or above 0.00001 ppm.
  • step c) determination of or establishment to the identity of a chemical compound and/or class of compounds in step c) is present in an amount at or above 7E-02 ng/ml in 50 ml. of said liquid fraction obtained from step b).
  • step c) a sample of the liquid fraction obtained from step b) is pre-treated in order to separate phases by centrifugation into supernatant and precipitate prior to determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds.
  • step c) a sample of the liquid fraction obtained from step b) is pre-treated in order to separate phases by centrifugation into supernatant and precipitate prior to determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds.
  • the separated precipitate fraction is further pre-treated by one or more of the following treatments: pressurized liquid extraction of the precipitate with silica and dichloromethane; pressurized liquid extraction of the precipitate with silica and dichloromethane followed by extraction with silica and methanol.
  • step c) determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds is done by one or more of the following methods: liquid chromatography, mass spectrometry, gas chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, gas chromatography-electrospray ionization-mass spectrometry, liquid chromatography- electrospray ionization-mass spectrometry.
  • step c) The method according to embodiment 28 wherein the liquid obtained from step b) is separated into precipitate and supernatant and wherein the compounds or classes of compounds in the precipitate is determined or established in step c) by one or more of the following: pre-treating with pressurized liquid extraction with silica and dichloromethane followed by gas-chromatography mass spectrometry; pre-treating with pressurized liquid extraction with silica and dichloromethane followed by pressurized liquid extraction with silica and methanol followed by liquid chromatography-mass spectrometry.
  • step c) The method according to embodiment 28 wherein the liquid obtained from step b) is separated into precipitate and supernatant and wherein the compounds or classes of compounds in the supernatant is determined or established in step c) by one or more of the following: liquid chromatography-mass spectrometry; pre-treating with liquid- liquid pentane extraction followed by gas chromatography-mass spectrometry; pre- treating with liquid- liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry; pre-treating with liquid-liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by adjusting the aqueous phase to pH 2 or 12 and subjecting to liquid- liquid dichloromethane extraction followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry.
  • step c) determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing a sample from the liquid fraction obtained in step b) with spiked samples comprising known amounts of one or more target compounds.
  • step c) determines the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is by comparing said sample from the waste batch by liquid-chromatography, and/ or gas-chromatography with one or more standards of known concentration analyzed by the same method.
  • step c) determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by identifying one or more chemical compounds from said sample from the waste batch by one or more of liquid- chromatography, mass-spectrometry, and/or gas-chromatography and comparing the obtained data with profiles of liquid-chromatography, mass-spectrometry, and/or gas- chromatography, respectively, from databases of known compounds.
  • step c) are ingredients or by-products of, or are related to the process for providing: fat, fatty acids and derivatives, compounds from biological sources, pharmaceuticals, plasticizers, plastic additives, pesticides and antimicrobial compounds, food additives, cosmetic additives and fluorinated compounds.
  • step c) is selected from organic acids, free fatty acids, esters, alcohols, alkanes, phthalates, amides, bisphenols, aromatic, and poly-aromatic compounds and their heterocyclic, alkyl-, hydroxyl-, and carboxylated varieties.
  • step c) is/are organic acid(s) selected from one or more of: carboxylic acids, sulfonic acids, alcohols, and organic compounds comprising an acidic thiol-, enol-, or phenol-group.
  • step c) is/are organic acid(s) selected from one or more of: 1 -adamantanecarboxylic acid, benzenepropanoic acid, 4-butylbenzoic acid, cyclohexanecarboxylic acid, cyclohexanepentanoic acid, dicyclohexylacetic acid, diphenic acid, 1 -hydroxy-2-naphthoic acid, linolenic acid, myristic acid, 1 -naphthoic acid, 2-naphthoic acid, oleic acid, palmitic acid, palmitoleic acid, pentadecanoic acid, phthalic acid, 1 -pyrenecarboxylic acid, salicylic acid, sorbic acid.
  • organic acid(s) selected from one or more of: 1 -adamantanecarboxylic acid, benzenepropanoic acid, 4-butylbenzoic acid, cyclohexanecarbox
  • step c) is one or more compound(s) selected from the following groups of compounds: Linear alkylbenzensulfonates (LAS); Polycyclic aromatic hydrocarbons such as ⁇ Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens, Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylat, nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2- ethylhexyl)phthalate; hormone-disturbing compounds such as bisphenol-A; drugs such as amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa; pesticides
  • LAS Linear alkylbenzensulfonates
  • step c) is selected from one or more of: Para- cresol, Skatole, N,N-dimethyl-1 -dodecanamine, Benzophenone, Oleic acid, 4- hydroxy-3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, , Methyl oleate, , 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13- docosenamide, 3-methylindole, 2-methylindole, 4-hydroxy-3,5-ditert- butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d-limonene, Dodecanoic acid
  • step b) Subjecting the treated MSW from step a) to step b);
  • step c) if the liquid obtained in step b) is subject to further treatment and separation steps and/or to anaerobic digestion
  • waste is derived from or comprises any one or more of waste from household, industry, agriculture, farming, county, or state activities.
  • waste is selected from one or more of unsorted municipal solid waste, centrally sorted municipal solid waste, source sorted municipal solid waste from households, municipal solid waste processed by shredding or pulping, organic fractions and paper rich fractions, Refuse-Derived-Fuel fractions.
  • biodegradable material in said waste municipal solid waste comprises a combination of one or more items selected from: food residues, paper, cardboard, and fines.
  • waste is sorted municipal solid waste not comprising items selected from one or more of the following: domestic appliances, glass, ceramics, batteries, newsprints, magazines, advertisements, books, plastics, fabrics, textiles, yard waste, electrical and electronic equipment, chemicals, pharmaceuticals, metals.
  • waste is selected from one or more of general industry waste fractions containing paper or other organic fractions, waste fractions from paper industry or recycling facilities, waste fractions from food and feed industry, waste fractions from the medicinal industry.
  • waste is selected from one or more of agriculture or farming, waste fractions from processes of sugar or starch rich products, contaminated or spoiled agricultural products not exploitable for food or feed purposes, manure, manure derived products.
  • waste is selected from one or more of waste fractions derived from municipal, county or state related or regulated activities, sludge from waste water treatment plants, fibre or sludge fractions from biogas processing, general waste fractions from the public sector containing paper or other organic fractions.
  • processing parameters to be adjusted are one or more of the parameters in step a) in accordance with embodiments 6, 7, 9, 10 to 20 and/or one or more of the parameters in step b) in accordance with embodiments 21 and 22.
  • processing parameter to be adjusted is the retention time of the waste in the bio-reactor and/or the retention time of the bioliquid in the anaerobic digestion process.
  • step b) polar and ionic chemical compounds primarily partition to the bioliguid and hydrophobic chemical compounds primarily partition to the solid fraction
  • step b) polar and ionic chemical compounds primarily partition to the bioliguid and hydrophobic chemical compounds primarily partition to the solid fraction
  • step b) polar and ionic chemical compounds primarily partition to the bioliguid and hydrophobic chemical compounds primarily partition to the solid fraction
  • said chemical compound is selected from one or more of the following groups of compounds: Linear alkylbenzensulfonates; Polycyclic aromatic hydrocarbons such as ⁇ Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens, Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylat, nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups
  • said chemical compound is selected from one or more of the following chemical compounds: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one , Para- cresol, Skatole, N,N-dimethyl-1 -dodecanamine, BenzophenoneOleic acid, 4-hydroxy- 3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, Methyl oleate, 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3- methylindole, 2-methylindole, 4-hydroxy-3,5-ditert-butylbenzaldehyde,
  • DIDP diisodecyl phthalate
  • the technology applied in the below examples is a process for treatment of municipal solid waste (MSW) by use of enzymatic degradation and microbial fermentation taking place in a bioreactor.
  • the bioliquid thus obtained comprises an organic fraction, which is suitable for anaerobic digestion leading to biogas production, while simultaneously recovering the solid fraction comprising e.g. plastics and metals for recycling or combustion.
  • the process includes addition of enzyme to the bioreactor to facilitate liquefaction and separation of the biodegradable material from the 2D and 3D fractions.
  • the bioliquid fraction obtained was subject to anaerobic digestion according to a process well known in the art wherein anaerobic microorganisms produces inter alia biogas.
  • the overall process applied at the large scale plant is illustrated in Figure 1 .
  • the plant was the Renescience plant situated at Amager in Denmark. Samples of the bioliquid fraction were collected at different steps in the process and the presence and/or abundance of various chemical compositions were measured using one or more of the methods as described in the specific examples
  • the waste subject to treatment was unsorted municipal solid waste (MSW) collected by vacuum truck in the Copenhagen area of Denmark.
  • the unsorted MSW was treated with the enzyme solution Cellic® CTec3 from Novozymes.
  • the enzyme was received in pallet tanks from which it was pumped into the bioreactor.
  • the enzyme was stored at room temperature and was not exposed to direct sunlight according to the manufacturer’s instructions.
  • the enzyme addition of Cellic CTec3 to the process is set to 9 kg per ton MSW (i.e. 0.9 %).
  • the bioreactor filling was 13.5 tonne.
  • the waste was processed in the bioreactor with a 12hr retention time and 80% rotation speed (corresponding to 0.8 turns per min). Water was added in a 2:1 waterwaste ratio and the enzyme dosage was 0.9% w/w relative to waste.
  • the temperature in the bioreactor was set to 50°C. However, we do not expect the settings of the bio-reactor to influence significantly the outcome of the chemical fingerprint of the bio-liquid as long as there is sufficient liquifaction of the MSW. Samples were collected at several steps in the process.
  • the output from the bio-reactor was separated using a ballastic separator into one liquid fraction (bioliquid) and two solid fractions denoted 2D and 3D. First, objects larger than 60mm x 60mm were removed from the liquid fraction. The liquid was then directed through a 3mm sieve and the rejected solid material from this sieve is denoted“3mm reject”, whereas the liquid going through the sieve is denoted “bioliquid”.
  • the 2D fraction consists mainly of large, flat objects such as plastic film and fabric. This fraction is washed and the resulting wash water was dewatered using a press. The solid, de watered fraction from this press is denoted“fiber fraction”.
  • Bioliquid samples (A1 ) were acquired for five consecutive days and pooled to even out day- to-day variations. In addition, one of the samples were split into three technical replicates (termed A1 a, A1 b, A1 c) to assess the quality of the sample preparation and analysis.
  • FIG. 2 shows a schematic overview of the pre-treatment from centrifugation to separation of a solid fraction from a liquid fraction.
  • the liquid-liquid extraction procedure where first a raw aqueous sample is taken out, then the same supernatant is extracted with pentane, then with DCM (dichloromethane), then the remaining aqueous phase is split into two, one part adjusted to pH 2 and the other to pH 12, and each of them extracted.
  • the pressurized liquid extraction of the solid part includes silica clean-up and dichloromethane as first extraction solvent, methanol as second extraction solvent, final extract volumes 50 ml_.
  • a set of 51 target compounds were quantified in 18 samples. Quantification was performed by a 1 - point calibration in either sample matrix S1 or AD1 , so that also matrix effects could be compensated for.
  • the spike-concentration used for quantification was either 10 ng/mL, 100 ng/mL or 1000 ng/mL depending on what was the native sample concentration. In special cases, i.e. when the analyte was present in high amount and thus the spike-levels were insignificant, or in cases where the spiked level was not detected for other reasons, non matrix match calibration in solvent was applied. In these cases matrix effects have not been compensated for.
  • Non-matrix match calibration was performed for 1 -adamantanecarboxylic acid (supernatant and PLE-DCM fraction), , cyclohexanepentanoic acid, and the four free fatty acids: linolenic acid, palmitoleic acid, linoleic acid and oleic acid.
  • the analyte is found in the PLE DCM or PLE MeOH fraction, then it originates from the solid part of the sample. Concentrations in PLE DCM and PLE MeOH are additive for the same reason as stated above. A concentration of 1 ng/mL in the PLE extracts can be converted to 10 ng/g of solid material (ww) since 5 g of solid material (ww) was extracted with a volume of 50 mL.
  • the LODs obtained in each fraction for the S1 sample (to represent the S matrix) and for the AD1 sample (to represent the AD matrix) and for each compound is given. S samples have been quantified using the calibration in S1 , and AD samples using the calibration in AD1. A, B and C samples we evaluated to resemble more the feed, thus calibration in S1 has been used.
  • 50 compounds were detected at elevated levels in samples compared to solvent blanks (all 6 fractions), plastic blanks (pentane and DCM fractions), and PLE blanks (PLE fractions). 22 of 50 compounds were discovered during the screening process, and were not previously included in the suspects screening list, but found in significantly elevated levels in selected samples. Peak areas and retention times for some of the compounds are listed in Table 4. Table 5 shows the suspect compounds found, examples of use and potential harmful effects. Information accessed through http://www.chemicalbook.com,
  • the chemical compounds which are released during the enzymatic liquifaction in the bio-reactor can be efficiently degraded during the ensuing anaerobic digestion.
  • An example of this is benzenepropanoic acid, which is found in high
  • FIG. 3 shows the areas obtained by GC-EI-MS analysis of benzenepropanoic acid of the fractions obtained during treatment of the bioliquid (S1 , S2, S3) and the effluent after anaerobic digestion (AD1 , AD2, AD3). Benzenepropanoic acid is found in several fractions in the bioliquid but after anaerobic digestion it has been completely degraded.
  • FIG. 5 shows the areas obtained by GC-EI-MS analysis of d-limonene of the fractions obtained during treatment of the bioliquid (A1 a) and the two solid fractions (fiber and 3mm reject) after various extractions d-limonene clearly distributes pre-dominantly to the solid fractions which is in line with the compound being hydrophobic.
  • Caffeine on the other hand can be found in much large concentrations in the bioliquid compared to the extractions of the solid fractions.
  • Figure 6 shows the areas obtained by GC- EI-MS analysis of caffeine of the fractions obtained during treatment of the bioliquid (A1 a) and the two solid fractions (fiber and 3mm reject) after various extraction. Caffeine clearly distributes pre-dominantly to the bioliquid which is in line with the compound being hydrophilic.
  • PCA principal component analysis
  • sample chromatograms Prior to the PCA, sample chromatograms were preprocessed in the following order: Baselines were removed by lower Convex Hull subtraction, retention time aligned using lcoshift and correlation optimized warping, chromatograms normalized to their Euclidean norm and each variable scaled using the inverse of the relative median absolute deviation (MAD/mean)-1 of the mixture samples. Finally, chromatograms of the four fractions were sample-wise concatenated and the PCA was performed.
  • PC1 describing 79% of the explained variation (EV), separates the AD from the S samples along the X-axis, with high negative score for the S samples and high positive for the AD samples.
  • PC2 (15% EV) plotted along the Y-axis mainly describes variation amongst the AD samples and it is included primarily to improve visual interpretation.
  • the PC1 loadings were depicted for the most important coefficients, with a probable compound identification from the NIST database. Negative loading coefficients are observed for most organic acids, free fatty acids and ethyl esters in three sections (pentane, DCM acidic and DCM neutral), while the DCM basic section has negative coefficients for alcohols. This indicates that these compounds are relatively more abundant in the S samples. Positive coefficients were observed for 15 compounds along the PC1 loadings. Inspection of the raw chromatograms was performed to assess, whether the positive coefficients indicate higher absolute abundances of these compounds in the AD samples, or if it is a result of normalization.
  • a non-target screening of the LC data was performed using Parallel Factor Analysis (PARAFAC), a multi-way decomposition method, which highlights the largest chemical variation through components, and extracts relevant information such as relative sample concentrations, retention time and pure mass spectra for each component.
  • PARAFAC Parallel Factor Analysis
  • PARAFAC models were established using the whole sample set (A1 a, A1 b, A1 c, A1 rep, A2, A3, B, C, PVC, HPDE, PPCO, S1 , S2, S3, AD1 , AD2, AD3, AD-Mix, S-Mix and AD+S-Mix) for five fractions (supernatant, DCM neutral, DCM alkaline and DCM acidic, PLE DCM and PLE MeOH). Prior to PARAFAC, chromatograms were binned to nominal masses and retention time aligned across samples. No normalization was done.
  • DP diagnostic power refers to: Standard deviation (samples)/ standard deviation (Mix-AD+S replicates) and limit of detection (LOD - Limit of detection is calculated as: 3 x standard deviation(blank samples) + mean(blank samples) were calculated and constant or non-chemical components were manually removed, e.g. components representing chromatographic baselines.
  • DP is used to assess the capability of a component to differentiate samples based on non-random variation. Spectral database search was performed for all components with DP >2.5 using the Norman MassBank and top 40 search matches were manually inspected for each component. Possible matches were further investigated in the raw chromatograms by exact mass of diagnostic ions, where 1 -10 ppm mass accuracy was considered acceptable.
  • Tables 6 to 10 provides information on relevant components (i.e. DP>2.5 and at least one of AD or S samples above LOD) for all five fractions. Components highlighted by * display systematic differences in relative intensities between AD and S samples when doing a pair-wise comparison (e.g. AD1 and S1 or AD3 and S3). Relative intensities cannot be translated to sample concentrations directly, as matrix effects are not compensated for by the non-target approach. If a compound name is given in column 4‘Compound ID’ then it can be considered annotated. The compound name given in column 12‘Normann Mass Bank Candidates’ represent the closest match, and most likely not the exact match.
  • a total of 57 components fulfilled the criteria of DP >2.5 and with levels detected above LOD for at least one AD or S sample (data not shown).
  • One component showed systematic difference between the 6 AD and S samples, but with only S3 having a relative intensity above LOD. No spectral id was obtained for this component. Positive identification was obtained for two compounds, both of which are drugs used for treatment of Parkinson’s disease and depressive disorder, respectively.
  • a total of 29 PARAFAC components fulfill the criteria of DP >2.5 and relative intensities above LOD for at least one AD or S samples (see Table 6, wherein some of the results are shown). 25 components showed systematic difference between the six AD and S samples; most displaying higher relative intensities in the S samples. 1 of the remaining 4 components were found at ⁇ LOD levels for all samples, while the remaining 3 component display relative intensities above >LOD levels for all 6 samples. A positive identification was obtained for the two components with highest diagnostic power; both fitted the exact mass and Norman MassBank spectra of L-nicotine, but the
  • Nicotine was also detected during the suspect screening of DCM alkaline fraction measured by GC-MS. Similar trends are observed between the two sets of data with increased relative intensities in the S samples compared to the AD samples.
  • L-nicotine (0.92 min) fits well with that observed in the DCM alkaline fraction (i.e. 0.82-1.03 min). Nicotine wasalso detected at elevated levels in all AD and S during the suspect screening. This fits with current findings as both AD and S samples are found at >LOD levels. AD and S samples have comparable nicotine intensity ranges, in accordance with the findings of the suspect screening.
  • PLE dichloromethane fraction a total of 81 PARAFAC components fulfilled the criteria of DP >2.5 and relative intensities above LOD for at least one AD or S samples (see Table 8, wherein some of the results are shown). 9 components discriminate systematically between the corresponding AD and S samples with relatively higher intensities displayed in the S samples.
  • a total of 64 PARAFAC components fulfilled the criteria of DP >2.5 and relative intensity above LOD for at least one AD or S samples (see Table 9, wherein some of the results are shown). 7 components discriminated between the paired AD and S samples with relative higher intensities found amongst the AD samples. This differs from the remaining fractions, where most discriminative components show higher concentrations for the S samples. Identification was only achieved for one component, which matches the Norman MassBank spectra and the exact mass of caffeine. The retention time and relative concentrations match quite well that found in the PLE DCM fraction, and thus serves as a confirmation of compound identity. Caffeine was also detected in the PLE MeOH fractions during the suspect screening by GC-EI-MS, with S samples exhibiting higher peak areas.
  • the relative pattern of GC-EI-MS peak areas does not match the relative pattern of intensities found for PARAFAC component, which only one S sample >LOD.
  • the discrepancy may be explained by the varying matrix effects imposed by the S and the AD matrix on analyte area and intensity, when using either the GC-EI-MS or the LC-ESI+-MS platform, respectively.
  • Example 10 Tracking of a chemical composition from a particular waste batch using marker Unsorted MSW is collected by vacuum truck in a registered part of the Copenhagen area in Denmark. The truck is selected prior to collecting the MSW and the truck-driver will register the arrival at the Renescience large scale plant situated at Amager in Copenhagen. While entering the waste into the plant the point of time for entering the waste and the amount of waste will be monitored. One or more of three different markers will be added to the waste bacth selected from a radioactive labelled marker, a specific chemical compound that is resistant to bio-degradation, and a maker that can be visualised during the various steps of the method such as a fluorescent marker.
  • the point in time when the waste is entered into the bioreactor will be monitored.
  • the waste will be treated with the enzyme solution Cellic® CTec3 from Novozymes.
  • the enzyme was received in pallet tanks from which it was pumped into the bioreactor.
  • the enzyme was stored at room temperature and was not exposed to direct sunlight according to the manufacturer’s instructions.
  • the enzyme addition of Cellic CTec3 to the process is set to 9 kg per ton MSW (i.e. 0.9 %).
  • the bioreactor filling is 13.5 tonne.
  • the waste will be processed in the bioreactor with a 12hr retention time and 80% rotation speed (corresponding to 0.8 turns per min). Water is added in a 2:1 waterwaste ratio and the enzyme dosage was 0.9% w/w relative to waste. The temperature in the bioreactor was set to 50°C.
  • Samples of bioliquid will be collected at different point in times after the marked waste batch was entered into the bioreactor.
  • One or more of the chemical compounds disclosed in Table 4 will be measured using one or more of the methods dislcosed in examples 7 and 8. These samples will also be screened for the presence of the marker.
  • Example 1 1 Tracking of a chemical composition from a particular waste batch
  • Unsorted MSW is collected by vacuum truck in a registered part of the Copenhagen area in Denmark.
  • the truck is selected prior to collecting the MSW and the truck-driver will register the arrival at the Renescience large scale plant situated at Amager in Copenhagen.
  • While entering the waste into the plant the point of time for entering the waste and the amount of waste will be recorded.
  • the waste is placed in a designated lot in the bunker and it is not allowed to mix with waste from other regions of the city before it is entered into the bio reactor.
  • the point in time when the waste is entered into the bioreactor is recorded.
  • the waste will be treated with the enzyme solution Cellic® CTec3 from Novozymes.
  • the enzyme was received in pallet tanks from which it was pumped into the bioreactor.
  • the enzyme was stored at room temperature and was not exposed to direct sunlight according to the manufacturer’s instructions.
  • the enzyme addition of Cellic CTec3 to the process is set to 9 kg per ton MSW (i.e. 0.9 %).
  • the bioreactor filling is 13.5 tonne.
  • the waste will be processed in the bioreactor with a 12hr retention time and 80% rotation speed (corresponding to 0.8 turns per min). Water is added in a 2:1 waterwaste ratio and the enzyme dosage was 0.9% w/w relative to waste. The temperature in the bioreactor was set to 50oC.
  • Samples of bioliquid will be collected at different point in times after the waste batch was entered into the bioreactor.
  • One or more of the chemical compounds disclosed in Table 4 will be measured using one or more of the methods disclosed in examples 7 and 8 and particular emphasis will be put on samples taken close to the retention time of the bio reactor. It can be advantageous to process all the waste from the same regionbefore starting to process waste from another region (AAABBB) rather than alternating between waste from different regions (ABABAB).
