WO2013000945A1 - Biogas from enzyme-treated bagasse - Google Patents
Biogas from enzyme-treated bagasse Download PDFInfo
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- WO2013000945A1 WO2013000945A1 PCT/EP2012/062430 EP2012062430W WO2013000945A1 WO 2013000945 A1 WO2013000945 A1 WO 2013000945A1 EP 2012062430 W EP2012062430 W EP 2012062430W WO 2013000945 A1 WO2013000945 A1 WO 2013000945A1
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- enzyme
- bagasse
- biogas
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/023—Methane
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Definitions
- the present invention relates to a process for treatment of a bagasse-derived material comprising lignocellulosic fibres in which the treatment increases the degradability of the lignocellulosic fibres.
- the invention relates to methane production from bagasse, where the enzyme-treatment of the invention is used to increase the methane production in comparison with untreated bagasse.
- Bagasse is the fibrous matter that remains after sugarcane or sorghum stalks are crushed to extract their juice. It is currently used as a biofuel and as a renewable resource in the manufacture of pulp and paper products and building materials. For each 10 tonnes of sugarcane crushed, a sugar factory produces nearly 3 tonnes of wet bagasse. Since bagasse is a by-product of the cane sugar industry, the quantity of production in each country is in line with the quantity of sugarcane produced. The moisture content of bagasse is typically 40 to 50% and the solid content is made up of about 45-50% cellulose, 20-25% hemicellulose, 18-24% lignin and 1 -4% ash on a washed and dried basis.
- the invention relates to a biogas production process based on bagasse-derived material, wherein said process comprises at least one enzymatic pre-treatment of the material prior to the anaerobic biogas-producing fermentation in the digester tank, or at least one enzymatic treatment step in the biogas digester tank either prior to or during the anaerobic fermentation.
- the invention relates to a biogas production process comprising the steps of providing a slurry comprising a bagasse-derived material and water, and:
- biogas is according to the invention intended to mean the gas obtained in a conventional anaerobic fermentor.
- the main component of biogas is methane and the terms "biogas” and “methane” are in this application and claims used interchangeably.
- primary digester is in this application and claims intended to mean the container wherein the first anaerobic fermentation takes place.
- second digester is in this application and claims intended to mean the container wherein the second anaerobic fermentation takes place.
- the primary digester may also serve as the secondary digester.
- Bagasse-derived material
- bagasse-derived material is in this application and claims intended to mean any material comprising bagasse or material derived therefrom, in any form, amount or ratio.
- the bagasse-derived material may comprise other plant derived components.
- the solids part of bagasse is typically made up of about 45-50% cellulose, 20-25% hemicellulose, 18-24% lignin and 1 -4% ash.
- Typical plant derived components are starch, glucans, arabans, galactans, pectins, mannans, galactomannans and hemicelluloses such as xylans.
- the bagasse-derived material may be any treated or untreated material derived from bagasse as well as any composition comprising such material. Pre-treatment:
- pre-treatment is intended to include any suitable treatment of the material prior to the actual biogas producing step.
- the bagasse-derived material which may simply be bagasse from sugar-cane, may be pre-treated in any suitable way.
- the pre-treatment is carried out before or at the same time as the enzymatic hydrolysis.
- the purpose of the pre- treatment is to reduce the particle size, separate and/or release cellulose; hemicellulose and/or lignin and in this way increase the rate of hydrolysis.
- Pre-treatment processes such as wet- oxidation and alkaline pre-treatment targets lignin, while dilute acid and auto-hydrolysis targets hemicellulose. Steam explosion is an example of a pre-treatment that targets lignin.
- the pre-treatment step may be a conventional pre-treatment step using techniques well known in the art, such as, milling or wet milling.
- pre-treatment takes place in a slurry of bagasse-derived material and water.
- the bagasse-derived material may during pre-treatment be present in an amount between 10-80 wt.%, preferably between 20-70 wt.-%, especially between 30-60 wt.-%, such as around 50 wt-%.
- the bagasse-derived material may according to the invention be chemically, mechanically and/or biologically pre-treated before hydrolysis in accordance with the process of the invention.
- Mechanical pre-treatment (often referred to as "physical"- pre-treatment) may be carried out alone or may be combined with other pre-treatment processes.
- the chemical, mechanical and/or biological pre-treatment is carried out prior to the hydrolysis.
- the chemical, mechanical and/or biological pre-treatment may be carried out simultaneously with hydrolysis, such as simultaneously with addition of one or more hydrolyzing enzymes, and/or other enzyme activities, to release fermentable sugars, such as glucose and/or maltose.
- chemical pre-treatment refers to any chemical pre-treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin.
- suitable chemical pre-treatments include treatment with; for example, dilute acid, lime, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide.
- wet oxidation and pH-controlled hydrothermolysis are also considered chemical pre-treatment.
- Other pre-treatment techniques are also contemplated according to the invention. Cellulose solvent treatment has been shown to convert about 90% of cellulose to glucose. It has also been shown that enzymatic hydrolysis could be greatly enhanced when the lignocellulose structure is disrupted.
- Alkaline H 2 0 2 , ozone, organosolv uses Lewis acids, FeCI 3 , AI 2 (S0 4 )3 in aqueous alcohols), glycerol, dioxane, phenol, or ethylene glycol are among solvents known to disrupt cellulose structure and promote hydrolysis (Mosier et al. Bioresource Technology 96 (2005), p. 673-686).
- Alkaline chemical pre-treatment with base e.g., NaOH, Na 2 C0 3 , NaHC0 3 , Ca(OH) 2 , lime hydrate, ammonia and/or KOH or the like
- base e.g., NaOH, Na 2 C0 3 , NaHC0 3 , Ca(OH) 2 , lime hydrate, ammonia and/or KOH or the like
- Pre- treatment processes using ammonia are described in, e.g., WO 2006/1 10891 , WO 2006/1 1899, WO 2006/1 1900, WO 2006/1 10901 , which are hereby incorporated by reference.
- the Kraft pulping process as described for example in "Pulp Processes" by Sven A. Rydholm, page 583- 648. ISBN 0-89874-856-9 (1985) might be used.
- the solid pulp (about 50% by weight based on the dry wood chips) is collected and washed before the enzymatic treatments.
- oxidizing agents such as: sulphite based oxidizing agents or the like.
- solvent pre-treatments include treatment with DMSO (Dimethyl Sulfoxide) or the like.
