Isolation and characterization of a new CO-utilizing strain, Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans, isolated from a geothermal spring in Turkey
TL;DR: Strain TLO has the capability to ferment a wide variety of mono-, di-, and polysaccharides and proteinaceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H2, and CO2 and was able to grow in an atmosphere of up to 25% of CO as sole electron donor.
Abstract: A novel anaerobic, thermophilic, Gram-positive, spore-forming, and sugar-fermenting bacterium (strain TLO) was isolated from a geothermal spring in Ayas, Turkey. The cells were straight to curved rods, 0.4–0.6 μm in diameter and 3.5–10 μm in length. Spores were terminal and round. The temperature range for growth was 40–80°C, with an optimum at 70°C. The pH optimum was between 6.3 and 6.8. Strain TLO has the capability to ferment a wide variety of mono-, di-, and polysaccharides and proteinaceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H2, and CO2. Remarkably, the bacterium was able to grow in an atmosphere of up to 25% of CO as sole electron donor. CO oxidation was coupled to H2 and CO2 formation. The G + C content of the genomic DNA was 35.1 mol%. Based on 16S rRNA gene sequence analysis and the DNA–DNA hybridization data, this bacterium is most closely related to Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacter siderophilus (99% similarity for both). However, strain TLO differs from Thermoanaerobacter thermohydrosulfuricus in important aspects, such as CO-utilization and lipid composition. These differences led us to propose that strain TLO represents a subspecies of Thermoanaerobacter thermohydrosulfuricus, and we therefore name it Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans.
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TL;DR: Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied.
Abstract: The distribution of membrane lipids of 17 different strains representing 13 species of subdivisions 1 and 3 of the phylum Acidobacteria, a highly diverse phylum of the Bacteria, were examined by hydrolysis and gas chromatography-mass spectrometry (MS) and by high-performance liquid chromatography-MS of intact polar lipids. Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied. This lipid has previously been encountered only in thermophilic Thermoanaerobacter species but bears a structural resemblance to the alkyl chains of bacterial glycerol dialkyl glycerol tetraethers (GDGTs) that occur ubiquitously in peat and soil and are suspected to be produced by acidobacteria. As reported previously, most species also contained iso-C15 and C16:1ω7C as major fatty acids but the presence of iso-diabolic acid was unnoticed in previous studies, most probably because the complex lipid that contained this moiety was not extractable from the cells; it could only be released by hydrolysis. Direct analysis of intact polar lipids in the Bligh-Dyer extract of three acidobacterial strains, indeed, did not reveal any membrane-spanning lipids containing iso-diabolic acid. In 3 of the 17 strains, ether-bound iso-diabolic acid was detected after hydrolysis of the cells, including one branched GDGT containing iso-diabolic acid-derived alkyl chains. Since the GDGT distribution in soils is much more complex, branched GDGTs in soil likely also originate from other (acido)bacteria capable of biosynthesizing these components.
360 citations
Cites background from "Isolation and characterization of a..."
...thermohydrosulfuricus (3), iso-diabolic acid was also detected only after hydrolysis of total cell material....
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...iso-Diabolic acid has previously been encountered only in thermophilic Thermoanaerobacter species (3, 24, 32), where it fulfills a role as a membranespanning lipid....
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TL;DR: It is demonstrated that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria.
Abstract: The cytoplasmic membrane of a prokaryotic cell consists of a lipid bilayer or a monolayer that shields the cellular content from the environment. In addition, the membrane contains proteins that are responsible for transport of proteins and metabolites as well as for signalling and energy transduction. Maintenance of the functionality of the membrane during changing environmental conditions relies on the cell’s potential to rapidly adjust the lipid composition of its membrane. Despite the fundamental chemical differences between bacterial ester lipids and archaeal ether lipids, both types are functional under a wide range of environmental conditions. We here provide an overview of archaeal and bacterial strategies of changing the lipid compositions of their membranes. Some molecular adjustments are unique for archaea or bacteria, whereas others are shared between the two domains. Strikingly, shared adjustments were predominantly observed near the growth boundaries of bacteria. Here, we demonstrate that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria.
249 citations
TL;DR: Three main types of fermentative CO metabolism can be distinguished: hydrogenogenesis, methanogenesis, and acetogenesis, generating hydrogen, methane and acetate, respectively, with emphasis on the potential enzymatic routes and bio-energetics involved.
