CN116457115A

CN116457115A - Method for sterilizing waste

Info

Publication number: CN116457115A
Application number: CN202180074562.9A
Authority: CN
Inventors: S·G·施塔尔胡特; H·R·瑟恩森
Original assignee: Renalson Co ltd
Current assignee: Renalson Co ltd
Priority date: 2020-11-04
Filing date: 2021-11-03
Publication date: 2023-07-18

Abstract

The invention relates to a method for disinfecting waste, whereby waste with a specific bacterial count is subjected to an enzyme composition at a pH value between 30 and 6.0 and a temperature between 40 and 60 ℃, the liquid is separated and the waste is subjected to the enzyme composition for 10 to 30 hours, in order to obtain at least a partial reduction of the bacterial count.

Description

Method for sterilizing waste

Technical Field

The invention relates to a method for disinfecting waste, whereby disinfected waste and biological fluid are produced and biogas is produced from the biological fluid.

Background

Methods have received great attention to the use of the energy stored in waste products comprising organic materials to the greatest extent. Agricultural waste, household waste and municipal waste are examples of waste that contain substantial amounts of dry matter and a certain content of organic matter, which are biodegradable. There has been a great interest in developing efficient and environmentally friendly methods of treating such waste to maximize the recovery of its inherent energy potential (biodegradable materials) and recovery of recyclable materials. One significant challenge of the "waste-to-energy" process is the heterogeneity of waste, such as Municipal Solid Waste (MSW).

Common methods of processing and subsequent disposal of waste (such as household, agricultural or municipal waste) include incineration, landfill, composting, and the like, where the method of choice often depends on, for example, the content of organic material as compared to the content of non-organic material. However, these methods do not directly provide optimal utilization of the energy stored in the organic material.

Pre-sorting of household waste may sometimes be provided by consumers or waste stations, which reduces pollution released by e.g. incineration and simplifies the process of degrading organic waste into valuable end products. However, the pre-sorting may not be effective in separating all non-biodegradable materials, such as metals and glass, from the organic waste.

In a process where the organic content of the waste is liquefied and/or saccharified while the non-organic content is maintained in its solid phase, then the solid and liquid phases are separated, pre-sorting may simplify the process, but is not required.

An example of an environmentally friendly waste treatment process is a bio-based process applied by renesecience, wherein waste comprising organic matter, such as common unclassified and/or classified/partially classified household waste, is mixed with water, enzymes and/or microorganisms to liquefy and/or saccharify organic waste, such as food waste, cardboard, paper, labels and the like. Such a process is described in international patent application WO 2013/185778, which describes a method and composition for the production of biomethane from MSW. The MSW, which may be unclassified, is processed simultaneously with enzymes and bacterial cultures to release the energy stored in the biodegradable material in the MSW and to change it into a biological fluid, which can be used to produce biogas by anaerobic digestion treatment.

Anaerobic Digestion (AD) can inactivate viable pathogens, including parasites, viruses, and pathogens that carry antibiotic resistance genes. The review article "Is anaerobic digestion a reliable barrier for deactivation of pathogens in bio-trudgeelsevier, vol.668, pages 893-902, june 10,2019" is intended to provide an important overview of AD's inactivation of sludge-related pathogens, which poses serious concerns about the effectiveness and rationality of AD's control of sludge pathogens. At the same time, potential inactivation mechanisms and influencing factors are discussed, with emphasis on pathogen-related modeling, engineering, and technical aspects of AD.

It was previously thought that the waste fraction should be hygienically treated by pre-processing at a temperature of 90-95 ℃ before being used for the production of biological fluids. The effect of the pre-processing is to sterilize/sanitize the waste portion, thereby killing undesirable microorganisms, such as pathogenic bacteria.

WO 2013/185778 teaches that preheating of the waste is not always necessary. This application shows that safe fermentation can be achieved for at least some pathogenic bacteria by adding microorganisms (inoculation of EC 12B) and enzymes to the waste and allowing simultaneous enzymatic processing and microbial fermentation at a temperature of 45-75 ℃ for 212 hours or more.

However, it is beneficial to perform enzymatic and/or microbiological processing of MSW in a safe, environmentally friendly and economical manner, without prior preheating, or at least by a method that requires less energy input, e.g. to raise the temperature.

By means of the present invention it has surprisingly been found that the total amount of most of the intestinal bacterial species found in MSW can be significantly reduced by a method of enzymatic and/or microbiological processing in a bioreactor at a pH value between 3.0 and 6.0, at a temperature between 40 ℃ and 60 ℃ for a period of only 10 to 30 hours, in particular e. The invention is therefore particularly advantageous in the application of lower temperatures and shorter enzymatic and/or microbiological processing durations than previously thought necessary.

Disclosure of Invention

The present invention relates to a method of disinfecting waste, the method comprising:

a) Subjecting waste comprising biodegradable and non-biodegradable materials and having a bacterial count of at least 2.5 x 10 to enzymatic and/or microbiological processing in a bioreactor at a pH between 3.0 and 6.0, a temperature between 40 ℃ and 60 ℃ and a period of 10 to 30 hours to obtain at least a partial reduction ⁸ Total bacterial count of CFU/gram waste of at least 1.5 x 10 ⁶ Bacterial count of E.coli of CFU/g waste, or at least 1.5X10 ⁸ Bacterial count of enterobacteriaceae of CFU/gram waste.

The method may further comprise:

b) Subjecting the processed waste from step a) to one or more separation steps to provide biological liquid and solid fractions (fractions);

c) Subjecting the biological liquid and/or solid fraction to downstream treatment.

The invention also relates to biological fluids and non-biodegradable materials obtainable by the treatment process of the invention.

The method of the invention is advantageous because it disinfects waste at low temperatures in a safe and economical manner.

Definition of the definition

"biodegradable material" refers to an organic substance that is capable of being partially or completely degraded by microorganisms and/or enzymes into simple compounds such as monosaccharides, disaccharides and/or oligosaccharides, amino acids and/or fatty acids. Biodegradable materials are typically organic materials that provide nutrients to microorganisms, such as monosaccharides, polysaccharides or oligosaccharides, fats and/or proteins. These numbers and types are so large that there are a large number of compounds that can be biodegraded, including hydrocarbons (oils), polycyclic Aromatic Hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and pharmaceutical substances. Microorganisms secrete biosurfactants, a cell surface active agent, to enhance this process.

"cellulose" is a homopolysaccharide consisting entirely of D-glucose linked together by [ beta ] -1, 4-glucosidic linkages and having a degree of polymerization of up to 10,000. The linear structure of cellulose is capable of forming intramolecular and intermolecular hydrogen bonds, which result in the aggregation of cellulose chains into microfibrils. Regions of high order within the microfibril filament are referred to as crystals, while regions of poor order are referred to as amorphous. The microfibrils are gathered into filaments, which form cellulose fibres.

"cellulosic material" refers to any material that contains cellulose. Cellulosic materials include agricultural residues, herbaceous materials (including energy crops), municipal solid waste, pulp and paper mill residues, waste paper, textiles including cotton materials, and wood (such as forestry residues).

"hemicellulose" is a complex heterogeneous polysaccharide consisting of several monomer residues: d-glucose, D-galactose, D-mannose, D-xylose, L-arabinose, D-glucuronic acid and 4-O-methyl-D-glucuronic acid. Hemicellulose has a degree of polymerization below 200, has side chains and may be acetylated. In cork like fir, pine and spruce, galactomannans and arabino-4-O-methyl-glucuronic acid are the main hemicellulose fractions. In hardwoods like birch, aspen (popar), aspen (aspen) or oak, 4-O-acetyl-4-methyl-glucuronic acid and glucomannan are the main components of hemicellulose.

"municipal solid waste" (MSW) refers to the portion of waste that is normally present in a city, but is not necessarily from any municipality in itself, i.e., MSW refers to every type of solid waste from any municipality, but is not necessarily typical household waste, and may be solid waste from airports, universities, campuses, canteens, general food waste, and the like. The MSW may be a combination of one or more of cellulose, plant, animal, plastic, metal, or glass waste, including, but not limited to, any one or more of the following: waste collected in a normal urban collection system, optionallyAlternatively, such as, for exampleOr (b)Is processed in a centralized classification, crushing or beating device; solid waste sorted from the home, including an organic fraction and a paper-rich fraction; generally, municipal solid waste in the western region of the world typically comprises one or more of the following: animal food waste, vegetable food waste, newspapers, magazines, advertisements, books and phone books, office papers, other clean papers, paper and carton containers, other cardboard, milk boxes and the like, fruit juice boxes and other aluminum foil-bearing cartons, kitchen towels, other dirty papers, other dirty cardboard, soft plastic, plastic bottles, other hard plastic, non-recyclable plastic, yard waste, flowers and the like, animals and excrement, diapers and tampons, cotton and the like, other cotton and the like, wood, textiles, shoes, leather, rubber and the like, office supplies, chemical empty bottles, plastic products, butts, other combustibles, vacuum cleaner bags, clear glass, green glass, brown glass, other glass, aluminum containers, aluminum trays, aluminum foils (including tea lamp candle foils), metal containers (-Al), metal foils (-Al), other various metals, soil, rock, and gravel, ceramics, cat litter, batteries (cotton cells, alkali, thermometers and the like), other non-combustibles and fines.

Oligosaccharides are carbohydrate polymers containing small amounts (typically three to ten) of monosaccharides. They are generally present in the form of glycans: oligosaccharide chains are linked to compatible amino acid side chains in lipids or proteins via N-or O-glycosidic linkages. The N-linked oligosaccharides are always pentasaccharides linked to asparagine by beta linkage to the amine nitrogen of the side chain. Alternatively, the O-linked oligosaccharides are typically attached to threonine or serine on the alcohol group of the side chain. Not all natural oligosaccharides are components of glycoproteins or glycolipids. Some oligosaccharides, such as the raffinose family, are used in plants for storage or transport of carbohydrates. Other oligosaccharides, such as maltodextrin or cellodextrin, are produced by microbial breakdown of larger polysaccharides, such as starch or cellulose.

"organic" refers to materials that include carbon and are biodegradable, and include materials from living organisms. The organic material may degrade in the presence of oxygen (oxygen containing) or anaerobically (without oxygen). The decomposition of the biodegradable material may include biological and non-biological steps.

Polysaccharides are polymeric carbohydrate molecules consisting of long chains of monosaccharide units joined together by glycosidic linkages, and under enzymatic processing, polysaccharides produce components of mono-or oligosaccharides. Their structures range from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as cellulose and chitin. The polysaccharide has a general formula of C _x (H2O) _y Where x is typically a large number between 200 and 2500. When the repeating units in the polymer backbone are six-carbon monosaccharides, the general formula is typically reduced to (C ₆ H ₁₀ O ₅ ) _n Wherein n is generally 40.ltoreq.n.ltoreq.3000. Polysaccharides contain more than ten monosaccharide units, but the precise demarcation varies by convention. The polysaccharide also includes calluses or laminarin, cordycepin, xylan, arabinoxylan, mannan, fucoidan, and galactomannans.

Starch is a polymeric carbohydrate, formed from a large number of glucose units linked by glycosidic linkages. Starch is the most common carbohydrate in the human diet and contains a large amount of carbohydrate in staple foods like potato, wheat, corn, rice and tapioca. Pure starch is a white, odorless and odorless powder that is insoluble in cold water or alcohol. It consists of two types of molecules: linear and helical amylose and branched amylopectin. Starch generally contains 20 to 25% by weight of amylose and 75 to 80% by weight of amylopectin, depending on the plant.

In industry, starch is converted to sugar, for example by making wheat, and fermented to produce ethanol when beer, whiskey and biofuels are made. Starch is treated to produce a number of sugars for use in processing food products. Mixing most starch in warm water produces a paste, such as wheat paste, which can be used as a thickener, hardener or adhesive. The largest industrial non-food use of starch is as a binder in papermaking processes. Starch may be applied prior to ironing portions of some garments to stiffen the portions of some garments.

Starch (a polymer of glucose) is used in plants to store polysaccharides, which exist in the form of amylose and branched amylopectin. In animals, a structurally similar glucose polymer is a more densely branched glycogen, sometimes referred to as "animal starch". The nature of glycogen allows it to be metabolized away more quickly, which is appropriate for the active life of the sports animal.