- Chemical pre-treatment is generally carried out for 1 to 60 minutes, such as from 5 to 30 minutes, but may be carried out for shorter or longer periods of time dependent on the material to be pre-treated.
- mechanical pre-treatment refers to any mechanical (or physical) pre- treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin from bagasse-derived material.
- mechanical pre-treatment includes various types of milling, irradiation, steaming/steam explosion, and hydrothermolysis.
- Mechanical pre-treatment includes comminution (mechanical reduction of the size).
- Comminution includes dry milling, wet milling and vibratory ball milling.
- Mechanical pre- treatment may involve high pressure and/or high temperature (steam explosion).
- high pressure means pressure in the range from 300 to 600 psi, preferably 400 to 500 psi, such as around 450 psi.
- high temperature means temperatures in the range from about 100 to 300°C, preferably from about 140 to 235°C.
- mechanical pre-treatment is carried out as a batch- process, in a steam gun hydrolyzer system which uses high pressure and high temperature as defined above.
- a Sunds Hydrolyzer available from Sunds Defibrator AB (Sweden) may be used for this.
- the bagasse-derived material is subjected to a irradiation pre- treatment.
- irradiation pre-treatment refers to any pre-treatment by microwave e.g. as described by Zhu et al. "Production of ethanol from microwave-assisted alkali pre-treated wheat straw” in Process Biochemistry 41 (2006) 869-873 or u ltrasonic pre-treatment, e.g. , as described by e.g. Li et al. "A kinetic study on enzymatic hydrolysis of a variety of pulps for its enhancement with continuous ultrasonic irradiation", in Biochemical Engineering Journal 19 (2004) 155-164.
- the bagasse-derived material prior to step (a) or (b) has been subjected to a microwave and/or an ultrasonic irradiation treatment.
- the bagasse-derived material or the slurry is homogenized; preferably by milling, wet-milling, grinding or wet-grinding prior to or during step (a) or prior to step (b).
- the bagasse-derived material is subjected to both chemical and mechanical pre-treatment.
- the pre-treatment step may involve dilute or mild acid treatment and high temperature and/or pressure treatment.
- the chemical and mechanical pre-treatments may be carried out sequentially or simultaneously, as desired.
- pre-treatment is carried out as a dilute and/or mild acid steam explosion step.
- pre-treatment is carried out as an ammonia fiber explosion step (or AFEX pre-treatment step).
- a base is added to the bagasse-derived material or the slurry prior to or while it is being homogenized; preferably the base is NaOH, Na 2 C0 3 , NaHC0 3 , Ca(OH) 2 , lime hydrate, ammonia and/or KOH.
- biological pre-treatment refers to any biological pre-treatment which promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the bagasse- derived material.
- Known biological pre-treatment techniques involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh, P., and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv. Appl. Microbiol.
- the bagasse-derived material has been chemically, mechanical and/or biologically treated prior to step (a) or (b).
- the invention relates to a biogas production process comprising the steps of providing a slurry comprising a bagasse-derived material and water, and:
- bagasse-derived material Before or while bagasse-derived material is fermented it is hydrolyzed enzymatically to break down especially hemicellulose and/or cellulose into fermentable sugars.
- polysaccharides like starch, hemicelluloses, mannan and cellulose are solubilised and converted mainly to oligosaccharides, any protein is hydrolysed mainly to peptides and cellulose is converted to cellodextrins.This may be achieved in an enzymatic pre-treatment.
- a milling of the biomass may be done, preferably a wet grinding, optionally facilitated by addition of the enzymes according to the invention. Temperature and pH is adjusted to allow the enzymes to function.
- the bagasse-derived material to be hydrolyzed typically constitutes above 2.5% wt-% DS (dry solids), preferably above 5 %wt-% DS, preferably above 10 %wt-% DS, preferably above 15 wt-% DS, preferably above 20 wt.-% DS, more preferably above 25 wt-% DS of a slurry.
- the content of bagasse-derived material in the slurry is adjusted by continuous or stepwise addition of material to the slurry during step (a) or (b).
- a solids separation step is performed in step (a) after the bagasse-derived material is degraded but before it is added to the digester tank, to purge not- solubilized solids and optionally feed them back into step (a) of the process.
- the bagasse-derived material may further be subjected to the action of one, or several or all enzyme activities selected from the group consisting of an amylolytic enzyme, a lipolytic enzyme, a proteolytic enzyme, a cellulolytic enzyme, an oxidoreductase and a plant cell-wall degrading enzyme.
- the one or more enzyme is selected from the group consisting of aminopeptidase, alpha-amylase, amyloglucosidase, arabinofuranosidase, arabinoxylanase, beta-g l u ca n a se , ca rbo h yd ra s e , ca rboxy pe pti d a s e , cata l a s e , cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, ferulic acid esterase, deoxyribonuclease, endo-cellulase, endo-glucanase, endo-xylanase, esterase, galactosidase, beta-galactosidase, glucoamylase, glucose oxidase, glucosi
- the one or more enzyme is a protease, a pectate lyase, a ferulic acid esterase and/or a mannanase.
- enzymatically liquefied material is fed to a biogas digester tank in a rate and ratio that fits with the conversion rate to gas.
- pH is kept at same pH as in the digester tank.
- the pre-treated biomass material should preferably have a neutral to basic pH value when it is added to the biogas digester, it is thought that addition of acidic biomass may halt the biogas conversion process due to inhibition of the common methanogenic microorganisms.
- the pH is between 7 and 10, such as from 7.6 to 10; preferably from 8 to 10, or from 8 to 9, preferably around pH 8.5.
- the pH may be adjusted using NaOH, Na 2 C0 3 , NaHC0 3 , Ca(OH) 2 , lime hydrate, ammonia and/or KOH.
- the temperature may be between 20-70°C, preferably 30-60°C, and more preferably 40-55°C, e.g., around 50°C.
- I n a hydrolysis step, cell walls are degraded and the cellulose fibrils are made accessible for further hydrolysis.
- the hydrolysis step may be carried out as a fed batch process where pre-treated bagasse-derived material is fed continuously/gradually or stepwise into a solution containing hydrolyzing enzymes.
- a pectate lyase, a ferulic acid esterase, and a mannanase is present in a second hydrolysis step in the pre-treatment.
- a pectate lyase, a ferulic acid esterase, mannanase and a cellulase is present.
