Compositions And Methods For Degrading Lignocellulosic Biomass

  • Published: Aug 25, 2011
  • Earliest Priority: Feb 17 2009
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AU 2010215597 A1
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 
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(10) International Publication Number (43) International Publication Date 
26 August 2010 (26.08.2010) PCT W O 2010/094665 A3 
(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every 
C12N 1/20 (2006.01) C12P 7/10 (2006.01) kind of national protection available): AE, AG, AL, AM, 
C12N15/01 (2006.01) C12N 1/22 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 
CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (21) International Application Number: DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM, GT, PCT/EP201/05F1885 HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 
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09305154.8 17 February 2009 (17.02.2009) EP kind of regional protection available): ARIPO (BW, GH, 
61/153,478 18 February 2009 (18.02.2009) US GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, 
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicants (for all designated States except US): TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, 
DEINOVE [FR/FR]; 4 Rue Tesson, F-75010 Paris (FR). ES, Fl, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 
CENTRE NATIONAL DE LA RECHERCHE SCIEN- MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, 
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(FR). ML, MR, NE, SN, TD, TG).  
(72) Inventors; and Declarations under Rule 4.17: 
(75) Inventors/Applicants (for US only): ISOP, Cathy - of inventorship (Rule 4.1 7(iv)) [FR/FR]; 79 rue Olivier de Serres, F-34400 Lunel (FR).  
JOSEPH, Pascale [FR/FR]; 18 rue Alcyone, "Le Capi- Published: 
tole", Appartement 331, F-34000 Montpellier (FR). - with international search report (Art. 21(3)) LEONETTI, Jean-Paul [FR/FR]; 9 Rue Jean Monet, 
F-34170 Castelnau Le Lez (FR). BITON, Jacques - before the expiration of the time limit for amending the 
[FR/FR]; 9 Impasse Dagobert, F-60610 Lacroix-Saint- claims and to be republished in the event of receipt of 
Ouen (FR). amendments (Rule 48.2(h)) 
(74) Agents: BECKER, Philippe et al.; Becker & Associes, (88) Date of publication of the international search report: 
rue Louis Le Grand, F-75002 Paris (FR). 21 October 2010 
O (54) Title: COMPOSITIONS AND METHODS FOR DEGRADING LIGNOCELLULOSIC BIOMASS 
(57) Abstract: The present invention relates to composition and methods of producing bioenergy products and metabolites of in
dustrial interest from lignocellulosic biomass. More specifically, the invention describes the identification, characterization and 
N 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, compo
sitions comprising such bacteria, and their uses for the modification of lignocellulosic biomass, with a view to producing bioener

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COMPOSITIONS AND METHODS FOR 
DEGRADING LIGNOCELLULOSIC BIOMASS 
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 biofuels 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

WO 2010/094665 PCT/EP2010/051885 
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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.  
W02009/063079 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.  
W097/10352 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.

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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.

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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

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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, Calditnonas, 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.  
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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.

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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.  
LEGEND TO THE FIGURES 
Figure 1 : growth results of UV-resistant thermophilic bacteria on MM+CMC 1% under 
anaerobiosis conditions.  
Figure 2 : growth results of UV-resistant thermophilic bacteria on MM+xylan1I% under 
anaerobiosis conditions.  
Figure 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.

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Figure 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.

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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.

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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).  
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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

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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.

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In an alternative method, the cell-destructing DNA damaging treatment comprises 
contacting the sample with a genotoxic agent, such as a solvent, mitomycin or H20 2. 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 10g/L, Yeast extract 5g/L, 
glucose lg/L) or LB (Bacto-tryptone lOg/L, Yeast extract 2.5g/L, Sodium chloride 
10g/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. Apple Environ Microbiol. 71 (9):5225-35; Ferreira, A.C., et al, 1997. Int JSyst

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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.