  • AAABBB alternating between waste from different regions
  • the results will show that in some of the samples both wherein the chemical composition measured and the marker will be present. It may be concluded that the chemical composition measured in these samples originated from the waste batch collected by the truck that was entered into the reactor at the specific point in time.
  • the specific geographic origin of the MSW collected by this truck will be established by consulting the route applied by that specific truck for collecting this batch of MSW.

Abstract

The invention relates to qualitative or quantitative determination of chemical compounds or classes of compounds in heterogeneous waste comprising biodegradable and non- biodegradable matter. The method may especially be used for identifying, tracking and mapping geographical origin of waste, such as e.g. municipal solid waste, agricultural waste or industry waste comprising one or more specified chemical compounds and for identifying, tracking and mapping any change in occurrence of one or more specified chemical compound(s) present in the waste from a certain geographic area. The current invention is well-suited for analyzing and/or monitoring waste being subjected to waste treatment in a large-scale waste plant.

Description

Method for determining chemical compounds in waste
Field of the invention
The present invention relates to the qualitative or quantitative determination of chemical compounds or classes of compounds in heterogeneous waste comprising biodegradable and non-biodegradable matter. The method may especially be used for identifying, tracking and mapping geographical origin of waste, such as e.g. municipal solid waste, agricultural waste or industry waste comprising one or more specified chemical compounds and for identifying, tracking and mapping any change in occurrence of one or more specified chemical compound(s) present in the waste from a certain geographic area. The current invention appears well-suited for analyzing and/or monitoring waste being subjected to waste treatment in a large-scale waste plant.
Background of the invention
Disposing of waste has huge environmental impacts. It is common to deposit waste in landfill sites - e.g. in holes in the ground. Some waste will eventually rot, but not all, and in the process it will smell or generate methane gas, which is explosive and contributes to the greenhouse effect. Incineration is one method which combines utilization of the energy stored within the organic material with disposal of waste such as household, agricultural or municipal waste. For example in Denmark, the heat generated by the incineration process is mainly used for district heating, but can also be converted into electricity. Incineration is, however, problematic if the material/waste to be incinerated includes non-organic parts like plastic, glass and metals. In order to optimally utilize the energy stored within organic material found in different types of material/waste by incineration, pre-sorting is required.
Pre-sorting may be provided by the consumers or by the waste station. Regardless of this, pre-sorting may not be efficient in separating all non-biodegradable material such as metal and glass from the organic waste. The waste separation process may time consuming, inefficient and expensive.
An alternative waste sorting method, is to liquefy the organic contents while maintaining the non-organic contents in their solid phase, and afterwards separate the solid and the liquid phases. The problem with pollution when e.g. incinerating the separated waste is thereby greatly diminished. Recently such enviromentaly friendly waste processing methods like the Renescience technology have been developed. With the Renescience technology, ordinary unsorted household waste is mixed with water, enzymes and optionally microorganisms in order to dissolve all food waste, cardboard, paper, labels and similar types of organic waste and turns it into a liquid that can for example be used for biogas.
Identifying individual chemical compounds in very heterogeneous matrix, such as municipal solid waste is a tedious approach involving presorting of the waste into individual fractions, in order to extract said chemical compounds. Normally, each chemical compound or class of compounds must be extracted individually, prior to analysis. Data about chemical composition of material fractions in waste such as household waste are scarce because of the difficult and costly procedures involved in waste characterization.
The waste that can be processed according to the present invention is heterogeneous waste, such as municipal solid waste (MSW), particularly including domestic household waste, waste from restaurants and food processing facilities, and waste from office buildings. Waste often comprises significant amounts of organic material that can be further processed to energy, fuels and other useful products. At present, only a small fraction of available waste, such as MSW, worldwide is recycled, the great majority being dumped into landfills. Due to the general heterogenity of waste, such as MSW, which comprises a mixture of diverse biodegradable and non-biodegradable matter, it has not previously been possible to sufficiently analyse the chemical composition of e.g. the soluble fraction of waste, including MSW.
Considerable interest has arisen in development of efficient and environmentally friendly methods of processing solid waste, to maximize recovery of their inherent energy potential and, also, recovery of recyclable materials. One significant challenge in“waste to energy” processing has been the heterogeneous nature of waste, such as MSW.
The degradable component of waste such as MSW can be used in“waste to energy” processing using both thermo-chemical and biological methods. Waste such as MSW can be subject to pyrolysis or other modes of thermo-chemical gasification.
Biological methods for conversion of degradable components of waste, such as MSW, include fermentation to produce specific useful end-products, such as ethanol. Biological conversion of waste into useful end-products can for example be achieved by anaerobic digestion to produce biomethane or biogas. Pre-sorted organic component of waste, such as MSW, can in some methods be converted to biomethane either directly or after a "pulping" process involving mincing in the presence of added water.
Pre-sorting of waste, such as MSW, to obtain the organic and bio-degradable component is typically costly, inefficient or impractical. Source-sorting requires large infrastructure and operating expenses as well as the active participation and support from the community from which waste are collected - an activity which has proved difficult to achieve in modern urban societies. Mechanical sorting is typically capital intensive and further associated with a large loss of organic material, on the order of at least 30% and often much higher.
Some of these problems with sorting systems have been successfully avoided through use of liquefaction of organic, degradable components in unsorted waste. Liquefied organic material can be readily separated from non-degradable materials. Once liquefied into a pumpable slurry, organic component can be readily used in thermo-chemical or biological conversion processes. Liquefaction of degradable components has been widely reported using high pressure, high temperature“autoclave” processes.
A radically different approach to liquefaction of degradable organic components is that this may be achieved using biological process, specifically through enzymatic hydrolysis and microbial fermentation. The method according to the present invention is suitable when waste has been subject to enzymatic degradation and microbial fermentation. Examples of such waste treatment processes are disclosed in W02006056838, W02007036795,
WO201 1032557, WO2013185778, WO2014198274, WO2016030480, WO2016030472, WO2016050893, WO2017/174093, which is hereby expressly incorporated by reference in entirety.
Enzymatic hydrolysis offers unique advantages over "autoclave" methods for liquefaction of degradable organic components. Using enzymatic liquefaction, processing of waste, such as MSW, can be conducted in a continuous manner, using comparatively cheap equipment and non-pressurized reactions run at comparatively low temperatures.
Enzymatic liquefaction may sometimes require thermal pre-treatment to a comparatively high temperature of at least 90- 95°C, in part to effect a "sterilization" of waste such as unsorted MSW and also so that degradable organic components can be softened and paper products "pulped." However, high temperature pre-treatment can be actively detrimental, since this kills ambient microorganisms which are thriving in the waste. Safe enzymatic liquefaction of unsorted MSW has been described without high temperature pre-treatment. Promoting microbial fermentation concurrently with enzymatic hydrolysis at thermophillic conditions >45°C improves "organic capture," either using "ambient" microorganisms or using selectively "inoculated" organisms. That is, concurrent thermophillic microbial fermentation safely increases the organic yield of "bioliquid," which is the term used herein for the liquefied degradable components obtained by enzymatic hydrolysis. Under these conditions, pathogenic microogranisms typically found in waste such as MSW do not thrive. Under these conditions, typical pathogens present in waste such as MSW-borne pathogens are easily outcompeted by e.g. lactic acid bacteria and other safe organisms present in the waste to be treated, and/or in the bioreactor, where the enzymatic and microbial liquefaction takes place. For the present invention, liquefaction and fermentation is performed at temperatures wherein ambient microoganisms survive, the specific temperature however depending on the specific kind of waste to be treated, and/or the end products to be obtained.
In addition to improving "organic capture" from enzymatic hydrolysis, concurrent microbial fermentation using any combination of lactic acid bacteria, or acetate-, ethanol-, formate-, butyrate-, lactate-, pentanoate- or hexanoate- producing microorganisms, "pre-conditions" the bioliquid so as to render it more efficient as a substrate for further processing, such as biomethane production. Microbial fermentation produces bioliquid having a generally increased percentage of dissolved compared with suspended solids, relative to bioliquid produced by enzymatic liquefaction alone. Higher chain polysaccharides are generally more thoroughly degraded due to microbial "pre-conditioning". Concurrent microbial fermentation and enzymatic hydrolysis degrades biopolymers into readily usable substrates and, further, achieves metabolic conversion of primary substrates to short chain carboxylic acids and/or ethanol. The resulting bioliquid comprising a high percentage of microbial metabolites provides a biomethane substrate, which effectively avoids the rate limiting "hydrolysis" step.
When waste is submitted to combined enzymatic and microbial treatment in a large-scale plant, substances suited for biological and enzymatic degration will be completely or partly degraded whereas some will not be degraded. Due to the wide range of chemical compositions present in the waste and the difficulties in measuring specific chemical compounds or screening for specific compounds or classes of compounds it has hitherto not been possible to investigate and track selected chemical compounds in large amounts of waste comprising heterogenous matter such as municipal solid waste. With the method according to the present invention, soluble chemical compounds can be identified and optionally measured and tracked.
This provide the possibility to establish a link between the specific chemical compound and geographic area from where the waste comprising the specific chemical compound originated. This information may be applied in several circumstances. For instance, the method according to the present invention makes it possible to track the geographic origin of specific waste compounds and to optionally initiate various acts directed at the geographic origin of the waste and to follow the occurrence of specific compounds of interest over time within the same geographic area or between areas.
Such acts could include setting op surveillance systems; directing campaigns regarding for instance sorting of waste; identifying areas with a high contend of i.e. drug or medicinal residues and optionally directing health campaigns and/or alter social compositions;
planning urban development; evaluating the impact of campaigns directed to reduce the presence of certain chemical compounds or classes of compounds. Moreover, the method according to the present invention makes it possible to compare the content of a certain chemical compound(s) or class(es) of compounds in waste from a given geographic area with the content of the same compound(s) or class(es) of compounds or in waste within the same geographic area or with other geographic areas. Such information makes it possible to more effectively address for instance sales campaigns, surveillance of activities that leaves chemical compounds in the waste. The above mentioned possibilities are suggestions of possible use of the information that may become available by use of the method according to the present invention. It is evident that it will be possible to apply the information obtained by use of the present method for other purposes also.
Summary of Invention
In a first aspect, the present invention relates to a method for determining the abundance of or establishing the identity of one or more chemical compound(s) or class(es) of compounds in waste, such as a waste batch collected by a garbage truck, said method comprising the steps of:
a) Subjecting said waste to a combined enzymatic and microbial treatment in a large- scale plant; b) Subjecting the treated waste from step a) to one or more separation step(s), whereby a liquid fraction is provided;
c) Determining the abundance of or establishing the identity of said chemical
compound(s) or class(es) of compounds in said liquid fraction obtained in step b).
In a specific embodiment, the method according to the first aspect of the invention further comprises one or more of the following steps:
Registering the geographic origin of a waste batch delivered for processing in said large scale plant;
Registering the point in time for the start of the processing of said waste batch in the plant;
Adding one or more marker(s) to said waste batch;
Registering the point in time when said waste batch enters and/or leaves one or more of treatment step(s) a) and/or separation step(s) b).
In a second aspect, the present invention relates to the use of the method according to the present invention for identifying the local geographical origin of specific chemical compounds present in the waste and optionally identifying the specific leaking or additive source(s) of said chemical compound.
In a third aspect, the present invention relates to the use of the method according to the present invention for identifying the local geographical origin of specific chemical compounds present in the waste and directing information campaigns, reward campaigns, sales activities, local sorting of waste, environmental or social improvement activities; to the geographic area or specific entity that has been identified as the source(s) of the waste comprising a specific chemical compound.
In a fourth aspect, the present invention relates to the use of the method according to the present invention for decreasing the abundance of or eliminating the presence of one or more chemical compound(s) in waste such as MSW before, during or after anaerobic digestion of the waste, by adjusting one or more of the processing parameters in step a) or step b)
In a fith aspect, the present invention relates to the use of the method according to the present invention for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste, such as MSW; based on the presence of one or more chemical compounds measured in step c).
Surprisingly and unexpectedly, the inventors have found that it is possible to determine and quantify the presence of individual chemical compounds in very heterogeneous waste, such as municipal solid waste, using a single extraction process, comprising enzymatic and/or microbial treatment. It is thus no longer necessary to use the conventional and tideous approach of presorting waste into individual fractions, in order to extract said chemical compounds, often individually, prior to analysis. The present invention appears well-suited for a large-scale environment, such as a municipal solid waste treatment plant.
One application of the present invention is to track the local geographical origin of one or more chemical compounds e.g. in relation to enforcement of regulations regarding storage, treatment and disposal of waste. Other applications include tracking of the local
geographical origin of one or more chemical compounds such as one or more drugs and/or narcotics e.g. in relation to a demographic study or a forensic analysis.
Brief description of the drawings
Figure 1 : Overview of general waste processing process.
Figure 2: Overview of sample treatment.
Figure 3: GC-EI-MS analysis of benzenepropanoic acid.
Figure 4: GC-EI-MS analysis of cholestan-3-one.
Figure 5: GC-EI-MS analysis of d-limonene.
Figure 6: GC-EI-MS analysis of caffeine.
Detailed description of the invention
The method of the present invention allows for determining the abundance of or
establishing the identity of one or more chemical compound(s) or class(es) of compounds present in waste subject to combined microbial and enzymatic hydrolysis of the organic fractions of waste, such as municipal solid waste, whereby a link to the geographical origin of the waste can be established. Use of the method accordingly allows for directing certain acts specifically to the source of the waste comprising the specific chemical composition(s). Moreover, use of the method of the present invention for adjusting the abundance of one or more chemical compound(s) during the process or for adjusting the processing parameters based on the presence of one or more of the chemical compound(s) measured in the waste. Definitions
In the context of the present invention, a“Chemical compound” means an entity consisting of two or more atoms, at least two from different chemical elements, which associate via chemical bonds.
In the context of the present invention, the term“Class(es) of chemical compound(s)” refers to one or more class(es) of chemical compound(s) classified by any kind of common classification of chemical compounds including classes based on the specific elements present in the compound, the types of bonds that the compound contains or the types of reactions that the chemical compound may undergo.
In the context of the present invention, the term“municipal solid waste” (MSW) refers to waste fractions which are typically available in a city, but that need not come from any municipality per se. MSW may be any combination of one or more of cellulosic, plant, animal, plastic, metal, or glass waste including, but not limited to, any one or more of the following: Garbage collected in normal municipal collections systems, optionally processed in central sorting, shredding or pulping device, such as e.g., a Dewaster® or a reCulture®; solid waste sorted from households, including both organic fractions and paper rich fractions; waste fractions derived from industry such as restaurant industry, food processing industry, general industry; waste fractions from paper industry; waste fractions from recycling facilities; waste fractions from food or feed industry; waste fraction from the medicinal industry; waste fractions derived from agriculture or farming related sectors; waste fractions from processing of sugar or starch rich products; contaminated or in other ways spoiled agriculture products such as grain, potatoes and beets not exploitable for food or feed purposes; or garden refuse.
In the context of the present invention, the term“unsorted” refers to a process in which waste or MSW is not substantially fractionated into separate fractions such that organic material is not substantially separated from plastic and/or other inorganic material, notwithstanding removal of some large objects or metal objects and notwithstanding some separation of plastic and/or other inorganic material may have taken place. The terms "unsorted waste" (or "unsorted MSW"), as used herein, refers to waste comprising a mixture of biodegradable and non-biodegradable material in which 15% by weight or greater of the dry weight is non-biodegradable material. In some embodiments of the method of the invention, waste that has been briefly sorted yet still produce a waste (or MSW) fraction that is unsorted. In the context of the present invention, the term“sorted”, refers to a process in which MSW is substantially fractionated into separate fractions such that organic material is substantially separated from plastic and/or other non-biodegradable material. The term "sorted waste" (or "sorted MSW") as used herein refers to waste in which less than 15% by weight of the dry weight is non-biodegradable material.
In the context of the present invention, the term "organic" refers to materials that comprises carbon and are bio-degradable and include matter derived from living organisms. When used herein the term“bio-degradable” refers to organic matter that can be partly or completely degraded by into simple chemical compounds such as mono or di-saccharides by micro-organisms and/or by enzymes.
In the context of the present invention, the term“liquid fraction”, means the mainly liquid slurry obtained after the waste to be processed has been subjected to a combined enzymatic and microbial treatment and thereafter has been subjected to one or more separation step(s), separating the treated waste into a liquid, i.e. a slurry, and a solid or semi-solid fraction.
In the context of the present invention, the term "bioliquid” is used for thehe liquefied material of the liquid fraction once separated from non-fermentable solids.
In the context of the present invention, the term“batch” refers to any defined amount of waste that is delivered from a specified geographic area to the waste plant. The amount of waste in a“batch” and the size of the geographic area will vary from plant to plant and depend of the specific renovation-collecting system and the specific size of the plant, such as a large scale plant. Each batch may be treated separately in each of step a), b) and c) of the present method or several batches may be treated continuously or at least having overlapping retention times at one or more step(s).Typically, a batch will be the amount of waste loaded into the waste plant by a single truck which usually comprises between 15 - 20 m3 waste disposal per load. Several batches from trucks may be collected, stored and entered into the treatment plant as one large batch. In such circumstances, the batch will usually comprise between 40 - 6000 m3 waste. In the context of the present invention,“large-scale plant” is a plant wherein waste is processed at a commercial scale; wherein the plant normally operates continuously for at least 24h periods typically followed by the next continous period; wherein waste collected from different sources are processed; and wherein each batch of waste entered into the plant can be defined by the way the waste enters the plant, such a one or more truck loads of a specific size, such as a truck load of 15 - 20 m3 waste disposal, or of a specific weight of the waste disposal, such as the weight of one or more truck loads of 15 - 20 m3 waste disposal. Optionally, the large scale plant also comprises means for“waste-to-energy” transformation or is connected to means for“waste-to-energy” transformation, such as means for the production of biogas, bioethanol, syngas, heat or electricity.
In the context of the present invention, the term “fermentation” or“fermentation process” refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.
In the context of the present invention, the term“fermentation medium” refers to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
In the context of the present invention, the term“fermenting microorganism” refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642. In the context of the present invention, the term a“Microbial consortium” refers to a consortium comprising one or more of any bacteria and/or yeasts capable of providing microbial fermentation.
In the context of the present invention, the term or“Auxiliary Activity 9 polypeptide” or“AA9 polypeptide” means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et al., 201 1 , Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips et al., 201 1 , ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20: 1051 -1061 ). AA9
polypeptides were formerly classified into the glycoside hydrolase Family 61 (GH61 ) according to Henrissat, 1991 , Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.
In the context of the present invention, the term“oversize particles” means particles above 600 mm.
In the context of the present invention, the term "anaerobic digestion system" refers to a fermentation system comprising one or more reactors operated under controlled aeration conditions in which methane gas is produced in each of the reactors comprising the system. Methane gas is produced to the extent that the concentration of metabolically generated dissolved methane in the aqueous phase of the fermentation mixture within the "anaerobic digestion system" is saturated at the conditions used and methane gas is emitted from the system. The "anaerobic digestion system" may be a fixed filter system. A "fixed filter anaerobic digestion system" refers to a system in which an anaerobic digestion consortium is immobilized, optionally within a biofilm, on a physical support matrix.
In a first aspect, the present invention relates to a method for determining the abundance of or establishing the identity of one or more chemical compound(s) or class(es) of compounds in waste, such as a waste batch collected by a garbage truck, said method comprising the steps of:
a) Subjecting said waste to a combined enzymatic and microbial treatment in a large- scale plant;
b) Subjecting the treated waste from step a) to one or more separation step(s),
whereby a liquid fraction is provided;
c) Determining the abundance of or establishing the identity of said chemical
compound(s) or class(es) of compounds in said liquid fraction obtained in step b). Relevant types of mono- and/or polysaccharide containing waste that is suitable for being processed by the combined enzymatic and microbial treatment in a large scale plant according to the present invention may include:
Waste fractions derived from households such as e.g.:
• Unsorted municipal solid waste (MSW)
• MSW processed in some central sorting, shredding or pulping device such as e.g.
Dewaster® or reCulture®
• Solid waste sorted from households, including both organic fractions and paper rich fractions
• RDF (Refuse-Derived-Fuel) fractions
Waste fractions derived from the industry such as e.g.:
• General industry waste fractions containing paper or other organic fractions now being treated as household waste
• Waste fraction from paper industry, e.g. from recycling facilities
• Waste fractions from food and feed industry
• Waste fraction from the medicinal industry
Waste fractions derived from agriculture or farming related sectors such as e.g.:
• Waste fractions from processes including sugar or starch rich products such as potatoes and beet
• Contaminated or in other ways spoiled agriculture products such as grain, potatoes and beet not exploitable for food or feed purposes
• Garden refuse
• Manure, or manure derived products
Waste fractions derived from municipal, county or state related or regulated activities such as e.g.:
• Sludge from waste water treatment plants
• Fibre or sludge fractions from biogas processing
• General waste fractions from the public sector containing paper or other organic fractions • The dry matter content of the mono- and/or polysaccharide containing waste fraction in the enzymatic hydrolysis and fermentation processes is in one embodiment above 20%, such as 20-80%, such as 20-50%, such as 20-45% and such as 20-40%.
Any waste comprising a mixture of biodegradable and non-biodegradable material could be used in the method of the invention
Waste, such as MSW, is typically heterogeneous. Statistics that provide firm basis for comparisons between countries concerning composition of waste materials are not widely known. Standards and operating procedures for correct sampling and characterization remain unstandardized. Indeed, only a few standardised sampling methods have been reported (see e.g. Riber et al., 2007). At least in the case of household waste, the composition exhibits seasonal and geographical variation, even over small distances of 200-300 km, see e.g., Dahlen et al., 2007; Hansen et al., 2007b; Muhle et al., 2010; Riber et al., 2009. As a general rule, the dry weight of modern urban waste from Western Europe typically comprise on the order of 25% by weight of "vegetable and food waste" In China, in contrast, the relative proportions of "food waste" are typically increased by a factor of at least two relative to MSW from Western Europe, see e.g. Zhang et al. 2010.
Municipal solid waste may in particular comprise one or more of kitchen putrecibles, garden putrecibles, paper, card, plastics, miscellaneous combustible and non-combustible matters, textiles, glass, ceramics, metals, electronic devises. Generally, municipal solid waste in the Western part of the world normally comprise one or more of: animal food waste, vegetable food waste, newsprints, magazines, advertisements, books and Phonebooks, office paper, other clean paper, paper and carton containers, other cardboard, milk cartons and alike, juice cartons and other carton with alu-foil, kitchen tissues, other dirty paper, other dirty cardboard, soft plastic, plastic bottles, other hard plastic, non-recyclable plastic, yard waste, flowers etc., animals and excrements, diapers and tampons, cottonsticks etc., other cotton etc., wood, textiles, shoes, leather, rubber etc., office articles, empty chemical bottles, plastic products, cigarette buts, other combustibles, vacuum cleaner bags, clear glass, green glass, brown glass, other glass, aluminium containers, alu-trays, alu-foil (including tealight candle foil), metal containers (-AI), metal foil (-AI), other sorts of metal, soil, rocks, stones and gravel, ceramics, cat litter, batteries (botton cells, alkali, thermometers etc.), other non-combustibles and fines. Unless otherwise specified, waste to be processed in the present invention may be sorted or unsorted.
In a preferred embodiment of the present invention the waste to be processed is MSW. In a specific embodiment, MSW is processed in the method according to the invention as unsorted MSW, i.e. in which greater than 15% by weight of the dry weight is non- biodegradable material. In some embodiments the dry weight of the non-biodegradable material is greater than 18% by weight, greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, or greater than 25% by weight. An increase in the percentage of non-biodegradable material may indicate a decrease in degree of sorting, but also geographical and/or seasonal variations.
