US20100015678A1 - Thermophilic Organisms For Conversion of Lignocellulosic Biomass To Ethanol - Google Patents

Thermophilic Organisms For Conversion of Lignocellulosic Biomass To Ethanol Download PDF

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US20100015678A1
US20100015678A1 US12/090,745 US9074506A US2010015678A1 US 20100015678 A1 US20100015678 A1 US 20100015678A1 US 9074506 A US9074506 A US 9074506A US 2010015678 A1 US2010015678 A1 US 2010015678A1
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gram
positive bacterium
ethanol
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bacterium
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Arthur Josephus Shaw, IV
Sunil G. Desai
Lee R. Lynd
Mikhail V. Tyurin
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N9/0004—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
    • 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.)
    • C12N9/12—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.
  • ⁇ -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 hemicellulose 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 (“ldh”), or to acetyl coenzyme A (“acetyl-CoA”) by the action of pyruvate dehydrogenase or pyruvate-ferredoxin oxidoreductase.
  • Acetyl-CoA is further converted to acetate by phosphotransacetylase and acetate kinase, or reduced to ethanol by acetaldehyde dehydrogenase (“AcDH”) and alcohol dehydrogenase (“adh”).
  • AcDH acetaldehyde dehydrogenase
  • adh alcohol dehydrogenase
  • the performance of ethanol-producing organisms is compromised by production of organic products other than ethanol, and particularly by ldh-mediated conversion of pyruvate to lactate, and by conversion of acetyl-CoA to acetate by phosphotransacetylase and acetate kinase.
  • 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.
  • ldh lactate dehydrogenase
  • ack acetate kinase
  • pta phosphotransacetylase
  • Thermoanaerobacter genus including Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium xylanolyticum.
  • 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 Thermoanaerobacterium 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 ALK1 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).
  • 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 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.
  • 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 ALK1.
  • FIG. 8 shows xylose, organic acid and ethanol concentrations during fermentation by strain ALK1.
  • 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 ALK1.
  • FIG. 11 shows xylose, organic acid and ethanol concentrations during fermentation by strain ALK2.
  • 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.
  • wild-type organisms may be considered to be in a native state.
  • ALK1 Thermoanaerobacterium saccharolyticum JW/SL-YS485 ALK1
  • ALK1 produces near theoretical amounts of ethanol at low substrate feedings in batch culture with a temperature in a range of about 30-66° C. and a pH in a range of about 3.85-6.5.
  • ethanol yield is at least about 90% of the theoretical maximum.
  • ALK1, 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 a simultaneous saccharification and co-fermentation (SSCF) process.
  • optimal cellulase activity parameters include a pH between 4-5 and temperature between 40-50° C. Additionally, it is unnecessary to adjust the pH of the fermentation broth when knockout organisms, which lack the ability to produce organic acids, are used.
  • ALK1, and similar organisms may also be suitable for a consolidated bioprocessing co-culture fermentation where the knockout organism would convert pentoses to ethanol, and cellulose would be degraded by a cellulolytic organism such as C. thermocellum.
  • thermophilic temperatures offer several important benefits over conventional mesophilic fermentation temperatures of 30-37° C.
  • costs for a process step dedicated to cellulase production are substantially reduced (e.g., 2-fold or more) for thermophilic 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 both thermophilic SSCF and CBP.
  • 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.
  • the lignocellulosic material may be any feedstock that contains one or more of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan and starch.
  • 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, Wyo. (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. Bacteriol. 178: 1539-1547, 1996; Mai, V.; Wiegel, J.
  • L-ldh lactate dehydrogenase
  • pta phosphotransacetylase
  • ack acetate kinase
  • the 6.2 kb suicide vector pSGD9 was based on pBLUESCRIPT II SK (+) (Stratagene) using a design approach similar to that reported earlier (Desai, 2004; Mai, 2000).
  • Gene fragments of the pta/ack sequence, pta-up ( ⁇ 1.2 kb) and ack-down ( ⁇ 0.6 kb), were amplified from genomic DNA using primer pairs SEQ ID NOS: 1-2, and SEQ ID NOS: 3-4.
  • PCR amplification was performed with pfu DNA polymerase and the fragments were extracted from a 1% electrophoresis gel.
