Isolation and characterization ofMethanobrevibacter oralis sp. nov.

A new coccobacillary, nonmotile, Gram-positive, methane-producing organism was isolated from human subgingival plaque. Both hydrogen and carbon dioxide were required for growth. No methane was produced from acetate, formate, or methanol. The optimum pH was 6.9–7.4, and the optimum temperature was 36–38°C. Fecal extract was required for growth, and a volatile fatty acid mixture was highly stimulatory. The DNA G+C content was 28 mol%. On the basis of these characteristics, DNA-DNA hybridization studies, and electrophoretic analysis of cellular proteins, the isolate was considered a new species and namedMethanobrevibacter oralis.     

Methanothrix soehngenii gen. nov. sp. nov., a new acetotrophic non-hydrogen-oxidizing methane bacterium

A new genus of methanogenic bacteria is described, which was isolated from a mesophilic sewage digester. It is most probably the filamentous bacterium, earlier referred to asMethanobacterium soehngenii, fat rod or acetate organism. The single non-motile, non-sporeforming cells are rod-shaped (0.8×2 m) and are normally combined end to end in long filaments, surrounded by a sheath-like structure. The filaments form characteristic bundles.Methanothrix soehngenii decarboxylates acetate, yielding methane and carbon dioxide. Other methanogenic substrates (H₂–CO₂, formate, methanol, methylamines) are not used for growth or methane formation. Formate is split into hydrogen and carbon dioxide. The temperature optimum for growth and methane formation is 37°C and the optimal pH range is 7.4–7.8. Sulfide and ammonia serve as sulfur and nitrogen source respectively. Oxygen completely inhibits growth and methane formation, but the bacteria do not loose their viability when exposed to high oxygen concentrations. 100 mg/l vancomycin showed no inhibition of growth and methanogenesis. No growth and methane formation was observed in the presence of: 2-bromoethanesulfonic acid, viologen dyes, chloroform, and KCN. The bacterium has a growth yield on acetate of 1.1–1.4 g biomass per mol acetate. The apparent K _S of the acetate conversion system to methane and carbon dioxide is 0.7 mmol/l. The DNA base composition is 51.9 mol% guanine plus cytosine. The nameMethanothrix is proposed for this new genus of filamentous methane bacterium. The type species,Methanothrix soehngenii sp. nov., is named in honor of N. L. Söhngen.     

Fervidobacterium nodosum gen. nov. and spec. nov., a new chemoorganotrophic,caldoactive, anaerobic bacterium

A new species of extremely thermophilic, glycolytic anaerobic bacterium, Fervidobacterium nodosum isolated from a New Zealand hot spring, is described. Fervidobacterium nodosum strains were Gram-negative, motile, non-sporulating obligately anaerobic rods that existed singly, in pairs or in chains. Electron micrographs of thin sections revealed a two-layered cell wall structure. The outer layer of the cell wall produced spheroids, which was a typical feature of this organism. The optimum temperature for growth was 65 to 70° C, the maximum 80° C and the minimum greater than 40° C. Growth occurred between a pH of 6.0 and 8.0 with the optimum being 7.0 to 7.5. The doubling time of Fervidobacterium nodosum at optimal temperature and pH was 105 minutes. The DNA base composition was 33.7% guanine plus cytosine as determined by thermal denaturation. A wide range of carbohydrates including glucose, sucrose, starch and lactose could be utilized by the organism. Lactate, acetate, hydrogen, and carbon dioxide were the major end products of glucose fermentation with lesser amounts of ethanol being formed. Growth was inhibited by tetracycline, penicillin and chloramphenicol indicating that the organism was a eubacterium.     

Isolation and characterization of "Methanosphaera cuniculi" sp. nov

A nonmotile, gram-positive, spherical organism was isolated from the intestinal tracts of rabbits. Both hydrogen and methanol were required for growth. No methane was produced from hydrogen-carbon dioxide, formate, acetate, methylamines, ethanol, or isopropanol. The optimum pH was 6.8, and the optimum temperature was 35 to 40 degrees C. The DNA G+C content is 23 mol%. The pseudomurein cell wall contained serine. These characteristics and the immunological fingerprinting results are consistent with its placement in the genus Methanosphaera as a new species.     