"sorting" refers to the process of substantially separating waste, such as MSW, into individual portions such that the organic material is substantially separated from the plastic and/or other non-biodegradable materials.

As used herein, "sorted waste" (or "sorted MSW") means that less than about 30%, preferably less than 20% and most preferably less than 15% by weight of dry weight of the waste is not biodegradable material.

By "unclassified" is meant that the waste or MSW is not substantially separated into individual parts, such that, for example, the organic material is not substantially separated from the plastic and/or other inorganic material, although some large or metallic objects are removed, and although some separation of the plastic and/or other inorganic material may have occurred, for example, before the bioreactor. The term "unclassified waste" (or "unclassified MSW") as used herein refers to waste comprising a mixture of biodegradable and non-biodegradable materials, wherein the non-biodegradable materials are 15% or more by weight of dry weight. Waste that has been simply sorted still produces an unclassified waste (or MSW) fraction. Typically, unclassified MSW may include organic waste, including one or more of food and kitchen waste; a paper and/or paperboard containing material; recyclable materials, including glass, bottles, jars, metals, and certain plastics; a combustible material; and inert materials including ceramics, rocks, and debris. The recyclable material may be before or after source sorting.

"waste" includes: classified and unclassified Municipal Solid Waste (MSW), agricultural waste, hospital waste, industrial waste, e.g., waste fractions from industries such as the catering industry, food processing industry, general industry, etc.; a waste portion of the paper industry; recovering a waste portion of the facility; waste parts of the food or feed industry; waste material of the pharmaceutical or pharmaceutical industry; waste parts of hospitals and clinics; a waste fraction of agriculture or agriculture-related departments; treating the waste portion of the sugar or starch-rich product; contaminated or otherwise spoiled agricultural products such as grains, potatoes and beets that cannot be used for food or feed purposes; or garden waste.

"waste fraction from home" includes unclassified Municipal Solid Waste (MSW); such as, for exampleOr->Is a part of the MSW processed in the centralized classification, crushing or pulping equipment; solid waste classified from the home, including an organic portion and a paper-rich portion; RDF (waste derived fuel); the parts obtained by post-processing, such as inert substances, organic parts, metal, glass and plastic parts. In a preferred embodiment, 2D and 3D portions are formulated. The 2D fraction may be further separated into recyclables and/or residues, such as SRF (solid recovery fuel), RDF (waste derived fuel) and/or inerts. The 3D fraction may also be further separated into recyclables and/or residues, such as metal, 3D plastic and/or RDF.

"waste fraction from industry" includes the general industrial waste fraction containing paper or other organic fraction, now considered household waste; waste fractions from the paper industry, for example from recovery facilities; waste fractions from the food and feed industry; waste fractions from the medical industry, hospital and clinic waste, airport waste, other public and private service sector waste.

"waste fraction from agriculture or farming related departments" includes waste fractions from the following processes, including: sugar or starch-rich products such as potatoes and sugar beets; contaminated or otherwise spoiled agricultural products such as grains, potatoes and beets that cannot be used for food or feed purposes; garden waste; faeces, or faeces derived products.

"waste fractions from municipal, county, or state related or regulatory activities" include sludge from wastewater processing plants; fiber or sludge fractions from biogas treatment; a general waste fraction from the public sector containing paper or other organic fractions.

Enzymes

An "enzyme" is a protein with catalytic function, meaning that it can increase the rate of a chemical reaction without itself undergoing any overall chemical change during this treatment. There are six major classes of enzymes that can catalyze different types of reactions, namely oxidoreductases (EC 1. X.x.x), transferases (EC 2. X.x.x), hydrolases (EC 3. X.x.x), lyases (EC 4. X.x.x), isomerases (EC 5. X.x.x) and ligases (EC 6. X.x.x), according to the Enzyme Committee (EC) classification. Enzymes involved in liquefaction and/or saccharification of organic materials mostly belong to the third class (EC 3. X.). These enzymes promote the processing reaction, i.e., cleavage of chemical bonds with the participation of water as a co-substrate. Enzymes of this class are generally named according to the substrate they hydrolyze: one or more amylase-hydrolyzed starches (amylose and amylopectin), one or more cellulases-hydrolyzed cellulose, one or more hemicellulases-hydrolyzed hemicellulose, one or more pectinases-hydrolyzed pectin, one or more lipases-hydrolyzed lipids, and one or more proteases-hydrolyzed proteins. Some of the one or more hemicellulases are one or more esterases that catalyze the ester linkage similarly to the case of one or more lipases. Some of the one or more pectinases are lyases, which use a non-hydrolytic reaction to remove chemical groups. Recently, a new class of enzymes has been discovered, known as the cleaving polysaccharide monooxygenases (LPMO), which have catalytic activity on cellulose (Quinlan et al, 2011,Proc.Natl.Acad.Sci.USA 208:15079-15084;Phillips et al, 2011,ACS Chem.6:1399-1406, lin et al, 2012,Structure 20:1051-1061). LPMO catalyzes the oxidative cleavage of cellulose with oxygen or hydrogen peroxide as co-substrates and is classified as a co-active 9 polypeptide. Another class of oxidases, such as catalase (EC 1.11.1.6), catalyzes the conversion of hydrogen peroxide to water and oxygen.

Starch degrading enzyme

An "amylase" is an enzyme that catalyzes the hydrolysis of starch to sugars. Important enzymes for hydrolyzing starch are alpha-amylase (1, 4- [ alpha ] -D-glucan glucohydrolase, (EC 3.2.1.1) these are endo-hydrolases that cleave 1,4- [ alpha ] -p-glucosidic bonds and can bypass but cannot hydrolyze the fulcrums of 1, 6-alpha-D-glucosidic, however, exo-acting saccharifying enzymes such as beta-amylase (EC 3.2.1.2) and Pullulan enzyme (EC 3.2.1.41) can also be used for starch hydrolysis, the result of starch hydrolysis is mainly glucose, maltose, maltotriose, q-dextrins and various amounts of oligosaccharides, amylases include but are not limited to alpha-amylases derived from the genus Rhizomucor such as for example Rhizomucor pusillus such as for example encoded by SEQ ID NO:5 as disclosed in WO 17076421 or homologues thereof.

Cellulose degrading enzyme

One or more "cellulases" are meant to include one or more enzymes that are capable of degrading cellulose and/or related compounds. Cellulases can also be used in any mixture or complex of various such enzymes that act continuously or synergistically to break down cellulosic material. Cellulases break down cellulose molecules into monosaccharides ("simple sugars"), such as glucose and/or shorter polysaccharides and oligosaccharides. Specific reactions may include hydrolysis of 1, 4-beta-D-glycosidic linkages in cellulose, hemicellulose, lichenin and cereal beta-D-glucose. Several different cellulases are known, which differ in structure and mechanism. Synonyms, derivatives and/or specific enzymes related to the "cellulase" name include endo-1, 4-beta-D-glucanases (beta-1, 4-glucanases, beta-1, 4-endoglucanases, endoglucanases D, 1,4- (1,3,1,4) -beta-D-glucans 4-glucohydrolases), carboxymethyl cellulases (CMCase), microcrystalline cellulases, cell dextrinases, cellulases A, cellulose APs, alkaline cellulases, cellulases A3, 9.5 cellulases and trypsin SS.

Cellulases can also be categorized based on the type of reaction catalyzed, with endocellulases (EC 3.2.1.4) cleaving internal bonds randomly at amorphous sites, which create new chain ends, and exocellulases or cellobiohydrolases (EC 3.2.1.91) cleaving two to four units from the end of the exposed chain produced by the endocellulases, resulting in tetraose, trisaccharide or disaccharides, such as cellobiase. The exocellulases are further classified as type I-acting continuously from the reducing end of the cellulose chain, and type II-acting continuously from the non-reducing end. Cellobiase (EC 3.2.1.21) or beta-glucosidase hydrolyzes the exocellulase products to individual monosaccharides. Oxidized cellulases depolymerize cellulose by free radical reactions, such as cellobiose dehydrogenase (acceptor). Cellulose phosphorylase uses phosphates rather than hydrolysis to depolymerize cellulose. The general understanding of cellulolytic systems divides cellulases into three classes: endo-1, 4- [ beta ] -D-glucanase (EG) (EC 3.2.1.4), which randomly hydrolyzes internal p-1, 4-glucoside linkages in the cellulose chain; exo-1, 4- [ beta ] -D-glucanase or Cellobiohydrolase (CBH) (EC 3.2.1.91), which cleaves cellobiose units from the ends of cellulose chains; 1,4- [ beta ] -D-glucosidase (EC 3.2.1.21), which hydrolyzes cellobiose to glucose, also cleaving glucose units from cellooligosaccharides.

"endoglucanase" refers to 4- (1, 3;1, 4) -beta-D-glucan 4-glucohydrolase (EC 3.2.1.4) which catalyzes the hydrolysis of cellulose, cellulose derivatives such as carboxymethyl cellulose and hydroxyethyl cellulose, lichenin, beta-1, 4-glycosidic linkages in mixed beta-1,3-1,4 glucans such as cereal beta-D-glucan or xyloglucan, and 1, 4-beta-D-glycosidic linkages in other plant materials containing cellulosic components. Endoglucanase activity may be determined by measuring a decrease in substrate viscosity or an increase in the reducing end as determined by a reducing sugar assay (Zhang et al 2006,Biotechnology Advances 24:452-481). Endoglucanase activity can also be determined according to Ghose,1987,Pure and Appl.59:257-268 using carboxymethyl cellulose (CMC) as substrate at pH 5, 40 ℃. Endoglucanases include, but are not limited to, one or more of the following: acidothermus cellulolyticus endoglucanases (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No.5,536,655; WO 00/70031; WO 05/093050), erwinia carotovara endoglucanases (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 include, but are not limited to, one or more of the following: 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 63:11-22), trichoderma reesei Cel A 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 (117 Bank: L29381), humicola grisea endoglucanase (Genk: AB 003107), melanocarpus albomyces endoglucanase (GenBank: MAL 515703), genBank: neurospora crassa endoglucanase (GenBank: XM 37), thermoascus aurantiacus-Thermoascus aurantiacus endoglucanase (Thermoascus aurantiacus), and (GenBank: XM: thermoascus aurantiacus).

"cellobiohydrolase" refers to 1, 4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176) which catalyzes the hydrolysis of 1, 4-beta-D-glucoside linkages in cellulose, cellooligosaccharides or any polymer containing beta-1, 4-linked glucose, releasing cellobiose from either the reducing (cellobiohydrolase I) or non-reducing (cellobiohydrolase II) ends of the chain (Teeri, 1997,Trends in Biotechnology 15:160-167; teeri et al 1998, biochem. Soc. Trans. 26:173-178). The cellobiohydrolase activity can be determined according to the procedure described below: lever et al, 1972, anal biochem.47:273-279; van tilbough et al 1982,FEBS Letters 149:152-156; van Tilbeurgh and Claeyssens,1985,FEBS Letters 187:283-288; and Tomme et al, 1988, eur.J.biochem.170:575-581. Cellobiohydrolases include, but are not limited to, one or more of the following: aspergillus aculeatus cellobiohydrolase II (WO 2011/059740), 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 occitanis cellobiohydrolase I (GenBank: AY 690482), talaromyces emersonii cellobiohydrolase I (GenBank: AF 439936), thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), thielavia terrestris cellobiohydrolase II (CEL 6A, WO 2006/074435), trichoderma reesei cellobiohydrolase I, trichoderma reesei cellobiohydrolase II and Trichophaea saccata cellobiohydrolase II (WO 2010/057086).

"Beta-glucosidase" refers to Beta-D-glucosidase hydrolase (EC 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing Beta-D-glucose residues and liberates Beta-D-glucose. Beta-glucosidase activity can be determined according to the procedure of Venturi et al 2002,J.Basic Microbiol.42:55-66 using p-nitrophenyl-Beta-D-glucopyranoside as substrate. One unit of beta-glucosidase is defined as containing 0.01% at 25℃and pH 4.8 with 1mM p-nitrophenyl-beta-D-glucopyranoside as substrate20 in 50mM sodium citrate, 1.0. Mu. Mole of p-nitrophenol anion was produced per minute. Beta-Glucosidase includes, but is not limited to, one or more of the beta-glucosidase 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 2011/035029) and Trichophaea saccata (WO 2007/019442).

