# Patent

## Title
Compositions and methods for degrading lignocellulosic biomass

## Abstract

The present invention relates to compositions and methods of producing bioenergy products and metabolites of industrial interest from lignocellulosic biomass. More specifically, the invention describes the identification, characterization and isolation of novel bacteria having the remarkable ability to transform lignocellulosic biomass into fermentable sugars and, even more remarkably, into bioenergy products and metabolites. The invention also discloses a method to isolate such bacteria, compositions comprising such bacteria, and their uses for the modification of lignocellulosic biomass, with a view to producing bioenergy.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage application of International Patent Application No. PCT/EP2010/051885, filed Feb. 16, 2010, which claims the benefit of U.S. Provisional Patent Application No. 61/153,478, filed Feb. 18, 2009, the disclosures of which are hereby incorporated by reference in their entireties, including all figures, tables and amino acid or nucleic acid sequences.


The present invention relates to composition and methods of producing bioenergy products and metabolites of industrial interest from lignocellulosic biomass. More specifically, the invention describes the identification, characterization and isolation of novel bacteria having the remarkable ability to transform lignocellulosic biomass into fermentable sugars and, even more remarkably, into bioenergy products and metabolites. The invention also discloses a method to isolate such bacteria, compositions comprising such bacteria, and their uses for the modification of lignocellulosic biomass, with a view to producing bioenergy.
BACKGROUND
The conversion of lignocellulosic biomass has been the subject of intense research efforts since the 1970s (Blumer-Schuette et al., 2008, Extremely thermophilic microorganisms for biomass conversion: status and prospects, Curr Opinion Biotechnol 19, pp. 210-217; Perez et al., 2002, Int Microbiol 5, pp 53-63).
In this regard, it is known to use microorganisms to conduct modification of transformed biomass, essentially pre-treated agricultural feedstocks, to produce bioenergy products such as ethanol. As reported in Mosier et al. (Bioresource Technology 96 (2005) 673-686), however, the pre-treatment of lignocellulosic biomass is required to alter the structure of cellulosic biomass to make cellulose more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars.
It is believed, however, that future bio fuels or bioenergy products should originate from raw lignocellulosic biomass, instead of from pre-treated agricultural feedstocks. The use of such raw biomass would require an effective method of degrading (e.g., hydrolysing) lignocellulosic biomass into fermentable sugars, which can then be transformed through fermentation into bioenergy products (e.g., alcohols and other metabolites of industrial value). The production of fermentable sugars (e.g., monomeric sugars) from raw lignocellulosic biomass, is therefore a major challenge, and various approaches have been proposed in this regard, such as thermochemical methods, acid hydrolysis and enzymatic hydrolysis.
However, due to the wide range of lignocellulosic biomass being considered, with each having a specific composition of cellulose, hemicellulose and lignin, the development of enzymes or enzymatic compositions for hydrolysing such a raw biomass does not appear cost-effective. In addition, lignin by-products remaining from such treatments of lignocellulosic biomass generally remain unmodified and lost.
WO2009/063079 describes the use of bacteria of the genus Deinococcus for the production of bioenergy products and metabolites through fermentation of biomass.
Taryn et al (ABB 45 (1994) 209) reports the ability of bacteria of the genus Clostridium for degrading fermentable sugars.
WO97/10352 relates to Pseudomonas bacterial strains that degrade cellulose. Similarly, Weon et al (Int. J. Systematic and Evolutionary Microbiology (2007), 57, pp 1685-1688) reports UV-resistant Deinococcus strains which hydrolyse cellulose.
W. Zimmermann (Journal of Biotechnology, 13 (1990) 119-130) provides a review of bacterial degradation of lignin.
Bacteria having the ability to hydrolyse the main constituents of lignocellulosic biomass, including lignin, xylan and cellulose, under conditions suitable for an industrial process, have never been reported. In particular, bacteria which can degrade lignin under industrial conditions have never been isolated.
Accordingly, there is an unmet need for a cost-effective and scalable process for the degradation of lignocellulosic biomass into valuable products such as fermentable sugars or bioenergy products and metabolites.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for producing valuable products from lignocellulosic biomass or derivatives thereof. More specifically, the invention relates to novel bacteria having the ability to transform lignocellulosic biomass or derivatives thereof into valuable products, including fermentable sugars and bioenergy products. The invention also relates to methods of producing valuable products and metabolites using such bacteria.
The invention derives inter alia from the identification of microorganisms having the unexpected and remarkable properties of transforming lignocellulosic biomass or derivatives thereof, with a view to obtaining compounds which can be used to produce bioenergy, ethanol in particular, on an industrial scale and both economically and reliably.
In this regard, the invention results from the design of an improved method of selecting or identifying bacterium from any sample. More particularly, the present invention discloses a method of selecting or identifying or isolating a bacterium, the method comprising the following steps:

    
    
        a) providing a sample comprising bacteria;
        b) subjecting the sample to a cell-destructing DNA damaging treatment; and
        c) selecting or isolating, from said treated sample, a bacterium which has the ability to live or grow in the presence of lignin, cellulose or xylan as a carbon source, e.g., to use lignin, cellulose or xylan as carbon source.
    
    