  • Fragments pta-up and ack-down were then A-tailed with Taq polymerase and cloned into TOPO pCR2.1 (Invitrogen, Carlsbad, Calif.).
  • a 1.5 kb fragment containing the kanamycin marker was obtained from a PstI/XbaI digest of pIKM1 and subcloned into pBLUESCRIPT II SK (+).
  • TOPO containing pta-up was digested with XhoI/BsiHK4I and subcloned into XhoI/PstI digested pBLUESCRIPT II SK (+), upstream of the previously subcloned kanamycin marker.
  • TOPO containing ack-down was digested with XbaI/SphI and subcloned into pUC19 (Invitrogen).
  • XbaI/A ⁇ III fragment containing ack-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 pIKM1 and the adenine methylase gene conferring erythromycin resistance from the plasmid pCTC1 (Klapatch, T. R.; Guerinot, M. L.; 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 XbaI/BamH1 (aph fragment) and BamHI/EcoRI (adenine methylase) and ligated into the multiple cloning site of pIKM1. 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 Thermoanaerobacterium sp. strain JW/SL-YS485 with plasmid PIKM1 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. 70(2): 883-890, 2004).
  • Cells were grown overnight using pre-reduced medium DSMZ 122 in sterile disposable culture tubes inside an anaerobic chamber in an incubator maintained at 55° C. Thereafter, cells were sub-cultured with 4 ⁇ g/ml isonicotonic acid hydrazide (isoniacin), a cell wall weakening agent (Hermans, J.; Boschloo, J. G.; de Bont, J. A. M. “Transformation of M. aurum by electroporation: the use of glycine, lysozyme and isonicotinic acid hydrazide in enhancing transformation efficiency” FEMS Microbiol. Lett. 72, 221-224, 1990), added to the medium after the initial lag phase.
  • isonicotonic acid hydrazide isoniacin
  • a cell wall weakening agent Hermans, J.; Boschloo, J. G.; de Bont, J. A. M. “Transformation of M. aurum by electroporation:
  • Exponential phase cells were harvested and washed with pre-reduced cold sterile 200 mM 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.
  • a voltage threshold corresponding to the formation of electropores in a cell sample was evaluated as a non-linear current change when pulse voltage was linearly increased in 200V increments.
  • a particular voltage that provided the best ratio of transformation yield versus cell viability rate at a given DNA concentration was used, which in this particular case was 25 kV/cm.
  • 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.
  • an organism where elimination of expression of all genes that confer the ability to produce organic acids was obtained by performing a second (sequential) transformation.
  • the second transformation was carried out as described above with the primary transformant substituted for the non-transformed cell suspension.
  • the secondary transformant, ALK1 was grown on medium containing both kanamycin and erythromycin.
  • Sequencing of the site directed knockout regions was done by PCR from genomic DNA using Taq polymerase (New England Biolabs) and primers outside the regions of homologous overlap between the genome and the suicide vectors. Primers inside the PCR products were used for sequencing with the BigDye Terminator kit v3.1 (ABI, Foster City, CA). Regions were arranged using the CAP3 software program (Huang, X. “An improved sequence assembly program” Genomics 33: 21-31, 1996) and compared to the expected DNA sequence using the CLUSTAL W algorithm (Higgins, D. G.; Bleasby, A. J.; Fuchs, R.
  • Identity refers to a comparison between pairs of nucleic acid or amino acid molecules. Methods for determining sequence identity are known. See, for example, computer programs commonly employed for this purpose, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482-489.
  • ALK1 Thermoanaerobacterium saccharolyticum JW/SL-YS485 ALK1
  • 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 Clostiridium 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, Calif.) at 55° C.
  • the mobile phase consisted of 5 mM sulfuric acid at a flow rate of 0.7 ml/min.
  • Detection was via refractive index using a Waters 410 refractometer (Milford, Mass.). 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 ALK1 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. 6 shows the ALK1 strain fermentation at time 0 hours and
  • FIG. 7 shows the same fermentation at 72 hours.
  • Time course fermentation plots of strain ALK1 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 ALK1 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 ALK1 yielded 0.46 mM ethanol.
  • FIG. 10 a continuous culture in which feed substrate concentration was increased over time was utilized to challenge ALK1.