Hemicellulose degrading enzyme

One or more "hemicellulases" are meant to include one or more enzymes capable of and/or contributing to the breakdown of hemicellulose, which is one of the major components of plant cell walls. Hemicellulose is a heterogeneous group of branched and linear polysaccharides that bind to cellulose microfibrils in the plant cell wall through hydrogen bonds, cross-linking the cellulose microfibrils into a strong network. Hemicellulose is also covalently linked to lignin, forming a highly complex structure with cellulose. Hemicellulose can be classified based on the carbohydrate monomers that build the backbone chain, i.e., dextran (polymer of glucose), glucomannan (polymer of glucose and mannose), mannan (polymer of mannose), and xylan (polymer of xylose). These backbone chains may have side chains of other carbohydrate monomers, acetyl groups and/or glucuronic acid. The glucan backbone without side chains and beta-1,3-1,4 linkages is called mixed-linked beta-glucan, as found in grasses. The glucan backbone with xylose side chains is called xylan, which stands out in hardwoods. Glucomannan backbones with galactose substitutes as found in cork are known as galactoglucomannans. The mannan backbone may be replaced with galactose and is therefore referred to as galactomannan. The xylan backbone, as found in hardwoods, which is predominantly substituted with glucuronic acid, is called glucuronic acid. Xylan backbones substituted with glucuronic acid, acetyl and arabinosyl groups which can be feruloylated are known as glucuronic acid arabinoxylans and are prominent in grasses.

Hemicellulose is structurally and organized diverse, and requires the synergistic action of many enzymes to degrade it completely. The catalytic module of hemicellulases is a Glycoside Hydrolase (GH) that hydrolyzes glycosidic linkages (EC 3.2. X), or an ester-linked Carbohydrate Esterase (CE) that hydrolyzes acetyl or ferulic acid side groups (EC 3.1. X). Hemicellulases are collectively referred to as their hydrolyzed backbone chains, and are specifically named according to their cleaved or removed bonds and side chains, respectively. One or more Beta-glucanases hydrolyze mixed linked (Beta-1, 3-1, 4) Beta-glucans, and one or more xyloglucanases hydrolyze xyloglucans. One or more glucomannanases and one or more mannanases hydrolyze (galactose) glucomannan and (galactose) mannan, respectively. In a similar manner, one or more glucuronidases and one or more xylanases are a generic term for enzymes that hydrolyze glucuronic acid and xylan, respectively. The enzyme that hydrolyses glucuronic acid may be referred to as arabinoxylase like the one or more glucuronidases, although it consists of one or more xylanases and other enzymes that remove the side chain groups. Arabinoxylases include alpha-arabinofuranosidases with the arabinose side chains removed, alpha-glucuronidases with the glucuronic acid side chains removed, and one or more esterases such as acetyl-and feruloyl-removing acetylxylan esterases and feruloyl esterases, respectively.

One or more "Beta-glucanases" refers to any type of endo-Beta-glucanase that hydrolyzes (1, 3) -or (1, 4) -linkages in Beta-D-glucan (EC 3.2.1.73) (EC 3.2.1.6). Beta-glucanases include, but are not limited to, members derived from Aspergillus such as, for example Aspergillus aculeatus, beta-glucanases encoded by the sequence encoded by SEQ ID NO. 4, as disclosed in, for example, WO 17076421, or homologues thereof.

One or more "xylanases" are meant to include one or more enzymes capable of degrading xylan and/or related compounds, including, for example, xylan-specific endo-beta-1, 4-glucanase (EC 3.2.1.151). Such enzymes belong to the family of hydrolases, in particular those glycosidases which hydrolyze O-and S-glycosyl compounds. Other commonly used names may include XEG, endo-xylanase-beta-1, 4-glucanase, xylanase, endo-xylanase, XH and 1, 4-beta-D-glucanohydrolase.

One or more "mannanases" refers to beta-mannanases and are defined as enzymes belonging to EC 3.2.1.78 or EC 3.2.1.25. Mannanases also include endo-mannanases and/or 1, 4-beta-mannanases. Mannanases have been identified in several Bacillus organisms. For example, talbot et al, appl.environ.Microbiol., vol.56, no.11, pp.3505-3510 (1990) describe a beta-mannanase derived from Bacillus stearothermophilus having an optimal pH of 5.5-7.5. Mendoza et al, world J.Microbiol. Biotech., vol.10, no.5, pp.551-555 (1994) describe a beta-mannanase derived from Bacillus subtilis having an optimal activity at pH 5.0 and 55 ℃. JP-03047076 discloses a beta-mannanase from Bacillus species having an optimum pH of 8-10.JP-63056289 describes the production of alkaline, thermostable beta-mannanases. JP-08051975 discloses alkaline beta-mannanases from the species Bacillus alcalophilus AM-001. Purified mannanases from Bacillus amyloliquefaciens are disclosed in WO 97/1 1164. WO 94/25576 discloses an enzyme from Aspergillus aculeatus, CBS 101.43 exhibiting mannanase activity and WO 93/24622 discloses a mannanase isolated from Trichoderma reesei.

One or more "glucomannanases" are meant to include one or more enzymes capable of degrading glucomannans and/or related compounds. This includes endo-1, 4- [ beta ] -D-mannanase (EC 3.2.1.78) which cleaves the bond between the mannose group in the backbone, beta-glucosidase (EC 3.2.1.21) which cleaves the bond between the glucosyl group and the mannose group in the backbone, alpha-D-galactosidase (EC 3.2.1.22) which removes the galactose side chains from the backbone.

One or more "mannosidases" refers to 1,4- [ beta ] -D-mannosidase (EC 3.2.1.25) that cleave mannooligosaccharides into mannose. The enzyme may be derived from the genus Bacillus, such as for example Bacillus bogoriensis, such as for example an endo-mannosidase encoded by SEQ ID NO:6 as disclosed in WO 17076421 or a homologue thereof.

One or more "xylanases" refers to a enzyme that catalyzes the internal hydrolysis of 1, 4-beta-D-xylan-hydroxylase (EC 3.2.1.8) in xylan. One unit of xylanase activity was defined as the production of 1.0. Mu. Mole of azurin per minute in 200mM sodium phosphate pH 6 at 37℃with 0.2% AZCL-arabinoxylan as substrate. Xylanases include one or more enzymes capable of degrading xylan and/or related compounds. Xylanase is any one of several enzymes, e.g., produced by a microorganism (such as yeast) that catalyzes the breakdown of xylan and/or related polysaccharides. Xylanases can also be used in any mixture or complex of various such enzymes, which act continuously or synergistically to break down xylan material. Synonyms, derivatives and specific enzymes related to the "xylanase" name may include EC 3.2.1.8, endo- (1- > 4) -beta-lignin 4-lignin hydrolase, endo-1, 4-lignin enzyme, endo-1, 4-beta-lignin enzyme, beta-1, 4-lignin enzyme, endo-1, 4-beta-D-lignin enzyme, 1, 4-beta-lignin hydrolase, beta-lignin enzyme, beta-1, 4-lignin hydrolase, beta-D-xylanase and/or xylan capable of degrading xylan, such as beta-1, 4-xylan, to xylose, thereby helping to break down hemicellulose, which is one of the major components of plant cell walls.

One or more "glucuronidases" are meant to include one or more enzymes capable of degrading glucuronic acid and/or related compounds.

"xylanase" refers to xylan 1, 4-beta-xylose (EC 3.2.1.37), also designated as xylanase, beta-xylanase, exo-1, 4-beta-D-xylanase or 4-beta-D-xylan hydrolase. This enzyme catalyzes the hydrolysis of (1, 4) -beta-D-xylan, removing consecutive D-xylose residues from the non-reducing end of the substrate (e.g., hemicellulose and disaccharide xylobiose). A unit of beta-xylosidase is a beta-xylosidase which is prepared at 40℃and pH 5 and contains 0.01%20.0. Mu. Mole of p-nitrophenol anion was produced per minute in 100mM sodium citrate.

"alpha-L-arabinofuranosidase" refers to an alpha-L-arabinofuranosidase (EC 3.2.1.55) which catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranosidic residues in alpha-L-arabinofuranosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinosides containing (1, 3) -and/or (1, 5) -linkages, arabinuronic acid and arabinuronic acid. The alpha-L-arabinofuranosidase is also known as an arabinosidase, an alpha-L-arabinosidase, an alpha-arabinofuranosidase, a polysaccharide alpha-L-arabinofuranosidase, an L-arabinosidase or an alpha-L-arabinoxylanase.

"alpha-glucuronidase" refers to alpha-D-glucuronidase (EC 3.2.1.139) which catalyzes the hydrolysis of alpha-D-glucuronate to D-glucuronate and alcohol. The activity of Alpha-glucuronidase can be determined according to de Vries,1998, J.Bacteriol. 180:243-249. One unit of alpha-glucuronidase is equivalent to an enzyme amount capable of releasing 1. Mu. Mole of glucuronic acid or 4-O-methylglucuronic acid per minute at pH 5, 40 ℃.

One or more "esterases" are meant to include one or more enzymes that catalyze the hydrolysis of organic esters to liberate alcohols or thiols and acids. The term is applicable to enzymes that hydrolyze carboxylic, phosphate and sulfate esters, but is more limited to the first category. Examples of esterases include acetyl esterases and feruloyl esterases, e.g., EC 3.1.X.x.

"Acetoxyesterase" refers to carboxylesterase (EC 3.1.1.72) which catalyzes the hydrolytic polymerization of acetyl groups in xylan, acetylated xylose, acetylated glucose, alpha-naphthalene acetate and p-nitrophenylacetate. One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1. Mu. Mole of p-nitrophenol anion per minute at pH 5, 25 ℃.

One or more "feruloyl esterases" refers to 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) which catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (ferulic acid) groups derived from esterified sugars, typically arabinose in natural biomass substrates, to produce ferulic acid (4-hydroxy-3-methoxycinnamic acid). Feruloyl esterase (FAE) is also known as feruloyl esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnamoyl esterase (cinneae), FAE-I or FAE-II. One unit of feruloyl esterase is equal to the amount of enzyme capable of releasing 1. Mu. Mole of p-nitrophenol anion per minute at pH 5, 25 ℃.

Pectin degrading enzyme

One or more "pectinases" refer to any enzyme that catalyzes the degradation of polysaccharides, i.e., pectin, in the plant cell wall, including 1) pectin lyase, otherwise known as pectin countereliminator; an endoprotease; a polymethylgalactose aldehyde reverse eliminating enzyme; pectate methyl counter elimination enzyme; pectolytic enzymes; PL; PNL; PMGL (EC 4.2.2.10) which performs an elimination cleavage of methyl (1- > 4) -alpha-D-galacturonate to give an oligosaccharide with a 4-deoxy-6-O-methyl-alpha-D-galacturonate group at the non-reducing end, 2) pectin fruit acyl hydrolase, other name pectin deoxyenzymes; pectate methoxylase; pectin methyl esterase; pectase; pectin methyl esterase; pectin esterases (EC 3.1.1.11) which hydrolyse methyl ester linkages in pectin, and 3) polygalacturonases (EC 3.2.1.15) which hydrolyse alpha-1, 4-glycosidic linkages in polygalacturonic acid chains.

Lipid degrading enzyme

"Lipase" refers to any enzyme that catalyzes the degradation of lipids and/or has hydrolytic activity in the class EC 3.1.1. RTM. As defined by the enzyme nomenclature. Particularly useful are triacylglycerol lipases (EC 3.1.1.3) and phospholipase A1 (EC 3.1.1.32) and phospholipase A2 (EC 3.1.1.4), but also other phospholipases (EC 3.1.1.5), (EC 3.1.4.4), (EC 3.1.4.11), (EC 3.1.4.50) and (EC 3.1.4.54). Lipases include, but are not limited to, lipases derived from the genus thermomyces sp. Such as for example Thermomyces lanuginosus, such as for example the lipase encoded by SEQ ID NO:2 as disclosed in WO17076421 (or homologues thereof), or lipases derived from the genus Humicola sp. Such as for example Humicola insolens (or homologues thereof).