- a pectate lyase, a ferulic acid esterase, mannanase, a cellulase and a protease is present.
- cellulose fibrils may be isolated and treated with an alkaline endo-glucanase composition under neutral to basic pH conditions.
- the dry solids (DS) is preferably above 10 wt.-% DS, preferably above 15 wt-% DS, preferably above 20 wt.-% DS, more preferably above 25 wt-% DS.
- the pH should be between 7 and 10, such as from 8 to 9, preferably around pH 8.5.
- the pH may be adjusted using NaOH, Na 2 C0 3 , NaHC0 3 , Ca(OH) 2 , lime hydrate, ammonia and/or KOH.
- the temperature may be between in range from 20-70°C, preferably 30-60°C, and more preferably 40-50°C.
- the cellulose fibrils may be treated with a cellulase composition comprising cellulolytic activity under neutral to acid pH conditions.
- the pH is between 4-7, preferably 5-7, such as around 5.5.
- the pH is preferably adjusted using phosphoric acid, succinic acid , hydrochloric acid and/or sulphuric acid.
- a temperature in the range of 20-70°C, preferably 30-60°C, and more preferably 40-50°C
- protease suitable for use under alkaline conditions can be used.
- Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically or genetically modified mutants are included.
- the protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease.
- alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
- trypsin- like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.
- Preferred commercially available protease enzymes include those sold under the trade names EverlaseTM, Kan naseTM, AlcalaseTM, SavinaseTM, PrimaseTM, Du razymTM, and EsperaseTM by Novozymes A/S (Denmark), those sold under the tradename Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the tradename Opticlean and Optimase by Solvay Enzymes. Hemicellulolvtic enzymes
- hemicellulase suitable for use in hydrolyzing hemicellulose may be used.
- Preferred hemicellulases include pectate lyases, xylanases, arabinofuranosidases, acetyl xylan esterase, ferulic acid esterase, glucuronidases, endo-galactanase, mannases, endo or exo arabinases, exo-galactanses, and mixtures of two or more thereof.
- the hemicellulase for use in the present invention is an endo-acting hemicellulase, and more preferably, the hemicellulase is an endo-acting hemicellulase which has the ability to hydrolyze hemicellulose under basic conditions of above pH 7, preferably pH 7-10.
- the hemicellulase is a xylanase.
- the xylanase may preferably be of microbial origin, such as of fungal origin (e.g., Trichoderma, Meripilus, Humicola, Aspergillus, Fusarium) or from a bacterium (e.g., Bacillus).
- the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeatus; or a strain of Humicola, preferably Humicola lanuginosa.
- the xylanase may preferably be an endo-1 ,4-beta-xylanase, more preferably an endo-1 ,4-beta-xylanase of GH10 or GH1 1 .
- Examples of commercial xylanases include SHEARZYME® 200L, SHEARZYME® 500L, BIOFEED WHEAT®, and PULPZYMETM HC (from Novozymes) and GC 880, SPEZYME® CP (from Genencor Int).
- the hemicellulase may be added in an amount effective to hydrolyze hemicellulose, such as, in amounts from about 0.001 to 0.5 wt.-% of total solids (TS), more preferably from about 0.05 to 0.5 wt.-% of TS.
- TS total solids
- Xylanases may be added in the amounts of 1 .0-1000 FXU/kg dry solids, preferably from 5-500 FXU/kg dry solids, preferably from 5-100 FXU/kg dry solids and most preferably from 10- 100 FXU/kg dry solids.
- Xylanases may alternatively be added in amounts of 0.001 -1.0 g/kg DS substrate, preferably in the amounts of 0.005-0.5 g/kg DS substrate, and most preferably from 0.05-0.10 g/kg DS substrate.
- Pectolytic enzymes or Pectinases
- pectinolytic enzyme that can degrade the pectin composition of plant cell walls may be used in practicing the present invention.
- Suitable pectinases include, without limitation, those of fungal or bacterial origin. Chemically or genetically modified pectinases are also encompassed.
- the pectinase used in the invention are recombinantly produced and are mono-component enzymes.
- Pectinases can be classified according to their preferential substrate, highly methyl- esterified pectin or low methyl-esterified pectin and polygalacturonic acid (pectate), and their reaction mechanism, beta-elimination or hydrolysis. Pectinases can be mainly endo-acting, cutting the polymer at random sites within the chain to give a mixture of oligomers, or they may be exo-acting, attacking from one end of the polymer and producing monomers or dimers.
- pectinase activities acting on the smooth regions of pectin are included in the classification of enzymes provided by Enzyme Nomenclature (1992), e.g., pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1 .15), exo-polygalacturonase (EC 3.2.1 .67), exo-polygalacturonate lyase (EC 4.2.2.9) and exo-poly-alpha-galacturonosidase (EC 3.2.1.82).
- pectate lyase EC 4.2.2.2
- pectin lyase EC 4.2.2.10
- polygalacturonase EC 3.2.1 .15
- exo-polygalacturonase EC 3.2.1 .67
- exo-polygalacturonate lyase EC 4.2.2.9
- the pectinase is a pectate lyase.
- Pectate lyase enzymatic activity refers to catalysis of the random cleavage of alpha-1 ,4-glycosidic linkages in pectic acid (also called polygalcturonic acid) by transelimination.
- Pectate lyases are also termed polygalacturonate lyases and poly(1 ,4-a-D-galacturonide) lyases.
- the Pectate lyase (EC 4.2.2.2) is an enzyme which catalyse the random cleavage of o
- pectic acid also called polygalacturonic acid
- Pectate lyases also include polygalacturonate lyases and poly(1 ,4-a-D-galacturonide) lyases.
- pectate lyases examples include those that have been cloned from different bacterial genera such as Erwinia, Pseudomonas, Klebsiella, Xanthomonas and Bacillus, especially Bacillus licheniformis (US patent application 6,124,127), as well as from Bacillus subtilis (Nasser et al. (1993) FEBS Letts. 335:319-326) and Bacillus sp. YA-14 (Kim et al.
- a preferred pectate lyase may be obtained from Bacillus licheniformis as described in
- pectate lyases could be those that comprise the amino acid sequence of a pectate lyase disclosed in Heffron et al., (1995) Mol. Plant-Microbe Interact. 8: 331 -334 and Henrissat et al., (1995) Plant Physiol. 107: 963-976.