A further object of this invention is a bacterium obtainable by such a method, or an extract of said bacterium.
Following the above method, the inventors have indeed isolated and identified from raw materials new microorganisms having the unexpected capacity of hydrolysing the three main components of the lignocellulosic biomass, i.e., cellulose, xylan and lignin.
This new finding that a microorganism is able to transform lignin-containing raw material for the production of bio-energy products represents a technological breakthrough by allowing a single-step production process of bioenergy products (e.g., ethanol) from raw biomass (lignocellulosic biomass), overcoming the previous drawbacks of using pre-treated agricultural feedstocks instead of lignocellulosic, raw biomass. Furthermore, the same bacteria of this invention may be used throughout the entire production process, i.e., to hydrolyse the lignocellulosic biomass and to produce bioenergy products by fermentation through simultaneous saccharification and fermentation process (SSF).
An object of this invention therefore resides in an isolated bacterium wherein said bacterium has the ability to grow in the presence of lignin or cellulose or xylan as a carbon source, at a temperature of 30° C. or more, and to resist an UV treatment of 4 mJ/cm2.
A particular object of this invention is an isolated bacterium wherein said bacterium has the ability to grow in the presence of lignin as sole carbon source, at a temperature of 30° C. or more, and to resist an UV treatment of 4 mJ/cm2.
Another particular object of this invention is an isolated bacterium wherein said bacterium has the ability to utilize cellulose and xylan as carbon source, at a temperature of 30° C. or more, and to resist an UV treatment of 4 mJ/cm2.
In a preferred embodiment, the bacterium of the present invention has the remarkable ability to use lignin, cellulose and xylan as carbon source. The invention indeed shows that bacteria having the ability to degrade all major constituents of lignocellulosic biomass can be identified and cultured. Such bacteria may be used to transform such biomass with unprecedented efficiency.
In a particular and preferred embodiment, the bacteria of the invention can be grown, or cultivated in both aerobic and anaerobic conditions. The invention indeed unexpectedly shows that bacteria of this invention can be operated under conditions, such as anaerobic conditions, suitable to produce high amounts of bioenergy products or metabolites from various substrates.
In a particular embodiment, the bacteria of this invention contain an exogenous nucleic acid molecule.
The bacteria of the invention belong, in a preferred embodiment, to a genus selected from Deinococcus, Bacillus, Microbacterium, Cellulosimicrobium, Methylobacterium, Sphingobacterium, Pseudomonas, Caldimonas, Paenibacillus, Gordonia, Rhodococcus, Stenotrophomonas, Novosphingobium, Sphingomonas, Flavobacterium, Sphingobium, Sphingopyxis, or Porphyrobacter. 
As illustrated in the examples, bacteria of the above genus which can be identified and cultured, are able to transform biomass and to resist a cell-destructing UV treatment of 4 mJ/cm2. These bacteria represent valuable means to convert biomass, including lignocellulosic biomass, into highly valuable products.
A further object of this invention is a composition comprising a bacterium of this invention and a culture medium.
A further object of this invention is an extract of a bacterium as disclosed above.
A further object of this invention resides in the use of a bacterium of the invention, or an extract thereof, to hydrolyse lignocellulosic biomass, or to convert or transform lignocellulosic biomass into fermentable sugars.
A further object of this invention is a method of degrading or converting lignocellulosic biomass into fermentable sugars, the method comprising a step of exposing a lignocellulosic biomass to a bacterium of this invention, or an extract thereof.
A further object of this invention is a method of producing fermentable sugars from lignocellulosic biomass, the method comprising a step of exposing a lignocellulosic biomass to a bacterium of this invention or an extract thereof.
The above methods can be performed under aerobic and/or anaerobic condition, at elevated temperatures (e.g., of at least 30° C. or above), and allow the efficient transformation of all major types of constituents of lignocellulosic biomass. Furthermore, in a particular embodiment, the methods of the invention comprise a further step of either collecting the fermentable sugars, or of transforming the fermentable sugars into bioenergy products or metabolites. Such transformation (fermentation) step may be carried out using a bacterium of the invention or a distinct bacterium, or a combination of bacteria, or extracts thereof.
In a preferred embodiment, both the hydrolysis and the fermentation are performed using a bacterium of this invention, most preferably the same bacterium. Indeed, in a preferred embodiment, the invention allows the production of bioenergy products directly from lignocellulosic biomass, by allowing both hydrolysis of lignocellulosic biomass and fermentation of sugars.
A further object of this invention thus resides in the use of a bacterium of this invention to produce bioenergy products or metabolites.
Another object of this invention is a method of producing a bioenergy product or metabolite, the method comprising exposing a lignocellulosic biomass to a bacterium of this invention, or an extract thereof, and recovering the bioenergy product or metabolite obtained.
In a preferred embodiment, both the hydrolysis and the fermentation are performed using a bacterium of this invention, most preferably the same bacterium. It should be noted that, in an alternative embodiment, two or more distinct bacteria may be used, sequentially or in combination, to produce bioenergy products from lignocellulosic biomass.
In a particular aspect, the present invention relates to a method comprising the following steps:

    
    
        providing (or culturing and/or growing) a bacterium of this invention, or an extract thereof,
        modifying lignocellulosic biomass or a derivative thereof, into bioenergy products or metabolites of industrial interest (e.g., bioenergy sources such as ethanol, chemical building blocks such as succinic acid) using said bacterium or an extract thereof, and
        collecting at least one bioenergy product or metabolite resulting from said modification.
    
    


The invention also relates to a composition comprising a bacterium as defined above and a lignocellulosic biomass or a derivative thereof.
The invention also relates to bioenergy products or metabolites produced using a method as described above.
A further object of this invention is a reactor or a fermentor comprising a lignocellulosic biomass and a bacterium of this invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: growth results of UV-resistant thermophilic bacteria on MM+CMC1% under anaerobiosis conditions.
FIG. 2: growth results of UV-resistant thermophilic bacteria on MM+xylan1% under anaerobiosis conditions.
FIG. 3: growth results of UV-resistant pink isolates on MM+lignin 0.1% under aerobiosis conditions at 30° C. after 5 days. Outlined are isolates M10-1D and M10-1E, which are able to use lignin as sole carbon source, and M10-8D, which is able to degrade lignin.
FIG. 4: Deinococcus strain DRH46 is able to degrade paper journal.