Protein degrading enzyme

"protease" refers to any protease or proteolytic enzyme suitable for use under neutral or acidic conditions. Suitable proteases include those of animal, plant or microbial origin. Including chemical or transgenic mutants. Suitable proteases include endo-metallo-proteases (EC 3.4.24.28) which hydrolyze internal peptide bonds, serine endo-proteases (EC 3.4.23.23) which hydrolyze internal peptide bonds, endo-proteases EC 3.4.21.4 which hydrolyze peptide bonds on the carboxy side of lysine and arginine residues, aminopeptidases (EC 3.4.11.1) and exo-proteases (EC 3.4.11.1) which release amino acids by hydrolysis of N-terminal peptide bonds. The protease may be derived from the genus Bacillus, such as for example Bacillus amyloliquefaciens, such as for example the protease encoded by SEQ ID NO. 1 as disclosed in WO 17076421 or a homologue thereof.

Oxidase enzyme

"helper activity 9 polypeptide" or "AA9 polypeptide" refers to a polypeptide that is classified as a lytic polysaccharide monooxygenase (Quinlan et al, 2011,Proc.Natl.Acad.Sci.USA 208:15079-15084;Phillips et al, 2011,ACS Chem.Biol.6:1399-1406; lin et al, 2012,Structure 20:1051-1061). According to Henrissat,1991, biochem. J.280:309-316, and Henrissat and Bairoch,1996, biochem. J.316:695-696, AA9 polypeptides were previously classified into the glycoside hydrolase family 61 (GH 61). AA9 polypeptides enhance the hydrolysis of cellulosic material by enzymes with cellulolytic activity. An increase in cellulolytic activity may be determined by measuring an increase in reducing sugars or an increase in the total amount of cellulose and glucose after hydrolysis of the cellulosic material by cellulolytic enzymes.

"catalase" refers to hydrogen peroxide: hydrogen peroxide oxidoreductase (EC 1.11.1.6). For the purposes of the present invention, catalase activity was determined according to U.S. Pat. No.5,646,025. One unit of catalase activity was equivalent to the amount of enzyme that catalytically oxidized 1. Mu. Mole hydrogen peroxide under the assay conditions.

Related terms of enzyme

"cellulase activity" refers to the enzymatic hydrolysis of 1,4- [ beta ] -D-glycosidic linkages in cellulose. In isolated cellulase formulations obtained from bacterial, fungal or other sources, cellulase activity typically comprises a mixture of different enzyme activities, including endoglucanases and exoglucanases (also known as cellobiohydrolases) that catalyze the endo-and exo-hydrolysis of 1,4- [ beta ] -D-glycosidic linkages, respectively, and [ beta ] -glucosidase enzymes that hydrolyze exo-glucosidase hydrolyzed oligosaccharide products to monosaccharides. Complete processing of insoluble cellulose generally requires a synergistic effect between the different activities.

"Cellulolytic Background Composition (CBC) or cellulolytic enzyme mixture" refers to an enzyme composition comprising a mixture of two or more cellulolytic enzymes. The CBC may comprise two or more cellulolytic enzymes selected from: i) Aspergillus fumigatus cellobiohydrolase I; (II) Aspergillus fumigatus cellobiohydrolysis II; (iii) Aspergillus fumigatus beta-glucosidase or variant thereof; and (iv) a Penicillium sp.gh61 polypeptide having cellulolytic enhancing activity; or a homologue thereof. The CBC may further comprise one or more enzymes selected from the group consisting of: (a) Aspergillus fumigatus xylanase or a homologue thereof; (b) Aspergillus fumigatus beta-xylanase or a homologue thereof; or (c) a combination of (a) and (b) (as described in further detail in WO 2013/028928). The primary activities of CBC may include: endo-1, 4-beta-glucanase (E.C. 3.2.1.4); endo-1, 4-beta-xylanase (E.C.3.2.1.8); endo-1, 4-beta-mannanase (E.C. 3.2.1.78), beta-mannosidase (E.C 3.2.1.25), but other enzyme activities may also be present in the CBC, such as from the following enzyme activities: glucanase, glucosidase, cellulolytic enzyme I, cellulolytic enzyme II; beta-glucosidase; beta-xylosidase; beta-L-arabinofuranosidase; amyloglucosidase; alpha-amylase; acetylxylose esterase. The CBC may be any CBC described in WO 2013/028928 (the contents of which are incorporated herein by reference). CBC may be from t.reesei. CBC may be from Myceliophtora thermophilae. The CBC may be one obtainable from Novozymes A/S (Bagsvaerd, denmark) CTec3. Cellulolytic enzyme activity may be determined by measuring the increase in sugar production/release during hydrolysis of a cellulosic material by one or more cellulolytic enzymes in the following conditions: 1-50 mg cellulolytic enzyme protein/g cellulose in pre-Processed Corn Stover (PCS) (or other pre-processed cellulosic material) at a suitable temperature and a suitable pH for 3-7 days, suitableTemperatures such as 40 ℃ to 80 ℃, e.g., 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or 80 ℃, and suitable pH values such as 4 to 9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, which is compared to a control process without the addition of cellulolytic enzyme protein.

"commercially available cellulase preparation optimized for biomass conversion" refers to a commercially available enzyme activity mixture sufficient for enzymatic processing of biomass, such as lignocellulosic biomass, which typically includes endocellulases (endoglucanases), exocellulases (exoglucanases), endo-cellulases, acetylxylanesterases, xylosidases and/or beta-glucosidase activities. The term "optimized for biomass conversion" refers to the selection and/or modification of enzyme mixtures during product development for the specific purpose of increasing yield and/or reducing enzyme consumption when processing biomass into fermentable sugars. Commercially available cellulase formulations optimized for bioconversion may be used, such as with the use provided by Genencor (now DuPont), DSM or Novozymes. Typically, these compositions include one or more cellulases and/or one or more hemicellulases, such as one or more of an exoglucanase, an endoglucanase, an endoxylanase, a xylanase, an acetylxylanesterase, and a beta-glucosidase, including any combination thereof. These enzymes can be isolated, for example, from the fermentation of the transgene Trichoderma reesei, such as, for example, ACCELLERASE TRIO in DuPont (and/or Genencor) ^TM A cellulase formulation commercially available under the trademark. Commercially available cellulase preparations useful for biomass conversion are provided by Novozymes and include exoglucanases, endoglucanases, endoxylanases, xylanases, acetylxylanesterases and beta-glucosidase enzymes, such as, for example, any of the trademarks from NovozymesCTec2 or->Commercial cellulase preparation of CTec 3.

It is believed that the specific enzyme activity present in the different commercially available cellulase formulations optimized for bioconversion can be analyzed in detail using methods known in the art to accurately measure degradation of a substrate directly related to enzyme activity/concentration, such as Glycospot ^TM 。

Cellulase formulations suitable for optimization for bioconversion typically include a variety of enzyme activities including exoglucanases, endoglucanases, hemicellulases (including xylanases) and beta-glucosidase. Enzyme formulations may be expressed in different activities/units, such as carboxymethyl cellulase (CMC U) units, acid birch ligninase units (ABXU) and pNP-glucosidase units (pNPG U). For example ACCELLERASE TRIO ^TM Comprising the following steps: endoglucanase activity: 2000-2600CMC U/g, xylanase activity: >3000ABX U/g, and beta-glucosidase activity:>2000pNPG U/g; wherein the activity of one CMC unit releases 1. Mu. Mol of reducing sugar (expressed as glucose equivalent) within one minute at 50℃and pH 4.8; one ABX unit refers to the amount of enzyme required to produce 1 μmol xylose reducing sugar equivalent per minute at 50 ℃ and pH 5.3; one pNPG unit represents the fraction of p-nitrobenzene- [ beta ] per minute at 50℃and pH 4.8]1. Mu. Mol of nitrophenol are released from the D-glucopyranoside. To find out how much enzyme of a given enzyme composition should be added, a solubilization test of the enzyme composition (described below) can be performed on the model waste to provide an optimal enzymatic liquefaction process.

"microbial enzymes" include any enzyme such as one or more cellulases, one or more hemicellulases and/or one or more starch degrading enzymes, which can be expressed in a suitable microbial host by methods known in the art. The enzyme is also commercially available, either in pure form or as an enzyme cocktail. The specific enzymatic activity may be purified from commercially available enzyme cocktails, again using methods known in the art-see, e.g. et al.(2005)"Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial fermentation residue "(Enzyme and Microbial Technology (2005) 773-784), wherein Trichoderma reesei beta-xylosidase is purified from Celluclast (Finizym), and additional commercially available enzyme formulations are disclosed.

"xylanolytic activity" or "xylanolytic activity" refers to the biological activity of hydrolyzing a xylan-containing material. Two basic methods for measuring xylanolytic activity include: (1) Measuring total xylanolytic activity, and (2) measuring individual xylanolytic activity (e.g., endoxylanase, beta-xylosidase, arabinofuranosidase, alpha-glucuronidase, acetylxylan esterase, feruloyl esterase, and alpha-glucuronidase). Several publications summarize the recent progress of xylanolytic enzyme assays, including Biely and Puchard,2006,Journal of the Science of Food and Agriculture 86 (11): 1636-1647; spanikova and Biely,2006,FEBS Letters 580 (19): 4597-4601; herrimann et al 1997,Biochemical Journal 321:375-381. The total xylanolytic activity can be measured by: determining reducing sugars formed from various types of xylan, including, for example, oat, beech and larch xylans; or photometrically determining the stained xylan fragments released from various covalently stained xylans. A common total xylanolytic activity assay is based on the production of reducing sugars from polymeric 4-O-methyl glucuronic acid, as described by Bailey et al 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23 (3): 257-270. Xylanase activity may also be determined by: 0.2% AZCL-arabinoxylans were used as substrate at 0.01% X-100 and 200mM sodium phosphate pH6 at 37 ℃. One unit of xylanase activity refers to the production of 1.0. Mu. Mole of azurin per minute in 200mM sodium phosphate pH6 at 37℃and pH6 with 0.2% AZCL-arabinoxylan as substrate. Xylan degrading activity can be determined by: measurement of birch wood aggregateSugar (Sigma Chemical co., inc., st.Louis, MO, USA) is increased by hydrolysis of one or more xylanolytic enzymes under the following typical conditions: 1ml of reaction, 5mg/ml of substrate (total solids), 5mg of xylanolytic protein per gram of substrate, 50mM sodium acetate pH 5, 50 ℃,24 hours, sugar was analyzed using a parahydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever,1972, anal. Biochem. 47:273-279.

"lactic acid producing bacteria" includes Lactic Acid Bacteria (LAB), wherein the presently accepted taxonomies are based on a list of names of prokaryotes with a naming authority (LPSN) -this is an online database maintaining information on the naming and taxonomies of prokaryotes, following the taxonomic requirements and regulations of the International bacteria naming convention. Phylogenetic purpose is based on 16S rRNA-based LTP release of the 'all species life tree' project. The term "lactic acid producing bacteria" as used herein includes, in addition to bacteria belonging to the order LAB, bacteria not belonging to the order LAB but still capable of producing lactic acid.

The amount of lactic acid bacteria can be measured by Assay II.

Terms relating to a process

"biological fluid" refers to a liquefied and/or saccharified degradable component obtained by enzymatic processing of waste containing organics. Biological fluids also refer to the liquid fraction obtained by enzymatic and/or microbiological processing of waste including organic matter once separated from non-fermentable solids. Biological fluids include water and organic substrates such as proteins, fats, galactose, mannose, glucose, xylose, arabinose, lactate, acetate, ethanol, and/or other ingredients, depending on the components of the waste (such as the components of proteins and fats may be in soluble and/or insoluble form). Biological fluids also include fibers, ash, and inert impurities. The produced biological fluid comprises a high ratio of microbial metabolites, provides a substrate for gas production, is suitable for anaerobic digestion, e.g. for biogas production.

"marsh gas" is a mixed gas produced by decomposing organic matters under anaerobic condition. Biogas can be produced from agricultural waste, manure, municipal waste, plant material, sewage, green waste or food waste. Biogas is a renewable energy source.

Biogas is produced by anaerobic digestion with methanogens or anaerobic organisms that digest material or ferment biodegradable material within a closed system. Such closed systems are known as anaerobic digesters, bio-digesters or bioreactors.

Biogas is mainly methane (CH) ₄ ) And carbon dioxide (CO) ₂ ) There may be a small amount of hydrogen sulfide (H ₂ S), moisture and silicone. The gases methane, hydrogen and carbon monoxide (CO) may be combusted with or oxidized by oxygen. This release of energy allows biogas to be used as fuel; it can be used for any heating purpose, such as cooking. It can also be used in gas engines to convert energy in gases into electrical and thermal energy.

