WO2010072220A1 - Paenibacillus macerans for the treatment of biomass - Google Patents

Abstract

Disclosed is a method for the treatment of biomass, wherein a microorganism of the type Paenibacillus macerans is added to the biomass.

Description

 Paenibacillus macerans for the treatment of biomass

Technical area

The invention relates to a method for the treatment of biomass using a microorganism of the species Paenibacillus macerans.

State of the art

Biofuels are mainly obtained by fermentation of plant substrates with the help of yeasts, bacteria or fungi. The produced liquid energy carriers, in particular bioethanol, can then be used as fuel in suitable combustion plants or as fuel or additive in motor vehicle engines or motors for power and heat generation. The biomass is thus converted into a liquid and easily transportable energy source with a relatively high energy density, which can then be used universally.

As raw materials for the production of liquid biofuels mostly locally available plants with a high content of sugar or starch are used. In Europe, these are mainly maize and cereal grains and sugar beet, maize in North America and sugar cane or molasses in Latin America. Other energy crops such as sugar millet (Sorghum), triticale or cassava (manioc) are also possible. Corn silage and cereal grain are currently used as starting material in the liquefaction of biomass. Due to the growing scarcity of raw materials, the rise in raw material prices, the emergence of competition for crops that serve both human and animal nutrition, as well as energy production, an improved CO ₂ balance and the conservation of resources, more and more people are trying, not just corn and to use crops, but rather to feed the entire plant to energy or to use plants that none intensive agricultural management require such as millet or miscanthus. The biofuels produced from these raw materials are so-called "second-generation" fuels, and the aim is also to increase the use of cheap plant residues such as straw, vegetable waste from the timber industry or gardening and landscape maintenance.

In order to ensure an efficient fermentation of the biomass, the plant material used as raw material and in particular the organic dry substance contained therein (oTS) must be opened for a subsequent utilization. This is done by hydrolysis of the organic dry matter, which also corresponds to a liquefaction of the organic dry matter. Ethanol or another biofuel is then produced from the liquid biomass substrate by fermentation. However, with the existing techniques, high energy efficiency is not achieved, which is due in particular to insufficient liquefaction and thus utilization of the raw material.

In the production of bioethanol, the ethanolic fermentation takes place primarily by added yeasts, which convert glucose into ethanol. Depending on the substrate, there may already be a relatively high level of sugar (for example molasses) or firstly high molecular weight substrates such as starch, cellulose or hemicellulose (for example in cereals, straw, whole plants) must be enzymatically or chemically cleaved to allow alcoholic fermentation. For this hydrolytic substrate digestion, which also corresponds to liquefaction, are primarily responsible microorganisms, especially bacteria.

If the conversion of organic raw materials does not lead to a liquid biofuel, but to a gaseous energy source, then biogas plants are used. In biogas plants, methane is produced by a microbial decomposition process of organic substances. The biogas is produced in a multi-stage process of fermentation or digestion by the activity of anaerobic or microaerophilic microorganisms, i. in the absence of air.

The organic material used as a fermentation substrate has, from a chemical point of view, a high molecular structure, which in the individual process steps of a Biogas plant is degraded by metabolic activity of microorganisms to low molecular weight building blocks. However, the populations of microorganisms which are active in the fermentation of the organic fermentation substrate have hitherto been insufficiently characterized.

According to the current state of knowledge, four sequential, but also parallel and interlocking biochemical metabolic processes can be distinguished, which enable the degradation of organic fermentation substrates to the end products methane and carbon dioxide: hydrolysis, acidogenesis, acetogenesis and methanogenesis.

In hydrolysis, high molecular weight, often particulate, organic compounds are converted to soluble fission products by exoenzymes (e.g., cellulases, amylases, proteases, lipases) of fermentative bacteria. For example, fats in fatty acids, carbohydrates, e.g. Polysaccharides, decomposed into oligo- and monosaccharides and proteins in oligopeptides or amino acids. The gaseous products formed besides consist predominantly of carbon dioxide.

Optional and obligate anaerobic bacteria, often identical to the hydrolyzing bacteria, metabolize in acidification the hydrolysis products (eg mono-, disaccharides, di-, oligopeptides, amino acids, glycerol, long chain fatty acids) intracellularly to short chain fatty or carboxylic acids such as butter -, propionic and acetic acid, to short-chain alcohols such as ethanol and the gaseous products hydrogen and carbon dioxide.

In the subsequent acetogenesis, the short-chain fatty acids and carboxylic acids formed in acidogenesis and the short-chain alcohols are taken up by acetogenic bacteria and excreted again as acetic acid after β-oxidation. By-products of acetogenesis are CO ₂ and molecular hydrogen (H ₂ ). The products of acetogenesis such as acetic acid but also other substrates such as methanol and formate are converted by methane-forming organisms in the obligate anaerobic methanogenesis to methane and CO ₂ . The resulting here CO ₂ and also during the other process steps such as hydrolysis, CO ₂ formed in turn can also be converted by microorganisms with the incurred H ₂ to methane.

The increase in the yield of end products from a given amount of starting materials, as in any chemical reaction, also in the case of liquefaction of biomass as well as in the production of biogas, a priority objective of the process management. From a given amount of biomass, the largest possible Amount of liquid organic compounds or the largest possible amount of methane are formed.

The Agency for Renewable Resources published the results of a biogas measurement program in 2005 ("Results of the Biogas Measurement Program", 2005, Publishing Agency for Renewable Resources, Gülzow), in which various biogas plants were compared.This measurement program represents a representative cross section of the existing plant diversity Since biogas plants of various types, different manufacturers and plants operated with different input materials were recorded, the measured total occupancy of the biogas plants was between 0.45 kgoTS / m ³ d (multi-stage plants) and 5.7 kgoTS / m ³ d For both single-stage and multi-stage systems, the space load was between 1 and 3 kgoTS / m ³ d.

The volume loading of a fermenter is the amount of substrate fed to the fermenter, expressed in kilograms of dry organic matter per cubic meter of fermenter volume and per day. The amount of biogas produced depends strongly on the volume load of the fermenter, with increasing space load an increasingly larger amount of biogas is generated. A high space load makes the process of biogas production increasingly economically viable, but on the other hand leads to an increasing destabilization of the biological processes of fermentation. Increasing the volume of space is one way to operate a biogas plant more efficiently. The biogas production is increased by a slow increase in the amount of feed in oTS per day. In practice, high space loads of more than 6 kgoTS / m ³ d are not yet available. The goal in plant operation is to operate a plant economically. The stability of the biological process is at the forefront, as plant failure causes extremely high costs.

In order to achieve this process stability, the existing systems are operated at low space loads, but this requires a correspondingly large fermentation volume for efficient operation. This increases the initial investment through additional construction costs. At high room loads, there is a continuous thickening of the fermenter contents due to a stronger load of existing in the fermenter biocenosis with organic material, especially in the form of solids. This is due to increasing dry matter contents and incomplete conversion of the biomass into biogas.

Substrates for biogas production have dry matter contents in the range of 5% to 90%. For wet fermentation processes in which pumpable substrates are required, substrates with a dry matter content of up to about 35 to at most 40% can be used. Since substrates with a higher dry matter content, in particular a higher proportion of organic dry matter, usually provide a higher energy content, they would preferably be used. In this case, however, there are higher costs due to increased expenditure of energy during pumping and stirring as well as higher ancillary costs due to wear or repair of pumps, agitators or the like with high viscosity or a high dry matter content. If substrate dilution is necessary because of a high dry matter content, additional water and wastewater costs as well as technical equipment for water supply, recovery or wastewater treatment are incurred. An increase in the dry matter content in continuous operation increasingly leads to problems in the stirring and pumpability of the fermenter content. With a further increase in the dry matter contents, this thickening can lead to the instability of the biocenosis and thus to extensive or complete cessation of the biological processes and thus also the gas production, which is referred to as "fermenter crash." To prevent thickening, by adding liquid substances, The problem with this is the legal framework (EEG), which does not allow the addition of substances that have a lower dry matter content than on average 30% when receiving the dry fermentation bonus. A liquefaction of the fermenter material by additional hydration can therefore rarely be used.

Considering in addition to the above considerations, the scarcity of the resource fresh water, so separates a liquefaction of the fermenter content by the addition of water. Manure as a liquefier is on the one hand because of the technical complexity associated with high costs, on the other not everywhere available and also of varying quality and quality. In addition, when using liquid manure, a hygienisation stage is required for legal reasons after the fermentation process, before the digestate can be returned to the field.

