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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • C—CHEMISTRY; METALLURGY
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
  • C12Y203/01008—Phosphate acetyltransferase (2.3.1.8)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
  • C12Y207/02001—Acetate kinase (2.7.2.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/145—Clostridium
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention pertains to the field of biomass processing to produce ethanol.
• novel thermophilic organisms that consume a variety of biomass derived substrates and produce ethanol in high yield are disclosed, as well as processes for the production and use of the organisms.
• Biomass represents an inexpensive and readily available cellulolytic substrate from which sugars may be produced. These sugars may be used alone or fermented to produce alcohols and other products. Among bioconversion products, interest in ethanol is high because it may be used as a renewable domestic fuel.
• SSF simultaneous saccharification and fermentation
• SSCF simultaneous saccharification and co-fermentation
• co-fermentation processes may also provide improved product yields because certain compounds that would otherwise accrue at levels that inhibit metabolysis or hydrolysis are consumed by the co-fermenting organisms.
• ⁇ -glucosidase ceases to hydrolyze cellobiose in the presence of glucose and, in turn, the build-up of cellobiose impedes cellulose degradation.
• An SSCF process involving co-fermentation of cellulose and hemi cellulose hydrolysis products may alleviate this problem by converting glucose into one or more products that do not inhibit the hydrolytic activity of ⁇ -glucosidase.
• CBP Consolidated bioprocessing
• Some bacteria have the ability to convert pentose sugars into hexose sugars, and to ferment the hexose sugars into a mixture of organic acids and other products by glycolysis.
• the glycolytic pathway begins with conversion of a six- carbon glucose molecule into two three-carbon molecules of pyruvate. Pyruvate may then be converted to lactate by the action of lactate dehydrogenase ("Idh”), or to acetyl coenzyme A (“acetyl-CoA”) by the action of pyruvate dehydrogenase or pyruvate- ferredoxin oxidoreductase.
• Idh lactate dehydrogenase
• acetyl-CoA acetyl coenzyme A
• Acetyl-CoA is further converted to acetate by phosphotransacetylase and acetate kinase, or reduced to ethanol by acetaldehyde dehydrogenase ("AcDH”) and alcohol dehydrogenase ( 1 WA").
• AcDH acetaldehyde dehydrogenase
• 1 WA alcohol dehydrogenase
• thermophilic, anaerobic bacteria that consume a variety of biomass derived substrates and produce ethanol in near theoretical yields.
• Methods for producing ethanol using the organisms are also disclosed.
• the instrumentalities reported herein result in the knockout of various genes either singly or in combination, where such genes in the native organism would otherwise result in the formation of organic acids.
• the methods and materials also apply to other members of the Thermoanaerobacter genus including Thermoanaerobacterium thermosulfurigenes , Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharo Iy ticum, Thermoanaerobacterium zeae, Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium xylanoly ticum.
• the methods and materials are useful generally in the field of metabolically engineered, thermophilic, Gram-positive bacteria.
• an isolated organism which does not express pyruvate decarboxylase, ferments a cellulolytic substrate to produce ethanol in a concentration that is at least 90% of a theoretical yield.
• a Gram-positive bacterium that in a native state contains at least one gene which confers upon the Gram-positive bacterium an ability to produce acetic acid as a fermentation product, is transformed to eliminate expression of the at least one gene.
• the bacterium may be a Thermoanaerobacter, such as Thermoanaerobacte ⁇ um saccharolyticum.
• the gene which confers upon the Gram-positive bacterium an ability to produce acetic acid as a fermentation product may code for expression of acetate kinase and/or phosphotransacetylase.
• the Gram-positive bacterium may be further transformed to eliminate expression of one or more genes that confer upon the Gram-positive bacterium the ability to produce lactic acid as a fermentation product.
• the gene that confers the ability to produce lactic acid may be lactate dehydrogenase.
• a method for producing ethanol includes transforming a native organism to produce a Gram-positive bacterium that has been transformed to eliminate expression of all genes that confer upon the Gram-positive bacterium the ability to produce organic acids as fermentation products, to produce a transformed bacterial host, and culturing the transformed bacterial host in medium that contains a substrate including a material selected from the group consisting of glucose, xylose, cellobiose, sucrose, xylan, starch, and combinations thereof under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the substrate.
• a biologically pure culture of a microorganism designated ALKl and deposited with the ATCC under Patent Deposit Designation No. PTA-7206 is described.
• an isolated polynucleotide comprises (a) a sequence of SEQ ID NO: 10, or (b) a sequence of SEQ ID NO: 9 and SEQ ID NO: 10, or (c) a sequence having at least about 90% sequence identity with the sequence of (a) or (b).
• a vector comprising the isolated polynucleotide of (a), (b), or (c) is described, as well as a host cell genetically engineered to express a compliment of the polynucleotide of (a), (b), or (c).
