Use of bacteria for the production of bioenergy

[0001] the present invention discloses compositions and methods of producing bioenergy. In particular, the invention discloses the use of bacteria of the genus Deinococcus and/or similar types, in the modification of biomass or derivatives of biomass for production of bioenergy products and metabolites.

BACKGROUND of the INVENTION [0002] it is Known to use microorganisms to conduct modification of biomass, particularly plant biomass for production of bioenergy products such as ethanol.

[0003] Currently used industrial processes only allow the reproduction and growth of microorganisms for fermentation and extraction of ethanol at a temperature of about 30°C, due to the volatility of used industrial microorganisms (yeast). Also due to the high costs of bioenergy, associated with seal of ethanol after fermentation, since the yeasts currently used for fermentation is not able to withstand high concentrations of 100 g/L. In addition, these fermentation, the yeast consume practically only C6 sugars, like glucose.

[0004] also Known is the processing of biological material between other bacterial strains to impart improved properties.

[0005] for Example, in the description of the patent US No. 6716631 reserved S. Del Cardayre etc., is described based on the repeated cycles of recombination and selection/screening for imparting desired properties to all cells and all organisms. These additional properties, e.g., increased compatibility for genetic recombination, increased the number of copies of the genome, the increased ability of expression and/or secretion of proteins and secondary metabolites.

[0006] With the aid of molecular genetics, the authors propose methods appropriate modification of the genomes of cells and organisms to give them new and improved properties.

[0007] In the method described in US6716631 apply to the population of different cells, the multiplication of these cells to create hybrid cells by fusion of protoplast, and then the screening or selection of cells that have evolved towards acquisition of the desired properties, and repeating these steps until obtaining at least one cell that has the desired modification. This method presented as the best alternative to the known methods based on the program of improving the strain.

[0008] the Protoplasts subjected to fusion can originate from organisms prokariotycznych.

[0009] One of the intended applications in the description of the patent US-a fermentation to produce, for example ethanol, and it was suggested that performance and cost are improved by using this method recombination via DNA shuffling used microorganisms. For example, the already mentioned homologiczną rekombinację Rhodococcus known that catalyzes between the two phases of the reaction.

[0010] In international patent application number WO01/023526 describes the production and application of bacteria that are resistant to radiation and able to conduct bioremediation, in particular of the genus Deinococcus (namely D. radiodurans and D. geothermalis) modified so that they were more effective in metabolizowaniu, destruction, or detoxification of pollutants of inorganic and organic, such as radionuclides, heavy metals and organic solvents. It is recommended that were possible such manipulation to these bacteria eksprymowały heterologiczne enzymes capable of detok2 sykacji these elements. Bacterial strains, as manipulated, to combine a number of functions encoded by different genes within the host organism.

[0011] In the patent application US, I. Narumi, etc., published 18 September 2003 under number 2003/0175977 described plazmid endogenous derived from a strain of D. radiopugnans, pUE30 that you can use as a vector capable of Autonomous replication in bacteria of the genus Deinococcus and which can be used to construct the vector suspension containing plazmid, capable of Autonomous replication in E. coli and derivatives, and capable of replication in bacteria of the genus Deinococcus and E. Coca-Cola.

[0012] In the description of the patent US No. 7160715, C. B. Fliermans, describes a method to measure the distribution and frequency of generation of cracks DNA strands in vivo. This method involves the application of SRPP protein derived from Deinococcus radiodurans.

[0013] In the patent application U.S., published under the number 2004/0224320 in favor of K. Akiko, etc. described bacteria Gram-positive (no access ATCC BAA-149 or its mutant), which is isolated and purified. This isolate is able to degrade various organic contaminants and is suitable for bioremediation of various organic impurities, in the presence of ionizing radiation.

[0014] in addition, recently published monograph on ąca ethanol production through fermentation of strains of microbes, called the "Ethanol Fermentation Strains" J. R. Hettenhaus under the auspices of the U.S. Department of Energy and National Laboratory of Renewable Energy (16 December 1998). In this document, which summed up the participation of the participants in this study reported that

the only strains of microorganisms that can be used in existing devices, should be similar to the one currently used, namely Saccharomyces, Zymomonas and E. coli;

- in the short term, the increased fermentation ksylozy and arabinozy should be the goal; while it is indicated that minor interest is the improved performance of converting other sugars on the type heksozy or oligomeru;

in the long term, you would have reached maximum ac advantages in the field of high operating temperatures and combining of the stages of production of the enzyme, scukrzania and hydrolysis.

[0015] Yes wi ec there was a demand for a method of fermentation of biomass and the production of ethanol and possibly other metabolites, which can be implemented in much better conditions than used on roads and which also could be easier to operate than the known methods, and could lead to food fermentation, which are cheaper and which improve easier.

[0016] the Present invention is able to provide a solution to these expectations and to provide improved ways in order to get the benefits from biomass for the production of alternative products, bio-energy, which are becoming increasingly necessary in connection with a significant reduction in energy sources of fossil origin.

Summary of the INVENTION [0017] the Present invention discloses methods and compositions for producing bioenergy products or metabolites. In particular, the invention relates to the use of special microorganisms for the production of bioenergy products or metabolites from biomass or its derivatives. The invention is based, inter alia, revealed that microbes of the genus Deinococcus have unexpected and useful properties which consists of changing or converting biomass or derivatives of biomass for the preparation of compounds that can be used for bioenergy production, particularly ethanol, on an industrial scale and in order, economical and reliable.

[0018] the Object of the invention is defined in the objection.

[0019] the Present invention discloses a method of production of bioenergy products or metabolites comprising contacting a biomass or biomass derivatives, and natural or modified bacteria with the ability to re-supply its genome, in whole or in part, after it has been damaged as a result of stress, preferably natural or modified bacteria of the genus Deinococcus, or an extract from it.

[0020] furthermore, the invention discloses a method of converting biomass or biomass derivatives into bioenergy products or metabolites comprising treating the biomass or derivatives of biomass in the presence of bacteria of the genus Deinococcus or bacteria, having the ability to resubmission of its genome, in whole or in part, after it has been damaged as a result of stress, or extract from it.

[0021] In a particular aspect, the present invention describes a method comprising the following steps:

a) breeding and/or growth of this bacterium in aerobic and/or anaerobic, b) modification of biomass or biomass derivatives with the formation of bioenergy products or metabolites of interest to industry (e.g., bioenergy sources such as ethanol, chemical building blocks such as succinic acid) using a composition containing the bacteria or extract from it, and

c) collecting at least one of bioenergetycznego product or metabolite obtained as a result of this modification of biomass or biomass derivatives.

[0022] the Present invention also discloses the use of bacteria of the genus Deinococcus, or an extract from it for the production of bioenergy products or metabolites from biomass or biomass derivatives.

[0023] the Invention also discloses a composition comprising a Deinococcus bacterium and biomass or derivatives of biomass.

[0024] the Invention also reveals the bio-energetic products produced using the above described method.

[0025] Method or use according to the invention can be accomplished using various natural or modified Deinococcus species, for example, without limitation, Deinococcus geothermalis, Deinococcus radiodurans, Deinococcus murrayi or Deinococcus cellulosilyticus. The present invention shows that Deinococcus bacteria can effectively contribute to the production of biofuels, such as ethanol, propanol, butanol, glycerin, butanodiol, PROPANEDIOL, or organic acids, which are of interest for applications in chemistry and their salts such as acetic acid, propyl alcohol, pyruvic acid, butyric acid, lactic acid and/or succinic acid, or esters, in particular esters created by the above-mentioned alcohols and acids.