DETAILED DESCRIPTION OF THE INVENTION
The invention describes the identification, characterization and isolation of novel bacteria having the remarkable ability to transform lignocellulosic biomass into fermentable sugars and, even more remarkably, into bioenergy products and metabolites. The invention also discloses a method to isolate such bacteria, compositions comprising such bacteria, and their uses for the modification of lignocellulosic biomass, with a view to producing bioenergy products and metabolites.
DEFINITIONS
The term lignocellulosic biomass according to the invention designates a raw biomass containing lignin, cellulose and/or xylan. The term lignocellulosic biomass thus essentially designates unprocessed material of biological origin, such as forestry products, including mature trees unsuitable for lumber or paper production, agricultural products, such as grasses, crops and animal manure, and aquatic products, such as algae and seaweed. Specific sources of biomass include, without limitation, hardwood or softwood stems, corn cobs, wheat straw, grass, leaves, seeds, paper, etc. (see for instance Sun et al., Bioresource Technology 83 (2002) 1-11). The term lignocellulosic biomass should be distinguished from transformed biomass or secondary biomass, which essentially contains hydrolysed pre-treated biomass products.
Examples of lignocellulosic biomass include wood or vegetal material derived from numerous types of plants, including miscanthus, hemp, sugarbeet, wheat, corn, poplar, willow, sorghum, sugarcane, and a variety of tree species, ranging from eucalyptus to oil palm.
As used herein, the term “biomass derivatives” designates a composition comprising molecules derived from lignocellulosic biomass, such as lignin, cellulose, hemicellulose.
In the context of the present application, the term bacteria includes wild type, or natural variant strains of a bacterium, e.g., strains obtained through accelerated evolution, by DNA-shuffling technologies, or recombinant strains obtained by insertion of eukaryotic, prokaryotic and/or synthetic nucleic acid.
An “extract of a bacterium” designates any fraction obtained from a bacterium, such as a cell supernatant, a cell debris, cell walls, DNA extract, enzymes or enzyme preparation or any preparation derived from a bacterium by chemical, physical and/or enzymatic treatment, which is essentially free of living bacteria. A bacterium extract preferably retains an enzymatic activity of the bacterium, most preferably the ability to hydrolyse lignocellulosic biomass.
Within the context of the present invention, the term “bioenergy” designates a renewable energy derived from biomass. More specifically, the term “bioenergy products” includes “biofuels” and all final products of modification of biomass or biomass derivatives that can be used as fuels, such as ethanol, propanol, butanol glycerol, butanediol and propanediol.
The term “metabolites” designates all possible intermediate molecules generated during the modification of biomass or biomass derivatives into bioenergy products, including but not limited to several chemical products of industrial interest, such as organic acids and building blocks, such as acetic acid, propionic acid, pyruvic acid, butyric acid, lactic acid and/or succinic acid.
The present invention relates to composition and methods of producing bioenergy from lignocellulosic biomass or lignocellulosic biomass derivatives. More specifically, the invention relates to the use of bacteria for the modification of lignocellulosic biomass with a view to producing bioenergy products and metabolites.
The invention now shows, for the first time, that bacteria can be isolated from environmental sources that are able to produce bioenergy products or metabolites from lignocellulosic biomass.
In this regard, an object of this invention resides in a method for isolating or identifying bacteria, comprising the following steps:

    
    
        a) providing a sample comprising bacteria;
        b) subjecting the sample to a cell destructing DNA damaging treatment; and
        c) isolating, from said treated sample, a bacterium which has the ability to live or grow in the presence of lignin, cellulose, hemicellulose or xylan as a carbon source.
    
    


