Isolation of Clostridium propionicum strain 19acry3 and further characteristics of the species

Two mixed cultures able to ferment acrylate to equimolar acetate and propionate were enriched from anaerobic sediments. From one of these mixed cultures a pure culture of a Gram-positive, obligately anaerobic bacterium was isolated. This strain, designated 19acry3 (= DSM 6251) was identified as belonging to the species Clostridium propionicum. Only a narrow range of organic compounds supported growth, including acrylate and lactate. Acrylate and lactate were fermented to acetate and propionate in a 1:2 molar ratio. When co-cultured with the non-acrylate-fermenting Campylobacter sp. strain 19gly1 (DSM 6222), the fermentation balance shifted to almost equimolar acetate and propionate. Strain 19acry3 was compared with Clostridium propionicum type strain X2 (DSM 1682). The two strains displayed similar phenotypic properties. The mol% G+C of DNA isolated from both strains was 36–37 (by thermal denaturation). Both strains displayed a characteristic fluorescence when observed by fluorescence microscopy. Cell-free extracts of both strains were examined by spectrophotofluorimetry. In both strains, two excitation peaks were observed at 378 and 470 nm. Excitation at either of these wavelengths resulted in an emission maximum at 511 nm.     

Fermentative degradation of glutarate via decarboxylation by newly isolated strictly anaerobic bacteria

Two strains of new strictly anaerobic, gramnegative bacteria were enriched and isolated from a freshwater (strain WoG13) and a saltwater (strain CuG11) anoxic sediment with glutarate as sole energy source. Strain WoG13 formed spores whereas strain CuG11 did not. Both strains were rod-shaped, motile bacteria growing in carbonate-buffered, sulfide-reduced mineral medium supplemented with 2% of rumen fluid. Both strains fermented glutarate to butyrate, isobutyrate, CO₂, and small amounts of acetate. With methylsuccinate, the same products were formed, and succinate was fermented to propionate and CO₂. No sugars, amino acids or other organic acids were used as substrates. Molar growth yields (Ys) were very small (0.5–0.9 g cell dry mass/mol dicarboxylate). Cells of strain WoG13 contained no cytochromes, and the DNA base ratio was 49.0±1.4 mol% guanine-plus-cytosine. Enzyme activities involved in glutarate degradation could bedemonstrated in cell-free extracts of strain WoG13. A pathway of glutarate fermentation via decarboxylation of glutaconyl-CoA to crotonyl-CoA is suggested which forms butyrate and partly isobutyrate by subsequent isomerization.     

Fermentation of glutamate by Selenomonas acidaminophila sp. nov.

From an anaerobic digester a novel type of strictly anaerobic, Gram-negative, non-sporeforming mesophilic bacterium was isolated. The cells were curved rods, motile by means of lateral flagella and contained b- and c-type cytochromes. The G+C content of the DNA was 48.0±1.0 mol%. The isolate was able to ferment only glutamate, aspartate, lactate and pyruvate. Organic fermentation products were acetate, propionate and succinate. Propionate was probably formed via a reductive succinate pathway. Strain DKglu16 is described as the type strain of a new species, Selenomonas acidaminophila sp. nov., in the family Bacteroidaceae.Abbreviations atm atmosphere - Pa Pascal - SSC standard saline citrate - G+C guanine+cytosine - _max maximum specific growth rate     

Fermentation of 2-methoxyethanol by Acetobacterium malicum sp. nov. and Pelobacter venetianus

Anaerobic bacteria degrading 2-methoxyethanol were enriched from freshwater sediments, and three strains were isolated in pure culture. Two of them were Grampositive non-spore-forming rods and grew strictly anaerobically by acetogenic fermentation. Optimal growth occurred at 30°C, initial pH 7.5–8.0. 2-Methoxyethanol and 2-ethoxyethanol were fermented to acetate and corresponding alcohols. Hydrogen plus carbon dioxide, formate, acetoin, l-malate, lactate, pyruvate, fructose, and methoxyl groups of 3,4,5-trimethoxybenzoate and 3,4,5-trimethoxycinnamate were fermented to acetate. 1,2-Propanediol was fermented to acetate, propionate, and propanol. Strain MuME1 was described as a new species, Actetobacterium malicum. It had a DNA base composition of 44.1 mol% guanine plus cytosine. The third strain, which was identified as Pelobacter venetianus, fermented 2-methoxyethanol to methanol, ethanol, and acetate.     