[0026] the Invention also unexpectedly shows that Deinococcus can work in such conditions as high temperatures, a wide pH range, in the presence of solvents, in the presence of the original substrates, therefore suitable for the production of a large number of bioenergy products or metabolites from various substrates.

[0027] Thus, the invention discloses new methods and compositions for producing bioenergy products or metabolites in very effective way.

EXPLANATION OF THE FIGURES [0028]

Figure 1: antibacterial Effect of ethanol on Deinococcus geothermalis DSM11301 in the exponential phase of growth: the antibacterial potential of ethanol is significant if the content is higher than 8.2% in the exponential phase of growth.

Figure 2: bactericidal Effect of ethanol on Deinococcus geothermalis DSM11301 at the stationary phase: the antibacterial potential of ethanol is significant in the case that the contents of above-from 11.7% in stationary phase.

Figure 3: bactericidal Effect of butanol on Deinococcus geothermalis DSM11300 in the exponential phase of growth: the bactericidal potential of butanol is significant in the case that the contents of above 1.5% in the exponential phase of growth.

Figure 4: bactericidal Effect of butanol on Deinococcus geothermalis DSM11300 at the stationary phase: the bactericidal potential of butanol is significant in the case that the contents of above 2% at the stationary phase.

Figure 5: Effect of ethanol on the growth of Deinococcus geothermalis DSM11300: black square, 0% ethanol; white square, 0.8% ethanol; black circles, 1.2% ethanol; white circle, 2,4% ethanol; black triangle, 3.1% ethanol.

Figure 6A: Effect of pH on the growth of D. geothermalis DSM 113000 (DRH05): black square, pH 8; black circle, pH 7; white square, pH 6; white circle, pH 5; black diamond, ph4.

Figure 6B: the Effect of pH on the growth of D. geothermalis HAMBI 2481 (DRH37): black square, pH 8; black circle, pH 7; white square, pH 6; white circle, pH 5; black diamond, ph4.

Figure 6C: the Effect of pH on the growth of D. geothermalis HAMBI 2480 (DRH38): black square, pH 8; black circle, pH 7; white square, pH 6; white circle, pH 5; black diamond, ph4.

Figure 6D: Effect of pH on the growth of D. geothermalis HAMBI 2411 (DRH39): black square, pH 8; black circle, pH 7; white square, pH 6; white circle, pH 5; black diamond, ph4.

Figure 7: Growth of D. cellulosilyticus in different liquid nutrient mediums. The bacteria were grown as described in materials and methods in example 9. Black circle, the increase in nutrient medium rich; a black square, an increase in the minimum culture medium containing CM-cellulose; white square, an increase in the minimum medium without carbon source.

DETAILED description of the INVENTION [0029] the Present invention discloses methods for the production of bioenergy products or metabolites using bacteria Deinococcus. In fact, the invention provides that the bacteria Deinococcus can produce products for the bioenergy of a person or metabolites from biomass in a very efficient way.

Definitions [0030] In the context of the present application, the term "bacteria of the genus Deinococcus" includes the Deinococcus strains wild-type, that is, natural change, as well as recombinant strains, the strains obtained using shuffling technology or DNA technology, directed evolution.

[0031] "Extract from bacteria" means of arbitrary and frakcj e uzyskan and bacteria, and as a supernatant from the cells, the remains of cells, cell walls, DNA extract, enzymes or enzyme preparation or any preparation derived from bacteria in the results of chemical treatment, physical and/or enzyme activity, which is essentially free of living bacteria.

[0032] In the context of the present invention, the term "bioenergy" refers to renewable energy derived from biomass. In particular, the term "bioenergy products" means "biofuels" and all final products of modification of biomass or biomass derivatives that can be used as fuels such as ethanol. The term "metabolites" refers to all possible intermediate particles arising in the process of modifying biomass or biomass derivatives to form products, bio-energy, including, but not exclusively, a number of interesting chemical products for industry, such as organic acids and building blocks.

[0033] In the context of the present invention, the term "biomass" refers to the survivors and recently dead biological material that can be used as fuel or for industrial production. Most often the biomass for the plant material is raised to generate electricity or biofuel, but also includes material of vegetable or animal used for production of fibers, chemicals or heat. Biomass may also include biodegradable wastes that can be burned as fuel. The term biomass does not include organic materials that have undergone transformation as a result of geological processes, including substances such as coal or oil.

[0034] industrial Biomass can be obtained from many plant species, including miskantu, prose rózgowego, hemp, sugar beet, wheat, corn, poplar, willow, sorghum, sugarcane, and various species of trees, from eucalyptus to palm.

[0035] the Biomass according to the invention includes untreated biomass and/or secondary biomass. Unprocessed biomass is a raw material of biological origin. Examples of biomass are forest products, such as Mature trees unsuitable for the manufacture of raw wood or paper, agricultural products such as herbs, cultivated plants and fertilizer of animal and aquatic products such as algae and seaweed. Secondary biomass is any material initially derived from raw biomass, which has undergone significant changes in chemical and physical characteristics. Examples include paper, leather, cotton, hemp, products of natural rubber, by-products from food processing and waste vegetable oils for cooking.

[0036] as Used herein, the term "biomass derivatives" means that all particles obtained from raw biomass and/or from secondary biomass, as defined above, and, in particular, each material, which were originally derived from raw biomass, which has undergone significant changes in chemical and physical characteristics, such as, for example, starch, cellulose, and lignin hemicelulozy.

[0037] is Used here, the term "intermediate platform" means that the particles obtained by fizykochemiczną or biochemical transformation of biomass derivatives, such as sugar, starch, and gas for the synthesis of biological origin (synthesis gas).

Detailed description [0038] the present invention provides the use of Deinococcus bacteria for the production of bioenergy products or metabolites from biomass. The present invention is, in fact, shows that bacteria of the genus Deinococcus exhibit unexpected properties that allow them to engage in the production of bioenergy products or metabolites by fermentation of biomass or derivatives of biomass.

[0039] it is Shown that Deinococcus bacteria may zdolno sc re evil part of its genome, in whole or in part, after it has been damaged as a result of stress (PCT/EP2006/005826 Radman-Zahradka). As mentioned earlier, it was suggested these bacteria, particularly D. radiodurans for bioremediation. However, never disclosed and it was not suggested that Deinococcus bacteria may be able to production of bioenergy products and metabolites from biomass. In addition, has never been suggested that Deinococcus bacteria having useful biological properties, can be isolated and grown.

[0040] the Present invention first shown that there is a possibility of selection or cultivation of Deinococcus bacteria having at least one of the following characteristics and that these bacteria are able to production of bioenergy products or metabolites:

- s but with live, or functional at high temperatures (e.g., about 40 to 70°C);

- they live or functional in the pH range from about 3 to about 9.5, preferably between about 4 and about 8;

- they live or functional in obecnoś you toxic substances, in particular organic solvents, for example ethanol;

- s a transformative C6 and C5 sugars;

- can contribute to the digestion of cellulose into glucose;

- can promote digestion hemicelulozy education ksylozy;

- capable of growth in aerobic and/or anaerobic in the presence of a suitable carbon source.

[0041] furthermore, the Deinococcus bacteria, as a rule, deprived of patogenności and związ6 ku this can be used without particular limitations.