WO 2010/094665 PCT/EP2010/051885 
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.  
WO 2010/094665 PCT/EP2010/051885 
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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,

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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 lOg/L, Yeast extract 5g/L, glucose lg/L) or LB

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(Bacto-tryptone lOg/L, Yeast extract 2.5g/L, Sodium chloride lOg/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.

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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.

WO 2010/094665 PCT/EP2010/051885 
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

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167 Thermus medium 
Tryptone 1 g 
Yeast extract 1 g 
Agar 28 g 
Nitrilotriacetic acid 100 mg 
CaSO4 x 2 H20 40 mg 
MgCl 2 x 6 H 20 200 mg 
0.01 M Fe citrate 0.5 ml 
Solution of trace elements (see below) 0.5 ml 
Phosphate buffer (see below) 100 ml 
H20 900 ml 
Adjust to pH 7.2 with NaOH, autoclave at 121 C for15 min.  
autoclave the phosphate buffer separately and add to the medium 
Phosphate buffer 
KH2PO 4  5.44 g 
Na2HPO4 x 12 H 20 43 g 
H20 1000 ml 
Adjust to pH 7.2 
Composition of minimum medium 
- MOPS buffer 1X (ph7) containing: acid MOPS buffer 40 mM, NH 4Cl 20mM, KOH 10 mM, 
NaOH 10 mM, CaCl 2 0,5gM, Na2 SO 4 0,276 mM, MgCl 2 0,528 mM.  
- A solution of micronutriments (pH5): (NH4)6 (MO7)24 3nM, H 3B0 3 400 nM, CoCl 2 30 nM, 
CuSO 4 10, nM, MnCl2 250 nM, ZnSO 4 10 nM.  
- Solution of vitamins, pH4.0, (1 gg/l each): D-biotin, niacin, pyridoxal-HCl, thiamin-HCl, 
vitamin B12 
- Source of phosphate: K2HPO4 5.7 mM 
- FeCl3 20 gM (prepared in a solution of sodium citrate then filtered).

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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: 
ADH 
Ethanol + NAD > 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 bv reverse phase high performance liquid chromatography 
Conditions: 
HPLC Gilson with automatic injector, detection by refractometry, 
Column: Phenomenex Rezex ROA, 300 mm x 7.8 mm 
Column temperature: 65 0C 
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

WO 2010/094665 PCT/EP2010/051885 
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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 (figure 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), NH 4Cl 20 mM, KOH 10 mM, NaOH 10 mM, CaCl2 0.5 gM, Na2 SO 4 
0,276 mM, MgCl 2 0.528 mM), a solution of micronutriments at pH5 ((NH4)6(MO 7)2 4 
3 nM, H3B0 3 400 nM, CoCl2 30 nM, CuSO 4 10 nM, MnCl2 250 nM, ZnSO 4 10 nM), a 
solution of vitamins at pH4 (1 gg/L of D-biotin, niacin, pyridoxal-HCl, thiamin-HCl 
and vitamin B 12), a solution of K2HPO 4 at 5.7 mM as well as a solution of FeCl3 at 
gM in NaH2(C3H 50(COO) 3).  
Conditions of anaerobiosis are ensured by the addition of a GENbag anaer (BioM6rieux, 
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 Figure 1).  
The UV resistant bacterium designated M1 1-2F 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.

