WO2009086811A2

WO2009086811A2 - Paenibacillus macerans for the generation of biogas

Info

Publication number: WO2009086811A2
Application number: PCT/DE2008/075016
Authority: WO
Inventors: Monika Reuter; Vera Duchow; Daniel Vater
Original assignee: Schmack Biogas Ag
Priority date: 2008-01-10
Filing date: 2008-12-23
Publication date: 2009-07-16
Also published as: WO2009086811A3

Abstract

A method is disclosed for generating biogas from biomass in a fermentation reactor, wherein the biomass is treated with a microorganism of the type Paenibacillus macerans.

Description

Paenibacillus macerans for the production of biogas

Technical area

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

State of the art

Biogas plants produce methane through 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 microorganisms, i. in the absence of air.

The organic material used as fermentation substrate has a high molecular structure from a chemical point of view, which is degraded in the individual process steps of a biogas plant 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, it is possible to distinguish between four consecutive, but also parallel and interlocking biochemical single processes 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 formed by exoenzymes (eg cellulases, amylases, proteases, lipases) fermentative bacteria converted into soluble fission products. For example, fats are broken down into fatty acids, carbohydrates, such as polysaccharides, into oligo- and monosaccharides and proteins into 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.

Increasing the yield of end products from a given amount of starting materials, as in any chemical reaction, is an urgent goal of process control even in the case of biogas production. For biogas production from biomass, this means that from a given amount of organic fermentation substrate a large amount of methane should be formed. In addition, the highest possible space load of the fermenter should be achieved. 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.

There is therefore still a need for methods for the production of biogas, which allow an increased space compared to the prior art.

Presentation of the invention

The object of the invention is to provide a method for the production of biogas, which allows a comparison with the prior art increased space load of the fermentation reactor.

This object is achieved by the method for producing biogas 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 for producing biogas from biomass in a fermentation reactor. The biomass is added to a microorganism of the species Paenibacillus macerans.

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 in experimental investigations - A -

could be shown, the addition of a microorganism of the species Paenibacillus macerans causes an increase in the space load of a fermenter by more than 50%, without any instability of the fermentation process would occur. Parallel to the increased volume of space, the amount of biogas produced is more than doubled. 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. By adding microorganisms of the species Paenibacillus macerans, the residence time of the fermentation substrate in the fermenter can be significantly shortened with constant gas yield, which makes it possible to increase the volume load. The use of microorganisms of the species Paenibacillus macerans therefore leads to a dramatic improvement in the efficiency and efficiency of biogas plants.

Fermentation for the purposes of the present invention includes both anaerobic and aerobic material conversions by the action of microorganisms that lead to the production of biogas. This explanation of the term "fermentation" is given under the heading "fermentation in the Römpp Chemistry Lexicon in the 9th, extended edition, published by Georg Thieme Verlag on page 1306, to which reference is hereby made in full.

The specific yield of biogas produced is calculated from the amount of biogas produced divided by the amount of organic dry matter. The question of whether the production of biogas in a given fermenter under certain conditions is satisfactory, can not be judged by the amount of biogas produced alone. Of course, the amount of biogas produced depends strongly on the amount of substrate supplied, ie on the amount of organic dry matter, which is given in kilograms of organic dry matter. If instabilities of the fermentation process occur, the specific gas yield decreases. With inventive addition of microorganisms of the species Paenibacillus macerans, the space load can be increased even beyond the usual level, whereby a significantly increased amount of gas is generated. By the raised Degradation can be achieved a significantly increased specific gas yield with improved substrate utilization.

In the context of the present invention, the term "type of microorganisms" is understood to mean the corresponding basic category of biological taxonomy.Species of microorganisms are identified and distinguished on the basis of their DNA sequences, not just microorganisms having a specific DNA But also, to some extent, their genetic variants, it is well known to those skilled in the art which strains of microorganisms fall within the term "species Paenibacillus macerans". Microorganisms of the species Paenibacillus macerans are not known in connection with the production of biogas by fermentation of organic substrates.

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. In fermentation substrates of biogas plants microorganisms of the species Paenibacillus could be detected only in small traces of less than 10 ^"4% share of the total number of present microorganisms macerans. Since the amount is not sufficient for the addition of the microorganism at isolated from their natural occurrence 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 species should be of microorganisms 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.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, using fluorescent-labeled oligosensors specifically the proportion of microorganisms of the species Paenibacillus macerans can be identified in a mixture. As already mentioned, 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.