- a single enzyme or a combination of pectate lyases may be used .
- a preferred commercial pectate lyase preparation suitable for the invention is BioPrep® 3000 L available from Novozymes A S.
- a mannanase is a beta- mannanase and defined as an enzyme belonging to EC 3.2.1 .78.
- Mannanases have been identified in several Bacillus organisms. For example, Talbot et al., Appl. Environ. Microbiol., Vol.56, No. 1 1 , pp. 3505-3510 (1990) describes a beta- mannanase derived from Bacillus stearothermophilus having an optimum pH of 5.5-7.5. Mendoza et al., World J. Microbiol. Biotech., Vol. 10, No. 5, pp.
- JP-03047076 discloses a beta-mannanase derived from Bacillus sp., having an optimum pH of 8-10.
- JP-63056289 describes the production of an alkaline, thermostable beta-mannanase.
- JP- 08051975 discloses alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001 .
- a purified mannanase from Bacillus amyloliquefaciens is disclosed in WO 97/1 1 164.
- 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.
- the mannanase may be derived from a strain of the genus Bacillus, such as the amino acid sequence having the sequence deposited as GENESEQP accession number AAY54122 or an amino acid sequence which is homologous to this amino acid sequence.
- a su itable commercial mannanase preparation is Mannaway® produced by Novozymes A/S.
- a ferulic esterase is defined as an enzyme belonging to EC 3.1.1.73.
- a suitable ferulic esterase preparation can be obtained from Malabrancea, e.g., from P. cinnamomea, such as e.g. a preparation comprising the ferulic esterase having the amino acid sequence shown in SEQ ID NO:2 in European patent application number 07121322.7, or an amino acid sequence which is homologous to this amino acid sequence.
- Another suitable ferulic esterase preparation can be obtained from Penicillium, e.g., from P. aurantiogriseum, such as e.g. a preparation comprising the ferulic esterase having the amino acid sequence shown in SEQ ID NO:2 in European patent application number 0815469.7, or an amino acid sequence which is homologous to this amino acid sequence.
- a suitable commercial ferulic esterase preparation preparation is NOVOZYM ® 342 L produced by Novozymes A/S. Alkaline endo-glucanases
- endoglucanases may be derived from a strain of the genus Trichoderma, preferably a strain of Trichoderma reesei; a strain of the genus Humicola, such as a stra i n of Humicola insolens; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense.
- the alkaline endo-glucanase composition is one of the commercially available products CAREZYM E®, EN DOLASE® and CELLU CLEAN® (Novozymes A/S, Denmark).
- the enzyme may be applied in a dosage of 1 -100 g/kg cellulose.
- acid cellulolytic activity as used herein are understood as comprising enzymes having cellobiohydrolase activity (EC 3.2.1 .91 ), e.g. , cellobiohydrolase I and/or cellobiohydrolase I I , as well as endo-glucanase activity (EC 3.2.1 .4) and/or beta-glucosidase activity (EC 3.2.1.21 ) having activity at pH below 6.
- the cellulolytic activity may, in a preferred embodiment, be in the form of a preparation of enzymes of fungal origin, such as from a strain of the genus Trichoderma, preferably a strain of Trichoderma reesei; a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense.
- a strain of the genus Trichoderma preferably a strain of Trichoderma reesei
- a strain of the genus Humicola such as a strain of Humicola insolens
- a strain of Chrysosporium preferably a strain of Chrysosporium lucknowense.
- the cellulolytic enzyme preparation contains one or more of the following activities: endoglucanase, cellobiohydrolases I and II, and beta-glucosidase activity.
- cellulolytic enzyme preparation is a composition disclosed in WO2008/151079, which is hereby incorporated by reference.
- the cellulolytic enzyme preparation comprising a polypeptide having cellulolytic enhancing activity, preferably a family GH61 A polypeptide, preferably those disclosed in WO 2005/074656 (Novozymes).
- the cellulolytic enzyme preparation may further comprise beta-glucosidase, such as beta-glucosidase derived from a strain of the genus Trichoderma, Aspergillus or Penicillium, including the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637 (Novozymes).
- the cellulolytic enzyme preparation may also comprises a CBH II enzyme, preferably Thielavia terrestris cellobiohydrolase I I (CEL6A).
- the cellulolytic enzyme preparation may also comprise cellulolytic enzymes; preferably those derived from Trichoderma reesei or Humicola insolens.
- the cellulolytic enzyme composition may also comprise a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; an Aspergillus oryzae beta- glucosidase fusion protein (WO 2008/057637), and cellulolytic enzymes derived from Trichoderma reesei.
- G61A cellulolytic enhancing activity
- WO 2005/074656 an Aspergillus oryzae beta- glucosidase fusion protein
- cellulolytic enzymes derived from Trichoderma reesei may also comprise a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; an Aspergillus oryzae beta- glucosidase fusion protein (WO 2008/057637), and cellulolytic enzymes derived from Trichoderma reesei.
- the cellulolytic enzyme composition may also comprise an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656).
- the cellulolytic enzyme composition may also comprise an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and/or a Trichoderma reesei cellulase preparation.
- the cellulolytic composition comprising a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; an Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637), Thielavia terrestris cellobiohydrolase II (CEL6A), and cellulolytic enzymes preparation derived from Trichoderma reesei.
- G61A cellulolytic enhancing activity
- WO 2008/057637 an Aspergillus oryzae beta-glucosidase fusion protein
- CEL6A Thielavia terrestris cellobiohydrolase II
- cellulolytic enzymes preparation preparation derived from Trichoderma reesei.
- the cellulolytic enzyme composition is the commercially available product CELLUCLASTTM 1 .5L, CELLUZYMETM, CellicTM CTec, CellicTM CTec2, CellicTM HTec, CellicTM HTec2 (all Novozymes A/S, Denmark) or ACCELLARASETM 1000 (Genencor Int, Inc., USA).
- the cellulolytic activity may be dosed in the range from 0.1 -100 FPU per gram total solids (TS), preferably 0.5-50 FPU per gram TS, especially 1 -20 FPU per gram TS.
- TS FPU per gram total solids
- a rolled filter paper strip (#1 Whatman; 1 X 6 cm; 50 mg) is added to the bottom of a test tube (13 X 100 mm).
- Enzyme dilutions are designed to produce values slightly above and below the target value of 2.0 mg glucose.
- a reagent blank is prepared by adding 1.5 mL of citrate buffer to a test tube.