Abstract: Carbon monoxide can act as a substrate for different modes of fermentative anaerobic metabolism. The trait of utilizing CO is spread among a diverse group of microorganisms, including members of bacteria as well as archaea. Over the last decade this metabolism has gained interest due to the potential of converting CO-rich gas, such as synthesis gas, into bio-based products. Three main types of fermentative CO metabolism can be distinguished: hydrogenogenesis, methanogenesis, and acetogenesis, generating hydrogen, methane and acetate, respectively. Here, we review the current knowledge on these three variants of microbial CO metabolism with an emphasis on the potential enzymatic routes and bio-energetics involved.
131 citations
Cites background from "Isolation and characterization of a..."
...No growth Kluyver and Schnellen, 1947 Methanosaeta concillii Aceticlastic Enzyme assay N.D. N.D....
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...Methanothermobacter thermoautotrophicus Hydrogenotrophic Cultivation/enzyme assay 50 kPa Methane, hydrogen ∼200 Daniels et al., 1977; Wasserfallen et al., 2000 Methanosarcina thermophila Aceticlastic Cultivation >2 kPa Hydrogen, Methane N.D. Zinder and Anguish, 1992 Methanothrix sp. Strain CALS-1 Aceticlastic Cultivation <2 kPa Methane No growth Zinder and Anguish, 1992 Archaeoglobus fulgidusB Sulfate reducer Cultivation >136 kPa Acetate, formate ∼10 Henstra et al., 2007a Not determined parameters are marked N.D. AForward arrows (>) indicate inhibitory levels have not been reached; numbers displayed are the maximal level tested....
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...Moorella thermoautotrophica EcH/ Cytochromes (H+) 7 (UM), 9 (DM) 33 (DM) 3.34 (UM) 2.53 (DM) 0.82 (DM) Savage and Drake, 1986; Savage et al., 1987; Collins et al., 1994 Moorella thermoacetica EcH/ Cytochromes (H+) 9 (DM) 16 (DM) 1.28 (UM) 0.46 (UM) Daniel et al., 1990; Collins et al., 1994; Pierce et al., 2008 Acetobacterium Woodii RnF (Na+ ) 13D (UM), 5.5A (UM + formate) 6.2A (UM) N.D. 1.05C (DM) Balch et al., 1977; Tschech and Pfennig, 1984; Genthner and Bryant, 1987; Hess et al., 2013; Bertsch and Müller, 2015 Blautia producta RnF (Na+ ) 1.5 (UM) 5 (UM) 2.13C (UM) 0.65C (UM) Lorowitz and Bryant, 1984; Geerligs et al., 1989 Clostridium aceticum RnF (N.D.) ∼10B (UM) 20 (UM) N.D. N.D. Braun et al., 1981; Sim et al., 2007...
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...…2007 Thermosinus carboxydivorans Hot spring, Norris Basin 60 1.15 Sokolova et al., 2004a Thermoanaerobacter thermohydrosulfuricus ssp. carboxydovorans Geothermal spring, Turkey 70 N.D. Balk et al., 2009 Desulfotomaculum carboxydivorans Paper mill wastewater sludge 55 N.D. Parshina et al., 2005b...
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...Methanobrevibacter arboriphilicus Hydrogenotrophic Enzyme assay N.D. N.D. N.D. Hammel et al., 1984 Methanosarcina acetivorans C2A Aceticlastic Cultivation/enzyme assay >150 kPa Methane, acetate, formate ∼20 Rother and Metcalf, 2004; Oelgeschläger and Rother, 2009 Methanosarcina barkeri Aceticlastic Cultivation/enzyme assay >100 kPa Hydrogen, Methane ∼65 O’Brien et al., 1984; Bott et al., 1986 Methanobacterium formicicum Hydrogenotrophic Cultivation N.D. N.D....
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TL;DR: Examination of lipid composition of seven phylogenetically divergent strains of subdivision 4 of the Acidobacteria, a bacterial group that is commonly encountered in soil, found the presence of ether bonds in the membrane lipids does not seem to be an adaptation to temperature, because the five mesophilic isolates contained a larger amount of ether lipids than the thermophile “Ca. Chloracidobacterium thermophilum.”
