CN114054481A

CN114054481A - Dissolving municipal solid waste with mixed enzymes

Info

Publication number: CN114054481A
Application number: CN202111269104.9A
Authority: CN
Inventors: H·R·索伦森; L·罗斯加尔德; H·B·尼尔森; L·巴克加尔德; J·瓦尔齐恩科齐克
Original assignee: Renescience AS
Current assignee: Renescience AS
Priority date: 2015-11-02
Filing date: 2015-11-02
Publication date: 2022-02-18
Also published as: WO2017076421A1; CN108136452A; WO2017076421A9

Abstract

The present invention relates to a method for the solubilization or hydrolysis of Municipal Solid Waste (MSW) with an enzyme mixture and an enzyme composition for the solubilization of Municipal Solid Waste (MSW) comprising a cellulolytic background composition and a protease, a lipase and/or a beta-glucanase.

Description

Dissolving municipal solid waste with mixed enzymes

This application is a divisional application of an invention patent application having an application date of 2015, 11/2, application number of 201580083419.0 and an invention name of "dissolving municipal solid waste with mixed enzyme".

Cross Reference to Related Applications

This application claims priority to european patent application 14182698.2 filed on 8/28 2014. The contents of this application are incorporated herein by reference in their entirety.

Reference to sequence listing

This application contains a sequence listing in computer readable form. This computer readable form is incorporated herein by reference.

Technical Field

The present invention relates to a method for dissolving or hydrolyzing Municipal Solid Waste (MSW) with an enzyme mixture, optionally for subsequent production of biogas and/or bioethanol.

Background

Municipal Solid Waste (MSW) is also commonly referred to as garbage (trash), garbage (garpage), garbage (reuse) or garbage (rubbishh). It consists of a solid waste fraction, usually from citizens, and comprises waste, for example from homes, schools, offices, hospitals, institutions, etc. MSW is produced in large quantities worldwide; thus 244 million tons (Eurostat, 2014) are produced in 2012 only in the european union. The challenges of MSW production are numerous and may include collection, sorting, processing, and disposal. Furthermore, well known environmental problems such as air and groundwater pollution of landfills are associated with MSW. As the world population continues to increase, resulting in an increasing production of waste, proper sustainable MSW management is a global challenge.

Although environmentally annoying, MSW also represents a large amount of unexplored resources available for energy production and recycling/reclamation of scarce resources. Incineration is a widely used technique in some european countries, such as denmark, sweden and germany. Energy production is efficient, but material recovery is limited. Furthermore, incineration of MSW results in the production of large amounts of slag (ash), which may have an environmental negative impact on certain types of waste fractions (Idris and Saed 2002, Journal of Hazardous Materials B93201-208). The capture of gases resulting from anaerobic digestion of organic matter at landfills is another method of generating energy from MSW, but this technique is also inefficient in terms of material recycling.

An integrated process with simultaneous energy production and material recovery is an attractive solution, which has recently attracted much attention. In such waste refineries, the organic portion of MSW represents a potentially useful resource for the production of bioenergy, for example in the form of biogas (Hartmann and Ahring,2006, Water Science & Technology Vol 53No 8 p.7-22). However, due to the very heterogeneous nature of MSW, sorting of MSW into organic fractions for bio-energy production and plastic/metal fractions for material recovery is not easy. As described by Jensen et al (2012) (WO2013/185777), pre-sorting of MSW is often expensive, inefficient or impractical, while source sorting requires large infrastructure and operational expenses, as well as active participation by the community from which waste is collected.

Enzymatic treatment of MSW has recently been described and appears to be a very interesting and innovative approach in MSW Management (Jensen et al 2010, Waste Management 30, p.2497-2503; Jensen et al 2011, Biochem Biotechnol 165, p.1799-1811; Tonini and Astrup 2012, Waste Management 32, p.165-176). The technique is based on the step of liquefying/dissolving the organic degradable fraction with a hydrolytic enzyme and subsequently separating the MSW into a biological liquid and a solid. The biological liquid can be used for biogas production, while the solids can be further sorted and used for recycling or burned depending on the composition of the material. This technique has proven to be very robust even at high dry matter concentrations (35%) and has been demonstrated in experimental/demonstration facilities, handling at most 1 ton MSW/hour.

The environmental sustainability of four different waste extraction scenarios using this enzymatic liquefaction technique was evaluated using life cycle assessment by tonii and Astrup (2012). Their evaluation was based on the Danish incinerator at Copenhagen, Denmark

Pilot plant facilities are set up. Different cases were compared with incineration. The authors conclude that "enzymatic extraction of waste and recovery of energy using products can represent a valuable alternative to incineration, both from an energy and environmental perspective. This is the case if the downstream energy source choice for developing the solid and liquid fractions is for co-combustion and anaerobic digestion of biogas production. The authors also concluded that the cost savings of waste extraction are associated with higher recovery of metals and energy. Furthermore, the improvement of the energy performance of the "environment as well as the waste extraction itself is mainly related to the optimization of the energy and enzyme consumption. "

The use of cellulase enzymes (e.g.Novozymes A/S) has been clearly demonstrated

1.5L，Novozymes A/S

Ctec2 and Novozymes A/S

Ctec3) to liquefy MSW and subsequently separate the unsorted waste into a biological liquid for biogas production and inorganic valuable products suitable for recovery (WO2013/185777a1, the content of which is incorporated herein by reference). However, MSW is a polymer containing other organic components besides cellulose (e.g. celluloseProteins and lipids), it seems reasonable to further improve the liquefaction process by supplementing cellulases with other enzymatic activities. Until now, this theory has not been proven. Jensen et al 2012 tried, which tested proteases (Novozymes A/S Alcalase 2.5L) and alpha-amylases (Novozymes A/S)

SC DC) as a single enzyme and with cellulase (Novozymes A/S)

1.5L) of a combination. "cellulolytic enzymes are key catalysts for liquefaction of degradable moieties, both in terms of reducing viscosity and particle size distribution. "the action of the alpha-amylase and the protease and the interaction between the alpha-amylase and the protease and the interaction with the cellulase are not found. Nevertheless, cellulases having similar properties to those previously tested are provided (

1.5L、

Ctec2 and

ctec3) compared to higher MSW liquefaction efficiency would be beneficial. Such an invention can contribute to global changes in MSW management practices and translate environmental issues into profitable and environmentally beneficial solutions.

Disclosure of Invention

The present invention relates to an enzyme composition for dissolving Municipal Solid Waste (MSW), comprising a Cellulolytic Background Composition (CBC), and a protease selected from the group consisting of (i) a protease; (ii) (ii) a lipase and (iii) a beta-glucanase. In one embodiment, the composition further comprises a second enzyme selected from (iv) a pectate lyase; (v) (vii) one or more enzymes of mannanase and (vi) amylase.

In one embodiment of the invention, the Cellulolytic Background Composition (CBC) comprises one or more enzymes selected from the group consisting of: (a) cellobiohydrolase I or a variant thereof; (b) cellobiohydrolase II or a variant thereof; (c) a β -glucosidase or a variant thereof; and (d) a polypeptide having cellulolytic enhancing activity; or a homologue thereof. In another embodiment of the invention, the cellulolytic background composition comprises one or more enzymes selected from the group consisting of: (a) aspergillus fumigatus (Aspergillus fumigatus) cellobiohydrolase I or a variant thereof; (b) an aspergillus fumigatus cellobiohydrolase II or variant thereof; (c) an aspergillus fumigatus beta-glucosidase or variant thereof; and (d) a Penicillium sp GH61 polypeptide having cellulolytic enhancing activity; or a homologue thereof.

In related embodiments of the invention, (i) the protease is derived from a Bacillus species, e.g., Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), e.g., the protease encoded by SEQ ID NO.1, or a protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO. 1.

In related embodiments of the invention, (ii) the lipase is derived from a thermophilic fungus, such as thermomyces lanuginosus (thermomyces lanuginosus), such as a lipase encoded by SEQ ID NO:2 (or a lipase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2), or wherein (ii) the lipase is derived from a Humicola, such as Humicola insolens (Humicola insolens) (or has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, or a lipase specific Humicola lipase, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity). In thatIn a related embodiment of the invention, (iii) the beta-glucanase is a beta-glucanase derived from a member of the genus Aspergillus, e.g.Aspergillus aculeatus (Aspergillus aculeatus), e.g.a beta-glucanase encoded by the sequence encoded by SEQ ID NO. 4 or a homologue thereof (e.g.a beta-glucanase having at least 60%, e.g.at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO. 4). In a related embodiment of the invention, (iv) the pectate lyase forms a multi-component enzyme composition comprising pectate lyase, xylanase and cellulase activities, e.g. Novozym 81243^TMPart (c) of (a). In a related embodiment of the invention, (v) the mannanase is an endo-mannosidase derived from Bacillus, such as Bacillus bogeniensis, e.g., an endo-mannosidase encoded by SEQ ID NO:6 or a homologue thereof (e.g., an endo-mannosidase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6). In a related embodiment of the invention, (vi) the amylase is an alpha-amylase derived from the genus Rhizomucor (Rhozimucor), e.g., Rhizomucor pusillus (Rhizomucor pusillus), e.g., an alpha-amylase encoded by SEQ ID NO:5 or a homolog thereof (e.g., an alpha-amylase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5).

In yet another related embodiment, the protease is present in a ratio between 0-20 wt/wt% of the total enzyme protein, such as 10 wt/wt%. In a related embodiment of the invention, the beta-glucanase is present in a ratio between 0-30 wt/wt% of the total enzyme protein, e.g. 15 wt/wt%. In yet another related embodiment of the invention, the pectate lyase is present in a proportion between 0-10 wt/wt% of the total enzyme protein, e.g. 5 wt/wt%. In a further related embodiment of the invention, the mannanase or amylase is present in a ratio between 0-10 wt/wt% of the total enzyme proteins, e.g. 5 wt/wt%. In yet another related embodiment of the invention, the cellulolytic enzyme mixture is present in a ratio of between 40-99 wt./wt. of total enzyme protein, such as between 50-90 wt./wt. such as 60-80 wt./wt. such as 65-75%. In yet another related embodiment of the invention, the enzyme composition further comprises one or more enzymes selected from the group consisting of cellulase, AA9 polypeptide, hemicellulase, Cellulose Inducible Protein (CIP), esterase, swollenin (expansin), ligninolytic enzyme, oxidoreductase, pectinase, protease, and swollenin (swellenin). In yet another related embodiment of the invention, the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetyl xylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase, and glucuronidase.

A related aspect of the invention relates to a method for dissolving waste comprising: contacting waste with the enzyme composition of the invention, wherein the waste may be Municipal Solid Waste (MSW).

Yet another related aspect of the invention relates to a method for producing a fermentation product, comprising: (a) treating MSW with an enzyme composition of the invention, (b) fermenting the solubilized and/or hydrolyzed MSW with one or more fermenting microorganisms to produce a fermentation product; and (c) recovering the fermentation product from the fermentation. The waste in the process may be pre-treated.

We have tested various commercial enzymes on a model-substrate of MSW and used dry matter dissolution of the substrate as a parameter for the liquefaction effect of the enzyme. The model Waste mimics the organic portion of MSW (publication based on the composition of Danish MSW; Riber et al 2009, Waste Management 29, p.1251-1257) and consists of a vegetable portion (e.g., carrots, potatoes, cereals, etc.), an animal by-product portion (e.g., cheese and meat), and a cellulose portion (e.g., paper, cardboard, textiles).

Screening experiments were performed on a 20 gram scale at 50 ℃ for 24 hours. Some specific enzymes (including some proteases, lipases and β -glucanases) improve the dry matter dissolution of model waste when they replace part of the Cellulolytic Background Composition (CBC). Subsequently, candidates were selected for further testing in a mixing experiment.

The present invention provides methods of solubilizing MSW by adding one or more enzymes (including acid protease, acid lipase and acid beta-glucanase) in combination with a cellulase composition to MSW at a suitable temperature and pH.

It is evident from the findings disclosed herein that surprisingly a synergistic effect in the dissolution of MSW is obtained when a mixture of different enzymes is added to a cellulolytic background composition (27.1%) compared to the individual contributions of e.g. the components b.a protease, t.i pholip and a.a BG (up to 5%, 8.5% and 8.2%, respectively).

Drawings

Figure 1 shows the dissolution of the model waste in a free fall experiment at two different dry matter concentrations. The figure shows the distribution of dry matter liquid (grey bars) and solid fraction (white bars).

Figure 2 shows a data plot from a dose response experiment using mixed enzyme and CBC and model waste. The figure illustrates the dry matter found in the liquid fraction (TS dissolution) at different enzyme concentrations.

Figure 3 shows a data plot from a dose response experiment using mixed enzyme and CBC and model waste. The figure illustrates the sum of glucose and xylose (g/l) at different enzyme concentrations.

Figure 4 shows a graph illustrating the effect of removing the b.a protease, t.i pholip or a.a BG components from the optimized mixture. The figure illustrates the dry matter found in the liquid fraction (TS dissolution).

Figure 5 shows a table of data from dose response experiments using mixed enzymes and CBC and model waste. The table illustrates the amounts of glucose, xylose and lactic acid at different enzyme concentrations.