The method can be implemented with various samples comprising uncharacterized bacteria, particularly with samples which are or derive from an environmental sample. Within the context of this invention, environmental samples include any sample containing (a plurality of) uncharacterized (micro)organisms, particularly uncultivated microorganisms (e.g., microorganisms that have not been purposely cultured and expanded in isolated form). The sample may be obtained or derived from natural environments or from artificial or specifically created environments.
As indicated, the sample may be any environmental sample, such as those obtained or derived from soil, water, vegetal extract, biological material, sediments, peatlands, industrial effluents or sites, mineral extracts, sand, and the like. The sample may be collected from various regions or conditions, such as but not limited to tropical regions, volcanic regions, forests, farms, industrial areas, etc. The sample usually contains various species of (uncharacterized, uncultivated) microorganisms, such as terrestrial microorganisms, marine microorganisms, freshwater microorganisms, symbiotic microorganisms, etc. Species of such environmental microorganisms include bacteria, algae, fungi, yeasts, moulds, viruses, etc. The microorganisms may include extremophile organisms, such as e.g., thermophiles. The sample typically comprises various species of such (uncultivated) microorganisms, as well as various amounts thereof. Furthermore, the sample may contain, in addition, known and/or cultivable microorganisms (e.g., prokaryotic or eukaryotic).
It should be understood that the present invention is not limited to any specific type of sample or environmental microorganism, but can be implemented using any sample comprising uncultivated microorganisms.
In a preferred embodiment, the sample is or derives from soil, water, hot springs, marine environment, mud, wood, stone, moss, vegetal extract, lichen, biological material, sediment, biofilm, industrial effluents, gas, sand, oil, sewage, or animal or human dejection.
For use in the present invention, the sample may be wet, soluble, dry, in the form of a suspension, paste, etc. Furthermore, prior to step b) of the method, the sample may be treated to improve the process, for instance to enrich for microorganisms, e.g., such as through filtration, washings, concentration, dilution, steering, drying, etc.
In a particular embodiment, the sample is in the form of a filtered suspension. More particularly, the sample may be sterile-filtered and/or placed in sterile water, prior to treatment step b).
Step b) of the process comprises subjecting the sample (i.e., microorganisms contained in the sample) to a cell destructing DNA damaging treatment.
The cell-destructing DNA damaging treatment designates a treatment that causes substantial cell death in the sample, as opposed to mere mutagenic treatments which introduce DNA modifications. In particular, the cell-destructing DNA damaging treatment is a treatment that is sufficient to cause 90% cell death, or more, in a culture of E. coli bacteria. Even more preferably, the cell destructing DNA damaging treatment is a treatment that is sufficient to reduce by at least 2 log the bacterial titer in a culture of E. coli. Surprisingly, the invention shows that such a treatment, which would normally be lethal to most cell populations, allows the efficient and rapid isolation of novel microorganisms from various types of samples, which microorganisms have unexpected properties. This result is particularly surprising since subjecting microorganisms to such cell destructing DNA damaging treatment would have been expected to prevent isolation of living microorganisms.
The DNA damaging treatment may comprise subjecting the sample to irradiation(s) and/or to one or several genotoxic agents. The treatment is conducted under conditions and/or for a period of time sufficient to induce substantial cell death in the microorganisms present in the sample.
In a particular embodiment, the DNA damaging treatment comprises subjecting the sample to one or several irradiations. A preferred treatment comprises subjecting the sample (i.e., microorganisms in the sample) to a repeated sequential irradiation treatment.
Irradiation may be selected from UV, gamma and/or X ray irradiation, either alone or in combinations, most preferably UV irradiation(s). Irradiation treatment typically comprises subjecting the microorganisms to one or several sequential irradiations (e.g., from 1 to 5), which may be of the same or different nature, preferably of the same nature.
A particularly preferred treatment comprises subjecting the sample to a cell-destructing UV irradiation. The invention indeed shows that such a treatment allows to isolate with high efficiency from environmental (e.g., soil or water) samples, under-represented bacteria species having remarkable enzymatic activities. Cell-destructing UV treatments are typically of between 0.5 and 400 mJ/cm2, more preferably of between 1 and 200 mJ/cm2, typically between 1 and 100 mJ/cm2. A preferred UV treatment is 4 mJ/cm2. Repeated irradiation treatments are typically carried out at an interval of between 1 and 8 hours, preferably 3 to 5 hours, and more preferably of about 4 hours.
In a specific embodiment, the cell-destructing DNA damaging treatment comprises subjecting the sample to at least 2, preferably at least 3 UV treatments of between 0.5 and 400 mJ/cm2 each, preferably of about 4 mJ/cm2 each, carried out at an interval of between 1 and 8 hours, preferably 3 to 5 hours, and more preferably of about 4 hours.
In an alternative method, the cell-destructing DNA damaging treatment comprises contacting the sample with a genotoxic agent, such as a solvent, mitomycin or H2O2. It should be understood that genotoxic agents may also be used in combination with irradiation.
During the treatment phase, the sample is preferably placed in a suitable culture medium such as, without limitation, PGY (Bacto-peptone 10 g/L, Yeast extract 5 g/L, glucose 1 g/L) or LB (Bacto-tryptone 10 g/L, Yeast extract 2.5 g/L, Sodium chloride 10 g/L). It should be understood that other suitable culture media are known to the skilled person (Buchanan et al, 1974, Difco, 1995) or may be prepared by the skilled person from such known media.
Treatment step b) is typically performed in a solid or semi-solid culture medium, such as in the presence of a gel (e.g., agar). A most preferred treatment medium comprises an agar culture medium, such as a soft agar culture medium. In a particular embodiment, a PGY agar medium is used to grow the bacteria. However, different solid media containing a carbon source, a nitrogen source and mineral salts can be used as well. Serial dilution techniques can also be used according to Schoenborn et al. (2004).
In step c), living or growing bacteria are identified or isolated from the treated sample. Living or growing bacteria may be identified by different means known per se in the art. In a particular embodiment, colonies which form in the culture media are identified. The living or growing bacteria can be isolated and placed in fresh medium for further culture or expansion.
In a preferred embodiment, the bacteria in step b) are cultured in a minimum medium comprising, as the sole carbon source, lignin (preferably 0.05-3% by weight), cellulose (preferably 0.5-3% by weight), and/or xylan (preferably 0.5-3% by weight). Lignin, cellulose and xylan may be obtained from commercial sources (lignin: Sigma, France; CMC and xylan: Fluka, France). Sources of minimal medium are provided in the examples. Other minimal media may be used as described previously (Rainey, F. A., et al. 2005. Appl Environ Microbiol. 71 (9):5225-35; Ferreira, A. C., et al, 1997. Int J Syst Bacteriol. 47(4):939-47; Kongpol A et al, 2008. FEMS Microbiol Lett. 286(2):227-235).
The methods of this invention can comprise one or several additional steps of selecting bacteria having particular properties. More particularly, in a preferred embodiment, the method further comprises one or several steps of selecting bacteria which are viable or grow under selected culture conditions, such as media, temperature, pH, salinity, nutrients, oxygenation or carbon source. For this purpose, the sample or bacteria can be placed under appropriate selection conditions during any one of steps b), or c), or during a prior or subsequent step, and the resulting property is selected for during any of these steps.
In a particular aspect of the present invention, the bacteria are cultured under particular temperature conditions in order to identify or isolate bacteria which are viable or can be grown in a temperature range from approximately 4 to 70° C. More particularly, the bacteria are maintained at the selected temperature during step b) and/or c), and/or during an additional step, in order to identify or isolate bacteria which are viable or can be grown at the desired temperature.
In another particular aspect of the present invention, the bacteria are cultured under particular saline conditions in order to identify or isolate bacteria which are viable or can be grown under concentration conditions of NaCl or equivalent salts possibly reaching around 5% weight/volume. More particularly, the bacteria are maintained at the selected salinity during step b) and/or c), and/or during an additional step, in order to identify or isolate bacteria which are viable or can be grown at the desired salinity.
In a further particular and preferred embodiment of the present invention, the bacteria are cultured under particular pH conditions in order to identify or isolate bacteria which are viable or can be grown in a pH interval between approximately 3 and 9.5, preferably between 4 and 8. More particularly, the bacteria are maintained at the selected pH during step b) and/or c); and/or during an additional step, in order to identify or isolate bacteria which are viable or can be grown at the desired pH.
In a further particular embodiment of the present invention, the bacteria are cultured under particular oxygenation conditions in order to identify or isolate bacteria which are viable or can be grown in aerobic and/or anaerobic conditions. More particularly, the bacteria are maintained under the selected oxygenation conditions during step b) and/or c); and/or during an additional step, in order to identify or isolate bacteria which are viable or can be grown at the desired conditions.
In a further particular embodiment of the present invention, the bacteria are cultured in a particular culture medium in order to identify or isolate bacteria which are viable or can be grown in the presence of a selected carbon source. More particularly, the bacteria are maintained under the medium during step b), c) and/or d) and/or during an additional step e), in order to identify or isolate bacteria which are viable or can be grown using the desired carbon source.
It should be understood that the above characteristics can be selected individually or in any combinations. For instance, the method can be used to identify bacteria which are viable or can be grown at a desired temperature and salinity, or at a desired temperature and pH, or at a desired temperature, pH and oxygenation condition.
Furthermore, the methods of this invention may comprise a further step of modifying, e.g., either biologically, genetically and/or chemically, the identified or isolated bacteria, or their DNA, by any process known per se in the art, said modification aiming e.g., to improve the viability, growth or functions of the said bacterium, e.g., enzymatic activity. Such modification step includes, without limitation, cell fusion, accelerated evolution, DNA shuffling, mutagenesis, insertion of eukaryote, prokaryote or synthetic nucleic acid (e.g., DNA) from another strain, or any genetic engineering technology. The modification may also include a step of introducing a marker gene (e.g., kanamycin resistance) in the bacterium. Said modification step can be carried out on the isolated bacteria, or at any earlier stage of the above process, e.g., on the sample of step a), for instance.
A further object of this invention is a bacterium obtainable by the above method.
More specifically, an object of this invention resides in an isolated bacterium, wherein said bacterium has the ability to grow in the presence of lignin or cellulose or xylan as the sole carbon source, at a temperature of at least 30° C., and to resist an UV treatment of 4 mJ/cm2. The combination of these features is unprecedented, and provides novel avenues in the exploitation of lignocellulosic biomass. In particular, the ability to resist the above UV treatment and to grow at 30° C. or more allows the use of the bacteria under special stringent industrial conditions compatible with the nature of biomass. The ability to grow in the presence of lignin, cellulose or xylan as the sole carbon source (i.e., to use lignin, cellulose or xylan as carbon source) makes the bacteria suitable to transform any type of lignocellulosic biomass.
In a further preferred embodiment, the bacterium has the ability to use lignin, cellulose and xylan as carbon source. Indeed, the inventors have identified bacteria which are able to degrade all major constituents of lignocellulosic biomass.
Furthermore, as illustrated in the experimental section, bacteria have been identified by the inventors which are able to grow either in aerobic or anaerobic condition, or both. In a particular and advantageous embodiment, the invention relates to bacteria as defined above which can be grown, or cultivated in both aerobic and anaerobic conditions. The invention indeed unexpectedly shows that bacteria can be operated under conditions, such as anaerobic conditions, suitable to produce high amounts of bioenergy products or metabolites from various substrates. The invention thus provides novel means and compositions for producing bioenergy products or metabolites from lignocellulosic biomass and/or lignocellulosic biomass components, namely cellulose, hemicellulose, and lignin, in a very efficient manner.
Preferred bacteria of this invention can hydrolyse lignocellulosic biomass to produce fermentable sugars, and can be cultivated in aerobic or anaerobic conditions.
Bacteria of this invention preferably belong to a genus selected from Deinococcus, Bacillus, Microbacterium, Cellulosimicrobium, Methylobacterium, Sphingobacterium, Pseudomonas, Caldimonas, Paenibacillus, Gordonia, Rhodococcus, Stenotrophomonas, Novosphingobium, Sphingomonas, Flavobacterium, Sphingobium, Sphingopyxis, or Porphyrobacter. 
Preferred bacteria of this invention exhibit the following characteristics:

    
    
        viable or functional at high temperatures (e.g., around 30-70° C.),
        viable or functional in anaerobic conditions,
        resist a UV treatment of 4 mJ/cm2; and
        able to promote cellulose digestion to yield glucose.
    
    


Other preferred bacteria of this invention exhibit the following characteristics:

    
    
        viable or functional at high temperatures (e.g., around 30-70° C.),
        viable or functional in anaerobic conditions,
        resist a UV treatment of 4 mJ/cm2; and
        able to use lignin as carbon source.
    
    


Other preferred bacteria of this invention exhibit the following characteristics:

    
    
        viable or functional at high temperatures (e.g., around 30-70° C.),
        viable or functional in anaerobic conditions,
        resist a UV treatment of 4 mJ/cm2; and
        able to use xylan as carbon source.
    
    


Other preferred bacteria of this invention exhibit the following characteristics:

    
    
        viable or functional at high temperatures (e.g., around 30-70° C.),
        viable or functional in anaerobic conditions,
        resist a UV treatment of 4 mJ/cm2;
        able to use lignin as carbon source; and
        able to promote cellulose digestion to yield glucose; and/or
        able to promote xylan digestion to yield xylose; and/or
        able to use xylan as carbon source.
    
    


The bacterium may be maintained or cultivated in suitable culture medium such, as without limitation, PGY (Bacto-peptone 10 g/L, Yeast extract 5 g/L, glucose 1 g/L) or LB (Bacto-tryptone 10 g/L, Yeast extract 2.5 g/L, Sodium chloride 10 g/L). It should be understood that other suitable culture media are known to the skilled person (Buchanan et al, 1974, Difco, 1995) or may be prepared by the skilled person from such known media.
Specific illustrative examples of bacteria of this invention are provided in the experimental section, together with their isolation technique and properties. It should be understood that other bacteria may be isolated or characterized following the teaching of the present invention.
A further object of this invention resides in the use of bacteria as provided by the inventors, or extracts thereof, to produce valuable products from lignocellulosic biomass.
In this regard, the invention relates to the use of such a bacterium, or an extract thereof, to hydrolyse lignocellulosic biomass.
The invention also relates to the use of such a bacterium, or an extract thereof, to transform lignocellulosic biomass into bioenergy products or metabolites.
The invention also relates to a method of degrading lignocellulosic biomass into fermentable sugars, the method comprising exposing said lignocellulosic biomass to a bacterium of this invention, or an extract thereof.
The method is particularly suited to produce fermentable sugars selected from glucose, cellobiose, mannose, xylose, arabinose or galactose.
A further object of this invention is a reactor or fermentor comprising lignocellulosic biomass and a bacterium of the invention or an extract thereof.
A further object of this invention is a method of producing bioenergy products or metabolites, particularly ethanol, the method comprising exposing a lignocellulosic biomass to a bacterium of the invention, or an extract thereof. The method preferably further comprises a step of collecting said bioenergy products or metabolites.
A further object of this invention is a bioenergy product or metabolite obtained by the above methods.
Culture or exposition can be made in any suitable condition or environment allowing modification of the lignocellulosic biomass to produce bioenergy products or metabolites. In this regard, the method can be performed in a reactor, in a fermentor, outdoor, in the presence of suitable nutrients or additives, if needed. The method is typically conducted under pH conditions, temperature above 40° C., and in the presence of suitable substrates.
In the above methods, the step of culturing and/or growing the bacterium and the step of transforming the biomass into bioenergy products or metabolites can be carried out either simultaneously, or sequentially; and the step of collecting bioenergy products or metabolites can be carried out simultaneously with the first and/or the second step, or sequentially. In this regard, the biomass can be contacted with the bacterium under suitable conditions to allow expansion of said bacterium, thereby increasing the efficiency of the process. Alternatively, bacterial strains can be expanded separately, under suitable culture conditions, and subsequently added to the biomass. It should be understood that the precise amounts of bacteria used initially in order to efficiently transform biomass into substantial bioenergy products or metabolites can be adjusted by the skilled artisan depending on the type of bacteria, the type of biomass, and the culture conditions.
In a particular embodiment of the method, the bacteria are grown separately from biomass conversion.
In a particular embodiment, the method of the invention is performed in a reactor of conversion of biomass. By “reactor” is meant a conventional fermentation tank or any apparatus or system for biomass conversion specially designed to implement the invention and therefore consisting in particular of bioreactors, biofilters, rotary biological contactors, and other gaseous and/or liquid phase bioreactors for the treatment of biomass. The apparatus which can be used according to the invention can be used continuously or in batch loads.
In the reactor, to implement the method of the invention, at least one bacterium or bacterial extract of the invention is used, whilst said reactor is arranged and supplied so that physicochemical conditions are set up and maintained therein so that said bacterium is operational for the application under consideration and so that, optionally, bacterial growth is possible and preferably promoted therein.
In another embodiment of the method of the invention, the bacteria are grown in a reactor, during the conversion of biomass, whilst suitable physicochemical conditions are set up and maintained for this bacterial growth to be possible, and preferably promoted.
In alternative embodiments of the invention, the conversion of biomass is conducted under aerobiosis, anaerobiosis or under microaerobiosis.
Further aspects and advantages of the invention will be disclosed in the examples, which are illustrative and do not limit the scope of protection.
EXAMPLES
Materials and Methods
Selection Tests and Culture Media Composition