Abstract: Recently, iso-diabolic acid (13,16-dimethyl octacosanedioic acid) has been identified as a major membrane-spanning lipid of subdivisions 1 and 3 of the Acidobacteria, a highly diverse phylum within the Bacteria. This finding pointed to the Acidobacteria as a potential source for the bacterial glycerol dialkyl glycerol tetraethers that occur ubiquitously in peat, soil, lakes, and hot springs. Here, we examined the lipid composition of seven phylogenetically divergent strains of subdivision 4 of the Acidobacteria, a bacterial group that is commonly encountered in soil. Acid hydrolysis of total cell material released iso-diabolic acid derivatives in substantial quantities (11 to 48% of all fatty acids). In contrast to subdivisions 1 and 3 of the Acidobacteria, 6 out of the 7 species of subdivision 4 (excepting “Candidatus Chloracidobacterium thermophilum”) contained iso-diabolic acid ether bound to a glycerol in larger fractional abundance than iso-diabolic acid itself. This is in agreement with the analysis of intact polar lipids (IPLs) by high-performance liquid chromatography-mass spectrometry (HPLC-MS), which showed the dominance of mixed ether-ester glycerides. iso-Diabolic acid-containing IPLs were not identified, because these IPLs are not released with a Bligh-Dyer extraction, as observed before when studying lipid compositions of subdivisions 1 and 3 of the Acidobacteria. The presence of ether bonds in the membrane lipids does not seem to be an adaptation to temperature, because the five mesophilic isolates contained a larger amount of ether lipids than the thermophile “Ca. Chloracidobacterium thermophilum.” Furthermore, experiments with Pyrinomonas methylaliphatogenes did not reveal a major influence of growth temperature over the 50 to 69°C range.
102 citations
Cites background from "Isolation and characterization of a..."
...(61), Sarcina ventriculi (62), members of the 304 Thermotogales (2,57,63-65), Thermoanaerobacter species (58,59,62) and Acidobacteria of 305 SD 1, 3 (14), and 4 (this work), respectively....
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...This lipid was identified 254 previously as an abundant lipid in Acidobacteria of SD 1 and 3 (14) and in thermophilic 255 Thermoanaerobacter species (58-60), in which they fulfill a role as membrane-spanning 256 lipids....
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TL;DR: Recent advances in the isolation of novel phylogenetic groups utilizing formate or CO, the remarkable genetic engineering that enhances H2 productivity, and the practical implementation of H2 production from C1 substrates are focused on.
63 citations
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TL;DR: Novel thermophilic, anaerobic, Gram-positive, rod-shaped bacteria, strains SL9 and OCA1, were isolated from oilfields in France and Australia, respectively and it is proposed to reassign them as a novel genus and species, Caldanaerobacter subterraneus gen. nov., with the creation of four novel subspecies.
Abstract: Novel thermophilic, anaerobic, Gram-positive, rod-shaped bacteria, strains SL9 and OCA1, were isolated from oilfields in France and Australia, respectively. Both strains, together with Thermoanaerobacter yonseiensis KB-1(T) (=DSM 13777(T)), Thermoanaerobacter tengcongensis MB4(T) (=DSM 15242(T)) and Carboxydibrachium pacificum JM(T) (=DSM 12653(T)), possessed genomic (DNA-DNA hybridization studies) and phylogenetic similarities with Thermoanaerobacter subterraneus SEBR 7858(T) (=DSM 13054(T)), which was isolated recently from an oilfield reservoir in south-west France. Marked phenotypic differences exist between the three oilfield isolates (T. subterraneus, strain OCA1 and strain SL9): they include temperature range for growth and substrates used. Differences were also observed in the DNA G+C contents of all organisms. Similarly to T. subterraneus, strains SL9 and OCA1, and also T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum, produced acetate and L-alanine as major end products of glucose metabolism [0.8-1.0 mol L-alanine produced (mol glucose consumed)(-1)] and reduced thiosulfate, but not sulfate, to sulfide. Because of these significant metabolic and phylogenetic differences between the oilfield isolates (T. subterraneus, strain OCA1 and strain SL9), T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum and other Thermoanaerobacter species, it is proposed to reassign them as a novel genus and species, Caldanaerobacter subterraneus gen. nov., sp. nov., comb. nov., with the creation of four novel subspecies, Caldanaerobacter subterraneus subsp. subterraneus subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. yonseiensis subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. tengcongensis subsp. nov., comb. nov. and Caldanaerobacter subterraneus subsp. pacificus subsp. nov., comb. nov.
118 citations
"Isolation and characterization of a..." refers background in this paper
...Utilization of CO has also been demonstrated for several representatives of a subdivision of the Thermoanaerobacter genus (Group 3) (Subbotina et al. 2003), Feature Strain TLO T. thermohydrosulfuricus T. siderophilus Source Geothermal spring Extraction juices of beet sugar factory Hydrothermal vent which was recently reassigned to the genus Caldanaerobacter (Fardeau et al. 2004)....
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...…of the Thermoanaerobacter genus (Group 3) (Subbotina et al. 2003), Feature Strain TLO T. thermohydrosulfuricus T. siderophilus Source Geothermal spring Extraction juices of beet sugar factory Hydrothermal vent which was recently reassigned to the genus Caldanaerobacter (Fardeau et al. 2004)....