• an isolated polynucleotide comprises a sequence having at least about 95% sequence identity with the sequence of (a) or (b).
• an isolated polynucleotide comprises a sequence having at least about 98%, or at least about 99%, sequence identity with the sequence of (a) or (b).
• a method of producing ethanol includes culturing a mutant bacterium expressing a compliment of the isolated polynucleotide of (a), (b), or (c) in medium containing a substrate selected from the group consisting of glucose, xylose, cellobiose, sucrose, xylan, starch, and combinations thereof under suitable conditions for a period of time sufficient to allow fermentation of the substrate to ethanol.
• a method for producing ethanol includes providing within a reaction vessel, a reaction mixture comprising lignocellulosic substrate, cellulase and a fermentation agent.
• the fermentation agent comprises a Gram-positive bacterium that has been transformed to eliminate expression of at least one gene that confers upon the Gram-positive bacterium, in a native state, an ability to produce acetic acid as a fermentation product.
• the reaction mixture is reacted under suitable conditions for a period of time sufficient to allow saccharif ⁇ cation and fermentation of the lignocellulosic substrate.
• Fig. 1 shows reactions of the glycolytic pathway.
• Fig. 2 shows hydrogen production in wild-type T. saccharolyticum compared to various knockout strains of T. saccharolyticum.
• Fig. 3 shows a comparison of experimental and expected polynucleotide sequences for the ldh region of the suicide vector pSGD9 (SEQ ID NO: 9) integrated into the genome of T. saccharolyticum.
• Fig. 4 shows a comparison of experimental and expected polynucleotide sequences for the pta/ack region of the suicide vector pSGD8-Erm (SEQ ID NO: 10) integrated into the genome of T. saccharolyticum.
• Figs. 5-7 show high-performance liquid chromotography (HPLC) traces of a fermentation broth at various time intervals during growth of ALKl.
• Fig. 8 shows xylose, organic acid and ethanol concentrations during fermentation by strain ALKl .
• Fig. 9 shows xylose, organic acid and ethanol concentrations during fermentation by wild-type T. saccharolyticum.
• Fig. 10 shows xylose, organic acid and ethanol concentrations during a continuous culture challenge of ALKl.
• Fig. 11 shows xylose, organic acid and ethanol concentrations during fermentation by strain ALK2.
• Fig. 12 shows ethanol production at various Avicel concentrations during thermophilic SSF reactions involving ALK2 and cellulase at 50°C.
• Fig. 13 shows ethanol yield for the thermophilic SSF reactions shown in Fig. 12.
• thermophilic, anaerobic, Gram-positive bacteria in the conversion of biomass to ethanol.
• an organism is in "a native state" if it is has not been genetically engineered or otherwise manipulated by the hand of man in a manner that intentionally alters the genetic and/or phenotypic constitution of the organism. For example, wild-type organisms may be considered to be in a native state.
• Complete elimination of organic acid production from a T. saccharolyticum in a native state was achieved using two site-directed DNA homologous recombination events.
• the mutant strain, Thermoanaerobacterium saccharolyticum JW/SL-YS485 ALKl (“ALKl ”) produces near theoretical amounts of ethanol at low substrate feedings in batch culture with a temperature in a range of about 30-66 0 C and a pH in a range of about 3.85-6.5. In one embodiment, ethanol yield is at least about 90% of the theoretical maximum.
• ALKl, and its decendents have the potential to contribute significant savings in the lignocellulosic biomass to ethanol conversion due to its growth conditions, which are substantially optimal for cellulase activity in SSF and SSCF processes.
• optimal cellulase activity parameters include a pH between 4-5 and temperature between 40-50 0 C.
• thermophilic temperatures offer several important benefits over conventional mesophilic fermentation temperatures of 30-37 0 C. In particular, enzyme concentrations necessary to achieve a given amount of conversion may be reduced due to higher enzyme activity at thermophilic temperatures.
• thermophilic SSF and SSCF costs for a process step dedicated to cellulase production are substantially reduced (e.g., 2-fold or more) for thermophilic SSF and SSCF, and are eliminated for CBP. Costs associated with fermentor cooling and also heat exchange before and after fermentation are also expected to be reduced for thermophilic SSF, SSCF and CBP. Finally, processes featuring thermophilic biocatalysts may be less susceptible to microbial contamination as compared to processes featuring conventional mesophilic biocatalysts.
• pyruvate may be metabolized to acetyl-CoA, carbon dioxide, and reduced ferredoxin by the enzyme pyruvate-ferredoxin oxidoreductase 2.
• the electrons carried by reduced ferredoxin must all be transferred to NAD via NAD: ferredoxin oxidoreductase 3 to form NADH.