There is therefore still a need for methods for the treatment of biomass, in particular for more efficient methods for liquefying biomass for the production of biofuels, as well as methods for producing biogas, which enable an increased yield of biogas compared to the prior art.

definitions

In the context of the present invention, the term "biofuel" is understood as meaning a liquid or gaseous fuel or energy carrier which is produced from biomass. Biofuels come for the operation of Internal combustion engines for both mobile (eg motor vehicles) and stationary (eg generation of electrical and thermal energy in a combined heat and power plant) applications are used. Examples of biofuels are biodiesel, bioethanol, biomethanol, biokerosene, biohydrogen or biogas.

In the context of the present invention, the term "bioethanol" refers to ethanol which has been produced by alcoholic fermentation from biomass as renewable carbon carrier or biodegradable fractions of waste and serves in various concentrations as an additive to mineral oil fuels such as biodiesel or biofuel.

In the context of the present invention, the term "biogas" is understood to mean the gaseous product of the anaerobic biodegradation of organic substrates, which generally contains about 45-70% of methane, 30-55% of carbon dioxide, and small amounts of nitrogen, hydrogen sulphide and other gases.

The terms "fermentation" or "fermentation" in the context of the present invention include both anaerobic and aerobic metabolic processes which, under the action of microorganisms in a technical process from the supplied substrate to produce a product, e.g. Lead biogas. A differentiation from the term "fermentation" is given in that it is exclusively anaerobic processes.

In the context of the present invention, a "fermenter" is understood to mean the container in which the microbiological degradation of the substrate takes place with simultaneous formation of biogas.The terms "reactor", "fermenter" and "digester" are used interchangeably.

The term "fermentation substrate" or "substrate" in the context of the present invention means organic and biodegradable material which is added to the fermenter for fermentation. Substrates can be renewable raw materials, organic fertilizers, substrates from the processing agricultural industry, municipal organic residues, slaughter residues or green waste. Examples of such substrates are corn silage, rye silage, Beet pulp, molasses, grass silage, cattle or pig slurry, beef, pork, chicken or horse manure, beer grains, apple, fruit or vine pomace, cereal, potato or fruit vinasse. Organic waste from bio-waste, leftovers, market wastes, grease from fat separators, rumen contents, pig stomach content. The terms "fermentation substrate" and "substrate" are used synonymously.

The term "fermentation residue" or "digestate" is understood to be the residue of biogas production which leaves the fermenter and is frequently stored in its own container.

In the context of the present invention, "volume load" is understood to mean the amount of dry organic matter (oTS) in kg supplied to the fermenter per day and cubic meter (m ³ ) working volume.

In the context of the present invention, "organic dry substance" (oTS) is understood to mean the anhydrous organic fraction of a substance mixture after removal of the inorganic constituents and drying at 105 ^° C. As a rule, the dry matter content is stated in% of the substrate.

In the context of the present invention, the term "residence time" is understood to mean the average residence time of the substrate in the fermenter.

In the context of the present invention, the term "specific biogas yield" or "specific methane yield" is used to denote the amount of biogas or methane produced (stated in standard cubic meters of Nm ³ gas) divided by the amount (as a rule per tonne) of organic dry substance used or substrate understood.

The term "space-time yield" is used to denote the amount of biogas produced in standard liters (Nl) normalized to one liter of fermentation volume, which serves to improve the comparability of the gas yield of various plants. The term "nucleotide sequence" encompasses both the DNA sequence and the corresponding RNA sequence, For example, RNA sequences given in the invention using bases A, U, C, G also refer to the corresponding DNA sequences using bases A, T, C, G and vice versa.

The determination of the nucleotide sequence of a microorganism by means of a standardized process by which the individual nucleotides of the DNA or RNA of the microorganism can be detected with high accuracy. In spite of the high accuracy of the sequencing methods, individual positions can repeatedly be present in a sequence for which the determination of the nucleotide present at the respective position was not possible with sufficient accuracy. In such cases, the letter "N" is indicated in the nucleotide sequence in the context of the present invention. If the letter "N" is subsequently used in a nucleotide sequence, this stands as an abbreviation for every conceivable nucleotide, that is to say for A, U, G or C. in the case of a ribonucleic acid or for A, T, G or C in the case of a deoxyribonucleic acid.

The term "nucleotide mutation" as used herein means a change in the starting nucleotide sequence, whereby individual nucleotides or several, directly following one another or interrupted by unmodified nucleotides

Nucleotide sequences are removed (deletion), added (insertion or addition) or replaced by others (substitution). The term also includes every possible one

Combination of the individual named changes.

The term "deletion" as used herein means removal of 1, 2 or more

Nucleotides from the respective starting sequence.

The term "insertion" or "addition" as used herein means the addition of 1, 2 or more nucleotides to the respective starting sequence.

The term "substitution" as used herein means the replacement of a nucleotide present at a particular position by another. The term "microorganism" is understood to mean microscopically small organisms, which as a rule are single-celled organisms but may also be multicellular organisms Examples of microorganisms are bacteria, microscopic algae, fungi or protozoa.

The term "genus" of microorganisms, "type" of microorganisms and "strain" of microorganisms is understood to mean the corresponding basic category of biological taxonomy, in particular the phylogenetic classification Among a particular genus, species or strain, not only are microorganisms having a particular RNA sequence but also, to a certain extent, their genetic variants, with genetic variance in the strain, species, genus increases.

The definition of a "species" of a bacterium is undergoing scientific change and is neither complete nor is the various concepts uncontroversial In the present invention, "species" is understood to be the species concept that relates to genomic and phylogenetic analyzes, and in the review of Staley (Philos. Trans. R. Soc., Lond., B. Biol., Sci., 2006, 361, 1899-1909). It is widely recognized that two bacterial species having more than 70% DNA-DNA hydride or more than 94% average nucleotide identity (ANI) belong to the same species, respectively two species having less than 97% identity 16S rRNA, are assigned to two different types.

In this context, the term "culture" refers to an accumulation of microorganisms under established conditions which ensure the growth or at least the survival of the microorganisms, for example enrichment cultures or pure cultures, liquid cultures as well as cultures on solid media such as nutrient media also permanent crops such as frozen glycerin cultures, immobilized cultures such as gel cultures or highly concentrated cultures such as cell pellets. The term "pure culture" of a microorganism is understood to mean the progeny of a single cell, which is isolated by a multi-step process from a mixture of different microorganisms.The multi-step process involves the separation of a single cell from a cell population and requires that also from the cell By careful separation of a colony, resuspension in liquid and repeated spreading, pure cultures of microorganisms can be selectively recovered By serial dilution of the suspension in the nutrient solution it can finally be achieved that in the last dilution stage there is only one cell left then the basis for a pure culture. The explanation of the term "pure culture" and methods for producing the same are in "General Microbiology" by Hans G. Schlegel, 7th revised edition, 1992, Georg Thieme Verlag, p is hereby incorporated by reference.

The term "mixed culture" is understood to mean a mixture of different microorganisms, but natural populations of microorganisms are usually mixed cultures, but mixed cultures can also be produced artificially, for example by combining several pure cultures.

Presentation of the invention

The object of the invention is to provide a method for the treatment of biomass, which allows a higher yield of usable biofuels than the prior art.

This object is achieved by the method for the treatment of biomass according to claim 1. Further advantageous details, aspects and Embodiments of the present invention will become apparent from the dependent claims, the description and the examples.

The present invention provides a method of treating biomass. The biomass is added to a microorganism of the species Paenibacillus macerans.

The method for treating biomass is preferably a method for liquefying biomass. From the prior art Paenibacillus macerans is known as an optionally anaerobically living organism. The use or occurrence of microorganisms of the species Paenibacillus macerans in the treatment of biomass or the production of biogas was previously unknown. Surprisingly, however, it has been shown that the addition of microorganisms of the species Paenibacillus macerans to a biomass substrate, the viscosity of the substrate can be greatly reduced. The use of microorganisms of the species Paenibacillus macerans leads to a significant increase in the liquefaction of the biomass and, as a result, to a significant improvement in the efficiency and efficiency of biofuel production plants.

Also preferably, the method for the treatment of biomass is a method for producing biogas from biomass. Surprisingly, it has been shown that the addition of microorganisms of the species Paenibacillus macerans to the fermentation substrate can both increase the volume load of the fermenter and significantly increase the amount of biogas formed. As shown in experimental studies, the addition of a microorganism of the species Paenibacillus macerans causes an increase in the volume load of a fermenter up to more than 50%, without any instability of the fermentation process would occur. Parallel to the increased space load, the amount of biogas produced is significantly increased. In addition, the specific yield of biogas increases, since significantly more of the organic dry matter is degraded than in the absence of addition of microorganisms of the species Paenibacillus macerans. Due to the increased degree of degradation, a significantly increased specific gas yield can be achieved with improved substrate utilization. In addition, by the Addition of microorganisms of the species Paenibacillus macerans the residence time of the fermentation substrate in the fermenter at constant gas yield can be significantly shortened, whereby the increase in the space load is possible. The use of microorganisms of the species Paenibacillus macerans therefore leads to a dramatic improvement in the efficiency and efficiency of biogas plants.