The term "bioreactor" refers to a device or system that supports a biologically active environment, such as an environment in which biological treatment is performed, involving the treatment of microorganisms or the treatment of biochemically active substances derived from microorganisms.

An example of a bioreactor is a container or vessel in which the microbiological and/or biochemical active is maintained under conditions such as aerobic or anaerobic conditions, temperature, etc. that are required to allow the biological reaction to operate.

"dry matter" is also referred to as "DM" and is meant to include both soluble and insoluble total solids, and in fact "non-water content". The dry matter content was measured by drying at about 60 ℃ for 48 hours as described in assay VIII (Assay VIII).

"hydrolysis" refers to the cleavage of chemical bonds with the participation of water as an auxiliary substrate. The term applies to the treatment of municipal solid waste material with an enzyme composition, breaking down cellulose and/or hemicellulose and other substrates into fermentable sugars such as glucose, cellobiose, xylose, arabinose, mannose, galactose and/or soluble oligosaccharides (also known as saccharification). Enzymatic processing is performed in one or more stages by one or more enzyme compositions. In this disclosure, the terms "hydrolysis," "liquefaction," "saccharification," and "solubilization" may be used interchangeably.

The enzymatic processing may be performed as a batch process or a series of batch processes. Enzymatic processing may be performed as a feed batch or continuous process, or a series of feed batches or continuous processes, wherein the municipal solid waste material is gradually fed into a solution, for example, containing an enzyme composition. The enzymatic process may be continuous, with the municipal solid waste material and enzyme composition added at different time intervals throughout the process, and the hydrolysate removed at different time intervals throughout the enzymatic process. The removal of the hydrolysate can be performed before, simultaneously with or after the addition of the cellulosic material and the cellulolytic enzyme composition.

An "effective amount" of one or more isolated enzyme compositions refers to an amount that, in combination, achieves sufficient solubilization of waste to provide a solution comprising a high ratio of sugars and other soluble degradation products, suitable for anaerobic digestion, such as substrates for biogas production. An effective amount can be determined by using the solubilization test described herein.

The enzymatic processing is preferably carried out in a suitable aqueous environment, the conditions of which can be readily determined by a person skilled in the art. In one aspect, the enzymatic processing is performed under conditions suitable for the activity of the one or more enzymes, i.e., optimal for the one or more enzymes.

The "solubilization test" is a test applied to find out how much of a particular enzyme composition should be added to the waste for adequate enzymatic processing. A solubilization test of the selected enzyme composition on the MSW model substrate can be applied to identify the optimal enzyme composition solubilization treatment. Solubilization of waste, such as municipal solid waste, can be determined by applying the following test methods:

a model substrate consisting of 41% mixed plant-derived food waste, 13% mixed animal-derived food waste, and 46% mixed cellulosic waste was minced, mixed, and ground several times until uniform, passed through a 3mm sieve, divided into smaller portions, and cryopreserved at less than or equal to-18 ℃.

A set of pre-tared 50mL centrifuge tubes, each containing 1.500.+ -. 0.010g TS of the model substrate described above (total solids at 60 ℃) was added with various amounts of enzyme to be tested (typically 5-60mg EP/g TS of model substrate) in 50mM sodium acetate buffer pH 4.50.+ -. 0.05, the final total weight in each tube being 20.000.+ -. 0.025g.

The tube was closed with a tightly fitting cap, the reaction mixture was incubated at 50.+ -. 1 ℃ for 24 hours.+ -. 10 minutes, and the tube was transposed (upside down) at a speed of 10.0.+ -. 0.5 revolutions per minute with stirring.

Immediately after the end of incubation, the tubes were centrifuged at 2100±10G for 10 minutes, and immediately after centrifugation (in less than 5 minutes) the supernatant was poured into another set of pre-tared tubes. The first set of tubes (including the cap) with residual undissolved model substrate and the second set of tubes with decanted supernatant containing solubilized model substrate were weighed on a 4-position decimal analytical balance and then placed in a well-ventilated dry box at 60±1 ℃ for 6 days.

After drying, the test tube (including the cap) was again weighed, the amount of TS in the pellet and supernatant was determined, and the mass balance was calculated as follows:

mass balance% = ((TS pellet + TS supernatant-TS enzyme)/TS model substrate) 100%.

The mass balance based on TS model substrate (1.500.+ -. 0.010 g) to ensure no material loss and proper drying will typically be in the range of 95-105%.

Based on the total amount of decanted supernatant and the amount of TS, the% TS in the decanted supernatant was calculated as follows:

TS% = (TS decanted supernatant/total decanted supernatant) ×100%

Finally, the solubilization is calculated as follows:

solubilisation% = (((TS%. Actual water/(1-TS%)) -tsenzyme)/TS model substrate) ×100%

The liquid phase trapped in the centrifugal precipitate will also be accounted for by calculating the solubilization based on the TS% of the decanted supernatant and the actual amount of water (the actual weight of the decanted supernatant and wet precipitate minus the initial weight of TS in the added model substrate).

The solubilization versus enzyme dosage graph will show the characteristics of the efficacy of the enzyme (maximum solubilization at high enzyme dosages) and the enzyme efficacy (dosages required to achieve a certain level of solubilization).

The enzyme efficacy may typically be 35-70% solubilization, depending on the model substrate composition and the enzyme composition to be tested. The dosage used can generally be defined as achieving 85-95% efficacy.

"isolation" refers to a substance in a form or environment that does not exist in nature. Non-limiting examples of educts include: (1) any non-naturally occurring material; (2) Any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, that is at least partially free of one or more or all naturally occurring components associated therewith in nature; (3) Any substance modified by the human hand relative to substances found in nature; or (4) any agent that is modified by increasing the amount of the agent relative to other components naturally associated therewith (e.g., recombinant production in a host cell; multiple copies of a gene encoding the agent; and use of a stronger promoter than that naturally associated with the gene encoding the agent).

"pretreatment" refers to any pretreatment treatment known in the art that can be used to destroy plant cell wall components of municipal solid waste material (Chandra et al, 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.9:1621-1651;Yang and Wyman,2008,Biofuels Bioproducts and Biorefining-Biofpr. 2:26-40). Conventional pre-processing includes, but is not limited to, steam pre-processing (with or without explosion), dilute acid pre-processing, hot water pre-processing, alkaline pre-processing, lime pre-processing, wet oxidation, wet explosion, ammonia fiber explosion, organic solvent pre-processing, and biological pre-processing. Other pretreatment methods include ammonia permeation, ultrasound, electroporation, microwaves, supercritical CO ₂ Supercritical H ₂ O, ozone, ionic liquid, brick making, granulating and gamma ray preprocessing.

"solubilisation" refers to the enzymatic processing of waste resulting in liquefaction and/or saccharification of organic matter. In this disclosure, the terms "hydrolysis," "liquefaction," "saccharification," and "solubilization" may be used interchangeably.

"sterilization" is a process that reduces the number of microorganisms to a level that has been formally approved as safe. It refers to controlling the bacteria level of equipment and vessels in dairy, other food processing plants, eating houses and other places where no specific pathogenic microorganism target is present. In this document, strains of E.coli are used as hygiene indicators, E.coli per gram of waste<10 ² The results of CFU were considered satisfactory, i.e. considered post-sterilization waste (sources: "Guidelines for assessing the microbiological safety of ready-to-eat foods placed on the market", health Protection Agency, nov 2009, p.24).

The "2D/3D separation" is achieved by one or more steps. In one embodiment, first, an impingement separator removes two strands of non-biodegradable material, resulting in a 2D portion consisting of plastic bags and other generally non-shaped materials, a 3D portion consisting of shaped bottles and containers, and a volume of a bio-derived liquid slurry of biodegradable components. In the second step of this embodiment, the 2D portion is further subjected to a screw press or similar device to further increase the yield of the biogenic liquid slurry. The 2D portion may be further subjected to washing to further recover the biodegradable material.

Drawings

FIG. 1 is a schematic illustration of an example of waste treatment including a bioreactor in which self-disinfecting enzymatic and microbial processes are performed.

Figure 2 shows bacterial counts on metals from mechanical bio-processing (MBT) (black bars) and from the treatment method of the invention (grey bars).

Figure 3 shows bacterial counts of waste derived fuel (RDF) from mechanical bio-processing (MBT) (black bars) and from the treatment process of the invention (grey bars).

Figure 4 shows a contour plot showing the negative base 10 log of the relative decrease in CFU count after 24 hours (versus 0 hours). Black colorThe dots show the conditions of the experimental tests and the numbers next to the dots show the number of repetitions performed on a particular combination of pH and temperature. This combination of pH and temperature was tested only once if no numbers were shown next to a certain point. Each contour line shows a relative decrease of 10 in CFU count after 24 hours based on the following model ⁿ Multiple (where n is the number in the box intersecting the contour): (-log ₁₀ (relative reduction of CFU count) ^0.59 ＝9.2615-0.9346×pH-0.1718×T+0.00274×T ² 。

Detailed Description

The method may further comprise the preliminary step of:

a) Removing large objects, crushing and/or beating.

The method may further comprise the subsequent step of:

b) Subjecting the processed waste from step a) to one or more separation steps, thereby providing a biological liquid and a solid fraction;

c) Subjecting the biological liquid and/or solid fraction to downstream processing.

The downstream treatment may be any treatment involving the solid or liquid fraction of the waste obtained from step b), which is carried out downstream of enzymatic and/or microbiological processes in the bioreactor in step a). Examples of downstream treatments are washing treatments, evaporation treatments, collection and anaerobic digestion of the biological liquid obtained from step b) or a part thereof. The downstream processing further includes: the solid and/or liquid fraction of the waste obtained from step b) is converted into biogas, which may be combusted to produce electricity and/or heat; and the solid and/or liquid fraction of the waste obtained from step b) is converted into renewable natural, biomethane gas and/or transportation fuel.

The inventors have surprisingly found that when the waste fraction is reacted with specific levels of naturally occurring bacteria and enzymes at low temperatures (40 ℃ -60 ℃), the amount of pathogenic bacteria in the produced biological fluid and non-biodegradable waste material is very low. Thus, biological fluids, waste and equipment for waste treatment do not expose the environment, e.g., workers, to undesirable bacteria.

Low temperatures during the enzymatic reaction are advantageous because fuel can be saved which heats the waste fraction to high temperatures, e.g. 75 ℃. In the case of a waste fraction reacted with enzymes for about 10 to 30 hours, considerable costs can be saved. Another advantage is that handling at low temperatures is easier than handling at high temperatures.

The inventors have found that even when the waste is reacted with enzymes at low temperatures, the amount of bacteria, e.g. pathogenic bacteria, in the produced biological fluid and non-biodegradable waste material is very low. The bacterial count present in the waste can be reduced to a bacterial count of E.coli below 20 CFU/g waste and/or to a bacterial count of Enterobacteriaceae below 10 ² CFU/gram waste.

The method for producing biological fluid according to the present invention comprises: in step a), the waste comprising biodegradable material and non-biodegradable material is subjected to enzymatic and/or microbiological processing. Waste includes biodegradable materials, which are organic materials that can be hydrolyzed by enzymes and/or microorganisms. The organic material may include carbohydrates, proteins, fats, and mixtures thereof, which are organic materials typically found in household waste. Waste also includes non-biodegradable materials such as plastics or metals.

Waste may be unclassified. In one embodiment of the invention, the unclassified waste comprises a mixture of biodegradable material and non-biodegradable material, wherein 15% or more by weight of the dry weight is non-biodegradable material.

In one embodiment of the invention, the waste comprises a mixture of biodegradable and non-biodegradable materials, wherein at least 20% w/w is non-biodegradable material, calculated on the weight of the waste. In one embodiment, at least 25% of the waste is non-biodegradable material, at least 30% of the waste is non-biodegradable material, at least 35% of the waste is non-biodegradable material, at least 40% of the waste is non-biodegradable material, at least 45% of the waste is non-biodegradable material, or at least 50% of the waste is non-biodegradable material.

The waste may be Municipal Solid Waste (MSW), for example, municipal waste or waste from household households and public facilities. The waste comprises a natural microbiota having at least 2.5X10 ⁸ Total bacterial count of CFU/gram waste of at least 1.5 x 10 ⁶ CFU/gram waste E.coli bacteria count and/or at least 1.5X10 ⁸ Bacterial count of enterobacteriaceae of CFU/gram waste. The natural microbiota may include lactic acid bacteria which proliferate during the time that the waste is subjected to the enzyme composition.