[0042] Thus, the invention for the first time shows the ability of Deinococcus bacteria for the production of bioenergy products or metabolites from biomass and their unexpected ability to grow and growing in special conditions subject to such use. The invention discloses also the use for production of bioenergy products or metabolites, and any bacteria having the ability resubmission of its genome, in whole or in part, after it has been damaged as a result of stress.

[0043] In a preferred form in accordance with the invention are applied thermophilic species Deinococcus, preferably selected from Deinococcus geothermalis, Deinococcus radiodurans and Deinococcus murrayi.

[0044] In a preferred form in accordance with the invention are applied Deinococcus bacteria living in the presence of toxic substances, in particular, in the presence of organic solvents such as ethanol. This statement, in fact, shows that Deinococcus strains can be grown in the presence of high concentrations of solvents, such as ethanol or butanol, providing a more efficient production of biofuels.

[0045] In another preferred form, in accordance with the invention are used bacteria that can multiply at a temperature in the range from about 40 to 70°C, preferably from 50°C to 60°C. In a more advantageous form, in accordance with the invention applied bacteria, which can be grown as elevated temperature (above 40°C), as in the presence of means toxic or organic solvent, as disclosed above.

[0046] next, In a special form, in accordance with the invention applied si e e Deinococcus bacteria, which can be alive or functional in the conditions of NaCl or equivalent salt, which can reach about 5% by weight-volume.

[0047] In another preferred form, in accordance with the invention are used bacteria that live in pH from about 3 to 9.5, preferably between 4 and 8. In fact, the inventors have discovered that Deinococcus strains can be kept in such harsh conditions that are particularly favorable for the conversion of biomass.

[0048] In a preferred form in accordance with the invention are applied Deinococcus bacteria, which is able to convert sugars C6 and/or C5 and/or may contribute to the digestion of cellulose into glucose and/or promote digestion hemicelulozy education ksylozy.

[0049] In a special form of the bacterium Deinococcus are capable of growth in the presence ksylanu and can promote digestion ksylanu.

[0050] Such enzymatic activity in combination with high heat resistance, a wide range of tolerance to pH and tolerance to toxic substances has not been described previously and never worthy of attention. As shown in the examples, Deinococcus bacteria having the above properties can be selected, bred and can produce large amounts of bioenergy products or metabolites from biomass.

[0051] In this regard, another advantage of the invention consists in the use in which these bacteria Deinococcus diluted in minimal nutrient medium containing a C6 sugar, preferably the glucose or more complex sugars, preferably sucrose, celobiozę or starch or sugar C5, preferably ksylozę as a carbon source. A further advantage of the present invention is that these bacteria Deinococcus can be grown in the presence of C3 carbohydrates, preferably glycerol or sodium pyruvate.

[0052] Specific examples of bacteria suitable for use in the present invention are Deinococcus geothermalis strains with the Deposit DSM11300, DSM11301, DSM11302, HAMBI2480, HAMBI2481 and HAMBI2411; Deinococcus murrayi strains with the Deposit DSM11303 and DSM11305; or a strain of Deinococcus cellu_T losilyticus with Deposit DSM18568 listed in Table 1), or strains, mostly similar to them, or their mutants.

<td colspan="4">Table 1: List of strains D</td><td colspan="3">einococcus</td>

<td>Marking</td><td>Type</td><td>Genre</td><td>Room</td><td>code</td><td>Temp °C</td><td>Link literaturowy</td>

<td>DRH 05</td><td>Deinococcus</td><td>geothermalis</td><td>DSM</td><td> 11300</td><td> 45-50</td><td>And in Ferreira, 1997 Int J Syst Bacteriol, 47(4):939-47</td>

<td>DRH 06</td><td>Deinococcus</td><td>geothermalis</td><td>DSM</td><td> 11301</td><td> 45-50</td><td>And in Ferreira, 1997 Int J Syst Bacteriol, 47(4):939-47</td>

<td>DRH 07</td><td>Deinococcus</td><td>geothermalis</td><td>DSM</td><td> 11302</td><td> 45-50</td><td>And in Ferreira, 1997 Int J Syst Bacteriol, 47(4):939-47</td>

<td>DRH 37</td><td>Deinococcus</td><td>geothermalis</td><td>HAMBI</td><td> 2481</td><td> 45-50</td><td>And in Kolari, 2003 J Ind Microbiol Biotechnol, 30 : 225-238</td>

<td>DRH 38</td><td>Deinococcus</td><td>geothermalis</td><td>HAMBI</td><td> 2480</td><td> 45-50</td><td>And in Kolari, 2003 J Ind Microbiol Biotechnol 30 : 225-238</td>

<td>DRH 39</td><td>Deinococcus</td><td>geothermalis</td><td>HAMBI</td><td> 2411</td><td> 45-50</td><td>Vaisanen and in 1997, Applied Microbiology 84: 1069-1084</td>

<td>DRH 08</td><td>Deinococcus</td><td>murrayi</td><td>DSM</td><td> 11303</td><td> 45-50</td><td>And in Ferreira, 1997 Int J Syst Bacteriol, 47(4):939-47</td>

<td>DRH 10</td><td>Deinococcus</td><td>murrayi</td><td>DSM</td><td> 11305</td><td> 45-50</td><td>And in Ferreira, 1997 Int J Syst Bacteriol, 47(4):939-47</td>

<td>DRH 46</td><td>Deinococcus</td><td>cellulosilyticus</td><td>DSM</td><td>18568 T</td><td> 45</td><td>Weon and in 2007, Int J of Syst &amp; Evolutionary Microbiol, 57, 16851688</td>

[0053] All strains listed in the table above may be grown in the growth medium of the type in PGY pH 7. Other relevant the growth medium identified in the experimental part.

[0054] it Should be understood that additional strains of Deinococcus having the characteristics represented currently 5 and is not provided may be subject to examination and was defined on the basis of information disclosed in the present application, for example, in accordance with the rules and tests described in the experimental part.

[0055] As mentioned above, the Deinococcus strains used in the present application, can be used in natural form or modified (e.g., chemically or genetycz10 no) to obtain improved properties. In this regard, in a particular form, the method used bacteria Deinococcus, which can be changed through the use of technology accelerated evolution or by DNA shuffling or gene insertion eukariotycznego, prokariotycznego or synthetic DNA that does not belong with a Deinococcus, or by introduction of a DNA gene from another strain of Deinococcus, and this modification affects żywot15 ek, the growth or action of this bacterium to contribute to changes in biomass. [0056] In another embodiment of the invention used by the bacteria can be recombinant or mo8 dyfikowany bacterial strain, preferably by a method such as described in international patent application number PCT/EP2006/005826.

[0057] As described above, the invention shows that bacteria of the genus Deinococcus or derivatives thereof, selected from, for example, D. geothermalis, D. radiodurans or D. murrayi, exhibit useful properties and is able to produce bioenergy products or metabolites from different initial substrates. Thus, the present invention discloses the use of bacteria of the genus Deinococcus for the production of bioenergy products or metabolites from biomass or biomass derivatives. The present invention also discloses a method of production of bioenergy products or metabolites from biomass or derivatives of biomass during the exposure or dilution of the biomass in the presence of bacteria of the genus Deinococcus, or an extract of them and recover the produced product bioenergetycznego or metabolite.