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...For example, Caldanaerobacter subterraneus subsp. tengcongensis and C. subterraneus subsp. pacificius are able to use CO....
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TL;DR: Alanine formation was shown to occur via an alanine aminotransferase, which exhibited a specific activity in cell-free extract of up to 6.0 U/mg (90°C; direction of pyruvate formation), enabling P. furiosus to adjust its fermentation, depending on the redox potential of the terminal electron acceptor.
Abstract: The hyperthermophilic archaeon Pyrococcus furiosus was found to form substantial amounts of l-alanine during batch growth on either cellobiose, maltose or pyruvate. Acetate, CO2 and H2 were produced next to alanine. The carbon- and electron balances were complete for all three substrates. Under standard growth conditions (N2/CO2 atmosphere) an alanine/acetate ratio of about 0.3 was found for either substrate. The alanine /acetate ratio was influenced, however, by the hydrogen partial pressure. In the presence of S0 or in coculture with Methanococcus jannaschii this ratio was only 0.07, whereas under a H2/CO2 atmosphere this ratio could amount up to 0.8. Alanine formation was also aflected by the NHinf4sup+concentration, i.e. below 4 mM, NHinf4sup+becomes limiting to alanine formation. Alanine formation was shown to occur via an alanine aminotransferase, which exhibited a specific activity in cell-free extract of up to 6.0 U/mg (90°C; direction of pyruvate formation). The alanine aminotransferase probably cooperates with glutamate dehydrogenase (up to 23 U/mg; 90°C) and ferredoxin: NADP+ oxidoreductase (up to 0.7 U/mg, using methyl viologen; 90°C) to recycle the electron acceptors involved in catabolism. Thus, the existence of this unusual alanine-forming branch enables P. furiosus to adjust its fermentation, depending on the redox potential of the terminal electron acceptor.
115 citations
Additional excerpts
...…and sugar cane extraction juices (Klaushofer and Parkkinen 1965; Wiegel and Ljungdahl 1981; Schmid et al. 1986; Cayol et al. 1995; Cook et al. 1996; Kozianowski et al. 1997; Larsen et al. 1997; Slobodkin et al. 1999; Fardeau et al. 2000; Kim et al. 2001; Onyenwoke et al. 2007; Wagner et al. 2008)....
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TL;DR: A new anaerobic, thermophilic, facultatively carboxydotrophic bacterium, strain Nor1(T), was isolated from a hot spring at Norris Basin, Yellowstone National Park and is proposed to be assigned to a new genus, Thermosinus gen. nov.
Abstract: A new anaerobic, thermophilic, facultatively carboxydotrophic bacterium, strain Nor1(T), was isolated from a hot spring at Norris Basin, Yellowstone National Park. Cells of strain Nor1(T) were curved motile rods with a length of 2.6-3 microm, a width of about 0.5 microm and lateral flagellation. The cell wall structure was of the Gram-negative type. Strain Nor1(T) was thermophilic (temperature range for growth was 40-68 degrees C, with an optimum at 60 degrees C) and neutrophilic (pH range for growth was 6.5-7.6, with an optimum at 6.8-7.0). It grew chemolithotrophically on CO (generation time, 1.15 h), producing equimolar quantities of H(2) and CO(2) according to the equation CO+H(2)O-->CO(2)+H(2). During growth on CO in the presence of ferric citrate or amorphous ferric iron oxide, strain Nor1(T) reduced ferric iron but produced H(2) and CO(2) at a ratio close to 1 : 1, and growth stimulation was slight. Growth on CO in the presence of sodium selenite was accompanied by precipitation of elemental selenium. Elemental sulfur, thiosulfate, sulfate and nitrate did not stimulate growth of strain Nor1(T) on CO and none of these chemicals was reduced. Strain Nor1(T) was able to grow on glucose, sucrose, lactose, arabinose, maltose, fructose, xylose and pyruvate, but not on cellobiose, galactose, peptone, yeast extract, lactate, acetate, formate, ethanol, methanol or sodium citrate. During glucose fermentation, acetate, H(2) and CO(2) were produced. Thiosulfate was found to enhance the growth rate and cell yield of strain Nor1(T) when it was grown on glucose, sucrose or lactose; in this case, acetate, H(2)S and CO(2) were produced. In the presence of thiosulfate or ferric iron, strain Nor1(T) was also able to grow on yeast extract. Lactate, acetate, formate and H(2) were not utilized either in the absence or in the presence of ferric iron, thiosulfate, sulfate, sulfite, elemental sulfur or nitrate. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. The DNA G+C content of the strain was 51.7+/-1 mol%. Analysis of the 16S rRNA gene sequence revealed that strain Nor1(T) belongs to the Bacillus-Clostridium phylum of the Gram-positive bacteria. On the basis of the studied phenotypic and phylogenetic features, we propose that strain Nor1(T) be assigned to a new genus, Thermosinus gen. nov. The type species is Thermosinus carboxydivorans sp. nov. (type strain, Nor1(T)=DSM 14886(T)=VKM B-2281(T)).