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 makes up at least 10% of the total number of microorganisms present in the culture, more preferably the The microorganism Paenibacillus macerans accounts for at least 50% of the total number of microorganisms present in the culture, and more preferably, the microorganism Paenibacillus macerans accounts for 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 of a microorganism comprises the progeny of a single cell, which is isolated by a multi-step process from a mixture of different microorganisms. This multi-step mechanism begins with the separation of a single cell from a cell population and requires that the colony resulting from the cell through growth and cell division also remain separate from other single cells or colonies. By careful separation of a colony, resuspension in liquid and repeated spreading, pure cultures of microorganisms can be selectively obtained.

However, the isolation of a pure culture can also be carried out in liquid nutrient media, provided that the desired organism outnumbered in the starting material. By serial dilution of the suspension in the nutrient solution, it can finally be achieved that only one cell remains in the last dilution stage. This cell then represents the basis for a pure culture. This explanation of the term "pure culture" and possible methods for producing this pure culture under the keyword "pure culture" in the work "General Microbiology" by Hans G. Schlegel in the 7th revised edition of 1992, published by Georg Thieme Verlag on page 205, to which reference is hereby incorporated by reference.

The pure culture thus obtained is also characterized biochemically 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 is added as a component of at least one 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 makes at least 10 ^"4 % of the total number of immobilized cultures added to the fermentation substrate

Microorganisms. Particularly preferred is the microorganism of the species

Paenibacillus macerans at least 10 ^"2 % of the total in the immobilized

Of the microorganisms present in the 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 makes the microorganism of the species Paenibacillus 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, polyvinylpyrrolidone, 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, polystyrenesulfonate, 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 of maleic anhydride and methyl vinyl ether, Cellulose acetate butyrate, chitosan, polydialkyldimethylammonium chloride, mixtures of polyacrylic acids and polydiallyldimethylammonium chloride and mixtures thereof. The polymer material can also be crosslinked using conventional crosslinkers such as glutaraldehyde, urea / formaldehyde resins or tannin compounds.

Alginates as Immobilisate prove to be particularly advantageous because they take on the one hand no negative impact on the activity of the microorganism Paenibacillus macerans and there on the other by microorganisms slowly be reduced. 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 exact procedures for immobilization are known to the person skilled in the art.

According to a further embodiment, in addition to the addition of a microorganism of the species Paenibacillus macerans, additional biomass is added to the fermentation reactor. In this context, "timely" refers to the period of time which elapses between the addition of new substrate following the addition of the microorganisms.This time span should be kept as short as possible, which is why the term "promptly" also includes a parallel addition of the microorganism Paenibacillus macerans and of the new substrate.

However, the said period may also extend to several hours to one or more days. In this case, the space load in the fermentation reactor can be continuously increased or kept approximately constant by continuous addition of new substrate, the fermentation at all room loads, preferably at a space load of ≥ 0.5 kg of organic dry matter per m ³ and day [kg oTS / m ³ d ], more preferably at a volume load of ≥4.0 kg oTS / m ³ d and particularly preferably at a volume load of ≥8.0 kg oTS / m ³ d can be performed, which in comparison to the current state of the art of increasing the space load by more than twice as much.

According to a further preferred embodiment, therefore, the space load in the fermentation reactor is continuously increased by the continuous addition of biomass. Particularly preferably, the production of biogas from biomass takes place at a volume load of ≥ 0.5 kg oTS / m ³ d, particularly preferably ≥ 4.0 kg oTS / m ³ d and very particularly preferably ≥ 8.0 kg oTS / m ³ d.

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.

According to a further embodiment, the production of biogas from 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 addition, the biogas formed 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 40 ° C to 50 ⁰ C is efficiently fermented. 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. Preference is therefore given to the production of biogas from biomass at a temperature of 20 ⁰ C to 80 ⁰ C and particularly preferably at a temperature of 40 ° C to 50 ⁰ C.

Of course, all embodiments of the present invention are not limited to single-stage 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.

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 to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Paenibacillus macerans is between 10 ^-8 % and 50% of the total number of microorganisms present in the fermentation substrate In particular, depending on the size of the fermenter and thus depending on the amount of Fermentation substrate may 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. Thus, for example, with the help of fluorescence-labeled oligosensors specifically the proportion of different microorganisms can be identified in the fermentation substrate

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 ^"40 Zo 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.

It should again be pointed out that the addition of microorganisms of the species Paenibacillus macerans can be done at any point in the fermentation process, in particular microorganisms of the species Paenibacillus macerans can also be used for inoculation of fermentation substrate during initial start-up or re-commissioning of a fermenter.

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 expiring 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 the organic dry matter in the fermentation substrate, the viscosity of the fermentation substrate and the volume loading of the fermentation reactor.