• NADH is subsequently oxidized back to NAD in the course of the two-step reduction of acetyl-CoA to ethanol by acetaldehyde dehydrogenase 7 and alcohol dehydrogenase 8.
• Evidence of the efficient utilization of NADH may be observed in Fig.
• Gram-positive bacteria such as members of the Thermoanaerober genus; Clostridium thermocellum and other thermophilic and mesophilic Clostridia; thermophilic and mesophilic Bacillus species; Gram-negative bacteria, such as Escherichia coli and Klebsiella oxytoca; Fibrobacter succinogenes and other Fibrobacter species; Thermoga neopolitana and other Thermotoga species; and anaerobic fungi including Neocallimatix and Piromyces species lack the ability to express PDC, and may benefit from the disclosed instrumentalities.
• lignocellulosic material that is saccharified to produce one or more of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan and starch may be utilized by the disclosed organisms.
• the lignocellulosic biomass comprises wood, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard, or combinations thereof.
• Thermoanaerobacterium saccharolyticum strain JW/SL-YS485 (DSM 8691) is a thermophilic, anaerobic bacteria isolated from the West Thumb Basin in Yellowstone National Park, Wyoming (Lui, S.Y; Gherardini, F. C; Matuschek, M.; Bahl, H.; Wiegel, J. "Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp strain JW/SL-YS485 in Escherichia coli" J. Bacterid.
• L-ldh lactate dehydrogenase
• pta phosphotransacetylase
• ack acetate kinase
• TOPO containing ⁇ cA:-down was digested with Xbal/Sphl and subcloned into pUC19 (Invitrogen).
• Xbal/AflIII fragment containing ⁇ c&-down was digested and subcloned downstream of the kanamycin marker to obtain the final construct pSGD9.
• Lactate dehydrogenase knockout vector with erythromycin resistance pSGD8-Erm
• the 5.5 kb suicide vector pSGD8-Erm was based on the plasmid pSGD8 as produced by Desai et al. 2004.
• a fusion gene based on the aph promoter from the plasmid pIKMl and the adenine methylase gene conferring erythromycin resistance from the plasmid pCTCl (Klapatch, T.R.; Guerinot, MX.; Lynd, L.R. "Electrotransformation of Clostridium thermosaccharolyticum” J. Ind. Microbiol. 16(6): 342-7, June 1996) were used for selection.
• PCR gene fragments were created using pfu polymerase (Stategene) and the primers SEQ ID NOS: 5-6 for the aph promoter and SEQ ID NOS: 7-8 for the adenine methylase open reading frame. Fragments were digested with Xbal/BamHl ⁇ aph fragment) and BamHI/EcoRI (adenine methylase) and ligated into the multiple cloning site of pIKMl . This fusion gene was then excised with BseRI/EcoRI and ligated into similarly digested pSGD8.
• Transformation of T. saccharolyticum was performed interchangeably with two methods, the first as previously described (Mai, V.; Lorenz, W.; Weigel, J. "Transformation of Thermoanaerobacte ⁇ um sp. strain JW/SL-YS485 with plasmid PIKMl conferring kanamycin resistance” FEMS Microbiol. Lett. 148: 163-167, 1997) and the second with several modifications following cell harvest and based on the method developed for Clostridium thermocellum (Tyurin, M. V.; Desai, S. G.; Lynd, L. R. "Electrotransformation of Clostridium thermocellum” Appl. Environ. Microbiol.
• Exponential phase cells were harvested and washed with pre-reduced cold sterile 20OmM cellobiose solution, and resuspended in the same solution and kept on ice. Extreme care was taken following the harvesting of cells to keep them cold (approximately 4°C) at all times including the time during centrifugation.
• Pulsed cells were initially diluted with 500 ⁇ l DSM 122 medium, held on ice for 10 minutes and then recovered at 55°C for 4-6 hrs. Following recovery, cells transformed with pSGD9 were mixed with 2% agar medium containing kanamycin at 75 ⁇ g/ml and poured onto petri plates and incubated in anaerobic jars for 4 days. Cells transformed with pSGD8-Erm were allowed to recover at 48°C for 4-6 hrs and were either plated in 2% agar medium at pH 6.0 containing erythromycin at 5 ⁇ g/ml or similar liquid media and incubated in anaerobic jars at 48°C for 6 days. Either of the transformed cell lines may be used without further manipulation.
• Genomic DNA from the mutant strain Thermoanaerobacterium saccharolyticum JW/SL-YS485 ALKl (“ALKl ”) showed the expected site-directed homologous recombination in the L-ldh and pta/ack loci through DNA sequencing. Both integration events were double integrations, which is a more genetically stable genotype.