Detailed Description

Definition of

Acetyl xylan ester acid: the term "acetylxylan esterase" refers to carboxylesterases (EC3.1.1.72) that catalyze the hydrolysis of acetyl groups from polymeric xylans, acetylated xylose, acetylated glucose, α -naphthyl acetate, and p-nitrophenylacetate. Acetylxylan esterase Activity Using 0.5mM p-nitrophenylacetate as substrate, a mixture containing 0.01% TWEEN^TM20 (polyoxyethylene sorbitan monolaurate) in 50mM sodium acetate pH 5.0. One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1. mu. mol of p-nitrophenolate anion per minute at pH5, 25 ℃.

Allelic variants: the term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variants arise naturally through mutation and may lead to polymorphism within populations. Gene mutations may be silent (no change in the encoded polypeptide) or may encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

α -L-arabinofuranosidase: the term "α -L-arabinofuranosidase" refers to an α 0-L-arabinofuranoside arabinofuranosidase (EC 3.2.1.55) which catalyzes the hydrolysis of the terminal non-reducing α 2-L-arabinofuranoside residue in α 1-L-arabinoside. The enzyme acts on alpha 3-L-arabinofuranoside, alpha 4-L-arabinoside containing (1,3) -and/or (1,5) -linkages, arabinoxylan, and arabinogalactan. The alpha-L-arabinofuranosidase is also referred to as arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidase hydrolase, L-arabinosidase, or alpha-L-arabinanase. The α -L-arabinofuranosidase activity can be determined as follows: 100mM sodium acetate pH5 in a total volume of 200. mu.l of 30 min per ml of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) was used at 40 ℃ followed by passage through

Arabinose analysis was performed by HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

α -glucuronidase: the term "α -glucuronidase" refers to an α -D-glucuronide ester (glucuronide) glucuronic acid hydrolase (EC 3.2.1.139) that catalyzes the hydrolysis of α -D-glucuronide (glucuronide) to D-glucuronide ester and alcohol. The alpha-glucuronidase activity can be determined according to de Vries,1998, J.Bacteriol.180: 243-249. One unit of alpha-glucuronidase is equal to the amount of enzyme capable of releasing 1. mu. mol of glucuronic acid or 4-O-methylglucuronic acid per minute at pH5, 40 ℃.

Helper-active 9 polypeptides: the term "helper active 9 polypeptide" or "AA 9 polypeptide" refers to a polypeptide classified as a catabolic polysaccharide monooxygenase (Quinlan et al, 2011, Proc. Natl. Acad. Sci. USA 208: 15079-. Polypeptides AA9 were previously classified as glycoside hydrolase family 61(GH61) according to Henrissat,1991, biochem.J.280: 309-.

AA9 polypeptide enhances hydrolysis of cellulosic material by enzymes having cellulolytic activity. Cellulolytic enhancing activity can be determined by measuring the increase in reducing sugars or the increase in the total amount of cellobiose and glucose produced by hydrolysis of a cellulosic material by a cellulolytic enzyme under the following conditions compared to a control hydrolysis with equal total protein loading but no cellulolytic enhancing activity (1-50mg of cellulolytic protein per g of cellulose in PCS): 1-50mg total protein per g of cellulose in Pretreated Corn Stover (PCS) at a suitable temperature, e.g. 40 ℃ -80 ℃, e.g. 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃,65 ℃, 70 ℃, 75 ℃ or 80 ℃, and a suitable pH, e.g. 4-9, e.g. 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0, at a suitable pH for 1-7 days, wherein the total protein comprises 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of AA9 polypeptide.

The AA9 polypeptide enhancing activity may be used

1.5L (Novozymes A/S, Bagsvaerd, Denmark) and a mixture of β -glucosidase enzyme, wherein the β -glucosidase enzyme is present at a weight of at least 2-5% protein loaded with cellulase protein, are determined as a source of cellulolytic activity. In one aspect, the β -glucosidase is aspergillus oryzae β -glucosidase (e.g., recombinantly produced in aspergillus oryzae according to WO 02/095014). In another aspect, the β -glucosidase is an aspergillus fumigatus β -glucosidase (e.g., recombinantly produced in aspergillus oryzae as described in WO 02/095014).

The activity of the AA9 polypeptide can also be enhanced by contacting the AA9 polypeptide with 0.5% phosphate-swollen cellulose (PASC), 100mM sodium acetate pH5, 1mM MnSO₄0.1% gallic acid, 0.025mg/ml aspergillus fumigatus beta-glucosidase and 0.01%

X-100(4- (1,1,3, 3-tetramethylbutyl) phenyl-polyethylene glycol) was assayed by incubation at 40 ℃ for 24-96 hours followed by determination of glucose release from PASC.

The enhancing activity of the AA9 polypeptide may also be determined on the high temperature composition according to WO 2013/028928.

The AA9 polypeptide enhances hydrolysis of a cellulosic material catalyzed by an enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to achieve the same degree of hydrolysis, preferably by at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

The AA9 polypeptide may also be used according to WO 2008/151043 or WO 2012/122518 in the presence of a soluble activating divalent metal cation, such as manganese or copper.

The AA9 polypeptide may be used in the presence of dioxy compounds, bicyclic compounds, heterocyclic compounds, nitrogen-containing compounds, quinone compounds, sulfur-containing compounds, or liquids obtained from pretreated cellulosic or hemicellulosic materials, such as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO 2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO 2012/021408, and WO 2012/021410).

Beta-glucanase: the term "β -glucanase" refers to any type of endo- β -glucanase which hydrolyses the (1,3) -or (1,4) -linkage (e.c.3.2.1.73) (e.c.3.2.1.6) in β -D-glucanase.

β -glucosidase: the term "β -glucosidase" refers to a β -D-glucoside glucohydrolase (e.c.3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing β -D-glucose residues with the release of β -D-glucose. Beta-glucosidase activity can be determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al 2001, J.basic Microbiol.42: 55-66. One unit of beta-glucosidase is defined as 0.01% at 25 ℃ at pH 4.8

1.0. mu. mol of p-nitrophenolate anion per minute was produced from 1mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50mM sodium citrate 20.

Beta-xylosidase: the term "β -xylosidase" refers to a β -D-xylosidase (e.c.3.2.1.37) that catalyzes the exo-hydrolysis of short β (1 → 4) -xylo-oligosaccharides to remove consecutive D-xylose residues from the non-reducing end. The beta-xylosidase activity can be determined using 1mM p-nitrophenyl-beta-D-xyloside as substrate at 0.01%

20 in 100mM sodium citrate, pH5, 40 ℃. One unit of beta-xylosidase is defined as containing 0.01% at pH5 at 40%

20 mM sodium citrate produced 1.0. mu. mol p-nitrophenolate anion per minute from 1mM p-nitrophenyl-beta-D-xyloside.

Binding domain: the term "binding domain", e.g., "cellulose binding domain", refers to a region of an enzyme that mediates binding of the enzyme to an amorphous region of a cellulose substrate. The Cellulose Binding Domain (CBD) is usually found at the N-or C-terminal end of the enzyme.

Catalytic domain: the term "catalytic domain" refers to a region of an enzyme that contains the catalytic machinery of the enzyme.

Carbohydrate binding module: the term "carbohydrate-binding module" refers to a domain within a carbohydrate-active enzyme that provides carbohydrate-binding activity (Boraston et al, 2004, biochem. J.383: 769-781). Most known Carbohydrate Binding Modules (CBMs) are continuous amino acid sequences with discrete folds. Carbohydrate-binding modules (CBM) are usually found at the N-or C-terminus of the enzyme. Some CBM's are known to be specific for cellulose.

Catalase: the term "catalase" refers to hydrogen peroxide, a hydrogen peroxide redox enzyme (EC 1.11.1.6), which catalyzes 2H₂O₂Conversion to O₂+2H₂And O. For the purposes of the present invention, catalase activity is determined according to U.S. Pat. No.5,646,025. One unit of catalase activity is equal to the amount of enzyme that catalyzes the oxidation of 1. mu. mol hydrogen peroxide under the conditions of the assay.

Cellobiohydrolase: the term "cellobiohydrolase" refers to a1, 4- β -D-glucan cellobiohydrolase (E.C.3.2.1.91 and E.C.3.2.1.176) which catalyzes the hydrolysis of the 1,4- β -D-glycosidic bond of cellulose, cellooligosaccharide or any β -1, 4-linked glucose-containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of the chain (Teeri,1997, Trends in Biotechnology 15: 160-. Cellobiohydrolase activity may be determined according to Lever et al, 1972, anal. biochem.47: 273-279; van Tilbeurgh et al, 1982, FEBS Letters 149: 152-; van Tilbeurgh and Claeussensens, 1985, FEBS Letters 187: 283-; and Tomme et al, 1988, Eur.J.biochem.170: 575-581.

Cellulose decomposition backLandscape composition (CBC) or cellulolytic enzyme mixture: the term "cellulolytic background composition" or "CBC" refers to an enzyme composition comprising a mixture of two or more cellulolytic enzymes. In one embodiment, the CBC comprises two or more cellulolytic enzymes selected from: i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) an aspergillus fumigatus beta-glucosidase or variant thereof; and (iv) a penicillium GH61 polypeptide having cellulolytic enhancing activity; or a homologue thereof. The CBC further comprises one or more enzymes selected from the group consisting of: (a) an aspergillus fumigatus xylanase or a homologue thereof; (b) an aspergillus fumigatus beta-xylosidase or a homolog thereof; or (c) a combination of (a) and (b) (as described in further detail in WO 2013/028928). The CBC may be any CBC described in WO2013/028928 (the contents of which are incorporated herein by reference). In one embodiment, the CBC is a Novozymes A/S (R) (B)

Denmark) obtained

Ctec3。

Cellulolytic enzymes or cellulases: the term "cellulolytic enzyme" or "cellulase" refers to one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanases, cellobiohydrolases, beta-glucosidases, or combinations thereof. Two basic methods for measuring cellulolytic enzyme activity include: (1) measuring total cellulolytic enzyme activity, and (2) measuring individual cellulolytic enzyme activities (endoglucanase, cellobiohydrolase, and beta-glucosidase) as reviewed by Zhang et al, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman No1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common determination of total cellulolytic activity is the use of Whatman NoFilter paper analysis with filter paper as substrate 1. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Pure appl. chem.59):257-68)。

Cellulolytic enzyme activity can be determined by measuring the increase in sugar production/release during hydrolysis of cellulosic material by cellulolytic enzymes under the following conditions compared to a control hydrolysis without added cellulolytic enzyme protein: 1-50mg cellulolytic enzyme protein/g cellulose in Pretreated Corn Stover (PCS) (or other pretreated cellulosic material) at a suitable temperature, e.g. 40 ℃ to 80 ℃, e.g. 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃,65 ℃, 70 ℃, 75 ℃ or 80 ℃, and a suitable pH, e.g. 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, for 3-7 days at a suitable temperature and a suitable pH. Typical conditions are 1ml of reacted, washed or unwashed PCS, 5% insoluble solids (dry weight), 50mM sodium acetate pH5, 1mM MnSO₄At 50 ℃, 55 ℃ or 60 ℃ for 72 hours by

Sugar analysis was performed by HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulose material: the term "cellulosic material" refers to any material containing cellulose.

In one embodiment, the cellulosic material is agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, or wood (including forestry residue).

cDNA: the term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription of a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor mRNA that is processed through a series of steps (including splicing) before it appears as mature spliced mRNA.

A coding sequence: the term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with an initiation codon, e.g., ATG, GTG, or TTG, and ends with a stop codon, e.g., TAA, TAG, or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

And (3) control sequence: the term "control sequences" refers to nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. To introduce specific restriction sites, the control sequences may have linkers to facilitate ligation of the control sequences with the coding region of the polynucleotide encoding the polypeptide.

Dissolved oxygen saturation: oxygen saturation was measured at a standard partial pressure (0.21 atm) of oxygen. The degree of saturation at standard partial pressure of oxygen depends on temperature and solute concentration. In embodiments where the temperature during hydrolysis is 50 ℃, the saturation level will typically be in the range of 5-5.5mg oxygen/kg slurry, depending on the solute concentration. Thus, a dissolved oxygen concentration at 50 ℃ at a saturation of 0.5 to 10% corresponds to a dissolved oxygen amount in the range of 0.025ppm (0.5 × 5/100) to 0.55ppm (10 × 5.5/100), for example 0.05 to 0.165ppm, and a dissolved oxygen concentration at 50 ℃ at a saturation of 10 to 70% corresponds to a dissolved oxygen amount in the range of 0.50ppm (10 × 5/100) to 3.85ppm (70 × 5.5/100), for example 1 to 2 ppm. In one embodiment, oxygen is added in an amount of 0.5 to 5ppm, such as 0.5 to 4.5ppm, 0.5 to 4ppm, 0.5 to 3.5ppm, 0.5 to 3ppm, 0.5 to 2.5ppm, or 0.5 to 2 ppm.

Endoglucanase: the term "endoglucanase" refers to 4- (1, 3; 1,4) - β -D-glucan 4-glucanohydrolase (E.C.3.2.1.4) which catalyzes the endo-hydrolysis of β -1,4 linkages in cellulose, cellulose derivatives (e.g., carboxymethylcellulose and hydroxyethylcellulose), lichenin, mixed β -1,3-1,4 glucans such as cereal β -D-glucans or xyloglucans, and 1,4- β -D-glycosidic linkages in other plant materials containing cellulosic components. Endoglucanase activity may be measured by measuring the decrease in viscosity of the substrate or the increase in reducing ends as determined by a reducing sugar assay (Zhang et al, 2006, Biotechnology Advances 24: 452-481). Endoglucanase activity may also be measured at pH5, 40 ℃ using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose,1987, Pure and appl. chem.59: 257-268.