Desulfotomaculum geothermicum sp. nov., a thermophilic,fatty acid-degrading,sulfate-reducing bacterium isolated with H2 from geothermal ground water

A strictly anaerobic, thermophilic, fatty acids-degrading, sporulating sulfate-reducing bacterium was isolated from geothermal ground water. The organism stained Gram-negative and formed gas vacuoles during sporulation. Lactate, ethanol, fructose and saturated fatty acids up to C₁₈ served as electron donors and carbon sources with sulfate as external electron acceptor. Benzoate was not used. Stoichiometric measurements revealed a complete oxidation of part of butyrate although growth with acetate as only electron donor was not observed. The rest of butyrate was oxidized to acetate. The strain grew chemolithoautotrophically with hydrogen plus sulfate as energy source and carbon dioxide as carbon source without requirement of additional organic carbon like acetate. The strain contained a c-type cytochrome and presumably a sulfite reductase P582. Optimum temperature, pH and NaCl concentration for growth were 54°C, pH 7.3–7.5 and 25 to 35 g NaCl/l. The G+C content of DNA was 50.4 mol %. Strain BSD is proposed as a new species of the spore-forming sulfate-reducing genus Desulfotomaculum, D. geothermicum.     

Isolation and physiological characterisation of Thiobacillus thyasiris sp. nov., a novel marine facultative autotroph and the putative symbiont of Thyasira flexuosa

A novel facultatively chemolithoautotropic Thiobacillus, isolated from the gill tissue of the marine bivalve Thyasira flexuosa, is described. It is believed to be the symbiont from this animal, providing the animal with carbon fixed by the Calvin cycle. The organism grows lithoautotrophically on thiosulphate, tetrathionate and elemental sulphur, which are oxidised to sulphate. It oxidizes sulphide, thiosulphate, trithionate, tetrathionate and hexathionate, but not thiocyanate. Kinetic constants for these substrates are presented. In autotrophic batch culture it produces yields that are among the lowest reported for thiosulphate or tetrathionate as energy substrates (1.25 and 2.5 g cell-carbon per mol substrate, respectively). Autotrophic cultures contain ribulose bisphosphate carboxylase and excreted 20% of their fixed carbon into the medium during growth. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of the carboxylase. The organism is slightly halophilic and markedly halotolerant, showing optimum growth at about pH 7.5 and maximum growth rate at 37° C. It contains ubiquinone Q-10 and its DNA contains 52 mol % G+C. These characteristics distinguish it from any other Thiobacillus or Thiomicrospira species previously described. The organism is formally described and named as Thiobacillus thyasiris.     

Isolation and characterization of a new thermophilic and autotrophic methane producing bacterium:Methanobacterium thermoaggregans spec. nov.

Methanobacterium thermoaggregans is a new thermophilic autotrophic rod-shaped methane producing bacterium. The organism likes to form aggregates during growth and utilizes only H₂ and CO₂ as substrates. Growth optimum is at 65°C with a doubling time of 3.5 h. Optimal growth occurs at pH-values between 7 and 7.5. The addition of yeast extract to the mineral salt medium stimulates growth. The DNA base composition is 42 mol% G+C. The organism was isolated from mud taken from a cattle pasture. Because of its optimal growth temperature and its tendency to form aggregates the nameMethanobacterium thermoaggregans is suggested.Abbreviations G+C Guanine+cytosine     

Isolation and characterization of a novel mesophilic,fresh-water methanogen from river sediment Methanoculleus oldenburgensis sp. nov.

A new mesophilic, irregular coccoid methanogen isolated from a river sediment is described. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. For growth acetate is strictly required. Elevated levels of sodium chloride were not required and were inhibitory at concentrations above 1.5% (w/v). The optimal growth temperature was at 45°C. The DNA base ratio was 48.6±1 mol% G+C. The polar lipid pattern and the polyamine content were similar to that found in several Methanoculleus species. The new isolate CB-1 was assigned as Methanoculleus oldenburgensis (DSM 6216).     

