WO2013017289A2

WO2013017289A2 - A method for producing biofuel and biogas from insoluble organics

Info

Publication number: WO2013017289A2
Application number: PCT/EP2012/003326
Authority: WO
Inventors: Bernd Peter ERNST; Petra KRACK
Original assignee: Sequence Laboratories Göttingen Gmbh
Priority date: 2011-08-04
Filing date: 2012-08-03
Publication date: 2013-02-07
Also published as: EP2739743A2; WO2013017289A3

Abstract

The present invention refers to a method for the production of biofuel and/or biogas, wherein (i) biomass comprising lignocellulose is decomposed with one or more microorganism(s) capable of decomposing said lignocellulose; and (ii) the biomass subjected to step (i) is converted to biofuel and/or biogas. Further, the present invention refers to the use of one or more microorganism(s) capable of decomposing lignocellulose for the production of biofuel and/or for the production of biogas, in particular methane. Moreover, the present invention refers to a method for the production of biofuel, wherein the moist rest product of a biogas plant is converted to biofuel by contacting said moist rest product with cellulase. Further, the present invention refers to the use of the moist rest product of a biogas plant for the production of biofuel, wherein said moist rest product is contacted with cellulase.

Description

A Method for Producing Biofuel and Biogas from Insoluble Organics The present invention refers to a biotechnological method for the production of biofuel and/or biogas from lignocellulose-comprising biomass. Further, it refers to the use of microorganisms capable of decomposing lignocellulose for the production of biofuel and/or biogas and to a method for the conversion of the moist rest product of a biogas plant to biofuel by means of cellulase.

Today, the prohibition or at least deceleration of climate change plays a crucial role in public perception and in science. In particular, the combustion of large amounts of fossil fuels such as, e.g., mineral oil, coal and domestic gas, leads to an immense annual carbon dioxide (C0₂) emission that severely intensifies undesired climate change. Further, the worldwide depletion of fossil fuels leads to severe political and economical conflicts.

Therefore, it is desired to change energy management increasingly from fossil fuels to renewable energies. The European Union, for instance, has established the serious ambition to increase the proportion of renewable energies to at least 10 % in the traffic sector until the year 2020 (Directive 2009/28/EC of the European Parliament and of the Council).

One renewable energy source of increasing importance is biogas and biofuel. In contrast to electric energy obtained from renewable energy sources, biogas and biofuel are well storable and universally usable. Biogas and biofuel are obtained from the conversion of biomass.

Today, the biomass mostly used for the production of biogas and biofuel predominantly originates from edible and forage crops. Biogas is often also generated by using stall manure. The production of biogas and biofuel by using edible and forage crops is also designated as biogas and biofuel production of the first generation. In temperate climate zones such as, e.g., in Europe and in the U.S.A., mostly corn, wheat, rye and sugar beets are used. In the tropical climate zone such as, e.g., in Brazil, mostly sugar cane is used. Therefore, the production of biogas and biofuel by production methods of the first generation directly competes with the production of foodstuff. In the past years, this led to perceptible price rises of foodstuff with severe consequences for the nutrition of the population of the Third World. Exemplarily, from 2010 to 2011, the wheat price increased by 44 % and the corn price even increased by 66 %. Further, the production of biogas and biofuel by using forage crops is comparably ineffective and large cultivated farming areas are typically used. Rain forest and other areas deserving protection are often severely negatively affected. Further, extensive fertilization and manuring of large areas is required.

For these reasons, several methods have been developed to generate biogas and biofuel by using lignocellulosic biomass. Here, not only the crops, but the most parts of a plant and even inedible plant are used. The production of biogas and biofuel by using lignocellulosic biomass is also designated as biogas and biofuel production of the second generation. The biogas and biofuel production of the second generation has the advantage that it is not in direct competition with food production and fertilizers are often abdicable. A large spectrum of biomass resources can be used as overall biomass averagely comprises approximately 70 % lignocellulose. Further, even waste, oddments and clippings of economical and agricultural plants such as, e.g., straw, husk, trester or splints may be used. Therefore, in principle, the production of biogas and biofuel by using lignocellulosic biomass is a promising approach to overcome many of the above-referenced problems.

However, the efficient production of biogas and biofuel by using lignocellulosic biomass is still hampered by the poor conversion of lignocellulose into sugars.

Lignocellulose is a typical insoluble organic. Typically, lignocellulose is composed of approximately 40-50 % cellulose, approximately 20-30 % hemicellulose and 20-30 % lignin. Cellulose is a linear polymer composed of glucose polymerized by an a- 1,4 glycosidic linkage. It typically forms microcrystalline structures which can only be poorly dissolved and hydrolyzed (Leschine, 1995). Hemicellulose is a heteropolysaccharide that is composed of different hexoses, pentoses and glucuronic acid. The hemicellulose xylane is often found in grass and wood. Lignin is an insoluble polymer. The cellulose microfibrils are conjugated with another by hemicellulose and/or lignin by covalent and non-covalent bonds (Tomme et al., 1995). These bonds, in particular the covalent bonds, are highly stable and nearly inert against chemical and biological hydrolysis.

Lignocellulose is poorly accessible for most of the cellulose degrading enzymes. Therefore, a pretreatment of lignocellulose is an important step to obtain higher yields of sugars that can be further converted into biogas and biofuel (see Figure 1).

However, during the pretreatment bears the risk that sugars, in particular pentoses, may be undesirably degraded and undesirable fermentation inhibiting agents may be generated. Further, lignin cannot be recovered by most of the methods employed in the art. The pretreatment of lignocellulose is typically the most expensive and laborious step of the production of bioethanol and the costs for said pretreatment step sums up to approximately 20-30 % of the total costs of bioethanol (Yang B und Wyman CE, 2008). However, in comparison with bioethanol production with no pretreatment, the pretreatment of lignocellulose-containing biomass can still reduce the costs per liter bioethanol approximately 6fold due to higher yields (Eggeman T und Richard T, 2005).

In general, the pretreatment can be enabled by physical, chemical and physicochemical means. Physical pretreatment may be, e.g., grinding, crushing, irradiation (e.g., gamma irradiation, cathode ray or microwave irradiation) and/or explosion (e.g., steam explosion, C0₂ explosion or S0₂ explosion). Chemical pretreatment may be, e.g., treatment with bases (basic hydrolysis) (e.g., sodium hydroxide solution and/or ammonia solution) or treatment diluted acids (acidic hydrolysis) (e.g., sulfuric acid, hydrochloric acid, phosphoric acid and/or nitric acid). Physicochemical pretreatment may be gas treatment (e.g., treatment with chlorine dioxide and/or sulfur dioxide), oxidation (e.g., hydrogen peroxide, active or oxygen or ozone treatment) and/or extraction of lignin (e.g., by a butyl alcohol solution and/or by an ethanol solution). Often, two or more of the aforementioned methods are also combined with another. Biomass subjected to grinding, crushing or the extraction of lignin is typically used for the production of biofuel, in particular ethanol. Biomass subjected to irradiation, explosion, any chemical treatment, gas treatment or oxidation is typically used for the production of biofuel or the production of biogas. However, all these pretreatment methods described above have severe drawbacks. The acidic hydrolysis has the disadvantage that the costs for the used acids and the acid- resistant containers are comparably high and that upon acidic hydrolysis inhibitors of the decomposition and fermentation of cellulose are generated that may potentially interfere with the further fermentation steps. These decomposition and fermentating inhibitors have to be removed by costly and laborious means. Basic hydrolysis has the disadvantage that the costs for the used bases are comparably high and that the lignin structure may be altered upon basic hydrolysis. Steam explosion may partly degrade the hemicellulose, acidic catalysts are required and toxic substances may be generated as a byproduct. The Liquid Hot Water (LHW) method has a high energy and water consumption and solid cellulose and lignin mass remains after treatment. The Ammonia Fiber Explosion (AFE) method requires the use of expensive ammonia and the regeneration of ammonia and it is comparably inefficient with samples of higher lignin content. Further, the lignin structure may be altered. The Ammonia Recycled Percolation (ARP) method results in high energy consumption. The use of Green Solvents has the disadvantage of high solvent costs and the solvents have to be recycled. For the use of supercritical fluids, high pressure is required and lignin and hemicellulose are not efficiently decomposed.

Additionally, acidic and heat-based pretreatment often leads to the production of inhibitors of the following fermentation steps, which may severely hamper the production of biofuel or biogas. These inhibitors are often weak acids (e.g., acetic acid, formic acid, ferulic acid), furan derivatives (e.g., furfural and 5-hydroxymethylfurfural) and/or lignin derivatives (e.g., vanillin, 4-hydroxybenzaldehyde, phenol). It has been shown that many yeasts are inhibited by phenol derivatives which may occure upon pretreatment with acids or steam (Palmqvist et al., 2000). Therefore, before further fermentation steps are conducted, the inhibitors have to be removed. This is sometimes performed by enzymes such as, e.g., laccases. However, the costs of said enzymes are comparably high and may approximately account for approximately 20-25 % of the final bioethanol price. Further, the incubation period is undesirably extended (Schubert, 2006). Alternatively, the generated inhibitors are sometimes removed by microorganisms (Naoyuki et. al., 2008; Nichols et. al., 2008; Palmqvist et. al., 1997). However, also this method is comparably laborious and costly as additional microorganisms have to be added to the pretreatment mixture that often tend to require conditions different to those used during the pretreatment procedure. Further, also here incubation period is undesirably extended. A further general disadvantage of the pretreatment methods used in the art is that the different forms of lignocelluloses found in nature require different decomposition methods. Exemplarily, the preferred decomposition method for corn leaves in the art is the treatment with 0.1 % sulfuric acid at a temperature in the range of approximately 160 to 200°C, whereas this method is not efficient when applied to corn stems (Donghai S. et al., 2006).