Expressing: the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: the term "expression vector" refers to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to regulatory sequences that provide for its expression.

Feruloyl esterase: the term "feruloyl esterase" refers to a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of a 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is typically arabinose in a natural biomass substrate, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase (FAE) is also known as feruloyl esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I or FAE-II. Feruloyl esterase activity can be determined using 0.5mM p-nitrophenyl ferulate as substrate in 50mM sodium acetate pH 5.0. One unit of feruloyl esterase is equal to the amount of enzyme capable of releasing 1. mu. mol of p-nitrophenol anion per minute at pH5, 25 ℃.

Fragment (b): the term "fragment" refers to a polypeptide or catalytic or binding domain having one or more (e.g., several) amino acids with a deletion of the amino and/or carboxy terminus of the mature polypeptide or domain; wherein the fragment has enzyme or substrate binding activity.

Hemicellulolytic or hemicellulase: the term "hemicellulolytic enzyme" or "hemicellulase" refers to one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, e.g., Shallom and Shoham,2003, Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, acetyl mannan esterase, acetyl xylan esterase, arabinanase, arabinofuranosidase, coumaric acid esterase, ferulic acid esterase, galactosidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. The substrate of these enzymes, hemicellulose, is a heterogeneous group of branched and linear polysaccharides that binds to cellulose microfibrils in the plant cell wall by hydrogen bonds, cross-linking them into a strong network. Hemicellulose is also covalently linked to lignin, forming a highly complex structure with cellulose. The variable structure and organization of hemicellulose requires the coordinated action of many enzymes to fully degrade it. The catalytic module of hemicellulases is a Glycoside Hydrolase (GH) which hydrolyzes glycosidic bonds or a Carbohydrate Esterase (CE) which hydrolyzes ester bonds of side groups of hexanoic or ferulic acid. These catalytic modules can be assigned to GH and CE families based on their primary sequence homology. Some families with generally similar folds may be further grouped into alphabetically labeled relatives (e.g., GH-a). The most informative and updated classification of these and other carbohydrate active enzymes is available in the carbohydrate-activating enzyme (CAZy) database. Hemicellulase activity may be measured according to Ghose and Bisaria,1987, Pure & Appl. chem.59:1739-1752 at a suitable temperature, e.g.40 ℃ to 80 ℃, e.g.40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃,65 ℃, 70 ℃, 75 ℃ or 80 ℃, and at a suitable pH, e.g.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.

Hemicellulose material: the term "hemicellulosic material" refers to any material comprising hemicellulose.

Host cell: the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" includes any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Separating: the term "isolated" refers to a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more or all of the naturally occurring components with which it is naturally associated; (3) any substance that is artificially altered with respect to substances found in nature; or (4) any substance that is altered by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a promoter that is stronger than the promoter with which the gene encoding the substance is naturally associated).

Lipase: the term "lipase" refers to any enzyme that catalyzes the hydrolysis of lipids and/or has hydrolytic activity in the EC3.1.1 class as defined by enzyme nomenclature. Particularly useful are triacylglycerol lipases (e.c.3.1.1.3) and phospholipase a1(EC 3.1.1.32) and phospholipase a2(e.c.3.1.1.4), but also other phospholipases (e.c.3.1.1.5), (e.c.3.1.4.4), (e.c.3.1.4.11), (e.c.3.1.4.50), (e.c. 3.1.4.54).

Mannanase: in the context of the present invention, a "mannanase" is a β -mannanase and is defined as an enzyme belonging to EC 3.2.1.78 or e.c.3.2.1.25. Mannanases have been identified in several bacillus organisms. For example, Talbot, appl.environ.Microbiol.Vol.56, No.11, pp.3505-3510(1990) describe a B-mannanase enzyme derived from Bacillus stearothermophilus, which has an optimum pH of 5.5-7.5. Mendoza et al, World J.Microbiol.Biotech., Vol.10, No.5, pp.551-555(1994) describe a B-mannanase from Bacillus subtilis which has optimal activity at pH 5.0 and 55 ℃. JP-03047076 discloses a beta-mannanase derived from Bacillus having an optimum pH of 8 to 10. JP-63056289 describes the production of alkaline thermostable beta-mannanases. JP-08051975 discloses an alkaline beta-mannanase from Bacillus alcalophilus AM-001. Purified mannanase from bacillus amyloliquefaciens is disclosed in WO 97/11164. WO 94/25576 discloses the enzyme CBS 101.43 from aspergillus aculeatus, which exhibits mannanase activity, and WO 93/24622 discloses mannanase isolated from trichoderma reesei.

Mature polypeptide: the term "mature polypeptide" refers to the final form of the polypeptide after its translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having enzymatic activity.

Municipal Solid Waste (MSW): the term "municipal solid waste" or "MSW" means the fraction of solid waste that is normally available in cities (cities, towns, villages). MSW can be a combination of plant material (fruit, vegetables, grain, corn, etc.), animal material (meat, etc.), cellulosic material (paper, cardboard, diapers, textiles, etc.), glass, plastic, metal. MSWs include, but are not limited to, any one or more of the following:

waste collected from homes, schools, hospitals, offices, businesses, industries such as restaurants and food processing industries. The MSW may have been processed through shredding or pulping equipment.

Nucleic acid construct: the term "nucleic acid construct" refers to a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that is modified to contain segments of nucleic acids in a manner that would not otherwise occur in nature, or that is synthetic, that comprises one or more control sequences.

Operatively connected to: the term "operably linked" refers to a construct in which control sequences are placed at appropriate positions relative to the coding sequence of a polynucleotide such that the control sequences direct the expression of the coding sequence.

Pretreated municipal solid waste material: the term "pretreated municipal solid waste material" refers to municipal solid waste material derived from biomass by treatment with heat and dilute sulfuric acid, alkaline pretreatment, neutral pretreatment, or any pretreatment known in the art.

Protease: the term "protease" refers to any protease or proteolytic enzyme suitable for use under neutral or acidic conditions. Suitable proteases include those of animal, vegetable or microbial origin. Chemically or genetically modified mutants are included. Suitable proteases include metalloendoproteases that hydrolyze internal peptide bonds (e.c.3.4.24.28), serine endoproteases that hydrolyze internal peptide bonds (E.C:3.4.23.23), endoproteases that hydrolyze peptide bonds on the carboxy side of lysine and arginine residues (e.c.3.4.21.4), aminopeptidases (e.c.3.4.11.1), and exopeptidases that release amino acids by hydrolysis of the N-terminal peptide bond (e.c. 3.4.11.1).

Sequence identity: the relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For The purposes of The present invention, sequence identity between two amino acid sequences is determined using The Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.biol.48:443-453) implemented in The Needle program of The EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al 2000, Trends Genet.16:276-277), preferably version 5.0.0 or more. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5 and an EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:

(same residue X100)/(alignment Length-Total number of vacancies in alignment)

For The purposes of The present invention, The sequence identity between two deoxyribonucleotide sequences is determined using The Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) implemented in The Needle program of The EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, supra), preferably version 5.0.0 or more. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and the EDNAFULL (EMBOSS version of NCBI NUC 4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:

(same deoxyribonucleotide X100)/(alignment length-total number of vacancies in alignment)

Dissolving: the term "solubilization" refers to the enzymatic treatment of a substrate. In the present disclosure, the terms "hydrolysis", "liquefaction", "saccharification" and "dissolution" may be used interchangeably.

Variants: the term "variant" refers to a polypeptide comprising an alteration (i.e., a substitution, insertion, and/or deletion) at one or more (e.g., several) positions. Substitution refers to the substitution of an amino acid occupying a position with a different amino acid; deletion refers to the removal of the amino acid occupying a position; an insertion refers to the proximity of an amino acid occupying a position followed by an addition of an amino acid.

Xylan-containing material: the term "xylan-containing material" refers to any material comprising plant cell wall polysaccharides comprising a backbone of β - (1-4) -linked xylose residues. Xylans from terrestrial plants are heteropolymers with a β - (1-4) -D-xylopyranose backbone, which branches into short carbohydrate chains. They contain D-glucuronic acid or its 4-O-methyl ether, L-arabinose and/or various oligosaccharides consisting of D-xylose, L-arabinose, D-or L-galactose and D-glucose. Xylan-type polysaccharides can be classified into homoxylans (homoxylans) and heteroxylans (heteroxylans), which include glucuronoxylan, (arabino) glucuronoxylan, (glucuronic) arabinoxylan, and complex heteroxylan. See, e.g., Ebrigrevova et al, 2005, adv. Polym. Sci.186: 1-67.

In the method of the invention, any xylan-containing material may be used. In a preferred aspect, the xylan-containing material is lignocellulose.

Xylan degrading activity or xylan decomposing activity: the term "xylan degrading activity" or "xylanolytic activity" refers to the biological activity of hydrolyzing 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, β -xylosidase, arabinofuranosidase, α -glucuronidase, acetylxylan esterase, ferulic acid esterase, and α -glucuronidase). Recent advances in the determination of xylanolytic enzymes are summarized in several publications, 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-; herrimann et al, 1997, Biochemical Journal 321: 375-.

Total xylan degrading activity can be determined by measuring the formation of xylan species from various xylan types including, for example, oat, beech and larch xylanGlycogen or stained xylan fragments released from various covalently stained xylans by photometric determination. A common assay for total xylanolytic activity is based on the production of reducing sugars from polymerized 4-O-methylglucuronoxyxylans as described by Bailey et al, 1992, intercalation testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270. The xylanase activity can also be measured at 0.01% using 0.2% AZCL-arabinoxylan as substrate

X-100 and 200mM sodium phosphate pH 6 at 37 ℃. One unit of xylanase activity was defined as 1.0 μmol azurin (azurine) produced per minute at 37 ℃, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase in hydrolysis of birch xylan by xylan degrading enzymes (Sigma Chemical co., inc., st.louis, MO, USA) under the following typical conditions: 1ml of reaction, 5mg/ml of substrate (total solids), 5mg of xylanolytic protein/g of substrate, 50mM sodium acetate pH5, 50 ℃ for 24 hours, as described by Lever,1972, anal. biochem.47: 273-.

Xylanase: the term "xylanase" refers to a1, 4- β -D-xylan hydrolase (e.c.3.2.1.8) that catalyzes the endo-hydrolysis of 1,4- β -D-xylosidic bonds in xylan. The xylanase activity can be 0.01% using 0.2% AZCL-arabinoxylan as substrate

X-100 and 200mM sodium phosphate pH 6 at 37 ℃. One unit of xylanase activity is defined as 1.0. mu. mol azurin produced per minute at 37 ℃ and pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6.

References herein to a "value or parameter of" about "includes reference to the value or parameter itself. For example, a description referring to "about X" includes the aspect "X".

As used herein and in the appended claims, the singular forms "a", "or" and "the" include plural referents unless the context clearly dictates otherwise. It is to be understood that aspects of the invention described herein include aspects "consisting of … …" and/or "consisting essentially of … …".

Unless defined otherwise or clear from context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In some embodiments of the invention, enzymatic solubilization of MSW is performed with naturally occurring microorganisms found in waste (concurrent enzymatic and microbial hydrolysis and fermentation) or found in the recovery process waste/solution.

In some embodiments, microbial growth has a pH-lowering effect, particularly when metabolites such as carboxylic acids and fatty acids (e.g., acetate, propionate, butyrate, lactate) are produced.

In other embodiments of the invention, it may be advantageous to inoculate the MSW with different microbial species. These may include microorganisms exhibiting extracellular cellulase activity, microorganisms capable of degrading lignin, acetate producing microorganisms, propionate producing microorganisms, butyrate producing microorganisms, ethanol producing microorganisms, and lactate producing microorganisms. Such embodiments are further described on pages 21-25 of the DONG patent (WO2013/185777, the contents of which are incorporated herein by reference).

In practicing embodiments of the invention, it may be advantageous to adjust the temperature and water and dry matter content of the MSW. Enzymes generally show an optimal temperature and dry matter range. The hydrolysis of MSW is usually carried out under stirring. This can be done in a reactor providing agitation by free fall mixing (as also described in DONG WO2006/056838 and WO 2011/032557), a stirred tank reactor or similar system. Suitable process time, temperature and pH conditions can be readily determined by one skilled in the art and depend on the MSW composition, dry matter concentration and enzyme.

The invention also relates to methods of using the compositions thereof.

The present invention also relates to a method for degrading municipal solid waste material, comprising: treating municipal solid waste material with an enzyme composition comprising a cellulolytic background composition in combination with one or more enzymes selected from the group consisting of: (i) protease: (ii) (ii) a lipase and (iii) a β -glucanase; and optionally in combination with one or more additional enzymes selected from: (iv) a pectate lyase; (v) (vii) mannanase and (vi) amylase. In one aspect, the method further comprises recovering the degraded municipal solid waste material. The degraded soluble products of the municipal solid waste material may be separated from the insoluble municipal solid waste material using methods known in the art, such as centrifugation, filtration or gravity settling. In a preferred embodiment, the dissolved compounds may be converted into biogas (mainly comprising CH) by anaerobic digestion₄And CO₂). In other embodiments, the dissolved sugars may be converted to ethanol by fermentation.