Typically, unsorted MSW may comprise organic waste, including one or more of food and kitchen waste; paper- and/or cardboard-containing materials; recyclable materials, including glass, bottles, cans, metals, and certain plastics; burnable materials; and inert materials, including ceramics, rocks, and debris.
In some embodiments of the method according to the invention, waste to be processed, such as MSW, can be processed as sorted MSW.
In some embodiments, waste to be processed, such as MSW, can be source-separated organic waste comprising predominantly fruit, vegetable and/or animal waste. A variety of different sorting systems may be applied to MSW, for instance source sorting, where individual households dispose off different waste materials separately. Source sorting systems are currently in place in some municipalities in Austria, Germany, Luxembourg, Sweden, Belgium, the Netherlands, Spain and Denmark. Alternatively, industrial sorting systems may be applied at the large scale plant prior to subjecting the waste to the combined enzymatic and micorbial treatment. Means of mechanical sorting and separation may include any methods known in the art including but not limited to the systems described in US2012/0305688; W02004/101 183; W02004/101098; W02001/052993; W02000/0024531 ; W01997/020643; W01995/0003139; CA2563845; US5465847.
The combined enzymatic and microbial treatment included in the method of the invention typically produce an organic slurry, which also may be known as a bioliquid, or a liquid fraction. As will be readily understood, the slurry is organic where it comprises predominantly organic material, but may also include inorganic contaminants. A slurry, as used herein, is a liquid to the extent that it is pumpable, notwithstanding substantial content of undissolved solids.
As will be readily understood by one skilled in the art, the capacity to render solid
components into a liquid slurry is increased with increased water content. For instance, effective pulping of paper and cardboard, which comprise a substantial fraction of MSW in some countries, is typically improved where water content is increased. Water content provides a medium in which the microbial preparation can propagate and which dissolves metabolites. Furthermore, enzyme activities may exhibit diminished activity when hydrolysis is conducted under conditions with low water content. For example, cellulases typically exhibit diminished activity in hydrolysis mixtures that have non-water content higher than about 10% by weight. In the case of cellulases, which degrade paper and cardboard, an effectively linear inverse relationship has been reported between substrate concentration and yield from the enzymatic reaction per gram substrate, see Kristensen et al. 2009.
The water content waste to be processed, such as e.g. MSW, may preferably have a non water content of between above 10% or more and below 45%, such as 10 - 45%, or in some embodiments between 12 and 40%, or between 13 and 35%, or between 14 and 30%, or between 15 and 25%. MSW may often comprise a considerable amount of water. However, the water content may be adjusted in order to achieve appropriate non-water content.
Table 1 describes the characteristic composition of unsorted MSW from Danish households, as reported by Riber et al., 2009:“Chemical composition of material fractions in Danish household waste,” Waste Management 29:1251. Riber et al. characterized the component fractions of household waste obtained from 2220 homes in Denmark on a single day in 2001 . It will be readily understood by one skilled in the art, that this reported composition is simply a representative example as large variations may occur even within the same country as described further herein above. In Table 1 the exemplified organic fraction comprising vegetable, paper and animal waste is reported to have approximately 47% non water content on average: [(absolute % non-water)/(% wet weight)=(7.15 + 18.76 +
4.23)/(31.08 + 23.18 + 9.88) = 47% non-water content]. Addition of a volume of water corresponding to one weight equivalent of the waste fraction being processed would reduce the non-water content of the waste itself to 29.1% (58.2%/2) while reducing the non-water content of the degradable component to about 23.5% (47%/2). Addition of a volume of water corresponding to two weight equivalents of the waste fraction being processed would reduce the non-water content of the waste itself to 19.4% (58.2%/3) while reducing the non water content of the degradable component to about 15.7% (47%/3).
Table 1 Summarised mass distribution of MSW fractions, Denmark 2001
Part of overall Part of overall waste expressed as
Waste fraction waste quantity absolute contribution to total non
%wet weight water content of 58.2%
Vegetable waste (a) 31.08 7.15
Paper waste (b) 23.18 18.76
Animal waste(a) 9.88 4.23
Plastic waste (c) 9.17 8.43
Diapers (a) 6.59 3.59
Non combustibles (d) 4.05 3.45
Metal (e) 3.26 2.9
Glass (f) 2.91 2.71
Other (g) 9.88 6.98
TOTAL 100.00% 58.20%
(a) Pure fraction.
(b) Sum of: newspaper, magazines, advertisements, books, office and clean/dirty paper, paper and carton containers, cardboard, carton with plastic, carton with Al foil, dirty cardboard and kitchen tissues.
(c) Sum of: Soft plastic, plastic bottles, other hard plastic and non-recyclable plastic.
(d) Sum of: Soil, Rocks etc., ash, ceramics, cat litter and other non combustibles.
(e) Sum of: Al containers, al foil, metal-like foil, metal containers and other metal.
(f) Sum of: Clear, green, brown and other glass.
(g) Sum of: The remaining 13 material fractions.
One skilled in the art will readily be able to determine an appropriate quantity of water content, if any, to add to waste in adjusting water content. Typically as a practical matter, notwithstanding some variability in the composition of MSW being processed, it is convenient to add a relatively constant mass ratio of water (which includes aqueous solution), in some embodiments between 0.8 and 1 .8 kg water per kg MSW, or between 0.5 and 2.5 kg water per kg MSW, or between 1.0 and 3.0 kg water per kg MSW. As a result, the actual non-water content of the waste (or MSW) during processing may vary within the appropriate range.
The waste to be processed by the method of the present invention is suited for being processed by the method of the present invention when it comprises a mixture of biodegradable and non-biodegradable matter. The method according to the invention has been applied to wet municipal solid waste, comprising above 20% biodegradable material by weight on a wet basis, such as 10 - 100%, 15 - 60%, such as 20 - 55%, such as 25 - 50%, such as 30 - 50%. In some embodiments, the content of the biodegradable matter has been determined after drying of the waste in accordance with standard methods known in the art. In such circumstances, the content of the biodegradable material in the waste processed by the method of the present invention has been found to be above 10% biodegradable material by weight on a dry basis, such as between 10-100%, such as 10 - 40%, such as 15 - 35%, such as 15 - 30% or such as 15 - 25%.
When the method according to the present invention is to be used for approaches directed to the geographic origin of the waste, it will be necessary to register or at least to keep track of which geographic area the waste was disposed off. Normally, the waste is disposed off in the vicinity of its final use prior to becoming a“waste subject”. In the Western part of the world, renovation-collecting systems are put in place where the waste is collected by trucks or by other collecting means. Alternatively, the waste is placed at waste disposal sites usually subject to some kind of sorting principle. Regardless of whether the waste is collected by a truck at the vicinity of its final use, or collected by a truck at a waste disposal site, the waste normally enters the large scale plant in batches that correspond to the amount of waste that can be loaded on the specific truck or other transportation means. Optionally, more than one load from one truck may be collected in a collecting container such as a vessel or tank prior to subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant.
When the waste has been entered into the large scale plant, it is in accordance with step a) of the method according to the invention subjected to a combined enzymatic and microbial treatment.
Step a) The combined enzymatic and microbial treatment in step a) is performed by adding hydrolytic enzymes, supplied in either native form or in form of microbial organisms giving rise to the accumulation of such hydrolytic enzymes; and by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing
biochemicals, ethanol, or biogas.
Enzymatic hydrolysis is initiated at that point in time at which isolated enzyme preparations are added. Alternatively, in the event that isolated enzyme preparations are not added, but instead microorganisms that exhibit desired extracellular enzyme activities are used, enzymatic hydrolysis is initiated at that point which the desired microorganism is added.
In practicing methods of the invention, enzymatic hydrolysis is conducted concurrently with microbial fermentation. Concurrent microbial fermentation can be achieved using a variety of different methods. In some embodiments, microorganisms naturally present in the waste, such as MSW, are simply allowed to thrive under the reaction conditions, where the processed waste has not previously been heated to a temperature that is sufficient to effect a "sterilization." Typically, microorganisms present in waste, such as MSW, will include organisms that are adapted to the local environment. The general beneficial effect of concurrent microbial fermentation is comparatively robust, meaning that a very wide variety of different organisms can, individually or collectively, contribute to organic capture through enzymatic hydrolysis of waste, such as MSW. Without wishing to being bound by theory, we consider that co-fermenting microbes individually have some direct effect on degradation of food waste that are not necessarily hydrolysed by cellulase enzymes. At the same time, carbohydrate monomers and oligomers released by cellulase hydrolysis, in particular, are readily consumed by virtually any microbial species. This gives a beneficial synergy with cellulase enzymes, possibly through release of product inhibition of the enzyme activities, and also possibly for other reasons that are not immediately apparent. The end products of microbial metabolism in any case are typically appropriate for biomethane substrates. The enrichment of enzymatically hydrolysed waste, such as MSW, in microbial metabolites is, thus, already, in and of itself, an improvement in quality of the resulting biomethane substrate. Lactic acid bacteria in particular are ubiquitous in nature and lactic acid production is typically observed where waste such as MSW, is enzymatically hydrolysed at non-water content between 10 and 45% within the temperature range 35-65 °C , preferably within 40-60 °C, most preferably within 45-55 °C. At higher temperatures, possibly other species of naturally occurring microorganisms may predominate and other microbial metabolites than lactic acid may become more prevalent.
Thus, in one embodiment the treatment in step a) is accomplished by the use of one or more species of microorganisms present in the waste.
In some embodiments, microbial fermentation can be accomplished by a direct inoculation using one or more microbial species. It will be readily understood by one skilled in the art that one or more bacterial species used for inoculation so as to provide simultaneous enzymatic hydrolysis and fermentation of waste, such as MSW, can be advantageously selected where the bacterial species is able to thrive at a temperature at or near the optimum for the enzymatic activities used.
In one embodiment said combined enzymatic and microbial treatment in step a) is performed by adding hydrolytic enzymes, supplied in either native form or in form of microbial organisms giving rise to the accumulation of such enzymes; and by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing biochemicals, ethanol, or biogas.
Some fermentation will occur concurrent with the hydrolysis of the waste, such as municipal solid waste. Fermentable sugars obtained from the hydrolyzed waste material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a fermentation products such as one or more of volatile fatty acids (e.g. acetate, propionate, butyrate), lactate and alcohols.
In the fermentation step, sugars, released from the waste such as municipal solid waste material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast. Hydrolysis and fermentation can be separate or simultaneous.
Examples of fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast. Yeast include strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae. Examples of fermenting organisms that can ferment pentose sugars in their native state include bacterial and fungal organisms, such as some yeast. Xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis ; and strains of Pichia, e.g., P. stipitis, such as P. stipitis CBS 5773. Pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus. Organisms not capable of fermenting pentose sugars, such as xylose and arabinose, may be genetically modified to do so by methods known in the art.
Examples of bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonas mobilis (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212).
Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans·, Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. scehatae·, Clostridium, such as C. acetobutylicum, C. thermocellum, and C. phytofermentans ; E. coli, especially E. coli strains that have been genetically modified to improve the yield of ethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala·, Klebsiella, such as K. oxytoca·, Kluyveromyces, such as K. marxianus, K. lactis, K. thermotolerans, and K. fragilis·, Schizosaccharomyces, such as S. pombe ; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum and Zymomonas, such as Zymomonas mobilis.
The fermenting microorganism may have been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.
The fermenting organism may comprise one or more polynucleotides encoding one or more cellulolytic enzymes, hemicellulolytic enzymes, and accessory enzymes described herein.
The fermenting microorganism is typically added to the degraded waste such as municipal solid waste material or hydrolysate and the fermentation is performed for about 8 to about 96 h, e.g., about 24 to about 60 h. The temperature is typically between about 26°C to about 60°C, e.g., about 32°C or 50°C, and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
In one embodiment, the yeast and/or another microorganism are applied to the degraded waste, such as municipal solid waste material, and the fermentation is performed for about 12 to about 96 h, such as typically 24-60 h. In one embodiment, the temperature is above 20°C such asbetween about 20°C to about 60°C, e.g., about 25°C to about 50°C, about 32°C to about 50°C, or about 32°C to about 50°C, and the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7 such as pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 6.5 or pH 7. However, some fermenting organisms, e.g., bacteria, have higher fermentation temperature optima. Yeast or another microorganism is applied in amounts of approximately 105 to 1012, such as from approximately 107 to 1010, such as approximately 2 x 108 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g.,“The Alcohol Textbook” (Editors K. Jacques, T.P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.
A fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of the fermenting microorganisms, in particular, yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E and any combinations thereof. See, for example, Alfenore et a!., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu and any combinations thereof.
Inoculation of the hydrolysis mixture so as to induce microbial fermentation can be accomplished by a variety of different means.
In one embodiment the treatment in step a) is accomplished by the use of one or more species of lactic acid bacteria, acetate-producing bacteria, propionate-producing bacteria, or butyrate-producing bacteria, including any combination thereof. In some embodiments, it can be advantageous to inoculate the waste, such as MSW, either before, after or concurrently with the addition of enzymatic activities or with the addition of microorganisms that exhibit extra-cellular cellulase activity.
In one embodiment the treatment step a) comprises subjecting the waste with a microbially- derived cellulase activity of at least 30 FPU/L that is provided by one or more
microorganisms, such as a microbial consortium, providing microbial fermentation.
In another embodiment the treatment step a) comprises addition of cellulase activity by inoculation with one or more microorganism(s) that exhibits extracellular cellulase activity.
The cellulase activity is preferably one or more (e.g., several) activities selected from the group comprising endoglucanases, cellobiohydrolases, and beta-glucosidases.
In some embodiments, it can be advantageous to inoculate using one or more species of LAB including but not limited to any one or more of the following, or genetically modified variants thereof: Lactobacillus plantarum, Streptococcus lactis, Lactobacillus casei, Lactobacillus lactis, Lactobacillus curvatus, Lactobacillus sake, Lactobacillus helveticus, Lactobacillus jugurti, Lactobacillus fermentum, Lactobacillus carnis, Lactobacillus piscicola, Lactobacillus coryniformis, Lactobacillus rhamnosus, Lactobacillus maltaromicus,
Lactobacillus pseudoplantarum, Lactobacillus agilis, Lactobacillus bavaricus, Lactobacillus alimentarius, Lactobacillus uamanashiensis, Lactobacillus amylophilus, Lactobacillus farciminis, Lactobacillus sharpeae, Lactobacillus divergens, Lactobacillus alactosus, Lactobacillus paracasei, Lactobacillus homohiochii, Lactobacillus sanfrancisco,
Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus ponti, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus viridescens, Lactobacillus confusus, Lactobacillus minor, Lactobacillus kandleri, Lactobacillus halotolerans, Lactobacillus hilgardi,
Lactobacillus kefir, Lactobacillus collinoides, Lactobacillus vaccinostericus, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactobacillus leichmanni, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus salicinus, Lactobacillus gasseri, Lactobacillus suebicus, actobacillus oris, Lactobacillus brevis, Lactobacillus vaginalis, Lactobacillus pentosus, Lactobacillus panis, Lactococcus cremoris, Lactococcus dextranicum,
Lactococcus garvieae, Lactococcus hordniae, Lactococcus raffinolactis, Streptococcus diacetylactis, Leuconostoc mesenteroides, Leuconostoc dextranicum, Leuconostoc cremoris, Leuconostoc oenos, Leuconostoc paramesenteroides, Leuconostoc
pseudoesenteroides, Leuconostoc citreum, Leuconostoc gelidum, Leuconostoc carnosum, Pediococcus damnosus, Pediococcus acidilactici, Pediococcus cervisiae, Pediococcus parvulus, Pediococcus halophilus, Pediococcus pentosaceus, Pediococcus intermedius, Bifidobacterium longum, Streptococcus thermophilus, Oenococcus oeni , Bifidobacterium breve, and Propionibacterium freudenreichii, or with some subsequently discovered species of LAB or with other species from the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, or Carnobacterium that exhibit useful capacity for metabolic processes that produce lactic acid.
In one embodiment, the treatment step a) comprises contacting the waste with a live lactic acid bacteria concentration of at least 1 .0 x 10L10 CFU/L.
It will be readily understood by one skilled in the art that a bacterial preparation used for inoculation may comprise a community of different organisms. In some embodiments, one or more naturally occurring bacteria which exist in any given geographic region and which are adapted to thrive in waste, such as MSW, from that region, can be used. As is well known in the art, LAB are ubiquitous and will typically comprise a major component of any naturally occurring bacterial community within waste, such as MSW.
In some embodiments, waste such as MSW, can be inoculated with naturally occurring bacteria, by continued recycling of wash waters or process solutions used to recover residual organic material from non-degradable solids. As the wash waters or process solutions are recycled, they gradually acquire higher microbe levels. In some embodiments, microbial fermentation has a pH lowering effect, especially where metabolites comprise short chain carboxylic acids/ fatty acids such as one or more of formate, acetate, butyrate, proprionate, or lactate. Accordingly, in some embodiments it can be advantageous to monitor and adjust pH of the concurrent enzymatic hydrolysis and microbial fermentation mixture. Where wash waters or process solutions are used to increase water content of incoming waste, such as MSW, prior to enzymatic hydrolysis, inoculation is advantageously made prior to addition of enzyme activities, either as isolated enzyme preparations or as microorganisms exhibiting extra-cellular cellulase activity. In some embodiments, one or more naturally occurring bacteria adapted to thrive on waste, such as MSW, from a particular region can be cultured on waste, such as MSW, or on liquefied organic component obtained by enzymatic hydrolysis of waste, such as MSW. In some embodiments, one or more cultured naturally occurring bacteria can then be added as an inoculum, either separately or supplemental to inoculation using recycled wash waters or process solutions. In some embodiments, bacterial preparations can be added before or concurrently with addition of one or more isolated enzyme preparations, or after some initial period of pre-hydrolysis.
In some embodiments, one or more specific strains can be cultured for inoculation, including strains that have been specially modified or“trained” to thrive under enzymatic hydrolysis reaction conditions and/or to emphasize or de-emphasize particular metabolic processes. In some embodiments, it can be advantageous to inoculate waste, such as MSW, using one or more bacterial strains which have been identified as capable of surviving on phthalates as sole carbon source. Such strains include but are not limited to any one or more of the following, or genetically modified variants thereof:
Chryseomicrobium intechense MW10T, Lysinibaccillus fusiformis NBRC 157175,
Tropicibacter phthalicus, Gordonia JDC-2, Arthrbobacter JDC-32, Bacillus subtilis 3C3, Comamonas testosteronii, Comamonas sp E6, Delftia tsuruhatensis, Rhodoccoccus jostii, Burkholderia cepacia, Mycobacterium vanbaalenii, Arthobacter keyseri, Bacillus sb 007, Arthobacter sp. PNPX-4-2, Gordonia namibiensis, Rhodococcus phenolicus, Pseudomonas sp. PGB2, Pseudomonas sp. Q3, Pseudomonas sp. 1131, Pseudomonas sp. CAT1-8, Pseudomonas sp. Nitroreducens, Arthobacter sp AD38, Gordonia sp CNJ863, Gordonia rubripertinctus, Arthobacter oxydans, Acinetobacter genomosp, and Acinetobacter calcoaceticus. See e.g. Fukuhura et al 2012; Iwaki et al. 2012A; Iwaki et al. 2012B; Latorre et al. 2012; Liang et al. 2010; Liang et al. 2008; Navacharoen et al. 201 1 ; Park et al. 2009; Wu et al. 2010; Wu et al. 201 1 . Phthalates, which are used as plasticizers in many commercial poly vinyl chloride preparations, are leachable and, in our experience, are often present in liquefied organic component at levels that are undesirable. In some
embodiments, strains can be advantageously used which have been genetically modified by methods well known in the art, so as to emphasize metabolic processes and/or de- emphasize other metabolic processes including but not limited to processes that consume one or more of glucose, xylose or arabinose.
In some embodiments, it can be advantageous to inoculate waste, such as MSW, using one or more bacterial strains which have been identified as capable of degrading lignin. Such strains include but are not limited to any one or more of the following, or genetically modified variants thereof: Comamonas sp B-9, Citrobacter freundii, Citrobacter sp FJ581023, Pandorea norimbergensis, Amycolatopsis sp ATCC 39116, Streptomyces viridosporous, Rhodococcus jostii, and Sphingobium sp. SYK-6. See e.g. Bandounas et al. 201 1 ; Bugg et al. 201 1 ; Chandra et al. 201 1 ; Chen et al. 2012; Davis et al. 2012. In our experience, waste such as MSW typically comprises considerable lignin content, which is typically recovered as undigested residual after AD.
In some embodiments, it can be advantageous to inoculate waste, such as MSW, using one or more acetate-producing bacterial strain, including but not limited to any one or more of the following, or genetically modified variants thereof: Acetitomaculum ruminis,
Anaerostipes caccae, Acetoanaerobium noterae, Acetobacterium carbinolicum,
Acetobacterium wieringae, Acetobacterium woodii, Acetogenium kivui, Acidaminococcus fermentans, Anaerovibrio lipolytica, Bacteroides coprosuis, Bacteroides propionicifaciens, Bacteroides cellulosolvens, Bacteroides xylanolyticus, Bifidobacterium catenulatum, Bifidobacterium bifidum , Bifidobacterium adolescentis, Bifidobacterium angulatum,
Bifidobacterium breve, Bifidobacterium gallicum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudolongum, Butyrivibrio fibrisolvens, Clostridium aceticum, Clostridium acetobutylicum, Clostridium acidurici, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricium, Clostridium cellobioparum, Clostridium formicaceticum, Clostridium histolyticum, Clostridium lochheadii, Clostridium methylpentosum, Clostridium pasteurianum, Clostridium perfringens, Clostridium propionicum, Clostridium putrefaciens, Clostridium sporogenes, Clostridium tetani, Clostridium tetanomorphum, Clostridium thermocellum, Desulfotomaculum orientis, Enterobacter aerogenes, Escherichia coli, Eubacterium limosum, Eubacterium ruminantium, Fibrobacter succinogenes, Lachnospira multiparus, Megasphaera elsdenii, Moorella thermoacetica, Pelobacter acetylenicus, Pelobacter acidigallici, Pelobacter massiliensis, Prevotella ruminocola, Propionibacterium freudenreichii, Ruminococcus flavefaciens, Ruminobacter amylophilus, Ruminococcus albus, Ruminococcus bromii, Ruminococcus champanellensis, Selenomonas ruminantium, Sporomusa paucivorans, Succinimonas amylolytica, Succinivibrio dextrinosolven,
Syntrophomonas wolfei, Syntrophus aciditrophicus, Syntrophus gentianae, Treponema bryantii and Treponema primitia.
In some embodiments, it can be advantageous to inoculate waste, such as MSW, using one or more of a butyrate-producing bacterial strain, including but not limited to one or more of the following, or genetically modified variants thereof: Acidaminococcus fermentans, Anaerostipes caccae, Bifidobacterium adolescentis, Butyrivibrio crossotus, Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Clostridium acetobutylicum, Clostridium aurantibutyricum, Clostridium beijerinckii, Clostridium butyricium, Clostridium cellobioparum, Clostridium difficile, Clostridium innocuum, Clostridium kiuyveri, Clostridium pasteurianum, Clostridium perfringens, Clostridium proteoclasticum, Clostridium sporosphaeroides, Clostridium symbiosum, Clostridium tertium, Clostridium tyrobutyricum, Coprococcus eutactus,
Coprococcus comes, Escherichia coli, Eubacterium barkeri, Eubacterium biforme,
Eubacterium cellulosolvens, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium hadrum, Eubacterium halii, Eubacterium limosum, Eubacterium moniliforme, Eubacterium oxidoreducens, Eubacterium ramulus, Eubacterium rectale, Eubacterium saburreum, Eubacterium tortuosum, Eubacterium ventriosum, Faecalibacterium prausnitzii,
Fusobacterium prausnitzii, Peptostreptoccoccus vaginalis, Peptostreptoccoccus tetradius, Pseudobutyrivibrio ruminis, Pseudobutyrivibrio xylanivorans, Roseburia cecicola, Roseburia intestinalis, Roseburia hominis and Ruminococcus bromii.