[0058] the Dilution or exposure can be carried out in any suitable conditions or environment for the modification of biomass or its derivatives with the formation of the product bioenergetycznego. In this regard, the method can be carried out in the reactor, in fermentorze, in the open air, if necessary, in the presence of appropriate nutrients or additives. It usually behaves in terms of pH, at a temperature not exceeding 40°C and in the presence of appropriate substrates.

[0059] Particular, the described subject matter is a method comprising the following steps:

a) breeding and/or growth of this bacterium in aerobic and/or anaerobic, b) modification (e.g., transformation or processing) of biomass or derivatives of biomass with the formation of bioenergy products or metabolites using a composition containing the bacteria or extract from it, and

c) collecting at least one of bioenergetycznego product or metabolite obtained as a result of this modification of biomass or biomass derivatives.

[0060] in Another of the described object is a method of converting biomass or derivatives of biomass using at least one bacterium or bacterial extract such as defined above or of a composition such as defined above, comprising coupling:

· at least one operation of placing in the breeding and development of this bacterial strain or extract of the bacterial strain under appropriate conditions for growth and development · at least one operation of converting a biomass or biomass derivatives with the participation of a sufficient number of the bacterial strain or extract of the bacterial strain under appropriate conditions for the conversion of biomass or derivatives of biomass and · collection of at least one of bioenergetycznego product or metabolite obtained in the conversion of biomass or biomass derivatives, in particular the collection, thus obtained ethanol.

[0061] In the above methods the first stage of selection and/or growth of this bacteria and the second stage of modification of biomass or biomass derivatives with the formation of bioenergy products or metabolites using a composition containing the bacteria or extract from it can be carried out simultaneously or sequentially; the third step of collecting bioenergy products or metabolites can be carried out simultaneously with the first and/or second step or sequentially. In this regard, you can contact the biomass with the bacterium under appropriate conditions, to ensure the multiplication of this bacteria, thus increasing the productivity of the process. Alternatively, you can bring a separate reproduction of bacterial strains under appropriate conditions of cultivation, and then add them into biomass. It should be understood that the exact number of bacteria used initially in order to effectively convert biomass into a vital bioenergy products or metabolites can be changed by a specialist, depending on the type of bacteria, type of biomass or derivatives and the growing conditions.

[0062] In a particular form described here of the method, the Deinococcus bacterium's and bred separately from biomass conversion.

[0063] In another particular form, the method used, the composition containing the bacteria Deinococcus or an extract from it, and at least one suitable package or auxiliary substances of the drug, preferably at least one means selected from the group including measures przeciwpieniące and nutrients. Appropriate means przeciwpieniącymi in particular, means, dispersants, detergents and surfactants, and more General relations amfifilowe.

[0064] In a particular form described here, the method is carried out in the reactor in which the conversion of biomass. "Reactor" means ordinary tank fermentacyjny or any camera or system for biomass conversion specially designed to implement the invention and therefore consisting in particular of bioreactors, biofiltrów, rotating fields biological and other reactors for gas phase and/or liquid form for the treatment of biomass or derivatives of biomass. The camera, which can be used according to the invention, can be used continuously or in busy areas.

[0065] In the reactor for the implementation of the enia with the invention, applied to si, at least one bacterium or bacterial extract of the invention and/or at least one composition such as defined above, while the reactor goes well and works so that prevailing circumstances, physico-chemical recorded and maintained so that the bacterium is functional to rozważanym application and so that may possible the growth of bacteria and favorably contributes to this.

[0066] the other, described here, form, bacteria's and bred in the reactor during the conversion process of biomass or derivatives of biomass, while the corresponding physico-chemical conditions recorded and maintained for this bacterial growth and is better for you to contribute to it. For example, you can use 500 ml Erlenmeyer flasks, in the presence of 100 ml of the nutrient solution or 167 Thermus minimal medium described below at a temperature of 50°C.

[0067] In alternative forms, the conversion of biomass or derivatives of biomass behaves in terms aerobiozy, anaerobiozy or mikroaerobiozy.

[0068] According to a further aspect of the leak is a reactor to convert biomass or biomass derivatives with at least one bacterium Deinococcus or an extract from bacteria such as defined above or of a composition such as defined above.

[0069] the Disclosed method can be used for bioenergy production from different types of biomass. In a preferred form, the biomass includes wood and wood residues, forest residues, residues from paper, a germ farm, the remnants of agriculture, edible and/or inedible plants, or parts thereof, straw, garden debris, aquatic plants, animal wastes, residues and livestock, natural fertilizer, organic waste and/or organic industrial waste. Biomass may also include biodegradable wastes.

[0070] In a particular form, the leak is a method of modifying biomass or biomass derivatives or intermediate platforms with the formation of bioenergy products or metabolites, wherein the biomass derivative is preferably the lignin, cellulose, hemiceluloza, starch, and intermediate platforms, preferably carbohydrates, such as ksylan, glukuronoksylan, arabinoksylan, glukomannan, ksyloglukan, starch, sucrose, lactose, maltose, trehaloza, glucose, xylose (wood sugar), mannose, arabinoza, ramnoza, galactose and/or fructose.

[0071] Particular, the described object is a method for the production of biofuels. In the context of the present invention, the term "biofuel" means fuel derived from living or recently dead biological source of carbon. Biofuels can be produced from renewable energy sources, in particular biomass of plant or animal or household wastes or industrial. Further, according to the invention includes a "first-generation biofuel" and/or "second generation biofuels".

[0072] the first-generation Biofuels, which are derived from material of vegetable or animal origin, preferably from sugar, starch, vegetable oils or animal fats. The main source for the production of first-generation biofuels are edible plants or their parts. First-generation biofuels include vegetable oil, biodiesel, bioalkohole, biogas, syngas and solid biofuels. Bioalkohole include ethanol, propanol and butanol. Cheaper disclosed method is used for the production of ethanol, propanol, butanol. The most preferred biofuel is ethanol.

[0073] second-generation Biofuels are made preferably from non-edible plants or non-edible parts of plants. They include growing niespożywcze, waste biomass, stalks of wheat, corn and wood. Further preferably, according to the invention includes cellulosic biofuels.

[0074] depending on the source of biomass, of bioenergy products or metabolites, such as biofuel, can require two successive phases: hydrolysis katalizowanej enzymes, beneficial celulazy or lakazy, which break down long, complex chains of carbohydrate, such as, respectively, cellulose or lignin, less sugar, susceptible of fermentation; and fermentation stage, where further decomposition of organic compounds such as sugars, alcoholic beverages. It should be emphasized that the Deinococcus strains of the present invention can be used in any or in both of these reactions. In fact, the invention shows that Deinococcus can hydrolizować long chain carbohydrate (e.g. cellulose or ksylan) and can also produce metabolites (e.g., ethanol, glycerol, butanodiol, PROPANEDIOL and acetic acid, propyl alcohol, pyruvic and butyric acid) from sugars, C3, C5 and C6. However, it should be emphasized that if desired the Deinococcus strains can be used in combination with any other bacterial strains.

[0075] the Following examples are given to illustrate and not limit the scope of the invention.

EXAMPLES

Example 1: Testing of selected [0076] in order to determine whether the microbe exhibits the properties necessary for the invention, it is necessary to conduct specific tests to determine whether a given type, genre, and/or bacterial strain is likely to have the necessary properties and act in the way that convert biomass or biomass derivatives, and to determine what significant improvements can be obtained in this way.