The present invention also relates to a method of producing a fermentation product comprising: (a) solubilizing municipal solid waste material with an enzyme composition comprising a cellulolytic background composition in combination with one or more enzymes selected from the group consisting of: (i) a protease; (ii) (ii) a lipase and (iii) a β -glucanase; and optionally in combination with one or more additional enzymes selected from: (iv) a pectate lyase; (v) (vii) mannanase and (vi) amylase; (b) fermenting the dissolved municipal solid waste material with one or more (e.g., several) fermenting microorganisms to produce a fermentation product; (c) recovering the fermentation product from the fermentation. In a preferred embodiment, the dissolved compounds may be converted into biogas (mainly comprising CH) by anaerobic digestion₄And CO₂). In other embodiments, the dissolved sugars may be converted to ethanol by fermentation.

The present invention also relates to a method of fermenting municipal solid waste material, comprising: fermenting municipal solid waste material with one or more (e.g. several) fermenting microorganisms, wherein the municipal solid waste material is saccharified with an enzyme composition comprising a cellulolytic background composition that is in contact with one selected from the group consisting ofOr a combination of enzymes: (i) a protease; (ii) (ii) a lipase and (iii) a β -glucanase; and optionally in combination with one or more additional enzymes selected from: (iv) a pectate lyase; (v) (vii) mannanase and (vi) amylase. In one aspect, fermentation of municipal solid waste material produces a fermentation product. In another aspect, the method further comprises recovering the fermentation product from the fermentation. In a preferred embodiment, the dissolved compounds may be converted into biogas (mainly comprising CH) by anaerobic digestion₄And CO₂). In other embodiments, the dissolved sugars may be converted to ethanol by fermentation.

The process of the present invention can also be used to solubilize municipal solid waste material into fermentable sugars and convert the fermentable sugars into a number of useful fermentation products, such as fuels (ethanol, n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals (e.g., acids, alcohols, ketones, gases, oils, etc.). The production of the desired fermentation product from municipal solid waste material typically involves enzymatic solubilization and fermentation.

The processing of municipal solid waste material according to the invention may be accomplished using methods conventional in the art. Further, the methods of the present invention may be practiced using any conventional biomass processing apparatus configured to operate in accordance with the present invention.

Separate or simultaneous solubilization and fermentation includes, but is not limited to, Separate Hydrolysis and Fermentation (SHF); simultaneous Saccharification and Fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); mixed hydrolysis and fermentation (HHF); hydrolysis alone and co-fermentation (SHCF); mixed hydrolysis and co-fermentation (HHCF); and Direct Microbial Conversion (DMC), sometimes also referred to as Coupled Bioprocessing (CBP). SHF uses separate process steps to first enzymatically hydrolyze municipal solid waste material to fermentable sugars, such as glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol. In SSF, enzymatic hydrolysis of municipal solid waste material and fermentation of sugars to ethanol are combined in one step (Philippidis, G.P.,1996, Cellulose biochemical technology, in Handbook on Bioethanol: Production and inactivation, Wyman, C.E., ed., Taylor & Francis, Washington, DC,179- & 212). SSCF involves the co-fermentation of multiple sugars (Sheehan and Himmel,1999, Biotechnol. prog.15: 817-827). HHF involves a separate hydrolysis step, and additional simultaneous saccharification and hydrolysis steps, which may be performed in the same reactor. The steps in the HHF process may be performed at different temperatures, such as high temperature enzymatic saccharification, followed by SSF at lower temperatures tolerated by the fermenting strain. DMC all three processes (enzyme production, hydrolysis and fermentation) are combined together in one or more (e.g., several) steps, using the same organism to produce enzymes for the conversion of municipal solid waste material into fermentable sugars and fermentable sugars into end products (Lynd et al, 2002, Microbiol. mol. biol. reviews66: 506-. It is understood herein that any method known in the art including pretreatment, enzymatic hydrolysis, fermentation, or a combination thereof, may be used to practice the methods of the present invention.

Conventional equipment may include fed-batch stirred reactors, continuous-flow stirred reactors with ultrafiltration and/or continuous plug-flow column reactors (de Castilhos Corazza et al, 2003, Acta scientific. technology 25: 33-38; Gusakov and Sinitsyn,1985, Enz. Microb. technology.7: 346-. Additional reactor types include fluidized bed reactors for hydrolysis and/or fermentation, upflow blanket reactors, immobilized reactors, and extrusion type reactors.

And (4) preprocessing.In practicing the methods of the invention, the plant cell wall components of municipal solid waste material may be disrupted using any pretreatment method known in the art (Chandra et al, 2007, adv. biochem. Engin./Biotechnology.108: 67-93; Galbe and Zachi, 2007, adv. biochem. Engin./Biotechnology.108: 41-65; Hendriks and Zeeman,2009, Bioresource Technology 100: 10-18; Mosier et al, 2005, Bioresource Technology 96: 673-.

In a preferred embodiment of the invention, the MSW is subjected to a mild to severe temperature pre-treatment in the range of 10-300 ℃ prior to hydrolysis. Heating will typically occur with mixing. Heating will typically be by the addition of water or steam. The pre-processing may also consist of MSW separation (manual or automatic) into different fractions. The municipal solid waste material may also be size reduced, sieved, pre-soaked, wetted, washed and/or conditioned prior to being pretreated using methods known in the art.

Conventional pretreatment includes, but is not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organic solvent pretreatment, and biological pretreatment. Additional pretreatment includes ammonia percolation, sonication, electroporation, microwave, supercritical CO₂Supercritical H₂O, ozone, ionic liquid and gamma irradiation pretreatment.

The municipal solid waste material may be pre-treated prior to hydrolysis and/or fermentation. The pretreatment is preferably carried out before the hydrolysis. Alternatively, pretreatment may be performed simultaneously with enzymatic hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases, the pretreatment step itself results in some degree of conversion of the biomass to fermentable sugars (even in the absence of enzymes).

Mechanical or physical pretreatment: the term "mechanical pretreatment" or "physical pretreatment" refers to any pretreatment that promotes particle size reduction. For example, such pre-treatment may involve various types of milling or grinding (e.g., dry milling, wet milling, or vibratory ball milling).

Municipal solid waste material may be subjected to both physical (mechanical) and chemical pretreatment. Mechanical or physical pretreatment may be combined with steaming/steam explosion, hydrothermal decomposition, dilute or weak acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure refers to a pressure preferably in the range of about 100 to about 400psi, for example about 150 to about 250 psi. In another aspect, elevated temperature refers to a temperature in the range of about 100 to about 300 ℃, such as about 140 to about 200 ℃. In a preferred aspect, the mechanical or physical pretreatment is carried out in a batch process using a high pressure and high temperature steam gun Hydrolyzer system as defined above (e.g., a Sunds hydrosizer available from Sunds Defibrator AB, Sweden). The physical and chemical pretreatments may be performed sequentially or simultaneously as desired.

Thus, in a preferred aspect, the municipal solid waste material is subjected to physical (mechanical) or chemical pretreatment, or any combination thereof, to facilitate the separation and/or release of cellulose, hemicellulose and/or lignin.

And (4) biological pretreatment. The term "biological pretreatment" refers to any biological pretreatment that facilitates the separation and/or release of cellulose, hemicellulose, and/or lignin from municipal solid waste material. The biological Pretreatment techniques may involve the application of lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T-A., 1996, Pretreament of bioglass, in Handbook on Bioethanol: Production and inactivation, Wyman, C.E., ed., Taylor & Francis, Washington, DC,179 212; Ghosh and Singh,1993, adv. appl. Microbiol.39: 295. 333; McMillan, J.D.,1994, Preeating lignocellulosic biological: a review, in enzymic Conversion of for Fuels Production, Himmel, M.E., Baker, J.O. and Oend, R.ACS.P., Symphosis, Securie, S.J.O. and Oceand. J.O. and R.ACS.P., Australin, C.R.F.G.R.R.G.G.R.G.R.G.R.D., Biochemical Engineering, C.S.S.R.G.G.G.R.G.J.R.S.R.J.R.S.F.J.R.R.F.S.A. No. 23, C.S.J.23, C.S.S.S.J.23, Biochemical Engineering, J.S.S.S.S.23, J.S.S.S.S.G.G.G.G.23, J.S.D.D.23, J.D.D.23, C.S.S.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D, 1990, adv.biochem.eng./biotechnol.42: 63-95).

In other embodiments, the MSW may be subjected to both physical (mechanical) and chemical pretreatment. Mechanical or physical pretreatment may be combined with steaming/steam explosion, hydrothermal decomposition, dilute or weak acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure refers to a pressure preferably in the range of about 100 to about 400psi, for example about 150 to about 250 psi. In another aspect, elevated temperature refers to a temperature in the range of about 100 to about 300 ℃, such as about 140 to about 200 ℃. In a preferred aspect, the mechanical or physical pretreatment is carried out in a batch process using a high pressure and high temperature steam gun Hydrolyzer system as defined above (e.g., a Sunds hydrosizer available from Sunds Defibrator AB, Sweden). The physical and chemical pretreatments may be performed sequentially or simultaneously as desired.

And (4) hydrolyzing.In the hydrolysis step, the municipal solid waste material, e.g. pretreated, is hydrolyzed to break down cellulose and/or hemicellulose and other substrates into fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose and/or soluble oligosaccharides (also referred to as saccharification). The hydrolysis is carried out enzymatically in one or more steps by one or more enzyme compositions. In the hydrolysis step, the municipal solid waste material, e.g. pre-treated, is hydrolysed to break down proteins and lipids (e.g. triglycerides) found in the waste.

The hydrolysis may be carried out as a batch process or a series of batch processes. The hydrolysis may be carried out as a fed-batch or continuous process or a series of fed-batch or continuous processes, wherein the municipal solid waste material is gradually fed into a hydrolysis solution, e.g. containing an enzyme composition. In one embodiment, the hydrolysis is a continuous hydrolysis, wherein the MSW material and the enzyme composition are added at different intervals throughout the hydrolysis process, and the hydrolysis products are removed at different intervals throughout the hydrolysis 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.

The enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art. In one aspect, the hydrolysis is carried out under conditions suitable for enzymatic activity (i.e., optimal for the enzyme).

In one aspect, saccharification is conducted in the presence of dissolved oxygen at a concentration of at least 0.5% saturation.

In one embodiment of the invention, the dissolved oxygen concentration during saccharification is in the range of at least 0.5% and at most 30%, such as at least 1% and at most 25%, at least 1% and at most 20%, at least 1% and at most 15%, at least 1% and at most 10%, at least 1% and at most 5% and at least 1% and at most 3% of saturation. In a preferred embodiment, the dissolved oxygen concentration is maintained at a concentration of at least 0.5% and at most 30% of saturation, such as at least 1% and at most 25%, at least 1% and at most 20%, at least 1% and at most 15%, at least 1% and at most 10%, at least 1% and at most 5%, and at least 1% and at most 3% during at least 25%, such as at least 50% or at least 75% of the saccharification period. When the enzyme composition comprises an oxidoreductase, the dissolved oxygen concentration may be higher, at up to 70% of saturation.

Oxygen is added to the vessel to achieve the desired dissolved oxygen concentration during saccharification. Maintaining the dissolved oxygen level within a desired range may be accomplished by adding compressed air through a diffuser or sparger or by aerating a vessel, tank, etc. by other known aeration methods. The aeration rate may be controlled based on feedback from a dissolved oxygen sensor placed in the vessel/tank, or the system may be run at a constant rate without feedback control. In the case of a hydrolysis train consisting of a plurality of vessels/tanks connected in series, aeration may be carried out in one or more or all of the vessels/tanks. Oxygen aeration systems are well known in the art. Any suitable inflation system may be used in accordance with the present invention. Commercial inflation systems are designed by, for example, Chemineer, Derby, England, and are manufactured by, for example, Paul Mueller Company, MO, USA.

The enzyme composition may comprise any protein useful for degrading municipal solid waste material.

In one aspect, the enzyme composition comprises or further comprises one or more (e.g., several) proteins selected from the group consisting of cellulases, AA9 polypeptides, hemicellulases, esterases, swollenins, ligninolytic enzymes, oxidoreductases, pectinases, proteases, and swollenins. In another aspect, the cellulase is preferably one or more (e.g., several) enzymes selected from endoglucanases, cellobiohydrolases, and beta-glucosidases. In another aspect, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetyl mannan esterase, an acetyl xylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a ferulic acid esterase, a galactosidase, a glucuronidase, a mannanase, a mannosidase, a xylanase and a xylosidase. In another aspect, the oxidoreductase is preferably one or more (e.g., several) enzymes selected from the group consisting of catalase, laccase, peroxidase.