A further severe drawback is that often merely a fraction of the cellulose is hydrolyzed. Exemplarily, the decomposition of Bermuda grass and rice straw with 1.5 % sulfuric acid only 76 % of all sugars and of rice straw even only 60 % of all sugars are released (Saha BC et al., 2005).

For wheat straw, the acidic decomposition by means of diluted sulfuric acid at 160°C is the most promising pretreatment method. In fact, sometimes, no cellulases are required and lignin and hemicellulose are released from the cellulose with minimal degradation and also the hemicellulose is decomposed into sugars. However, after the pretreatment step, the acids have to be removed by extensive washing procedures, the used acid has to be recycled and inhibitors of the following fermentation steps are generated. Further, typical biogas plants mostly produce comparably large amounts of remnants such as moist rest product as a byproduct. These moist rest products account for approximately 8-12 % of the dry mass introduced into the biogas plant. Typically, this moist rest product still comprises large amounts of cellulose, hemicellulose, lignin and lignocellulose. Today, this moist rest is mostly used as a natural fertilizer in agriculture. However, today, the cultivated farming areas the nearer environment of biogas plants are often already overfertilized what leads to the need to transport the moist rest products over long distances to other agricultural areas. This consequently leads to a severe increase in cost and operating expenses and, thus, to a lower efficacy of the biogas plants. In the view of the above, the artisan will recognize that the methods known in the art are comparably inefficient, laborious and costly. Therefore, there is still an unmet need for further methods for pretreating and decomposing insoluble organics such as lignocelluloses. Surprisingly, in the context of the present invention, it has been found that the decomposition of biomass by means of one or more microorganism(s) capable of decomposing said lignocellulose leads to sufficient rates of the production of biofuel and/or biogas and that biofuel can also be produced by converting of the moist rest product of a biogas plant by contacting said moist rest product with cellulase.

In a first aspect, the present invention refers to a method for the production of biofuel and/or biogas, wherein

(i) biomass comprising lignocellulose is decomposed with one or more microorganism(s) capable of decomposing said lignocellulose; and

(ii) the biomass subjected to step (i) is converted to biofuel and/or biogas.

As used in the context of the present invention, the term "biofuel" may be understood in the broadest sense as any type of fuel which is derived in any way from biomass. Preferably, the biofuel in the context of the present invention is a liquid biofuel. The biofuel may mainly comprise an extensively pure compound, thus, may be a biofuel comprising more than 95 % of said compound and less than 5 % of one or more other compound(s), of more than 80 % of said compound and less than 20 % of one or more other compound(s) or of more than 75 % of said compound and less than 25 % of one or more other compound(s). Alternatively, the biofuel may be a mixture of different compounds.

Preferably the biofuel comprises one or more alcohol(s), one or more ester(s), one or more carbonic acid(s), one or more ketone(s), one or more aldehyde(s) or one and/or more terpene(s). More preferably, the biofuel comprises one or more alcohol(s), one or more ketone(s) (e.g., acetone), one or more aldehyde(s) and/or comprises one or more ester(s). Even more preferably, the biofuel comprises one or more alcohol(s) and/or comprises one or more ester(s). Even more preferably, the biofuel may comprise more than 50 % (v/v), more then 70 % (v/v), more than 80 % (v/v), more than 90 % (v/v) or more than 95 % (v/v) of one or more alcohol(s). These alcohols are preferably aliphatic alcohols (e.g., methanol, ethanol, n- propanol, isopropanol and/or butanol), more preferably aliphatic alcohols of the general molecular formula H-C_nH_2n-OH, even more preferably, one of the first four aliphatic alcohols with n = 1-4 (i.e., methanol, ethanol, propanol and/or butanol). In the context of the present invention these alcohols may also be designated as "bioalcohols" (i.e., as "biomethanol", "bioethanol", "biopropanol" and "biobutanol"). Due to its chemical and technical characteristics, in the context of biofuel, butanol is sometimes also designated as "biogasoline". Alternatively or additionally, the alcohol may be a di-, tri or polyalcohol such as, e.g., glycerol. Even more preferably, the biofuel in the context of the present invention comprises more than 50 % (v/v), more then 70 % (v/v), more than 80 % (v/v), more than 90 % (v/v), or more than 95 % (v/v) ethanol. Most preferably, the biofuel of the present invention comprises at least 90 % (v/v) ethanol.

Alternatively or additionally, the biofuel may also be any other biofuel known in the art such as, e.g., biodiesel, green diesel, vegetable oil, one or more bioethers or another biological diesel or gasoline substitute.

In the context of the present invention, biodiesel may be understood in the broadest sense as a vegetable oil- or animal fat-based diesel-like fuel mainly composed of one or more long-chain alkyl alcohol ester(s). These alcohols are preferably methanol, ethanol and/or propanol. Biodiesel is typically made by chemically reacting fatty acids obtained from any fat known in the art such as, e.g., vegetable oil and/or animal fat esterified with an alcohol.

Green diesel, also designated as "renewable diesel", may be understood as a form of diesel-like fuel which is derived from renewable feedstock from any biomass used for this purpose in the art such as, e.g., plant material such as, e.g, canola, algae, tallow, jatropha and/or salicornia. In contrast to biodiesel, green diesel uses traditional fractional distillation (Shi S et al., 201 1) to process the oils instead of transesterification.

Vegetable oil in the context of the present invention may be understood as any biological oil known in the art such as, e.g., palm oil, soybean oil, rapeseed oil, canola oil, sunflower oil, peanut oil, cottonseed oil, coconut oil, olive oil or a mixture of two or more thereof. Preferably, in the context of the present invention, the vegetable oil is filtered waste vegetable oil. Vegetable oil may be waste vegetable oil (WVO) if it is oil that was discarded from a restaurant or straight vegetable oil (SVO) or pure plant oil (PPO) or a mixture thereof. Bioethers are also designated as "fuel ethers" or "oxygenated fuels".

Another biological diesel substitute may preferably be hydrogenated oils and/or fat such as, e.g., hydrogenated vegetable oil. The resulting product may then be a straight chain hydrocarbon with a high cetane number, low in aromatics and sulfur and does extensively not contain oxygen.

It will further be understood that all the biofuels listed above may be mixed, blended and/or naturally combined with another in any ration of mixture. Further, it will be understood that the biofuel obtained by the present invention may also be blended with biofuel obtained from other sources and/or may be mixed with one or more mineral oil(s) and/or may be blended with other component such as, e.g., one or more anti-freezing agent(s) and/or may be blended with one or more mineral oil product(s), such as, e.g., refined oils such as, e.g., diesel and/or gasoline. The biofuel may also contain traces of water, wherein these traces preferable account for a water content of below 25 % (v/v), below 10 % (v/v), below 5 % (v/v), below 4 % (v/v), below 3 % (v/v), below 2 % (v/v), below 1 % (v/v), or below 0.5 % (v/v). In the context of the present invention, the term "biogas" may be any inflammable gas obtained from biological sources. Preferably, biogas comprises more than 40 % (v/v), more than 50 % (v/v), more than 60 % (v/v), more than 70 % (v/v), more than 80 % (v/v), or even more than 90 % (v/v) methane. Typically, biogas comprises between 50 and 75 % (v/v) methane. Further, biogas may comprise one or more other component(s) such as, e.g, carbon dioxide, nitrogen, hydrogen, hydrogen sulfide and/or oxygen.

The obtained biogas may be subjected to a drying procedure, to a desulfurization process and/or to the abscission of the carbon dioxide fraction as known in the art (Kobayashi T et al., 2011). Further, optionally, siloxanes and/or carbohydrates may be removed from the biogas. The biogas may or may not be compressed.

As used throughout the present invention, the term "biomass" refers to any biological material. It may be biological material from living, or recently living organisms. Herein, the term "recently living" may be understood as biological material from any biological source that results from organisms that died less than 100 years, less than 20 years, less than ten years, less than 5 years, less than 1 year, less than six months, less than three months or less than 1 month. Therefore, the term "recently living" as used herein does not refer to fossil sources of organisms that stopped living thousands or millions of years ago. Biological material may comprise cellular and extracellular matrix of one or more organism(s) as well as material formed by these organisms including but not limited to any material originating from plants (e.g., wood, bark, stem, dead tree, branch, tree stump, leaves, husk, liquid pitch, tree gum, crops, stover, bagasse, fruit, seed, pit, peels, seed coat, pollen, plant oils, sawmill and paper mill discards, trester and/or other plant extracts) and/or any material originating from animals (feces, excrements, urine, gland secretions, eggs, cytoplasm, sanies, tallow and/or other animal extracts), clearing sludge, biosolids, compost, any material originating from fungals and/or any material originating from bacteria (eubacteria and/or archaebacteria). Plants or animals may be unicellular and/or multicellular organisms.