Propionigenium modestum gen. nov. sp. nov. a new strictly anaerobic,nonsporing bacterium growing on succinate

From marine and freshwater mud samples and from human saliva new strictly anaerobic, Gram-negative, nonsporeforming bacteria were isolated growing with succinate as sole source of carbon and energy. All strains grew in defined mineral media containing at least 1% sodium chloride. Succinate was stoichiometrically transformed to propionate und carbon dioxide; the growth yield varied between 2.1 and 2.4 g cell dry weight per mol of succinate fermented. In addition to succinate, only fumarate, l-aspartate, l-malate, oxaloacetate and pyruvate, were utilized and were stoichiometrically fermented to propionate and acetate. Yeast extract was not fermented but enhanced growth rates and yields. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 33.9±0.3 mol % guanine plus cytosine. A marine isolate, strain Gra Succ 2, is described as type strain of a new species, Propionigenium modestum gen. nov. sp. nov., in the family Bacteroidaceae.     

A new purple sulfur bacterium from saline littoral sediments,Thiorhodovibrio winogradskyi gen. nov. and sp. nov.

Two strains of a new purple sulfur bacterium were isolated in pure culture from the littoral sediment of a saline lake (Mahoney Lake, Canada) and a marine microbial mat from the North Sea island of Mellum, respectively. Single cells were vibrioid-to spirilloid-shaped and motile by means of single polar flagella. Intracellular photosynthetic membranes were of the vesicular type. As photosynthetic pigments, bacteriochlorophyll a and the carotenoids lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin were present.Hydrogen sulfide and elemental sulfur were used under anoxic conditions for phototrophic growth. In addition one strain (06511) used thiosulfate. Carbon dioxide, acetate and pyruvate were utilized by both strains as carbon sources. Depending on the strain propionate, succinate, fumarate, malate, tartrate, malonate, glycerol or peptone may additionally serve as carbon sources in the light. Optimum growth rates were obtained at pH 7.2, 33 °C, 50 mol m^-2 s^-1 intensity of daylight fluorescent tubes and a salinity of 2.2–3.2% NaCl. During growth on sulfide, up to ten small sulfur globules were formed inside the cells. The strains grew microaerophilic in the dark and exhibited high specific respiration rates. No vitamins were required for growth. The DNA base composition was 61.0–62.4 mol% G+C.The newly isolated bacterium belongs to the family chromatiaceae and is described as a member of a new genus and species, Thiorhodovibrio winogradskyi gen. nov. and sp. nov. with the type strain SSP1, DSM No. 6702.     

Selenomonas acidaminovorans sp. nov., a versatile thermophilic proton-reducing anaerobe able to grow by decarboxylation of succinate to propionate

A moderately thermophilic anaerobic bacterium (strain Su883), which decarboxylated succinate to propionate, was isolated from granular methanogenic sludge. The bacterium appeared to ferment a number of amino acids including glutamate, histidine, arginine, ornithine, citrulline, and threonine to propionate, acetate and hydrogen. Propionate was formed via the oxidative decarboxylation of -ketoglutarate to succinyl-CoA. In addition, the strain degraded glucose, fructose, glycerol, pyruvate, serine, alanine, citrate and malate to acetate, carbon dioxide and hydrogen, and branched-chain amino acids to branched-chain fatty acids. With all single substrates solely hydrogen was formed as reduced fermentation product. Mixed cultures of strain Su883 and Methanobacterium thermoautotrophicum H showed a more rapid conversion of substrates and with some substrates a shift from acetate to propionate formation.Strain Su883 is a motile, gram-negative, non-sporeforming, slightly curved rod with a DNA base ratio of 56.5 mol% guanine-plus-cytosine. Selenomonas acidaminovorans Su883 is proposed as type strain for the new species within the genus Selenomonas.     

Anaerobic degradation of 3-hydroxypropanoate by a newly isolated Clostridium sp.