167 Thermus medium

















Tryptone
1
g


Yeast extract
1
g


Agar
28
g


Nitrilotriacetic acid
100
mg


CaSO4 × 2 H2O
40
mg


MgCl2 × 6 H2O
200
mg


0.01M Fe citrate
0.5
ml


Solution of trace elements (see below)
0.5
ml


Phosphate buffer (see below)
100
ml


H2O
900
ml


Adjust to pH 7.2 with NaOH, autoclave at 121° C. for 15 min.


autoclave the phosphate buffer separately and add to the


medium



















Phosphate buffer



















KH2PO4
5.44
g



Na2HPO4 × 12 H2O
43
g



H2O
1000
ml



Adjust to pH 7.2










Composition of Minimum Medium

    
    
        MOPS buffer 1× (ph7) containing: acid MOPS buffer 40 mM, NH4Cl 20 mM, KOH 10 mM, NaOH 10 mM, CaCl2 0.5 μM, Na2SO4 0.276 mM, MgCl2 0.528 mM.
        A solution of micronutriments (pH5): (NH4)6(MO7)24 3 nM, H3BO3 400 nM, CoCl2 30 nM, CuSO4 10, nM, MnCl2 250 nM, ZnSO4 10 nM.
        Solution of vitamins, pH4.0, (1 μg/1 each): D-biotin, niacin, pyridoxal-HCl, thiamin-HCl, vitamin B12
        Source of phosphate: K2HPO4 5.7 mM
        FeCl3 20 μM (prepared in a solution of sodium citrate then filtered).

Detection of the Cellulase Activity of the Bacterium:

Principle:

    
    


The test is based on follow-up of the conversion of NAD into NADH during degradation of the cellulose. An increase in absorbency is then monitored at 340 nm following the supplier's instructions,
Detection of Ethanol Production:
Ethanol can be quantified using two methods.
Enzymatic Method:




  
    Ethanol
    +
    NAD
  
  ⁢
  
    →
    ADH
  
  ⁢
  
    Acetaldehyde
    +
    NADH
  




This method is based on follow-up of the conversion of NAD into NADH in the presence of ethanol and alcohol dehydrogenase.
This reaction translates as in increase in absorbency at 340 nm. For this measurement, the Sigma N7160, kit can be used following the manufacturer's instructions.
Measurement by Reverse Phase High Performance Liquid Chromatography


    
    
        Conditions:

HPLC Gilson with automatic injector, detection by refractometry,

Column: Phenomenex Rezex ROA, 300 mm×7.8 mm

Column temperature: 65° C.

Mobile phase: 0.005 N sulphuric acid

Flow rate: 0.600 ml/min

    
    