One possible explanation for the positive characteristics of the microorganisms of the species Paenibacillus macerans lies in a hydrolysis rate which is increased by their metabolic activity, so that as a result of the metabolic activity a part of the present hardly degradable organic substances (eg cellulose, hemicellulose) is converted into soluble acids and CO ₂ . Cellulose is insoluble in water and aqueous solutions, therefore degradation of the cellulose in the fermentation process leads to liquefaction. The stirring and pumpability of the material is maintained because the dry matter content does not increase further. This allows a constant mixing of the material and the gas can be better removed from the process.

Due to the increased degradation or metabolic performance of microorganisms of the species Paenibacillus macerans in the hydrolysis stage, a liquefaction of the fermenter material is brought about without an acidification, or a deterioration of the methane content of the resulting biogas is observed. The space load is significantly increased and the stability of the process improved.

The inventive addition of microorganisms of the species Paenibacillus macerans thus provides a method which ensures increased stability of the fermentation process and in which liquefaction of the fermentation substrate occurs. The liquefaction of substrate by the decomposition of insoluble constituents can also be used for the production of biofuel from biomass.

According to a preferred embodiment of the present invention, a microorganism of the species Paenibacillus macerans is added in the form of a culture of microorganisms consisting predominantly of a microorganism of the species Paenibacillus macerans persists. In fermentation substrates of biogas plants, microorganisms of the species Paenibacillus macerans could only be detected in minute traces of less than 10% ⁴ % of the total number of microorganisms present, since the amount of microorganisms isolated from their natural occurrence is generally sufficient for the addition of the microorganisms In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Paenibacillus macerans can be carried out in the form of a culture suspension, in the form of dry, freeze-dried or moist cell pellets or also in the form of spore suspensions, spore preparations or dry, freeze-dried or moist spore pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Paenibacillus macerans, this type of microorganisms should be present in the added culture in a quantity exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. The only requirement is that the species Paenibacillus macerans is present in an amount that is enriched in relation to the natural occurrence.

Preference is given to mixed cultures of microorganisms of the species Paenibacillus macerans with microorganisms of the species Clostridium sartagoformum and / or Clostridium sporosphaeroides. Particularly preferred are mixed cultures of microorganisms of the strain Paenibacillus macerans SBG2 with microorganisms of the strains Clostridium sartagoformum SBGI a and / or Clostridium sporosphaeroides SBG3. Microorganisms of the species Clostridium sartagoformum and Clostridium sporosphaeroides have similar properties with respect to the treatment of biomass as the microorganisms of the species Paenibacillus macerans, which is why they are predestined for use in the treatment of biomass. Microorganisms of the species Clostridium sartagoformum and Clostridium Sporosphaeroides can therefore also be used in the methods and applications described herein for the treatment of biomass.

Microorganisms of the species Paenibacillus macerans are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Paenibacillus macerans. If, in addition to the determination of the number of microorganisms of the species Paenibacillus macerans, the total number of microorganisms is also determined, the proportion of microorganisms of the species Paenibacillus macerans in the culture may be expressed as a percentage. In a mixed culture, microorganisms of the species Paenibacillus macerans are the predominant species of microorganisms when they have the highest percentage of the various types of microorganisms present in the mixed culture.

The composition of the microbial populations in the various fermentation substrates as well as the development of the organism composition during the fermentation process is largely unknown, but very variable and subject to a complicated dynamic process, which is also influenced by the respective process conditions. To determine the microbial composition and the total number of microorganisms in a fermentation substrate as well as the determination of the proportions of different types of microorganisms in the fermentation substrate, various methods are known to those skilled in the art, for example, in the review article by Amann et al. (Microbiol. Review., 59, 143-169, 1995).

A preferred method for determining the microorganism composition independently of a previous cultivation of the microorganisms is, for example, the preparation of a rDNA clone library (eg based on 16S rRNA) after nucleic acid extraction and PCR, which can then be sequenced. Using this clone library, the composition of the microbial population in the fermentation substrate can be determined, for example, by in situ hybridization with specific fluorescence-labeled oligonucleotide probes. Suitable rRNA-based oligonucleotide probes are known from the review mentioned above or may be prepared, for example, by probe base (Loy et al., 2003, Nucleic Acids Res. 31, 514-516. Loy et al., Nucleic Acids Res. 35: D800-D804) or the ARB program package (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). A quantitative determination of the proportion of individual microorganisms in the total population can be carried out in a suitable manner with the methods of quantitative dot blot, in situ hybridization or whole cell hybridization.

According to preferred embodiments of the present invention, the microorganism Paenibacillus macerans accounts for at least 10 ^"4 % of the total number of microorganisms present in the culture added to the fermentation substrate." More preferably, the microorganism Paenibacillus macerans accounts for at least 10 ^"2 % of the total number of microorganisms present in the culture and particularly preferably, the microorganism Paenibacillus macerans accounts for at least 1% of the total number of microorganisms present in the culture.

According to further preferred embodiments, the microorganism Paenibacillus macerans accounts for at least 10% of the total number of microorganisms present in the culture, more preferably the microorganism Paenibacillus macerans accounts for at least 50% of the total number of microorganisms present in the culture, and more preferably the microorganism makes Paenibacillus macerans at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Paenibacillus macerans is added. The pure culture is biochemically characterized by specific metabolic processes and activities, as well as by special growth conditions. Due to the specific metabolic processes and activities, the addition of a pure culture of a fermentative microorganism can especially contribute to an improved control of the complex biogas production process.

According to another preferred embodiment of the present invention, a microorganism of the species Paenibacillus macerans as a component at least added to an immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Paenibacillus macerans, this type of microorganisms should be present in the added immobilized culture in an amount enriched in comparison to the natural occurrence. Of course, immobilized mixed cultures of any composition can be used for the addition. The only requirement is that microorganisms of the species Paenibacillus macerans are present in an amount that exceeds their natural occurrence.

According to preferred embodiments of the present invention, the microorganism of the species Paenibacillus macerans accounts for at least 10.sup.- ⁴ % of the total number of microorganisms present in the immobilized culture added to the fermentation substrate.Particularly, the microorganism of the species Paenibacillus macerans accounts for at least 10.sup.- ² % of the total number In microorganisms present in the immobilized culture, and particularly preferably, the microorganism of the species Paenibacillus macerans accounts for at least 1% of the total number of microorganisms present in the immobilized culture.

According to further preferred embodiments, the microorganism of the species Paenibacillus macerans makes up at least 10% of the total number of microorganisms present in the immobilized culture, more preferably the microorganism of the species Paenibacillus macerans makes up at least 50% of the total number of microorganisms present in the immobilized culture, and is particularly preferred the microorganism of the species Paenibacillus macerans makes up at least 90% of the total number of microorganisms present in the immobilized culture. According to a very particularly preferred embodiment, at least one immobilized pure culture of a microorganism of the species Paenibacillus macerans is added.

As support materials on which the microorganism Paenibacillus macerans is immobilized, natural or synthetic polymers can be used. Gel-forming polymers are preferably used. These have the advantage that bacteria can be taken up or stored within the gel structure. Preferably, those materials are used which dissolve slowly in water or are degraded, so that the release of the microorganism Paenibacillus macerans takes place over a longer period of time.

Examples of suitable polymers are polyaniline, polypyrrole, polyvinyl pyrolidone, polystyrene, polyvinyl chloride, polyvinyl alcohol, polyethylene, polypropylene,

Epoxy resins, polyethyleneimines, polysaccharides such as agarose, alginate or cellulose,

Ethylcellulose, methylcellulose, carboxymethylethylcellulose, cellulose acetates,

Alkali cellulose sulfate, copolymers of polystyrene and maleic anhydride,

Copolymers of styrene and methyl methacrylate, polystyrene sulfonate, polyacrylates and polymethacrylates, polycarbonates, polyesters, silicones, cellulose phthalate, proteins such as gelatin, gum arabic, albumin or fibrinogen, mixtures of gelatin and water glass, gelatin and polyphosphate, gelatin and copolymers

Maleic anhydride and methyl vinyl ether, cellulose acetate butyrate, chitosan,

Polydialkyldimethylammonium chloride, mixtures of polyacrylic acids and Polydiallyldimethylammoniumchlorid and mixtures thereof. The polymeric material may also be prepared by conventional crosslinkers such as glutaraldehyde,

Urea / formaldehyde resins or tannin compounds are crosslinked.