In a preferred embodiment of the invention, the waste comprises a natural microbiota having at least 3.0X10 s ⁸ Total bacterial count of CFU/gram waste of at least 1.6x10 ⁶ CFU/gram waste E.coli bacteria count and/or at least 1.9x10 ⁸ Bacterial count of enterobacteriaceae of CFU/gram waste.

It has previously been thought necessary to inoculate the waste portion with bacteria in order to produce biological fluids from the waste. The inventors have found that naturally occurring bacteria in the waste portion are sufficient to control the microbiota during the reaction time in which the waste portion is exposed to the enzyme composition. Examples show that the number of enterobacteriaceae and escherichia coli in biological fluids is very low.

In one embodiment of the invention, the waste provided contains lactic acid bacteria. The ratio between lactic acid bacteria and total bacterial count in the waste may be at least 1:1, such as at least 1:1.5, at least 1:2, at least 1:3, at least 1:4, at least 1:5, or at least 1:10.

The dry matter content of the waste fraction provided in the process of the invention may be in the range of 10-90% w/w. The dry matter content in the waste fraction can be measured by assay VIII (Assay VIII). In one embodiment of the invention, the dry matter content of the waste fraction may be in the range of 30-80% w/w, preferably in the range of 50-70% w/w.

In one embodiment of the invention, the dry matter content of the waste fraction provided in the method of the invention may be about 10% w/w, such as about 15% w/w, about 20% w/w, about 25% w/w, about 30% w/w, about 35% w/w, about 40% w/w, about 45% w/w, about 50% w/w, about 55% w/w, about 60% w/w, about 65% w/w, about 70% w/w, about 75% w/w, about 80% w/w, about 85% w/w, or about 90% w/w.

In one embodiment of the method of the invention, the waste processing in step a) may be subjected to the action of water. The dry matter content of the waste fraction can be measured according to assay VIII. Depending on the dry matter content, water may be added to the waste portion. For example, when the waste fraction provided is Municipal Solid Waste (MSW), the waste fraction may conveniently be subjected to water in an amount of from about 0.5 to about 3.0 kg of water per kg of MSW. In one embodiment of the invention, each kilogram of MSW may be subjected to about 0.5 to about 2.5 kilograms of water. In a preferred embodiment of the present invention, about 0.8 to about 1.8 kg of water may be subjected to per kg of municipal solid waste. As a result of the addition of water to the waste portion, the dry matter content of the waste portion including water is lower than the dry matter content of the waste portion prior to the addition of water.

In a preferred embodiment of the invention, the waste fraction is subjected to the action of water, such that a water to waste ratio in the range of about 0.1:1 to 5:1, preferably in the range of 0.5:1 to 3:1, more preferably in the range of 1:1 to 2:1, even more preferably in the range of 1:1 to 1.5:1 is obtained.

The method of the invention comprises subjecting the waste to an enzyme composition in step a). The purpose of the enzyme composition is to process biodegradable materials present in the waste fraction. Biodegradable materials are thus degraded into smaller parts, for example by enzymes capable of hydrolysing carbohydrates into sugar molecules.

Suitable enzyme compositions are well known in the art and are commercially available. For example, a suitable enzyme composition is a composition comprising a Cellulolytic Background Composition (CBC) in combination with one or more enzymes.

When added to the treatment, the Cellulolytic Background Composition (CBC) comprises a commercially available cellulolytic enzyme composition. Examples of commercially available cellulolytic enzyme compositions suitable for use in the method according to the invention include, but are not limited to, e.g.CTec(Novozymes A/S)、/>CTec2(Novozymes A/S)、/>CTec3(Novozymes A/S)、/>(Novozymes A/S)、NOVOZYM ^TM 188(Novozymes A/S)、SPEZYME ^TM CP(Genencor Int.)、ACCELLERASE ^TM TRIO(DuPont)、/>NL(DSM)、S/L 100(DSM)、ROHAMENT ^TM 7069W(/>GmbH), orCMAX3 ^TM (Dyadic International,Inc.)。

When the enzyme composition comprises a further enzyme activity in addition to the activity present in the CBC, such enzyme activity may be added from a separate source or added together as part of the enzyme mixture. Suitable mixtures include, but are not limited to, commercially available enzyme compositions, namely Cellulase PLUS, xylanase PLUS, brewzyme LP, fibre zyme G200 and NCE BG PLUS from Dyadic International (Jupiter, FL, USA), or Optimash BG from Genencor (Rochester, N.Y., USA).

CBC may include the following enzymatic activities:

cellobiohydrolase I:

endo-1, 4-beta-glucanase

beta-glucosidase

Endo-1, 4-beta-xylanases

Beta-xylosidase

Beta-L-arabinofuranosidase

Amyloglucosidase

Alpha-amylase

Acetylxylitol esterase

In a preferred embodiment, the activity of the CBC is as followsTRIO ^TM (Genencor Int.), cellular CTec2 (Novozymes A/S) or cellular CTec3 (Novozymes A/S).

The enzyme composition may comprise about 40-99% w/w of an enzyme having cellulolytic activity. In one embodiment, the enzyme composition comprises about 50-90% w/w of an enzyme having cellulolytic activity, such as about 60-80% w/w of an enzyme having cellulolytic activity or about 65-75% w/w of an enzyme having cellulolytic activity. The enzyme composition may comprise about 0-20% w/w protease, e.g. about 10% w/w enzyme composition. The enzyme composition may comprise about 0-30% w/w beta-glucanase, e.g. about 15% w/w enzyme composition. The enzyme composition may comprise about 0-10% w/w pectate enzyme, e.g. 5% w/w enzyme composition. The enzyme composition may comprise about 0-10% w/w of the mannanase or amylase, e.g. about 5% w/w of the enzyme composition.

The waste may be subjected to the enzyme composition at a concentration of about 10-20kg of enzyme composition per ton of waste, preferably about 12-19kg of enzyme composition per ton of waste, more preferably about 14-17kg of enzyme composition per ton of waste. In a preferred embodiment, the waste may be subjected to the enzyme composition at a concentration of about 16kg enzyme composition per ton of waste.

The treatment method of the invention comprises subjecting the waste fraction to the action of an enzyme composition in step a) and performing the reaction at a pH value between 3.0 and 6.0 and a temperature between 40 ℃ and 60 ℃ to obtain a biological fluid.

In one embodiment of the invention the pH in step a) is below 6.0, preferably below 5.0, more preferably below 4.5, even more preferably below 4.4, most preferably below 4.2. The pH may be in the range of 3.0-6.0, such as in the range of 3.0-5.8, such as in the range of 3.5, 4.0-5.5, 4.0-5.0, in the range of 4.0-4.5, or in the range of 4.0-4.4.

The temperature in step a) of the process of the invention is 55℃or less, 50℃or less or 45℃or less. In one embodiment of the invention, the temperature is in the range of 40-55 ℃, in the range of 40-50 ℃, or in the range of 40-45 ℃.

In one embodiment of the invention, the pH in step a) is in the range of 3.0-6.0 and the temperature is in the range of 40-55 ℃. In another embodiment, the pH is in the range of 4.0-5.8 and the temperature is in the range of 40-55deg.C. More preferably, the pH is in the range of 4.0-5.5 and the temperature is in the range of 40-50 ℃. More preferably, the pH is in the range of 4.0-5.0 and the temperature is in the range of 40-45 ℃.

In one embodiment of the invention, the waste is subjected to the enzyme composition for a period of time ranging from 10 to 30 hours, preferably from 20 to 25 hours, and more preferably about 18 hours.

In a preferred embodiment, the present invention relates to a method of sanitizing waste, the method comprising:

a) Allowing the waste to flow in the bioreactor at a pH of between 4.0 and 5.0, a temperature of between 40 ℃ and 50 ℃ and for 18 to 25 hoursSubjecting the time period to enzymatic and/or microbiological processing to obtain at least a partial reduction of bacterial count, said waste comprising biodegradable and non-biodegradable materials and having a value of at least 2.5x10 ⁸ Total bacterial count of CFU/gram waste of at least 1.5 x 10 ⁶ Bacterial count of E.coli of CFU/g waste, or at least 1.5X10 ⁸ Bacterial count of enterobacteriaceae of CFU/gram waste.

In another preferred embodiment, the present invention relates to a method of sanitizing waste comprising:

a) Subjecting waste comprising biodegradable and non-biodegradable materials and having a bacterial count of at least 2.5 x 10 to enzymatic and/or microbiological processing in a bioreactor at a pH between 4.0 and 5.0, a temperature between 40 ℃ and 50 ℃ and a period of 18 to 25 hours ⁸ Total bacterial count of CFU/gram waste of at least 1.5 x 10 ⁶ Bacterial count of E.coli of CFU/g waste, or at least 1.5X10 ⁸ Bacterial count of enterobacteriaceae of CFU/gram waste;

b) Subjecting the processed waste from step a) to one or more separate steps to provide a biological liquid and a solid fraction;

Low temperatures during the enzymatic reaction are advantageous because fuel can be saved which heats the waste fraction to high temperatures, e.g. 75 ℃. In the case of waste material reacting with enzymes for about 10 to 30 hours, substantial costs can be saved. Another advantage is that the workers handling the method of the invention are not exposed to high temperatures.

It was previously thought that the waste fraction should be pre-processed at a temperature of 90-95 ℃ before being used to produce biological fluids. The effect of the pre-processing is to disinfect the waste portion, thereby killing undesirable microorganisms, such as pathogenic bacteria. WO 2013/185778 teaches that preheating of the waste is not necessary. The application shows that safe fermentation can be achieved by adding microorganisms (inoculation of EC 12B) and enzymes to the waste and allowing simultaneous enzymatic processing and microbial fermentation at a temperature of 45-75 ℃.

The inventors of the present invention have surprisingly found that when the waste fraction is reacted with specific levels of naturally occurring bacteria and enzymes at low temperatures (40-60 ℃), the bacteria (enterobacteriaceae and escherichia coli) in the produced biological fluid and non-biodegradable waste material, which are considered to be excellent indicators, have very low bacterial numbers. Thus, biological fluids, non-biodegradable materials, and devices used do not expose the environment to undesirable bacteria, e.g., pathogens. Thus, a safer environment is achieved, especially for workers handling the method of the invention and for sorting the waste after separation from the biological fluid.

Depending on the waste, various food-borne viruses, blood viruses, and faecal-transmitted viruses may also be present in the waste. However, the treatment conditions described in step a) and/or step c) of the present invention completely inactivate or reduce viruses, such as coronaviruses, adenoviruses, herpesviruses, measles, aids viruses and influenza viruses, to harmless levels during the treatment. In one embodiment of the invention, the disinfection effort comprises reducing or inactivating viruses.

The treatment process of the present invention further comprises recovering the biological fluid by separating the biological fluid from the non-biodegradable material. The biological fluid may be separated by one or more separation devices, such as one or more impact separators, one or more screens, one or more wash cylinders, one or more presses, and/or one or more hydraulic presses. In one embodiment of the invention, the biological fluid is separated from the waste portion by using an impact separator.

One or more separation devices separate the biological fluid from the waste. The waste may include several types of non-biodegradable materials, such as textiles and aluminum foil (2D) and cans and plastic bottles (3D).

The water used for flushing the non-biodegradable waste may be recycled, heated and act on the waste fraction in step a) of the method of the invention.

Inert materials, such as sand and glass, are typically removed, e.g., sieved, from biological fluids. Metals are typically removed from all waste fractions. The 2D portion may be further separated into recyclables and/or residues, such as Solid Recovery Fuel (SRF), waste derived fuel (RDF), and/or inerts. The 3D fraction may also be further separated into recyclables and/or residues, such as metal, 3D plastic and/or RDF.

In one embodiment of the invention, the biological fluid produced by the method of the invention is treated to a biofuel, e.g., biogas.

According to the health protection agency (Guidelines for assessing the microbiological safety of ready-to-eat foods placed on the marked, health Protection Agency, london, november 2009,

https:// webarchive.localized. Gov. Uk/20140714111812/http:// www.hpa.org.uk/webc/HPA webFile/HPA web_C/1259151921557), the amount of enterobacteriaceae in the ready-to-eat food should be less than 1 x 10 ² CFU/ml is satisfactory. The amount of Enterobacteriaceae in the instant food exceeds 1×10 ⁴ CFU/ml is unsatisfactory, 1X 10 ² -1×10 ⁴ The amount of CFU/ml is at the edge level.