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

Bacteria of the species Paenibacillus macerans can be isolated from the fermentation substrate 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 sequence 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. The DNA sequence SEQ ID NO: 1 comprises 1476 nucleotides. The next relative was Paenibacillus sp. H10-05 identified. A comparison of the sequences reveals that there are a total of 32 exchanges of nucleotides or gaps. With a length of the sequence of Paenibacillus macerans SBG2 of 1476 nucleotides, the FASTA algorithm gives a 97% match.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97% sequence identity with the nucleotide sequence of SEQ ID NO: 1. More preferably, the nucleotide sequence contains a sequence range greater than 97.1% or greater than 97.2% or greater than 97.3% or greater than 97.4% or greater than 97.5% or greater than 97.6%. or more than 97.7% or more than 97.8% or more than 97.9% sequence identity with the nucleotide sequence SEQ ID NO: 1, and more preferably the nucleotide sequence contains a sequence region having greater than 98% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 1, and more preferably the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence Nucleotide sequence SEQ ID NO.

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

Thus, the present invention also encompasses all microorganisms having a nucleic acid whose nucleotide sequence has at least one sequence region which has nucleotide exchange in only one position relative to the nucleotide sequence SEQ ID No. 1. Also included are all microorganisms whose DNA sequence has at least one sequence region which is opposite to the Nucleotide sequence SEQ ID NO. 1 only at five positions has a nucleotide exchange. In addition, all microorganisms are encompassed whose DNA sequence has at least one sequence region which, compared to the nucleotide sequence SEQ ID No. 1, has only one nucleotide exchange at ten positions.

In addition, the present invention also encompasses all microorganisms whose DNA sequence has at least one sequence region which has nucleotide exchanges in 15 positions relative to the nucleotide sequence SEQ ID No. 1. Also included are all microorganisms whose DNA sequence has at least one sequence region which has nucleotide exchanges with the nucleotide sequence SEQ ID No. 1 at 20 positions. In addition, all microorganisms are encompassed whose DNA sequence has at least one sequence region which has nucleotide exchanges with respect to the nucleotide sequence SEQ ID No. 1 at 25 positions.

In addition, the present invention also encompasses all microorganisms whose DNA sequence has at least one sequence region which has nucleotide exchanges in 30 positions relative to the nucleotide sequence SEQ ID No. 1. Also included are all microorganisms whose DNA sequence has at least one sequence region which has nucleotide exchanges with the nucleotide sequence SEQ ID No. 1 at 31 positions.

It goes without saying that the exchanges of the nucleotides can be present at any position of the DNA sequence. In particular, the exchanges can be present at arbitrarily far apart positions. Are compared to

Nucleotide sequence SEQ ID NO: 1, for example, six nucleotides exchanged, these six exchanged nucleotides may be adjacent to each other.

In this case, a six nucleotide portion of the nucleotide sequence of SEQ ID NO: 1 would be altered. Likewise, however, the six exchanged nucleotides may each be, for example, 100 nucleotides apart. The

Exchanges can be in any combination. Likewise, the exchanges mentioned may also be in the form of gaps. The present invention therefore also encompasses all microorganisms having a nucleic acid whose nucleotide sequence has at least one sequence region in which, compared to the nucleotide sequence SEQ ID No. 1, only one nucleotide is missing at one position. Also included are all microorganisms whose DNA sequence has at least one sequence region in which, compared to the nucleotide sequence SEQ ID No. 1, only five positions lack nucleotides. In addition, all microorganisms are included whose DNA sequence has at least one sequence region in which, compared to the nucleotide sequence SEQ ID No. 1, only ten positions lack nucleotides.

In addition, the present invention also encompasses all microorganisms whose DNA sequence has at least one sequence region in which, compared to the nucleotide sequence SEQ ID No. 1, nucleotides are absent at 15 positions. Also included are all microorganisms whose DNA sequence has at least one sequence region in which nucleotides are missing at 20 positions in comparison to the nucleotide sequence SEQ ID No. 1. In addition, all microorganisms are encompassed whose DNA sequence has at least one sequence region in which nucleotides are absent from the nucleotide sequence SEQ ID No. 1 at 25 positions.

In addition, the present invention also encompasses all microorganisms whose DNA sequence has at least one sequence region in which, compared to the nucleotide sequence SEQ ID No. 1, nucleotides are absent at 30 positions. Also included are all microorganisms whose DNA sequence has at least one sequence region in which the nucleotide sequence SEQ ID No. 1 at 31 positions lacks nucleotides.