In some embodiments, it can be advantageous to inoculate waste, such as MSW, using one or more of a propionate-producing bacterial strain, including but not limited to any one or more of the following, or genetically modified variants thereof: Anaerovibrio lipolytica, Bacteroides coprosuis, Bacteroides propionicifaciens, Bifidobacterium adolescentis, Clostridium acetobutylicum, Clostridium butyricium, Clostridium methylpentosum,
Clostridium pasteurianum, Clostridium perfringens, Clostridium propionicum, Escherichia coli, Fusobacterium nucleatum, Megasphaera elsdenii, Prevotella ruminocola,
Propionibacterium freudenreichii, Ruminococcus bromii, Ruminococcus champanellensis, Selenomonas ruminantium and Syntrophomonas wolfei.
The present invention also relates to a method wherein in step a) the waste is treated with an enzyme composition comprising a cellulolytic background composition combined with one or more enzymes selected from (i) a protease; (ii) a lipase and (iii) a beta-glucanase; and optionally combined with one or more further enzymes selected from (iv) a pectate lyase; (v) a mannanase and (vi) an amylase.
In one embodiment the treatment in step a) is accomplished by treating the waste with an enzyme composition comprising a cellulolytic background composition and one or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase. In another embodiment the treatment in step a) is accomplished by treating the waste with an enzyme composition comprises a cellulolytic background composition and two or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase (e.g. a protease and a lipase; a protease and a beta-glucanase; or a lipase and a beta-glucanase).
In a further embodiment treatment in step a) is accomplished by treating the waste with an enzyme composition comprising a cellulolytic background composition and (i) a protease,
(ii) a lipase, and (iii) a beta-glucanase.
In one embodiment the treatment in step a) is accomplished by treating the waste with an enzyme composition a cellulolytic background composition and (i) a protease, (ii) a lipase, and (iii) a beta-glucanase and further comprising one or more enzymes selected from (iv) a pectate lyase, (v) a mannanase, and (vi) an amylase.
In one embodiment the cellulolytic background composition comprises one or more enzymes selected from the group comprising: cellobiohydrolases I or variants thereof; cellobiohydrolases II or variants thereof; beta-glucosidases or variants thereof; polypeptides having cellulolytic enhancing activity; and/or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
In one embodiment the cellulolytic background composition comprises a cellobiohydrolase I or a variant thereof; a cellobiohydrolase II or a variant thereof; a beta-glucosidase or a variant thereof; and a polypeptide having cellulolytic enhancing activity; or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
In one embodiment of the invention, the cellulolytic background composition (CBC) comprises one or more enzymes selected from a) a cellobiohydrolase I or variant thereof;
(b) cellobiohydrolase II or variant thereof; (c) beta-glucosidase or variant thereof; and (d) a polypeptide having cellulolytic enhancing activity; or homologs thereof.
The cellulolytic background composition may comprise one or more enzymes selected from (a) an Aspergillus fumigatus cellobiohydrolase I or variant thereof; (b) an Aspergillus fumigatus cellobiohydrolase II or variant thereof; (c) an Aspergillus fumigatus beta- glucosidase or variant thereof; and (d) a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof. The cellulolytic background composition may comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLUCLAST® (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP (Genencor Int.),
ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Rohm GmbH), or ALTERNAFUEL® CMAX3™ (Dyadic
International, Inc.). The cellulolytic enzyme preparation is added in an amount effective from about 0.001 to about 5.0 wt. % of solids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 to about 2.0 wt. % of solids.
Examples of bacterial endoglucanases that can be used in the processes of the present invention, include, but are not limited to one or more of: Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551 ; U.S. Patent No. 5,536,655; WO 00/70031 ; WO 05/093050), Erwinia carotovara
endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), and Thermobifida fusca endoglucanase V (WO 05/093050).
Examples of fungal endoglucanases that can be used in the present invention, include, but are not limited to one or more of: Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichoderma reesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reesei endoglucanase II (Saloheimo et al., 1988, Gene 63:1 1 -22),
Trichoderma reesei Ce\5A endoglucanase II (GenBank:M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563,
GenBank:AB003694), Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228, GenBank:Z33381 ), Aspergillus aculeatus
endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusarium
oxysporum endoglucanase (GenBank:L29381 ), Humicola grisea var. thermoidea
endoglucanase (GenBank:AB003107), Melanocarpus albomyces endoglucanase
(GenBank:MAL515703), Neurospora crassa endoglucanase (GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthora thermophila CBS 1 17.65
endoglucanase, Thermoascus aurantiacus endoglucanase I (GenBank:AF487830), Trichoderma reesei strain No. VTT-D-80133 endoglucanase (GenBank:M15665), and Penicillium pinophilum endoglucanase (WO 2012/062220). Examples of cellobiohydrolases useful in the present invention include, but are not limited to one or more of: Aspergillus aculeatus cellobiohydrolase II (WO 201 1/059740), Aspergillus fumigatus cellobiohydrolase I (WO 2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO 2013/028928), Chaetomium thermophilum cellobiohydrolase I, Chaetomium
thermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolase I, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871 ), Penicillium occitan is cellobiohydrolase I (GenBank:AY690482), Talaromyces emersonii cellobiohydrolase
Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielav
cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and Trichophaea saccata cellobiohydrolase II (WO 2010/057086).
Examples of beta-glucosidases useful in the present invention include, but are not limited to one or more of: beta-glucosidases from Aspergillus aculeatus (Kawaguchi et al., 1996,
Gene 173: 287-288), Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO 201 1/035029), and Trichophaea saccata (WO 2007/019442).
Other useful endoglucanases, cellobiohydrolases, and beta-glucosidases are disclosed in numerous Glycosyl Hydrolase families using the classification according to Henrissat, 1991 , Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.
In the processes of the present invention, any“Auxiliary Activity 9 polypeptide” or“AA9” polypeptide can be used as a component of the enzyme composition.
Examples of AA9 polypeptides useful in the processes of the present invention include, but are not limited to one or more of: AA9 polypeptides from Thielavia terrestris (WO
2005/074647, WO 2008/148131 , and WO 201 1/035027), Thermoascus aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344), Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868, and WO 2009/033071 ), Aspergillus fumigatus (WO 2010/138754), Penicillium pinophilum (WO 201 1 005867), Thermoascus sp. (WO
201 1/039319), Penicillium sp. emersoni (WO 201 1/041397 and WO 2012/000892),
Thermoascus crustaceous (WO 201 1/041504), Aspergillus aculeatus (WO 2012/125925), Thermomyces lanuginosus (WO 2012/1 13340, WO 2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporus alborubescens (WO 2012/122477), Trichophaea saccata (WO 2012/122477), Penicillium thomii (WO 2012/122477), Talaromyces stipitatus (WO 2012/135659), Humicola insolens (WO 2012/146171 ), Malbranchea cinnamomea (WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), and Chaetomium
thermophilum (WO 2012/101206), and Talaromyces thermophilus (WO 2012/129697 and WO 2012/130950).
The (i) a protease may be derived from the genus Bacillus, such as e.g. Bacillus
amyloliquefaciens such as e.g. the protease encoded by SEQ ID NO:1 as disclosed in W017076421 , or a protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 as disclosed in W017076421 .
In a related embodiment of the invention the (ii) a lipase is derived from the genus
Thermomyces sp. such as e.g. Thermomyces lanuginosus such as e.g. the lipase encoded by SEQ ID NO: 2 as disclosed in W017076421 (or a lipase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 as disclosed in W017076421 ) or wherein the (ii) a lipase is derived from the genus Humicola sp. such as e.g. Humicola insolens (or a lipase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the Humicola insolens lipase). In a related embodiment of the invention the (iii) a beta-glucanase is derived from a member of the genus Aspergillus such as e.g. Aspergillus aculeatus such as e.g. the beta-glucanase encoded by the sequence encoded by SEQ ID NO: 4 as disclosed in W017076421 or homologs thereof (e.g., a beta- glucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 156875at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 as disclosed in W017076421 ). In a related embodiment of the invention the (iv) a pectate lyase forms part of a multicomponent enzyme composition comprising pectate lyase, xylanase and cellulase activities such as e.g. Novozym 81243™. In a related embodiment of the invention the (v) a mannanase is an endo-mannosidase derived from the genus Bacillus such as e.g. Bacillus bogoriensis such as e.g. the endo- mannosidase encoded by SEQ ID NO: 6 as disclosed in W017076421 or homologs thereof (e.g., an endo-mannosidase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6 as disclosed in W017076421 ). In a related embodiment of the invention the (vi) an amylase is an alpha-amylase derived from the genus
Rhizomucor such as e.g. Rhizomucor pusillus such as e.g. the alpha-amylase encoded by SEQ ID NO: 5 as disclosed in W017076421 or homologs thereof (e.g., an alpha-amylase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 as disclosed in W017076421 )..
In yet a related embodiment, the protease is present at a ratio between 0-20% w/w, such as e.g. 10% w/w of the total enzyme protein. In a related embodiment of the invention the the beta-glucanase is present at a ratio between 0-30% w/w, such as e.g. 15% w/w of the total enzyme protein. In a yet another related embodiment of the invention, the pectate-lyase is present at a ratio between 0-10% w/w, such as e.g. 5% w/w of the total enzyme protein. In a yet another related embodiment of the invention, the mannanase or amylase is present at a ratio between 0-10% w/w, such as e.g. 5% w/w of the total enzyme protein. In a yet another related embodiment of the invention the cellulolytic enzyme blend is present at a ratio between 40%-99% w/w, such as e.g. between 50%-90% w/w, such as e.g. 60%-80% w/w, such as e.g. 65-75% of the total enzyme protein. In a yet another related embodiment of the invention the enzyme composition further comprises one or more enzymes selected from a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein (CIP) an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin. In a yet another related embodiment of the invention the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
The enzymatically hydrolysing of the biodegradable parts of the waste concurrently with microbial fermentation according to step a) of the present invention may be performed at a temperature above 20°C and up to 75°C resulting in liquefaction of biodegradable parts of the waste and accumulation of microbial metabolites.
In one embodiment, the method according to treatment step a) is performed at a
temperature between 20 and 75°C, 30 and 70°C, 40 and 60°C, 45 and 55°C, or around 50°C. In practicing embodiments of the invention, it can be advantageous to adjust the
temperature of the waste such as MSW prior to initiation of enzymatic hydrolysis. As is well known in the art, cellulases and other enzymes typically exhibit an optimal temperature range. While examples of enzymes isolated from extreme thermohillic organisms are certainly known, having optimal temperatures on the order of 60 or even 70° C, enzyme optimal temperature ranges typically fall within the range 35°C to 55°C. In some
embodiments, enzymatic hydrolysis are conducted within the temperature range 30 to 35°C, or 35°C to 40° C, or 40°C to 45°C, or 45°C to 50°C, or 50°C to 55°C, or 55°C to 60°C, or 60°C to 65°C, or 65°C to 70°C, or 70°C to 75°C. In some embodiments it is advantageous to conduct enzymatic hydrolysis and concurrent microbial fermentation at a temperature of at least 45°C, because this is advantageous in discouraging growth of waste-borne pathogens, such as MSW-borne pathogens.
As used herein, the temperature to which waste such as MSW is heated is the highest average temperature of waste such as MSW achieved within the reactor. In some embodiments, the highest average temperature may not be maintained for the entire period. In some embodiments, the heating reactor may comprise different zones such that heating occurs in stages at different temperatures. In some embodiments, heating may be achieved using the same reactor in which enzymatic hydrolysis is conducted. The object of heating is simply to render the majority of cellulosic waste and a substantial fraction of the plant waste in a condition optimal for enzymatic hydrolysis. To be in a condition optimal for enzymatic hydrolysis, waste should ideally have a temperature and water content appropriate for the enzyme activities used for enzymatic hydrolysis.
In some embodiments, it can be advantageous to agitate during heating so as to achieve evenly heated waste. In some embodiments, agitation can comprise free-fall mixing, such as mixing in a reactor having a chamber that rotates along a substantially horizontal axis or in a mixer having a rotary axis lifting the waste such as MSW or in a mixer having horizontal shafts or paddles lifting the waste such as MSW. In some embodiments, agitation can comprise one or more of shaking, stirring or conveyance through a transport screw conveyor. In some embodiments, agitation continues after waste such as MSW has been heated to the desired temperature. In some embodiments, agitation is conducted for between 1 and 5 min, or between 5 and 10 min, or between 10 and 15 min, or between 15 and 20 min, or between 20 and 25 min, or between 25 and 30 min, or between 30 and 35 min, or between 35 and 40 min, or between 40 and 45 min, or between 45 and 50 min, or between 50 and 55 min, or between 55 and 60 min, or between 60 and 120 min.
The enzymatic liquefaction and fermentation in step a) is performed continuously at a certain residence time, at the optimal temperature and pH for enzyme performance.
In one embodiment the treatment step a) is conducted for a period of time in the range of 1 - 10Oh, in another embodiment the treatment step a) is conducted for a period of time in the range of 2.5 - 48h, in another embodiment the treatment step a) is conducted for a period of time in the range of 4 - 36h, and in a preferred embodiment, the treatment step a) is conducted for a period of time in the range of 5 - 25h, 6 - 18h, 8 - 15h or around 12h.
In treatment step a), the microbial fermentation is conducted under pH conditions that is adjusted to the optimum pH for the enzymatic activity, which is generally a pH at 7 or below. The pH may be adjusted in order to discourage methane production by methanogens, for example, by adjusting the pH to less than 6.0, or less than 5.8, or less than 5.6, or less than 5.5. In one embodiment treatment step a), is conducted at pH conditions that is
below 7.0, such as at pH 6.5, or at pH 6.0, or at pH 5.5, or at pH 5.0. The pH may be adjusted by any suitable means known in the art.
Step b)
Step b) in the method of the present invention is a separation step where the bio-liquid is separated from the non-degradable fractions. Clean and efficient use of the degradable component of waste, such as MSW, combined with recycling typically requires some method of sorting to separate degradable from non-degradable material. The separation in step b) may be performed by any means known in art, such as in a ballistic separator, washing drums or hydraulic presses.
In a preferred aspect of the invention, the liquid fraction is separated from the solid fraction of the waste at one or more of the following steps:
Prior to subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant;
After said enzymatic treatment but prior to said microbial treatment in a large- scale plant; After subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant.
After some period of enzymatic hydrolysis and concurrent microbial fermentation, the liquefied, fermentable parts of the waste are separated from non-fermentable solids. In some embodiments, at least 40% of the non-water content of this bioliquid comprises dissolved volatile solids, or at least 35%, or at least 30%, or at least 25%. In some embodiments, at least 25% by weight of the dissolved volatile solids in the bioliquid comprise any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate. In some embodiments, at least 70% by weight of the dissolved volatile solids comprises lactate, or at least 60%, or at least 50%, or at least 40%, or at least 30%, or at least 25%.
In one embodiment the liquid fraction in step b) has a pH below 7.0, such as a pH at 6.5, or a pH at 6.0, or a pH at 5.5, or a pH at 5.0, or a pH at 4.5. In other embodiments the liquid fraction in step b) has a pH below 6.5, a pH below 6.0, a pH below 5.5, a pH below 5.0, or a pH below 4.5.
In some embodiments, separation of non-fermentable solids from liquefied, fermentable parts of the waste, such as MSW, so as to produce a bioliquid characterized in comprising dissolved volatile solids of which at least 25% by weight comprise any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate is conducted in less than 16 h after the initiation of enzymatic hydrolysis, or in less than 18 h, or in less than 20 h, or in less than 22 h, or in less than 24 h, or in less than 30 h, or in less than 34 h, or in less than 36 h.
In one embodiment, the non-water content of the liquid fraction in step b) comprises at least 40% by weight dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate.
Separation of liquefied, fermentable parts of the waste from non-fermentable solids can be achieved by a variety of means. In some embodiments, this may be achieved using any combination of at least two different separation operations, including but not limited to screw press operations, ballistic separator operations, vibrating sieve operations, or other separation operations known in the art. In some embodiments, the non-fermentable solids separated from fermentable parts of the waste comprise at least about 20% of the dry weight of the waste such as MSW, or at least 25%, or at least 30%. In some embodiments, the non-fermentable solids separated from fermentable parts of the waste comprise at least 20% by dry weight of recyclable materials, or at least 25%, or at least 30%, or at least 35%. In some embodiments, separation using at least two separation operations produces a bioliquid that comprises at least 0.15 kg volatile solids per kg waste, such as MSW processed, or at least 0.10 kg volatile solids per kg waste, such as MSW, processed. It will be readily understood by one skilled in the art that the inherent biogenic composition of waste, such as MSW, is variable. Nevertheless, the figure 0.15 kg volatile solids per kg waste, such as MSW, processed reflects a total capture of biogenic material in typical unsorted waste, such as MSW, of at least 80%. The calculation of kg volatile solids captured in the bioliquid per kg waste, such as MSW, processed can be estimated over a time period in which total yields and total waste, such as total MSW, processed are determined.
The ballistic separator separates the enzymatic treated waste such as MSW into the bio liquid, a fraction of 2D non-degradable materials and a fraction of 3D non-degradable materials. The 3D fraction (physical 3 dimensional objects as cans and plastic bottles) does not bind large amounts of bio-liquid, so a single washing step is sufficient to clean the 3D fraction. The 2D fraction (textiles and foils as examples) binds a significant amount of bio liquid. Therefore, the 2D fraction is pressed using a screw press, washed and pressed again to optimize the recovery of bio-liquid and to obtain a“clean” and dry 2D fraction. Inert material which is sand and glass is sieved from the bio-liquid. The water used in all the washing drums can be recirculated, heated and then used as hot water in the first step for heating.
Step c)
In step c) of the method of the present invention the abundance of one or more chemical compound(s) or class(es) of compound(s) in the liquid fraction obtained in step b) is/are determined, or the identity of said chemical compound(s) or class(es) of compounds in the liquid fraction obtained in step b) is/are established.
By use of the present method, the identity of a chemical compound can be detected if it is present in the liquid fraction obtained in step b) in an amount above 0.00001 ppm. In one embodiment, any chemical compound measured in step c) is present in the liquid fraction obtained in step b) in an amount ranging from 0.00001 ppm to 90 ppm, such as from 0.0001 ppm to 90 ppm, or such as from 0.001 ppm to 90 ppm, or such as from 0.01 ppm to 90 ppm, or such as from 0.1 ppm to 10 ppm.
Accordingly, by use of the present method, the identity of a chemical compound can be detected if it is present in the liquid fraction obtained in step b) in an amount above 7E-02 ng/ml in 50 ml. of said liquid fraction obtained from step b).
In order for the chemical compound to be measured by any of the below-mentioned means, it may be necessary to prepare the sample of bioliquid obtained from step b) in order to separate the phases.
Thus, in one embodiment, in step c), a sample of the liquid fraction obtained from step b) is prepared in order to separate phases by centrifugation into supernatant and precipitate prior to determining the abundance of or establishing the identity of the chemical compound(s) or class(es) of compounds.
It may also be necessary to prepare the precipitate fraction of the separated sample of bioliquid obtained from step b) further by one or more means.
Thus, in step c), the separated precipitate fraction may be further prepared by one or more of the following treatments: pressurized liquid extraction of the precipitate with silica and dichloromethane; pressurized liquid extraction of the precipitate with silica and
dichloromethane followed by extraction with silica and methanol.
In a preferred embodiment the separated precipitate fraction is further prepared by pressurized liquid extraction of the precipitate with silica and dichloromethane. In another preferred embodiment, the separated precipitate fraction may be further prepared pressurized liquid extraction of the precipitate with silica and dichloromethane followed by extraction with silica and methanol.
It may also be necessary to prepare the supernatant fraction of the separated sample of bioliquid obtained from step b) further by one or more means. Thus, in step c), the separated supernatant fraction may further be prepared by one or more of the following treatments: liquid-liquid extraction with pentane, liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane, liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane followed by liquid- liquid extraction with dichloromethane, optionally adjusting the sample pH to 2 or 12.
In one preferred embodiment, the separated supernatant fraction is further prepared by liquid-liquid extraction with pentane. In another preferred embodiment the separated supernatant fraction is further prepared by liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane. In another preferred embodiment the separated supernatant fraction is further prepared by liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane followed by liquid-liquid extraction with dichloromethane. In any of the embodiments, the pH may optionally be adjusted to pH 2 or to pH 12.
Once the samples of bioliquid has optionally been prepared, the abundance of the chemical compound(s) or class(es) of compound(s) present in either the supernatant or the precipitate fraction may the determined, or the identity of the chemical compound(s) or class(es) of compound(s) may be established by one or more of the following methods: liquid chromatography, mass spectrometry, gas chromatography, liquid chromatography- mass spectrometry, gas chromatography-mass spectrometry, gas chromatography- electrospray ionization-mass spectrometry, liquid chromatography-electrospray ionization- mass spectrometry.
In a preferred embodiment the liquid obtained from step b) is separated into precipitate and supernatant and the compound(s) or class(es) of compound(s) in the precipitate is determined or established in step c) by preparing with pressurized liquid extraction with silica and dichloromethane followed by gas-chromatography mass spectrometry. In another preferred embodiment the liquid obtained from step b) is separated into precipitate and supernatant and the compound(s) or class(es) of compound(s) in the precipitate is determined or established in step c) by preparing with pressurized liquid extraction with silica and dichloromethane followed by pressurized liquid extraction with silica and methanol followed by liquid chromatography-mass spectrometry. In a preferred embodiment, the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by liquid chromatography-mass spectrometry. In another preferred embodiment, the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by prepareing with liquid-liquid pentane extraction followed by gas chromatography-mass spectrometry. In another preferred embodiment the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by prepareing with liquid-liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry. In another preferred embodiment the liquid obtained from step b) is separated into precipitate and supernatant and the compounds or classes of compounds in the supernatant is determined or established in step c) by preparing with liquid-liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by adjusting the aqueous phase to pH 2 or to pH12 and subjecting to liquid-liquid dichloromethane extraction followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry.
The identity/abundance of the chemical compound(s) or class(es) of compound(s) may be determined using one or more strategies. If the scope is to determine the
identity/abundance of a specific known chemical compound, the results obtained from the bioliquid sample may be compared to spiked samples containing a known amount of one or more target compounds. If the scope is to screen the bioliquid for several compounds, the results obtained from the samples comprising the bioliquid may be compared with one or more pre-selected chromatography signatures of known compounds. If the scope is a broader screen for various different compounds, the results obtained from the samples comprising the bioliquid may be compared with profiles of liquid-chromatography, mass- spectrometry, and/or gas-chromatography, respectively, from databases of known compounds.