Alginates as Immobilisate prove to be particularly advantageous because they do not have a negative impact on the activity of the microorganism Paenibacillus macerans and on the other hand they are slowly degraded by microorganisms. Due to the slow degradation of the alginate immobilizates, the trapped microorganisms of the species Paenibacillus macerans are gradually released. For immobilization, the microorganisms are mixed with a polymer gel and then cured in a suitable hardener solution. For this they are first mixed with a gel solution and then dropped into a hardener solution of suitable height. The detailed procedures for immobilization are known to the person skilled in the art.

According to a further preferred embodiment, additional biomass is added to the fermentation reactor in a timely manner to the addition of the microorganisms described below. Timely addition of additional biomass may occur within a period of 1 second to 3 days after addition of microorganisms, or it may occur simultaneously with the addition of microorganisms. The space load in the fermentation reactor can be continuously increased or kept approximately constant by the continuous addition of new substrate, wherein the fermentation at all room loads, preferably at a space load of ≥ 0.5 kg of organic dry matter per m ³ and day [kgoTS / m ³ d] , more preferably at a space load of ≥ 4.0 kgoTS / m ³ d and particularly preferably at a room load of ≥ 6.0 kgoTS / m ³ d can be performed, which corresponds to an increase in the space load to about double compared to the current state of the art.

The fermentation substrate used can in particular also have a high proportion of solid constituents. By adding a hydrolytically active, fermentative microorganism of the species Paenibacillus macerans, these solid constituents are at least partially liquefied. Due to the liquefaction of the fermentation substrate due to the addition of the microorganism Paenibacillus macerans, a thickening of the fermenter material can be prevented and targeted counteracted. Another liquid entry into the fermentation substrate in the form of water or manure during fermentation can be avoided. Thus, there is another advantage in conserving the resource freshwater. Another advantage is the thus obtained obtaining the stirring and pumpability of the substrate. As a result, agitators and pumps are spared and significantly less energy is required for the stirring process. Thus, microorganisms of the species Paenibacillus macerans can also be used for the liquefaction of biomass in alcoholic fermentation with the aim of producing biofuel.

According to a further embodiment, the treatment of biomass takes place with constant mixing of the fermentation substrate. Due to the constant mixing of the fermentation substrate, the cultures of Paenibacillus macerans can be better distributed in the fermentation substrate. In the case of biogas production, moreover, the biogas produced can be better removed from the fermentation process.

The constant mixing of the fermentation substrate also leads to a uniform heat distribution in the fermentation reactor. Measurements of the temperature in the fermentation reactor, which were carried out at periodic intervals, but also continuously, showed that the fermentation substrate in a temperature range of 20 ⁰ C to 80 ⁰ C, preferably at about 35 ⁰ C to 60 ⁰ C, particularly preferably at 40 ⁰ C is fermented efficiently to 50 ⁰ C. These temperature ranges are therefore preferred in the context of the present invention. In addition to the hydrolysis, in particular the last stage of the fermentation process, namely the formation of methane by methanogenic microorganisms, particularly efficient at elevated temperatures. For multi-stage biogas plants, it is also useful to operate the different reactors at different temperatures, eg the first stage under mesophilic conditions and the downstream stages, especially methanogenesis, under thermophilic conditions. Suitable conditions for this are known from the prior art (eg DE 10 2005 012367 A1).

All embodiments of the present invention are not limited to one-step processes for the production of biogas. The use of microorganisms of the species Paenibacillus macerans can also be carried out in two or more stages. Likewise, microorganisms of the species Paenibacillus macerans can be used in processes for the production of liquid biofuels. According to another embodiment, fermentation substrate and a microorganism of the species Paenibacillus macerans are added continuously. The continuous operation of a fermentation reactor should result in a stable microbial biocenosis to a continuous production of biogas, the exposure of the substrate addition to the fermentation should be reduced as a result of a process disturbance.

It is also conceivable to carry out this fermentation process and the processes associated therewith in a discontinuous operation, for example "batch" fermentation, For example, according to a further embodiment, during the fermentation, the microorganism of the species Paenibacillus macerans may be added at regular intervals to the fermentation substrate Addition of the microorganism of the species Paenibacillus macerans at regular intervals leads to an increase in the Lebendzellzahl and thus to an improved sequence of fermentative processes, such as hydrolysis with a simultaneous improved utilization of the fermentation substrate for fermentation.

According to a preferred embodiment of the present invention, the microorganism of the species Paenibacillus macerans is added in an amount to the

Fermentation substrate added so that after addition of the proportion of the microorganism of

Species Paenibacillus macerans between 10 ^"8 % and 50% of the total in the

Fermentation substrate present microorganisms. In particular in

Depending on the fermenter size and thus depending on the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

Both the determination of the total number of microorganisms in the fermentation substrate as well as the determination of the proportions of different types of microorganisms in the fermentation substrate is not a problem for the expert. For example, with the help of fluorescence-labeled oligosensors specifically the proportion of different microorganisms in the fermentation substrate after the identified above A microorganism of the species Paenibacillus macerans is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Paenibacillus macerans between 10 ^"6% and 25% of the total number of present in the fermentation substrate microorganisms. Especially preferred is the microorganism of the species Paenibacillus macerans is added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Paenibacillus macerans between 10 ^"4% and 10% of the total number present in the fermentation substrate microorganisms. Most preferably, the microorganism of the species Paenibacillus macerans is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Paenibacillus macerans is between 10 ^-3 % and 1% of the total number of microorganisms present in the fermentation substrate.

In principle, the addition of microorganisms of the species Paenibacillus macerans can be carried out at any point in the fermentation process; in particular, microorganisms of the species Paenibacillus macerans can also be used to inoculate fermentation substrate upon first use or restart of a fermenter. Microorganisms of the species Paenibacillus macerans may be added in the form of a culture once or several times at regular or irregular intervals, but preferably weekly or monthly, more preferably daily or twice to five times per week in an appropriate concentration and amount. Suitable concentrations of microorganisms and added amounts have already been mentioned or are described in the working examples.

It is also possible to add microorganisms of the species Paenibacillus macerans in case of disturbances in the fermentation process to stabilize the fermentation. Such disorders can be detected early by monitoring certain characteristic parameters of the fermentation. Characteristic parameters provide information about the quality of an ongoing fermentation process for the production of biogas. Such characteristic parameters are not only the amount of biogas produced and the methane content of the biogas produced but also, for example, the hydrogen content of the biogas produced, the pH of the fermentation substrate, the redox potential of the Fermentation substrate, the carboxylic acid content of the fermentation substrate, the proportions of various carboxylic acids in the fermentation substrate, the hydrogen content of the fermentation substrate, the proportion of dry matter in the fermentation substrate, the proportion of organic dry matter in the fermentation substrate, the viscosity of the fermentation substrate and the volume loading of the fermentation.

The present invention also encompasses the use of a microorganism of the species Paenibacillus macerans for the treatment of biomass.

The present invention also includes the use of a microorganism of the species Paenibacillus macerans for the liquefaction of biomass.

The present invention also encompasses the use of a microorganism of the species Paenibacillus macerans for the fermentative production of biogas from biomass.

The present invention also encompasses the use of the liquefied biomass for biofuel production obtained by one of the described processes. The liquefied biomass obtained by one of the processes described is preferably used for the production of bioethanol.

Likewise, the present invention comprises the strain of the microorganism Paenibacillus macerans SBG2, as deposited under No. DSM 22569. The microorganism Paenibacillus macerans SBG2 was deposited in a pure culture at the German Collection of Microorganisms and Cell Cultures GmbH in Braunschweig under the Budapest Treaty. The name is Paenibacillus macerans SBG2 with the accession number DSM 22569.

Likewise, the present invention comprises the strain of the microorganism Clostridium sartagoformum SBGIa, as deposited under No. DSM 22578. The microorganism Clostridium sartagoformum SBGIa was deposited in a pure culture at the German Collection of Microorganisms and Cell Cultures GmbH in Braunschweig under the Budapest Treaty. The name is Clostridium sartagoformum SBGIa with the accession number DSM 22578. Bacteria of the species Paenibacillus macerans can be isolated from the fermentation substrate or fermentation residue of a fermenter with the aid of methods known to those skilled in the art. In this case, a suitable substrate is introduced from a fermenter into a selection medium, cultured for a long time and finally isolated individual colonies of microorganisms from the selection medium. After amplification of the microbial DNA obtained therefrom by PCR, microorganisms of the species Paenibacillus macerans can be selected on the basis of the DNA.

Bacteria Paenibacillus macerans SBG2 were isolated from the fermentation substrate of a post-fermenter. For this purpose, nitrogen and carbon dioxide were passed through a liquid selection medium, then Na ₂ S was added to the selection medium and autoclaved (20 min. At 121 ⁰ C). Then, the biomass obtained from the secondary digester was introduced into the selection medium and cultured for at least one week at a temperature status of at least 30 ⁰ C. A sample obtained from the liquid selection medium was applied to a solid selection medium and subsequently the colonies of microorganisms grown on the solid selection medium were selected. After amplification of the obtained microbial DNA by PCR, a comparison with known DNA sequences could be performed.