Preferably, the biomass comprises insoluble organics. More preferably, the biomass comprises at least 25 % (w/w) insoluble organics, at least 50 % (w/w) insoluble organics or at least 75 % (w/w) insoluble organics. As used herein, an insoluble organic may be any organic substance that is insoluble or poorly soluble in water. Typically, an insoluble organic may have a solubility in water of less than 10 mg/ml, less than 1 mg/ml, less than 0.1 mg/ml, less than 0.05 mg/ml, less than 0.01 mg/ml, less than 0.005 mg/ml or even less than 0.001 mg/ml. The insoluble organic may have a polar or a nonpolar surface. The insoluble organic may be charged or uncharged. The insoluble organic may be a polymeric molecule of a molecular mass of more than 1000 Da, more than 10,000 Da, more than 50,000 Da or even more than 100,000 Da. Alternatively, the insoluble organic may also be a small molecule of a molecular mass of less than 5,000 Da or even less than 1,000 Da. Preferably, the insoluble organic is a polymeric molecule, more preferably a polymeric molecule with a polar surface, even more preferably a polymeric molecule bearing hydroxyl moieties at the surface, most preferably a polymeric carbohydrate. Typically, the insoluble organic may be a fiber material or a mixture of two or more fiber materials, in particular cellulose, hemicellulose, lignin and/or lignocellulose.

Preferably, at least 25 % (w/w), at least 50 % (w/w), at least 75 % (w/w) or at least 80 % (w/w) of the biomass originates from plant material, in particular stems, leaves and/or husk of one or more edible and/or forage plant(s). Alternatively or additionally, parts of the biomass may originate from stall manure.

As used in the context of the present invention the term "edible plant" may be understood in the broadest sense as any plant of which at least parts can be consumed by humans and/or fed by animals.

As used in the context of the present invention the term "forage plant" may be understood in the broadest sense as any plant that may be used to produce food for humans and/or feed for animals.

It will be understood by a person skilled in the art that the choice of the biomass depends on the desired product. Typically, for the production of biofuel, preferably, more than 50 % (w/w) of biomass originated from plant material will be used. In particular, discards from food and brin production are preferably used in the context of the present invention such as e.g., straw, stems, seed coat, husk and leaves may be used. Exemplarily, crop straw, hemp stems, crop husk, grass cut may be used.

In contrast, for the production of biogas, biomass originated from plant material and/or biomass originated from animal, fungal or bacterial material may be used. Typically considerable amounts of stall manure and/or discards from forage plants will be used.

It will be understood by the person skilled in the art that biomass of different sources may be combined with another in any ratio.

In the content of the present invention, biomass is preferably biomass comprising lignocellulose. In this context, the term "lignocellulose" may be understood in the broadest sense as a material comprising high contents of cellulose and lignin. Typically, lignocellulose will mainly comprise cellulose, hemicellulose and lignin. Herein, the carbohydrate polymers, i.e., cellulose and/or hemicelluloses, are typically covalently and/or non-covalently conjugated with lignin. Typically, lignocellulose is comprised in many parts of a multicellular plant, in particular in the leaf, stem, wood, bark material, in straw, in seed coats and/or in husk. According to the present invention, the lignocellulose is decomposed with one or more microorganism(s) capable of decomposing said lignocellulose.

Throughout the whole invention, the term "microorganism", "micro-organism", "micro organism", "microorganism" and "microbe" may be understood interchangeably in the broadest sense as any organism of microscopic size, thus, every organism that has a diameter size smaller than 1 m^"3, preferably smaller than 5 m^"4, more preferably smaller than 1 m^"4, even more preferably smaller than 5 m^"5, even more preferably smaller than 1 m^"5, even more preferably smaller than 5 m^"6 and even more preferably smaller than 1 m^"6.

The microorganism may be a unicellular or multicellular organism. Preferably, the microorganism is unicellular or lives in a colony of unicellular organisms. The microorganism in the sense of the present invention may be a prokaryotic microorganism (i.e., a bacterium (eubacterium or archaebacterium (archaeum))) or a eukaryotic microorganism (protist) (i.e., a fungal microorganism, a plant microorganism or an animal microorganism). Preferably, a microorganism in the sense of the present invention is a bacterium or a fungal or animal microorganism. The microorganism may be an anaerobe microorganism, an aerobe microorganism or may be a microorganism that can adapt its metabolism to anaerobe and aerobe conditions.

In the context of the one or more microorganism(s) capable of decomposing lignocellulose, a microorganism is preferably an anaerobe bacterium or a fungal or animal microorganism that can have an anaerobe metabolism. Exemplarily, in the context of the one or more microorganism(s) capable of decomposing lignocellulose, an anaerobic bacterium may be selected from the group consisting of but may not be limited to Bacillus fusiformis, Lysinibacillus sphaericus, Spirochaetes, Bacillus amylobacter, Plectridium friebes and/or Clostridia. These bacteria are commercially available, e.g., at the Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig, Germany.

Additionally or alternatively, a microorganism in the context of the one or more microorganism(s) capable of decomposing lignocellulose may be a microorganism that may originate from a rumen of any ruminant animals (e.g. cattle, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn, or nilgai). Herein, the terms "rumen" and "paunch" may be understood interchangeably. Typically, these one or more microorganism(s) will prefer a slightly acidic pH such as, e.g., a pH between pH 7.0 and 6.0, a pH between pH 6.5 and 5.5 or a pH between pH 5.5 and 4. However, there may also be microorganisms that prefer another pH.

Additionally or alternatively, a microorganism in the sense of the present invention may be a microorganism that may originate from the gastrointestinal tract of any animal that is able to decompose plant material comprising lignocellulose fibers (e.g., leaves and/or stems)). This microorganism will typically prefer a slightly basic pH such as, e.g., a pH between pH 7.0 and 8.0, a pH between pH 7.5 and 8.5 or a pH between pH 8.0 and 9.5. However, there may also be microorganisms that prefer another pH.

In this context, it will be understood that different microorganisms may be combined with another in any proportion.

As used in the context of decomposition of lignocellulose, the term "decompose" may be understood in the broadest sense as the biochemical degradation, digestion, fibrolysis or cleavage of lignocellulose. In particular, the term "decompose" refers to the hydrolysis of ester linkages between lignin and hemicellulose or cellulose. This hydrolysis may liberate the cellulose and/or hemicellulose polymers in a way that cellulose- and/or hemicellulose metabolizing enzyme(s) may have access to the cellulose and/or hemicellulose molecules and may, therefore, be able to metabolize these.

In the context of the step of converting said biomass to biofuel and/or biogas, the term "microorganism" may be understood in the broadest sense as any microorganism that can convert the biomass subjected to the decomposition of lignocellulose to biofuel and/or biogas. Preferably, in this context, the microorganism can metabolize one or more polysaccharide(s), in particular one or more polysaccharide(s) comprising cellulose. In this context, the microorganism may be an anaerobe microorganism, an aerobe microorganism or may be a microorganism that can adapt its metabolism to anaerobe and aerobe conditions. Preferably, the microorganism may be an anaerobe microorganism or may be a microorganism that can adapt its metabolism to anaerobe and aerobe conditions. Exemplarily, the microorganism may be a microorganism converting cellulose into glucose and/or ethanol. Alternatively, the microorganism may be a microorganism converting cellulose into biogas, in particular methane. Exemplarily, the microorganism may be yeast or may be a methanogenic bacterium. In this context, it will be understood that different microorganisms may be combined with another in any ratio and that also one or more microorganism(s) and one or more enzyme(s) may be combined with another.

As used in the context of the present invention, the terms "methanogenic microorganism" and "methanogen" may be understood interchangeably in the broadest sense as any microorganism capable of producing methane from an organic biomass feedstock. Typically, a methanogenic microorganism produces methane as a metabolic byproduct in anoxic conditions. Preferably, a methanogenic microorganism is a methanogenic bacterium, more preferably an anaerobe methanogenic bacterium, most preferably an anaerobe methanogenic archaeum. Exemplarily, methanogenic microorganisms may originate from wetlands, where they are responsible for marsh gas and/or from the guts of herbivore animals such as, e.g., ruminants, insects and humans, where they are responsible for the methane content of belching in ruminants and flatulence in humans. Some other methanogenic microorganisms are extremophiles which are exemplarily extensively inert to high temperatures. Many methanogenic microorganisms comprise coenzyme F430, coenzyme B, coenzyme M, methanofuran and/or methanopterin. In the context of the step of converting said biomass to biofuel and/or biogas, an enzyme may be used. Herein, the term "enzyme" may be understood in the broadest sense as any polypeptide that catalyzes one or more chemical reaction(s). Herein, the term "catalyze" refers to the increase the rates of the reaction(s). In the context of the present invention, enzymes may preferably be able to catalyze the conversion of one or more polysaccharide(s) into one or more monosaccharide(s), catalyze the conversion of one or more monosaccharide(s) into one or more intermediate product(s), catalyze the conversion of said one or more intermediate product(s) into one or more alcohol(s) and/or catalyze the conversion of said one or more intermediate product(s) into one or more biogas component(s). More preferably, the enzymes may be enzymes catalyzing the conversion of cellulose into glucose, catalyzing the conversion of glucose into the intermediate products (e.g., glucose-6-phosphate, fructose-6-phosphate, fructose- 1 ,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 1,3-bisphosphoglycerate, 2,3- bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate) and/or catalyzing the conversion of said one or more intermediate product(s), in particular pyruvate, into ethanol and/or catalyzing the conversion of said one or more intermediate product(s) into methane.

It will be understood by a person skilled in the art that typically more than one enzyme may be used and/or the combination of one or more enzyme(s) and one or more microorganism(s) may be used.