A strain (ToHP1) of anaerobic bacteria which degrades 3-hydroxypropanoate was isolated in pure culture from the sludge of an anaerobic digestor at a Tokyo sewage treatment facility. The strain grew by degrading 3-hydroxypropanoate, lactate, and pyruvate to propionate and acetate. It also grew auxotrophically by degrading 2-hydroxybutanoate to butyrate and propionate in the presence of acetate. Cells were lemon-shaped rods; 1.4 to 3.6 μm in length and 0.5 to 1.1 μm in width. Spores with a diameter of 0.8 to 1.1 μm were formed subterminally. The strain occurred singly or in pairs. It stained gram-positive. The strain grew optimally at 30 to 35°C and ca. pH 7.2. It required yeast extract for growth. The strain did not utilize nitrate, sulfate, sulfite, thiosulfate, or elemental sulfur as an electron acceptor. The guanine plus cytosine content of the DNA was 43 mol%. Strain ToHP1 was identified as a member of the genus Clostridium.     

d-arabinose metabolism byBacteroides fragilis strain 2044

During growth in the presence of 1-14C-d-arabinose,Bacteroides fragilis strain 2044 formed labeled succinic, acetic, and propionic acis. Degradation of the acids by the Schmidt reaction revealed that at least 89% of the succinate radioactivity was found in the methylene carbons and that 75% and 84% of the label in propionate and acetate were found in the noncarboxyl carbons of these molecules. No label was found in acetate, propionate, or succinate during growth of strain 2044 in the presence of 5-3H-d-arabinose. Strain 2044 converted radioactivity from 1- or 2-labeled glycolic acid and glycine to succinate by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons. Label from 1- or 2-labeled glycine and 2 but not 1-labeled glycolic acid was found in acetate. Uniformly labeled 14C-glyoxalate gave rise to labeled acetate, but not succinate.Bacteroides fragilis strain 2044 metabolizesd-arabinose by a mechanism involving a 32 cleavage of the molecule.     

Propionate acts as carboxylic group acceptor in aspartate fermentation by Propionibacterium freudenreichii

Cells of Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii did not show significant growth or product formation in a mineral medium with 10 mM aspartate or 10 mM fumarate, vitamins, and a small amount (0.05% w/v) of yeast extract. In the presence of added propionate, growth with aspartate or fumarate was possible, and depended strictly on the amount of propionate provided, according to the equation: 3 aspartate + propionate 3 succinate + acetate + CO₂+3 NH₃. Cocultures of P. freudenreichii with the succinate-decarboxylating strain Ft2 converted 3 aspartate stoichiometrically to acetate and 2 propionate. High activity of methylmalonyl-CoA: pyruvate transcarboxylase, and lack of methylmalonyl-CoA decarboxylase and oxaloacetate decarboxylase activity in cell-free extracts of aspartate-grown cells indicated that failure to use aspartate as sole substrate was due to the inability of these strains to catalyze a net decarboxylation of C₄-dicarboxylic acids.Dedicated to Prof. Dr. Norbert Pfennig on occasion of his 65th birthday     

Clostridium grantii sp. nov., a new obligately anaerobic,alginolytic bacterium isolated from mullet gut

A gram-positive, motile, rod-shaped, strictly anaerobic, sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C and pH6.5, and at a salinity of 1–10³. Out of a variety of polysaccharides tested as growth substrates, only alginate supported growth in either semidefined or complex culture medium. The organism also grew on a variety of mono- and disaccharides. Moles product per 100mol of alginate monomer degraded were: acetate, 186; ethanol, 19; formate, 54; and CO₂, 0.19. Moles product per 100mol of hexose in cellobiose or glucose degraded were: acetate, 135; ethanol,61; formate, 63: and CO₂, 61. Hydrogen was not detectable during the incubations (detection limit, <10^-5atm) and propionate, butyrate, lactate, or succinate were not produced as fermentation end products (<2 mol per 100 mol of monomer). The G+C content of DNA from the bacterium was 30.2±0.3 mol%, and the cell walls contained the peptidoglycan component meso-diaminopimelic acid. A phylogenetic analysis of the 16S rDNA sequence indicated that the organism grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. However, it differed from other species of the genus with regard to morphology, growth temperature optimum, substrate range, and fermentation pattern and is therefore designated as a new species of Clostridium; the type strain is A-1 (DSM 8605).     