After the bacteria Paenibacillus macerans SBG2 had been successfully isolated from the fermentation substrate of the postgrader, these microorganisms were subjected to sequence analysis. A partial sequence of the 16S rRNA was determined, which was then transformed into the corresponding DNA sequence. The DNA sequence SEQ ID NO: 1 comprises 1476 nucleotides. The next relative was the microorganism Paenibacillus sp. H10-05 identified (1503 nucleotides) isolated from various habitats in Korea (Genbank Identification Number AM162315). A comparison of the sequences shows that in comparison to SEQ ID No. 1 in the microorganism Paenibacillus sp. H10-05 contains a total of 35 nucleotide or gap replacements. With a length of the sequence of Paenibacillus macerans SBG2 of 1476 nucleotides, an identity of 97.63% is calculated. The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.63% sequence identity with the nucleotide sequence of SEQ ID NO: 1. More preferably, the nucleotide sequence contains a sequence range greater than 97.64% or greater than 97.66% or greater than 97.68% or greater than 97.70% or greater than 97.75% or greater than 97.80%. or more than 97.90% sequence identity with the nucleotide sequence of SEQ ID NO: 1.

Particularly preferably, the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 1. More preferably, the nucleotide sequence contains a sequence range greater than 98.1% or greater than 98.2% or greater than 98.3% or greater than 98.4% or greater than 98.5% or greater than 98.6%. or more than 98.7% or greater than 98.8% or greater than 98.9% sequence identity with the nucleotide sequence SEQ ID NO: 1, and more preferably the nucleotide sequence contains a sequence region having greater than 99% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 1, and more preferably the nucleotide sequence contains a sequence region having greater than 99.8% sequence identity having the nucleotide sequence of SEQ ID NO: 1.

According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 1.

In preferred embodiments of the present invention, SEQ ID NO: 1 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve Positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or 22 positions or 23 positions or 24 positions or at 25 positions or at 26 positions or at 27 positions or at 28 positions or at 29 positions or at 30 positions or at 31 positions or at 32 positions or at 33 positions or at 34 positions or at 35 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for treating biomass, in particular a process for liquefying biomass and / or a process for the fermentative production of biogas from biomass, wherein a microorganism is present in the culture of microorganisms which has a nucleotide sequence containing a sequence region having at least 97.63% sequence identity with the nucleotide sequence of SEQ ID NO: 1, wherein the microorganism constitutes at least 10 ^"4 % of the total number of microorganisms present in the culture.

Preference is given in the suitable for use in a method for the treatment of biomass, in particular a method for liquefying biomass and / or a method for fermentative production of biogas from biomass culture of microorganisms present a microorganism having a nucleotide sequence with a sequence region, the more than 97.64% or more than 97.66% or more than 97.68% or more than 97.70% or more than 97.75% or more than 97.80% or more than 97.90% or more as 98.0% or more than 98.1% or more than 98.2% or more than 98.3% or more than 98.4% or more than 98.5% or more than 98.6% or more 98.7% or greater than 98.8% or greater than 98.9% sequence identity with the nucleotide sequence SEQ ID NO: 1. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 1. According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the treatment of biomass, in particular a process for the liquefaction of biomass and / or a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 1. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to a very particularly preferred embodiment, a microorganism which has a nucleotide sequence is present in the culture of microorganisms suitable for use in a process for the treatment of biomass, in particular a process for liquefying biomass and / or a process for the fermentative production of biogas from biomass containing a sequence region corresponding to the nucleotide sequence of SEQ ID NO: 1.

According to further preferred embodiments, the microorganism Paenibacillus macerans accounts for at least 10.sup.- ² %, preferably at least 1% of the total number of microorganisms present in the culture.Preferably, the microorganism Paenibacillus macerans makes at least 10%, more preferably at least 25% of the total number in the Culture of existing microorganisms.

Most preferably, the microorganism Paenibacillus macerans accounts for at least 50%, in particular at least 90%, of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a method for the treatment of biomass, in particular a method for liquefying biomass and / or a method for fermentative production of biogas from biomass, which is a pure culture of the microorganism Paenibacillus macerans SBG2 as characterized above with respect to its nucleotide sequence. Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

The present invention also encompasses an immobilized culture of microorganisms suitable for use in a method for the treatment of

Biomass, in particular a process for the liquefaction of biomass and / or a process for the fermentative production of biogas from biomass, wherein in the immobilized culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region containing at least 97.63% Sequence identity with the nucleotide sequence SEQ ID

No. 1 has.

Preferably, in the method for the treatment of biomass, in particular a method for liquefying biomass and / or a method for the fermentative production of biogas from biomass suitable immobilized culture of microorganisms present a microorganism having a nucleotide sequence with a sequence region, more than 97.64% or more than 97.66% or more than 97.68% or more than 97.70% or more than 97.75% or more than 97.80% or more than 97.90% or more than 98.0% or more than 98.1% or more than 98.2% or more than 98.3% or more than 98.4% or more than 98.5% or more than 98.6% or greater than 98.7% or greater than 98.8% or greater than 98.9% sequence identity to the nucleotide sequence of SEQ ID NO: 1. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to further preferred embodiments, a microorganism which contains a nucleotide sequence is present in the immobilized culture of microorganisms suitable for use in a process for the treatment of biomass, in particular a process for the liquefaction of biomass and / or a process for the fermentative production of biogas from biomass Has sequence region having more than 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 1. Very particularly preferably contains the Nucleotide sequence has a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to a very particularly preferred embodiment, the invention is for use in a method for the treatment of biomass, in particular a method for liquefying biomass and / or a method for fermentative

Production of biogas from biomass suitable immobilized culture of

Microorganisms include a microorganism having a nucleotide sequence containing a sequence region corresponding to the nucleotide sequence of SEQ ID NO: 1.

The present invention also encompasses the use of microorganisms as characterized above with respect to their nucleotide sequence in a method of treating biomass. These microorganisms are preferably used in one of the methods for the treatment of biomass explained in greater detail above.

The present invention also encompasses the use of microorganisms as characterized above with respect to their nucleotide sequence in a process for liquefying biomass. These microorganisms are preferably used in one of the above-explained methods for liquefying biomass.

The present invention also encompasses the use of microorganisms as characterized above with respect to their nucleotide sequence in a process for the fermentative production of biogas from biomass. These microorganisms are preferably used in one of the methods explained in more detail above for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms as characterized above with respect to their nucleotide sequence in a method of treating biomass. These cultures of microorganisms are preferably used in one of the methods for the treatment of biomass explained in more detail above. The present invention also encompasses the use of a culture of microorganisms as characterized above with respect to their nucleotide sequence in a process for liquefying biomass. These cultures of microorganisms are preferably used in one of the above-explained methods for liquefying biomass.

The present invention also encompasses the use of a culture of microorganisms as characterized above with respect to their nucleotide sequence in a process for the fermentative production of biogas from biomass. These cultures of microorganisms are preferably used in one of the methods explained in more detail above for the fermentative production of biogas from biomass.

Brief description of the drawings

To illustrate the invention and to illustrate its advantages, embodiments are given below. The exemplary embodiments will be explained in more detail in connection with FIGS. 1 to 3. It goes without saying that the statements made in connection with the exemplary embodiments are not intended to limit the invention. Show it:

Fig. 1 shows a substrate flow test for investigating the degree of liquefaction of fermentation substrate;

Fig. 2 Results of a fermentation: Plotted is the gas yield as space-time yield (N l / l) and the space load of the fermenter against time;

Fig. 3 results of a further fermentation: Plotted is the gas yield as space-time yield (Nl / I) and the specific gas yield (Nm ³ A) of the fermenter against time; Ways to carry out the invention

Bacteria Paenibacillus macerans SBG2 were successfully isolated from the fermentation substrate of a post-fermenter. The deposit of the organism was carried out in a pure culture at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) according to the Budapest Treaty (Paenibacillus macerans SBG2 with the accession number DSM 22569).

Bacteria Clostridium sartagoformum SBGIa were also successfully isolated from the fermentation substrate of a post-fermenter. The deposit of the organism was carried out in a pure culture at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) according to the Budapest Treaty (Clostridium sartagoformum SBGIa with the accession number DSM 22578).

As can be seen in the following examples, bacteria of the strain Clostridium sartagoformum SBGIa both in mixed culture and in pure culture properties according to the invention similar to those to bacteria of the strain Paenibacillus macerans SBG2, such. an increase in the volume of space, an increase in gas yield and a stabilization of the biogas process as well as the ability to liquefy biomass.