[0077] These special tests, according to the invention is carried out in seq e the following conditions:

Soybean breeding:

[0078] D. geothermalis (D. G.) grows si e at 50°C under stirring with oxygen. To maintain the strain used in the nutrient medium 167. In the experiments of fermentation applied minimal nutrient medium, in particular, in order to characterize the metabolites. In this case, 500 ml of a nutrient medium for cultivation is kept for from 1 to 7 days, in the process of mixing in 1-liter Erlenmeyer flasks and then inoculated 5 ml konfluentnej breeding D. G.

Soybean breeding 167 [0079] _

<td>Trypton</td><td> 1</td><td>g</td>

<td>Yeast extract</td><td> 1</td><td>g</td>

<td>Agar</td><td> 28</td><td>g</td>

<td>Acid nitrylotrioctowy</td><td> 100</td><td>mg</td>

<td>CaSO4 x 2 H₂On</td><td> 40</td><td>mg</td>

<td>MgCl₂ x 6 H2O</td><td> 200</td><td>mg</td>

<td>0.01 M Fe citrate</td><td> 0,5</td><td>ml</td>

<td>The solution of trace elements (see below)</td><td> 0,5</td><td>ml</td>

<td>Phosphate buffer (see below)</td><td> 100</td><td>ml</td>

<td>H2O</td><td> 900</td><td>ml</td>

<td>The pH was adjusted to 7.2 with NaOH, autoklawować at 121°C for 15 minutes. Autoklawować phosphate buffer separately and add to the nutritive medium</td><td></td><td></td>

Phosphate buffer

<td>KH₂AFTER₄</td><td> 5,44</td><td>g</td>

<td>Na2HPO4 x 12 H2O</td><td> 43</td><td>g</td>

<td>H2O</td><td> 1000</td><td>ml</td>

<td colspan="3">The pH was adjusted to 7.2</td>

The solution of trace elements:

<td>H2SO4</td><td> 0,5</td><td>ml</td>

<td>MnSO₄ x H₂On</td><td> 2,28</td><td>g</td>

<td>ZnSO4 x 7 H2O</td><td> 0,5</td><td>g</td>

<td>H3BO3</td><td> 0,5</td><td>g</td>

<td>CuSO4 x 5 H2O</td><td> 25</td><td>mg</td>

<td>On₂Mu₄ x 2 H₂On</td><td> 25</td><td>mg</td>

<td>CoCl2 x 6 H2O</td><td> 45,00</td><td>mg</td>

<td>H2O</td><td> 1000</td><td>ml</td>

Soy is minimal [0080]

MOPS buffer

<td>Acid pug</td><td> 400</td><td>mm</td>

<td>NH4Cl</td><td> 200</td><td>mm</td>

<td>NaOH</td><td> 100</td><td>mm</td>

<td>KOH</td><td> 100</td><td>mm</td>

<td>CaCl2</td><td> 5</td><td>M</td>

<td>K2SO4</td><td> 276</td><td>mm</td>

<td>MgCl2</td><td> 5,28</td><td>mm</td>

<td colspan="3">pH 7, filtered, sterilized</td>

The carbon source

<td>Glucose</td><td> 160</td><td>mm</td>

<td colspan="3">Filtered, sterilized</td>

Phosphate

<td>K₂HPO₄</td><td> 12,3</td><td>mm</td>

<td>KH2PO4</td><td> 7,7</td><td>mm</td>

<td colspan="3">Filtered, sterilized</td>

Vitamins

<td>D-Biotin</td><td> 10</td><td>µm</td>

<td>Niacin</td><td> 10</td><td>µm</td>

<td>Pirydoksal-HCl</td><td> 10</td><td>µm</td>

<td>Thiamine-HCl</td><td> 10</td><td>µm</td>

<td colspan="3">Store in pH 4, filtered, sterilized</td>

The solution of trace elements

<td>H2SO4</td><td> 5</td><td>ml</td>

<td>MnSO4 x H2O</td><td> 22,8</td><td>g</td>

<td>ZnSO4 x 7 H2O</td><td> 5</td><td>g</td>

<td>H3BO3</td><td> 5</td><td>g</td>

<td>CuSO4 x 5 H2O</td><td> 250</td><td>mg</td>

<td>On₂Mu₄ x 2 H₂On</td><td> 250</td><td>mg</td>

<td>CoCl2 x 6 H2O</td><td> 450</td><td>mg</td>

<td>H2O</td><td> 1000</td><td>ml</td>

<td colspan="3">Filtered, sterilized</td>

Source of iron

<td>FeCl3</td><td> 200</td><td>M</td>

<td>Sodium citrate</td><td> 200</td><td>M</td>

<td colspan="3">Filtered, sterilized</td>

Amino acids

<td>Cheese</td><td> 100</td><td>mm</td>

<td>Gln</td><td> 100</td><td>mm</td>

<td colspan="3">Filtered, sterilized</td>

These solutions are stored in the form of a 10-fold articulation diluted immediately before use.

Determination of the activity of bacterial lakazy

Principle:

[0081]

Syringaldazyna + O₂ -► Hydrogen peroxide syringaldazyna + H₂On

Lakaza

Reagents:

[0082]

A. 100 mm Buffer based on potassium phosphate, pH 6.5 at 30°C

B. 0,216 mm Syringaldazyna (derived 3 ml solution in absolute ethanol with syringaldazyny obtained from Sigma Edition No. S-7896.)

C. The Enzyme

<td></td><td>test</td><td>attempt blind</td>

<td>H2O</td><td>0.50 ml</td><td>Niesfermentowana soybeans, 0.5 ml or dilution</td>

<td>Reagent And</td><td>2,20 ml</td><td>2,20 ml</td>

<td>Reagent B</td><td>0.3 ml</td><td>0.3 ml</td>

<td>Reagent C</td><td>Fermented soybeans, 0.5 ml or dilution</td><td> 0</td>

[0083] the Increase in optical density is recorded at 530 nm.

[0084] under these conditions one unit of enzyme produces an increase in optical density of 0.001 per minute at pH 6.5 and at a temperature of + 30°C.

Determination of the activity of bacterial celulazy

Principle:

[0085] This test is based on monitoring the conversion of NAD to NADH in the process of decomposition of cellulose. Then you can control the growth of absorption values at 340 nm in accordance with the supplier's instructions available on the website:

(<a href="http://www.sigmaaldrich.com/img/assets/18160/Cellulase.pdf">http://www.sigmaaldrich.com/img/assets/18160/Cellulase.pdf</a>)

Detection of ethanol production:

[0086] Ethanol means quantitatively using two methods.

Fermentation method:

[0087]

ADH ethanol + NAD -► aldehyde acetic acid + NADH [0088] This method is based on monitoring the conversion of NAD to NADH in the presence of ethanol and pyruvate alcoholic.

[0089] This reaction is expressed in the increase of the absorption values at 340 nm. To perform this measurement used a set of Sigma N7160, in accordance with the manufacturer's instructions available on the website:

(<a href="http://www.sigmaaldrich.com/sigma/bulletin/N7160BUL.pdf">http://www.sigmaaldrich.com/sigma/bulletin/N7160BUL.pdf</a>).

Measurement by high performance liquid chromatography in inverted system~ phases.

Conditions:

[0090]

HPLC Gilson with automatic injector, detection refraktometryczna, Column: Phenomenex Rezex ROA, 300 mm x 7.8 mm column Temperature: 65°C

Mobile phase of 0.005 N sulfuric acid flow rate: 0,600 ml/min [0091] At the beginning of a calibration curve prepared by introducing to the column a nutrient medium containing known concentration of ethanol. Measured peak area eluowanego 22,26 min after the defendant etanolowi. Given the calibration curve.