In a preferred embodiment, determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing a sample from the liquid fraction obtained in step b) with spiked samples comprising known amounts of one or more target compounds. In another preferred embodiment, determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing a sample from the liquid fraction obtained in step b) with spiked samples comprising known amounts of one or more target compounds wherein said spiked sample used for quantification comprises e.g. at or above 10 ng/mL, such at 10 ng/ml, 100 ng/mL or 1000 ng/mL of the target compound to be quantified.
In a preferred embodiment determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing said sample from the waste batch by liquid-chromatography, and/ or gas-chromatography with one or more standards of known concentration analyzed by the same method.
In a preferred embodiment determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by identifying one or more chemical compounds from said sample from the waste batch by one or more of liquid- chromatography, mass-spectrometry, and/or gas-chromatography and comparing the obtained data with profiles of liquid-chromatography, mass-spectrometry, and/or gas- chromatography, respectively, from databases of known compounds.
The chemical compounds that can be detected in step c) of the present method have proven to be of very various character, and it is believed that the method will be applicable to most if not all chemical compounds that are present in the bioliquid fraction obtained from step b) according to the present invention
The method according to the present method has shown to be suitable for measuring ingredients and/or by-products of fat, fatty acids and derivatives, compounds from biological sources, pharmaceuticals, plasticizers, plastic additives, pesticides and anitmicrobial compounds, food additives, cosmetic additives and fluorinated compounds.
In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing fat and fatty acids. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to compounds from biological sources. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing pharmaceuticals. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing cosmetics. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing plastic additives. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing plasicisers. In one embodiment, the chemical compounds identified in the method according to the present invention are ingredients or by-products of, or are related to the process for providing fluorinated compounds.
The method of the present invention has proven suitable for measuring chemical compound(s) or class(es) of compound(s) selected from organic acids, free fatty acids, esters, alcohols, alkanes, phthalates, amides, bisphenols, aromatic, and poly-aromatic compounds and their heterocyclic, alkyl-, hydroxyl-, and carboxylated variants.
In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s). In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are free fatty acids. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are esters. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are alcohols. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are alkanes. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are phthalates. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are amides. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are bisphenols. In one embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are aromatic, and poly aromatic compounds and their heterocyclic, alkyl-, hydroxyl-, and carboxylated varieties.
In one embodiment, the chemical compound(s) or class(es) of compound(s)s determined or established in step c) is/are organic acid(s) selected from one or more of: carboxylic acids, sulfonic acids, alcohols, and organic compounds comprising an acidic thiol-, enol-, or phenol-group. In another embodiment, the chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s) selected from one or more of: 1 - adamantanecarboxylic acid, benzenepropanoic acid, 4-butylbenzoic acid,
cyclohexanecarboxylic acid, cyclohexanepentanoic acid, dicyclohexylacetic acid, diphenic acid, 1 -hydroxy-2-naphthoic acid, linolenic acid, myristic acid, 1 -naphthoic acid, 2-naphthoic acid, oleic acid, palmitic acid, palmitoleic acid, pentadecanoic acid, phthalic acid, 1 - pyrenecarboxylic acid, salicylic acid, sorbic acid.
In another embodiment, the chemical compound determined or established in step c) is selected from one or more of the following groups of compounds: Linear
alkylbenzensulfonates (LAS); Polycyclic aromatic hydrocarbon (PAH); å PAH = å
Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens,
Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylates (NPE). NPE comprise nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2-ethylhexyl)phthalate (DEHP); hormone-disturbing compounds e.g. bisphenol-A; drugs (amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa); pesticides; ingredients from personal care products (limonene, parabens); plasticisers other than DEHP; biocides (e.g. triclocarban, 2-phenylphenol, DEET); problematic compounds from paper and packaging industry (PFOS, PFOA).
In a preferred embodiment, the chemical compound determined or established in step c) is selected from one or more of: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one N,N-dimethyl-1 -dodecanamine, Benzophenone, Oleic acid, 4-hydroxy-3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, Methyl oleate, 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3- methylindole, 2-methylindole, 4-hydroxy-3,5-di-tert-butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d-limonene, Dodecanoic acid propyl ester, Ethyl oleate, Eugenol, Ibuprofen, Isopropyl palmitate, Methenamine, Methyl oleate, Myristic acid, N,N-dimethyl-1 - dodecanamine, Nicotine, Palmitic acid, Para-cresol, Pentadecanoic acid, Pentadecanoic acid methyl ester, Sorbic Acid, Triethyl citrate, (-)-Nicotine,
In a preferred aspect of the method according to the present invention, the method further comprises steps for linking the origin of the waste to the specific chemical compounds found in the waste. These steps comprise registering the geographic origin of a waste batch; the point in time wherein a given batch is delivered to the large scale plant and the point in time for processing the batch. This may sometimes require that a marker is added to the waste. The steps may also be a registration of the point in time when a batch enters and/or leaves one or more separation step(s).
In a further aspect of the invention, the method according to the present invention comprises in addition to step a), b) and c) one or more of:
Registering the geographic origin of a waste batch delivered for processing in said large-scale plant;
Registering the point in time for the start of the processing of said waste batch in the plant;
Adding one or more marker(s) to said waste batch;
Registering the point in time when said waste batch enters and/or leaves one or more treatment step(s) a) and/or separation step(s) b).
Anaerobic digestion of the bioliquid obtained from step b).
Production of biogas from anaerobic digestion of the bioliquid obtained from step b).
Anaerobic digestion is a series of biological processes in which microorganisms break down biodegradable material in the absence of oxygen. One of the end products is biogas, which can be combusted to generate electricity and heat, or can be processed into renewable natural gas and transportation fuels. A range of anaerobic digestion technologies exists in the state of the art for converting waste, such as municipal solid waste, municipal wastewater solids, food waste, high strength industrial wastewater and residuals, fats, oils and grease (FOG), and various other organic waste streams into biogas. Many different anaerobic digester systems are commercially available and the skilled persion will be familiar with how to apply and optimize the anaerobic digestions process. The metabolic dynamics of microbial communities engaged in anaerobic digestion are complex. In typical anaerobic digestion (AD) for production of methane biogas, biological processes mediated by microorganisms achieve four primary steps - hydrolysis of biological marcomolecules into constituent monomers or other metabolites; acidogenesis, whereby short chain hydrocarbon acids and alcohols are produced; acetogenesis, whereby available nutrients are catabolized to acetic acid, hydrogen and carbon dioxide; and methanogenesis, whereby acetic acid and hydrogen are catabolized by specialized archaea to methane and carbon dioxide. The hydrolysis step is typically rate-limiting It has been shown that the method according to the invention is particularly applicable when the waste is municipal solid waste. Under such circumstance, a preferred aspect of the invention is a method comprising the following steps:
Registering the geographic origin of a MSW batch delivered for processing in step a) and the point in time for the start of the processing of said waste batch in the plant;
Optionally adding a marker to said MSW batch;
Subjecting MSW to step a);
Monitoring the point in time when the material of said MSW batch leaves step a);
Subjecting the treated MSW from step a) to step b);
Monitoring the point in time when the material of said MSW batch enters and leaves each treatment step a) and b);
Subjecting the liquid obtained from step b) to step c);
Repeating step c) if the liquid obtained in step b) is subject to further treatment and separation steps and/or to anaerobic digestion.
The markers used in the method can be any marker known in the art that can be tracked and the choice of specific marker may depend on the waste to be processed.
The marker may be selected from one or more of: a radioactive labelled marker, a specific chemical compound that is resistant to bio-degradation, or a maker that can be visualised during the various steps of the method such as a fluorescent marker.
In a forth aspect, the present invention relates to the use of the method according to the present invention for decreasing the abundance of or eliminating the presence of one or more chemical compound(s) in waste such as MSW before, during or after anaerobic digestion of the waste, by adjusting one or more of the processing parameters in step a) or step b).
In a related fith aspect, the present invention relates to the use of the method according to the present invention for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste, such as MSW; based on the presence of one or more chemical compounds measured in step c).
For both the forth and the fifh aspect, said chemical compound(s) could be any
compound(s) of interest, preferably a compound(s) within one or more of the following groups of compounds: Linear alkylbenzensulfonates (LAS); Polycyclic aromatic
hydrocarbon (PAH); å PAH = å Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens, Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylates (NPE). NPE comprise nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2-ethylhexyl)phthalate (DEHP); hormone-disturbing compounds e.g.
bisphenol-A; drugs (amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa); pesticides; ingredients from personal care products (limonene, parabens);
plasticisers other than DEHP; biocides (e.g. triclocarban, 2-phenylphenol, DEET);
problematic compounds from paper and packaging industry (PFOS, PFOA).
In one embodiment of the forth and/or fitht aspect, said chemical compound(s) is selected from one or more of the following compound(s): butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one, Para-cresol, Skatole, N,N-dimethyl-1 - dodecanamine, Benzophenone, Oleic acid, 4-hydroxy-3,5-ditert-butylbenzaldehyde,
Phthalic acid, Pentadecanoic acid, Squalene, Methyl oleate, 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3-methylindole, 2-methylindole, 4-hydroxy- 3,5-di-tert-butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid,
Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d- limonene, Dodecanoic acid propyl ester, Ethyl oleate, Eugenol, Ibuprofen, Isopropyl palmitate, Methenamine, Methyl oleate, Myristic acid, N,N-dimethyl-1 -dodecanamine, Nicotine, Palmitic acid, Pentadecanoic acid, Pentadecanoic acid methyl ester, Triethyl citrate, (-)-Nicotine.
In one embodiment, the chemical compound measured is selected from one or more of: lactic acid, acetate, propionate andbutyrate. In another embodiment, the chemical compound measured is selected from one or more of: glucose, xylose, arabinose, lactate, acetale and ethanol.
Also, for both the forth and fifth aspects, the processing parameters to be adjusted could be any of the parameters mentioned above in relation to step a) and/or step c) of the method of the invention i.e the ratio of biodegradable waste to non-biodegradable waste, the amount of enzyme added to the waste, the identity of the specific enzyme composition added to the waste, the addition of microorganism to the waste, the temperature applied in step a), the retention time of the waste in step a), the pH applied in step a), the non-water content of the liquid fraction in step b),
Particularly, for both the forth and the fifth aspects, the processing parameter to be adjusted could be the retentiontime of the waste in bio-reactor and/or the retentiontime of the bioliqued in the subsequent AD process.
Preferred waste compositions
The method according to the present invention can be applied to any kind of waste comprising a mixture of biodegradable and non-biodegradable matter.
In one embodiment, the waste is derived from or comprises any one or more of waste from household, industry, agriculture, farming, county, or state activities.
The method according to the invention has been applied to wet municipal solid waste, comprising above 20% biodegradable material by weight on a wet basis, such as 15 - 60%, such as 20 - 55%, such as 25 - 50%, such as 30 - 50%. In some embodiments, the content of the biodegradable matter has been determined after drying of the waste in accordance with standard methods known in the art. In such circumstances, the content of the biodegradable material in the waste processed by the method of the present invention has been found to be above 10% biodegradable material by weight on a dry basis, such as between 10 - 40%, such as 15 - 35%, such as 15 - 30% or such as 15 - 25%.
In a particularly preferred aspect of the invention, the waste is municipal solid waste. Such waste may be sorted or unsorted. In one embodiment, the waste is unsorted municipal solid waste. In one embodiment, the waste is centrally sorted municipal solid waste. In one embodiment, the waste is source sorted municipal solid waste from households. In one embodiment, the waste is municipal solid waste processed by shredding or pulping. In one embodiment, the waste is organic fractions and paper rich fractions. In one embodiment, the waste is Refuse-Derived-Fuel fractions. In a preferred embodiment, the biodegradable material in said municipal solid waste comprises a combination of one or more items selected from: food residues, paper, cardboard, and fines.
In one embodiment the waste is sorted municipal solid waste not comprising items selected from one or more of the following: domestic appliances, glass, ceramics, batteries, newsprints, magazines, advertisements, books, plastics, fabrics, textiles, yard waste, electrical and electronic equipment, chemicals, pharmaceuticals, metals.
In one embodiment one or more of the following groups of items are removed from the waste prior to the combined enzymatic and microbial treatment in step a): leaves, grasses, wood, fabrics, stones, plastics, metals.
In another embodiment, the waste is selected from one or more of general industry waste fractions containing paper or other organic fractions, waste fractions from paper industry or recycling facilities, waste fractions from food and feed industry, waste fractions from the medicinal industry.
In another embodiment, the waste is selected from one or more of agriculture or farming, waste fractions from processes of sugar or starch rich products, contaminated or spoiled agricultural products not exploitable for food or feed purposes, manure, manure derived products.
In another embodiment, the waste is selected from one or more of waste fractions derived from municipal, county or state related or regulated activities, sludge from waste water treatment plants, fibre or sludge fractions from biogas processing, general waste fractions from the public sector containing paper or other organic fractions.
Pre-treatment of waste
Depending on the specific waste material, it may be advantageous to pre-treat the waste prior to subjecting the waste to step a) of the method according to the present invention.
Thus, in one embodiment, the said waste is subjected to pre-treatment prior to step a). The pre-treatment may depend upon the dry-matter content of the waste to be processed. The method according to the present invention is suitable for waste with high dry-matter content, such as waste having a dry-matter content above 20%. The dry-matter content may be even higher if the waste to be processed is municipal solid waste. In such cases, the dry- matter of the waste could be up to 75%, such as waste comprising 20 - 75% dry-matter, such as 30 - 75% dry-matter, such as 40 - 75% dry matter, such as 50 - 75% dry-matter, or such as 55 - 70% dry-matter.
Any pre-treatment process known in the art can be used to disrupt plant cell wall components of the municipal solid waste material (Chandra et a!., 2007, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv. Biochem. Engin./Biotechnol. 108: 41 -65; Hendriks and Zeeman, 2009, Bioresource Technology 100: 10-18; Mosier et al., 2005, Bioresource Technology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci. 9: 1621 -1651 ; Yang and Wyman, 2008, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).
In one embodiment of the invention waste, such as MSW, is subject to a mild to severe temperature pretreatment in the range 10-300°C prior to hydrolysis. Heating will normally occur together with a mixing. Heating will normally be carried out by addition of water or steam. Pretreatment might also consist of a separation (manual or automatic) of waste such as MSW in different fractions. The municipal solid waste material can also be subjected to particle size reduction, sieving, pre-soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art. For example, such pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
When the waste is municipal solid waste, the MSW may be pretreated both physically (mechanically) and chemically. Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi. In another aspect, high temperature means temperature in the range of about 100 to about 300°C, e.g., about 140 to about 200°C. In a one embodiment, mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired. The municipal solid waste material may accordingly be subjected to physical (mechanical) or chemical pre-treatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose, and/or lignin.
Mechanical or physical pre-treatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi. In another aspect, high temperature means temperature in the range of about 100 to about 300°C, e.g., about 140 to about 200°C. In one embodiment, mechanical or physical pre-treatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.
The waste may also be subjected to any biological pre-treatment known in the art that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the municipal solid waste material. Biological pretreatment techniques can involve applying lignin- solubilizing microorganisms and/or enzymes
Pre-treatment in general is required if subsequent hydrolysis (e.g. enzymatic hydrolysis) of the polysaccharides requires the break down of an otherwise protecting structure (e.g. lignin) of plant materials. Several pre-treatment techniques are known within e.g. the field of bioethanol production. Pre-treatment-processes may be based on e.g. acidic hydrolysis, steam explosion, oxidation, extraction with alkali or ethanol etc. A common feature of the pre treatment techniques is that combined with the action of possible added reactants they take advantage of the softening and loosening of plant materials that occurs at temperatures above 100°C, i.e. a process requiring the application of pressure.
The waste to be treated in accordance with the present method may optionally be subjected to pre-treatment prior to step a) and the pre-treatment is selected from one or more of: acid hydrolysis, steam explosion, oxidation, extraction with alkali, extraction with ethanol, sorting, shredding, pulping, pressure, size fractionation, bag opening, free fall mixing, stirring, rotation. In one embodiment, the waste is subjected to acid hydrolysis pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to steam explosion pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to oxidation pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to extraction with alkali pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to extraction with ethanol pre-treatment prior to step a) and optionally one or more other pre treatment means. In one embodiment, the waste is subjected to sorting pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to shredding pre-treatment prior to step a) and optionally one or more other pre treatment means. In one embodiment, the waste is subjected to pulping pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to pressure pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to size fractionation pre treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to bag opening pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to free fall mixing pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to stirring pre-treatment prior to step a) and optionally one or more other pre-treatment means. In one embodiment, the waste is subjected to rotation pre-treatment prior to step a) and optionally one or more other pre treatment means.
When the waste is municipal solid waste, sorting at the source, or alternatively some kind of central sorting of organic and inorganic fractions is believed to enhance performance of processes according to the present invention. For other fractions a rough shredding of the waste to open up bags and reduce volume is believed to enhance performance of processes according to the present invention.
Pre-treatment of municipal solid waste may be performed at atmospheric pressure which reduces the energy cost, the equipment cost and the mechanical difficulties significantly. Further more no chemical addition is needed in the pre-treatment. This is applicable if the waste comprises fractions where the polysaccharides mainly are sugars, starch or already pre-treated cellulose as paper, cardboard or similar. Hereby the costs for enzymes also are kept low, as amylases in general are cheaper than cellulases. This is a low-priced extraction of monosaccharides. Unconverted lignocellulosics can possibly be sorted out after the fermentation and used for instance in a process with high pressure pre-treatment.
The purpose of the pre-treatment may be to minimize the amount of unwanted
microorganisms through biocidal activities prior to the actual enzymatic treatment.
Examples of such activities are radioactive radiation, UV-radiation and electroporation. By using thermal treatment as an option, softening of processed biomass such as various paper fractions, break down of the internal structure of biomass with high water content and low lignin content such as vegetables, and opening of starch structures occurs in addition to the biocidal effect. If a proper performance of the subsequent hydrolysis of the
polysaccharides requires the decomposition of an otherwise protecting structure (e.g. lignin) of the original lignocellulosic material, a pressurised pre-treatment might be required.
However, the purpose of the pre-treatment in this is non-pressurised is not to break down protecting structures of non-processed biomass or the like, but rather to delimit microbial activity and to soften the processed waste fraction. The non-pressurised pre-treatment process is preferably based on steam admission, but can optionally be supplemented with acidic or alkali compounds. The non-pressurised pre-treatment of mono- and/or
polysaccharide containing waste fractions preferably utilises steam to heat up the waste to approximately 1005C.
If a thermal non-pressurised pre-treatment is chosen the following technical data is preferred:
• Pre-treatment temperature: 60-1 10°C, such as 65-105°C, such as 70-105°C, such as 75- 105°C and such as 80-100°C
• Pre-treatment time: 0-120 min, such as 5-100 min, such as 10-90 min, such as 20-80 min and such as 30-60 min.
• Pre-treatment steam admission: 0-2 kg/kg dry matter, such as 0.01 -1.5 kg/kg dry matter, such as 0.02-1.0 kg/kg dry matter, such as 0.03-0.8 kg/kg dry matter and such as 0.05- 0.5 kg/kg dry matter.
In one embodiment of the invention, the pre-treatment is a non-pressurised pre-treatment for up to 120 min with a temperature ranging between 60-1 10°C and a steam admission of up to 2 kg/kg dry matter. The non-pressurised pre-treatment described above is particularly suitable for waste having a relatively high dry matter content, preferably above 20%, and including relatively large particles. The treatment of said material can be carried out without additional water supply, detoxification or mechanical shredding.
However, it may also be beneficial to add additional water before adding enzymes or enzyme producing microorganisms in step a) of the present invention. Cooling and heating of the mash could optionally be performed by circulating cold or hot water (e.g. district heating water) through a vessel jacket or by injecting steam or cold water directly into the vessel. Prior to the enzymatic hydrolysis it may also be necessary to add additional water in order to reach an appropriate dry matter content, for the enzymes.
In one embodiment of the invention, 0.5 - 2.5 kg water per kg waste having a DM of up to 75% is added to the waste prior to subjecting said waste to step a).
In one embodiment of the invention, part or all of the water added to the waste prior to subjecting the waste to step a) is recycling wash water that is added to the incoming waste stream.
In addition to the above it should be noted that enzymatic hydrolysis and fermentation has traditionally been conducted in stirred tank reactors equipped with impellers, e.g. Rushton turbine or Intermig impeller, mounted on a centrally placed impeller shaft similar to what is used in the grain fermentation industry. Using this equipment, solutions of high viscosity, very sticky or very dry material cannot be stirred efficiently but will result in zones with very poor or no mixing. Furthermore, stirrings of such mixtures require very large energy inputs, which is detrimental to the process economy. Operating with mono- and/or polysaccharide containing waste fractions this has previously restricted the upper limit of the dry matter content to approximately 20%. The free fall based mixing principle overcomes this problem and may be used for poly- and/or monosaccharide containing waste fractions with dry matter content above 20%.
In one embodiment of the invention, waste with a dry matter content above 20% is processed mechanically, e.g. by free fall mixing while subjected to pre-treatment and/or to step a).
Waste, such as MSW, from big cities is often collected as is in plastic bags. The waste, such as MSW, is typically transported to the large scale waste refinery plant where it may become stored in a silo until processing. Depending on the character of the waste, a sorting step can be installed in front of the system to take out oversize particles.
In one embodiment of the invention, waste, such as sorted or unsorted MSW is size fractionated into fractions, providing a fraction with a size range of e.g. 0 to 60 cm, and/or providing an oversize fraction (bulk refuse refraction), such as a fraction comprising waste with a size exceeding 60 or more cm.
The oversize fraction may be pre-treated mechanically by any known means in order to reduce the size of the particles. When the waste in the oversize fraction has been de- fractionized, it may be subject to further pre-treatment as described herein or directly enter into step a) of the invention.
Use of the method according to the invention
An important aspect of the present invention is the use of the information that is obtained in step c) of the method of the invention.
Various compounds may be if interest to identify or quantify in step c) of the method of the invention in a given batch of waste and link this to the geographic origin of the waste. This is possible with the present invention in those embodiments where the method of the invention in addition to step a), b) and c) also comprises the following steps:
Registering the geographic origin of a waste batch delivered for processing in said large- scale plant;
Registering the point in time for the start of the processing of said waste batch in the plant;
Adding one or more marker(s) to said waste batch;
Registering the point in time when said waste batch enters and/or leaves one or more treatment step(s) a) and/or separation step(s) b).
And/or when the method of the invention comprises the following steps:
Registering the geographic origin of a waste batch, such as a MSW batch, delivered for processing in step a) and the point in time for the start of the processing of said waste batch in the plant; Optionally adding a marker to said waste batch;
Subjecting waste to step a);
Monitoring the point in time when the material of said waste batch leaves step a);
Subjecting the treated waste from step a) to step b);
Monitoring the point in time when the material of said waste batch enters and leaves each treatment step a) and b);
Subjecting the liquid obtained from step b) to step c);
Repeating step c) if the liquid obtained in step b) is subject to further treatment and separation steps.
Chemical compounds that may be of interest to idenity and/or quantify for use of the method according to the invention is for identifying the local geographical origin of specific chemical compounds present in the waste and optionally identifying the specific leaking or additive source(s) of said chemical compound (s).
In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of chemical compounds present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of fat, fatty acids or derivatives thereof present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of compounds from biological sources present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of medicinal drugs/pharmaceuticals and degradation products thereof present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of plasticizers present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of plastic additives present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of pesticides or antimicrobial compounds present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of food additives present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of cosmetic additives present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of fluorinated compounds present in the waste. In one embodiment of the present invention, use of the method according to the invention is for identifying the local geographical origin of chemical compounds subject to legal restrictions regarding their manufacture, import, use or disposal present in the waste. For all the embodiments mentioned above, the leaking or additive source(s) of said chemical compound (s) may optionally be identified.