The health protection agency recommends that it is desirable for the bacterial count of E.coli to be below 20CFU/ml for ready-to-eat foods. The bacterial count of the Escherichia coli is 20-1×10 ² The CFU/ml range is at marginal level, and the bacterial count of E.coli exceeds 1X 10 for ready-to-eat foods ² Is unsatisfactory.

In another aspect, the invention relates to a biological fluid produced by the method of the invention. By the method of the invention it is possible to produce a biological fluid that meets the microbiological requirements of the ready-to-eat food.

In one embodiment of the invention, the biological fluid produced comprises a very low number of pathogenic bacteria, e.g., E.coli.

In one embodiment of the invention, the bacterial count of the enterobacteriaceae family in the biological fluid is measured as Assay I (Assay I)The amount is less than 1X 10 ² -1×10 ⁴ CFU/ml, preferably below 1X 10 ² CFU/ml。

In one embodiment of the invention the bacterial count of E.coli in the biological fluid is measured as Assay II (Assay II) below 20-100CFU/ml, preferably below 20CFU/ml, more preferably below 10CFU/ml.

In one embodiment of the invention, the bacterial count of the lactic acid bacteria of the biological fluid is at least 1X 10 as measured by assay III (Assay III) ⁵ CFU/ml, preferably at least 1X 10 ⁶ CFU/ml。

The invention also relates to non-biodegradable waste materials obtainable from the process of the invention. The non-biodegradable waste material may be a 2D or 3D material that can be washed after separation from the biological fluid. In one embodiment of the invention, the non-biodegradable is a 2D waste material.

In one embodiment of the invention, the non-biodegradable waste material has a bacterial count of enterobacteriaceae of less than 1X 10 as measured by Assay IV (Assay IV) ² -1×10 ⁴ CFU/ml, preferably below 1X 10 ² CFU/ml。

In one embodiment of the invention, the bacterial count of E.coli of the non-biodegradable waste material is measured in assay II below 20-100CFU/ml, preferably below 20CFU/ml, and more preferably below 10CFU/ml.

In one embodiment of the invention, the non-biodegradable waste material has a bacterial count of at least 1X 10 as measured in assay III ⁵ CFU/ml, preferably at least 1X 10 ⁶ CFU/ml。

In one embodiment of the invention, the ratio between the bacterial count (CFU/ml) of the lactic acid bacteria and the total bacterial count (CFU/ml) is at least 1:2 to 1:1.

In one aspect, the invention relates to biogas produced from biological fluids obtained by the method of the invention.

Fig. 1 is a schematic overview of a waste treatment process and will be explained in more detail below.

During this first stage, means (not shown in fig. 1) are typically provided to open the plastic bag and thoroughly pulp or pulverize the degradable component, preparing the waste into a more uniform organic phase prior to enzyme addition. In some cases, the initial components, such as metals or other materials, may be removed prior to placing the waste into the bioreactor. In some cases, the particle size distribution is reduced or the material is pre-classified. Water, enzymes and/or microorganisms are added. Enzymatic and/or saccharification and/or microbial fermentation is carried out continuously at optimal residence time, temperature and pH values to achieve enzymatic and microbial performance. By this enzymatic processing and fermentation, the biogenic portion of MSW is liquefied and/or saccharified to biological fluids, including monosaccharides, disaccharides, and/or oligosaccharides.

The method of the present invention allows for the disinfection of waste, such as MSW, comprising objects of different sizes, and in one embodiment of the present invention, the large solid objects are pre-sorted before the waste enters the bioreactor. The method according to the invention is effective for objects of various particle sizes. In one embodiment, the method according to the invention is suitable for objects having a maximum particle size of 600mm, such as 500mm, such as 400mm, such as 300mm, such as 200mm, such as 100mm, such as 80mm, such as 70mm, such as 60mm or such as 50 mm.

In the separation step, the biological fluid is separated from the non-biodegradable fraction. Separation is typically performed by one or more separation means, such as one or more impact separators, one or more screens, one or more wash cylinders, one or more presses, and/or one or more hydraulic presses. The biological fluid may be cleaned and then further processed into biogas in a biogas plant.

The one or more separation devices separate waste, such as MSW, that has been enzymatically and/or microbiologically processed into biological fluid, a portion of 2D material, such as non-biodegradable material, and a portion of 3D material, such as non-biodegradable material. The 3D parts (such as pop cans and plastic bottles) do not bind large amounts of biological fluids, so a single washing step is usually sufficient to wash the 3D parts. The 2D part (e.g. textile and foil) typically incorporates a large amount of biological fluid. Thus, the 2D fraction is typically pressed, washed and pressed again using, for example, a screw press to optimize the recovery of biological fluids and obtain a cleaner and drier 2D fraction. Inert materials, such as sand and glass, are typically removed, e.g., screened, from biological fluids. The metal is typically removed from all mentioned parts. The water used in the one or more cleaning drums may be recycled, heated, and then used in a first step to heat the waste. The 2D fraction may be further separated into recyclables and/or residues, such as SRF (solid recovery fuel), RDF (spent derived fuel) and/or inerts. The 3D fraction may also be further separated into recyclables and/or residues, such as metal, 3D plastic and/or RDF.

Example

Assay

Assay I: total bacterial count

1ml from each diluted biological fluid was coated on petrifilm sheets, i.e. "3M ^TM Petrifilm ^TM Aerobic Count Plate "for total bacterial count. Petrifilm plates were incubated at 30 ℃ for 48 hours, after which Colony Forming Units (CFU) were counted according to manufacturer's instructions.

Assay II: lactic acid bacteria count

1ml from each diluted biological fluid was coated on petrifilm sheets, i.e. "3M ^TM Petrifilm ^TM Lactic Acid Bacteria Count Plate "for lactobacillus counting. Petrifilm plates were incubated at 37 ℃ for 48 hours, after which Colony Forming Units (CFU) were counted according to manufacturer's instructions.

Assay III: enterobacteriaceae count

1ml from each diluted biological fluid was coated on petrifilm sheets, i.e. "3MPetrifilm ^TM Enterobacteriaceae Count Plate "for enterobacteriaceae counting. Petrifilm plates were incubated at 37 ℃ for 48 hours, after which Colony Forming Units (CFU) were counted according to manufacturer's instructions.

Assay IV: coli count

1ml from each diluted biological fluid was coated on petrifilm sheets, i.e. "3MPetrifilm ^TM type E.coli and Coliform Count "for e.coli counts. Petrifilm plates were incubated at 37 ℃ for 48 hours, after which Colony Forming Units (CFU) were counted according to manufacturer's instructions.

Assay V: counting aerobic bacteria

The total amount of aerobic bacteria count was performed using Yeast Extract Agar (YEA). 1ml of sample from each diluted biological fluid was coated on an empty petri dish (1 petri dish per sample). The molten YEA cooled to about 47 ℃ was then poured into a petri dish and mixed with the sample to equalize the bacterial growth distribution in the agar. Once the agar solidified, the coating was incubated at 30℃for 72 hours, after which the CFU was counted.

Assay VI: enterobacteriaceae count

Enterobacteriaceae counts were performed using mauve bile glucose agar (VRBGA). 1ml of sample from each diluted biological fluid was coated in an empty petri dish (1 petri dish per sample). The molten VRBGA cooled to about 47 ℃ was then poured into a petri dish and mixed with the sample to equalize the bacterial growth distribution in the agar. Once the agar solidified, a cover of VRBGA was added and the coating was then incubated at 37 ℃ for 24 hours before counting CFU.

Assay VII: coli count

Coli counts were performed using mauve bile agar (VRBA). 1ml of sample from each diluted biological fluid was coated in an empty petri dish (1 petri dish per sample). The molten VRBA cooled to about 47 ℃ was then poured into a petri dish and mixed with the sample to equalize the bacterial growth distribution in the agar. Once the agar solidified, VRBA was added and the coating was then incubated at 44 ℃ for 24 hours before counting CFU. All colonies counted had to go through a validation process using MacConkey agar, YEA agar, oxidase test, lactopeptide water and trypsin water (containing Kovacs reagent).

Assay VIII: dry matter content

The dry matter content of the waste can be determined by drying the sample at 60 ℃ for 48 hours. The weight of the sample before and after drying should be measured and the dry matter content (percent) can be calculated by the following formula:

weight of dried sample _x100=% dry matter percentage in the sample

Weight of sample before drying

Example 1

This example investigates the bacterial count of the output samples sorted according to the method of the invention and compares it with the bacterial count of MBT (mechanical biological treatment) plant output samples. The RDF (waste derived fuel) fraction and metals processed according to the treatment process of the present invention were compared with RDF and metals obtained at MBT facilities in the uk.

The dry matter content of the waste (MSW) subjected to the method according to the invention is 50-70%. The MSW is then transported to the bioreactor. Water is added to the MSW to obtain a slurry of waste and water with a water to MSW ratio of 1.5-2 to 1. Cellic at a concentration of 0.9-2.3% w/w (based on MSW weight before adding water)The (Novozymes a/S) enzyme composition was added to the MSW slurry, which was then allowed to react at a temperature between 40 ℃ and 60 ℃ at a pH between 4.0 and 6.0 for 24 hours.

The dry matter content of the waste entering the MBT plant is 50-70%. MSW is classified into RDF, metals, and biodegradable materials before the biodegradable materials are delivered into the bioreactor.

RDF from the process and MBT facilities of the present invention was sampled and analyzed as follows: 5 separate and isolated samples obtained from different locations within the RDF output container were pooled and 1g thereof was mixed with 9ml of sterile 0.9% NaCl. The mixture was vortexed and inverted for 1 minute to form a diluent 10 ^-1 . RDF samples were thereafter serially diluted 10 with sterile 0.9% NaCl ⁸ Multiple times. According to the above assays I, II, III and IV, 1ml from each dilution was coated on petrifilm sheets.

The metals from the treatment process and MBT facility of the present invention were sampled and analyzed as follows:

wiping a sample from a standard can (containing, for example, tuna or roasted beans) with 5 sterile swab sticks of size 77cm ² The swab is then placed in 1ml of an appropriate medium. Then 5ml were pooled and sterilized with 0.9% NaCl H ₂ Serial dilution of O to 10 ^-8 . 1ml from each dilution was coated on petrifilm sheets according to assays I, II, III and IV above.

The bacterial count on the metal can lids obtained from the processing method according to the invention (test 1) and the bacterial count on the metal can lids obtained from MBT (test 2) were compared, respectively. Tests 3 and 4 investigate the bacterial count on RDF obtained from the processing method according to the invention and the bacterial count on RDF obtained from MBT, respectively. The results are shown in table 1 and discussed below.

Table 1.

Test 1

On average, the total amount of bacteria was 3.5X10 ⁵ CFU/can lid, and pathogenicity index bacteria count is: enterobacteriaceae count is 5.11×10 ² CFU/can lid, accounting for about 1/700 of the whole viable flora, and E.coli count of 6.6X10 ⁰ CFU/can lid, accounting for about 1/5000 of the total viable flora. Lactic acid bacteria count was 1.78X10 ⁵ CFU// can lid thus represents about 1/2 of the total viable flora in the metal sample classified according to the method of the invention (fig. 2). In the classified metals processed according to the treatment method of the present invention, the amounts of the index bacteria enterobacteriaceae and escherichia coli are surprisingly low.

Test 2

On average, the total amount of bacteria was 2.21×10 ⁷ CFU/can lid, and pathogenicity index bacteria count is: enterobacteriaceae count is 4.10X10 ⁵ CFU/can lid, accounting for about 1/54 of the whole viable flora, and E.coli count of 2.1X10 ⁴ CFU/can lid, accounting for about 1/1000 of the total viable bacteria. Lactic acidThe bacterial count was 1.25X10 ⁵ CFU/can lid, and thus about 1/175 of the total viable flora in MBT classified metals (fig. 2). Therefore, in MBT classified metals, the amount of enterobacteriaceae as an indicator bacterium is about 4 times the amount of lactic acid bacteria.