In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes and one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) enzymes selected from cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises an endoglucanase. In another aspect, the enzyme composition comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises a β -glucosidase. In another aspect, the enzyme composition comprises an AA9 polypeptide. In another aspect, the enzyme composition comprises an endoglucanase and an AA9 polypeptide. In another aspect, the enzyme composition comprises a cellobiohydrolase and an AA9 polypeptide. In another aspect, the enzyme composition comprises a β -glucosidase and an AA9 polypeptide. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises an endoglucanase and a beta-glucosidase. In another aspect, the enzyme composition comprises a β -glucosidase and a cellobiohydrolase. In another aspect, the enzyme composition comprises a β -glucosidase and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises an endoglucanase, an AA9 polypeptide, and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase, an AA9 polypeptide, a cellobiohydrolase I, a cellobiohydrolase II, or a combination of cellobiohydrolase I and cellobiohydrolase II. In another aspect, the enzyme composition comprises an endoglucanase, a beta-glucosidase, and an AA9 polypeptide. In another aspect, the enzyme composition comprises a β -glucosidase, an AA9 polypeptide, and a cellobiohydrolase. In another aspect, the enzyme composition comprises a β -glucosidase, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises an endoglucanase, a beta-glucosidase, and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase, a beta-glucosidase, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, and an AA9 polypeptide. In another aspect, the enzyme composition comprises an endoglucanase, a β -glucosidase, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetyl mannan esterase. In another aspect, the enzyme composition comprises an acetylxylan esterase. In another aspect, the enzyme composition comprises an arabinase (e.g., an alpha-L-arabinase). In another aspect, the enzyme composition comprises an arabinofuranosidase (e.g., an alpha-L-arabinofuranosidase). In another aspect, the enzyme composition comprises a coumarate esterase. In another aspect, the enzyme composition comprises a feruloyl esterase. In another aspect, the enzyme composition comprises a galactosidase (e.g., an alpha-galactosidase and/or a beta-galactosidase). In another aspect, the enzyme composition comprises a glucuronidase (e.g., an alpha-D-glucuronidase). In another aspect, the enzyme composition comprises a glucuronidase. In another aspect, the enzyme composition comprises a mannanase enzyme. In another aspect, the enzyme composition comprises a mannosidase (e.g., a β -mannosidase). In another aspect, the enzyme composition comprises a xylanase. In one embodiment, the xylanase is a family 10 xylanase. In another embodiment, the xylanase is a family 11 xylanase. In another aspect, the enzyme composition comprises a xylosidase (e.g., a β -xylosidase).

At another placeIn one aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises swollenin. In another aspect, the enzyme composition comprises a ligninolytic enzyme. In one embodiment, the ligninolytic enzyme is a manganese peroxidase. In another embodiment, the lignin degrading enzyme is lignin peroxidase. In another embodiment, the ligninolytic enzyme is H-producing₂O₂An enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises an oxidoreductase. In one embodiment, the oxidoreductase is a catalase. In another embodiment, the oxidoreductase is a laccase. In another embodiment, the oxidoreductase is a peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises a swellin.

In the process of the invention, the enzyme may be added before or during saccharification, saccharification and fermentation, or fermentation.

One or more (e.g., several) components of the enzyme composition can be a native protein, a recombinant protein, or a combination of a native protein and a recombinant protein. For example, one or more (e.g., several) components can be native proteins of a cell used as a host cell to recombinantly express one or more (e.g., several) other components of the enzyme composition. It is understood herein that a recombinant protein may be heterologous (e.g., foreign) and/or native to the host cell. One or more (e.g., several) components of the enzyme composition can be produced as a single component, which is then combined to form the enzyme composition. The enzyme composition may be a combination of multi-component and single-component protein preparations.

The enzyme used in the process of the invention may be in any form suitable for use, e.g.a fermentation broth preparation or a cell composition, a cell lysate with or without cell debris, a semi-purified or purified enzyme preparation, or a host cell from which the enzyme is derived. The enzyme composition may be a dry powder or granules, a non-sprayed granule, a liquid, a stabilized liquid, or a stabilized protected enzyme. The liquid enzyme preparation may be stabilized, for example, according to established methods by adding stabilizers such as sugars, sugar alcohols or other polyols, and/or lactose or another organic acid.

The optimal amount of enzyme and polypeptide having enzymatic activity depends on several factors including, but not limited to, a mixture of cellulolytic and/or hemicellulolytic enzymes, municipal solid waste material, concentration of municipal solid waste material, pretreatment of the municipal solid waste material, temperature, time, pH, and inclusion of fermenting organisms (e.g., for simultaneous saccharification and fermentation).

In one aspect, the effective amount of the enzyme composition on the municipal solid waste material is from about 0.5 to about 50mg, for example from about 0.5 to about 40mg, from about 0.5 to about 25mg, from about 0.75 to about 20mg, from about 0.75 to about 15mg, from about 0.5 to about 10mg, or from about 2.5 to about 10mg per gram of the municipal solid waste material. In related aspects, the protease is present in a proportion of between 0-20 wt/wt% of the total enzyme protein, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 wt/wt%.

In one aspect, the β -glucanase is present in a ratio between 0-30 wt/wt% of the total enzyme protein, e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 wt/wt%. In a related aspect, the pectate lyase is present in a ratio between 0-10 wt/wt% of the total enzyme protein, e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, 10 wt/wt%. In a related aspect, the mannanase or amylase is present in a ratio between 1-10 wt/wt% of the total enzyme protein, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 wt/wt%. In a further related aspect, the cellulolytic enzyme mixture is present in a ratio of between 40% -99% w/w of the total enzyme protein, such as between 50% -90% w/w, such as 60% -80% w/w, such as 65-75%.

Polypeptides having cellulolytic or hemicellulolytic enzyme activity and other proteins/polypeptides useful for the degradation of municipal solid waste material, such as the AA9 polypeptide, may be derived or obtained from any suitable source, including archaeal, bacterial, fungal, yeast, plant or animal sources. The term "obtained" herein also refers to an enzyme which may be recombinantly produced in a host organism using the methods described herein, wherein the recombinantly produced enzyme is native or foreign to the host organism or has an altered amino acid sequence, e.g. one or more (e.g. several) amino acids with deletions, insertions and/or substitutions, i.e. a recombinantly produced enzyme which is a mutant and/or fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling (shuffling) methods as known in the art. Included within the meaning of a native enzyme are native variants and within the meaning of a foreign enzyme are variants obtained, for example, by site-directed mutagenesis or shuffling.

Each polypeptide may be a bacterial polypeptide. For example, each polypeptide can be a gram-positive bacterial polypeptide having enzymatic activity or a gram-negative bacterial polypeptide having enzymatic activity.

Each polypeptide may also be a fungal polypeptide, such as a yeast polypeptide or a filamentous fungal polypeptide.

Chemically modified or protein engineered mutants of polypeptides may also be used.

One or more (e.g., several) components of the enzyme composition may be recombinant components, i.e., produced by cloning a DNA sequence encoding a single component, followed by transformation of the cell with the DNA sequence and expression in a host (see, e.g., WO 91/17243 and WO 91/17244). The host may be a heterologous host (the enzyme is foreign to the host), but the host may also be a homologous host (the enzyme is native to the host) under certain conditions. Monocomponent cellulolytic proteins may also be prepared by purifying such proteins from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example

CTec(Novozymes A/S)、

CTec2(Novozymes A/S)、

CTec3(Novozymes A/S)、

(Novozymes A/S)、NOVOZYM^TM188(Novozymes A/S)、SPEZYME^TMCP(Genencor Int.)、ACCELLERASE^TMTRIO(DuPont)、

NL(DSM)、

S/L 100(DSM)、ROHAMENT^TM7069W(

GmbH) or

CMAX3^TM(Dyadic International, Inc.). The cellulolytic enzyme preparation is added in an effective amount of about 0.001 to about 5.0 wt% solids, for example about 0.025 to about 4.0 wt% solids or about 0.005 to about 2.0 wt% solids.

Examples of bacterial endoglucanases useful in the methods of the invention include, but are not limited to, Acidothermus cellulolyticus endoglucanases (WO91/05039, 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 carotovora endoglucanase (Saarilahti et al, 1990, Gene 90:9-14), Thermoactinomyces thermophilus (Thermobifida fusca) endoglucanase III (WO 05/093050), and Thermoactinomyces thermophilus endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases useful in the present invention include, but are not limited to, 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 Cel5A endoglucanase II (GenBank: M19373), Trichoderma reesei endoglucanase III (Okada et al, 1988, applied. environ. Microbiol.64:555-563, GenBank: AB003694), Trichoderma reesei endoglucanase V (Saloheimo et al, 1994, Molecular biology 13:219-228, GenBank: Z33381), Aspergillus oryzae endoglucanase (Otoi et al, 1990, Trichoderma reesei endoglucanase V: 5884), Fusarium strain Aspergillus niger endoglucanase I (Aspergillus 2927: Aspergillus niger L.29381, Aspergillus niger strain I (Aspergillus niger I: 2927: Aspergillus niger, Humicola grisea variant Thermoidea endoglucanase (GenBank: AB003107), Melanocarpus albomyosam endoglucanase (GenBank: MAL515703), Neurospora crassa endoglucanase (GenBank: XM-324477), Humicola insolens endoglucanase V, myceliophthora thermophila CBS 117.65 endoglucanase, Thermoascus aurantiacus (Thermoascus aurantiacaus) endoglucanase I (GenBank: AF487830), Trichoderma reesei strain No. VTT-D-80133 endoglucanase (GenBank: M65), and Penicillium pinophilum endoglucanase (WO 2012/062220).

Examples of cellobiohydrolases useful in the present invention include, but are not limited to, 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 (WO 2009/042871), penicillium occipitans cellobiohydrolase I (GenBank: AY690482), Talaromyces emersonii cellobiohydrolase I (GenBank: AF439936), Thielavia hyricane cellobiohydrolase II (WO 2010/141325), Thielavia hycanariee cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and Trichoderma saccharose cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include, but are not limited to, those 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 brasiliensis IBT 20888(WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO 2011/035029), and Trichophaea saccata (WO 2007/019442).

Other useful endoglucanases, cellobiohydrolases and beta-glucosidases are disclosed in a number of glycosyl hydrolase families using the classification according to Henrissat,1991, biochem.J.280:309-316 and Henrissat and Bairoch,1996, biochem.J.316: 695-696.

Any AA9 polypeptide may be used as a component of the enzyme composition in the methods of the invention.

Examples of AA9 polypeptides that may be used in the methods of the invention include, but are not limited to, those from Thielavia terrestris (WO 2005/074647, WO 2008/148131 and WO 2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344), myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868 and WO 2009/033071), Aspergillus fumigatus (WO 2010/138754), Penicillium pinophilum (WO 2011/005867), Thermoascus thermosiphenesis (WO 2011/039319), Penicillium emersonii 0(WO 2011/041397 and WO 2012/000892), Thermoascus scleroderma (Thermoascus crusteous) (WO 2011/041504), Aspergillus aculeatus (WO 2012/125925), Thermomyces lanuginosus (WO 2012/113340, Thermomyces lanuginosus, WO 2012/129699, WO 2012/130964 and WO 2012/129699), Aurantiporus alborubescens (WO 2012/122477), Trichophaea saccharocata (WO 2012/122477), Penicillium chrysium (Penicillium thomii) (WO 2012/122477), Talaromyces stipitis (WO 2012/135659), Humicola insolens (WO 2012/146171), Cladosporium camphorata (Malbranchea cinmamomera) (WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), and Chaetomium thermophilum (WO 2012/101206) and Chaetomium thermophilum (WO 2012/129697 and WO 2012/130950).

In one aspect, the AA9 polypeptide is used in the presence of a soluble activating divalent metal cation (e.g., manganese or copper) according to WO 2008/151043.

In another aspect, the AA9 polypeptide is used in the presence of a dioxygen compound, a bicyclic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquid obtained from pretreated municipal solid waste material, such as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO 2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO 2012/021408, and WO 2012/021410).

The term "liquor" refers to the aqueous, organic, or combination thereof solution phase resulting from treatment of lignocellulosic and/or hemicellulosic material or monosaccharides thereof such as xylose, arabinose, mannose, etc. in a slurry under the conditions described in WO 2012/021401, and the soluble content thereof. Liquids for cellulolytic enhancement of AA9 polypeptides may be produced by treating lignocellulosic or hemicellulosic material (or feedstock), optionally in the presence of a catalyst such as an acid, optionally in the presence of an organic solvent, by applying heat and/or pressure, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids. Such conditions determine the degree of cellulolytic enhancement obtainable by the combination of a liquid and an AA9 polypeptide during hydrolysis of a cellulosic substrate by a cellulolytic enzyme preparation. The liquid may be separated from the treated material using standard methods in the art, such as filtration, sedimentation or centrifugation.

In one aspect, an effective liquid-to-cellulose amount is about 10 per gram of cellulose^-6To about 10 grams, e.g., about 10 per gram of cellulose^-6To about 7.5 grams, about 10^-6To about 5 grams, about 10^-6To about 2.5 grams, about 10^-6To about 1 gram, about 10^-5To about 1 gram, about 10^-5To about 10^-1Gram, about 10^-4To about 10^-1Gram, about 10^-3To about 10^-1Grams, or about 10^-3To about 10^-2And g.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME^TM(Novozymes A/S)、

HTec(Novozymes A/S)、

HTec2(Novozymes A/S)、

HTec3(Novozymes A/S)、

(Novozymes A/S)、

(Novozymes A/S)、

HC(Novozymes A/S)、

Xylanase(Genencor)、

XY(Genencor)、

XC(Genencor)、

TX-200A(AB Enzymes)、HSP 6000Xylanase(DSM)、DEPOL^TM333P(Biocatalysts Limit,Wales,UK)、DEPOL^TM740L. (Biocatalysts Limit, Wales, UK) and DEPOL^TM762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), and ALTERNA FUEL 200P (Dyadic).