- a substrate control is prepared by placing a rolled filter paper strip into the bottom of a test tube, and adding 1 .5 mL of citrate buffer.
- Enzyme controls are prepared for each enzyme dilution by mixing 1 .0 mL of citrate buffer with 0.5 mL of the appropriate enzyme dilution.
- a 100 mL stock solution of glucose (10.0 mg/mL) is prepared, and 5 mL aliquots are frozen. Prior to use, aliquots are thawed and vortexed to mix.
- Glucose standard tubes are prepared by adding 0.5 mL of each dilution to 1 .0 mL of citrate buffer.
- the glucose standard tubes are assayed in the same manner as the enzyme assay tubes, and done along with them.
- a glucose standard curve is prepared by graphing glucose concentration (mg/0.5 mL) for the four standards (G1 -G4) vs. A540. This is fitted using a linear regression (Prism Software), and the equation for the line is used to determine the glucose produced for each of the enzyme assay tubes.
- a line is drawn between the enzyme dilution that produced just above 2.0 mg glucose and the dilution that produced just below that. From this line, it is determined the enzyme dilution that would have produced exactly 2.0 mg of glucose.
- cellulolytic enhancing activity is defined herein as a biological activity that enhances the hydrolysis of a bagasse-derived material by proteins having cellulolytic activity.
- cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or in the increase of the total of cellobiose and glucose from the hydrolysis of a bagasse-derived material, e.g., pre-treated bagasse-derived material by cellulolytic protein under the following conditions: 1 -50 mg of total protein/g of cellulose in PCS (pre-treated corn stover), wherein total protein is comprised of 80-99.5% w/w cellulolytic protein/g of cellulose in PCS and 0.5-20% w/w protein of cellulolytic enhancing activity for 1 -7 day at 50°C compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1 -50 mg of cellulolytic protein/g of cellulose in PCS).
- the polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a bagasse-derived material catalyzed by proteins having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 0.1 -fold, more at least 0.2-fold, more preferably at least 0.3-fold, more preferably at least 0.4-fold, more preferably at least 0.5-fold, more preferably at least 1 -fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 10-fold, more preferably at least 20-fold, even more preferably at least 30-fold, most preferably at least 50-fold, and even most preferably at least 100-fold.
- the hydrolysis and/or fermentation is carried out in the presence of a cellulolytic enzyme in combination with a polypeptide having cellulolytic enhancing activity.
- the polypeptide having cellulolytic enhancing activity is a family GH61A polypeptide.
- WO 2005/074647 discloses isolated polypeptides having cellulolytic enhancing activity and polynucleotides thereof from Thielavia terrestris.
- WO 2005/074656 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Thermoascus aurantiacus.
- U.S. Published Application Serial No. 2007/0077630 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Trichoderma reesei.
- any alpha-amylase may be used, such as of fungal, bacterial or plant origin.
- the alpha-amylase is an acid alpha-amylase, e.g., acid fungal alpha-amylase or acid bacterial alpha-amylase.
- the term "acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1 .1 ) which added in an effective amount has activity optimum at a pH in the range of 3 to 7, preferably from 3.5 to 6, or more preferably from 4-5.
- bacterial alpha-amylase is preferably derived from the genus Bacillus.
- Bacillus alpha-amylase is derived from a strain of Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus stearothermophilus, but may also be derived from other Bacillus sp.
- contemplated alpha-amylases include the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase SEQ I D NO: 5 in WO 99/19467 and the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 (all sequences hereby incorporated by reference).
- the alpha-amylase may be an enzyme having a degree of identity of at least 60%, preferably at least 70%, more preferred at least 80%, even more preferred at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the seq uences shown i n S EQ I D NOS: 1 , 2 or 3 , respectively, in WO 99/19467.
- the Bacillus alpha-amylase may also be a variant and/or hybrid, especially one described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby incorporated by reference).
- WO 96/23873 WO 96/23874
- WO 97/41213 WO 99/19467
- WO 00/60059 WO 02/10355
- Specifically contemplated alpha-amylase variants are disclosed in US patent nos. 6,093,562, 6,297,038 or US patent no.
- BSG alpha-amylase Bacillus stearothermophilus alpha- amylase (BSG alpha-amylase) variants having a deletion of one or two amino acid in positions R179 to G182, preferably a double deletion disclosed in WO 1996/023873 - see e.g., page 20, lines 1 -10 (hereby incorporated by reference), preferably corresponding to delta(181 -182) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids R179 and G180 using SEQ ID NO:3 in WO 99/19467 for numbering (which reference is hereby incorporated by reference).
- BSG alpha-amylase Bacillus stearothermophilus alpha- amylase
- Bacillus alpha-amylases especially Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta(181 -182) and further comprise a N193F substitution (also denoted 1181 * + G182 * + N193F) compared to the wild-type BSG alpha- amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467.
- the bacterial alpha-amylase is dosed in an amount of 0.0005-5 KNU per g DS, preferably 0.001 -1 KNU per g DS, such as around 0.050 KNU per g DS.
- Fungal alpha-amylases include alpha-amylases derived from a strain of the genus Aspergillus, such as, Aspergillus oryzae, Aspergillus niger and Aspergillis kawachii alpha- amylases.
- a preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylase which is derived from a strain of Aspergillus oryzae.
- the term "Fungamyl-like alpha-amylase” indicates an alpha-amylase which exhibits a high identity, i.e. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.
- Another preferred acid alpha-amylase is derived from a strain Aspergillus niger.
- the acid fungal alpha-amylase is the one from Aspergillus niger disclosed as "AMYA_ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in WO 89/01969 (Example 3 - incorporated by reference).
- a commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark).
- wild-type alpha-amylases include those derived from a strain of the genera Rhizomucor and Meripilus, preferably a strain of Rhizomucor pusillus (WO 2004/055178 incorporated by reference) or Meripilus giganteus.
- the alpha-amylase is derived from Aspergillus kawachii and disclosed by Kaneko et al. J. Ferment. Bioeng. 81 :292-298(1996) "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii.”; and further as EMBL:#AB008370.
- the fungal alpha-amylase may also be a wild-type enzyme comprising a starch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e., none-hybrid), or a variant thereof.
- SBD starch-binding domain
- alpha-amylase catalytic domain i.e., none-hybrid
- the wild-type alpha-amylase is derived from a strain of Aspergillus kawachii.