• ALKl Thermoanaerobacterium saccharolyticum JW/SL-YS485 ALKl
• T. saccharolyticum was grown in partially defined MTC media containing 2.5 g/L Yeast Extract (Zhang, Y.; Lynd, L. R. "Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation: development of an enzyme-linked immunosorbent assay-based method with application to Clostridium thermocellum batch cultures" Anal. Chem. 75: 219-222, 2003).
• Glucose, xylose, acetate, lactate and ethanol were analyzed by HPLC on an Aminex 87H column (BioRad Laboratories, Hercules, CA) at 55°C.
• the mobile phase consisted of 5mM sulfuric acid at a flow rate of 0.7ml/min. Detection was via refractive index using a Waters 410 refractometer (Milford, MA). The minimum detection level for acetate was 1.0 mM.
• a standard trace containing 5 g/L xylose, 5 g/L lactic acid, 5 g/L acetic acid and 5 g/L ethanol is shown in Fig. 5.
• Strain ALKl produced only ethanol with up to 17 g/L xylose, or with 5 g/L xylose and 5 g/L glucose, with no organic acids or other products detected by HPLC. Fig.
• FIG. 6 shows the ALKl strain fermentation at time 0 hours and Fig. 7 shows the same fermentation at 72 hours.
• Time course fermentation plots of strain ALKl and wild-type on xylose media buffered with 8 g/L MES at an initial pH of 6.0, 55°C and 100 rpm show that strain ALKl is able to convert over 99% of xylose to ethanol (Fig. 8), while the wild-type under similar conditions becomes pH limited due to organic acid production and is unable to consume all the xylose present (Fig. 9).
• the wild-type organism yielded 0.15 mM ethanol, while ALKl yielded 0.46 mM ethanol.
• FIG. 10 a continuous culture in which feed substrate concentration was increased over time was utilized to challenge ALKl .
• Fig. 10 shows xylose, xylulose and ethanol concentrations during the continuous culture. After more than 1000 hours of exposure to this stress-evolution cycle, an improved strain, ALK2, was isolated from the fermentation broth. ALK2 was able to initiate growth at 50 g/L xylose in batch culture.
• Fig. 11 shows xylose, organic acid, optical density (OD) and ethanol concentrations during fermentation by strain ALK2.
• thermotolerant yeast strains have been tested for ethanol production via SSF at temperatures of 40-45°C, with reduced yield above these temperatures (Banat, I. M.; Nigam, P.; Singh, D.; Marchant, R.; McHaIe, A.P. "Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I - Yeasts in general.” World Journal of Microbiology and Biotechnology 14: 809-821, 1998).
• Patel et al. have demonstrated SSF at 50°C for the production of lactic acid using a Bacillus strain (Patel, M. A.; Ou, M. S.; Ingram, L. O.; Shanmugam, K. T.
• thermophilic organisms transformed according to the present disclosure have the potential to contribute significant savings in lignocellulosic biomass to ethanol conversion due to their growth conditions, which are substantially optimal for cellulase activity in SSF processes.
• ALKl and ALK2 are anaerobic thermophiles that can grow at 50°C and pH 5.0, while optimal cellulase activity parameters include a pH between 4-5 and temperature between 40-50 0 C.
• optimal cellulase activity parameters include a pH between 4-5 and temperature between 40-50 0 C.
• tSSF tSSF reaction performed on a 1.5 L scale in a batch reactor at 50 0 C
• ALK2 was used in conjunction with 4 FPU/g T. reesei cellulase (Genencor, Palo Alto, CA) to produce ethanol from the solid substrate Avicel (20 g/L, 50 g/L and 80 g/L). There were no soluble sugars measured after sixteen hours of fermentation and less than 0.5 g/L lactic acid were produced in any of the trials.
• Fig. 12 shows that 30 g/L ethanol were produced in 140 hours when the tSSF reaction was performed on 80 g/1 Avicel, with 25 of the 30g/L ethanol produced in the first 50 hours.
• Fig. 13 shows ethanol yields for the reactions illustrated in Fig.
• T. saccharolyticum can ferment both pentose and hexose sugars.
• the disclosed organisms can therefore be used in Simultaneous Saccharification and Co-Fermentation (SSCF) reactions, where an enzyme that converts hemicellulose into pentose sugars (e.g., xylase) may be utilized in combination with cellulase.
• SSCF Simultaneous Saccharification and Co-Fermentation
• ALKl has been deposited with the American Type Culture Collection, Manassas, VA 20110-2209. The deposit was made on November 1, 2005 and received Patent Deposit Designation Number PTA-7206. This deposit was made in compliance with the Budapest Treaty requirements that the duration of the deposit should be for thirty (30) years from the date of deposit or for five (5) years after the last request for the deposit at the depository or for the enforceable life of a U.S. Patent that matures from this application, whichever is longer. ALKl will be replenished should it become non-viable at the depository.
• Gram-positive bacterium and especially members of the Thermoanaerobacter genus including Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium xylanolyticum.

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