This use may be directed to approaching the residents of the geographic area where the waste originated from with the purpose of preventing materials/compositions that was found in the waste to be present in future waste or to direct information or sales campaigns to a specific geographic area. The use may also be directed at a public affairs level with the purpose of urban planning, public health, detection of activities etc.
Use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste includes directing information campaigns, reward campaigns, fees for disposal of waste, sales activities, local sorting of waste, ; to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
In one embodiment if the invention, use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing information campaigns to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
In one embodiment of the invention, use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing reward campaigns to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
In one embodiment of the invention, use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing sales activities to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s). In one embodiment of the invention, use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing local sorting of waste to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
In one embodiment of the invention, use of a method according to the invention for identifying the local geographical origin of specific chemical compounds present in the waste is for directing environmental or social improvements to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
In one embodiment of the invention, the identity and/or abundance of a certain chemical compound(s) or class(es) of compounds from a certain geographic area is registered and compared over time so that the occurrence of chemical compounds of interest can be followed or surveilled.
A furhter embodiment of the invention is the use of a method according to the invention for decreasing the abundance of or eliminating the presence of a chemical compound measured in step c) in waste before, during or after fermentation of the waste by adjusting one or more of the processing parameters in step a) or step b) as described above.
In a related embodiment, the present invention relates to the use of the method according to the present invention for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste, such as MSW; based on the presence of one or more chemical compounds measured in step c).
A preferred embodiment of the invention is the use of a method according to the invention for degrading one or more of benzenepropanoic acid, benzyl benzoate, caffeine, ethyl oleate, eugenol, myristic acid, which are released during step a) by subsequent anaerobic digestion . Another preferred embodiment of the invention is the use of a method according to the invention further comprising an anaerobic digestion step wherein cholestan-3-one is generated.
Another preferred embodiment of the invention is the use of a method according to the present invention wherein in step b) polar and ionic chemical compounds primarily distribute to the bioliquid and hydrophobic chemical compounds primarily partition to the solid fraction.
In a preferred embodiment of the present invention, use of the method according to the invention is for identifying one or more of the following chemical compounds: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one, Para-cresol, Skatole, N,N-dimethyl-1 -dodecanamine, Benzophenone, Oleic acid, 4-hydroxy-3,5-ditert- butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, Methyl oleate, 13- Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3-methylindole, 2- methylindole, 4-hydroxy-3,5-ditert-butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d-limonene, Dodecanoic acid propyl esterEthyl oleate, Eugenol, Ibuprofen, Isopropyl palmitate, Methenamine, Methyl oleate, Myristic acid, N,N-dimethyl-1 - dodecanamine, Nicotine, Palmitic acid, Pentadecanoic acid, Pentadecanoic acid methyl ester, Sorbic Acid, Squalene, Triethyl citrate, (-)-Nicotine.
Use of the method according to the invention is possible within the limits of the method as described above for each of step a), b) and c). In particular, this includes waste that comprises both biodegradable and non-biodegradable material and particularly when the waste comprises 10-60% biodegradable material on a wet basis. The use of the method and method as such has proven to be efficient when the waste is municipal solid waste.
In one embodiment of the invention, the use of the method according to the invention is for waste comprising both biodegradable and non-biodegradable material.
In a further embodiment of the invention, the use of the method according to the invention is for waste comprising 10-60% biodegradable material on a wet basis.
In a preferred embodiment of the invention, the use of the method according to the invention is when the waste to be processed is municipal solid waste. Detailed description of the drawings
Figure 1 : Overview of the process applied at the large scale plant for processing of the waste in the examples disclosed herein. The plant was the Renescience plant situated at Amager in Denmark. Unsorted MSW was treated with enzymes in the bioreactor. The generated bioliquid was suitable for biogas production e.g. in a conventional biogas plant. Solids can be recoved in 2D and 3D fraction for recycling purposes.
Figure 2: Overview of the different sample pre-treatment conditions used in the examples herein. A schematic overview of the pre-treatment from centrifugation to separation of a solid fraction from a liquid fraction. The liquid-liquid extraction procedure where first a raw aqueous sample is taken out, then the same supernatant is extracted with pentane, then with DCM (dichloromethane), then the remaining aqueous phase is split into two, one part adjusted to pH 2 and the other to pH 12, and each of them was extracted. The pressurized liquid extraction of the solid part includes silica clean-up and dichloromethane as first extraction solvent, methanol as second extraction solvent, final extract volumes 50 ml_. * indicates samples for LC-MS analysis, # indicates samples for GC-MS analysis and n indicates samples for both LC-MS and GC-MS analysis. Fraction names are given in brackets.
Figure 3: GC-EI-MS analysis of benzenepropanoic acid. In the figure is shown the areas obtained by GC-EI-MS analysis of benzenepropanoic acid of the fractions obtained during treatment of the bioliquid (S1 , S2, S3) and the effluent after anaerobic digestion (AD1 , AD2, AD3). Benzenepropanoic acid is found in several fractions in the bioliquid but after anaerobic digestion it is not detected.
Figure 4: GC-EI-MS analysis of cholestan-3-one. In the figure is shown the areas obtained by GC-EI-MS analysis of cholestan-3-one of the fractions obtained during treatment of the bioliquid (S1 , S2, S3) and the effluent after anaerobic digestion (AD1 , AD2, AD3).
Cholestan-3-one is not detected in the bioliquid but after the after anaerobic digestion it was found in the DCM fraction following PLE.
Figure 5: GC-EI-MS analysis of d-limonene. In the figure is shown the areas obtained by GC-EI-MS analysis of d-limonene of the fractions obtained during treatment of the bioliquid (A1 a) and the two solid fractions (fiber and 3mm reject) after various extraction d-limonene clearly partitions pre-dominantly to the solid fractions which is in line with the compound being hydrophobic.
Figure 6: GC-EI-MS analysis of caffeine. In the figure is shown the areas obtained by GC- EI-MS analysis of caffeine of the fractions obtained during treatment of the bioliquid (A1 a) and the two solid fractions (fiber and 3mm reject) after various extraction. Caffeine clearly partitions pre-dominantly to the bioliquid which is in line with the compound being
hydrophilic.
The invention thus relates to the following embodiments:
1. A method for determining the abundance of or establishing the identity of one or more chemical compound(s) or class(es) of compounds in waste, such as a waste batch collected by a garbage truck, said method comprising the steps of:
a) Subjecting said waste to a combined enzymatic and microbial treatment in a large- scale plant;
b) Subjecting the treated waste from step a) to one or more separation step(s), whereby a liquid fraction is provided;
c) Determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in said liquid fraction obtained in step b).
2. The method according to embodiment 1 , wherein said waste is delivered to said large- scale plant in waste batches of a defined size.
3. The method according to embodiment 1 or 2, wherein the waste comprises both biodegradable and non-biodegradable material.
4. The method according to any one of the preceding embodiments, wherein said waste is unsorted or sorted municipal solid waste (MSW).
5. The method according to any one of the preceding embodiments, further comprising one or more of:
Registering the geographic origin of a waste batch delivered for processing in said large-scale plant; Registering the point in time for the start of the processing of said waste batch in the plant;
- Adding one or more marker(s) to said waste batch;
Registering the point in time when said waste batch enters and/or leaves one or more treatment step(s) a) and/or separation step(s) b)
- Anaerobic digestion of the bioliquid obtained from step b).
Production of biogas from anaerobic digestion of the bioliquid obtained from step b).
6. The method according to any one of the preceding embodiments, wherein said combined enzymatic and microbial treatment in step a) is performed by adding hydrolytic enzymes, supplied in either native form or in form of microbial organisms giving rise to the accumulation of such enzymes; and by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing biochemicals, ethanol, or biogas.
7. The method according to any one of the preceding embodiments, wherein the treatment in step a) is accomplished by the use of one or more species of lactic acid bacteria, acetate-producing bacteria, propionate-producing bacteria, or butyrate- producing bacteria, including any combination thereof.
8. The method according to any one of the preceding embodiments, wherein the treatment in step a) is accomplished by the use of one or more species of microorganisms present in the waste.
9. The method according to any one of the preceding embodiments, wherein the treatment step a) comprises contacting the waste with a live lactic acid bacteria concentration of at least 1 .0 x 10L10 CFU/L.
10. The method according to any one of the preceding embodiments, wherein the treatment step a) comprises subjecting the waste with a microbially-derived cellulase activity of at least 30 FPU/L that is provided by one or more microorganisms, such as a microbial consortium, providing microbial fermentation. 1 1 . The method according to any one of the preceding embodiments, wherein the treatment step a) comprises addition of cellulase activity by inoculation with one or more microorganism(s) that exhibits extracellular cellulase activity.
12. The method according to any one of the preceding embodiments, wherein the treatment in step a) is accomplished by treating the waste with an enzyme composition comprising a cellulolytic background composition and one or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase.
13. The method according to embodiment 12, wherein the composition comprises two or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase (e.g. a protease and a lipase; a protease and a beta-glucanase; or a lipase and a beta-glucanase).
14. The method according to any of embodiments 12 to 13, wherein the enzyme composition comprises (i) a protease, (ii) a lipase, and (iii) a beta-glucanase.
15. The method according to any of embodiments 12 to 14, wherein the enzyme composition further comprises one or more enzymes selected from (iv) a pectate lyase, (v) a mannanase, and (vi) an amylase.
16. The method according to any of embodiments 12 to 15, wherein the cellulolytic background composition comprises one or more enzymes selected from the group comprising: cellobiohydrolases I or variants thereof; cellobiohydrolases II or variants thereof; beta-glucosidases or variants thereof; polypeptides having cellulolytic enhancing activity; and/or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
17. The method according to any of embodiments 1 1 to 16, wherein the cellulolytic background composition comprises a cellobiohydrolase I or a variant thereof; a cellobiohydrolase II or a variant thereof; a beta-glucosidase or a variant thereof; and a polypeptide having cellulolytic enhancing activity; or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned. 18. The method according to any one of the preceding embodiments, wherein the treatment step a) is performed at a temperature between 20 and 75°C, 30°C and 70°C, 40°C and 60°C, 45 and 55°C, or around 50°C.
19. The method according to any one of the preceding embodiments, wherein the treatment step a) is conducted for a period of time in the range of 2.5-48h, 4-36h, 5- 24h, 6-18h, 8-15h, 9-14h or around 12h.
20. The method according to any one of the preceding embodiments, wherein the treatment step a) is performed at a pH below 7.0, 6.5, 6.0, 5.5, or 5.0.
21 . The method according to any one of the preceding embodiments, wherein the liquid fraction in step b) has a pH below 7.0, 6.5, 6.0, 5.5, 5.0, or 4.5.
22. The method according to any one of the preceding embodiments, wherein the non water content of the liquid fraction in step b) comprises at least 40% by weight dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate.
23. The method according to any of the preceding embodiments, wherein said determination of or establishment to the identity of a chemical compound and/or class of compounds in step c) is present in an amount at or above 0.00001 ppm.
24. The method according to any of the preceding embodiments, wherein said determination of or establishment to the identity of a chemical compound and/or class of compounds in step c) is present in an amount at or above 7E-02 ng/ml in 50 ml. of said liquid fraction obtained from step b).
25. The method according to any of the preceding embodiments, wherein in step c) a sample of the liquid fraction obtained from step b) is pre-treated in order to separate phases by centrifugation into supernatant and precipitate prior to determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds. The method according to embodiment 25 wherein the separated precipitate fraction is further pre-treated by one or more of the following treatments: pressurized liquid extraction of the precipitate with silica and dichloromethane; pressurized liquid extraction of the precipitate with silica and dichloromethane followed by extraction with silica and methanol. The method according to embodiment 25 wherein the separated supernatant fraction is further pre-treated by one or more of the following treatments: liquid-liquid extraction with pentane, liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane, liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane followed by liquid-liquid extraction with dichloromethane, optionally adjusting the sample pH to 2 or 12. The method according to any of the preceding embodiments, wherein in step c) determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds is done by one or more of the following methods: liquid chromatography, mass spectrometry, gas chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, gas chromatography-electrospray ionization-mass spectrometry, liquid chromatography- electrospray ionization-mass spectrometry. The method according to embodiment 28 wherein the liquid obtained from step b) is separated into precipitate and supernatant and wherein the compounds or classes of compounds in the precipitate is determined or established in step c) by one or more of the following: pre-treating with pressurized liquid extraction with silica and dichloromethane followed by gas-chromatography mass spectrometry; pre-treating with pressurized liquid extraction with silica and dichloromethane followed by pressurized liquid extraction with silica and methanol followed by liquid chromatography-mass spectrometry. The method according to embodiment 28 wherein the liquid obtained from step b) is separated into precipitate and supernatant and wherein the compounds or classes of compounds in the supernatant is determined or established in step c) by one or more of the following: liquid chromatography-mass spectrometry; pre-treating with liquid- liquid pentane extraction followed by gas chromatography-mass spectrometry; pre- treating with liquid- liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry; pre-treating with liquid-liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by adjusting the aqueous phase to pH 2 or 12 and subjecting to liquid- liquid dichloromethane extraction followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry.
31 . The method according to any one of the preceding embodiments, wherein determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing a sample from the liquid fraction obtained in step b) with spiked samples comprising known amounts of one or more target compounds.
32. The method according to embodiment 31 , wherein said spiked sample used for quantification comprises either 10 ng/mL, 100 ng/ml_ or 1000 ng/ml_ of the target compound to be quantified.
33. The method according to any one of the preceding embodiments, wherein determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is by comparing said sample from the waste batch by liquid-chromatography, and/ or gas-chromatography with one or more standards of known concentration analyzed by the same method.
34. The method according to any one of the preceding embodiments, wherein determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by identifying one or more chemical compounds from said sample from the waste batch by one or more of liquid- chromatography, mass-spectrometry, and/or gas-chromatography and comparing the obtained data with profiles of liquid-chromatography, mass-spectrometry, and/or gas- chromatography, respectively, from databases of known compounds.
35. The method according to any of the preceding embodiments, wherein said chemical compound or class(es) of compounds determined or established in step c) are ingredients or by-products of, or are related to the process for providing: fat, fatty acids and derivatives, compounds from biological sources, pharmaceuticals, plasticizers, plastic additives, pesticides and antimicrobial compounds, food additives, cosmetic additives and fluorinated compounds.
36. The method according to any of the preceding embodiments, wherein said chemical compound or class(es) of compounds determined or established in step c) is selected from organic acids, free fatty acids, esters, alcohols, alkanes, phthalates, amides, bisphenols, aromatic, and poly-aromatic compounds and their heterocyclic, alkyl-, hydroxyl-, and carboxylated varieties.
37. The method according to embodiment 36 wherein said chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s) selected from one or more of: carboxylic acids, sulfonic acids, alcohols, and organic compounds comprising an acidic thiol-, enol-, or phenol-group.
38. The method according to embodiment 37 wherein said chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s) selected from one or more of: 1 -adamantanecarboxylic acid, benzenepropanoic acid, 4-butylbenzoic acid, cyclohexanecarboxylic acid, cyclohexanepentanoic acid, dicyclohexylacetic acid, diphenic acid, 1 -hydroxy-2-naphthoic acid, linolenic acid, myristic acid, 1 -naphthoic acid, 2-naphthoic acid, oleic acid, palmitic acid, palmitoleic acid, pentadecanoic acid, phthalic acid, 1 -pyrenecarboxylic acid, salicylic acid, sorbic acid.
39. The method according to embodiment 37 wherein said chemical compound or class(es) of compounds determined or established in step c) is one or more compound(s) selected from the following groups of compounds: Linear alkylbenzensulfonates (LAS); Polycyclic aromatic hydrocarbons such as å Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens, Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylat, nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2- ethylhexyl)phthalate; hormone-disturbing compounds such as bisphenol-A; drugs such as amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa; pesticides; ingredients from personal care products such as limonene and parabens; plasticisers other than DEHP; biocides such as triclocarban, 2- phenylphenol, DEET; problematic compounds from paper and packaging industry such as PFOS and PFOA. The method according to any of the preceding embodiments, wherein said chemical compound determined or established in step c) is selected from one or more of: Para- cresol, Skatole, N,N-dimethyl-1 -dodecanamine, Benzophenone, Oleic acid, 4- hydroxy-3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, , Methyl oleate, , 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13- docosenamide, 3-methylindole, 2-methylindole, 4-hydroxy-3,5-ditert- butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d-limonene, Dodecanoic acid propyl ester, Ethyl oleate, Eugenol, Ibuprofen, Myristic acid, N,N- dimethyl-1 -dodecanamine, Nicotine, Palmitic acid, Pentadecanoic acid, Pentadecanoic acid methyl ester, Triethyl citrate, (-)-Nicotine,
The method according to any of the preceding embodiments, wherein the liquid fraction is separated from the solid fraction of the waste at one or more of the following steps:
Prior to subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant;
- After said enzymatic treatment but prior to said microbial treatment in a large-scale plant;
- After subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant.
- Anaerobic digestion of the bioliquid obtained from step b).
Production of biogas from anaerobic digestion of the bioliquid obtained from step b). The method according to any of the preceding embodiments comprising the steps of:
Registering the geographic origin of a MSW batch delivered for processing in step a) and the point in time for the start of the processing of said waste batch in the plant;
- Optionally adding a marker to said MSW batch;
- Subjecting MSW to step a);
- Monitoring the point in time when the material of said MSW batch leaves step a);
- Subjecting the treated MSW from step a) to step b);
Monitoring the point in time when the material of said MSW batch enters and leaves each treatment step a) and b); - Subjecting the liquid obtained from step b) to step c);
Repeating step c) if the liquid obtained in step b) is subject to further treatment and separation steps and/or to anaerobic digestion
43. The method according to any of the preceding embodiments, wherein said waste is derived from or comprises any one or more of waste from household, industry, agriculture, farming, county, or state activities.
44. The method according to any of the preceding embodiments, wherein said waste comprises 10-100% biodegradable material on a dry basis.
45. The method according to any of the preceding embodiments, wherein said waste comprises 20-30% biodegradable material on a dry basis.
46. The method according to any of the preceding embodiments, wherein said waste comprises 10-100% biodegradable material on a wet basis.
47. The method according to any of the preceding embodiments, wherein said waste comprises 25-60% such as 35-50% biodegradable material on a wet basis.
48. The method according to any of the preceding embodiments, wherein said waste is selected from one or more of unsorted municipal solid waste, centrally sorted municipal solid waste, source sorted municipal solid waste from households, municipal solid waste processed by shredding or pulping, organic fractions and paper rich fractions, Refuse-Derived-Fuel fractions.
49. The method according to any of the preceding embodiments wherein the biodegradable material in said waste municipal solid waste comprises a combination of one or more items selected from: food residues, paper, cardboard, and fines.
50. The method according to any of the preceding embodiments wherein said waste is sorted municipal solid waste not comprising items selected from one or more of the following: domestic appliances, glass, ceramics, batteries, newsprints, magazines, advertisements, books, plastics, fabrics, textiles, yard waste, electrical and electronic equipment, chemicals, pharmaceuticals, metals.
51 . The method according to any of the preceding embodiments, wherein one or more of the following groups of items are removed from the waste prior to the combined enzymatic and microbial treatment in step a): leaves, grasses, wood, fabrics, stones, plastics, metals.
52. The method according to any of the preceding embodiments, wherein said waste is selected from one or more of general industry waste fractions containing paper or other organic fractions, waste fractions from paper industry or recycling facilities, waste fractions from food and feed industry, waste fractions from the medicinal industry.
53. The method according to any of the preceding embodiments, wherein said waste is selected from one or more of agriculture or farming, waste fractions from processes of sugar or starch rich products, contaminated or spoiled agricultural products not exploitable for food or feed purposes, manure, manure derived products.
54. The method according to any of the preceding embodiments, wherein said waste is selected from one or more of waste fractions derived from municipal, county or state related or regulated activities, sludge from waste water treatment plants, fibre or sludge fractions from biogas processing, general waste fractions from the public sector containing paper or other organic fractions.
55. The method according to any of the preceding embodiments, wherein said waste is subjected to pre-treatment prior to step a).
56. The method according to embodiment 55 wherein said pre-treatment is selected from one or more of: acid hydrolysis, steam explosion, oxidation, extraction with alkali, extraction with ethanol, sorting, shredding, pulping, pressure, size fractionation, bag opening, free fall mixing, stirring, rotation. 57. The method according to embodiment 55 or 56, wherein 0.5 - 2.5 kg water per kg waste having a DM of up to 50% is added to the waste prior to subjecting said waste to step a).
58. The method according to embodiment 57 wherein part or all of said water is recycling wash water that is added to the incoming waste stream.
59. The method according to any of embodiments 55 to 58 wherein sorted or unsorted MSW is size fractionated into fractions, providing a fraction with a size range of e.g. 0 to 60 cm, and/or providing an oversize fraction (bulk refuse refraction), such as a fraction comprising waste with a size exceeding 60 or more cm.
60. The method according to any of embodiments 55 to 59 wherein said pre-treatment is a non-pressurised pre-treatment for up to 120 minutes with a temperature ranging between 60-1 10°C and a steam admission of up to 2 kg/kg dry matter.
61 . The method according to any of embodiments 55 to 60 wherein the waste with a dry matter content above 20% is processed mechanically, e.g. by free fall mixing while subjected to pre-treatment and/or to step a).
62. Use of a method according to any of embodiments 1 to 61 for identifying the local geographical origin of specific chemical compounds present in the waste and optionally identifying the specific leaking or additive source(s) of said chemical compound (s).
63. Use of a method according to any of embodiments 1 to 61 for identifying the local geographical origin of specific chemical compounds present in the waste and directing one or more of information campaigns, reward campaigns, sales activities, local sorting of waste, fees for disposal of waste; to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
64. Use of a method according to any of embodiments 1 to 61 for decreasing the abundance of or eliminating the presence of a chemical compound measured in step c) in waste before, during or after fermentation of the waste by adjusting one or more of the processing parameters in step a) and/or one or more of the parameters in step b). Use of the method according to any of embodiments 1 to 61 for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste; based on the presence of one or more chemical compounds measured in step c). Use according to embodiment 64 or 65 wherein said processing parameters to be adjusted are one or more of the parameters in step a) in accordance with embodiments 6, 7, 9, 10 to 20 and/or one or more of the parameters in step b) in accordance with embodiments 21 and 22. Use according to embodiment 64 or 65 wherein said processing parameter to be adjusted is the retention time of the waste in the bio-reactor and/or the retention time of the bioliquid in the anaerobic digestion process. Use of a method according to any of embodiments 1 to 61 for degrading one or more chemical composition such as benzenepropanoic acid, benzyl benzoate, caffeine, ethyl oleate, eugenol, myristic acid, which are released during step a) by subsequent anaerobic digestion. Use of a method according to any of embodiments 1 to 61 further comprising an anaerobic digestion step wherein a chemical composition is generated, such as cholestan-3-one. Use of a method according to any of embodiments 1 to 61 wherein in step b) polar and ionic chemical compounds primarily partition to the bioliguid and hydrophobic chemical compounds primarily partition to the solid fraction Use according to any of embodiments 62 to 70 wherein said chemical compound is selected from one or more of the following groups of compounds: Linear alkylbenzensulfonates; Polycyclic aromatic hydrocarbons such as å Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens, Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylat, nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2-ethylhexyl)phthalate; hormone-disturbing compounds such as bisphenol-A; drugs such as amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa; pesticides; ingredients from personal care products such as limonene and parabens; plasticisers other than DEHP; biocides such as triclocarban, 2-phenylphenol, DEET; problematic compounds from paper and packaging industry such as PFOS and PFOA.