- An acid alpha-amylases may according to the invention be added in an amount of 0.001 to 10 AFAU/g DS, preferably from 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.
- Commercial Alpha-Amylase Products preferably from 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.
- compositions comprising alpha-amylase include MYCOLASETM from DSM (Gist Brocades), BANTM, TERMAMYLTM SC, FU NGAMYLTM, LIQUOZYMETM X, LIQUOZYMETM SC and SANTM SUPER, SANTM EXTRA L (Novozymes MS) and CLARASETM L-40,000, DEX-LOTM, SPEZYMETM FRED, SPEZYMETM AA, and SPEZYMETM DELTA AA (Genencor Int.), and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A S, Denmark).
- SP288 available from Novozymes A S, Denmark
- carbohydrate-source generating enzyme includes glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators) and also pullulanase and alpha-glucosidase.
- a carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol.
- the generated carbohydrate may be converted directly or indirectly to the desired fermentation product, preferably ethanol.
- a mixture of carbohydrate-source generating enzymes may be used.
- mixtures are mixtures of at least a glucoamylase and an alpha-amylase, especially an acid amylase, even more preferred an acid fungal alpha-amylase.
- the ratio between acid fungal alpha-amylase activity (FAU-F) and glucoamylase activity (AGU) may in an embodiment of the invention be between 0.1 and 100, in particular between 2 and 50, such as in the range from 10-40.
- a glucoamylase used according to the invention may be derived from any suitable source, e.g., derived from a microorganism or a plant.
- Preferred glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1 102), or variants thereof, such as those disclosed in WO 92/00381 , WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A.
- Aspergillus oryzae glucoamylase disclosed in WO 84/02921 , Aspergillus oryzae glucoamylase (Agric. Biol. Chem. (1991 ), 55 (4), p. 941 -949), or variants or fragments thereof.
- Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J.
- glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see US patent no. 4,727,026 and (Nagasaka,Y. et al.
- Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831 ) and Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289; or Peniophora rufomarginata disclosed in PCT/US2007/066618; or a mixture thereof.
- hybrid glucoamylase are contemplated according to the invention. Examples the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference).
- glucoamylases which exhibit a high identity to any of above mention glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzymes sequences mentioned above.
- compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANTM SUPER, SANTM EXTRA L, SPIRIZYMETM PLUS, SPIRIZYMETM FUEL, SPIRIZYMETM B4U and AMGTM E (from Novozymes A/S); OPTI DEXTM 300 (from Genencor Int.); AMIGASETM and AMIGASETM PLUS (from DSM); G-ZYMETM G900, G-ZYMETM and G990 ZR (from Genencor Int.).
- Glucoamylases may in an embodiment be added in an amount of 0.0001 -20 AGU/g DS, preferably 0.001 -10 AGU/g DS, especially between 0.01 -5 AGU/g DS, such as 0.1 -2 AGU/g DS.
- Microorganisms for additional biological treatment or pre-treatment may be selected among bacteria, yeasts or fungi, or mixtures thereof.
- the microorganisms or mixtures of two or more microorganisms may provide for an improved methane production in the anaerobic fermentation step of the biogas production process.
- microorganisms includes strains of the genus: Bacillus, Pseudomonas, Enterobacter, Rhodococcus, Acinetobacter, and Aspergillus such as Bacillus licheniformis, Pseudomonas putida, Enterobacter dissolvens, Pseudomonas fluorescens, Rhodococcus pyridinivorans, Acinetobacter baumanii, Bacillus amyloliquefaciens, Bacillus pumilus, Pseudomonas plecoglossicida, Pseudomonas pseudoacaligenes, Pseudomonas antarctica, Pseudomonas monteilii, Pseudomonas mendocina, Bacillus subtilis, Aspergillus niger and Aspergillus oryzae and any combinations or two or more thereof.
- Bacillus licheniformis Bacillus lichen
- Particular preferred strains include: Bacillus subtilis (NRRL B-50136), Pseudomonas monteilii (NRRL B-50256), Enterobacter dissolvens (NRRL B-50257), Pseudomonas monteilii (NRRL B-50258), Pseudomonas plecoglossicida (ATCC 31483), Pseudomonas putida (NRRL B-50247), Pseudomonas plecoglossicida (NRRL B-50248), Rhodococcus pyridinivorans (NRRL 50249), Pseudomonas putida (ATCC 49451 ), Pseudomonas mendocina (ATCC 53757), Acinetobacter baumanii (NRRL B-50254), Bacillus pumilus (NRRL B-50255), Bacillus licheniformis (NRRL B-50141 ), Bacillus amyloliquefaciens (
- strains are added in amounts in the range of 1 .0x10 6 to 5.0x10 9 CFU/g.
- microorganisms or mixtures of two or more microorganisms can be mentioned:
- microorganism or mixture of two or more microorganisms commercially available from Novozymes Biological Inc. under the trade names: BI-CHEM ABR-Hydrocarbon, BI-CHEM DC 1008 CB and Manure Degrader are also suitable.
- Incubation under aerobic conditions may be performed as batch process, fed batch process or continuous process.
- a batch process the container is filled, a suitable inoculum of the microorganisms is added and the process proceeding for a desired time.
- a fed batch process a initial volume of bagasse-derived material is added into the container, typically 25- 75% of the total operational volume of the container, a suitable inoculum of the microorganism is added and the process is proceeding until a certain conversion /cell density is reached where additional feed in form of bagasse-derived material is added at a suitable rate and the process is continued until the container is full and optionally for an additional time without additional feed.
- the process is started by adding the material into the container and a suitable inoculum of the microorganism is added, when a desired cell density is reached a stream of the composition in the container is removed and simultaneously a stream of the material is added to the container so that the volume remains essentially constant and the process is continued in principle as long as desired. It may even be possible to use a combination of these techniques. These techniques are known within the art and the skilled person will appreciate how to find suitable parameters for a particular process depending on the particular dimensions and properties of the container.
- Means for aeration are well known in the art and it is within the capabilities of the skilled person to select suitable means for aeration for the present invention.
- aeration is performed by blowing atmospheric air through the composition typically via one or more tube(s) or pipe(s) located in the lower part of the container said one or more tube(s) or pipe(s) is/are provided with holes at regular intervals to provide for an even distribution of the air in the composition.
- Other means for aerating may also be used according to the invention.