First a calibration curve is made by injecting culture medium containing known concentrations of ethanol into the column. The peak area eluted at 22.26 min corresponding to ethanol is measured. A calibration curve is plotted.
Next, the quantity of ethanol produced by the bacterium is measured by injecting the culture supernatant into the column. The peak area eluted at 22.26 min and corresponding to ethanol is measured. The concentration of ethanol present in the supernatant is deduced by comparison with the calibration curve.
The detection and quantification of the other metabolites possibly produced in diverse proportions can be made following conventional methods of analysis and evaluation.
Example 1
Selection of Thermophilic UV-Resistant Bacteria which Use Cellulose as Carbon Source and Grow in Anaerobiosis (FIG. 1)
Thermophilic bacteria selected for their resistance to UV rays and isolated from environmental samples (3 treatments of 4 mJ/cm2 with an interval of 4 hours) are inoculated on a solid minimal culture medium sterilized by autoclaving (15 minutes at 120° C.) containing the carbon source of interest at 1%: carboxymethylcellulose (CMC; Fluka, France) or xylan from birchwood (Fluka, France). The minimal culture medium is made up of a MOPS buffer solution at pH7 and filtered: acid MOPS buffer 40 mM (Sigma, France), NH4Cl 20 mM, KOH 10 mM, NaOH 10 mM, CaCl2 0.5 μM, Na2SO4 0.276 mM, MgCl2 0.528 mM), a solution of micronutriments at pH5 ((NH4)6(MO7)24 3 nM, H3BO3 400 nM, CoCl2 30 nM, CuSO4 10 nM, MnCl2 250 nM, ZnSO4 10 nM), a solution of vitamins at pH4 (1 μg/L of D-biotin, niacin, pyridoxal-HCl, thiamin-HCl and vitamin B12), a solution of K2HPO4 at 5.7 mM as well as a solution of FeCl3 at 20 μM in NaH2(C3H5O(COO)3).
Conditions of anaerobiosis are ensured by the addition of a GENbag anaer (BioMérieux, France). After incubation in anaerobiosis at 45° C. for 4 to 5 days, the colonies using the carbon source present in the culture medium are visible (see FIG. 1).
The UV resistant bacterium designated M11-2F is able to grow in anaerobiosis and at 45° C. by using CMC as sole carbon source.
This bacterium is thus able to transform lignocellulosic biomass into valuable products under industrial conditions.
Example 2
Selection of Thermophilic UV-Resistant Bacteria which Use Xylan in Anaerobiosis (FIG. 2)
Thermophilic bacteria selected for their resistance to UV rays from environmental samples (3 treatments of 4 mJ/cm2 with an interval of 4 hours) are inoculated on a solid minimal culture medium sterilized by autoclaving (15 minutes at 120° C.) containing the carbon source of interest at 1%: carboxymethylcellulose (CMC; Fluka, France) or xylan from birchwood (Fluka, France). The minimal culture medium is made up of a MOPS buffer solution at pH7 and filtered: acid MOPS buffer 40 mM (Sigma, France), NH4Cl 20 mM, KOH 10 mM, NaOH 10 mM, CaCl2 0.5 μM, Na2SO4 0.276 mM, MgCl2 0.528 mM), a solution of micronutriments at pH5 ((NH4)6(MO7)24 3 nM, H3BO3 400 nM, CoCl2 30 nM, CuSO4 10 nM, MnCl2 250 nM, ZnSO4 10 nM), a solution of vitamins at pH4 (1 μg/L of D-biotin, niacin, pyridoxal-HCl, thiamin-HCl and vitamin B12), a solution of K2HPO4 at 5.7 mM as well as a solution of FeCl3 at 20 μM in NaH2(C3H5O(COO)3).
Conditions of anaerobiosis are ensured by the addition of a GENbag anaer (BioMérieux, France). After incubation in anaerobiosis at 45° C. for 4 to 5 days, the colonies using the carbon source present in the culture medium are visible (see FIG. 2).
The UV resistant bacterium designated M11-3C is able to grow in anaerobiosis and at 45° C. by using xylan from birch wood as sole carbon source.
The UV resistant bacterium designated M11-9D is able to grow in anaerobiosis and at 45° C. on a culture medium containing either CMC or xylan as source of carbon.
These bacteria are thus able to transform lignocellulosic biomass into valuable products under industrial conditions.
Example 3
Selection of UV-Resistant Bacteria which are Able to Use Lignin as Carbon Source (FIG. 3)
Bacteria selected for their resistance to UV rays from environmental samples (3 treatments of 4 mJ/cm2 with an interval of 4 hours) are inoculated on a solid minimal culture medium containing lignin at 0.1% (Sigma, France). Composition of the minimum culture medium is described in the previous examples. After incubation at 30° C. for 4 to 5 days, the colonies using the lignin present in the culture medium as carbon source are visible (bacteria which degrade the lignin decolorize the medium). The results are depicted in FIG. 3 and in the following Tables 1 and 2.













TABLE 1





Biotope
Name

Isolation

aerobiosis
anaerobiosis


Environment
Strain
Isolation t° C.
medium
cultured closest neighbors
Lignin
CMC + Xylan