72. Use according to any of embodiments 62 to 71 , wherein said chemical compound is selected from one or more of the following chemical compounds: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one , Para- cresol, Skatole, N,N-dimethyl-1 -dodecanamine, BenzophenoneOleic acid, 4-hydroxy- 3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalene, Methyl oleate, 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3- methylindole, 2-methylindole, 4-hydroxy-3,5-ditert-butylbenzaldehyde,
Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d-limonene, Dodecanoic acid propyl ester, Ethyl oleate, Eugenol, Ibuprofen, Isopropyl palmitate, Methenamine, Methyl oleate, Myristic acid, N,N-dimethyl-1 -dodecanamine, Nicotine, , Palmitic acid, Para-cresol, Pentadecanoic acid, Pentadecanoic acid methyl ester, Sorbic Acid, Triethyl citrate, (-)-Nicotine,
73. Use according to any one of embodiments 62 to 72, wherein said waste comprises both biodegradable and non-biodegradable material.
74. Use according to any one of embodiments 62 to 73, wherein said waste comprises 25-60% biodegradable material on a wet basis.
75. Use according to any one of embodiments 62 to 74, wherein said waste is municipal solid waste. References
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Examples
Materials and methods applied in the below examples
The technology applied in the below examples is a process for treatment of municipal solid waste (MSW) by use of enzymatic degradation and microbial fermentation taking place in a bioreactor. The bioliquid thus obtained comprises an organic fraction, which is suitable for anaerobic digestion leading to biogas production, while simultaneously recovering the solid fraction comprising e.g. plastics and metals for recycling or combustion. The process includes addition of enzyme to the bioreactor to facilitate liquefaction and separation of the biodegradable material from the 2D and 3D fractions. The bioliquid fraction obtained was subject to anaerobic digestion according to a process well known in the art wherein anaerobic microorganisms produces inter alia biogas. The overall process applied at the large scale plant is illustrated in Figure 1 . The plant was the Renescience plant situated at Amager in Denmark. Samples of the bioliquid fraction were collected at different steps in the process and the presence and/or abundance of various chemical compositions were measured using one or more of the methods as described in the specific examples.
In all of the examples, the waste subject to treatment was unsorted municipal solid waste (MSW) collected by vacuum truck in the Copenhagen area of Denmark.
The unsorted MSW was treated with the enzyme solution Cellic® CTec3 from Novozymes. The enzyme was received in pallet tanks from which it was pumped into the bioreactor. The enzyme was stored at room temperature and was not exposed to direct sunlight according to the manufacturer’s instructions. The enzyme addition of Cellic CTec3 to the process is set to 9 kg per ton MSW (i.e. 0.9 %). The bioreactor filling was 13.5 tonne.
The waste was processed in the bioreactor with a 12hr retention time and 80% rotation speed (corresponding to 0.8 turns per min). Water was added in a 2:1 waterwaste ratio and the enzyme dosage was 0.9% w/w relative to waste. The temperature in the bioreactor was set to 50°C. However, we do not expect the settings of the bio-reactor to influence significantly the outcome of the chemical fingerprint of the bio-liquid as long as there is sufficient liquifaction of the MSW. Samples were collected at several steps in the process. The output from the bio-reactor was separated using a ballastic separator into one liquid fraction (bioliquid) and two solid fractions denoted 2D and 3D. First, objects larger than 60mm x 60mm were removed from the liquid fraction. The liquid was then directed through a 3mm sieve and the rejected solid material from this sieve is denoted“3mm reject”, whereas the liquid going through the sieve is denoted “bioliquid”.
The 2D fraction consists mainly of large, flat objects such as plastic film and fabric. This fraction is washed and the resulting wash water was dewatered using a press. The solid, de watered fraction from this press is denoted“fiber fraction”.
The samples in questions and their labelling is described in Table 2.
Bioliquid samples (A1 ) were acquired for five consecutive days and pooled to even out day- to-day variations. In addition, one of the samples were split into three technical replicates (termed A1 a, A1 b, A1 c) to assess the quality of the sample preparation and analysis.
A suitable amount of bioliquid was stored (frozen) until it could be processed in the pilot-scale anaerobic digestion reactor (SEAD) at the technical university of Denmark. To ascertain the effect of the AD process samples were acquired before (S1 , S2, S3) and after the anaerobic digestion of the bioliquid (AD1 , AD2, AD3). In principle, the S1 -S3 samples should resemble bioliquid samples (A1 -A3) but due to storage, transport, thawing as well as variations due to changes in bio-reactor setting and incoming waste they are not identical.
The collected samples were subject to different sample pre-treatment conditions as illustrated in Figure 2. This Figure shows a schematic overview of the pre-treatment from centrifugation to separation of a solid fraction from a liquid fraction. The liquid-liquid extraction procedure where first a raw aqueous sample is taken out, then the same supernatant is extracted with pentane, then with DCM (dichloromethane), then the remaining aqueous phase is split into two, one part adjusted to pH 2 and the other to pH 12, and each of them extracted. The pressurized liquid extraction of the solid part includes silica clean-up and dichloromethane as first extraction solvent, methanol as second extraction solvent, final extract volumes 50 ml_. * indicates samples for LC-MS analysis, # indicates samples for GC-MS analysis and n indicates samples for both LC-MS and GC-MS analysis. Fraction names are given in brackets in Figure 2. The sample A1 (bioliquid) was split into three portions, each of which were extracted (termed A1 a, A1 b, A1 c). The samples carry information on intra-sample variation as well as analytical variation. The top seven samples were extracted in the spring 2016 along with a PPCO plastic container from University of Copenhagen. The PPCO, PVC and HPDE extracts served as plastic blanks. The remainder of the samples was extracted during summer 2016 along with an extra replicate of the A1 sample (termed A1 rep). All samples were analysed or re-analyzed together in the examples described below.
Example 1 : Quantification of target compounds
A set of 51 target compounds were quantified in 18 samples. Quantification was performed by a 1 - point calibration in either sample matrix S1 or AD1 , so that also matrix effects could be compensated for. The spike-concentration used for quantification was either 10 ng/mL, 100 ng/mL or 1000 ng/mL depending on what was the native sample concentration. In special cases, i.e. when the analyte was present in high amount and thus the spike-levels were insignificant, or in cases where the spiked level was not detected for other reasons, non matrix match calibration in solvent was applied. In these cases matrix effects have not been compensated for. Non-matrix match calibration was performed for 1 -adamantanecarboxylic acid (supernatant and PLE-DCM fraction), , cyclohexanepentanoic acid, and the four free fatty acids: linolenic acid, palmitoleic acid, linoleic acid and oleic acid.
Sample extracts were not pre-concentrated nor diluted prior to analysis in order to mitigate the introduction of excess variation.
In Table 3, compounds detected above the LOD by method (GC-EI-MS, LC-ESI- -MS or LC- ESI+-MS) and by fraction (supernatant, pentane, DCM, DCM alkaline, DCM acidic, PLE DCM, PLE MeOH) are reported along with their concentration. If the analyte is found in the supernatant, pentane, DCM, DCM alkaline or the DCM acidic fraction, then it originates from the liquid part of the sample. Concentrations found in pentane, DCM, DCM alkaline, DCM acidic are additive because these extractions are performed in a consecutive way on the same supernatant portion. The supernatant corresponds to the liquid part of the sample. If the analyte is found in the PLE DCM or PLE MeOH fraction, then it originates from the solid part of the sample. Concentrations in PLE DCM and PLE MeOH are additive for the same reason as stated above. A concentration of 1 ng/mL in the PLE extracts can be converted to 10 ng/g of solid material (ww) since 5 g of solid material (ww) was extracted with a volume of 50 mL. In column 4 and 5 of Table 3, the LODs obtained in each fraction for the S1 sample (to represent the S matrix) and for the AD1 sample (to represent the AD matrix) and for each compound is given. S samples have been quantified using the calibration in S1 , and AD samples using the calibration in AD1. A, B and C samples we evaluated to resemble more the feed, thus calibration in S1 has been used.
Levels detected below the corresponding LOD or below the maximum blank injection signal were discarded. For levels detected between LOD and LOQ (3.3 c LOD) no concentration was given, but they were highlighted by“<LOQ”.
Example 2: Spike experiments
From the early spike experiments (not disclosed) it was clear that analytes do not distribute in the same relative pattern, depending on if they are present in an S matrix or an AD matrix. Especially more of the carboxylic acids seem to be in their neutral form in the S matrix (they distribute into more non- polar fractions), whereas more carboxylic acids seem to be in their charged form in the AD matrix (they participate into acidic or more polar fraction). Most likely, this is due to the different pH of the S samples (below 7) and higher pH in the AD samples (above 7). If a similar analyte behavior across sample matrices are required, then pH could be adjusted prior to both PLE and LLE.
It was found that some analytes, such as bisphenol A behaves differently in feed (S) and anaerobic digest (AD), e.g. distribute differently between fractions. Behaviour of e.g.
bisphenol A suggest that the negatively charged specie prevail in AD samples while the neutral form prevail in S samples, thus AD have higher pH than S samples.
Example 3: Suspect screening
208 suspect compounds were looked for in GC-EI-MS chromatograms: samples A1 a, A1 b,
A1 c, A1 rep, A2, A3, B, C, PVC, HPDE, PPCO, S1 , S2, S3, AD1 , AD2, and AD3 and fractions pentane, DCM, DCM acidic, DCM alkaline, PLE-DCM and PLE-MeOH. 50 compounds were detected at elevated levels in samples compared to solvent blanks (all 6 fractions), plastic blanks (pentane and DCM fractions), and PLE blanks (PLE fractions). 22 of 50 compounds were discovered during the screening process, and were not previously included in the suspects screening list, but found in significantly elevated levels in selected samples. Peak areas and retention times for some of the compounds are listed in Table 4. Table 5 shows the suspect compounds found, examples of use and potential harmful effects. Information accessed through http://www.chemicalbook.com,
https://pubchem.ncbi.nlm.nih.gov and safety data sheets. Compounds discovered during the screening procedure, and which were not already part of the suspect list, have been marked by *.
Example 4: Degradation of compounds during AD
In some cases the chemical compounds which are released during the enzymatic liquifaction in the bio-reactor can be efficiently degraded during the ensuing anaerobic digestion. An example of this is benzenepropanoic acid, which is found in high
concentrations before the AD process (S1 , S2, S3) but has almost completely disappeared after the AD (AD1 , AD2, AD3). Figure 3 shows the areas obtained by GC-EI-MS analysis of benzenepropanoic acid of the fractions obtained during treatment of the bioliquid (S1 , S2, S3) and the effluent after anaerobic digestion (AD1 , AD2, AD3). Benzenepropanoic acid is found in several fractions in the bioliquid but after anaerobic digestion it has been completely degraded. Other examples of this behavior is benzyl benzoate, caffeine, ethyl oleate, eugenol, methylparaben, myristic acid, myristic acid ethyl ester, phenol and stearic acid propyl ester.
Example 5: Generation of new compounds during AD
In some cases chemical compounds which are not present in the waste nor in the bioliquid can be generated during anaerobic digestion. An example of this is Cholestan-3-one which is generated during oxidation of cholesterol and only present in the AD effluent. In Figure 4 is shown the areas obtained by GC-EI-MS analysis of cholestan-3-one of the fractions obtained during treatment of the bioliquid (S1 , S2, S3) and the effluent after anaerobic digestion (AD1 , AD2, AD3). Cholestan-3-one is not present in the bioliquid but after the after anaerobic digestion it can be found in the DCM fraction following PLE.
Example 6: Distribution between solid and liquid fractions
During the solid/liquid separation each compound will distribute between the two phases depending on their physicochemical properties. We expect polar and ionic compounds mainly to distribute to the bioliquid whereas hydrophobic compounds to a significantly larger extent will distribute to the solid fiber and 3mm reject fraction. Figure 5 shows the areas obtained by GC-EI-MS analysis of d-limonene of the fractions obtained during treatment of the bioliquid (A1 a) and the two solid fractions (fiber and 3mm reject) after various extractions d-limonene clearly distributes pre-dominantly to the solid fractions which is in line with the compound being hydrophobic.
Caffeine on the other hand can be found in much large concentrations in the bioliquid compared to the extractions of the solid fractions. Figure 6 shows the areas obtained by GC- EI-MS analysis of caffeine of the fractions obtained during treatment of the bioliquid (A1 a) and the two solid fractions (fiber and 3mm reject) after various extraction. Caffeine clearly distributes pre-dominantly to the bioliquid which is in line with the compound being hydrophilic.
Example 7: Pixel based analysis of GC-EI-MS chromatograms
Pentane and dichloromethane fractions
GC-EI-MS chromatograms of samples S1 -3 and AD1 -3 from the pentane and DCM fractions (neutral, alkaline and acidic) were evaluated using principal component analysis (PCA). PCA highlights the largest variation between these samples in the first principal component (PC1 ), the second largest variation between these samples in PC2 etc. Three types of mixture samples (mixture of both AD and S samples, and mixtures of either AD or S samples) were analyzed three times in each sequence and predicted on the PCA model for quality control. Prior to the PCA, sample chromatograms were preprocessed in the following order: Baselines were removed by lower Convex Hull subtraction, retention time aligned using lcoshift and correlation optimized warping, chromatograms normalized to their Euclidean norm and each variable scaled using the inverse of the relative median absolute deviation (MAD/mean)-1 of the mixture samples. Finally, chromatograms of the four fractions were sample-wise concatenated and the PCA was performed. PC1 , describing 79% of the explained variation (EV), separates the AD from the S samples along the X-axis, with high negative score for the S samples and high positive for the AD samples. PC2 (15% EV) plotted along the Y-axis, mainly describes variation amongst the AD samples and it is included primarily to improve visual interpretation.
The PC1 loadings were depicted for the most important coefficients, with a probable compound identification from the NIST database. Negative loading coefficients are observed for most organic acids, free fatty acids and ethyl esters in three sections (pentane, DCM acidic and DCM neutral), while the DCM basic section has negative coefficients for alcohols. This indicates that these compounds are relatively more abundant in the S samples. Positive coefficients were observed for 15 compounds along the PC1 loadings. Inspection of the raw chromatograms was performed to assess, whether the positive coefficients indicate higher absolute abundances of these compounds in the AD samples, or if it is a result of normalization.
Comparable signal intensities (accepting up to ±100 % variation) between AD and S samples were observed for 13 of the compounds, which indicate similar absolute abundances between the samples. For para-cresol significantly higher signal intensity (approx. 10 times higher) was observed for AD1 relative to S1 , suggesting an increase in absolute abundance, while other AD and S samples displayed comparable levels. Some compounds could not be identified in the S samples, indicating an increase in absolute abundance in the AD samples. Other compounds were identified in both AD and S samples with identical spectra, but with a systematic shift in retention time observed between the two groups of samples. This variation is expressed in the PC1 loadings as two set of coefficients of opposite sign. Inspection of the raw data indicates that the abundances of these compounds are similar in AD and S samples. The significance of this retention time shift, may question whether these are the same compounds found in both AD and S samples, or if they are isomeric compounds given rise to similar spectral match but different retention times.
PLE dichloromethane and PLE methanol fractions
GC-EI-MS chromatograms of samples AD1 -3 and S1 -3 from the PLE fractions (PLE-DCM and PLE- MeOH) were evaluated using PCA. Prior to PCA sample chromatograms where preprocessed in asimilar fashion as in the previous model. Once again PC1 (89%) separates the AD and S samples, while PC2 (6%) mainly describes variation amongst the S samples.
The PC1 loadings were depicted for the most important coefficients, and with a probable compound identification from the NIST database. From the PLE-DCM section, we observe negative coefficients for fatty acids and alkylated esters, while alkanes and phthalates have positive coefficients. In the PLE-MeOH section, negative coefficients are observed for alcohols, methyl esters, lactic acid, and caffeine. These negative coefficients represent compounds that are relatively more abundant in the S samples. Positive coefficients are observed for other fatty acid methyl esters and amides,. Comparable signal intensities (accepting up to ±100 % variation) between AD and S samples were observed for all compounds with positive coefficients, with exception of one compound that could not be identified in any of the S samples, which imply higher abundance of this compoind in the AD samples. Example 8: PARAFAC analysis of LC-ESI+-MS chromatograms
A non-target screening of the LC data was performed using Parallel Factor Analysis (PARAFAC), a multi-way decomposition method, which highlights the largest chemical variation through components, and extracts relevant information such as relative sample concentrations, retention time and pure mass spectra for each component.
PARAFAC models were established using the whole sample set (A1 a, A1 b, A1 c, A1 rep, A2, A3, B, C, PVC, HPDE, PPCO, S1 , S2, S3, AD1 , AD2, AD3, AD-Mix, S-Mix and AD+S-Mix) for five fractions (supernatant, DCM neutral, DCM alkaline and DCM acidic, PLE DCM and PLE MeOH). Prior to PARAFAC, chromatograms were binned to nominal masses and retention time aligned across samples. No normalization was done. For each component the diagnostic power (DP - diagnostic power refers to: Standard deviation (samples)/ standard deviation (Mix-AD+S replicates) and limit of detection (LOD - Limit of detection is calculated as: 3 x standard deviation(blank samples) + mean(blank samples) were calculated and constant or non-chemical components were manually removed, e.g. components representing chromatographic baselines. DP is used to assess the capability of a component to differentiate samples based on non-random variation. Spectral database search was performed for all components with DP >2.5 using the Norman MassBank and top 40 search matches were manually inspected for each component. Possible matches were further investigated in the raw chromatograms by exact mass of diagnostic ions, where 1 -10 ppm mass accuracy was considered acceptable. Tables 6 to 10 provides information on relevant components (i.e. DP>2.5 and at least one of AD or S samples above LOD) for all five fractions. Components highlighted by * display systematic differences in relative intensities between AD and S samples when doing a pair-wise comparison (e.g. AD1 and S1 or AD3 and S3). Relative intensities cannot be translated to sample concentrations directly, as matrix effects are not compensated for by the non-target approach. If a compound name is given in column 4‘Compound ID’ then it can be considered annotated. The compound name given in column 12‘Normann Mass Bank Candidates’ represent the closest match, and most likely not the exact match. Keep in mind that we rely on spectra obtained in the scan-mode in this non target approach, thus they are noisier compared to those found in the Normann Mass bank database. The latter originate from MSMS experiments, where the precursor ion has been isolated and fragmented by induced collisions. One way of getting closer to a compound identity could be to perform MSMS experiments on selected precursor ions, this would also improve the likelihood of finding a spectral match in the Normann Mass Bank. Focus here has been on components present in S and AD samples alone.
For the supernatant fraction a total of 57 components fulfilled the criteria of DP >2.5 and with levels detected above LOD for at least one AD or S sample (data not shown). One component showed systematic difference between the 6 AD and S samples, but with only S3 having a relative intensity above LOD. No spectral id was obtained for this component. Positive identification was obtained for two compounds, both of which are drugs used for treatment of Parkinson’s disease and depressive disorder, respectively.
Alkaline dichloromethane fraction
For the DCM alkaline fraction a total of 29 PARAFAC components fulfill the criteria of DP >2.5 and relative intensities above LOD for at least one AD or S samples (see Table 6, wherein some of the results are shown). 25 components showed systematic difference between the six AD and S samples; most displaying higher relative intensities in the S samples. 1 of the remaining 4 components were found at <LOD levels for all samples, while the remaining 3 component display relative intensities above >LOD levels for all 6 samples. A positive identification was obtained for the two components with highest diagnostic power; both fitted the exact mass and Norman MassBank spectra of L-nicotine, but the
components exhibited different retention times - most likely because a portion of the positively charged analyte eluted in the dead-volume. Nicotine was also detected during the suspect screening of DCM alkaline fraction measured by GC-MS. Similar trends are observed between the two sets of data with increased relative intensities in the S samples compared to the AD samples.
Neutral dichloromethane fraction
For the DCM neutral fraction a total of 53 PARAFAC components fulfilled the criteria of DP >2.5 and relative intensities above LOD for at least one AD or S samples (see Table 7 wherein some of the results are shown). Only 4 components displayed systematic differences in relative intensity between the S and AD samples, within these 4 components most samples have relative intensities below LOD. One component had a very high diagnostic power with S2 having relative intensities 992 times higher than LOD, while the remaining samples have relative intensities <2 times LOD. No positive identification was obtained for this component. Exact mass identification was achieved for L-nicotine, which did not provide a clear discrimination between AD and S samples.
The retention time of L-nicotine (0.92 min) fits well with that observed in the DCM alkaline fraction (i.e. 0.82-1.03 min). Nicotine wasalso detected at elevated levels in all AD and S during the suspect screening. This fits with current findings as both AD and S samples are found at >LOD levels. AD and S samples have comparable nicotine intensity ranges, in accordance with the findings of the suspect screening.
PLE dichloromethane fraction
For the PLE dichloromethane fraction a total of 81 PARAFAC components fulfilled the criteria of DP >2.5 and relative intensities above LOD for at least one AD or S samples (see Table 8, wherein some of the results are shown). 9 components discriminate systematically between the corresponding AD and S samples with relatively higher intensities displayed in the S samples.
One component displayed high relative intensity in both AD and S samples having levels up 766 times LOD, not clearly discriminating between the two sample classes. One component was identified as caffeine. Caffeine was also detected in the PLE DCM fractions during the suspect screening, although with a lower sample-wise correlation.
PLE methanol fraction
For the PLE MeOH fraction a total of 64 PARAFAC components fulfilled the criteria of DP >2.5 and relative intensity above LOD for at least one AD or S samples (see Table 9, wherein some of the results are shown). 7 components discriminated between the paired AD and S samples with relative higher intensities found amongst the AD samples. This differs from the remaining fractions, where most discriminative components show higher concentrations for the S samples. Identification was only achieved for one component, which matches the Norman MassBank spectra and the exact mass of caffeine. The retention time and relative concentrations match quite well that found in the PLE DCM fraction, and thus serves as a confirmation of compound identity. Caffeine was also detected in the PLE MeOH fractions during the suspect screening by GC-EI-MS, with S samples exhibiting higher peak areas. The relative pattern of GC-EI-MS peak areas does not match the relative pattern of intensities found for PARAFAC component, which only one S sample >LOD. The discrepancy may be explained by the varying matrix effects imposed by the S and the AD matrix on analyte area and intensity, when using either the GC-EI-MS or the LC-ESI+-MS platform, respectively.
Example 9: Variations related to sampling
It was found that it is possible to smooth out batch variations by this specific batch sampling strategy: sampling at three specific time-points within 24 h, and then pooling five days of sampling for each time-point (results not shown).
Example 10: Tracking of a chemical composition from a particular waste batch using marker Unsorted MSW is collected by vacuum truck in a registered part of the Copenhagen area in Denmark. The truck is selected prior to collecting the MSW and the truck-driver will register the arrival at the Renescience large scale plant situated at Amager in Copenhagen. While entering the waste into the plant the point of time for entering the waste and the amount of waste will be monitored. One or more of three different markers will be added to the waste bacth selected from a radioactive labelled marker, a specific chemical compound that is resistant to bio-degradation, and a maker that can be visualised during the various steps of the method such as a fluorescent marker.