- the rate of aeration during the aerobic fermentation step is selected to provide for a convenient growth rate of the microorganisms.
- Rate of aeration may be measured in volume air per volume ferment per minute (v/v/m) and usually aeration in the range of 0.01 v/v/m to 10 v/v/m is suitable, preferably 0.05 v/v/m to 5 v/v/m, more preferred 0.1 v/v/m to 2 v/v/m, more preferred 0.15 v/v/m to 1 .5 v/v/m and most preferred 0.2 v/v/m to 1 v/v/m.
- the duration of this step will be decided taking into account that on one side the incubation under aerobic conditions should be continued for a sufficient long time to make a satisfactory part of the lignocellulosic soluble and available for the following microbial or biological process, on the other side the aerobic step should not be extended so long that a too large fraction of the fibre fraction is combusted.
- the aerobic fermentation is continued for 5 to 30 days, preferably from 7 to 25 days, more preferred from 10 to 20 days and most preferred around 15 days. It has been found that using such an incubation period a suitable high fraction of the lignocellulosic fibres is converted into a form that can be converted in a following microbial or biological process.
- the temperature in this step should be selected taking into account the particular requirements of the microorganism or mixture of two or more microorganisms used according to the invention. Usually the temperature is selected in the range of 10°C to 60°C, preferably in the range of 15°C to 50°C, more preferred in the range of 20°C to 45°C, even more preferred in the range of 25°C to 40°C and most preferred about 35°C.
- the method according to the invention increases the degradability of the bagasse- derived material making it more accessible for a following microbial or biological process such as for example a biogas production process leading to a higher yield than would have been possible without the method of the invention.
- the incubation under aerobic conditions is continued until the degradability of the lignocellulosic fibres has been increased in a satisfactory extent so that a considerable high fraction of lignocellulosic fibres has been made accessible for a following microbial or biological process.
- the accessible fibres or part thereof will be available for the following microbial or biological process, meaning that the accessible fibres or part thereof can be converted in the following microbial or biological process.
- a material comprising lignocellulosic fibres can be treated using a method of the invention, followed by a usual anaerobic biogas forming process and the yield of the biogas using the material comprising lignocellulosic fibres treated according to the invention can be determined and compared with the same biogas forming process but without the method of the invention. If the yield of biogas is higher using the method of the invention, according to the invention, the accessibility of the lignocellulosic fibres has increased. The skilled person will appreciate that the increased accessibility according to the invention can be determined in other ways using different following microbial or biological methods.
- a method of the invention relates to the production of methane.
- the production of methane may be conducted as a two step process comprising a microbiological aerobic step and/or an enzymatic pre-treatment followed by a process for biogas production.
- the production of methane may be conducted as a two step process comprising a microbiological aerobic step and/or a pre- treatment followed by a process for biogas production with a simultaneous enzymatic treatment before or during the biogas production process.
- any process for biogas formation as known within the art may be used herein.
- the production of methane may be conducted as a process comprising a first process for biogas formation, followed by a microbiological aerobic step and/or an enzymatic treatment, again followed by a second process for biogas formation.
- Example 1 Substrate characterization of raw and steam-exploded bagasse
- Tests were performed on sugarcane bagasse which is a difficult biodegradable lignocellulosic rich substrate.
- the bagasse sample originated from a Brazilian sugarmill and was treated in an industrial scale steam explosion process (STEX).
- Table 1 Characterization of the raw bagasse sample and of the bagasse sample after steam explosion.
- TS total solids
- Ash was determined in a muffle furnace by heating the sample to 600 degree Celsius in a crucible until no further weight change occurred.
- Volatile solids (VS) was calculated by subtracting total solids with ash content.
- Total volatile fatty acids was determined by the distillation and subsequent pH titration according to standard methods in the art (Standard Methods for the Examination of Water and Wastewater, Eaton et al. , Amer Public Health Assn; 21 , October 15, 2005).
- Chemical oxygen demand was measured by the potassium dichromate method as described by Greenberg et al. in Standard Methods for the examination of water and wastewater. 18th Edition, 1992, p. 5 - 7.
- the pH was measured by adding 1 g wet weight material to 100ml distilled water and stirring for 24 hours, followed by pH measurement by calibrated pH electrode.
- thermophilic seeding sludge All the fermentation tests were started with the same type of thermophilic seeding sludge. For each test series, a fresh sample was collected. The latter originated from different thermophilic full-scale biogas installation plants, treating manure, slaughterhouse waste and/or by-products from food-processing industries.
- this seeding sludge was characterized in terms of biomass concentration as total suspended solids (TSS) and volatile suspended solids (VSS), residual soluble compounds (soluble COD and VFA) and specific methanogenic activity (SMA).
- the specific methanogenic activity was measured according to a standard procedure: In an Erienmeyer flask of 1.0 L, 800 ml of the sludge was brought together with a synthetic, easily biodegradable feed, consisting of 1 .5 ml ethanol and 4 g sodium acetate, which cooresponds to approximatily 5 g of COD. The reactor was connected to a biogas column and placed in a hot water bath at 53°C. The volume and rate of biogas production were followed over time.
- VSS concentration was considered as the best approach to measure the active anaerobic biomass concentration. Yet, it should be noted that for this measurement, there is interference of residual non-degraded organic particular matter from the substrate, which was previously fed to this biomass. Moreover, centrifugation was required to separate the biomass from the liquid phase.
- thermophilic seeding sludge used for the anaerobic experiments, had a satisfying specific methanogenic activity.
- the thermophilic sludge was highly pH buffered.
- thermophilic anaerobic sludge obtained from a full scale biogas plant running on manure and industrial waste.
- Substrate either in the form of raw or steam-exploded bagasse, was added to the bottles which were then flushed with nitrogen and sealed with butyl rubber stoppers to ensure anaerobic conditions and an airtight seal.
- the bottles were incubated at 53°C until no further methane production was observed. During the course of the experiment the bottles were vented on a regular basis to avoid the build-up of pressure in the bottles. Before venting, the precise amount of methane in each flask was determined to allow for accurate quantification of the methane produced throughout the experiment.
- samples of the bottle headspace volumes were taken with a Hamilltion gas syringe and subjected to GC analysis on a Varian 3900 gas chromatograph with a PoraPLOT Q (10 ⁇ ) 25 m x 0.32 mm fused silica separation column (Varian, Agilent Technologies, USA).