An enzyme may be obtained from any source known in the art. Exemplarily, it can be obtained from any animal, plant, fungal or bacterial organism. Alternatively, it may be excreted by any animal, plant, fungal or bacterial organism. It may be obtained from its natural source or may be obtained from any molecular biology cloning method known in the art or may be of synthetical origin or a combination thereof. The enzyme may or may not have been subjected to a posttranscriptional modification. Therefore, exemplarily, the enzyme may or may not be lipidated, phosphorylated, sulfated, cyclized, oxidated, reduced, decarboxylated, acetylated, acylated, amidated, deamidated, biotinylated or bound to one or more other small molecule(s) and/or terpene(s). Further, the enzyme may or may not comprise one or more intramolecular disulfide bond(s) or one or more intermolecular disulfide bond(s). Optionally, an enzyme of the present invention may further bear one or more other modification(s). Exemplarily, the enzyme may or may not be amidated or capped at its C-terminus (e.g., by a non-amino acid moiety auch as, e.g., a bead and/or a polymer, in particular a hydrophilic polymer). As used herein, the terms "C-terminus", "C- terminal end", "carboxy terminus", "carboxyterminus" and "carboxyterminal end" may be understood interchangeably. Further, the N-terminus may or may not be capped (e.g., by an acetyl moiety, a methyl moiety, a pyroglutamyl moiety, a bead and/or or a polymer, in particular a hydrophilic polymer). As used herein, the terms "N-terminus", "N-terminal end", "amino terminus" and "amino terminal end" may be understood interchangeably. The capping and/or amidation at one terminus or both termini may render the enzyme more stable against endo- and/or exopeptidases. Further, optionally, the enzyme may or may not be stabilized by immobilization to a surface (e.g., the surface of the reaction container, the fermenter, a column and/or beads). Alternatively or additionally, the enzyme may optionally be conjugated with one or more polymer(s), in particular one or more hydrophilic polymer(s). Exemplarily, such hydrophilic polymer may be polyethylene glycol (PEG) or derivatives thereof, polyethylene imine (PEI) or derivatives thereof, polyacrylic acid or derivatives thereof, in particular hydroxypropyl methacrylate (HPMA), polysaccharide(s) or derivatives thereof, preferably amino polysaccharide(s), aminoalkyl polysaccharide(s), hydroxyalkyl polysaccharide(s) and/or alkyl polysaccharide(s), lipopolysaccharide(s), hydrophilic polypeptide(s) and/or conjugate(s) or blockpolymer(s) comprising two or more of the aforementioned.

However, a unit of the one or more employed enzyme(s) may optionally also comprise salts, cofactors, one or more filling material(s) and/or one or more solvent(s) (e.g., water, dimethyl sulphoxide (DMSO), dimethylformamide (DMF)). The employed unit of the one or more employed enzyme(s) may be directly isolated or may be stored. It may be of commercial origin. It may be present in a solution or may be frozen, dried or freeze dried.

Optionally, an enzyme in the context of the present invention may also be one or more commercially provided enzyme(s) and/or one or more commercially provided mixture(s) of enzymes. Exemplarily, in the context of the present invention, a commercially obtainable mixture of cellulases may be used (such as, e.g., a Biogazym mixture (ASA Spezialenzyme GmbH, Wolfenbiittel, Germany). These enzymes may optionally comprise or may be free of Tricoderma reseei spores.

In the context of the present invention, the term "converting" may be understood in the broadest sense as an alteration of a chemical structure into another chemical structure, thus a chemical reaction, preferably by means of the metabolism of one or more microorganism(s) and/or by means of one or more enzyme(s) catalyzing said reaction. Preferably, the conversion in the sense of the present invention is a fermentation step.

In the context of the present invention, the decomposition of biomass comprising lignocellulose with one or more microorganism(s) may be considered as a pretreatment. This pretreatment may be conducted before the conversion of the pretreated biomass to biofuel and/or biogas or concomitant with the conversion of the pretreated biomass to biofuel and/or biogas.

After or concomitant to the pretreatment, the production of biofuel from complex organic biomass is typically characterized by the subsequent steps of hydrolyzing the biomass to monomers, fermentation of the monomers and purification of the fermentation products, thus, the biofuel. In a first step, the hydrolysis of the biomass into monomers, typically one or more polysaccharide(s) are hydrolyzed into one or more monosaccharide(s). Additionally, one or more fat(s) may be hydrolyzed into fatty acids and glycerol and one or more protein(s) may be hydrolyzed into amino acids. Exemplarily, cellulose may be converted into glucose by means of one or more cellulase(s). Starch (e.g., amylose, amylopectin) may be converted into glucose by one or more starch-converting enzyme(s) (e.g., amylase(s)). Pectin may be converted into galacturonic acid by one or more pectinase(s). Other complex structures, such as, e.g, one or more terpene(s), one or more cholesterol(s) and one or more steroid(s) and/or one or more derivative(s) thereof, may also be converted into their one or more monomer(s) such as, e.g., acetate.

The one or more monosaccharide(s) obtained from the preceding step and likewise also the one or more amino acid(s), fatty acids, glycerol and the one or more other degradation product(s), may be converted to biofuel, preferably, one or more alcohol(s) by any conversion method known in the art. As laid out above, conversion is preferably conducted with one or more microorganism(s) such as one or more anaerobe bacterium/bacteria (e.g., Zymomonas mobilis) and/or yeast under anaerobe conditions. Alternatively or additionally, also one or more isolated enzyme(s) and/or one or more extract(s) from one or more fermentating microorganism(s) may be used for the generation of biofuel.

In a further step, the biofuel obtained from the method described above is isolated by any means known in the art. Exemplarily, the biofuel may be isolated by distillation, filtration, centrifugation, isolation via a membrane, in particular a semipermeable membrane, skimming and/or decantation. Preferably, the biofuel is isolated by distillation.

Most preferably, the employed biomass has high polysaccharide content, is converted by one or more microorganism(s) and/or one or more enzyme(s) converting the polysaccharide into monosaccharides, in particular glucose. Then, the glucose may be converted into ethanol, which is finally separated by the crude mixture by distillation.

The processes described above, in particular the hydrolysis and fermentation may take place subsequently or chronologically simultaneously and concomitantly. The delineation depicted above merely represents the flow of the biochemical processes. The reactions described and depicted above may be catalyzed by various microorganism(s) and/or enzyme(s). This/these microorganism (s) and/or enzyme(s) may be added to the reaction mixture at once or subsequently. Alternatively, several or even all reactions described above may be catalyzed by a single microorganism. Even the isolation, in particular when conducted via a semipermeable membrane can also take place concomitantly to one or even both of the steps of hydrolysis and fermentation.

As used throughout the present invention, the term "monosaccharide" may be any single sugar known in the art. Exemplarily, a monosaccharide may be a triose (e.g., an aldotriose (e.g. glyceraldehyde) or a ketotriose (e.g., dihydroxyacetone)), a tetrose (e.g., an aldotetrose (e.g., erythrose, threose) or a ketotetrose (e.g., erythrulose)), a pentose (e.g., an aldopentose (e.g., ribose, deoxyribose, arabinose, xylose, lyxose) or a ketopentose (e.g., ribulose, xylulose)), a hexose (e.g., an aldohexose (e.g., allose, altrose, glucose, mannose, gulose, idose, galactose, talose), other hexoses (e.g., glucuronic acid, galacturonic acid, N- acetylglucosamine/N-acetylchitosamine, glucosamine/chitosamine, N- acetylgalactosanine/N-acetylchondrosamine, fucose, desoxygalactose, rhamnose, deoxymannose, chinovose, deoxyglucose) or a ketohexose (e.g., fructose)) or a higher monosaccharide unit such as, e.g., heptulose, sedoheptulose, desoxymannooctulosonic acid/ketodesoxyoctonic acid, sialic acid/ N-acetylneuraminic acid. Preferably, the monosaccharide is a hexose or a pentose, more preferably the monosaccharide is a hexose, even more preferably the monosaccharide is glucose or fructose. Most preferably, the monosaccharide is glucose. It will be understood by a person skilled in the art that the monosaccharide of the present invention may also be any modified form of a monosaccharide that can still be converted to biofuel and/or biogas by the employed fermentating one or more enzyme(s) and/or one or more fermentating microorganism(s). Exemplarily, the monosaccharide may be lipidated, phosphorylated, sulfated, cyclized (e.g. as lactone, lactame, amine and/or imine), oxidated (e.g., into an aldehyde, ketone and/or carbonic acid), reduced (e.g., into a reduced sugar), decarboxylated, acetylated, acylated, amidated, biotinylated or bound to one or more other small molecule(s) and/or terpene(s).

After pretreatment, the production of biogas typically starts with the same step as the production of biofuel, namely, the hydrolysis of the employed biomass to monomers. Then, the steps of acidogenesis and acetogenesis may follow. Finally, in a last step the biogas is produced. Hydrolysis is typically followed by the acid-forming phase of acidogenesis. In this process, acidogenic bacteria turn the products of hydrolysis into simple organic compounds, mostly short chain (volatile) acids (e.g., propionic, formic, lactic, butyric, or succinic acids), ketones (e.g., acetone) and alcohols (e.g., ethanol, methanol, glycerol). The specific concentrations of products formed in this stage may vary with the type of bacteria as well as with culture conditions, such as temperature and pH. Typical reactions in the acid- forming stages may be the conversion of glucose to ethanol: C₆H₁₂0₆ «→ 2CH₃CH₂OH + 2C0₂ and the transformation of glucose to propionate:

C₆H₁₂0₆ + 2H₂ «→ 2CH₃CH₂COOH + 2H₂0

The next stage of acetogenesis is often considered with acidogenesis to be part of a single acid forming stage. Biological oxygen demand (BOD) and chemical oxygen demand (COD) may be reduced through these pathways. Acetogenesis may occur through carbohydrate fermentation, through which acetate is the main product and other metabolic processes. The result may be a combination of acetate, C0₂ and H₂. The role of hydrogen as an intermediary may be of critical importance to AD reactions. Long chain fatty acids, formed from the hydrolysis of lipids, are oxidized to acetate or propionate and hydrogen gas may be formed. Under standard conditions, the presence of hydrogen in the solution may inhibit the oxidation. Typically, the reaction only proceeds if the hydrogen partial pressure is low enough to thermodynamically allow the conversion. The presence of hydrogen scavenging bacteria (HMBs) that consume hydrogen, thus, lowering the partial pressure, may ensure thermodynamic feasibility and thus the conversion of all the acids. As a result, the concentration of hydrogen, measured by partial pressure, may be used as an indicator of the health of a digester. Exemplarily, the free energy value of the reaction that converts propionate to acetate:

CH₃CH₂COO^" + 3H₂0 «→ CH₃COO^" + H⁺ + HC0₃ ^" + 3H₂ is +76.1 kJ, so that this reaction is thermodynamically impractical. When acetate and hydrogen are consumed by bacteria, however, the free energy may become negative. In general, for reactions producing H₂, hydrogen should typically have a low partial pressure for the reaction to proceed. Other important reactions in the acidogenic stage may involve the conversion of glucose, ethanol and bicarbonate to acetate:

C₆H,₂0₆ + 2H₂0 <→ 2CH₃COOH + 2C0₂ + 4H₂

CH₃CH₂OH + 2H₂0 «→ CH₃COO^" + 2H₂ +H⁺

2HC0₃ ^" + 4H₂ + H⁺ «→ CH₃COO^" + 4H₂0

The transition of the substrate from organic material to organic acids in the acid forming stages may cause the pH of the system to drop. This may be beneficial for the acidogenic and acetagenic bacteria that typically prefer a slightly acidic environment, with a pH of 4.5 to 5.5 and are typically less sensitive to changes in the incoming feed stream.

In a last step, the acids of the step described above may be converted into the biogas, in particular methane. The step of methane production is also designated as methanogenesis. Methanogenesis is mostly catalyzed by methanogenic bacteria, in particular methanogenic anaerobic bacteria. The methanogenic anaerobic bacteria involved in this stage, also known as methanogenesis or methane formation, may be the same bacteria that occur naturally in deep sediments or in the rumen of herbivores. Exemplarily, this population may convert the soluble matter into methane, about two thirds of which is derived from acetate conversion: 2CH₃CH₃OH+ C0₂ <→ 2CH₃COOH + CH₄

CH₃COOH <→ CH₄ + C0₂ and/or the fermentation of an alcohol, such as methyl alcohol: CH₃OH + H₂ <→ CH₄ + H₂0 and/or may be the result of carbon dioxide reduction by hydrogen:

C0₂ + 4H₂ <→ CH₄ + 2H₂0 Alternatively, methane may also be produced by the conversion of formic acid, methanol, methylamine(s), dimethyl sulfide and/or methanethiol and salts thereof. Methanogens often tend to be comparably sensitive to changes and prefer a neutral to slightly alkaline environment. If the pH is allowed to fall below 6, many methanogenic bacteria may loose viability. Therefore, typically the pH of the fermenter may be increased before the step of methanogenesis. Methanogenesis is the rate-controlling portion of the process of production of biogas because methanogens typically have a much slower growth rate than acidogenesis. Therefore, the kinetics of the entire process can often be described by the kinetics of methanogenesis. Further, also sulfuric and nitrogen components of the biomass may be converted into gas, such as, e.g., hydrogen sulfide and ammonia.

In a most preferred embodiment, the employed biomass comprises a high content of polysaccharides such as, e.g., cellulose and/or starch and is converted into gas with a high content of methane.

It will be understood by a person skilled in the art that all processes, thus, the hydrolysis, acidogenesis, acetogenesis and methanogenesis, may take place subsequently or chronologically simultaneously and concomitantly. The delineation depicted above merely represents the flow of the biochemical processes. The reactions described and depicted above may be catalyzed by one or more microorganism(s) and/or one or more enzyme(s). This/these microorganism(s) and/or enzyme(s) may be added to the reaction mixture at once or subsequently. Alternatively, several or even all reactions described above may be catalyzed by a single microorganism.

In a preferred embodiment of the present invention, the one or more microorganism(s) capable of decomposing lignocellulose are anaerobic microorganism(s), preferably anaerobic bacteria, in particular anaerobic bacteria selected from the group consisting of (i) Bacillus fusiformis,

(ϋ) Lysinibacillus sphaericus,

(iii) Spirochaetes,

(iv) Bacillus amylobacter,

(v) Plectridium friebes, and/or (vi) Clostridia.

As used throughout the present invention, the terms "anaerobic microorganism" and "anaerobe" may be understood interchangeably in the broadest sense as any organism that does not require oxygen for growth. Often, the presence of oxygen may even have a negative impact on the viability of the anaerobic microorganism. Exemplarily, the anaerobic microorganism may be an obligate anaerobic microorganism, an aerotolerant microorganism or a facultative anaerobic microorganism. In this context an obligate anaerobic microorganism cannot use oxygen for growth and is typically harmed by the presence of oxygen. An aerotolerant microorganism cannot use oxygen for growth, but tolerates the presence of oxygen. A facultative anaerobic microorganism may grow without oxygen but can utilize oxygen if it is present. Typically, obligate anaerobic microorganisms may use fermentation or anaerobic respiration, whereas aerotolerant microorganisms are strictly fermentative. In the presence of oxygen, facultative anaerobic microorganisms prefer to use aerobic respiration, whereas, without oxygen, some of them ferment, some use anaerobic respiration.

The term "anaerobic bacterium" as used herein refers to an anaerobic microorganism that is a bacterium.

Highly preferably, the one or more microorganism(s) comprises a species of anaerobe Clostridia bacteria. As throughout the present invention, Clostridia preferably are Clostridia producing no or low amounts of toxic metabolites. Particularly preferably, Clostridia are Clostridium acetobutylicum producing only minor amounts of toxic metabolites compared to other Clostridia.

Most preferably, the composition of the present comprises Spirochaetes and Clostridia, in particular at least one species of Spirochaetes and Clostridium acetobutylicum, and, optionally, one or more other species of bacteria. Spirochaetes and Clostridia may be also found in the gastrointestinal tract of termites.

Preferably, the biomass to be subjected to the methods of the present invention is extensively free of Candida yeasts such as Candida albigans. More preferably, the biomass to be subjected to the methods of the present invention is entirely free of Candida yeasts such as Candida albigans.

In a preferred embodiment of the present invention, the step of biomass comprising lignocellulose is decomposed with one or more microorganism(s) capable of decomposing said lignocellulose (step (i)) and/or the step of converting said biomass to biofuel and/or biogas (step (ii)) is/are conducted in a moistened environment at a temperature suitable for the activity of said one or more microorganism(s), preferably wherein said temperature is below 100°C, more preferably wherein said temperature is below 75°C, even more preferably wherein said temperature is below 60°C, in particular wherein said temperature is below 50°C.

In this context, the term "moistened environment" may be understood in the broadest sense as an environment with high humidity.

As used in the context of the present invention, the term "fermenter" may be understood in the broadest sense as any bioreactor that is suitable for the one or more microorganism(s) employed in the context of the present invention. Therefore, in general, a bioreactor may refer to any manufactured or engineered device or system that supports a biologically active environment. The fermenter may be composed of any material known for this purpose in the art such as, e.g., coated or uncoated stainless steal, another alloy that is coated or uncoated, glass material or plastic material. The environmental conditions of the fermenter such as, e.g., gas in- and output (i.e., air, oxygen, nitrogen, carbon dioxide, biogas), temperature, pH and dissolved oxygen levels and/or agitation speed/circulation may be adapted to the employed microorganism(s). In the context of the present invention, there will typically be no oxygen input and no or a low agitation rate. Further, there will typically be no input of light, but the fermenter will be light impermeable. The fermenter may have any shape. In order to disburden cleaning and to improve pressure resistance, it will typically have rounded edges or will be extensively spherical. The fermenter may comprise a heat exchanger to maintain the bioprocess at a constant temperature and/or to avoid overheating of the fermenter. Therefore, the fermenter may optionally need refrigeration by, e.g., an external jacket and/or with internal coils. This heat may also be needed for technical processes. Most preferably, the biomass is present in a liquid environment, mainly comprising water. The mixture of water, biomass and microorganism(s) may also be understood as culture broth. Herein, the water may be added or may originate by the added biomass.

Alternatively, the biomass can be moisturized and be present in an environment mainly composed of gas with a relative humidity of more than 50 %, more preferably more than 60 %, even more preferably more than 70 %, even more preferably more than 80 %, even more preferably more than 90 % even more preferably more than 95 % or even closed to 100 %.

The temperature suitable for the activity of the employed one or more microorganism(s) may be controlled and adapted or may be not controlled and adapted. In a highly preferred embodiment, the temperature is between 20°C and 50°C, even more preferably between 25°C and 45°C, most preferably between 25°C and 40°C.

In another preferred embodiment of the present invention, the step of biomass comprising lignocellulose is decomposed with one or more microorganism(s) capable of decomposing said lignocellulose (step (i)) and/or the step of converting said biomass to biofuel and/or biogas (step (ii)) is/are conducted in an extensively anaerobic environment, in particular wherein step (i) and step (ii) are conducted in an extensively anaerobic environment.