Examples of xylanases that can be used in the methods of the invention include, but are not limited to, xylanases from Aspergillus aculeatus (GeneSeqP: AAR 63790; WO 94/21785), Aspergillus fumigatus (WO 2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium penicillium (WO 2010/126772), Thermomyces lanuginosus (GeneSeqP: BAA22485), Talaromyces thermophilus (GeneSeqP: BAA22834), Thielavia terrestris NRRL 8126(WO 2009/079210), and Trichophaea saccata (WO 2011/057083).

Examples of beta-xylosidases that may be used in the methods of the present invention include, but are not limited to, beta-xylosidases from Neurospora crassa (SwissProt: Q7SOW4), Trichoderma reesei (UniProtKB/TrEMBL: Q92458), Talaromyces emersonii (SwissProt: Q8X212), and Talaromyces thermophilus (GeneSeqP: BAA 22816).

Examples of acetylxylan esterases that may be used in the method of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprot: Q2GWX4), Chaetomium gracile (GeneSeqP: AAB82124), Humicola insolens DSM 1800(WO 2009/073709), Hypocrea jecorina (WO 2005/001036), myceliophthora thermophila (WO 2010/014880), Neurospora crassa (Uniprot: Q7s259), Phaeospheria nodorum (Uniprot: Q0UHJ1) and Thielavia terrestris NRRL 8126(WO 2009/042846).

Examples of feruloyl esterases (feruloyl esterases) that may be used in the method of the invention include, but are not limited to, feruloyl esterases from Humicola insolens DSM 1800(WO 2009/076122), Fusarium fischeri (Neosartorya fischeri) (Uniprot: A1D9T4), Neurospora crassa (Uniprot: Q9HGR3), Penicillium fulvum (Penicillium aurantiagriseum) (WO 2009/127729) and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases that may be used in the method of the present invention include, but are not limited to, arabinofuranosidases from Aspergillus niger (GeneSeqP: AAR94170), Humicola insolens DSM 1800(WO 2006/114094 and WO 2009/073383) and M.giganteus (WO 2006/114094).

Examples of alpha-glucuronidases that may be used in the methods of the invention include, but are not limited to, alpha-glucuronidases from Aspergillus clavatus (UniProt: alcc12), Aspergillus fumigatus (SwissProt: Q4WW45), Aspergillus niger (UniProt: Q96WX9), Aspergillus terreus (Aspergillus terreus) (SwissProt: Q0CJP9), Humicola insolens (WO 2010/014706), Penicillium chrysogenum (WO 2009/068565), Talaromyces emersonii (Unit: Q8X211), and Trichoderma reesei (UniProt: Q99024).

Examples of oxidoreductases that may be used in the method of the invention include, but are not limited to, Aspergillus lentilus catalase, Aspergillus fumigatus catalase, Aspergillus niger catalase, Aspergillus oryzae catalase, Humicola insolens catalase, Neurospora crassa catalase, Penicillium emersonii catalase, Serratia thermophila catalase, Scytalidium thermophilum catalase, Talaromyces catalase, Thermoascus aurantiacus catalase, Coprinus cinereus laccase, myceliophthora thermophila laccase, Polyporus pinsitus laccase, Micrococcus cinnabarinus laccase, Rhizoctonia solani laccase, Streptomyces coelicolor laccase, Coprinus cinerea peroxidase, soybean peroxidase, and Rongtonia awamori peroxidase.

The enzymatically active polypeptides used in the methods of the present invention can be produced by fermentation of the above-described microbial strains on nutrient media containing suitable carbon and nitrogen sources and inorganic salts using procedures known in the art (see, e.g., Bennett, j.w. and LaSure, L. (eds.), More Gene industries in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American type culture Collection). Temperature ranges and other conditions suitable for growth and enzyme production are known in the art (see, e.g., Bailey, j.e. and olivis, d.f., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

Fermentation may be any cell culture process that results in the expression or isolation of an enzyme or protein. Thus, fermentation may be understood to include shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The resulting enzyme produced by the above-described process can be recovered from the fermentation medium and purified by conventional procedures.

And (5) fermenting.In a preferred embodiment, some fermentation will occur simultaneously with hydrolysis of MSW. Fermentable sugars obtained from hydrolyzed municipal solid waste material may be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a fermentation product, e.g., volatileFatty acids (e.g., acetate, propionate, butyrate), lactate esters, and alcohols.

"fermentation" or "fermentation process" refers to any fermentation process or any process that includes a fermentation step. Fermentation processes also include fermentation processes for the consumer alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can be readily determined by the person skilled in the art.

In the fermentation step, the sugars released from the municipal solid waste material as a result of the pretreatment and enzymatic hydrolysis steps are fermented by a fermenting organism, such as yeast, into a product, such as ethanol. Hydrolysis and fermentation may be separate or simultaneous.

Any suitable hydrolyzed municipal solid waste material may be used in the fermentation step in the practice of the invention. The materials are generally chosen on the basis of economics, i.e., cost per equivalent of sugar potential, and recalcitrance to enzymatic conversion.

The term "fermentation medium" is understood herein to mean the medium prior to the addition of the fermenting microorganism, e.g. the medium resulting from a saccharification process, as well as the medium used for a simultaneous saccharification and fermentation process (SSF).

"fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism may be a hexose and/or pentose fermenting organism, or a combination thereof. Both hexose and pentose sugar fermenting organisms are well known in the art. Suitable fermenting microorganisms are capable of fermenting, i.e. converting, sugars such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose and/or oligosaccharides directly or indirectly into the desired fermentation product. Examples of ethanol producing bacterial and fungal fermenting organisms are described by Lin et al, 2006, appl. Microbiol. Biotechnol.69: 627-642.

Examples of fermenting microorganisms that can ferment hexoses include bacterial and fungal organisms, such as yeast. Yeasts (yeast) include strains of Candida, Kluyveromyces, and Saccharomyces (Saccharomyces), such as Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars in their native state include bacterial and fungal organisms, such as some yeasts. Xylose fermenting yeast includes strains of candida, preferably c.sheatae or c.sonorensis; and pichia stipitis strains, e.g., pichia stipitis, such as pichia stipitis CBS 5773. Pentose fermenting yeasts include strains of pachysolen, preferably p. Organisms that are unable to ferment pentoses (e.g., xylose and arabinose) may be genetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexoses and pentoses to ethanol include, for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus, Thermoanaerobacter saccharolyticum, and Zymomonas mobilis (Philippidis, G.P.,1996, cell biocompression technology, in Handbook on Bioethanol: Production and inactivation, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212).

Other fermenting organisms include strains of bacillus, such as bacillus coagulans; candida species, such as c.sonorensis, c.methanosbosa, c.diddensiae, c.parapsilosis, c.naedodenra, c.blankii, c.entomophilia, c.brassicae, candida pseudotropicalis (c.pseudotropicalis), c.boidinii, candida utilis (c.utilis), and c.scelhatae; clostridia, such as clostridium acetobutylicum, clostridium thermocellum, and clostridium phytofermentans; coli, particularly strains of escherichia coli that have been genetically modified to increase ethanol production; geobacillus sp.geotrichum; hansenula, such as Hansenula anomala; klebsiella, such as klebsiella oxytoca (k. oxytoca); kluyveromyces, such as kluyveromyces marxianus, kluyveromyces lactis, kluyveromyces thermotolerans, and kluyveromyces fragilis (k.fragilis); fission yeast, such as Schizosaccharomyces pombe; thermoanaerobacters, such as thermoanaerobacter saccharolyticum; and zymomonas, such as zymomonas mobilis.

In one aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose-utilizing, arabinose-utilizing, and xylose and arabinose co-utilizing microorganisms.

In another aspect, the fermenting organism comprises one or more polynucleotides encoding one or more cellulolytic enzymes, hemicellulolytic enzymes, and helper enzymes described herein.

As described herein, it is well known in the art that the above organisms can also be used to produce other substances.

The fermenting microorganisms are typically added to the degraded municipal solid waste material or hydrolysate and the fermentation is carried out for about 8 to about 96 hours, for example about 24 to about 60 hours. The temperature is typically between about 26 ℃ to about 60 ℃, e.g., about 32 ℃ or 50 ℃, and about pH 3 to about pH 8, e.g., pH 4-5, 6 or 7.

In one aspect, yeast and/or additional microorganisms are applied to the degraded municipal solid waste material and fermentation is carried out for about 12 to about 96 hours, for example typically 24-60 hours. In another aspect, the temperature is preferably between about 20 ℃ to about 60 ℃, e.g., about 25 ℃ to about 50 ℃, about 32 ℃ to about 50 ℃, or about 32 ℃ to about 50 ℃, and the pH is typically about pH 3 to about pH 7, e.g., about pH 4 to about pH 7. However, some fermenting organisms, such as bacteria, have a higher optimum condition for fermentation temperature. The yeast or the further microorganism is preferably present at about 10 per ml fermentation broth⁵To 10¹²Preferably about 10⁷To 10¹⁰In particular about 2X 10⁸The amount of viable cell count is administered. Further guidance regarding The use of yeast for fermentation can be found, for example, in "The Alcohol Textbook" (Editors k. jacques, t.p. lyons and d.r. kelsall, Nottingham University Press, United Kingdom 1999), which is incorporated herein by reference.

The fermentation stimulant can be used in combination with any of the methods described herein to further improve the performance, e.g., rate increase and ethanol production, of the fermentation process, particularly the fermenting microorganism. "fermentation stimulant" refers to a stimulant used for the growth of fermenting microorganisms, particularly yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-creatine, thiamine, pyridoxine, p-aminobenzoic acid, folic acid, riboflavin, and vitamins A, B, C, D and E. See, for example, Alfenore et al, Improving ethanol production and reliability of Saccharomyces cerevisiae by a vitamin feeding process felt-batch process, Springer-Verlag (2002), which is incorporated herein by reference. Examples of minerals include minerals and mineral salts that can provide nutrients including P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation product: the fermentation product may be any material derived from fermentation. The fermentation product can be, but is not limited to, an alcohol (e.g., arabitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1, 3-propanediol [ propylene glycol ]]Butylene glycol, glycerin, sorbitol, and xylitol); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), alkenes (e.g., pentene, hexene, heptene, and octene); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); gases (e.g. methane, hydrogen (H)₂) Carbon dioxide (CO)₂) And carbon monoxide (CO)); isoprene; ketones (e.g., acetone); organic acids (e.g., acetic acid, pyruvic acid, adipic acid, ascorbic acid, citric acid, 2, 5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketides.

In one aspect, the fermentation product is an alcohol. The term "alcohol" includes materials that contain one or more hydroxyl moieties. The alcohol can be, but is not limited to, n-butanol, isobutanol, ethanol, methanol, arabitol, butanediol, ethylene glycol, glycerol, 1, 3-propanediol, sorbitol, xylitol. See, e.g., Gong et al, 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology; scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany,65: 207-241; silveira and Jonas,2002, appl.Microbiol.Biotechnol.59: 400-; nigam and Singh,1995, Process Biochemistry 30(2): 117-; ezeji et al, 2003, World Journal of Microbiology and Biotechnology 19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane may be an unbranched or branched alkane. The alkane may be, but is not limited to, pentane, hexane, heptane, octane, nonane, decane, undecane, or dodecane.

In another aspect, the fermentation product is a cycloalkane. The cycloalkane may be, but is not limited to, cyclopentane, cyclohexane, cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The olefin may be an unbranched or branched olefin. The olefin may be, but is not limited to, pentene, hexene, heptene, or octene.

Fermentation broth preparation or cell composition

The invention also relates to a fermentation broth formulation or a cellular composition comprising a polypeptide of the invention. The fermentation broth product also comprises additional components used in the fermentation process, such as cells (including host cells containing a gene encoding a polypeptide of the invention, which are used to produce the polypeptide), cell debris, biomass, fermentation medium, and/or fermentation product. In some embodiments, the composition is a cell killing whole liquid comprising an organic acid, killed cells and/or cell debris, and culture medium.

The term "fermentation broth" refers to a preparation produced by fermentation of a cell that has undergone no or little recovery and/or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation, incubated under carbon-limited conditions to allow protein synthesis (e.g., by expression of an enzyme of the host cell), and secreted into the cell culture medium. The fermentation broth may contain an unfractionated or fractionated content of the fermented material obtained at the end of the fermentation. Typically, the fermentation broth is unfractionated and contains spent media and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, for example, by centrifugation. In some embodiments, the fermentation broth comprises spent cell culture medium, extracellular enzymes, and viable and/or non-viable microbial cells.

In embodiments, the fermentation broth formulation and cell composition comprises a first organic acid component comprising at least one organic acid of 1-5 carbons and/or a salt thereof and a second organic acid component comprising at least one organic acid of 6 or more carbons and/or a salt thereof. In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing, and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

In one aspect, the composition contains an organic acid and optionally also killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from the whole cell killing solution to provide a composition free of these components.

The fermentation broth formulation or cell composition may also include preservatives and/or antimicrobial agents (e.g., bacteriostats) including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

The fermentation broth formulation or cell composition may further comprise a plurality of enzyme activities, for example one or more (e.g. several) enzymes selected from the group consisting of cellulase, hemicellulase, Cellulose Inducible Protein (CIP), esterase, swollenin, laccase, ligninolytic enzyme, pectinase, peroxidase, protease and swollenin. The fermentation broth preparation or the cell composition may also comprise a compound selected from the group consisting of hydrolases, isomerases, ligases, lyases, oxidoreductases, or transferases, such as alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The cell killing whole liquid or composition may contain an unfractionated content of the fermented material obtained at the end of the fermentation. Typically, the cell killing whole liquid or composition contains spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of cellulase and/or glucosidase). In some embodiments, the cell killing whole liquid or composition comprises a used cell culture medium, an extracellular enzyme, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell killing whole liquid or composition may be permeabilized and/or lysed using methods known in the art.