The point in time when the waste is entered into the bioreactor will be monitored. The waste will be treated with the enzyme solution Cellic® CTec3 from Novozymes. The enzyme was received in pallet tanks from which it was pumped into the bioreactor. The enzyme was stored at room temperature and was not exposed to direct sunlight according to the manufacturer’s instructions. The enzyme addition of Cellic CTec3 to the process is set to 9 kg per ton MSW (i.e. 0.9 %). The bioreactor filling is 13.5 tonne.
The waste will be processed in the bioreactor with a 12hr retention time and 80% rotation speed (corresponding to 0.8 turns per min). Water is added in a 2:1 waterwaste ratio and the enzyme dosage was 0.9% w/w relative to waste. The temperature in the bioreactor was set to 50°C.
Samples of bioliquid will be collected at different point in times after the marked waste batch was entered into the bioreactor. One or more of the chemical compounds disclosed in Table 4 will be measured using one or more of the methods dislcosed in examples 7 and 8. These samples will also be screened for the presence of the marker.
The results will show that in some of the samples both wherein the chemical composition measured and the marker will be present. It may be concluded that the chemical composition measured in these samples originated from the waste batch collected by the truck that was selected for marking of the waste. The specific geographic origin of the MSW collected by this truck will be establihed by consulting the route applied by that specific truck for collecting this batch of MSW.
Example 1 1 : Tracking of a chemical composition from a particular waste batch
Unsorted MSW is collected by vacuum truck in a registered part of the Copenhagen area in Denmark. The truck is selected prior to collecting the MSW and the truck-driver will register the arrival at the Renescience large scale plant situated at Amager in Copenhagen. While entering the waste into the plant the point of time for entering the waste and the amount of waste will be recorded. The waste is placed in a designated lot in the bunker and it is not allowed to mix with waste from other regions of the city before it is entered into the bio reactor. The point in time when the waste is entered into the bioreactor is recorded. The waste will be treated with the enzyme solution Cellic® CTec3 from Novozymes. The enzyme was received in pallet tanks from which it was pumped into the bioreactor. The enzyme was stored at room temperature and was not exposed to direct sunlight according to the manufacturer’s instructions. The enzyme addition of Cellic CTec3 to the process is set to 9 kg per ton MSW (i.e. 0.9 %). The bioreactor filling is 13.5 tonne.
The waste will be processed in the bioreactor with a 12hr retention time and 80% rotation speed (corresponding to 0.8 turns per min). Water is added in a 2:1 waterwaste ratio and the enzyme dosage was 0.9% w/w relative to waste. The temperature in the bioreactor was set to 50oC.
Samples of bioliquid will be collected at different point in times after the waste batch was entered into the bioreactor. One or more of the chemical compounds disclosed in Table 4 will be measured using one or more of the methods disclosed in examples 7 and 8 and particular emphasis will be put on samples taken close to the retention time of the bio reactor. It can be advantageous to process all the waste from the same regionbefore starting to process waste from another region (AAABBB) rather than alternating between waste from different regions (ABABAB). The results will show that in some of the samples both wherein the chemical composition measured and the marker will be present. It may be concluded that the chemical composition measured in these samples originated from the waste batch collected by the truck that was entered into the reactor at the specific point in time. The specific geographic origin of the MSW collected by this truck will be established by consulting the route applied by that specific truck for collecting this batch of MSW.
Table 2 o
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Table 3
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Table 3 continued
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Table 3 continued
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Table 3 continued
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Table 3 continued
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Table 6
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Claims

Claims
1 . A method for determining the abundance of or establishing the identity of one or more chemical compound(s) or class(es) of compounds in waste, such as a waste batch collected by a garbage truck, said method comprising the steps of: a) Subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant;
b) Subjecting the treated waste from step a) to one or more separation step(s), whereby a liquid fraction is provided;
c) Determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in said liquid fraction obtained in step b).
2. The method according to claim 1 , wherein said waste is delivered to said large- scale plant in waste batches.
3. The method according to claim 1 or 2, wherein the waste comprises both biodegradable and non-biodegradable material.
4. The method according to any one of the preceding claims, wherein said waste is unsorted or sorted municipal solid waste (MSW).
5. The method according to any one of the preceding claims, further comprising one or more of:
Registering the geographic origin of a waste batch delivered for processing in said large-scale plant;
Registering the point in time for the start of the processing of said waste batch in the plant;
- Adding one or more marker(s) to said waste batch;
Registering the point in time when said waste batch enters and/or leaves one or more treatment step(s) a) and/or separation step(s) b)
- Anaerobic digestion of the bioliquid obtained from step b).
Production of biogas from anaerobic digestion of the bioliquid obtained from step b).
6. The method according to any one of the preceding claims, wherein said combined enzymatic and microbial treatment in step a) is performed by adding hydrolytic enzymes, supplied in either native form or in form of microbial organisms giving rise to the accumulation of such enzymes; and by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing biochemicals, ethanol, or biogas.
7. The method according to any one of the preceding claims, wherein the treatment in step a) is accomplished by the use of one or more species of lactic acid bacteria, acetate-producing bacteria, propionate-producing bacteria, or butyrate- producing bacteria, including any combination thereof.
8. The method according to any one of the preceding claims, wherein the treatment in step a) is accomplished by the use of one or more species of microorganisms present in the waste.
9. The method according to any one of the preceding claims, wherein the treatment step a) comprises contacting the waste with a live lactic acid bacteria concentration of at least 1 .0 x 10L10 CFU/L.
10. The method according to any one of the preceding claims, wherein the treatment step a) comprises subjecting the waste with a microbially-derived cellulase activity of at least 30 FPU/L that is provided by one or more microorganisms, such as a microbial consortium, providing microbial fermentation.
1 1. The method according to any one of the preceding claims, wherein the treatment step a) comprises addition of cellulase activity by inoculation with one or more microorganism(s) that exhibits extracellular cellulase activity. 12. The method according to any one of the preceding claims, wherein the treatment in step a) is accomplished by treating the waste with an enzyme composition comprising a cellulolytic background composition and one or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta-glucanase. 13. The method according to claim 12, wherein the composition comprises two or more enzymes selected from (i) a protease, (ii) a lipase, and (iii) a beta- glucanase (e.g. a protease and a lipase; a protease and a beta-glucanase; or a lipase and a beta-glucanase).
14. The method according to any of claims 12 to 13, wherein the enzyme composition comprises (i) a protease, (ii) a lipase, and (iii) a beta-glucanase. 15. The method according to any of claims 12 to 14, wherein the enzyme composition further comprises one or more enzymes selected from (iv) a pectate lyase, (v) a mannanase, and (vi) an amylase.
16. The method according to any of claims 12 to 15, wherein the cellulolytic background composition comprises one or more enzymes selected from the group comprising: cellobiohydrolases I or variants thereof; cellobiohydrolases II or variants thereof; beta-glucosidases or variants thereof; polypeptides having cellulolytic enhancing activity; and/or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
17. The method according to any of claims 1 1 to 16, wherein the cellulolytic background composition comprises a cellobiohydrolase I or a variant thereof; a cellobiohydrolase II or a variant thereof; a beta-glucosidase or a variant thereof; and a polypeptide having cellulolytic enhancing activity; or homologs of any of the aforementioned enzymes, including any combination of any of the aforementioned.
18. The method according to any one of the preceding claims, wherein the treatment step a) is performed at a temperature between 20 and 75°C, 30°C and 70°C, 40°C and 60°C, 45 and 55°C, or around 50°C.
19. The method according to any one of the preceding claims, wherein the treatment step a) is conducted for a period of time in the range of 2.5-48h, 4-36h, 5-24h, 6-18h, 8-15h, 9-14h or around 12h.
20. The method according to any one of the preceding claims, wherein the treatment step a) is performed at a pH below 7.0, 6.5, 6.0, 5.5, or 5.0.
21. The method according to any one of the preceding claims, wherein the liquid fraction in step b) has a pH below 7.0, 6.5, 6.0, 5.5, 5.0, or 4.5.
22. The method according to any one of the preceding claims, wherein the non-water content of the liquid fraction in step b) comprises at least 40% by weight dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate. 23. The method according to any of the preceding claims, wherein said determination of or establishment to the identity of a chemical compound and/or class of compounds in step c) is present in an amount at or above 0.00001 ppm.
24. The method according to any of the preceding claims, wherein said determination of or establishment to the identity of a chemical compound and/or class of compounds in step c) is present in an amount at or above7E-02 ng/ml in 50 ml. of said liquid fraction obtained from step b).
25. The method according to any of the preceding claims, wherein in step c) a sample of the liquid fraction obtained from step b) is pre-treated in order to separate phases by centrifugation into supernatant and precipitate prior to determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds. 26. The method according to claim 25 wherein the separated precipitate fraction is further pre-treated by one or more of the following treatments: pressurized liquid extraction of the precipitate with silica and dichloromethane; pressurized liquid extraction of the precipitate with silica and dichloromethane followed by extraction with silica and methanol.
27. The method according to claim 25 wherein the separated supernatant fraction is further pre-treated by one or more of the following treatments: liquid-liquid extraction with pentane, liquid-liquid extraction with pentane followed by liquid- liquid extraction with dichloromethane, liquid-liquid extraction with pentane followed by liquid-liquid extraction with dichloromethane followed by liquid-liquid extraction with dichloromethane, optionally adjusting the sample pH to 2 or 12.
28. The method according to any of the preceding claims, wherein in step c) determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds is done by one or more of the following methods: liquid chromatography, mass spectrometry, gas chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, gas chromatography-electrospray ionization-mass spectrometry, liquid chromatography-electrospray ionization-mass spectrometry.
29. The method according to claim 28 wherein the liquid obtained from step b) is separated into precipitate and supernatant and wherein the compounds or classes of compounds in the precipitate is determined or established in step c) by one or more of the following: pre-treating with pressurized liquid extraction with silica and dichloromethane followed by gas-chromatography mass spectrometry; pre-treating with pressurized liquid extraction with silica and dichloromethane followed by pressurized liquid extraction with silica and methanol followed by liquid chromatography-mass spectrometry.
30. The method according to claim 28 wherein the liquid obtained from step b) is separated into precipitate and supernatant and wherein the compounds or classes of compounds in the supernatant is determined or established in step c) by one or more of the following: liquid chromatography-mass spectrometry; pre treating with liquid-liquid pentane extraction followed by gas chromatography- mass spectrometry; pre-treating with liquid-liquid pentane extraction followed by liquid-liquid dichloromethane extraction of the aqueous phase followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry; pre-treating with liquid-liquid pentane extraction followed by liquid- liquid dichloromethane extraction of the aqueous phase followed by adjusting the aqueous phase to pH 2 or 12 and subjecting to liquid-liquid dichloromethane extraction followed by gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry.
31. The method according to any one of the preceding claims, wherein determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by comparing a sample from the liquid fraction obtained in step b) with spiked samples comprising known amounts of one or more target compounds.
32. The method according to claim 31 , wherein said spiked sample used for quantification comprises either 10 ng/mL, 100 ng/ml_ or 1000 ng/ml_ of the target compound to be quantified.
33. The method according to any one of the preceding claims, wherein determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is by comparing said sample from the waste batch by liquid-chromatography, and/ or gas-chromatography with one or more standards of known concentration analyzed by the same method.
The method according to any one of the preceding claims, wherein determining the abundance of or establishing the identity of said chemical compound(s) or class(es) of compounds in step c) is done by identifying one or more chemical compounds from said sample from the waste batch by one or more of liquid- chromatography, mass-spectrometry, and/or gas-chromatography and comparing the obtained data with profiles of liquid-chromatography, mass- spectrometry, and/or gas-chromatography, respectively, from databases of known compounds.
The method according to any of the preceding claims, wherein said chemical compound or class(es) of compounds determined or established in step c) are ingredients or by-products of, or are related to the process for providing: fat, fatty acids and derivatives, compounds from biological sources, pharmaceuticals, plasticizers, plastic additives, pesticides and anitmicrobial compounds, food additives, cosmetic additives and fluorinated compounds.
The method according to any of the preceding claims, wherein said chemical compound or class(es) of compounds determined or established in step c) is selected from organic acids, free fatty acids, esters, alcohols, alkanes, phthalates, amides, bisphenols, aromatic, and poly-aromatic compounds and their heterocyclic, alkyl-, hydroxyl-, and carboxylated varieties.
The method according to claim 36 wherein said chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s) selected from one or more of: carboxylic acids, sulfonic acids, alcohols, and organic compounds comprising an acidic thiol-, enol-, or phenol-group.
The method according to claim 37 wherein said chemical compound or class(es) of compounds determined or established in step c) is/are organic acid(s) selected from one or more of: 1 -adamantanecarboxylic acid, -, , , benzenepropanoic acid, 4-butylbenzoic acid, cyclohexanecarboxylic acid, cyclohexanepentanoic acid, dicyclohexylacetic acid, diphenic acid, 1 -hydroxyl- naphthoic acid, - linoleic acid, linolenic acid, , myristic acid, 1 -naphthoic acid, 2- naphthoic acid, oleic acid, palmitic acid, palmitoleic acid, pentadecanoic acid, phthalic acid, 1 -pyrenecarboxylic acid, salicylic acid, sorbic acid.
The method according to claim 37 wherein said chemical compound or class(es) of compounds determined or established in step c) is one or more compound(s) selected from the following groups of compounds: Linear alkylbenzensulfonates (LAS); Polycyclic aromatic hydrocarbons such as å Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens, Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylat, nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2- ethylhexyl)phthalate; hormone-disturbing compounds such as bisphenol-A; drugs such as amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa; pesticides; ingredients from personal care products such as limonene and parabens; plasticisers other than DEHP; biocides such as triclocarban, 2-phenylphenol, DEET; problematic compounds from paper and packaging industry such as PFOS and PFOA.
The method according to any of the preceding claims, wherein said chemical compound determined or established in step c) is selected from one or more of: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one Para-cresol, Skatol, N,N-dimethyl-1 -dodecanamine, Benzophenon, Oleic acid, 4-hydroxy-3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalen, Methyl oleate, 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3-methylindole, 2-methylindole, 4- hydroxy-3,5-ditert-butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d-limonene, Dodecanoic acid propyl ester, Ethyl oleate, Eugenol, Ibuprofen, Isopropyl palmitate, Methenamine, Methyl oleate, Myristic acid, N,N- dimethyl-1 -dodecanamine, Nicotine, Palmitic acid, Para-cresol, Pentadecanoic acid, Pentadecanoic acid methyl ester, Sorbic Acid, Squalene, Triethyl citrate, L- Nicotine, (-)-Nicotine,
The method according to any of the preceding claims, wherein the liquid fraction is separated from the solid fraction of the waste at one or more of the following steps:
Prior to subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant;
- After said enzymatic treatment but prior to said microbial treatment in a large- scale plant; - After subjecting said waste to a combined enzymatic and microbial treatment in a large-scale plant.
- Anaerobic digestion of the bioliquid obtained from step b).
Production of biogas from anaerobic digestion of the bioliquid obtained from step b).
42. The method according to any of the preceding claims comprising the steps of:
Registering the geographic origin of a MSW batch delivered for processing in step a) and the point in time for the start of the processing of said waste batch in the plant;
- Optionally adding a marker to said MSW batch;
- Subjecting MSW to step a);
Monitoring the point in time when the material of said MSW batch leaves step a);
- Subjecting the treated MSW from step a) to step b);
Monitoring the point in time when the material of said MSW batch enters and leaves each treatment step a) and b);
- Subjecting the liquid obtained from step b) to step c);
Repeating step c) if the liquid obtained in step b) is subject to further treatment and separation steps and/or to anaerobic digestion
43. The method according to any of the preceding claims, wherein said waste is derived from or comprises any one or more of waste from household, industry, agriculture, farming, county, or state activities.
44. The method according to any of the preceding claims, wherein said waste comprises 10-100% biodegradable material on a dry basis. 45. The method according to any of the preceding claims, wherein said waste comprises 20-30% biodegradable material on a dry basis.
46. The method according to any of the preceding claims, wherein said waste comprises 10-100% biodegradable material on a wet basis.
47. The method according to any of the preceding claims, wherein said waste comprises 25-60% such as 35-50% biodegradable material on a wet basis.
48. The method according to any of the preceding claims, wherein said waste is selected from one or more of unsorted municipal solid waste, centrally sorted municipal solid waste, source sorted municipal solid waste from households, municipal solid waste processed by shredding or pulping, organic fractions and paper rich fractions, Refuse-Derived-Fuel fractions.
49. The method according to any of the preceding claims wherein the biodegradable material in said waste municipal solid waste comprises a combination of one or more items selected from: food residues, paper, cardboard, and fines.
50. The method according to any of the preceding claims wherein said waste is sorted municipal solid waste not comprising items selected from one or more of the following: domestic appliances, glass, ceramics, batteries, newsprints, magazines, advertisements, books, plastics, fabrics, textiles, yard waste, electrical and electronic equipment, chemicals, pharmaceuticals, metals.
51. The method according to any of the preceding claims, wherein one or more of the following groups of items are removed from the waste prior to the combined enzymatic and microbial treatment in step a): leaves, grasses, wood, fabrics, stones, plastics, metals.
52. The method according to any of the preceding claims, wherein said waste is selected from one or more of general industry waste fractions containing paper or other organic fractions, waste fractions from paper industry or recycling facilities, waste fractions from food and feed industry, waste fractions from the medicinal industry.
53. The method according to any of the preceding claims, wherein said waste is selected from one or more of agriculture or farming, waste fractions from processes of sugar or starch rich products, contaminated or spoiled agricultural products not exploitable for food or feed purposes, manure, manure derived products. 54. The method according to any of the preceding claims, wherein said waste is selected from one or more of waste fractions derived from municipal, county or state related or regulated activities, sludge from waste water treatment plants, fibre or sludge fractions from biogas processing, general waste fractions from the public sector containing paper or other organic fractions.
55. The method according to any of the preceding claims, wherein said waste is subjected to pre-treatment prior to step a).
56. The method according to claim 55 wherein said pre-treatment is selected from one or more of: acid hydrolysis, steam explosion, oxidation, extraction with alkali, extraction with ethanol, sorting, shredding, pulping, pressure, size fractionation, bag opening, free fall mixing, stirring, rotation.
57. The method according to claim 55 or 56, wherein 0.5 - 2.5 kg water per kg waste having a DM of up to 50% is added to the waste prior to subjecting said waste to step a).
58. The method according to claim 57 wherein part or all of said water is recycling wash water that is added to the incoming waste stream.
59. The method according to any of claims 55 to 58 wherein sorted or unsorted MSW is size fractionated into fractions, providing a fraction with a size range of e.g. 0 to 60 cm, and/or providing an oversize fraction (bulk refuse refraction), such as a fraction comprising waste with a size exceeding 60 or more cm.
60. The method according to any of claims 55 to 59 wherein said pre-treatment is a non-pressurised pre-treatment for up to 120 min with a temperature ranging between 60-1 10°C and a steam admission of up to 2 kg/kg dry matter.
61. The method according to any of claims 55 to 60 wherein the waste with a dry matter content above 20% is processed mechanically, e.g. by free fall mixing while subjected to pre-treatment and/or to step a).
62. Use of a method according to any of claims 1 to 61 for identifying the local geographical origin of specific chemical compounds present in the waste and optionally identifying the specific leaking or additive source(s) of said chemical compound (s).
63. Use of a method according to any of claims 1 to 61 for identifying the local geographical origin of specific chemical compounds present in the waste and directing one or more of information campaigns, reward campaigns, sales activities, local sorting of waste, fees for disposal of waste; to the geographic area or specific entity that has been identified as the source(s) of the waste comprising said specific chemical compound(s).
64. Use of a method according to any of claims 1 to 61 for decreasing the abundance of or eliminating the presence of a chemical compound measured in step c) in waste before, during or after fermentation of the waste by adjusting one or more of the processing parameters in step a) and/or one or more of the parameters in step b).
65. use of the method according to any of claims 1 to 61 for adjusting the processing parameters in step a) and/or step b) or for adjusting the processing parameters in the subsequent anaerobic digestion of the waste; based on the presence of one or more chemical compounds measured in step c).
66. Use according to claim 64 or 65 wherein said processing parameters to be adjusted are one or more of the parameters in step a) in accordance with claims 6, 7, 9, 10 to 20 and/or one or more of the parameters in step b) in accordance with claims 21 and 22.
67. Use according to claim 64 or 65 wherein said processing parameter to be adjusted is the retention time of the waste in the bio-reactor and/or the retention time of the bioliquid in the anaerobic digestion process.
68. Use of a method according to any of claims 1 to 61 for degrading one or more chemical composition such as benzenepropanoic acid, benzyl benzoate, caffeine, ethyl oleate, eugenol, , myristic acid, which are released during step a) by subsequent anaerobic digestion.
69. Use of a method according to any of claims 1 to 61 further comprising an anaerobic digestion step wherein a chemical composition is generated, such as cholestan-3-one. 70. Use of a method according to any of claims 1 to 61 wherein in step b) polar and ionic chemical compounds primarily partition to the bioliquid and hydrophobic chemical compounds primarily partition to the solid fraction Use according to any of claims 62 to 69 wherein said chemical compound is selected from one or more of the following groups of compounds: Linear alkylbenzensulfonates; Polycyclic aromatic hydrocarbons such as å Acenaphthene, Phenathrene, Fluoren, Fluoranthene, Pyren, Benzfluoranthens, Benz(a)pyren, Benz(ghi)perylen, lndeno(1 ,2,3-cd)pyren; Nonylphenol and ethoxylat, nonylphenol and nonylphenolethoxylates with 1 -2 ethoxy groups; di(2- ethylhexyl)phthalate; hormone-disturbing compounds such as bisphenol-A; drugs such as amitriptyline, dronabinol, salicylic acid, carbamazepine, ibuprofene, carbidopa; pesticides; ingredients from personal care products such as limonene and parabens; plasticisers other than DEHP; biocides such as triclocarban, 2-phenylphenol, DEET; problematic compounds from paper and packaging industry such as PFOS and PFOA.
Use according to any of claims 62 to 71 , wherein said chemical compound is selected from one or more of the following chemical compounds: butylbenzene, catechol, propyl stearate, methyl palmitate, methyl lineoleate, cholestan-3-one Para-cresol, Skatol, N,N-dimethyl-1 -dodecanamine, Benzophenon, Oleic acid, 4-hydroxy-3,5-ditert-butylbenzaldehyde, Phthalic acid, Pentadecanoic acid, Squalen, Methyl oleate, 13-Docosenamide, Linolenic acid, Glycerol tricaprylate, 13-docosenamide, 3-methylindole, 2-methylindole, 4-hydroxy-3,5-ditert- butylbenzaldehyde, Benzeneacetic acid, Benzenepropanoic acid, Benzophenone, Benzyl benzoate, Caffeine, Cholestan-3-one, Cholesterol, Cotinine, d-limonene, Dodecanoic acid propyl ester, Ethyl oleate, Eugenol, Ibuprofen, Isopropyl palmitate, Methenamine, Methyl oleate, Myristic acid, N,N- dimethyl-1 -dodecanamine, Nicotine, Palmitic acid, Para-cresol, Pentadecanoic acid, Pentadecanoic acid methyl ester, Sorbic Acid, Squalene, Triethyl citrate, L- Nicotine, (-)-Nicotine,
Use according to any one of claims 62 to 72, wherein said waste comprises both biodegradable and non-biodegradable material.
Use according to any one of claims 62 to 73, wherein said waste comprises 25- 60% biodegradable material on a wet basis.
Use according to any one of claims 62 to 74, wherein said waste is municipal solid waste.
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