As used in the context of the present invention, the term "extensively anaerobic environment" refers to an environment that merely comprises a very low oxygen concentration. The oxygen concentration may preferably be below 10 % (v/v) referred to the total gas content of the environment of the one or more microorganism(s), more preferably below 5 % (v/v) referred to the total gas content of the environment of the one or more microorganism(s), even more preferably below 2 % (v/v) referred to the total gas content of the environment of the one or more microorganism(s), even more preferably below 1 % (v/v) referred to the total gas content of the environment of the one or more microorganism(s), even more preferably below 0.5 % (v/v) referred to the total gas content of the environment of the one or more microorganism(s) and most preferably below 0.1 % (v/v) referred to the total gas content of the environment of the one or more microorganism(s). As oxygen is poorly soluble in water and is rapidly metabolized by aerobic and facultative anaerobic microorganisms and is even converted by numerous enzymes, a liquid fermenter broth will often be extensively free of oxygen.

In another preferred embodiment of the present invention, the one or more microorganism(s) capable of decomposing lignocellulose are capable of cleaving the interconnection of lignocellulose mediated by hemicellulose and/or lignin.

The term "hemicellulose" as used herein refers to the polymeric material hemicellulose as commonly used and well-known in the art. Hemicellulose is a heteropolymer composed of different monomers such as, e.g., glucose, xylose, mannuronic acid, galacturonic acid, mannose, galactose, rhamnose and/or arabinose. Hemicellulose is present along with cellulose in many plant cell walls found in nature. Hemicellulose typically has a random, amorphous structure. Hemicellulose is typically mainly composed of D-pentose sugars and occasionally small amounts of L-sugars as well. Typically, xylose is found in comparably high amounts. Optionally, also mannuronic acid and galacturonic acid may be present in higher amounts. Often, hemicellulose may have a chain length of from approximately 500 to approximately 3,000. In addition, hemicellulose may be a branched polymer. Hemicelluloses are regularly embedded in the cell walls of plants and bind with pectin to cellulose to form a network of cross-linked fibers.

The terms "lignin" and "lignen" may be understood interchangeably as generally used and understood by those skilled in the art. Lignin is a complex chemical compound most commonly derived from wood and an integral part of the secondary cell walls of plants and some algae. However, it may also be chemically synthesized. Typically, lignin is a cross- linked racemic macromolecule with molecular masses up to more than of 10,000 Da. A lignin polymer is composed of aromatic monomers. There are different monomers of lignol (monolignol) known in nature methoxylated to various degrees such as, e.g., p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These monomers may be incorporated into lignin in the form of the phenylpropanoids p-hydroxyphenyl (H), guaiacyl (G) and syringal (S), respectively. Often, a lignin polymer tends to be comparably hydrophobic. The polymer is often polymerized in a haphazard manner.

The interconnection between cellulose and lignin and/or hemicellulose may be a covalent or a non-covalent connection. A covalent connection bound may exemplarily be an ester bond or an ether bond. These bounds may typically be cleavable by hydrolysis catalyzed by an enzyme and/or enabled by a microorganism.

In a preferred embodiment, the step of converting said biomass to biofuel and/or biogas (step (ii)) comprises the following steps:

(a) hydrolysis of cellulose, hemicellulose, lignin, starch and/or other polysaccharides to monosaccharides and/or disaccharides, preferably by one or more anaerobic microorganism(s) and/or one or more enzyme(s);

(b) fermentation of the monosaccharides and/or disaccharides generated by step (a) to an alcohol, in particular ethanol, preferably by one or more anaerobic microorganism(s) and/or one or more enzyme(s); and

(c) isolation of the alcohol generated by step (b), in particular by distillation.

As used herein, the term "hydrolysis" may be understood in the broadest sense as the decomposition of the cellulose, hemicellulose, lignin, starch and/or other polysaccharide strand into its one or more monomer(s). Exemplarily, the alcohol may be isolated by distillation, filtration, centrifugation, isolation via a membrane, in particular a semipermeable membrane, skimming and/or decantation. Preferably, the biofuel is isolated by distillation. Exemplarily, distillation may be extraction of ethanol performed by vacuum distillation at a pressure of approximately 40 mbar and a temperature of approximately 37°C. The residual rest of the distillation process including the microorganisms and dead fragments thereof may be used as fertilizer and/or animal feed. Preferably, the alcohol is methanol, ethanol, propanol and/or butanol. Most preferably, the alcohol is ethanol.

The steps (a), (b) and/or (c) may be conducted consecutively or concomitantly. Steps (a) and (b) may be conducted by many of different microorganisms and/or enzymes or by a single microorganism capable of performing step (a) and (b).

In an alternative preferred embodiment, the step of converting said biomass to biofuel and/or biogas (step (ii)) comprises the following steps:

(a) hydrolysis of cellulose, hemicellulose, lignin, starch and/or other polysaccharides to monosaccharides and/or disaccharides, preferably by one or more anaerobic microorganism(s) and/or one or more enzyme(s); (b) converting the monosaccharides and/or disaccharides of step (a) to acids, ketones and/or alcohols, preferably by one or more anaerobic microorganism(s) and/or one or more enzyme(s); and

(c) converting the acids, ketones and/or alcohols of step (b) to methane, preferably by methanogenic anaerobic bacteria.

Herein, the acids, ketones and/or alcohols may be any acids, ketones and/or alcohols known in the art to enable microorganisms to produce methane. Exemplarily, these molecules may be acetic acid, carbon dioxide, formic acid, methanol, methylamine(s), dimethyl sulfide and/or methanethiol and salts thereof.

In another preferred embodiment, the biomass comprises at least 25 % (w/w) lignocellulose in the dry mass, preferably at least 50 % (w/w) lignocellulose in the dry mass, more preferably at least 60 % (w/w) lignocellulose in the dry mass, in particular at least 70 % (w/w) lignocellulose in the dry mass.

In another preferred embodiment, the biomass is plant material, preferably stem material, straw, cereal material, husk, bark material, splint, leaves, woody material and/or fiber material.

In general, the biomass may preferably comprise large amounts of material that is any discard from processes such as, e.g., food production, feeding stuff production, wood processing and/or fiber processing (e.g., processing of cotton fiber, hem fiber etc.).

However, as described above, it will be understood that the biomass may also comprise other material such as, e.g., stall manure.

In a preferred embodiment, the one or more microorganism(s) capable of decomposing lignocellulose produce one or more lignocellulose digesting enzyme(s), in particular one or more lignase(s) and/or one or more phenoloxidase(s).

A lignocellulose digesting enzyme may be any enzyme capable of decomposing lignocellulose, preferably decomposing lignocellulose in a way that cellulose is rendered accessible by microorganism(s) and/or enzyme(s) capable of converting said cellulose into its monomer(s).

In a preferred embodiment, biomass of the present invention is hemp material, more preferably hemp stem material, in particular stem material from oil hemp and/or fiber hemp. Oil hemp may comprise 30 to 44 % (w/w) oily crops. Therefore, this plant is particularly energy rich and may serve a biological source for high energy raw material leading to comparably high yields of biofuel and/or biogas. For industrial purposes, it is particularly preferably to use a hemp plant for (i) producing hemp oil (e.g., a oil for engines), mainly from the crops; and (ii) producing organic solvents (e.g., ethanol), mainly from the residual parts of the plant. This double usage makes hemp and other oil-producing plants, in particular fast growing oil-producing plants, particularly preferable in the context of the present invention.

A second aspect of the present invention refers to the use of one or more microorganism(s) capable of decomposing lignocellulose for the production of biofuel and/or for the production of biogas, in particular methane. All embodiments of the method for the production of biofuel and/or biogas described above also apply to the use of the one or more microorganism(s) biofuel and/or for the production of biogas.

In a preferred embodiment, the biofuel is ethanol.

In another preferred embodiment, the biogas has a methane content of at least 50 % (v/v), preferably a methane content at least 60 % (v/v), in particular a methane content at least 70 % (v/v). In another preferred embodiment, the one or more microorganism(s) is/are anaerobic microorganism(s), preferably anaerobic bacteria, in particular anaerobic bacteria selected from the group consisting of

(i) Bacillus fusiformis,

(ii) Lysinibacillus sphaericus, (iii) Spirochaetes,

(iv) Bacillus amylobacter,

(v) Plectridium friebes, and/or

(vi) Clostridia.

In another preferred embodiment, the one or more microorganism(s) capable of decomposing lignocellulose is/are capable of cleaving the interconnection of lignocellulose mediated by hemicellulose and/or lignin. In another preferred embodiment, the one or more microorganism(s) capable of decomposing lignocellulose produce(s) one or more lignocellulose digesting enzyme(s), in particular one or more lignase(s) and/or one or more phenoloxidase(s).

Another aspect of the present invention refers to a method for the production of biofuel, wherein the moist rest product of a biogas plant is converted to biofuel by contacting said moist rest product with cellulase.

In the context of the present invention, the terms "moist rest product" and "biorest" may be understood interchangeably in the broadest sense as the remnant obtained by the production of biogas. Preferably, the moist rest product comprises at least 25 % (w/w) insoluble organics, at least 50 % (w/w) insoluble organics, at least 75 % (w/w) insoluble organics, at least 80 % (w/w) insoluble organics, at least 85 % (w/w) insoluble organics, at least 90 % (w/w) insoluble organics or at least 95 % (w/w) insoluble organics in its dry mass. Typically, this moist rest product comprises large amounts of fiber material, in particular cellulose, hemicellulose, lignin and/or lignocellulose. Further, the moist rest product may comprise other remnants such as, e.g., microorganisms, salts, silicates and/or other fiber material. It will be understood by a person skilled in the art that the amount and exact composition of a moist rest product will depend on the biomass used in the biogas plant.