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Example 2 - Selection of thermophilic UV-resistant bacteria which use xylan in 
anaerobiosis (figure 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), 
NH 4Cl 20 mM, KOH 10 mM, NaOH 10 mM, CaCl2 0.5 gM, Na 2 SO 4 0,276 mM, MgCl 2 
0.528 mM), a solution of micronutriments at pH5 ((NH 4)6(MO 7)24 3 nM, H3B0 3 
400 nM, CoCl2 30 nM, CuSO 4 10 nM, MnCl2 250 nM, ZnSO 4 10 nM), a solution of 
vitamins at pH4 (1 gg/L of D-biotin, niacin, pyridoxal-HCl, thiamin-HCl and vitamin 
B12), a solution of K2HPO 4 at 5.7 mM as well as a solution of FeCl 3 at 20 gM in 
NaH2(C3H 50(COO) 3) 
Conditions of anaerobiosis are ensured by the addition of a GENbag anaer (BioM6rieux, 
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 Figure 2).  
The UV resistant bacterium designated M1 1-3C 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 MI 1-9D 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.  
WO 2010/094665 PCT/EP2010/051885 
Example 3 - Selection of UV-resistant bacteria which are able to use lignin as 
carbon source figuree 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 figure 3 and in the following Tables 1 and 2.  
Table 1 
Isolation 
Biotope Name Isolation tC medium cultured closest neighbors 
aerobiosis anaerobiosis 
Environment Strain Lignin CMC+Xylan 
Muck M5-6H 30'C PGY B acillus + + 
animal dejection M6-2H 30'C PGY Microbacterium + + 
stones M6-4H 30'C PGY Cellulosimicrobium + + 
M8-1B 30'C PGY Methylo bacterium + + 
M8-1H 30'C PGY Methylo bacterium + + 
M8-2B 30'C PGY Methylo bacterium + + 
M8-3A 30'C PGY Methylo bacterium + + 
M8-3C 30'C PGY Methylo bacterium + + 
M8-4A 30'C PGY Me thylo bacterium + + 
M8-4B 30'C PGY Me thylo bacterium + + 
M8-4H 30'C PGY Me thylo bacterium + + 
M8-5A 30'C PGY Methylo bacterium + + 
M8-5B 30'C PGY Methylo bacterium + + 
M8-6G 30'C PGY Me thylo bacterium + + 
M8-7 E 30'C PGY Me thylo bacterium + + 
moss M8-9E 30'C PGY Methylo bacterium + + 
M8-9H 30'C PGY Methylo bacterium + + 
M8-11C 30'C PGY Methylo bacterium + + 
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 + + 
M9-7 E 30'C PGY Uncultured bacterium isolated + + 
from A rgalis sheep feces

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Table 2 
cultured 
t Is olation closest 
Biotope Name isolation medium neighbors Carbon source 
Aerobiosis Anaerobiosis 
CMC Xylane Lignin CMC Xylane Lignin 
Water MX 1-1B 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 MC 1-1G 45'C MM+CMC 1% Paenibacillus + + + - -
Water MC1-2A 45'C MM+CMC 1% Bacillus + + + - + 
S ediment MC1-2D 45'C MM+CMC 1% Paenibacillus + + + - + 
Water M10-1D 3 OC PGY Rhodococcus + + + nd nd nd 
M10-1E 3 OC PGY Rhodococcus + + + nd nd nd 
M10-8D 3 OC 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 gM, Na 2SO 4 0,276 mM, MgCl2 0.528 mM), a solution of micronutriments at 
pH5 ((NH 4)6(MO7)24  3 nM, H3B0 3  400 nM, CoCl2 30 nM, CuSO 4 10 nM, 
MnCl2 250 nM, ZnSO4 10 nM), a solution of vitamins at pH4 (1 gg/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 gM in NaH2(C 3H 50(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).

WO 2010/094665 PCT/EP2010/051885 
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The results are depicted in figure 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 450 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 450C, 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 450 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.  
Strain EtOH Species Starch Sucrose Xylan Whatman I Glucose production 
name 
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.