[0092] Then, a measured amount of ethanol produced by bacteria, introducing the supernatant was diluted in column. Measured peak area eluowanego 22,26 min after the defendant etanolowi. The concentration of ethanol present in supernatancie is determined by comparison with a calibration curve.

[0093] Other metabolites possibly produced in different proportions, can be detected and indicate that the quantity in accordance with known methods of analysis and evaluation.

[0094] the Bacteria are haploidalnymi organisms that multiply by binary fission and who eat mineral and organic substances in the environment.

[0095] Their demand for gas, especially oxygen, and a variety of applied techniques of cultivation and fermentation should be adapted to the fact whether these microbes are absolutely of oxygen, strictly anaerobic or facultative hydroxy-anaerobic. [0096] Activity celulazy, preferably required for the invention involved in the breakdown of cellulose, while activity lakazy allows or facilitates the distribution of lignin.

[0097] the Production of bioenergy products, such as, in particular, ethanol and/or other metabolites by fermentation of biomass is performed under the operating conditions given in the repeated test conditions and engineering parameters of the present invention, which in particular are: the amount of a nutrient medium for cultivation of bacteria, the operating conditions of temperature and/or pressure, and the ability of the fermentation, aerobic, anaerobic or mikrotlenowej.

[0098] After performing the above special testing and marking of selected natural or genetically modified strains are used in accordance with the archives of ways.

Example 2: Production of ethanol in the presence of Deinococcus geothermalis [0099] In 500 ml Erlenmeyer flasks containing 100 ml of the minimum nutrient medium in 50°C, inoculum 10¹⁰ D. geothermalis (DG) adds to 50°C. the Cultivation is maintained in the mixing process, to contribute to aeration.

[0100] Then, the dilution ready for use in conventional tank fermentation of biomass, which, in the best conditions, it is possible to obtain ethanol and other metabolites with excellent performance at 55°C.

[0101] For 1 to 7 days in the reactor with the treated biomass, the presence of the above mentioned ethanol and metabolites quantified by HPLC (in accordance with the described above Protocol). Revealed the disappearance of glucose and the simultaneous production of ethanol, the concentration of which is evaluated analytically. Was discovered with other interesting metabolites. Replacement ksylozą glucose in the nutrient medium also allows the growth of bacteria and production of ethanol.

[0102] In one embodiment of this example, similar results can be obtained by carrying out dilutions, and bacterial fermentation in the same container.

Example 3: bactericidal Effect of ethanol and butanol on Deinococcus geothermalis Material and methods [0103] This method allows to evaluate the bactericidal effect of organic solvents on bacteria in the growth phase or in stationary phase. Those solvents are ethanol and butanol. Studied bacteria belonging to the genus Deinococcus:

- develop at temperatures between 40 and 70°C

- they are functional at pH 3 to pH 9.5

- have the ability to re-introduce its genome, in whole or in part, after it has been damaged as a result of stress, namely radiation, especially UV or gamma rays, drying, action of enzymes, ultrasound or by chemical stress.

[0104] the Test should be performed at a temperature optimal for growth of the examined strains. Using primary culture in stationary phase in an enriched culture medium, inoculated in 10 ml of enriched nutrient medium in the amount of 1 vol.%/OTK. Enriched nutrient medium contains 2 g/l peptonu, 5 g/l yeast extract and 10 g/l glucose: sterile solution for autoklawowanie (20 minutes at 120°C). To this solution was added the following solutions: MOPS buffer (10 x) pH 7 [acid 400 mm MOPS, 200 mm NH4Cl, 1000 mm NaOH, 100 mm KOH, 5 ?? CaCl₂, Of 2.76 mm At₂SO₄, 5.28 mm MgCl₂]; micronutrients (10000 x) [300 mm (NH4)6(Mo7)24, H3BO3 4 mm, CoCl2 0.3 mm, CuSO4 0.1 mm, MnCl2 2.5 mm, ZnSO4 0.1 mm]; FeCl3 20 mm (100 x) in C6H5Na3O7 20 mm; 1 g/l K2HPO4: the solutions were sterilized by filtration (45 gm).

[0105] 200 gl of dilution applied to 96-studzienkowej mikropłytce. To avoid the phenomenon of evaporation of the solvent, mikropłytkę coated impervious barren film.

[0106] After reaching the exponential growth phase (optical density at 600 nm of 0.5) or stationary phase (plateau), added solvent. Article investigates the content of ethanol is from 0 to 31%, and butanol 0 to 2.5%. Then breeding is kept under stirring for one hour.

[0107] Calculation: After completion of the incubation for each concentration of the solvent, 20 gl of dilution is transferred to another mikropłytkę and was diluted in a cascading manner (dilution 1/10 9 wells). Soybean breeding is used to dilute the nutrient medium is supplemented. 5 gl of each dilution is placed in three repetitions on nutrient agar PGY: 5 g/l peptonu, 2.5 g/l yeast extract, 0.5 g/l glucose, 14 g/l agar: soybean, autoklawowanie through sterilized 20 minutes at 120°C. If growth is pozala, for each of the studied percentage concentration of solvent, records are maintained in order to assess the effect of organic solvents on the strain.

Results [0108] the Concentration of the solvent in which it was recognized that it comes to loss of viability of bacteria, which corresponds to the minimum concentration of solvent at which a decrease by one unit logarytmiczną compared to control.

[0109] the Studied strains (Figures 1 to 4), shows a satisfactory resistance to solvents from the point of view of industrial applications, in fermentorze.

Example 4: Growth of bacteria in the presence of sources of carbon, C3, C5 and C6

Material and methods [0110] the Preliminary culture was performed in medium A containing pepton (2 g/l), yeast extract (5 g/l), glucose (10 g/l) or in a nutrient medium PGY. After centrifugation of the nutrient medium for cultivation, the sediment of the bacteria washed twice with minimal medium And to remove all sources of nutrients from the inoculum. Then this inoculum is used for implantation (1/66) nutrient media for cultivation A (200?^, containing one of the following carbon sources at a concentration of 1% (wt./vol.): D(+)glucose and D(+)celobiozę, sucrose, starch, D(+)ksylozę, ksylan from birch wood, glycerin, sodium pyruvate. In the case of strains DRH07, DRH39, DRH08 and DRH10, in the environment of selection added glutamate (10 mm). Cultivation of bacteria was carried out at 45°C in 96-studzienkowych mikropłytkach in the process of mixing, and then measured the optical density at 544 nm using a spectrophotometer (Chameleon multilabel detection Platform plate, ScienceTec) or at 600 nm with a reader mikropłytek Spectrostar OMEGA (BMG Labtech). [0111] References to sources of carbon: Ksylan from birch wood (95588, Fluka), celobioza (22150, Fluka), D(+)xylose (wood sugar) (95730, Fluka), glucose (G8270-1KG, Sigma), sucrose (S9378-1KG, Sigma), starch (S9765-500G, Sigma), glycerol (453752, CarloErba), sodium pyruvate (Sigma).

The composition and preparation of nutrient media for cultivation [0112] Soybean PGY: Pepton (10 g/l), glucose (1 g/l), yeast extract (5 g/l), the mixture autoklawowana for 20 minutes at 120°C.