Comparison of test 1 and test 2

The bacterial count of MBT classified metals (test 1) was compared with the bacterial count of classified metals processed by the treatment method of the present invention (test 2). The total amount of bacteria in MBT classified metals was >60 times or more compared to the total amount of bacteria of classified metals obtained from the treatment method of the present invention (fig. 2). In contrast, the enterobacteriaceae count in MBT-classified metals was > 800-fold or more compared to the enterobacteriaceae count in the present classified metals, and the escherichia coli count in MBT-classified metals was > 1800-fold or more compared to the escherichia coli count in the present classified metals (fig. 2). Finally, lactic acid bacteria are similar between MBT-classified metals and the present classified metals.

These findings indicate two points: 1) Compared with the classified metals of the present invention, the growth conditions of bacteria in the MBT classified metals are better, including pathogenic index bacteria (enterobacteriaceae and escherichia coli), but not including lactic acid bacteria; 2) The conditions in the bioreactor using the method according to the invention create a unique environment capable of combating pathogenic bacteria.

Test 3

On average, the total bacterial count was 2.59X10 ⁷ CFU/g RDF, while the pathogenicity index bacteria count is: enterobacteriaceae count is 4.88×10 ² CFU/g RDF, and E.coli count was 0CFU/g RDF. Lactic acid bacteria count was 3.37X10 ⁶ CFU/g RDF and thus account for about 1/7 of the total viable flora in the classified RDF of the invention (FIG. 3). The amount of pathogenicity index flora grouped by enterobacteriaceae and escherichia coli is surprisingly low in the classification RDF of the invention.

Test 4

On average, the total bacterial count was 7.46×10 ⁷ CFU/g RDF, while the pathogenicity index bacteria count is: enterobacteriaceae count is 3.48×10 ⁵ CFU/g RDF, accounting for about 1/138 of the whole viable flora, and E.coli count was 2.94X10 ⁴ CFU/g RDF, represents about 1/1500 of the total viable population. Lactic acid bacteria count was 1.31X10 ⁷ CFU/g RDF and thus account for about 1/7 of the total viable flora in MBT-classified RDF (FIG. 3).

Comparison of bacterial counts in MBT Classification RDF and RDF obtained from the treatment methods of the invention

The bacterial count of MBT-classified RDF (test 3) was compared with the bacterial count of classified RDF obtained from the treatment method of the present invention (test 4). The total amount of bacteria in MBT-classified RDF was > 2-fold or more compared to classified RDF obtained from the treatment method of the invention (fig. 3). In contrast, the enterobacteriaceae count in MBT-classified RDF was > 700-fold or more than that obtained from the treatment method of the present invention, and the escherichia coli count in MBT-classified RDF was > 29000-fold or more than that obtained from the treatment method of the present invention (fig. 3). Finally, the lactobacillus count in MBT-classified RDF is > 3-fold or more compared to classified RDF obtained from the treatment method of the invention.

These findings indicate two points: 1) The growth conditions of bacteria in MBT class RDF are better than those obtained from the treatment method of the invention, including pathogenic index bacteria (enterobacteriaceae and escherichia coli); 2) The conditions in the bioreactor using the method according to the invention create a unique environment capable of combating pathogenic bacteria.

Example 2-pH and temperature test

In order to establish specific limits (ranges) in terms of pH and temperature for destroying pathogenic bacteria in the treatment method according to the invention, experiments were carried out using model waste (model MSW).

The model MSW is used to simulate the MSW, as described below.

To simulate the composition of real municipal solid waste, a "model MSW" may be prepared. The composition of the model MSW consisting of 3 parts is described below:

41% vegetable portions (see Table 2)

13% protein/fat fraction (animal origin) (see Table 3)

-46% cellulose fraction (see table 4).

Table 2: vegetable portions in model MSW

Table 3: protein/fat fraction of model MSW (animal origin)

Table 4: cellulose part of model MSW

Composition of model MSW	% cellulose fraction (wt%)
		Milk packing box	30.0
Newspaper and its production process	8.0
		Magazine	2.8
Advertising material	9.7
		Telephone directory	0.7
Printing paper	2.2
		Gift package	6.2
Paperboard	9.8
		Paper towel	22.5
Cotton pad	1.7
		Wood	1.2
Textile (napkin)	5.3

The fermentation was performed under the conditions listed in table 5.

Table 5.

In Sartorius equipped with mechanical stirrer, heating jacket, cooling tower for exhaust gas and pH meter ^TM Fermentation was performed in 1L. Using electrically-heated or cooled jackets to change temperature(see Table 5) and stirred at 600rpm. By Sartorius ^TM The automatic pump system adds 1M HCl or 1M NaOH to adjust the pH to the appropriate value. The added ingredients (solids and liquids) were not preheated prior to addition to the fermentor.

166 grams of model MSW, 1L deionized water andCTec3 (Novozymes A/S) performed fermentation work of model MSW. The water and model MSW were first heated or cooled to the appropriate temperature (see Table 5) while stirring (300 rpm). At the same time, the pH was adjusted to an appropriate state by adding HCl or NaOH (see Table 5). After reaching the desired temperature and pH value, cellic +.>(4g) And stirring was increased to 600rpm. Then 1.3ml of E.coli (DSM 498 strain) were added at a concentration of about 8X 10 ⁸ CFU/ml。

Coli was grown overnight in nutrient broth and added to the fermenter after shaking overnight at 37 ℃. Furthermore, the E.coli overnight culture was centrifuged at 5000rpm for 5 minutes and the pellet was resuspended in 0.9% NaCl H ₂ O to OD ₆₀₀ ＝1。

Sample collection and analysis

About 10ml of sample was withdrawn from the fermenter and sterile 0.9% NaCl H was used ₂ O serially dilutes the produced biological fluid to 10 ^-8 . 1ml of each dilution was coated on petrifilm sheets. The bacterial count of the indicator bacteria, i.e. of the enterobacteriaceae and of the escherichia coli, was measured according to assays III and IV.

The counting of indicator bacteria is to verify the specific environment for the growth capacity of pathogenic bacteria. Enterobacteriaceae (such as escherichia coli) live in the intestinal tract of mammals as symbiotic bacteria and have the ability to become pathogenic bacteria.

Coli has been considered an excellent indicator bacteria for decades. If these organisms are found in the environment, this may indicate that generally pathogenic bacteria are able to grow in that particular environment.

TABLE 6 Escherichia coli counts measured at various pH and temperature

The experimental data obtained were analyzed in Design-Expert software version 11 (Stat-Ease, inc.). To model the results obtained, the data were transformed as follows:

1. calculating the ratio of CFU counts after 24 hours and at the start of the experiment;

2. if the ratio from point 1 is equal to 0, then 10 is used ^-4 Replacing the ratios so as to apply logarithmic conversion to all ratios;

3. Calculating the negative logarithm of the ratio, base 10;

4. to make the data more normally distributed, a power conversion is applied to the value at point 3, the power being 0.59;

5. the final model includes pH, temperature (T) and square of temperature (T ² ) An item. The model proved to be significant (p<0.0001)，pH(p<0.0001 And T) ² The term (p=0.0341) is also significant. The T term (p=0.0635) is because of T ² Items are listed. The lack of fitness was not significant (p= 0.6876). R for model ² Is 0.84, predicted R ² Is 0.695.

FIG. 4 depicts the predicted limits for the growth of non-pathogenic bacteria in terms of pH and temperature when running the method according to the invention. These lines represent a relative decrease of 10log of the E.coli at that temperature and pH, i.e., line 2 equals 10log2 = 100 relative decrease of E.coli CFU, line 5 equals 10log5 = 100.000 relative decrease of E.coli CFU, and so on. Thus, pathogenic E.coli is reduced by 10log5 to the left of line 5. The dots represent the experiments performed.

Example 3: model waste liquefaction on a laboratory scale, without prior sanitation

Using 166 grams of model MSW (formulated as described in example 2), 1L deionized water, and 2, 4, 6, or 8 gramsCtec3 (available from Novozymes A/S) was subjected to a series of separate fermentations in which a fixed amount of inoculum (166 g) from the CSTR digester mentioned earlier, from the Foulum biogas plant of Denmark was added. First, water, inoculum (166 g) and model MSW were mixed in 5L Sartorius ^TM The fermenter was heated to 50℃while stirring (300 rpm). After reaching the desired temperature, the enzyme (cellular CTec3 is added ^TM ) (2, 4, 6 or 8 g) and stirring was increased to 1200rpm for 5 minutes and thereafter to 900rpm for 1 hour. After stirring for 1 hour, the stirring was reduced to 600rpm until the end of the experiment. After enzyme addition by HPLC, the concentrations of glucose, xylose, lactic acid, acetic acid and ethanol were measured at time points 18.25, 25.50, 42.50, 47.50 and 114 h.

Table 7 data obtained using 2g of cellular Ctec3 (all units are g/L)

Table 8 data obtained using 4 g of cellular Ctec3 (all units are g/L)

Table 9 data obtained using 6 g of cellular Ctec3 (all units are g/L)

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Table 10 uses 8g of Cellic Ctec3 data (all units are g/L)

In all four experiments, model MSW was solubilized and glucose was released. In experiments with low enzyme load, methanogenic bacteria from the inoculum resulted in a large amount of ethanol production. Thus, less glucose is available to lactic acid bacteria, the acidification rate is slower, and the time at pH values above 5 is about 40 hours. The acidification rate was progressively faster as the enzyme dosage increased, and the pH dropped below 4.5 in 24 hours at the high enzyme dosage (8 g). This also effectively limits the production of ethanol and acetic acid to 0.3 and 1.2g/L, respectively. These experiments show that if the lactic acid producing community inherent in the waste is limited, the sanitation of the waste water may be beneficial, whereas if a sufficiently large lactic acid community is present in the waste, the sanitation of the waste water is not necessary, as lactic acid bacteria are able to outperform the methanogens inherent in the waste water.

Claims

1. A method of sanitizing waste, the method comprising:

2. The method of claim 1, further comprising:

3. A method according to any one of the preceding claims, wherein the dry matter content of the waste is in the range 50-70% wt.

4. A method according to any one of the preceding claims, wherein the waste in step a) is subjected to the action of water to reduce the dry matter content of the waste.

5. A method according to any one of the preceding claims, wherein the waste is subjected to enzymatic and/or microbiological processes in the bioreactor for a period of about 20-25 hours, preferably about 18 hours.

6. A process according to any one of the preceding claims, wherein the pH in step a) is below 6.0, preferably below 5.0, more preferably below 4.5, even more preferably below 4.4 and most preferably below 4.2.

7. The method according to any of the preceding claims, wherein the temperature in step a) is 55 ℃ or less, 50 ℃ or less or 45 ℃ or less.

8. A method according to any one of the preceding claims, wherein the waste is subjected to enzymatic and/or microbiological processes in the bioreactor in step a) at a pH of 24 hours and 4-6, and at a temperature in the range of 40-60 ℃.

9. A biological fluid obtainable by the method according to claims 1-8.

10. The biological fluid of claim 9, wherein the bacterial count of enterobacteriaceae in the biological fluid is less than 1 x 10 as measured by Assay III ² -1×10 ⁴ CFU/ml, preferably below 1X 10 ² CFU/ml, and/or wherein the bacterial count of e.coli in the biological fluid is below 20-100CFU/ml, preferably below 20CFU/ml, more preferably below 10CFU/ml measured as Assay IV.

11. Biological fluid according to any of claims 9-10, wherein the bacterial count of lactic acid bacteria in the biological fluid is at least 1 x 10 as measured by Assay II ⁵ CFU/ml, preferably at least 1X 10 ⁶ CFU/ml。

12. A non-biodegradable waste material obtainable by the method according to claims 1-8.

13. The non-biodegradable waste material of claim 12, wherein the enterobacteriaceae bacteria count in the material is less than 1 x 10 as measured by Assay IV ² -1×10 ⁴ CFU/ml, preferably below 1X 10 ² CFU/ml。

14. The non-biodegradable waste material according to any one of claims 12-13, wherein the bacterial count of e.coli in the material is lower than 20-100CFU/ml, preferably lower than 20CFU/ml, more preferably lower than 10CFU/ml measured as Assay II.

15. The non-biodegradable waste material according to any one of claims 12-14, wherein the lactic acid bacteria in the material have a bacterial count of at least 1 x 10 as measured by Assay III ⁵ CFU/ml, preferably at least 1X 10 ⁶ CFU/ml。