Biogas plants typically produce moist rest products having a pH of approximately 8 to 12. At such pH values, the bacteria that are preferably used in the context of the present invention, in particular Clostridia, lose their viability. By inserting C0₂ into the moist rest product, the pH may be decreased to a pH of approximately pH 5. Therefore, for using the moist rest product from a biogas plant in the context of the present invention, the generated C0₂ is preferably collected to optimize the pH of the moist rest product subsequently. This allows the conversion of comparably high amounts of the cellulose residues into sugar. In the context of the present invention, the term "cellulase" may be understood in the broadest sense as any enzyme that is capable of digesting cellulose, preferably digesting cellulose into its monomers, thus, glucose.

The EC number for cellulases typically is EC 3.2.1.4. In the art, cellulases are sometimes also designated as "cellulase enzyme", "endo-l,4-beta-glucanase", "endo-l,4-beta-D- glucanase", "beta-l,4-glucanase", "beta-l,4-endoglucan hydrolase" or "celludextrinase".

Typically, a cellulase will be present in an organism or may be excreted by an organism along with numerous other components. In contrast, the isolated cellulase will be extensively free of other proteins, thus, will account for more than 50 % (w/w) of the whole protein content, more than 60 % (w/w) of the whole protein content, more than 70 % (w/w) of the whole protein content, more than 80 % (w/w) of the whole protein content, or even more than 90 % (w/w) of the whole protein content of the added protein. However, a unit of the isolated cellulase comprise salts, cofactors, one or more filling material(s) and/or one or more solvent(s) (e.g., water, dimethyl sulphoxide (DMSO), dimethylformamide (DMF)).

It will be understood that the terms "cellulase" comprises the salts and modifications thereof. A modification may be any modification described for enzymes in general above or any combination thereof. The employed unit of cellulase may be directly isolated or may be stored. It may be of commercial origin. It may be present in a solution or may be frozen, dried or freeze dried.

As described above, for the production of biofuel, the fermenter will typically comprise one or more further microorganism(s) and/or one or more further enzyme(s) to convert the obtained glucose to biofuel, in particular into alcohol. The cellulase may be obtained from any source known in the art, such as, e.g., from any organism, from ne or more molecular cloning method(s) and/or from one or more synthetical method(s). In the context of the present invention, the terms "biogas plant", "digester", "converter", "bioconverter" and "fermentation plant" may be understood interchangeably as any device capable for a controlled conversion of biomass into biogas.

In a preferred embodiment, the cellulase is contacted with the moist rest product by contacting said moist rest product with an isolated cellulase enzyme and or with a microorganism producing cellulase.

As used in the present context, the term "isolated" may be understood as the separation of the cellulase out of its natural context.

A cellulase in the context of the present invention may also be a carboxymethyl cellulase (CMCase) enzyme.

In another preferred embodiment, the moist rest product further comprises lignocellulose, hemicellulose and/or lignin.

In another preferred embodiment, further at least one of the group consisting of

(i) hemicellulase enzyme or a microorganism producing hemicellulase;

(ii) lignase enzyme or a microorganism producing lignase; and/or

(iii) phenoloxidase enzyme or a microorganism producing phenoloxidase

is contacted with the moist rest product.

In another preferred embodiment, the biogas plant is operated with stall manure and/or plant material, in particular stems, leaves and/or husk of forage plants.

In another preferred embodiment, further one or more microorganism(s) capable of decomposing lignocellulose is/are contacted with the moist rest product.

In another preferred embodiment, the one or more microorganism(s) capable of decomposing lignocellulose is/are anaerobic microorganism(s), preferably anaerobic bacteria, in particular anaerobic bacteria selected from the group consisting of (i) Bacillus fusiformis,

(ii) Lysinibacillus sphaericus,

(iii) Spirochaetes,

(iv) Bacillus amylobacter,

(v) Plectridium friebes, and/or

(vi) Clostridia.

In another preferred embodiment, the method is conducted in a moistened environment at a temperature suitable for the activity of said one or more microorganism(s), preferably wherein said temperature is below 100°C, more preferably wherein said temperature is below 75°C, even more preferably wherein said temperature is below 60°C, in particular wherein said temperature is below 50°C.

In another preferred embodiment, the method is conducted in an extensively anaerobic environment.

In another preferred embodiment, further one or more microorganism(s) capable of decomposing lignocellulose is/are contacted with the moist rest product, in particular wherein said one or more microorganism(s) is/are capable of cleaving the interconnection of lignocellulose mediated by hemicellulose and/or lignin.

In another preferred embodiment, the method further comprises the following steps:

(a) fermentation of the monosaccharides and/or disaccharides generated by cellulase to alcohols, in particular ethanol, preferably by one or more anaerobic microorganism(s) and/or one or more enzyme(s); and

(b) isolation of the alcohol generated by step (a), in particular by distillation.

Another aspect of the present invention refers to the use of the moist rest product of a biogas plant for the production of biofuel, wherein said moist rest product is contacted with cellulase.

All embodiments of the method for the production of biofuel described above also apply to the use of the moist rest product. In a preferred embodiment, the biofuel is ethanol.

In another preferred embodiment, the cellulase is contacted with the moist rest product by contacting said moist rest product with an isolated cellulase enzyme and/or with a microorganism producing cellulase.

Optionally, the products obtained from the means and methods of the present invention may also be (re-)inserted into a biogas plant. As it is well-known that many types of biogas plants are comparably sensitive with respect to pH values which typically are in the range of approximately pH 8, the person skilled in the art will notice that, when (re-)inserting material into such biogas plant, the pH may preferably be thoroughly controlled and/or adjusted to the desired pH level. When the pH is too low, for instance, the material may preferably be (re-)inserted via a comparably slow and/or continuous process. Alternatively or additionally, the pH of the material to be (re-)inserted into the biogas plant may also be increased by any means known in the art such as, e.g., by addition of basic material (e.g., calcium carbonate) before being (re-)inserted.

The invention is further explained by the following examples and figures, which are intended to illustrate, but not to limit the scope of the present invention.

Brief Description of the Figures

Figure 1 depicts the schematic process of decomposing of lignocellulose with and without pretreatment. Without pretreatment, the degrading enzymes have limited access to the lignocellulose fibers. The figure has been adapted from Taherzadeh MJ and Karimi , 2008.

Figure 2 depicts a utilizable workflow for the production of biogas from insoluble organics. In the context of the present invention, the insoluble organics may be present in the moist rest product of a biogas plant. The figure has been adapted from Ostrem K, 2004. Examples Example 1

Method for the Production of Bioethanol using Lignocellulose-Containing Biomass

After harvesting hemp stems, 5 g of these stems were cut into pieces of between approximately 5 and 6 cm in length. The stem pieces had a diameter of between approximately 1 and 2 cm. In addition, 25 g of hemp fibers were added. The mixture was mixed with 900 ml liter bidest water and 50 mg cellulase enzyme. The mixture was incubated at 30°C for 7 days. During the incubation, the pH was measured and several of the microorganisms were identified. Bacteria in the fermenter were identified by sequencing the 16srRNA by a sequencing method widely known in the art (cf, e.g., Riesenfeld CS et al., 2004; F. Sanger et al., 1977).

The mixture had a pH value between 3 and 4. In the culture, the anaerobic bacteria Bacillus fusiformis, Lysinibacillus sphaericus, Spirochaetes, Bacillus amylobacter, Plectridium friebes and Clostridia were detected by sequencing the 16srRNA.

After an incubation time of 7 days at 30°C, C0₂ and ethanol could be detected. This shows that the above referenced organisms can be used for the production of biofuel from lignocellulose-containing biomass.

Example 2

Method for the Production of Bioethanol from a Moist Rest Product of a Biogas Plant

Moist rest product was obtained from a biogas plant. The production of biogas in the used fermenter produced a moist rest product accounting for approximately 8 % (w/w) of the dry matter. The moist rest product was analyzed. Then, 0.2 g of a mixture of cellulases (Biogazym, product No. 3050 (ASA Spezialenzyme GmbH, Wolfenbuttel, Germany)(1000 units cellulase (=1 g enzyme) per kg moist rest product) were added to approximately 200 ml of moist rest product. The sample was incubated at 30°C for 7 days. After the incubation period of 7 days, the material has been analyzed.

Before incubation, the moist rest product contained the nutrients (ammonia, potassium and phosphorus), stable organic matter and biomass. Further, it still contained cellulose and lignocellulose.

The product contained 500 mg/1 ethanol and 15.484 g 1 glucose. This shows that the biorest of biogas plants can be used to produce bioethanol.

Example 3

Method for the Production of Bioethanol using Lignocellulose-Containing Plant Raw Material

Fresh oil hemp stems and fiber hemp stems were harvested before ligniflcation and proceeded by dew retting for three weeks at temperatures of approximately 20-30°C. 500 g of these stems were cut and pressed into pieces of approximately 2-3 cm in length and filled in a 5000 ml one neck flask. The stem pieces had a diameter of between approximately 0.5 and 2 cm. In addition, 10 g of commercially available hemp fibers derived from water retting (OBI Baumarkt, Gottingen) were added. The flask was filled up with water to a total volume of 5000 ml and 250 mg cellulase enzymes (ASA GmbH) were added. The suspension was saturated with C0₂ and mixed thoroughly. The mixture was incubated under anaerobe conditions at 30°C for 7 days. During the incubation, the pH was measured and several types of microorganisms were identified by sequencing the 16srRNA.