Unless otherwise stated below, standard molecular biology methods have been used, e.g. by Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2nd Edition, Col. Spring Harbor Laboratory Press, Col. Spring Harbor, New York.

Isolation and accumulation of microorganisms

Bacteria of the strain Paenibacillus macerans SBG2 were successfully isolated from the fermentation substrate of a post-fermenter. The microorganisms were isolated using a selection medium containing carboxymethylcellulose (CMC) as the sole carbon source. Carboxymethylcellulose is very similar to the cellulose contained in fermentation substrates of biogas plants and, moreover, has an improved solubility in an aqueous medium due to the linking of the hydroxyl groups with carboxymethyl groups (-CH ₂ -COOH-). That for selection Medium used by Paenibacillus macerans SBG2 (DSMZ medium 520 plus 1% CMC and 0.2% yeast extract) was gassed with N ₂ for selection to be made under anaerobic conditions. Contained residual oxygen was reduced by means of 0.5 g / l Na ₂ S.

The selection medium was then inoculated with the supernatant of material from a post fermenter (diluted 1: 2000). After one week of cultivation at 40 ^° C., single rods were observed under microscopic analysis. Further selection of liquid cultures and isolation to pure cultures was achieved by smearing on anaerobic carboxymethylcellulose plates.

For larger amounts of bacteria of the strain Paenibacillus macerans SBG2 (eg for the addition in the fermenter type 3), the cultivation was carried out under the specified conditions in a 1 m ³ fermenter, which was inoculated with 100 ml preculture. Since growth is quite fast with a doubling time of about 2 h, Paenibacillus macerans SBG2 is particularly suitable for biotechnological application. Smaller amounts of bacteria (eg for addition to fermenter types 1, 2 and 4) were cultured in 500 ml to 1 l scale. Cultivation for the addition to a fermentation process took place over a period of 1 to 2 days. Cell densities in the range of 10 ⁸ to 10 ¹⁰ cells per ml of culture medium were achieved. The bacterial cells were harvested by centrifugation and taken in the smallest possible volume of fresh medium before they were used in the fermentation process. For interim storage, the cells were frozen.

Microorganisms of the species Clostridium sartagoformum and Clostridium sporosphaeroides, e.g. for the use of mixed cultures with approximately equal proportions of the 3 mentioned microorganisms could be cultured under the same conditions.

DNA isolation and nucleotide sequence determination

The cell material of the grown sporadic colonies was used for amplification of the microbial DNA by the Colony PCR method according to a standard program. To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the cell DNA by PCR. For this purpose, the primers with the sequences GRGTTTGATCCTGGCTCAG and ACGGHTACCTTGTTACGACTT were used (indicated in 5 ^" → 3 ^" direction, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning - Handbook) and examined by colony PCR. The obtained colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

Sequenzanalvse

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371) and classified as an organism of the species Paenibacillus macerans become. An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov the clone Paenibacillus sp. H 10-05 determined as the next relative.

Liquefaction of cellulose-containing medium

Experiments with a pure culture of the hydrolytically active, fermentative microorganism Paenibacillus macerans SBG2 showed that the addition of the highly viscous, viscous carboxymethylcellulose-containing selection medium leads to a successive liquefaction of the medium to a water-like consistency during bacterial growth, which by shaking the culture flask and visual inspection can be clearly observed. Liquefaction of fermentation substrate - Substrate flow test

The liquefaction of a fermentation substrate with a high dry matter content or the

Maintaining a liquid-pulpy consistency in a wet fermentation process is essential to ensure a smooth and cost-effective process of the technical process. In some cases continuous fermentation also leads to an increase in the fermenter content, which also has a negative effect on the biogas production process.The effect that the addition of microorganisms has on the consistency of the fermentation substrate was determined by a test in which the flow behavior of substrate samples was measured on an inclined plane (substrate flow test).

Starting material for the flow test was material from a biogas plant with a dry matter content of about 8-12%. Each 500 ml of material from a fermenter was filled into 1 I Schott bottles and incubated for 1 day at 40 ⁰ C. Subsequently, each of the bacterial cell mass from a 500 ml preculture (equivalent to approximately 4 x 10 ¹¹ cells) of Paenibacillus macerans SBG2, Clostridium sartagoformum SBGIa, Clostridium sporosphaeroides SBG3 or a mixture of the three bacteria was resuspended in equal proportions in 1 ml of medium, and added for further Incubated for 5 days at 40 ^° C. with shaking. For the control sample, only 1 ml of medium was added. Each 1, 3 g of the samples were placed at the starting point of the inclined plane of metal and read on a cm-scale, the flow rate (distance traveled per 10 min) of the respective samples, which is a measure of the liquefaction or the viscosity of the fermentation substrate , The results of the flow test are shown in FIG.

The following traces are shown in FIG. 1: Lane 1: control without addition of microorganisms (comparative example); Lane 2: Clostridium sartagoformum SBGIa (comparative example); Lane 3: Clostridium sporosphaeroides SBG3 (comparative example); Lane 4: Paenibacillus macerans SBG2; Lane 5: Mixture of Clostridium sartagoformum SBGI a, Clostridium sporosphaeroides SBG3 and Paenibacillus macerans SBG2 in equal parts. FIG. 1A shows the traces directly after the application of the samples, FIG. 1B after 2 min. and Figure 1 C after 18 min. It was found that the viscosity of the fermenter content decreased sharply due to the addition of live bacteria of the strain Paenibacillus macerans SBG2. With the addition of the organisms Clostridium sartagoformum SBGI a, the viscosity decreased to a lesser extent, while organisms Clostridium sporosphaeroides SBG3 exerted an even stronger effect. By adding a mixture of the 3 microorganisms, it was possible to achieve a liquefaction of the substrate which was approximately as strong as when the species Paenibacillus macerans SBG2 was added, indicating that the three microorganisms exert an additive effect. In a further experiment it could be shown that the addition of dead cells (dead autoclaving) had practically no effect. A visual inspection of the samples also showed that when adding live cells of the strain Paenibacillus macerans SBG2, the proportion of slimy substances decreased. It was surprising that the added bacteria, even in the case of a substrate which has already undergone a fermenter and thus a fermentation process brought about by microorganisms, still brought about further liquefaction. This means that in a fermentation process without external addition of microorganisms, the substrate is not completely degraded, but harbors additional energy potential, which can be used by the addition of the microorganisms of the invention.

Determination of cell density

In order to determine the cell density with respect to living bacteria, in each case 10 μl of bacterial sample (eg preculture, bacterial culture batch, supernatant from fermenter sample) were mixed with 2 μl of BacLight ™ dye (Invitrogen) and microscoped at 1000 × magnification (AXIO Imager A1, Zeiss, Jena). Under UV light and using two different filters, the Bacl_ight ™ system can be used to determine the proportion of living cells in the sample. The total number of cells was counted in a Thomazählkammer. The proportion of live cells from the precultures was generally higher than 90%.

Determination of the cellular content of Clostridium sporosphaeroides SBG3 or Paenibacillus macerans SBG2 or Clostridium sartaαoformum SBGI a in a sample - "Fishing" The proportion of bacteria of the species Clostridium sporosphaeroides SBG3 or Paenibacillus macerans SBG2 or Clostridium sartagoformum SBGI a was determined by whole cell hybridization according to the method described in Amann et al., (1995, Microbiol Rev 59, 143-169) Probe for fishing was used for Clostridium sporosphaeroides SBG3 a labeled oligonucleotide with the sequence Cy3-CCACAGCTCTCACGCCCG (indicated in 5 ^" → 3 ^" direction), for Paenibacillus macerans SBG2 a labeled oligonucleotide having the sequence Cy3- GCAACCCGAACTGAGACC (indicated in 5 ^" → 3 ^" direction) and for Clostridium sartagoformum SBGIa a labeled oligonucleotide having the sequence Cy3 CTTCATGCGAAAATGTAA (indicated in 5 ^" → 3 ^" direction). The oligonucleotides carried as marker at the 5 ^" end of the dye indocarbocyanine (Cy3).

fermenter types

Experiments were carried out on a pilot plant scale in different fermenter types. The fermenters were operated at operating temperatures of about 40 ⁰ C. The substrate used was mainly corn silage with an organic dry matter (OTS) content of 30-35%. In order to ensure the supply of trace elements for the metabolically active microorganisms involved in the fermentation, commercially available trace element mixtures were generally added (eg Novodyn®).

Fermenter type 1: Lying plug fermenter rectangular, volume 150 1, subdivision of the fermenter compartment by retracted wall with small-area passage for the substrate flow, division of the fermenter space in VA directly after substrate addition nozzle and ³ A to pinhole, 2-stage system.

Fermenter Type 2: Lying plug fermenter rectangular, volume 150 I as 1st stage plus totally mixed round fermenter, volume 200 I as 2nd stage; Total volume 350 I. 2-stage plant with recirculation between round fermentor and plug-flow fermenter.