It goes without saying that the nucleotides can be absent at any position of the DNA sequence. In particular, the gaps may be present at arbitrarily far apart positions. For example, if there are six nucleotides missing from the nucleotide sequence of SEQ ID NO: 1, these six missing nucleotides in the nucleotide sequence SEQ ID NO: 1 may be adjacent to each other. In this case, a six nucleotide portion of the nucleotide sequence SEQ ID NO: 1 would be missing. Likewise, the six missing ones However, nucleotides in the nucleotide sequence SEQ ID No. 1, for example, are each 100 nucleotides apart. The gaps can therefore be present in any combination.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region having at least 97% 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.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.1% or greater than 97.2% or greater than 97 , 3% or more than 97.4% or more than 97.5% or more than 97.6% or more than 97.7% or more than 97.8% or more than 97.9% Sequence identity with the nucleotide sequence SEQ ID No. 1. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 98% 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 fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range of more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO. 1 has. Most 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 a very particularly preferred embodiment, in the suitable for use in a process for the fermentative production of biogas from biomass culture of microorganisms, a microorganism is present, the one A nucleotide sequence 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 accounts for at least 10%, particularly preferably at least 25% of the total number in the Culture of existing microorganisms.

Most preferably, the microorganism Paenibacillus macerans makes up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the microorganism Paenibacillus macerans accounts for at least 90% of the total number of microorganisms present in the culture. More preferably, it is a pure culture of microorganisms suitable for use in a process for the 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 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 which is at least 97% Sequence identity with the nucleotide sequence SEQ ID NO. 1 has.

Preference is given in the immobilized culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass a microorganism having a nucleotide sequence having a sequence range of more than 97.1% or more than 97.2% or more than 97.3% or more than 97.4% or more than 97.5% or more 97.6% or greater than 97.7% or greater than 97.8% or greater than 97.9% sequence identity to the nucleotide sequence of SEQ ID NO: 1. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to further preferred embodiments, in the immobilized culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which has a sequence range of more than 98.5%.

Sequence identity with the nucleotide sequence SEQ ID NO. 1 has. Most 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 a very particularly preferred embodiment, in the immobilized culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region corresponding to the nucleotide sequence SEQ ID No. 1.

Ways to carry out the invention

Bacteria of the species Paenibacillus macerans SBG2 were successfully isolated from the fermentation substrate of a post-fermenter. The microorganisms were isolated using a selection medium containing carboxymethylcellulose 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-). The medium used to select Paenibacillus macerans SBG2 was supplemented with N ₂ and CO ₂ fumigated so that the selection could take place under anaerobic conditions. Contained residual oxygen was subsequently 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. After one week of cultivation at 40 ^° C., single rods were observed under microscopic analysis. Further selection of the liquid cultures was made by smearing on anaerobic carboxymethylcellulose plates. The cell material of the grown colonies was used to amplify the microbial DNA by the Colony PCR method according to a standard program. After the sequence analysis of the colonies, the phylogenetic comparison of the sequence data generated therefrom by means of the BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that the sequence obtained can be assigned to the organism Paenibacillus macerans as the closest relative ,

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.

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions over a period of several months, the time course of the volume load of the fermenter in kilograms of organic dry matter per cubic meter per day (kg oTS / m ³ d) and the time course of Total generated biogas in standard liters (gas volume at 273.15 K and 1013 mbar) per day (Nl / d) determined. In addition, the time course of the theoretical gas production in [Nl / d] was calculated.

The Kuratorium für Technik und Bauwesen in Landwirtschaft (KTBL) as well as the Landesanstalt für Landwirtschaft have issued guideline values which roughly indicate which amounts of biogas are to be expected depending on the substrate used with a stable fermentation. These theoretical guidelines can thus reflect the amount of theoretically generated biogas. Alternatively, may also be issued by other institutes in other countries Guide values are used. The time course of the theoretical gas production in [Nl / d] was calculated according to such guideline values.

The fermentation process was carried out under otherwise identical conditions once without the addition of microorganisms and once with multiple addition of microorganisms Paenibacillus macerans SBG2.

Without the addition of microorganisms Paenibacillus macerans SBG2, a continuous and stable fermentation process takes place over several weeks, which is characterized by an increase in the volume load to about 6.5 kg oTS / m ³ d and by a continuously increasing formation of biogas. Because of an uncontrolled high acid concentration in the fermentation substrate, the load on the room was not further increased by suspending the increase in the substrate supply after a certain time for several days. However, since the acid concentrations still remained uncontrollable, the substrate feed was then completely suspended. It was found that a maximum volume load of 5.5 kg oTS / m ^s d could be achieved. At a higher space load due to the collapse of the fermentation process no stable operation is possible.