Acetonema longum gen.nov.sp.nov., an H2/CO2 acetogenic bacterium from the termite,Pterotermes occidentis

A previously undescribed, H₂-oxidizing CO₂-reducing acetogenic bacterium was isolated from gut contents of the wood-feeding termite, Pterotermes occidentis. Cells of representative strain APO-1 were strictly anaerobic, Gram-negative, endospore-forming motile rods which measured 0.30–0.40×6–60 m. Cells were catalase positive, oxidase negative, and had 51.5 mol percent G+C in their DNA. Optimum conditions for growth on H₂+CO₂ were at 30–33°C and pH (initial) 7.8, and under these conditions cells formed acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other energy sources supporting good growth of strain APO-1 included glucose, ribose, and various organic acids. Acetate and butyrate were major fermentation products from most organic compounds tested, however propionate, succinate, and 1,2-propanediol were also formed from some substrates. Based on comparative analysis of 16S rRNA nucleotide sequences, strain APO-1 was related to, but distinct from, members of the genus Sporomusa. Moreover, physiological and morphological differences between strain APO-1 and the six known species of Sporomusa were significant. Consequently, it is proposed herewith that a new genus, Acetonema, be established with strain APO-1 as the type strain of the new species, Acetonema longum. A. longum may contribute to the nutrition of P. occidentis by forming acetate, propionate and butyrate, compounds which are important carbon and energy sources for termites.     

<Emphasis Type="Italic">Halobacillus locisalis</Emphasis> sp. nov., a halophilic bacterium isolated from a marine solar saltern of the Yellow Sea in Korea

A Gram-positive, motile, endospore-forming and rod-shaped halophilic bacterial strain MSS-155 (KCTC 3788 and KCCM 41687) was isolated from a marine solar saltern of the Yellow Sea in Korea and was subjected to a polyphasic taxonomic study. This organism grew at temperature of 10.0–42.0°C with an optimum of 35°C. Strain MSS-155 grew optimally in the presence of 10% NaCl and did not grow in the absence of NaCl. The cell wall peptidoglycan type of strain MSS-155 was A4 based on l-Orn-d-Asp. Strain MSS-155 was also characterized chemotaxonomically by having menaquinone-7 (MK-7) as the predominant isoprenoid quinone and anteiso-C_15:0 as the major fatty acid. The DNA G+C content was 44.0 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain MSS-155 falls within the radiation of the cluster comprising Halobacillus species. Levels of 16S rDNA sequence similarity between strain MSS-155 and the type strains of four Halobacillus species were in the range 97.6–98.8%. Strain MSS-155 exhibited levels of DNA-DNA relatedness of 6.2–11.2% to the type strains of Halobacillus species described previously. On the basis of phenotypic properties, phylogeny, and genomic data, strain MSS-155 should be placed in the genus Halobacillus as a member of a novel species, for which we propose the name Halobacillus locisalis sp. nov.Communicated by W.D. Grant     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Fermentation of Inulin by Clostridium thermosuccinogenes sp. nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65°C, with optimum growth at 58°C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H₂, and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species, Clostridium thermosuccinogenes sp. nov., with a G+C content of 35.9 mol%.     

Sporomusa termitida sp. nov., an H2/CO2-utilizing acetogen isolated from termites

H₂-oxidizing CO₂-reducing acetogenic bacteria were isolated from gut contents of Nasutitermes nigriceps termites. Isolates were strictly anaerobic, Gram negative, endospore-forming, straight to slightly curved rods (0.5–0.8×2–8 m) that were motile by means of lateral flagella. Cells were oxidase negative, but catalase positive and possessed a b-type cytochrome(s) associated with the cell membrane. Cells grew anaerobically with H₂+CO₂ as energy source and catalyzed a total synthesis of acetate from this gas mixture. H₂ uptake by a representative isolate (strain JSN-2) displayed a K _m=6 M and V _max=380 nmol x min^-1 x mg protein^-1. Other substrates used as energy sources for growth and acetogenesis included CO, methanol, betaine, trimethoxybenzoate, and various other organic acids. Succinate was also fermented, but propionate was formed from this substrate instead of acetate. Of a variety of sugars and sugar alcohols tested, only mannitol supported growth. Cells grew optimally at 30° C and pH 7.2 and required yeast extract or a source of amino acids (e.g. Casamino acids) for good growth. During initial enrichment and isolation, cells appeared sensitive to various reducing agents commonly employed in media for anaerobes. The DNA base composition of strain JSN-2 was 48.6 mol% G+C. On the bases of cell morphology, substrate utilization spectrum, and DNA base composition, strain JSN-2 is here-with proposed as the type strain of the new species Sporomusa termitida.Journal article no. 12513 from the Michigan Agricultural Experiment Station     