Fermenter type 3: horizontal cylindrical plug-flow fermenter, volume 30 m ³ , single-stage plant. Distribution of microorganisms within the fermenter - "Tracer test" Since the mixing of the viscous substrate and thus also other additives in a fermenter takes some time and also depends on the stirring technique used, a test was established with which the period to a uniform In this "tracer test", instead of microorganisms, powdered LiCI was added at a concentration of 10 mg lithium / kg starting substrate (double the amount for fermenter type 2) at the site of substrate addition. Thereafter, at various times (added on day 1) at various points of the fermenter (at the beginning of the fermenter after substrate entry and at the end before the residual substrate exit) substrate samples taken and this by means of inductively coupled plasma atomic emission spectroscopy according to the methods DIN EN ISO 13346 (S7a) and DIN EN ISO 11885 (E22) for their Li content. After complete mixing of the fermenter contents, the measured Li content at the front end of the fermenter and at the rear end of the fermenter should be the same and have a value of about 10 mg / kg starting substrate (about 20 mg / kg starting substrate for fermenter type 2) , The results of such "tracer tests" for 3 different fermenter types are shown in Table 1.

Table 1 :

It was observed that a complete mixing of the fermenter content and, consequently, a uniform distribution of the tracer in the fermenters types 2 and 3 was achieved five days after the addition of the tracer, in the fermenter type 1 only after 8 days. It can be assumed that the period of time until a uniform distribution of added microorganisms also amounts to several days.

Long-term fermentation for biogas production with or without the addition of microorganisms according to the invention

On a technical scale, at least a doubling of the average room load known from practice could be stably achieved. Stable operation was achieved at a volume loading of 6 to 7 kgoTS / m ³ d by adding about ¹ x 10 ¹³ cells per m ³ and week. In doing so, nothing was changed in the usual process parameters, such as temperature (40 ^° C.) and pH value (6-9), or buffer capacity.

During the fermentation process in different test fermenters with a volume of 150 l to 30,000 l under realistic plant conditions (T ~ 40 ⁰ C, pH 6 - 8, continuous feed, continuous mixing) was over a period of several months, the time course of the space load of the fermenter in kilograms of organic dry matter per cubic meter per day (kgoTS / m ³ d) and the time course of the biogas produced. For better comparability of the various experimental plants, the space-time yield (standard liters of produced biogas per liter fermentation volume, [Nl / I]) was given for the gas yield instead of the specific biogas yield, in which the amount of biogas produced was normalized to the respective fermentation volume. In addition, the time course of the theoretically expected gas production was calculated.

The Board of Trustees for Engineering and Construction in Agriculture e. V. (KTBL) but also the Federal Research Center for Agriculture and the Agency for Renewable Resources have published guidelines that roughly indicate which amounts of biogas, depending on the substrate used in a stable fermentation are expected. These theoretical guidelines can thus reflect the amount of theoretically generated biogas. Alternatively, benchmarks issued by other institutes in other countries may be used. For maize silage, whose shares in OTS were between 22% and 40%, expected gas yields in the range of 533 NI / kgoTS and 650 NI / kgoTS were mentioned (in "Biogas Production and Utilization Guide", 2006, edited by the Fachagentur Nachwachsende Rohstoffe eV, Gülzow and KTBL Homepage, www.ktbl.de) The time course of the theoretical gas production in [Nl / d] was calculated according to such guideline values, whereby in all experiments already here the very high value for a theoretical gas yield of 663 NI / The actually measured values for the proportions of oTS in the maize silage used varied between 30 and 40% oTS depending on the batch.

In addition to gas yield and volume load, a number of other characteristic parameters of the fermentation process such as dry matter content of the fermenter content, pH, acid concentration (eg acetic acid, propionic acid, butyric acid, valeric acid, acetic acid equivalent), temperature, specific gas yield, composition of the biogas, viscosity, conductivity, redox potential and concentration measured on nutrients and trace elements.

Lanqzeitfermentation in a fermenter type 3 with the addition of microorganisms Paenibacillus macerans SBG2

This pilot plant is a large test facility on a pilot plant scale with a volume of 30 m ³ , which is constructed as a horizontal cylindrically shaped plug-flow fermenter. It is operated as a 1-stage system.

FIG. 2 shows measurement results of various characteristic parameters during a fermentation process in a type 3 experimental fermenter with and without the addition of microorganisms of the strain Paenibacillus macerans SBG2. The curve provided with the reference numeral 10 shows the time course the volume load of the fermenter in kilograms of organic dry matter per cubic meter per day (kgoTS / m ³ d), the curve indicated by the reference numeral 20 the time course of the measured space-time yield (Nl / I), the curve indicated by the reference numeral 30, the time course of measured space-time yield, averaged over 6 days. With the reference numeral 40, the time course of the theoretical gas production (Nl / I) is marked. The reference numeral 50 marks the symbols each representing the one-time addition of microorganisms of the strain Paenibacillus macerans SBG2.

The biogas plant was operated from day 70 to day 102 without external addition of microorganisms at an average volume load of about 4 kgoTS / m ³ d and provided in a stable fermentation process space-time yields averaging 2.8 Nl / I, slightly above the expected theoretical Gas yield lay. In anticipation of the planned addition of Paenibacillus macerans SBG2 microorganisms, 20 l of water were added each day instead of a bacterial culture. From day 102 to day 138, 20 l of a pure culture of Paenibacillus macerans SBG2 (bacterial concentration about 2 × 10 ⁹ cells / ml) were added daily. The addition of the microorganisms according to the invention caused that in the fermentation process, the volume load of 4 kgoTS / m ³ d could be increased to about 7 kgoTS / m ³ d without the fermentation process would become unstable. This proved the observation of further process parameters such as pH value (stable between pH 7.5 to 7.7), gas quality (methane content stable at 53 to 54%) or the measured values for various free fatty acids (no increase to critical values despite increased substrate supply) , The space-time yield could be increased from values of almost 3 Nl / l to values above 5 Nl / l, so that the energy efficiency of the fermentation process increased considerably. From the beginning of the addition of microorganisms Paenibacillus macerans SBG2, the gas production was significantly higher than the theoretically expected values.

FIG. 3 shows measurement results of various characteristic parameters during a fermentation process in a type 3 experimental fermenter with and without the addition of microorganisms of the species Paenibacillus macerans SBG2. The curve provided with the reference numeral 10 shows the time course of measured specific gas yield (Nm ³ / t), the curve indicated by the reference numeral 20, the time course of the measured specific gas yield (Nm ³ A), averaged over 6 days. The curve provided with the reference numeral 30 shows the time course of the measured space-time yield (Nl / I), the curve indicated by the reference numeral 40, the time course of the measured space-time yield (Nl / I), averaged over 6 days. The arrow marks the beginning of the addition of microorganisms of the strain Paenibacillus macerans SBG2 from day 102.

As already shown in FIG. 2, the space-time yield (NI / I) in the Type 3 fermenter rose very sharply after the addition of microorganisms of the strain Paenibacillus macerans SBG2 because the volume load could be greatly increased. With increased substrate supply thus increased in a still stable fermentation process, the gas yield achieved per fermenter volume. It can be seen from the experimental results shown in FIG. 3 that the specific gas yield (Nm ³ A) also increased significantly after the addition of microorganisms of the strain Paenibacillus macerans SBG 2 (see curves with the reference numbers 10 and 20). Before the addition of the microorganisms of the strain Paenibacillus macerans SBG2, the average specific gas yield was about 720 Nm ³ / t substrate, after addition of microorganisms of the strain Paenibacillus macerans SBG2 about 780 Nm ³ / t substrate. The measured variable "specific gas yield" is not normalized to the fermentation volume, but to the amount of substrate used, ie an increase in the specific gas yield means a higher energy efficiency in the substrate conversion, but an economic advantage for the user is an increase in the space-time yield as well an increase in the specific gas yield.

By adding the pure cultures of Paenibacillus macerans SBG2, the volume load in fermentations could be increased to a maximum value of about 8 kgoTS / m ³ d.

Parallel to the increasing volume load, an increase in biogas yield was observed. It was a coincidence of the amount of biogas produced in standard liters / day [Nl / d] with the theoretical gas production in standard liters / day [Nl / d] or a gas production increased compared to the theoretical gas production.

The space load of the plant could be further increased as a result of the addition of Paenibacillus macerans SBG2. The percentage content of dry substance or organic dry matter remained almost constant. This observation suggests that no accumulation of non-fermented organic dry substance occurs during the fermentation of the fermentation substrate. The addition of pure cultures of Paenibacillus macerans SBG2 thus contributes to a continuous conversion of the dry matter contained in the fermentation substrate, which in turn leads to a continuous fermentation by the accumulation of dry matter is reduced.