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CLAIMS 
1. A method of selecting or identifying a bacterium, the method comprising the 
following steps: 
a) providing a sample comprising bacteria; 
b) subjecting the sample to a repeated irradiation treatment; and 
c) isolating, from said treated sample, a bacterium which has the ability to live or 
grow in the presence of lignin, cellulose and/or xylan as a carbon source.  
2. The method of claim 1, wherein the sample is or derives from an environmental 
sample, preferably obtained or derived from soil, water, hot springs, marine 
environment, mud, wood, stone, moss, vegetal extract, lichen, biological material, 
sediment, peatlands, biofilm, industrial effluents, gas, sand, oil, sewage, animal or 
human dejection.  
3. The method of claim 1 or 2, wherein the irradiation treatment is a treatment that 
reduces by at least 2 log the bacterial titer in a culture of E. coli.  
4. The method of any one of claims 1 to 3, wherein said treatment comprises several 
irradiations selected from UV-, gamma- and/or X ray-irradiation(s), either alone or in 
combinations, most preferably UV irradiation(s).  
5. The method of claim 4, wherein the treatment comprises subjecting the sample to at 
least 2, preferably at least 3 UV treatments of between 0.5 and 400 mJ/cm2, more 
preferably of between 1 and 200 mJ/cm2, typically between 1 and 100 mJ/cm2 each, 
carried out at an interval of between 1 and 8 hours.  
6. The method of any one of claims 1 to 4, wherein step b) is performed in a solid 
culture medium.  
7. The method of any one of claims 1 to 5, wherein steps b) and c) are performed 
sequentially, in any order, or simultaneously.

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8. The method of any one of claims 1 to 7, which further comprises a step of modifying, 
either biologically, genetically and/or chemically, the identified or isolated bacteria, or 
their DNA, in order to improve the viability, growth or functions of the said bacterium.  
9. The method of any one of claims 1 to 8, wherein step c) comprises the selection of a 
bacterium which grows in the presence of lignin as the sole carbon source, 
10. A bacterium obtainable by a method of any one of claims 1 to 9, or an extract 
thereof.  
11. An isolated bacterium, or an extract thereof, wherein said bacterium has the ability 
to grow in the presence of lignin as sole carbon source, at a temperature of at least 30'C, 
and to resist an UV treatment of 4 mJ/cm2.  
12. An isolated bacterium, or an extract thereof, wherein said bacterium has the ability 
to utilize cellulose and xylan as carbon source, at a temperature of at least 30'C, and to 
resist an UV treatment of 4 mJ/cm2.  
13. The bacterium of claim 11 or 12, which has the ability to use lignin, cellulose and 
xylan as carbon source.  
14. The bacterium of claim 11 or 12, which can hydrolyse lignocellulosic biomass to 
produce fermentable sugars.  
15. The bacterium of any one of claims 10 to 14, wherein said bacterium can be 
cultivated in aerobic and anaerobic conditions.  
16. The bacterium of any one of claims 10 to 15, which belongs to a genus selected 
from Deinococcus, Bacillus, Microbacterium, Cellulosimicrobium, Methylobacterium, 
Sphingobacterium, Pseudomonas, Caldimonas, Paenibacillus, Gordonia, Rhodococcus,

WO 2010/094665 PCT/EP2010/051885 
Stenotrophomonas, Novosphingobium, Sphingomonas, Flavobacterium, Sphingobium, 
Sphingopyxis, or Porphyrobacter.  
17. The use of a bacterium of any one of claims 10 to 16, or an extract thereof, to 
hydrolyse lignocellulosic biomass.  
18. A method of degrading lignocellulosic biomass into fermentable sugars, the method 
comprising exposing said lignocellulosic biomass to a bacterium of any one of claims 
to 16, or an extract thereof.  
19. The method of claim 18, wherein the fermentable sugar is selected from glucose, 
cellobiose, mannose, xylose, arabinose or galactose.  
20. A reactor or fermentor comprising lignocellulosic biomass and a bacterium of any 
one of claims 10 to 16, or an extract thereof.  
21. The use of a bacterium of any one of claims 10 to 16, or an extract thereof, to 
produce bioenergy products or metabolites.  
22. A method of producing bioenergy products or metabolites, the method comprising 
exposing a lignocellulosic biomass to a bacterium of any one of claims 10 to 16, or an 
extract thereof, to produce fermentable sugars, and fermenting said sugars into 
bioenergy products or metabolites.  
23. The method of claim 22, wherein the fermentation is performed using a bacterium 
of any one of claims 10 to 16.  
24. The method of claim 22 or 23, which further comprises collecting said bioenergy 
products or metabolites.  

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