During the incubation under anaerobe conditions vigorous formation of C0₂ was observed. The mixture had a pH value between 3 and 4. In the culture, the anaerobic bacteria Bacillus fusiformis, Lysinibacillus sphaericus, Spirochaetes, Bacillus amylobacter, Plectridium friebes and Clostridia were detected by sequencing the 16srRNA. After an incubation time of 7 days at 30°C, ethanol could be detected. The mixture was then treated for 3 months under anaerobe conditions at 30°C. All cellulose fibers and hemicellulose were degraded during this fermentation time. A yellow colored smear of small particles and thin-walled residual stem artifacts remained on the ground. The yield of direct ethanol production accounts for an ethanol content of between 1 and 4 % (w/v) in the broth. When the glucose is converted into ethanol via fermentation with yeasts, the total ethanol yield accounts for an ethanol content of between 3 and 4 % (w/v) in the broth.

This experiment further confirms that the above referenced organisms can be used for the production of biofuel from lignocellulose-containing plant raw material. Moreover, this experiment confirms that the production of biofuel according to the present invention is scalable up to industrial production scales.

Example 4

Method for the Production of Bioethanol using Lignocellulose-Containing Plant Raw Material and Cellulase

500 g hemp fibers were filled in a 5000 ml one neck glass flask. 250 mg cellulase enzymes (ASA GmbH, Wolfenbuttel), 3 g (NH₄)₂S0₄, 4 g Na₂HP0₄, 4 g CaC0₃, 1.5 g MgS0₄, 0.05 g FeS0₄, 1 g KH₂P0₄ and 0.25 g NaCL were added and, finally, the flask was filled up with bidest water up to a total volume of 5000 ml. The suspension was mixed and saturated with C0₂ (resulting in a pH of approximately 5.5) and incubated under anaerobe conditions at 30°C. The sample further comprises Clostridium acetobutylicum.

Within 24 hours, vigorous formation of C0₂ was observed and the pH decreased to a pH of approximately 3-4. After an incubation time of 60-90 days, the hemp fibers and structures have been completely dissolved, only a yellow-brown smear remained at the bottom of the glass flask.

The chemical analysis of the resulting solutions comprised: glucose: 77.5 g / 1 (= 387.5 g / 5000

butanol: 10-20 g / 1

lactic Acid: 6 g /l

xylose: 4.3 g /l

acetic acid: 2.7 g /l ethanol: 2.5 g / 1

cellobiose: 2.3 g / 1

In this experiment, the conversion of lignocellulose and cellulose into glucose shows reaction yields of approximately 77 %. Then, the total ethanol yield accounts for an ethanol content of between 3 and 4 % (w/v) in the broth. The reaction was mainly driven by Clostridium acetobutylicum. The resulting glucose can be fermented to ethanol by yeasts.

Furthermore, it could be shown that comparable results could also be obtained at an incubation time of 40 days at 37°C and an incubation time of 90 days at 20°C.

This experiment further confirms that cellulose can be used for the production of biofuel from lignocellulose-containing plant raw material. Moreover, this experiment confirms that the production of biofuel according to the present invention is scalable up to industrial production scales.

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Claims

A method for the production of biofuel and/or biogas, wherein

(ii) the biomass subjected to step (i) is converted to biofuel and/or biogas.

The method of claim 1, wherein said one or more microorganism(s) capable of decomposing lignocellulose is/are anaerobic microorganism(s), preferably anaerobic bacteria, in particular anaerobic bacteria selected from the group consisting of

(i) Bacillus fusiformis,

(ii) Lysinibacillus sphaericus,

(iii) Spirochaetes,

(iv) Bacillus amylobacter,

(v) Plectridium friebes, and/or

(vi) Clostridia.

The method of claim 1 or 2, wherein step (i) and/or step (ii) is/are conducted in a moistened environment at a temperature suitable for the activity of said one or more microorganism(s), preferably wherein said temperature is below 100°C, more preferably wherein said temperature is below 75°C, even more preferably wherein said temperature is below 60°C, in particular wherein said temperature is below 50°C.

4. The method of at least one of claims 1 to 3, wherein step (i) and/or step (ii) is/are conducted in an extensively anaerobic environment, in particular wherein step (i) and step (ii) are conducted in an extensively anaerobic environment.

5. The method of at least one of claims 1 to 4, wherein said one or more microorganism(s) capable of decomposing lignocellulose is/are capable of cleaving the interconnection of lignocellulose mediated by hemicellulose and/or lignin.

The method of at least one of claims 1 to 5, wherein step (ii) comprises the following steps:

(b) fermentation of the monosaccharides and/or disaccharides generated by step (a) to alcohols, in particular ethanol, preferably by one or more anaerobic microorganism(s) and/or one or more enzyme(s); and

(b) converting the monosaccharides and/or disaccharides of step (a) to acids, ketones and/or alcohols, preferably by one or more anaerobic microorganism(s) and/or one or more enzyme(s); and

The method of at least one of claims 1 to 7, wherein said biomass comprises at least 25 % (w/w) lignocellulose in the dry mass, preferably at least 50 % (w/w) lignocellulose in the dry mass, more preferably at least 60 % (w/w) lignocellulose in the dry mass, in particular at least 70 % (w/w) lignocellulose in the dry mass.

The method of at least one of claims 1 to 8, wherein said biomass is plant material, preferably stem material, straw, cereal material, husk, bark material, splint, leaves, woody material and/or fiber material.

10. The method of at least one of claims 1 to 9, wherein said one or more microorganism(s) capable of decomposing lignocellulose produce(s) one or more lignocellulose digesting enzyme(s), in particular one or more lignase(s) and/or one or more phenoloxidase(s).

1 1. The method of at least one of claims 1 to 10, wherein said biomass is hemp material, preferably hemp stem material, in particular stem material from oil hemp and/or fiber hemp.

12. Use of one or more microorganism(s) capable of decomposing lignocellulose for the production of biofuel and/or for the production of biogas, in particular methane.

13. The use of claim 12, wherein the biofuel is ethanol.

14. The use of claim 12 or 13, wherein the biogas has a methane content of at least 50 % (v/v), preferably a methane content at least 60 % (v/v), in particular a methane content at least 70 % (v/v).

15. The use of at least one of claims 12 to 14, wherein said one or more microorganism(s) is/are anaerobic microorganism(s), preferably anaerobic bacteria, in particular anaerobic bacteria selected from the group consisting of

(i) Bacillus fusiformis,

(ii) Lysinibacillus sphaericus,

(iii) Spirochaetes,

(iv) Bacillus amylobacter,

(v) Plectridium friebes, and/or

(vi) Clostridia.

16. The use of at least one of claims 12 to 15, wherein said one or more microorganism(s) capable of decomposing lignocellulose is/are capable of cleaving the interconnection of lignocellulose mediated by hemicellulose and/or lignin.

17. The use of at least one of claims 12 to 16, wherein said one or more microorganism(s) capable of decomposing lignocellulose produce(s) one or more lignocellulose digesting enzyme(s), in particular one or more lignase(s) and/or one or more phenoloxidase(s).

18. A method for the production of biofuel, wherein the moist rest product of a biogas plant is converted to biofuel by contacting said moist rest product with cellulase.

19. The method of claim 18, wherein the cellulase is contacted with the moist rest product by contacting said moist rest product with an isolated cellulase enzyme and/or with a microorganism producing cellulase.

20. The method of claim 18 or 19, wherein said moist rest product further comprises lignocellulose, hemicellulose and/or lignin.

21. The method of at least one of claims 18 to 20, wherein further at least one of the group consisting of

(i) hemicellulase enzyme or a microorganism producing hemicellulase;

(ii) lignase enzyme or a microorganism producing lignase; and/or

(iii) phenoloxidase enzyme or a microorganism producing phenoloxidase, is contacted with the moist rest product.

22. The method of at least one of claims 18 to 21, wherein said biogas plant is operated with stall manure and/or plant material, in particular stems, leaves and/or husk of forage plants.

23. The method of at least one of claims 18 to 22, wherein further one or more microorganism(s) capable of decomposing lignocellulose is/are contacted with the moist rest product.

24. The method of claim 23, wherein the one or more microorganism(s) capable of decomposing lignocellulose is/are anaerobic microorganism(s), preferably anaerobic bacteria, in particular anaerobic bacteria selected from the group consisting of

(i) Bacillus fusiformis,

(ii) Lysinibacillus sphaericus, (iii) Spirochaetes,

(iv) Bacillus amylobacter,

(v) Plectridium friebes, and/or

(vi) Clostridia.

25. The method of at least one of claims 18 to 24, wherein said method is conducted in a moistened environment at a temperature suitable for the activity of said one or more microorganism(s), preferably wherein said temperature is below 100°C, more preferably wherein said temperature is below 75°C, even more preferably wherein said temperature is below 60°C, in particular wherein said temperature is below 50°C.

26. The method of at least one of claims 18 to 25, wherein said method is conducted in an extensively anaerobic environment.

27. The method of at least one of claims 18 to 26, wherein further one or more microorganism(s) capable of decomposing lignocellulose is/are contacted with the moist rest product, in particular wherein said one or more microorganism(s) is/are capable of cleaving the interconnection of lignocellulose mediated by hemicellulose and/or lignin.

28. The method of at least one of claims 18 to 27, wherein said method further comprises the following steps:

(a) fermentation of the monosaccharides and/or disaccharides generated by cellulase to an alcohol, in particular ethanol, preferably by one or more anaerobic microorganism (s) and/or one or more enzyme(s); and

29. Use of the moist rest product of a biogas plant for the production of biofuel, wherein said moist rest product is contacted with cellulase.

30. The use of claim 29, wherein the biofuel is ethanol.

31. The use of claim 29 or 30, wherein the cellulase is contacted with the moist rest product by contacting said moist rest product with an isolated cellulase enzyme and/or with a microorganism producing cellulase.

32. The use of at least one of claims 29 to 31, wherein said biogas plant is operated with stall manure and/or plant material, in particular stems, leaves and/or husk of forage plants.