Muck
M5-6H
30° C.
PGY
Bacillus
+
+


animal dejection
M6-2H
30° C.
PGY
Microbacterium
+
+


stones
M6-4H
30° C.
PGY
Cellulosimicrobium
+
+



M8-1B
30° C.
PGY
Methylobacterium
+
+



M8-1H
30° C.
PGY
Methylobacterium
+
+



M8-2B
30° C.
PGY
Methylobacterium
+
+



M8-3A
30° C.
PGY
Methylobacterium
+
+



M8-3C
30° C.
PGY
Methylobacterium
+
+



M8-4A
30° C.
PGY
Methylobacterium
+
+



M8-4B
30° C.
PGY
Methylobacterium
+
+



M8-4H
30° C.
PGY
Methylobacterium
+
+



M8-5A
30° C.
PGY
Methylobacterium
+
+



M8-5B
30° C.
PGY
Methylobacterium
+
+



M8-6G
30° C.
PGY
Methylobacterium
+
+



M8-7E
30° C.
PGY
Methylobacterium
+
+


moss
M8-9E
30° C.
PGY
Methylobacterium
+
+



M8-9H
30° C.
PGY
Methylobacterium
+
+



M8-11C
30° C.
PGY
Methylobacterium
+
+


animal dejection
M9-2E
30° C.
PGY
Sphingobacterium
+
+



M9-3E
30° C.
PGY
Sphingobacterium
+
+



M9-3F
30° C.
PGY
Sphingobacterium
+
+


cow dejection
M9-7D
30° C.
PGY
Pseudomonas
+
+



M9-7E
30° C.
PGY
Uncultured bacterium isolated
+
+






from Argalis sheep feces



















TABLE 2









cultured
Carbon source













t°
Isolation
closest
Aerobiosis
Anaerobiosis

















Biotope
Name
isolation
medium
neighbors
CMC
Xylane
Lignin
CMC
Xylane
Lignin





Water
MX1-1B
45° C.
MM + Xyl 1%
Bacillus
+
+
+
+
+
+


Water
MX1-6G
45° C.
MM + Xyl 1%
Caldimonas
+
+
+

+
−


Water
MX1-7B
45° C.
MM + Xyl 1%
Paenibacillus
+
+
+
+
+
+


Water
MX1-7C
45° C.
MM + Xyl 1%
Paenibacillus
+
+
+
+
+
+


Water
MC1-1D
45° C.
MM + CMC 1%
Gordonia
+
+
+
+
+
−


Water
MC1-1G
45° C.
MM + CMC 1%
Paenibacillus
+
+
+
−
−
−


Water
MC1-2A
45° C.
MM + CMC 1%
Bacillus
+
+
+
−
+
−


Sediment
MC1-2D
45° C.
MM + CMC 1%
Paenibacillus
+
+
+
−
+
−


Water
M10-1D
30° C.
PGY
Rhodococcus
+
+
+
nd
nd
nd



M10-1E
30° C.
PGY
Rhodococcus
+
+
+
nd
nd
nd



M10-8D
30° C.
PGY
Stenotrophomonas
+
+
+
nd
nd
nd









All these bacteria are thus able to transform lignocellulosic biomass into valuable products under industrial conditions. Using of lignin as carbon source by bacteria belonging to Sphingobacterium, Microbacterium or Cellulosimicrobium genera has never been described until now.
Example 4
Lignocellulosic Biomass Conversion
A bacteria sample from a liquid culture performed in rich medium, is washed with deionized water before inoculating ( 1/10) 20 ml-minimal medium containing acid MOPS buffer 40 mM (Sigma, France), NH4Cl 20 mM, KOH 10 mM, NaOH 10 mM, CaCl2 0.5 μM, Na2SO4 0.276 mM, MgCl2 0.528 mM), a solution of micronutriments at pH5 ((NH4)6(MO7)24 3 nM, H3BO3 400 nM, CoCl2 30 nM, CuSO4 10 nM, MnCl2 250 nM, ZnSO4 10 nM), a solution of vitamins at pH4 (1 μg/L of D-biotin, niacin, pyridoxal-HCl, thiamin-HCl and vitamin B12), a solution of K2HPO4 at 5.7 mM as well as a solution of FeCl3 at 20 μM in NaH2(C3H5O(COO)3). Paper journal and/or Whatman paper is used as source of lignocellulosic biomass and carbon source. About 140 mg of paper journal is added to the culture medium described above. The degradation of the paper journal and/or Whatman paper is monitored during several weeks and compare to the control tube (without any bacteria in the culture medium).
The results are depicted in FIG. 4. The Deinococcus strain DRH46 is able to degrade paper journal and/or Whatman paper. The degradation is visualized with the release of paper fibers in the culture medium which become cloudy after several weeks of growth (compare to the control tube).
Example 5
Selection of UV-Resistant Cellulolytic and Xylanolytic Deinococci Producing Detectable Level of Ethanol
Bacteria selected for their resistance to UV rays from environmental samples (3 treatments of 4 mJ/cm2 with an interval of 4 hours)) are inoculated at 45° C. on a minimal liquid medium containing Whatman I filter paper or xylan, as sole carbon source of lignocellulosic substrate. Composition of the minimum culture medium is described in the previous examples. The paper whatman I is degraded after several weeks of incubation with bacteria at 45° C., whereas no degradation was seen of whatman I filter paper treated in the same way as described above but containing no bacteria (control). In addition, an increase in opacity of culture medium containing bacteria grown in xylan as sole carbon source was seen after several days of incubation at 45° C. compared to the control medium containing no bacteria indicating a bacterial growth in such conditions. The metabolic properties of the strains are then determined to assess their ability to produce ethanol. Among various strains identified, the following may be mentioned, which is cellulolytic, xylanolytic, and produces ethanol.
























EtOH



Strain





production


Species
name
Starch
Sucrose
Xylan
Whatman I
Glucose
(%)







D. murrayi
M13-8D
+
+
+
+
+
0.016









This example further illustrates the efficacy of the method of the present invention, and the ability to isolate bacteria having the selected remarkable properties when applying the claimed method to various samples.

## Claims

1. A method of producing bioenergy products or metabolites, the method comprising exposing a lignocellulosic biomass to a bacterium to produce fermentable sugars, and fermenting said sugars into bioenergy products or metabolites, wherein said bacterium is selected from:
a) a bacterium which can grow in the presence of lignin as a sole carbon source, at a temperature of at least 30° C., and resist 3 UV treatments of 4 mJ/cm2 carried out at an interval of 4 hours; or
b) a bacterium which has the ability to utilize cellulose and xylan as carbon sources, at a temperature of at least 30° C., and to resist 3 UV treatments of 4 mJ/cm2 carried out at an interval of 4 hours.

2. The method of claim 1, further comprising collecting said bioenergy products or metabolites.

3. The method of claim 1, wherein said bacterium can use lignin, cellulose and xylan as carbon sources.

4. The method of claim 1, wherein said bacterium can be cultivated in aerobic and anaerobic conditions.

5. The method of claim 1, wherein said bacterium belongs to a genus selected from Deinococcus, Bacillus, Microbacterium, Cellulosimicrobium, Methylobacterium, Sphingobacterium, Pseudomonas, Caldimonas, Paenibacillus, Gordonia, Rhodococcus, Stenotrophomonas, Novosphingobium, Sphingomonas, Flavobacterium, Sphingobium, Sphingopyxis, or Porphyrobacter.

6. The method of claim 1, wherein said bacterium is obtained by a method comprising:
a) providing a sample comprising bacteria;
b) subjecting the sample to a cell-destructing UV irradiation comprising at least 3 UV treatments of 4 mJ/cm2 carried out at an interval of 4 hours; and
c) culturing said UV treated sample in/on a selection medium containing: lignin as the sole carbon source, cellulose as the sole carbon source, xylan as the sole carbon source or a combination of cellulose and xylan as the sole carbon sources; and
d) isolating from said cultured UV treated sample, a bacterium which utilizes lignin as the sole carbon source in the selection medium, utilizes cellulose as the sole carbon source in the selection medium, utilizes xylan as the sole carbon source in the selection medium or utilizes both cellulose and xylan as sole carbon sources in the selection medium.

7. The method of claim 1, wherein the lignocellulosic biomass comprises unprocessed material of biological origin.

8. The method of claim 7, wherein the unprocessed material of biological origin is selected from forestry products, agricultural products, or aquatic products.

9. The method of claim 1, wherein the fermentable sugar is selected from glucose, cellobiose, mannose, xylose, arabinose or galactose.

10. The method of claim 1, wherein the bioenergy product is a biofuel.

11. A method of producing a biofuel, the method comprising exposing a lignocellulosic biomass to a bacterium to produce fermentable sugars, and fermenting said sugars into biofuel, wherein said bacterium is selected from:
a) a bacterium which can grow in the presence of lignin as a sole carbon source, at a temperature of at least 30° C., and resist 3 UV treatments of 4 mJ/cm2 carried out at an interval of 4 hours;
b) a bacterium which has the ability to utilize cellulose as a sole carbon source, at a temperature of at least 30° C., and to resist 3 UV treatments of 4 mJ/cm2 carried out at an interval of 4 hours;
c) a bacterium which has the ability to utilize xylan as a sole carbon source, at a temperature of at least 30° C., and to resist 3 UV treatments of 4 mJ/cm2 carried out at an interval of 4 hours; or
d) a bacterium which has the ability to utilize both cellulose and xylan as carbon sources, at a temperature of at least 30° C., and to resist 3 UV treatments of 4 mJ/cm2 carried out at an interval of 4 hours.