- Sample vials used for all gas samples were Supelco, Precleaned 2 cm 3 Clear Screw Cap Vials (Supelco, Bellafonte, PA, USA). Gas samples were quantified by comparison to a standard curve obtained with methane gas standards (Mikrolab, Aarhus, Denmark).
- thermophilic (53°C) short-term batch-tests were performed on lab-scale.
- Each reactor set-up consisted of a 1 .0 L Erlenmeyer flask placed in a thermostatic hot waterbath (temperature regulated at 53°C) and connected to a biogas column for the collection and measurement of biogas produced.
- the reactors were seeded with the same amount of fresh thermophilic anaerobic sludge.
- the feeding of the seeding sludge with a certain amount of different lignocellulosic substrates was done manually batchwise. After the feeding and pH- measurement of the mixed liquors, each reactor was connected to a column to follow the biogas production. At the end of the digestion period (about 1 week or more per feeding cycle), samples were taken to analyze the methane concentration of the produced biogas by gas chromatography. For each treatment, three to four successive feedings with the same substrate were performed.
- Enzymatic treatment was carried out either as an enzymatic pretreatment prior to anaerobic digestion or by direct enzyme addition to the reactor. In both cases, the enzymes were a mixture of a two enzyme products A and B.
- Enzyme A is a a Trichoderma reesei cellulase preparation containing Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656).
- Enzyme B is an Aspergillus aculeatus GH10 xylanase (WO 94/021785).
- Test 1 Seeding sludge + enzymatically pre-treated steam-exploded bagasse
- Test 2 Seeding sludge + steam-exploded bagasse + enzyme mix.
- bagasse in tap- water at a concentration of about 5 % TS (1 10 g wet weight pre-treated bagasse + 1 L tap- water) was applied as feeding.
- the 4 reactors were seeded with the same amount of thermophilic sludge, namely 800 ml, corresponding with a biomass concentration of about 17 g VSS.
- Steam-exploded bagasse was added as substrate.
- the input of bagasse in the control reactor and in the two test reactors amounted to about 5 g of total solids per feeding cycle.
- the corresponding amount of total COD input ranged around 6.2 g COD per feeding cycle.
- Table 4 Summary of the digestion tests with steam-exploded bagasse as substrate; the upper part summarizes results for steam-exploded bagasse at 5g TS and 6.2g COD; the lower part summarizes results for steam-exploded bagasse at 20g TS and 24.8g COD
- the enzyme mix used in this experiment was a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656).
- the dosage was 55 mg enzyme blend/ g TS (or 7.8 mg enzyme protein/g TS).
- the feedstream for the second test reactor was previously submitted to a pre-treatment:
- the bagasse was brought in with tap water (about 8 % TS), the same dosage of enzyme was added, the pH was adjusted to about 5 and the influent vessel was placed on a magnetic stirrer with continuous mixing and in an incubator at 50°C during a period of 2 days.
- the tests with the pre-treatment and enzyme addition were carried out over a period of 4 months.
- the three reactors were operated under stable conditions at the same relativly low volumetric loading rate.
- the three CSTR reactors were started at a volumetric loading rate of about 2.3 g TS/L reactor per day, corresponding with a hydraulic and sludge retention time of 35 days.
- the volumetric loading rate of the reactors was stepwise increased by augmentation of the amount of STEX (steam exploded) bagasse and tap water.
- the reactors were supplemented with nitrogen(N H 4 CI) and phosphorous (KH 2 P0 4 ) because of the low N and P content of the steam-exploded bagasse substrate.
- nitrogen and phosphorous suplements a mixture of trace elements was also added to the reactors to ensure that no micronutrients would become limiting.
- the total test period was divided into 5 sub-periods on the basis of the volumetric loading rate.
- Table 5 a general overview of the process parameters and resulting biogas production of the three continuous reactors is shown for each sub-period in the experiment.
- Reactor 2 (enz. pretreat)
- Reactor 2 6993 ⁇ 368 801 1 ⁇ 708 8880 ⁇ 1356 6824 ⁇ 248 7746
- Reactor 1 193 ⁇ 269 71 ⁇ 105 28 ⁇ 1 22 ⁇ 8 0
- Reactor 2 463 ⁇ 432 183 ⁇ 138 192 ⁇ 223 90 ⁇ 74 754 - 1900
- thermophilic digestion of the STEX bagasse in a CSTR reactor was investigated in a long-term lab-scale test.
- Three similar reactors (1.8 L of active reactor volume) were operated at gradually increasing loading rates, starting from 2.25 g TS/L reactor per day and stepwise augmented up to 5.56 g TS/I reactor per day. It concerned a control reactor with no enzyme addition, a test reactor with enzyme addition directly in the anaerobic reactor and a test reactor with enzyme addition in a specific pre-treatment (2 days stirring at 50°C and pH of about 5). The performance of these reactors was followed by measurements of biogas and methane production on the one hand and characterization of the anaerobic effluents.
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MX2013014236A MX2013014236A (en) | 2011-06-28 | 2012-06-27 | Biogas from enzyme-treated bagasse. |
BR112013032543A BR112013032543A2 (en) | 2011-06-28 | 2012-06-27 | biogas production process |
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EP2871231A1 (en) | 2013-11-08 | 2015-05-13 | Senzyme GmbH | Method for generating biogas |
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CN104744090B (en) * | 2015-03-25 | 2018-01-16 | 杨健 | The method and device of ecological cultivation matrix is prepared using urban waste |
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Cited By (4)
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EP2871231A1 (en) | 2013-11-08 | 2015-05-13 | Senzyme GmbH | Method for generating biogas |
DE102013018695A1 (en) | 2013-11-08 | 2015-05-13 | Senzyme Gmbh | Process for the production of biogas |
DE102016013620A1 (en) | 2016-11-15 | 2018-05-17 | Christine Apelt | Process for the material and energetic utilization of residues of sugarcane processing and arrangement for carrying out the process |
WO2018091004A1 (en) | 2016-11-15 | 2018-05-24 | Apelt, Christine | Process for material and energy recovery of residues from sugar cane processing and arrangement for performing the process |
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MX2013014236A (en) | 2014-01-23 |
US20140106427A1 (en) | 2014-04-17 |
CN103620043A (en) | 2014-03-05 |
IN2014CN00599A (en) | 2015-04-03 |
BR112013032543A2 (en) | 2017-01-17 |
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