  • 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.
  • ALK1 has been deposited with the American Type Culture Collection, Manassas, Va. 20110-2209. The deposit was made on Nov. 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. ALK1 will be replenished should it become non-viable at the depository.
  • the foregoing examples may be suitably modified for use upon any Gram-positive bacterium, and especially members of the Thermoanaerobacter genus including Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermanaerobacterium polysaccharolyticwum, Thermoanaerobacterium zeae, Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium xylanolyticum.
  • members of the Thermoanaerobacter genus including Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermanaerobacterium polysaccharolyticwum, Thermoanaerobacterium zeae, Thermoanaerobacterium thermosaccharolyticum, and Thermoan

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102433293A (zh) * 2011-12-29 2012-05-02 华南理工大学 一种可利用木糖发酵生产光学纯l-乳酸的工程菌及其构建

Families Citing this family (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0618074A2 (pt) * 2005-10-31 2011-08-16 Dartmouth College organismos termofìlicos para conversão de biomassa lignocelulósica em etanol
US8969033B2 (en) * 2005-11-02 2015-03-03 Battelle Energy Alliance, Llc Alteration and modulation of protein activity by varying post-translational modification
US9234228B2 (en) 2008-02-27 2016-01-12 Battelle Energy Alliance, Llc Thermophilic and thermoacidophilic glycosylation genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods
CA2640429C (en) * 2006-01-27 2014-04-01 University Of Massachusetts Systems and methods for producing biofuels and related materials
BRPI0712490A2 (pt) * 2006-05-22 2012-10-09 Biogasol Ipr Aps cepa bg1 de trehmoanaerobacter mathranii
US9121040B2 (en) 2006-12-18 2015-09-01 University Of Maryland Process for rapid anaerobic digestion of biomass using microbes and the production of biofuels therefrom
KR20100037031A (ko) * 2007-05-09 2010-04-08 마스코마 코포레이션 유전자 녹아웃 중온성 및 호열성 생물체, 및 이의 사용 방법
WO2009079584A1 (en) * 2007-12-17 2009-06-25 The Trustees Of Dartmouth College Modification of hydrogenase activities in thermophilic bacteria to enhance ethanol production
NZ585947A (en) * 2008-01-25 2012-01-12 Batttelle Energy Alliance Llc Thermal and acid tolerant beta-xylosidases, genes encoding, related organisms, and methods
US8492114B2 (en) * 2008-01-25 2013-07-23 Battelle Energy Alliance, Llc Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes
US9732330B2 (en) 2008-01-25 2017-08-15 Battelle Energy Alliance, Llc Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes
CN101932702A (zh) 2008-01-31 2010-12-29 巴特勒能源同盟有限公司 来自酸热脂环酸杆菌和相关生物体的耐热和耐热耐酸的生物聚合体降解基因和酶，方法
US8426185B2 (en) * 2008-01-31 2013-04-23 Battelle Energy Alliance, Llc Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods
US8557557B2 (en) * 2008-01-31 2013-10-15 Battelle Energy Alliance, Llc Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods
US8497110B2 (en) * 2008-01-31 2013-07-30 Battelle Energy Alliance, Llc Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods
BRPI0907407A2 (pt) 2008-02-22 2019-09-24 Battelle Energy Alliance Llc controle transcricional em alicyclobacillus acidocaldarius e genes, proteínas e métodos associados
MX2010008246A (es) 2008-02-26 2010-11-12 Battelle Energy Alliance Llc Genes transportadores de azucar termofilicos y termoacidofilicos y enzimas de alicyclobacillus acidocaldarius y organismos relacionados, metodos.