Cytophaga xylanolytica sp. nov., a xylan-degrading,anaerobic gliding bacterium

Gliding bacteria attached in masses to, and dominated the fermentation of, xylan powder in methanogenic and sulfidogenic enrichments from various freshwater sediments. Isolates of such bacteria were all gram-negative, slender rods (0.4×4-24 m) that formed no endospores, microcysts or fruiting bodies. Representative strain XM3 was a mesophilic, aeroduric anaerobe that grew by fermentation of mono-, di-, and poly-saccharides (but not cellulose) in a mineral medium containing up to 3% NaCl. However, CO₂/HCO _inf3 ^sup- was required in media for consistent initiation of growth. Fermentation products included acetate, propionate, succinate, CO₂, and H₂. Xylan-grown cells had xylanase and various glycosidase activities that were mainly or almost entirely cell-associated, respectively. Strain XM3 was weakly catalase positive, but oxidase negative; it possessed sulphonolipids and carotenoid, but not flexirubin, pigments; and its total cellular fatty acids were dominated by C_15:0 anteiso (75%), n (13%) and iso (2%) isomers. Strain XM3 had 45.5 mol% G+C in its DNA, and partial sequencing of its 16S rRNA placed XM3 within the Bacteroides-Flavobacterium phylogenetic group. Similar strains were isolated from marine sediments. Strain XM3 is herewith proposed as the type strain of the new species, Cytophaga xylanolytica. Results, which are discussed in terms of our current concept of the genus Cytophaga, suggest that the importance of C. xylanolytica in anaerobic biopolymer decomposition has not been fully appreciated.Dedicated to Professor Norbert Pfennig, in whose laboratory this project was initiated and who recently retired after more than four decades of contributions to microbiology     

Desulforhabdus amnigenus gen. nov. sp. nov., a sulfate reducer isolated from anaerobic granular sludge

From granular sludge of an upflow anaerobic sludge bed (UASB) reactor treating paper-mill wastewater, a sulfate-reducing bacterium (strain ASRB1) was isolated with acetate as sole carbon and energy source. The bacterium was rod-shaped, (1.4–1.9×2.5–3.4 μm), nonmotile, and gram-negative. Optimum growth with acetate occurred around 37°C in freshwater medium (doubling time: 3.5–5.0 days). The bacterium grew on a range of organic acids, such as acetate, propionate, and butyrate, and on alcohols, and grew autotrophically with H₂, CO₂ and sulfate. Fastest growth occurred with formate, propionate, and ethanol (doubling time: approx. 1.5 days). Strain ASRB1 clusters with the delta subdivision of Proteobacteria and is closely related toSyntrophobacter wolinii a syntrophic propionate oxidizer. Strain ASRB1 was characterized as a new genus and species:Desulforhabdus amnigenus.     

d-Pentose metabolism byBacteroides vulgatus strain 8482

Bacteroides vulgatus strain 8482 metabolizedd-arabinose by a mechanism involving a 32 (top to bottom) cleavage of the arabinose carbon skeleton. During growth in the presence of 1-¹⁴C-d-arabinose, acetate, propionate, and succinate were labeled, but during growth in the presence of 5-labeledd-arabinose, only labeled acetate and succinate were formed. The metabolism ofd-ribose by strain 8482 differed from that ford-arabinose. Strain 8482 converted glycolic acid and glycine to acetate and succinate, but not propionate, by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons and equilibration of carbons 1 and 2 of glycolic acid and glycine with nonequivalent metabolic pools. The metabolism ofd-arabinose,d-ribose,d-glycine, andd-glycolic acid by strain 8482 was similar, in some respects, to that ofBacteroides fragilis strain 2044, but differed substantially from the metabolism of the same substances byBacteroides ruminicola strain B₁4.     

A new 3-hydroxybutyrate fermenting anaerobe,Ilyobacter polytropus,gen. nov. sp. nov., possessing various fermentation pathways

From marine anoxic mud, a new strictly anaerobic, Gram-negative, non-sporeforming bacterium was isolated with 3-hydroxybutyrate as substrate. 3-Hydroxybutyrate and crotonate were fermented to acetate and butyrate. Glycerol was fermented to 1,3-propanediol and 3-hydroxypropionate. Acetate and formate were the only products of pyruvate or citrate fermentation. Glucose and fructose were fermented to acetate, formate and ethanol. Malate and fumarate were fermented to acetate, formate and propionate. Neither sulfate, sulfur, nor nitrate was reduced. The DNA base ratio was 32.2±0.5 mol% guanine plus cytosine. Strain CuHbu1 is described as type strain of a new genus and species, Ilyobacter polytropus gen. nov. sp. nov., in the family Bacteroidaceae.