[0113] Soybean, A: Different solutions used for obtaining nutrients And is made from basic solutions sterilized by filtration through a e:

- Solution (pH 7) containing: 40 mm buffer on the basis of acid MOPS, 20 mm NH4Cl, 10 mm KOH, 10 mm NaOH, 0.5 mm CaCl₂, 0,276 mm₂SO₄, 0,528 which mm MgCl₂.

the solution of trace elements (pH 5): 3 nm (NH4)6(MO7)24, 400 nm, H3BO3, 30 nm CoCl2, 10 nm CuSO4, 250 nm MnCl2, 10 nm ZnSO4.

- A solution of vitamins, pH 4, (1 gg/l each): D-Biotin, Niacin, pirydoksal-HCl, tiaminaHCl, vitamin B12.

The source of phosphate: K2HPO4 5.7 mm.

- 20 gM FeCl₃ (prepared in sodium citrate solution and then, after filtration). Results [0114] the Bacteria listed in Table 2 (below) able to reproduce in a minimal nutrient medium (nutrient solution A) containing the tap as the sole carbon source, a C6 sugar, such as glucose, sucrose, and starch celobioza. It should be emphasized that the strains DRH37 and DRH06 they are also capable of growth in the presence of glycerol and sodium pyruvate (carbohydrates C3).

[0115] the Bacteria listed in Table 3 are also capable of growth on minimal nutrient medium containing a sugar crane C5 (ksylozę or ksylan) as the sole carbon source, the output ątkiem strains DRH06 and DRH07 that are not capable of growth in the presence of, respectively, ksylanu and ksylozy.

Table 2: Test of assimilation of various carbon sources C6 and C3 carried out for different types of D. geothermalis and D. murrayi : - ΔOD OF&lt;0.2 ; + ΔOD =0,2; 0,3 ++ &gt; ΔOD&gt; 0,4; +++ 0,4 &gt; ΔOD &gt; 0,5 ; ++++ ΔOD &gt;0,6. ΔOD corresponds to the difference between the value at 544 nm for the initial time T0 of growth and time T196 hours (approximately 8 days).

<td rowspan="2">Carbon sources</td><td></td><td></td><td colspan="2">D. geothermalis</td><td></td><td></td><td>D. murrayi</td>

<td rowspan="3">DRH05</td><td rowspan="3">DRH06</td><td rowspan="3">DRH07</td><td rowspan="3">DRH37</td><td rowspan="3">DRH38</td><td rowspan="3">DRH39</td><td rowspan="3">DRH08 DRH10</td>

<td>at a concentration of</td>

<td> 1%</td>

<td>(wt./Rev.) C6 Carbohydrates: D-(+)-glucose</td><td> +++</td><td> +</td><td> ++</td><td> ++</td><td> +++</td><td> +++</td><td> + +</td>

<td>D-(+)-celobio-</td><td> ++</td><td> -</td><td> +++</td><td> +++</td><td> ++</td><td> +++</td><td> ++ -</td>

<td>for</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Sucrose</td><td> +++</td><td> ++</td><td> ++</td><td> ++</td><td> +++</td><td> +++</td><td> ++ -</td>

<td>Starch</td><td> +++</td><td> ++</td><td> ++</td><td> ++</td><td> +++</td><td> -</td><td> ++ -</td>

<td>Carbohydrates C3: Glycerol</td><td></td><td></td><td></td><td> ++</td><td></td><td></td><td></td>

<td>Pyruvate sodium</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td><td> - -</td>

Table 3: test of assimilation of various carbon sources C5 and C6, were carried out for various species of D. geothermalis - ΔOD OF&lt;0.2; + ΔOD = 0,2; ++ 0,3 &gt;? &gt; 0,4; +++ 0,4 &gt; ? FROM &gt; 0,5; ++++ ? With &gt;0,6. ? FROM meets the difference between the value at 600 nm for the initial time T0 of growth and time T64 hours (approximately 2.5 days).

<td>Carbon source at a concentration of</td><td>DRH05</td><td>DRH06</td><td>DRH07</td><td>DRH37</td><td>DRH38</td><td>DRH39</td>

<td>1% (wt./Rev.)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>D-(+)-glucose</td><td> +++</td><td> ++</td><td> +</td><td> ++++</td><td> +++</td><td> +++</td>

<td>Ksylan</td><td> +++</td><td> -</td><td> +</td><td> ++++</td><td> ++++</td><td> ++++</td>

<td>Xylose (wood sugar)</td><td> +++</td><td> +++</td><td> -</td><td> ++++</td><td> +++</td><td> +</td>

Example 5: bacterial Growth at high concentration of ethanol

Material and methods [0116] This method allows to evaluate the ability of the germ to develop in the presence of high ethanol concentrations. Studied bacteria belonging to the genre of Deinococcus geothermalis:

- develop at temperatures between 40 and 70°C,

- function pH 3 to pH 9,5,

- have the ability to re-introduce its genome, in whole or in part, after it has been damaged as a result of stress, namely radiation, especially UV or gamma rays, the drying action of the enzyme, ultrasound or stress che15 micznego.

[0117] Test should be performed at a temperature optimal for growth of the examined strains. Using primary culture in stationary phase in an enriched culture medium, for each of the investigated concentrations of ethanol, inoculated with 20 ml of enriched nutrient medium in the amount of 1 vol.%/OTK. Enriched environment development includes: 2 g/l peptonu, 5 g/l yeast extract and 10 g/l glucose: sterile solution for autoklawowanie (20 minutes in

120°C). To this solution was added the following solutions: MOPS buffer (10 x) pH 7 [400 mm buffer on the basis of acid MOPS, 200 mm NH4Cl, 1000 mm NaOH, 100 mm KOH, 5 ?? CaCl₂, Of 2.76 mm At₂SO₄, 5.28 mm MgCl₂]; micronutrients (10000 x) [300 mm (NH4)6(Mo7)24, H3BO3 4 mm, CoCl2 0.3 mm, CuSO4 0.1 mm, MnCl2 2.5 mm, ZnSO4 0.1 mm]; FeCl3 20 mm (100 x) in C6H5Na3O7 20 mm; 1 g/l K2HPO4: the solutions were sterilized by filtration (45 gm).

[0118] the Ethanol is added at time T0, its content varies from 0% to 31%. The increase in tracks for each of the investigated ethanol. FROM reading at 600 nm with a spectrophotometer (UV Light XS5, SECOMAM). 1 ml portion of dilutions are charged in time: T0, T0+1 h T0+3 h T0+18 h, T0+20 h, T0+22 h, T0+24 hours.

[0119] When it is necessary to read the culture was diluted ten times in an enriched nutrient medium. Growth curves can be constructed for each of the investigated ethanol. At the end of the incubation period and for each study ethanol, performs counting to assess the effect of ethanol on strain.

Results [0120] studied Some strains, such as Deinococcus geothermalis DSM11300, capable of growing on a nutrient medium containing ethanol (see Figure 5). Some strains, such as Deinococcus geothermalis DSM11300, are resistant to high ethanol content in the growth medium (see figure 6A).

Example 6: Preparation of interesting metabolites using Deinococcus murrayi

Material and methods [0121] This method allows to evaluate the ability of the microbe to create interesting metabolites (from the group including glycerin, butanodiol, PROPANEDIOL and acetic acid, propyl alcohol, pyruvate and butyric acid) from biomass or derivatives of biomass. Studied bacteria belonging to the genre of Deinococcus geothermalis:

- develop at temperatures between 40 and 70°C,

- function pH 3 to pH 9,5,

- have the ability to re-introduce its genome, in whole or in part, after it has been damaged as a result of stress, namely radiation, especially UV or gamma rays, drying, action of enzymes, ultrasound or by chemical stress.