CN101981199A (zh) * 2008-02-27 2011-02-23 奎特罗斯公司 通过两种微生物的顺序作用将植物材料转化为燃料和化学产品的方法
EP2245143A4 (en) 2008-02-28 2013-07-31 Battelle Energy Alliance Llc THERMOPHILIC AND THERMOACIDOPHILIC ACID CHALK AND ENZYMES FROM ALICYCLOBACILLUS ACIDOCALDARIUS AND CORRESPONDING ORGANISMS AND METHODS
US8268600B2 (en) 2008-03-05 2012-09-18 Council Of Scientific & Industrial Research Strain and a novel process for ethanol production from lignocellulosic biomass at high temperature
WO2009152362A2 (en) * 2008-06-11 2009-12-17 University Of Massachusetts Methods and compositions for regulating sporulation
JP2011528565A (ja) 2008-07-24 2011-11-24 バイオガソル・イーペーエア・アンパルトセルスカブ 組換え細菌における上昇したエタノール産生
CA2732078A1 (en) 2008-07-28 2010-02-04 University Of Massachusetts Methods and compositions for improving the production of products in microorganisms
US9193979B2 (en) 2008-11-11 2015-11-24 Richard Allen Kohn Process for producing lower alkyl alcohols from cellulosic biomass using microorganisms
CA2743505A1 (en) * 2008-11-12 2010-05-20 Mascoma Corporation Gene knockout mesophilic and thermophilic organisms, and methods of use thereof
CA2748135A1 (en) 2008-12-23 2010-07-01 Mascoma Corporation Heterologous biomass degrading enzyme expression in thermoanaerobacterium saccharolyticum
US20100086981A1 (en) * 2009-06-29 2010-04-08 Qteros, Inc. Compositions and methods for improved saccharification of biomass
CA2759726A1 (en) * 2009-04-20 2010-10-28 Qteros, Inc. Compositions and methods for fermentation of biomass
CA2783533A1 (en) * 2009-12-07 2011-06-16 Mascoma Corporation Heterologous expression of urease in anaerobic, thermophilic hosts
CN102770526A (zh) * 2009-12-22 2012-11-07 比奥咖索尔Ipr有限公司 具有高醇产率的嗜热性意大利热厌氧杆菌marato亚种
GB2478791A (en) * 2010-03-19 2011-09-21 Qteros Inc Ethanol production by genetically-modified bacteria
US20110275135A1 (en) 2010-05-05 2011-11-10 Battelle Energy Alliance, Llc Genetic elements, proteins, and associated methods including application of additional genetic information to gram (+) thermoacidophiles
CN103201395B (zh) 2010-06-26 2016-03-02 威尔迪亚有限公司 糖混合物及其生产和使用方法
IL207945A0 (en) 2010-09-02 2010-12-30 Robert Jansen Method for the production of carbohydrates
WO2012109578A2 (en) * 2011-02-11 2012-08-16 The Trustees Of Dartmouth College Clostridium thermocellum strains for enhanced ethanol production and method of their use
US9512495B2 (en) 2011-04-07 2016-12-06 Virdia, Inc. Lignocellulose conversion processes and products
WO2013055785A1 (en) 2011-10-10 2013-04-18 Virdia Ltd Sugar compositions
CA3163204A1 (en) 2012-05-03 2013-11-07 Virdia, Llc Methods for treating lignocellulosic materials
GB201215505D0 (en) 2012-08-31 2012-10-17 C5 Labs Aps Process for the production of ethanol
CN105358608B (zh) 2013-05-03 2018-11-16 威尔迪亚公司 用于制备热稳定的木质素级分的方法
CN112226466A (zh) 2015-01-07 2021-01-15 威尔迪亚公司 萃取和转化半纤维素糖的方法
CN114717174B (zh) * 2022-04-26 2023-10-20 苏州聚维元创生物科技有限公司 一种产高品质还原糖的工程菌株、构建方法及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182199A (en) * 1987-05-27 1993-01-26 Hartley Brian S Thermophilic ethanol production in a two-stage closed system
CA2424890C (en) * 2000-10-06 2014-06-03 Elsworth Biotechnology Limited Ethanol production in gram-positive bacteria with a stabilized mutation in lactate dehydrogenase
CA2491145A1 (en) * 2002-07-03 2004-01-15 Basf Aktiengesellschaft Microorganisms and processes for enhanced production of pantothenate
FI118012B (fi) * 2004-06-04 2007-05-31 Valtion Teknillinen Menetelmä etanolin valmistamiseksi
BRPI0618074A2 (pt) * 2005-10-31 2011-08-16 Dartmouth College organismos termofìlicos para conversão de biomassa lignocelulósica em etanol

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102433293A (zh) * 2011-12-29 2012-05-02 华南理工大学 一种可利用木糖发酵生产光学纯l-乳酸的工程菌及其构建

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