In phases in which the biogas plant is operated at a constant volume load, even a decrease in the dry matter is observed, which suggests that the microorganisms Paenibacillus macerans SBG2 with their hydrolytic metabolic activity not only reduce the accumulation of dry matter in the fermenter, but also the improve the hydrolytic conversion of this dry substance.

In the described embodiments, pure cultures of Paenibacillus macerans but also mixed cultures with a proportion of Paenibacillus macerans were used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The addition of the hydrolytically active, fermentative microorganism Paenibacillus macerans SBG2 has a positive effect on the hydrolysis of organic dry matter. By adding microorganisms of the species Paenibacillus macerans, the volume load of a fermenter can be increased from about 4 to 5 kgoTS / m ³ d to about 6 to 8 kgoTS / m ³ d under otherwise identical conditions, without even instability of the fermentation process would suggest. Parallel to the increased space load, the amount of biogas produced is significantly increased. In addition, the specific yield of biogas increases, since significantly more of the organic dry matter is degraded than in the absence of addition of microorganisms of the species Paenibacillus macerans. The use of microorganisms of the species Paenibacillus macerans leads to a dramatic improvement in the efficiency and efficiency of biogas plants.

Expression (original in electromscher format)

0-1 Form PCT / RO / 134 (SAFE) Information on a deposited microorganism and / or other stored biological material

0-1-1 created with

0-2 International File PCT / DE2009 / 075036

0-3 file number of the applicant or lawyer SCM067WO

1 The following information concerns the

Microorganism and / or other biological material used in the

Description is called

1-1 paragraph number Paenibacillus macerans page 1, line 1

1-3 Details concerning deposit

1-3-1 Name of depository DSMZ-Deutsche Sammlung von Mikroorganismen and

Cell Culture GmbH

1-3-2 Address of the depository Inhoffenstr. 7B

38124 Braunschweig, Germany

1-3-3 Date of filing 15. May 2009 (15.05.2009)

1-3-4 input number DSM 22569

1-4 Further information

1-5 States of destination for which special information is given all the countries of destination

1-6 Separately submitted information

These details are given to the International

Office transmitted later

TO BE COMPLETED BY THE REGISTRATION OFFICE

0-4 This form has been received with the international application

(Yes or no)

0-4-1 authorized agent

TO BE COMPLETED BY THE INTERNATIONAL OFFICE

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Claims

claims

1. A process for the treatment of biomass, characterized in that the biomass is added to a microorganism of the species Paenibacillus macerans.

2. The method according to claim 1, characterized in that it is a method for liquefying biomass.

3. The method according to claim 1, characterized in that it is a method for producing biogas from biomass.

4. The method according to at least one of claims 1 to 3, characterized in that the microorganism of the species Paenibacillus macerans is added in the form of a culture of microorganisms, wherein the microorganism of the species Paenibacillus macerans at least 10 ^"4 % of

Total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that in the culture of microorganisms, the microorganism of the species Paenibacillus macerans accounts for at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that in the culture of microorganisms, the microorganism of the species Paenibacillus macerans makes up at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that in the culture of microorganisms of the microorganism of the species Paenibacillus macerans accounts for at least 90% of the total number of microorganisms present in the culture.

8. The method according to at least one of claims 4 to 7, characterized in that a pure culture of the microorganism of the species Paenibacillus macerans is added.

9. The method according to at least one of claims 4 to 8, characterized in that the microorganism of the species Paenibacillus macerans biomass is added as part of at least one immobilized culture of microorganisms.

10. The method according to at least one of claims 1 to 9, characterized in that in time for the addition of the microorganism of the species Paenibacillus macerans additional biomass is added to the fermentation reactor.

1 1. The method according to at least one of claims 1 to 10, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

12. The method according to at least one of claims 3 to 11, characterized in that the production of biogas from biomass in a

Room load of ≥ 0.5 kgoTS / m ³ d, preferably ≥ 4.0 kgoTS / m ³ d, particularly preferably ≥ 6.0 kgoTS / m ³ d is performed.

13. The method according to at least one of claims 1 to 12, characterized in that the addition of substrate and the addition of the

Microorganism of the species Paenibacillus macerans is carried out continuously.

14. The method according to at least one of claims 1 to 13, characterized in that the microorganism of the species Paenibacillus macerans is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Paenibacillus macerans between 10 ^"8 % and 50% the total number of microorganisms present in the fermentation substrate.

15. The method according to claim 14, characterized in that the microorganism of the species Paenibacillus macerans is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Paenibacillus macerans between 10 ^"3 % and 1% of the total in the Fermentation substrate present microorganisms.

16. The method according to at least one of claims 1 to 15, characterized in that in addition microorganisms of the species Clostridium sartagoformum are added.

17. The method according to claim 16, characterized in that it is microorganisms of the strain Clostridium sartagoformum SBGI a.

18. The method according to at least one of claims 1 to 17, characterized in that in addition microorganisms of the species Clostridium sporosphaeroides are added.

19. The method according to claim 18, characterized in that it is microorganisms of the strain Clostridium sporosphaeroides SBG3.

20. The method according to at least one of claims 1 to 19, characterized in that it is in the microorganism of the species Paenibacillus macerans to a microorganism of the strain Paenibacillus sp. H10-05.

21. Use of a microorganism of the species Paenibacillus macerans for the treatment of biomass.

22. Use of a microorganism of the species Paenibacillus macerans for the liquefaction of biomass.

23. Use of a microorganism of the species Paenibacillus macerans for the fermentative production of biogas from biomass.

24. Use according to at least one of claims 21 to 23, characterized in that it is in the microorganism of the species Paenibacillus macerans to a microorganism of the strain Paenibacillus sp. H10-05.

25. Use of the obtained by a method according to at least one of claims 1 to 20 liquefied biomass for the production of biofuel.

26. Use according to claim 25, characterized in that the liquefied biomass is used for the production of bioethanol.

27. Strain of the microorganism Paenibacillus macerans SBG2 deposited with the DSMZ under No. 22569.

28. Strain of the microorganism Clostridium sartagoformum SBGI a deposited with the DSMZ under No. 22578.

29. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.63% sequence identity with the nucleotide sequence SEQ ID No. 1.

Microorganism according to claim 29, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 1.

31. Microorganism according to claim 30, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 1.

32. The microorganism according to claim 31, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 1.

33. The microorganism according to claim 32, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 1.

34. Microorganism according to claim 33, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 1.

35. Culture of microorganisms suitable for use in a process for the treatment of biomass, characterized in that in the culture of

Microorganisms a microorganism Paenibacillus macerans according to claims 29 to 34 is present, wherein the microorganism Paenibacillus macerans at least 10 ^" accounts for ⁴ % of the total number of microorganisms present in the culture.

36. Culture of microorganisms according to claim 35, characterized in that the microorganism Paenibacillus macerans constitutes at least 1% of the total number of microorganisms present in the culture.

37. Culture of microorganisms according to claim 36, characterized in that the microorganism Paenibacillus macerans constitutes at least 25% of the total number of microorganisms present in the culture.

38. Culture of microorganisms according to claim 37, characterized in that the microorganism Paenibacillus macerans makes up at least 50% of the total number of microorganisms present in the culture.

39. Culture of microorganisms according to claim 38, characterized in that the microorganism Paenibacillus macerans makes up at least 90% of the total number of microorganisms present in the culture.

40. Culture of microorganisms according to claim 39, characterized in that it is a pure culture of the microorganism Paenibacillus macerans.

41. Culture of microorganisms according to at least one of claims 35 to 40, characterized in that it is an immobilized culture of microorganisms.

42. Use of a microorganism according to at least one of claims 27 to 34 for the treatment of biomass.

43. Use according to claim 42, characterized in that it is a method for the treatment of biomass according to at least one of the claims

1 to 20 acts.

44. Use of a microorganism according to at least one of claims 27 to 34 for the liquefaction of biomass.

45. Use according to claim 44, characterized in that it is a method for liquefying biomass according to at least one of claims 2 to 20.

46. Use of a microorganism according to at least one of claims 27 to 34 for the fermentative production of biogas from biomass.

47. Use according to claim 46, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 3 to 20.

48. Use of a culture of microorganisms according to at least one of claims 35 to 41 for the treatment of biomass.

49. Use according to claim 48, characterized in that it is a method for the treatment of biomass according to at least one of claims 1 to 20.

50. Use of a culture of microorganisms according to at least one of claims 35 to 41 for the liquefaction of biomass.

51. Use according to claim 50, characterized in that it is a method for liquefying biomass according to at least one of

Claims 2 to 20 is.

52. Use of a culture of microorganisms according to at least one of claims 35 to 41 for the fermentative production of biogas from biomass.

53. Use according to claim 52, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 3 to 20.