The fermentation process described above was carried out under otherwise identical conditions with addition of the microorganism Paenibacillus macerans to the fermentation substrate. Was added, the cell mass from 11 of a preculture of Paenibacillus macerans SBG2, which had been incubated for 5 days at a temperature of about 40 ⁰ C was first. The cell count of this preculture had a cell density of about 2.0 × 10 ⁸ cells / ml with a proportion of living cells of more than 90%. While the addition was initially made on an 11-scale twice a week, the amount of addition was doubled at a later time, so that twice a week the cell mass was added from 21 of a preculture of Paenibacillus macerans SBG2. Finally, cell mass from 21 of a preculture of Paenibacillus macerans SBG2 was added three times weekly. In addition, the incubation period of the preculture was extended to at least 7 days, whereby the cell density increased to an average of 1 × 10 ⁹ cells / ml. By adding the pure cultures of Paenibacillus macerans SBG2, the

RRaauummbbeelastung be increased to a maximum value of about 8.5 kg oTS / m ³ d.

Parallel to the increasing volume load, an increase in biogas yield was observed. A coincidence of the produced biogas quantity in standard liters / day [Nl / d] with the theoretical gas production in standard liters / day [Nl / d] was to be observed.

At the time of the first addition of a pure culture of Paenibacillus macerans SBG2, the fermenter was operated at a volumetric loading of about 4.5 kg of dry organic matter / m ³ and day. 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 matter 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 Paenibacillus macerans SßG2 microorganisms with their hydrolytic metabolic activity not only reduce the accumulation of dry matter in the fermenter, but also improve the hydrolytic conversion of this dry substance.

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

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 5.5 kg oTS / m ³ d to about 8.5 kg oTS / m ³ d, ie by more than 50%, under otherwise identical conditions. without any instability of the fermentation process even hinting. Parallel to the increased volume of space, the amount of biogas produced is more than doubled. 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.

Claims

claims

1. A process for the production of biogas from biomass in a fermentation reactor, 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 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 ^" accounts for ⁴ % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that in the culture of microorganisms of the microorganism of the species Paenibacillus macerans at least 10 ^"2 % of the total number of existing in the culture

Constitutes microorganisms.

4. The method according to claim 3, characterized in that in the culture of microorganisms, the microorganism of the species Paenibacillus macerans makes up at least 1% of the 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 at least 75% of the total number of microorganisms present in the culture.

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

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species

Paenibacillus macerans is added.

10. The method according to at least one of claims 2 to 9, characterized in that the microorganism of the species Paenibacillus macerans the biomass as part of at least one immobilized culture of

Microorganisms is added.

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

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

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ⁰ C to 80 ⁰ C, preferably at a temperature of 40 ° C to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Paenibacillus macerans carried out continuously.

17. The method according to at least one of claims 1 to 16, 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.

18. The method according to claim 17, 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

Microorganism of the species Paenibacillus macerans accounts for between 10 ^"6 % and 25% of the total number of microorganisms present in the fermentation substrate.

19. The method according to claim 18, characterized in that the microorganism of the species Paenibacillus macerans in an amount to the

Fermentation substrate is added, that after addition of the proportion of the microorganism of the species Paenibacillus macerans accounts for between 10 ^"4 % and 10% of the total number of present in the fermentation substrate microorganisms.

20. The method according to claim 19, 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.

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

22. 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% sequence identity with the nucleotide sequence SEQ ID No. 1.

23. The microorganism according to claim 22, characterized in that the nucleotide sequence contains a sequence region which has more than 97.2% sequence identity with the nucleotide sequence SEQ ID No. 1.

24. Microorganism according to claim 23, characterized in that the nucleotide sequence contains a sequence range which exceeds 97.4%.

Sequence identity with the nucleotide sequence SEQ ID NO. 1 has.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 97.5% sequence identity with the nucleotide sequence SEQ ID No. 1.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 97.6% sequence identity with the nucleotide sequence SEQ ID No. 1.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 97.8% sequence identity with the nucleotide sequence SEQ ID No. 1.

28. Microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 1.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which has more than 99% 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 99.5% 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 corresponds to the nucleotide sequence

SEQ ID NO: 1 corresponds.

32. culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms a microorganism Paenibacillus macerans according to claims 22 to 31 is present, wherein the microorganism Paenibacillus macerans at least 10 ^"4 % of the total number of microorganisms present in the culture.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism Paenibacillus macerans at least 10 ^" accounts for ² % of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism Paenibacillus macerans makes up at least 1% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism Paenibacillus macerans constitutes at least 10% 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 makes up at least 25% 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 makes up at least 50% 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 constitutes at least 75% 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 32 to 40, characterized in that it is an immobilized culture of microorganisms.