A whole liquid or cellular composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, media components, and/or insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

The whole liquid preparations and cell compositions of the present invention can be produced by the methods described in WO 90/15861 or WO 2010/096673.

Examples of the use of the compositions of the present invention are given below. The dosage of the composition and other conditions under which the composition is used can be determined based on methods known in the art.

Enzyme composition

The invention also relates to compositions comprising the polypeptides of the invention. Preferably, the composition is enriched for such polypeptides. The term "enriched" means that the cellobiohydrolase activity of the composition has been increased, e.g., has an enrichment factor of at least 1.1.

The composition may comprise as a major enzyme component a polypeptide of the invention, e.g. a single component composition. Alternatively, the composition may comprise a plurality of enzyme activities, for example one or more (e.g. several) enzymes selected from the group consisting of cellulase, hemicellulase, AA9 polypeptide, CIP, esterase, swollenin, laccase, ligninolytic enzyme, pectinase, peroxidase, protease, and swollenin. The composition may further comprise one or more (e.g., several) enzymes selected from a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The compositions may be prepared according to methods known in the art, and may be in the form of liquid or dry compositions. The composition may be stabilized according to methods known in the art.

In still other embodiments, the enzyme of the composition of the invention may be a protease derived from bacillus amyloliquefaciens, a triacylglycerol lipase having phospholipase activity derived from thermomyces lanuginosus, a triacylglycerol lipase derived from humicola insolens, a triacylglycerol lipase derived from thermomyces lanuginosus, NZ81243 (a multicomponent enzyme with pectate lyase, xylanase and cellulase activity commercially available as Novozym 81243), a β -glucanase derived from aspergillus aculeatus. Alpha-amylase derived from Rhizomucor miehei and/or endo-mannosidase derived from Bacillus bogoriensis.

Materials and methods

1. Substrate preparation

Unsorted MSW essentially consists of two solid fractions: 1) inorganic non-degradable substances composed of plastics, glass, metals, etc.; and 2) degradable organic matter consisting of vegetables, animal waste, food waste, paper, cardboard, etc. For example, the latter fraction typically constitutes about 65-70% of the Waste produced in Denmark (see Riber et al 2009, Waste Management 29, p.1251-1257 for detailed compositional analysis).

The scope of the experiments described below is to optimize the dissolution of the degradable organic portion of MSW. Therefore, we chose to use a model waste that reflects the composition of degradable organics in MSW as described by Riber et al 2009. The model waste consists of 3 parts based on fresh product supplemented with water:

-40.1% vegetable origin. Mixtures of fresh vegetables (onion, cabbage, carrot, cucumber, etc.), cereals (oatmeal, corn flakes, etc.), breads, cakes, flowers, cooked rice, cooked pasta, fruits, tomato paste, etc.

11.3% animal origin. Meat paste from chicken, pig, and cattle, sausage, hot wing, sparerib, and coarse meat.

36.2% cellulose source. Newsprint, office paper, magazines, cardboard, juice cartons, kitchen paper, cotton, wood, textiles, and the like.

Water for obtaining consistency of model waste suitable for assay (described later).

A total of 3 different batches were used, prepared and used for the experiments. Batches 1 and 2 were prepared from RENeScience (DONG A/S). Batch 3 was prepared in our own laboratory. The composition of the model waste is given in the following table:

total solids/Dry matter (TS)	25.2-28.3％
		And TS comprises:
krason (Klason) lignin	12％
		Cellulose, process for producing the same, and process for producing the same	29-32％
Xylan
		10％
Arabinoglycan			1％
	Galactan	1.7％
Mannan
		4％
Fat			7％
	Protein	7-8％
Starch			3-10％
	Ash of	15％

2. Determination of enzymatic solubilization of laboratory-scale model waste.

Experiments were performed on a 20g scale if not otherwise stated. Standard assays for enzymatic solubilization of model waste were used as follows:

1. weigh 50ml centrifuge tube + lid.

2. Model waste was added to the tube (1.5 g TS total).

3. 50mM citrate buffer (pH 5) (about 14.7ml) and enzyme were added (total weight 20g and 7.5% TS) and the tube was shaken vigorously.

4. The tubes were incubated in an oven at 50 ℃ for 24 hours at 12rpm on a Stuart Rotator SB 3.

5. After incubation, 0.4ml of 10% proxel was added to kill lactic acid bacteria and other microorganisms.

6. The tubes were centrifuged at 2090Xg for 10 minutes.

7. A new set of 50ml tubes without caps was weighed and labeled as supernatant.

8. The supernatant was poured into the tube and the tube + supernatant was weighed.

9. Approximately 2X 1.5ml of supernatant was removed and poured into eppendorf tubes for analysis. The 50ml tube containing the supernatant was weighed again.

11. The first tube + pellet was weighed again.

12. The eppendorf tubes containing the supernatant were stored in a refrigerator.

13. The TS of the supernatant and the pellet was determined by drying the tubes at 50 ℃. The tubes were weighed, then dried at 105 ℃ and weighed again.

And (3) analysis: mass balance was performed before and after incubation on a weight basis. If more than 5% of the substrate has been lost, the sample is discarded. The ratio of the dry matter content in the supernatant and the pellet after centrifugation was calculated for all samples. The frozen samples were analyzed on HPLC for sugar content, acetic acid and lactate, if necessary.

Examples

Example 1

Screening of individual enzymes and selection of enzyme candidates for mixing.

First, a very broad range of enzymes (> 50; Novozymes A/S, Bagsvaerd, Denmark) was screened for the potential to improve hydrolysis of model waste. Enzymes tested included alpha-amylase, glucoamylase, pullulanase, protease, lipase, cellulase, xylanase, pectinase and beta-glucanase.

The standard assay was used. Control vials were charged with 2.4%/TS (product to dry)Mass) of CBC (C: (C)

Ctec 3; novozymes A/S, Bagsvaerd, Denmark). In the test vials, portions of the CBC were replaced with other enzymes at different ratios in the range of 1-50% based on protein. The density of the CBC and different products is taken into account. In addition, a blank with substrate and buffer but no enzyme was prepared. All tests were performed at least twice.

The main success criterion for improved hydrolysis was defined as an increase in soluble total solids (TS in supernatant). During the screening process, for samples with CBC, the proportion of TS dissolved is typically about 25-28%, but changes are seen. The TS dissolution of the enzyme-free tubes was always about 10-11%. Candidates were selected for further testing:

a protease (SEQ ID NO: 1): a protease derived from bacillus amyloliquefaciens that gives a TS lysis increase of up to 5% at 30% substitution of CBC.

T.I pholip (SEQ ID NO: 2): a triacylglycerol lipase with phospholipase activity derived from Thermomyces lanuginosus which gives a TS solubilization increase of at most 4.4-8.5% at 2-20% substitution of CBC.

H.i trilip (H.i trilip): triglycerides lipase derived from Humicola insolens, which gives a TS lysis increase of up to 4-7% at 5-20% replacement of CBC.

I trilip (SEQ ID NO: 3): triacylglycerol lipase derived from thermomyces lanuginosus which gives a TS lysis increase of up to 10-15% at 1-10% substitution of CBC.

NZ 81243: multicomponent enzymes with pectate lyase, xylanase and cellulase activity are commercially available as Novozym 81243. At 5-20% replacement of CBC gives up to 4% increase in TS dissolution. 2:1

A.a BG: (SEQ ID NO: 4): beta-glucanase from aspergillus aculeatus. Contains additional activities (cellulase, xylanase, pectinase). At 20-40% replacement of the CBC gives a TS dissolution increase of up to 6.2-8.2%. When mixed with NZ81243 at a 2:1 ratio, gives a TS dissolution of up to 16% at 30% replacement of CBC.

R.p Alam (SEQ ID NO: 5): alpha-amylase from rhizomucor pusillus, which improves glucose yield in supernatant, at most 20% at 5-20% replacement of CBC.

B.b Enma: an endomannosidase from Bacillus bogoriensis. At 2.5-10% replacement of the CBC gives a TS dissolution increase of up to 6-10%.

Example 2

By mixing selected candidates, an optimized enzyme mixture is designed

Statistical experiments were set up to find the optimal ratio between CBC and selected enzyme candidates in a multi-component enzyme mixture. Based on the screening experiments, we decided to use two different templates, which specify the ratio of enzymes to be used:

for both templates, it was decided to use 0-20% b.a protease (protease) and 0-10% lipase. This was supplemented with a) 0-30% a.a BG (β -glucanase) +/-NZ81243 (pectate lyase) or b) 0-20% b.b Enma (endomannosidase) or 0-20% r.a. palam (α -amylase). A total of 6 mixing experiments were performed.

Experiments a.1-a.3.The first three experiments were performed to select the most appropriate lipase when 0-20% b.a protease and 0-30% a.a BG were combined. The template is as follows:

hybrid template a.1

Enzyme name	Ratio of total enzyme protein
		A protease	0-20％
T.I pholip	0-10％
		A.a BG	0-30％
CBC	Make up to 100%

Hybrid template a.2

Enzyme name	Ratio of total enzyme protein
		A protease	0-20％
T.I trilip	0-10％
		A.a BG	0-30％
CBC	Make up to 100%

Hybrid template a.3

Enzyme name	Ratio of total enzyme protein
		A protease	0-20％
H.i trilip	0-10％
		A.a BG	0-30％
CBC	Make up to 100%

Use of

The Design Of Experiments (DOE) to Design the dose in the experiment (see table below). As in example 1, the enzyme concentration in the control vials (vials 17 and 39) was 2.4% CBC/TS. In the test vials, portions of the CBC were replaced with other enzymes based on protein as described in the table below. Each test mixture was performed in duplicate.

Enzyme dosage in tube. For the templates a.1, a.2, a.3, a.4, a.5

The bright spots for tests a.1, a.2 and a.3 are given in the following table:

as shown, the best improvement in TS dissolution over CBC was found in experiment a.1 to obtain a 27.1 increase in the ratio of b.a protease t.i pholip: a.a BG: CBC of 10:5:15: 70. Interestingly, TS dissolution in individual vials showed significant differences ranging from slight negative effects caused by the enzyme mixture to a significant increase of + 30.4% in tube No. 23. As described in example 1, individual improvements of b.a protease, t.i pholip and a.a BG were at most 5%, 8.5% and 8.2%, respectively. The increase in individual effect amounted to only 21.7%. Thus, a synergistic effect was clearly obtained in experiment a.1 when mixing the enzymes.

Significant improvement in TS dissolution was also seen in experiments a.2 (combination of B.a protease: T.I trilip: A.aBG: CBC at a ratio of 20:10:30:40, up to 23.5%) and a.3 (combination of B.a protease: H.i trilip: A.a BG: CBC at a ratio of 10:5:15:70, up to 24.5%). However, it was decided to continue the use of t.i pholip in the following experiment.

Experiment a.4.This experiment was used to determine if a.a BG should be supplemented with NZ 81243. When the two enzymes are combined, the initial screen (example 1) has shown increased TS dissolution.

Hybrid template a.4

Enzyme name	Ratio of total enzyme protein
		A protease	0-20％
T.I pholip	0-10％
		A.a BG + NZ81243(2:1 mixture)	0-30％
CBC	Make up to 100%

As shown in the table below, TS dissolution can be increased to 22.1%. However, this is a smaller improvement than in experiment a.1 where a.a BG was not supplemented with NZ 81243.

Experiments b.1 and b.2. this experiment was used to determine if the alpha-amylase R.p Alam or endo-mannosidase b.b Enma "matched" better than A.a BG for protease B.a protease and lipase T.I pholip.

The following mixing and dosing templates were used:

hybrid template b.1

Enzyme name	Ratio of total enzyme protein
		A protease	0-20％
T.I pholip	0-10％
		B.b Enma	0-10％
CBC	Make up to 100%

Hybrid template b.2

Enzyme name	Ratio of total enzyme protein
		A protease	0-20％
T.I pholip	0-10％
		R.p Alam	0-10％
CBC	Make up to 100%

Enzyme dosage in tube. For forms b.1, b.2

In both mixing experiments, improved performance in TS dissolution compared to CBC was found, but all tests were inferior to the mixing ratio found in experiment a.1, where b.a protease, t.i pholip, a.a BG and CBC were mixed in a ratio of 10:5:15: 70.

Based on these observations, it was decided to use this combination of enzymes in further development.

Example 3

Testing selected enzyme mixtures and in free fall experiments at elevated dry matter concentrations

Ctec3。

Experiments were performed to test the efficiency of selected enzyme mixtures at dry matter concentrations above during the screening and mixing experiments. However, due to the high viscosity of the model waste, mixing in a 50ml tube was not optimal when the experiment was performed at dry matter concentrations above 7.5%. Instead, the experiment was performed in 100ml Kautex bottles. The model waste was mixed with water to a volume of 50ml, and to a TS concentration of 7.5% and 15%. The CBC and selected mixture (B.a protease: T.I pholip: A.a BG: CBC in a ratio of 10:5:15: 70) were added in an amount of 2.4%/TS (product to dry matter CBC). The tubes were incubated in a drum reactor at 50 ℃ for 24 hours. The results are shown in FIG. 1.