[0122] the Test should be performed at a temperature optimal for growth of the examined strains. Using primary cultures (in stationary phase) prepared in enriched nutrient medium, inoculated with 20 ml of enriched nutrient medium: zaszczepianie in the amount of 1 vol.%/OTK.

[0123] Contains soy, a selection includes: 2 g/l peptonu, 5 g/l yeast extract and 10 g/l glucose: sterile solution for autoklawowanie (20 minutes at 120°C). To this solution was added the following solutions: solution a buffer of MOPS (10 x) pH 7 [acid 400 mm MOPS, 200 mm NH₄Cl 1000 mm NaOH, 100 mm KOH, 5 gM CaCl₂, Na2SO4 was 2.76 mm, MgCl2 5.28 mm]; micronutrients (10000 x) [300 mm (NH4)6(Mo7)24, H3BO3 4 mm, CoCl2 0.3 mm, CuSO4 0.1 mm, MnCl2 2.5 mm, ZnSO4 0.1 mm]; FeCl3 20 mm (100 x) in C6H5Na3O7 20 mm; 1 g/l K2HPO4: the solutions were sterilized by filtration (45 gm).

[0124] culture of leaves in a thermostat at 45°C under stirring until reaching the stationary phase. After ociągnięciu the stationary phase, the farming itself tsentrifugirujut for 10 minutes at 4000 rpm./minutes. The supernatant is decanted into another test tube and placed at a temperature of -80°C. before HPLC Analysis with UV and refraktometria (column jonowymienna (H+)BioRad, mobile phase of 5 mm H2SO4, flow rate mobile phase 0.6 ml/min, mode izokratyczny) allow to identify interesting metabolites.

Results [0125] Some of the studied strains to produce specific, desirable, interesting metabolites (table 4).

Table 4: Metabolites produced by Deinococcus murrayi DSM11305 (concentration expressed in g/l).

<td></td><td>GLUCOSE</td><td>ACID OC-</td><td>ACID PRO-</td><td>ACID PYRO-</td>

<td></td><td></td><td>TOVA</td><td>VERTICAL</td><td>GRONOWY</td>

<td>DRH10 CM</td><td> 7,76</td><td> 0,138</td><td> 1,044</td><td> 0,043</td>

Example 7: Growth of Deinococcus geothermalis in various pH conditions

Material and methods [0126] Strains are grown at 45°C in a nutrient medium at different pH PGY. nastawiano pH using 10% NH3 (Rev./Rev.) or 10 N HCl. Growth control by measuring the optical density at 600 nm with a reader mikropłytek Spectrostar OMEGA, BMG Labtech.

Results [0127] Four strains (D. geothermalis) were able to reproduce in a pH range from 5 to 8 (see Figures 6A, 6B, 6C and 6D).

Example 8: Isolation of bacteria termofilnych, resistant to UV from the environment

Processing of samples hot water [0128] the Samples compacted hot water, filtration through a 0.22 µm nitrocellulose filter (Millipore, France), and then put in suspension in 10 ml of sterile water. Then the filtered solution is treated with ultrasound for about 60 seconds to re bacteria in suspension.

Processing of samples of wood and pebbles [0129] Samples of wood and stones immersed in sterile water, and then worteksuje and it seems to ultrasound for about 60 seconds.

Processing of samples of stones, moss, lichen, dirt, silt, biological membranes, soil and manure [0130] Samples of moss, lichens, wetlands, soil and manure placed in suspension in sterile water (vol./vol.), and then worteksuje. The samples were then treated with ultrasound for about 60 seconds.

Isolation of bacteria resistant to UV termofilnych [0131] After treatment with ultrasound, between the 500 µl and 2 ml of suspension is applied on the solid agar enriched nutrient medium PGY, pickled in autoklawowanie (20 minutes at 120°C) containing tap 1 g/l glucose (Sigma-Aldrich, France), 10 g/l peptonu (Fluka, France) and 5 g/l yeast extract (Fluka, France). Inoculated nutrient medium is then subjected to 3 times of effects of UV radiation with the camera BLX-E254 biolink (VilberLourmat, France) with an energy of 4 MJ/cm² each performed with an interval of 4 hours. After incubation at 45°C for 3 to 4 days visible colonies of thermophilic interesting.

Example 9: Digestion of cellulose by Deinococcus cellulosilyticus

Material and methods [0132] Pre-cultivation of the strain D. cellulosilyticus was carried out in enriched nutrient medium (composition, see below). This pre-dilution is applied to the implant (1% vol./Rev.) 10 ml of enriched nutrient medium, the minimum culture medium containing karboksymetylocelulozę (CM-cellulose) or the same nutrient medium without carbon source.

[0133] bacterial Growth occurred at 30°C in 50 ml Falcon tubes, in the mixing process (110 rpm./minutes), and then measured the optical density at 600 nm with a spectrophotometer (WPA Biowave, the counter of the number of cells).

[0134] Contains the nutrient solution: 2 g/l peptonu; 5 g/l yeast extract; 10 g/l glucose; solution (pH 7) containing 40 mm MOPS acid, 20 mm NH₄Cl, 10 mm KOH, 10 mm NaOH, 0.5 mm CaCl₂, 0,276 mm₂SO₄, 0,528 which mm MgCl₂; the solution of trace elements (pH 5): 3 nm (NH4)6(MO7)24, 400 nm, H3BO3, 30 nm CoCl2, 10 nm CuSO4, 250 nm MnCl₂, 10 nm ZnSO₄; solution of vitamins, pH 4, (1 gg/l each): D-Biotin, Niacin, pirydoksal-HCl, thiamine-HCl, vitamin B12; source of phosphate: 5.7 mm K₂HPO₄; 20 µm FeCl3.

[0135] Soybean minimum: a solution (pH 7) containing: 40 mm acid MOPS, 20 mm NH4Cl, 10 mm KOH, 10 mm NaOH, 0.5 mm CaCl₂, 0,276 mm₂SO4, 0,528 which mm MgCl2; the solution of trace elements (pH 5): 3 nm (NH4)6(MO7)24, 400 nm, H3BO3, 30 nm CoCl₂, 10 nm CuSO₄, 250 nm MnCl₂, 10 nm ZnSO₄; solution of vitamins, pH 4, (1 gg/l each): D-Biotin, Niacin, pirydoksal-HCl, thiamine-HCl, vitamin B12; source of phosphate:

5.7 mm K₂HPO₄; 20 µm FeCl₃.

Results [0136] it was Shown that the strain D. cellulosilyticus DSMZ noted in number DSM _T

18568 (Weon and in 2007) shows the activity against CM-cellulose (Weon et al, 2007, international journal of Systematic and Evolutionary Microbiology, 57, 1685-1688.) [0137] As shown in Figure 7, D. cellulosilyticus is able to multiply in a nutrient medium containing the tap CM-cellulose as the sole carbon source; the change in optical density at 600 nm after 10 days of cultivation in this environment was significant (ADO _{Maximum torque of 600 nm} = 0,5) compared with breeding control (nutrient medium without carbon source; (ADO _{Maximum torque of 600 nm} = 0,18). This result indicates that D. cellulosilyticus is not only capable of decay (depolymerization) CM-cellulose, but also for the assimilation of products resulting from this decomposition (celobiozy and glucose).