111 citations
Additional excerpts
...CO2 (Svetlichnyi et al. 1994, 2001; Sokolova et al. 2001, 2002, 2004, 2005, 2007; Slepova et al. 2006)....
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TL;DR: A novel anaerobic, thermophilic, CO-utilizing bacterium, strain 41(T), was isolated from a terrestrial hot vent on the Kamchatka Peninsula and proposed that this isolate represents a new genus and species, Carboxydocella thermautotrophica gen. nov.
Abstract: A novel anaerobic, thermophilic, CO-utilizing bacterium, strain 41(T), was isolated from a terrestrial hot vent on the Kamchatka Peninsula. Strain 41(T) was found to be a Gram-positive bacterium, its cells being short, straight, motile rods. Chains of three to five cells were often observed. The isolate grew only chemolithoautotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O --> CO2+H2). Growth was observed in the temperature range 40-68 degrees C, with an optimum at 58 degrees C, and in the pH range 6.5-7.6, with an optimum at pH 7.0. The generation time under optimal conditions for chemolithotrophic growth was 1.1 h. The DNA G+C content was 46 +/- 1 mol%. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. On the basis of the phenotypic and phylogenetic features, it is proposed that this isolate represents a new genus and species, Carboxydocella thermautotrophica gen. nov., sp. nov. (type strain 41(T) = DSM 12356(T) = VKM B-2282(T)).
93 citations
Additional excerpts
...CO2 (Svetlichnyi et al. 1994, 2001; Sokolova et al. 2001, 2002, 2004, 2005, 2007; Slepova et al. 2006)....
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TL;DR: The results of 16S rRNA sequence comparisons indicated that strain SEBR 5268T was closely related to Thermoanaerobacter brockii and Thermoanobacter finnii, and these three organisms exhibited levels of ribosomal DNA sequence homology of 98 to 99%.
Abstract: A strictly anaerobic, thermophilic, gram-positive, spore-forming eubacterium designated strain SEBR 5268T (T = type strain) was isolated from an oil field at a depth of 2,100 m, where the temperature was 92°C. The cells of this organism were gram-positive, straight, motile rods (0.5 by 2 to 3 μm) with peritrichous flagella. The cells occurred singly or in pairs during the logarithmic growth phase, but were pleomorphic and filamentous (length, 15 μm) in old cultures. Growth occurred at temperatures of 40 to 75°C, and optimum growth occurred at temperatures between 55 and 60°C. The fermentable substrates included glucose, fructose, galactose, mannose, cellobiose, maltose, sucrose, lactose, d-xylose, d-ribose, mannitol, pyruvate, and starch. The products of fermentation of glucose were lactate, acetate, ethanol, H2, and CO2. The DNA base composition was 35 mol% G+C. The results of 16S rRNA sequence comparisons indicated that strain SEBR 5268T was closely related to Thermoanaerobacter brockii and Thermoanaerobacter finnii, and these three organisms exhibited levels of ribosomal DNA sequence homology of 98 to 99%. The results of DNA-DNA hybridization studies performed with the three organisms confirmed this close affiliation, and as base pairing values of > 70% were obtained, these organisms belong to the same species. Therefore, we propose that T. finnii should be reclassified as a subspecies of T. brockii, Thermoanaerobacter brockii subsp. finnii comb. nov. This automatically creates Thermoanaerobacter brockii subsp. brockii. We also propose that strain SEBR 5268T should be classified as a member of a new subspecies of T. brockii, Thermoanaerobacter brockii subsp. lactiethylicus. The latter differs from T. brockii subsp. brockii and T. brockii subsp. finnii by its 16S rRNA sequence, DNA sequence diversity, lower temperature optimum, G+C content, and carbohydrate utilization spectrum. Strain SEBR 5268T has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 9801T.
92 citations
"Isolation and characterization of a..." refers background in this paper
...…aquifers, soil, sugar beet, and sugar cane extraction juices (Klaushofer and Parkkinen 1965; Wiegel and Ljungdahl 1981; Schmid et al. 1986; Cayol et al. 1995; Cook et al. 1996; Kozianowski et al. 1997; Larsen et al. 1997; Slobodkin et al. 1999; Fardeau et al. 2000; Kim et al. 2001;…...
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