The TS dissolution of the original biomass in the control tube (no enzyme) was 10.9% and 3.2% at 7.5% and 15%, respectively. Addition of CBC improved TS dissolution to 29.6% and 25.8. Supplementation of CBC with other enzymes further improved the solubilization to 37.1 and 30.7%. This corresponds to a relative improvement of 25.7% (7.5% dry matter) and 18.9% (15% dry matter) when compared to the mixture with CBC. The figures obtained at 7.5% dry matter demonstrate the improvement seen in the 20g scale (50ml tube), which is at most 27.3%.

Example 4 comparable dose response experiments with CBC and enzyme mixtures

The experiments were performed in 100ml Kautex bottles. The model waste was mixed with water to a volume of 50ml and a TS concentration of 7.5%. CBC and selected mixtures were added in amounts corresponding to 0%, 25%, 50%, 75%, 100% and 200% of the concentration (2.4% enzyme protein/TS) that had been used as default in previous experiments (b.a protease: t.i pholip: a.a BG: CBC in a ratio of 10:5:15: 70). The bottles were incubated on a Stuart Rotator SB3 and placed in an oven at 50 ℃ for 24 hours.

A significant improvement in TS dissolution was seen at all applied enzyme concentrations when compared to the mixture with CBC. The TS solubility of the default setting (2.4% CBC/TS) is about 25%. This is achieved with only about 0.9% of the mixture, which corresponds to a reduction of the enzyme dosage by about 2.5 to 2.7 times (see fig. 2). At the same time, we found a significant increase in hydrolysis and fermentation products such as glucose, xylose, lactic acid (fig. 3 and 5). This was an unexpected finding, as 15% of the CBC (cellulase and xylanase activities) was replaced by lipase and protease.

Example 5

Omitting individual components from the mixture

Experiments were performed to test the optimized mixture (B.a protease: T.I pholip: A.a BG: CBC in a ratio of 10:5:15:70) Correlation of individual components in (A). The control vial settings were as described in example 1 (2.4%

Ctec, 7.5% TS, 20 gram scale). In the test vials, the optimized mixture, including all enzymes, was applied. At the same time, test vials were prepared in which either b.a protease, t.i pholip or a.a BG had been excluded from the mixture.

Figure 4 illustrates the effect on TS dissolution and clearly shows that removal of any individual enzyme results in lower TS dissolution compared to vials with all enzymes. However, when only two (2) selected enzymes were used to supplement CBC (t.i pholip + a.a BG, b.a protease + t.i pholip), we still observed improved TS lysis compared to the vials of CBC alone, even though the total enzyme protein concentration had been reduced by removal of the individual enzymes.

Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to those skilled in the art that any equivalent aspects or modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

The invention may be further described by the following numbered paragraphs:

[1] an enzyme composition for solubilizing Municipal Solid Waste (MSW), the enzyme composition comprising: (i) a cellulolytic background composition and (ii) a protease; and/or (iii) a lipase.

[2] The composition of paragraph [1], further comprising (iv) a β -glucanase; (v) a pectate lyase; (vi) (vii) mannanase and/or (vii) amylase.

[3] The composition of paragraphs [1] or [2], wherein the cellulolytic background composition comprises a) a cellobiohydrolase I or a variant thereof; (b) cellobiohydrolase II or a modification thereof; (c) a β -glucosidase or a variant thereof; and (d) a polypeptide having cellulolytic enhancing activity; or a homologue thereof.

[4] The composition of any of paragraphs [1] to [3], wherein the cellulolytic background composition comprises (a) Aspergillus fumigatus cellobiohydrolase I or a variant thereof; (b) an aspergillus fumigatus cellobiohydrolase II or variant thereof; (c) an aspergillus fumigatus beta-glucosidase or variant thereof; and (d) a penicillium GH61 polypeptide having cellulolytic enhancing activity; or a homologue thereof.

[5] The composition of any of paragraphs [1] to [4], wherein (ii) the protease is derived from Bacillus, e.g., Bacillus amyloliquefaciens, e.g., the protease encoded by SEQ ID NO: 1.

[6] The composition of any of paragraphs [1] to [5], wherein (iii) the lipase is derived from a thermophilic fungus, such as Thermomyces lanuginosus, e.g., the lipase encoded by SEQ ID NO:2, or wherein (ii) the lipase is derived from a Humicola, e.g., Humicola insolens.

[7] The composition of any of paragraphs [1] to [6], wherein (iv) the beta-glucanase is a beta-glucanase derived from a member of the genus Aspergillus, e.g., Aspergillus aculeatus, e.g., a beta-glucanase encoded by the sequence encoded by SEQ ID NO. 4 or a homolog thereof.

[8]Paragraph [1]To [7]]The composition of any one of (a) wherein (v) the pectate lyase forms a multi-component enzyme composition comprising pectate lyase, xylanase and cellulase activities, e.g. Novozym 81243^TMPart (c) of (a).

[9] The composition of any of paragraphs [1] to [8], wherein (vi) the mannanase is an endo-mannosidase derived from Bacillus, e.g., Bacillus bogoriensis, e.g., the endo-mannosidase encoded by SEQ ID NO:6 or a homolog thereof.

[10] The composition of any of paragraphs [1] to [9], wherein (vii) the amylase is an alpha-amylase derived from a Rhizomucor, e.g., Rhizomucor pusillus, e.g., an alpha-amylase encoded by SEQ ID NO:5 or a homolog thereof.

[11] The composition of any of paragraphs [1] to [10], wherein the protease is present at a ratio between 0-20 wt/wt% of total enzyme protein, e.g., about 10 wt/wt%.

[12] The composition of any of paragraphs [1] to [11], wherein the beta-glucanase is present at a ratio of between 0-30 wt/wt% of total enzyme proteins, e.g., 15 wt/wt%.

[13] The composition of any of paragraphs [1] to [12], wherein the pectate lyase is present in a ratio between 0-10 wt/wt% of the total enzyme protein, e.g., 5 wt/wt%.

[14] The composition of any of paragraphs [1] to [13], wherein the mannanase or amylase is present at a ratio between 0-10 wt/wt% of total enzyme proteins, e.g., 5 wt/wt%.

[15] The composition of any of paragraphs [1] to [14], wherein the cellulolytic enzyme mixture is present in a ratio of between 40% -99% w/w of total enzyme protein, such as between 50% -90% w/w, such as between 60% -80% w/w, such as 65-75%.

[16] The composition of any of paragraphs [1] to [15], wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of cellulases, AA9 polypeptides, hemicellulases, Cellulose Inducible Proteins (CIPs), esterases, swollenins, ligninolytic enzymes, oxidoreductases, pectinases, proteases, and swollenins.

[17] The composition of any of paragraphs [1] to [16], wherein the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase, and glucuronidase.

[18] A method for dissolving waste, comprising:

(a) contacting the waste with the enzyme composition of any one of paragraphs [1] to [17 ].

[19] The method of paragraph [18], wherein the waste is Municipal Solid Waste (MSW).

[20] A method for producing a fermentation product, comprising:

(a) treating MSW with an enzyme composition according to any one of paragraphs [1] to [17 ];

(b) fermenting the solubilized and/or hydrolyzed MSW with one or more fermenting microorganisms to produce a fermentation product; and

(c) recovering the fermentation product from the fermentation.

[21] The method of any of paragraphs [18] to [20], wherein the waste is pretreated.

Claims

1. A method for dissolving waste, comprising:

(a) contacting the waste with an enzyme composition comprising a cellulolytic background composition and a protease selected from (i) a protease; (ii) (ii) a lipase and (iii) a beta-glucanase.

2. The method of claim 1, wherein the waste is Municipal Solid Waste (MSW).

3. A method for producing a fermentation product, comprising:

(a) treating Municipal Solid Waste (MSW) with an enzyme composition comprising a cellulolytic background composition and a protease selected from the group consisting of (i) a protease; (ii) (ii) a lipase and (iii) a β -glucanase;

(c) recovering the fermentation product from the fermentation.

4. The method of any one of claims 1-3, wherein the waste is pre-treated.

5. The method of any one of claims 1-4, wherein the composition comprises two or more enzymes selected from the group consisting of: (i) a protease; (ii) (ii) a lipase and (iii) a beta-glucanase (e.g., a protease and a lipase; a protease and a beta-glucanase; or a lipase and a beta-glucanase).

6. The method of any one of claims 1-4, wherein the enzyme composition comprises (i) a protease; (ii) (ii) a lipase and (iii) a beta-glucanase.

7. The method of any one of claims 1-6, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of: (iv) a pectate lyase; (v) a mannanase enzyme; and (vi) an amylase.

8. The method of any one of claims 1-7, wherein the cellulolytic background composition comprises one or more enzymes selected from the group consisting of: (a) cellobiohydrolase I or a variant thereof; (b) cellobiohydrolase II or a variant thereof; (c) a β -glucosidase or a variant thereof; and (d) a polypeptide having cellulolytic enhancing activity; or a homologue thereof.

9. The method of any one of claims 1-7, wherein the cellulolytic background composition comprises (a) a cellobiohydrolase I or a variant thereof; (b) cellobiohydrolase II or a variant thereof; (c) a β -glucosidase or a variant thereof; and (d) a polypeptide having cellulolytic enhancing activity; or a homologue thereof.

10. The process of claim 8 or 9, wherein the cellobiohydrolase I of (a) is aspergillus fumigatus cellobiohydrolase I or a variant thereof; (b) the cellobiohydrolase II of (a) is aspergillus fumigatus cellobiohydrolase II or a variant thereof; (c) the beta-glucosidase of (a) is an aspergillus fumigatus beta-glucosidase or a variant thereof; the polypeptide having cellulolytic enhancing activity of (a) and (d) is a penicillium GH61 polypeptide having cellulolytic enhancing activity; or a homologue thereof.

11. The method of any one of claims 1 to 10, wherein the protease (i) of the enzyme composition is derived from a bacillus, e.g., a bacillus amyloliquefaciens, e.g., a protease encoded by SEQ ID No.1 (e.g., a protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 1).

12. The method of any one of claims 1 to 11, wherein the lipase (ii) of the enzyme composition is derived from a thermophilic fungus, such as thermomyces lanuginosus, such as a lipase encoded by SEQ ID No. 2 (e.g., a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 2), or wherein the lipase (ii) is derived from a humicola, such as humicola insolens (e.g., having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, or both a humicola insolens-specific lipase, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity).

13. The process of any of claims 1-12, wherein the beta-glucanase (iii) in the enzyme composition is derived from a member of the genus aspergillus, such as aspergillus aculeatus, such as a beta-glucanase encoded by the sequence encoded by SEQ ID No. 4 or a homologue thereof (e.g. a beta-glucanase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 4).

14. The method of any of claims 7 to 13, wherein the pectate lyase (iv) of the enzyme composition forms a multi-component enzyme composition comprising pectate lyase, xylanase and cellulase activities, such as Novozym 81243^TMPart (c) of (a).

15. The method of any one of claims 7 to 14, wherein the mannanase (v) of the enzyme composition is an endo-mannosidase derived from Bacillus, e.g., Bacillus borgoriensis, e.g., an endo-mannosidase encoded by SEQ ID No. 6 or a homologue thereof (e.g., an endo-mannosidase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 6).

16. The method of any one of claims 7 to 15, wherein the amylase (vi) of the enzyme composition is an alpha-amylase derived from rhizomucor, e.g., rhizomucor pusillus, e.g., an alpha-amylase encoded by SEQ ID No.5 or a homolog thereof (e.g., an alpha-amylase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 5).

17. The method of any one of claims 1 to 16, wherein the protease of the enzyme composition is present in a ratio between 0-20 wt/wt% of total enzyme protein, such as about 5-15 wt/wt%, or about 10 wt/wt%.

18. The method of any one of claims 1 to 17, wherein the lipase of the enzyme composition is present in a ratio between 0-10 wt/wt% of total enzyme protein, such as about 2.5-7.5 wt/wt%, or about 5 wt/wt%.

19. The method of any one of claims 1 to 18, wherein the beta-glucanase of the enzyme composition is present in a ratio between 0-30 wt/wt% of the total enzyme proteins, such as 10-20 wt/wt%, or about 15 wt/wt%.

20. The method of any one of claims 7 to 19, wherein the pectate lyase of the enzyme composition is present in a ratio between 0-10 wt/wt% of the total enzyme protein, such as 2.5-7.5 wt/wt%, or about 5 wt/wt%.

21. The method of any one of claims 7 to 20, wherein the mannanase or amylase of the enzyme composition is present in a ratio of between 0-10 wt/wt% of total enzyme protein, such as about 2.5-7.5 wt/wt, or about 5 wt/wt%.

22. The method of any one of claims 1 to 21, wherein the cellulolytic background composition is present in the enzyme composition in a proportion of between 40-99 wt./wt.% of total enzyme protein, such as between 50-90 wt./wt.%, such as 60-80 wt./wt.%, such as 65-75 wt./wt.%.

23. The method of any one of claims 1 to 22, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of cellulases, AA9 polypeptides, hemicellulases, Cellulose Inducible Proteins (CIPs), esterases, swollenins, ligninolytic enzymes, oxidoreductases, pectinases, proteases, and swollenins.

24. The method of claim 23, wherein the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase, and glucuronidase.