Physiology of a Methanobacterium strain AZ

A methanogenic bacterium using H₂ and CO₂ as sole energy and carbon source has been isolated in pure culture from digested sludge. Its colonies on mineral agar are translucent, convex, circular with entire margins and yellow to brownish in colour. Cells are gram-positive, non motile and appear as straight cods, normally about 3 m long. A marked pleomorphism depending on the media was observed. The organism is chemolithoheterotrophic, has a pH optimum of 7.0 and an optimal temperature for growth of 33–40°C; no growth occurs above 45°C. The generation time at optimal conditions is less than 5 h. Cysteine must be supplied in the growth medium. It can act as sole sulfur source. The addition of sulfide accelerates the growth at an optimum concentration of 10^-4 to 10^-5 molar. A growth factor, not identical with SH-coenzyme M, occurring in anaerobic sewage sludge and yeast extract shows a stimulatory effect. 7.0–8.2% of the total carbon dioxide uptake is assimilated and 11.2% of the energy obtained from the reduction of carbon dioxide to methane is refound in the caloric value of the biomass. 0.01 ppm of dissolved oxygen completely inhibits growth and methane production. However, the bacteria do not loose their viability when exposed to high oxygen concentrations. Further informations are needed before this organism (DSM 744) is specifically identified.List of Abbreviations TOC total organic carbon - DOC dissolved organic carbon - POC particulate organic carbon     

Acetogenium kivui,a new thermophilic hydrogen-oxidizing acetogenic bacterium

Hydrogen-oxidizing acetogenic bacteria in pure culture are presently represented by the two mesophilic species, Acetobacterium woodii and Clostridium aceticum. From Lake Kivu we have isolated a Gram negative, chemolithotrophic, thermophilic anaerobe (LKT-1) that oxidizes hydrogen and reduces carbon dioxide to acetic acid. It is a non-motile, non-sporeforming rod, about 0.7m in width and 2–7.5m in length, often occuring in pairs or chains. The cell wall has a banded appearance; the surface layer contains a regular array of particles with six-fold rotational symmetry. No outer membrane is present. The temperature optimum for growth is 66°C, and the pH optimum is 6.4. Organic growth substrates include glucose, mannose, fructose, pyruvate, and formate; acetate is the principal product. The doubling time for growth on hydrogen and carbon dioxide is about 2h. Vitamins are neither required nor stimulatory. Yeast extract and Trypticase enhance the final yield but do not affect the growth rate. Cysteine or sulfide are required and cannot be replaced by thioglycolate or dithiothreitol. LKT-1 was mass cultured on hydrogen and carbon dioxide in a 24.1 fermentor with a yield of 34g (wet weight) of cells. The DNA base composition as determined by buoyant density is 38 mol % guanine plus cytosine. LKT-1 appears only distantly related to physiologically similar bacteria. A new genus Acetogenium is proposed, and the species is Acetogenium kivui.     

Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulphur-oxidising autotrophic eubacterium from a Western Australian hypersaline lake

The isolation of a novel obligately chemolithotrophic, halophilic and extremely halotolerant Thiobacillus from a hypersaline lake is described. Attempts to demonstrate sulphur- and ferrous iron-oxidizing chemolithotrophs in neighbouring hypersaline lakes were unsuccessful. The organism isolated differs from any other Thiobacillus species previously described and is formally named as Thiobacillus halophilus. It possesses ribulose bisphosphate carboxylase and grows chemolithoautotrophically on thiosulphate, tetrathionate and sulphur, oxidising them to sulphate. Kinetic constants for oxidation of sulphide, thiosulphate, trithionate and tetrathionate are presented. The organism is obligately halophilic, growing best with 0.8–1.0 M NaCl, and tolerating up to 4 M NaCl. Optimum growth was obtained at about 30° C and pH 7.0–7.3. It contains ubiquinone Q-8 and its DNA contains 45 mol % G+C. Organisms of this type might contribute significantly to the autotrophic fixation of carbon dioxide in some hypersaline extreme environments of the kind described.     

Bacteroides xylanolyticus sp. nov., a xylanolytic bacterium from methane producing cattle manure

As part of a study of the biogas production from cattle waste, xylanolytic bacteria were isolated from enrichments of fermenting cattle manure. From 34 isolates, mostly Gram-negative rods, a typical strain was investigated in more detail. It was an anaerobic non-sporeforming, Gramnegative rod, which was motile with peritrichous flagella. This organism fermented xylan and many soluble sugars (glucose, cellobiose, mannose, xylose, arabinose). Other hemicelluloses such as gum xanthan, laminaran, locust bean gum, and gum arabic were not utilized. It also could not use cellulose. Fermentation products were carbon dioxide, hydrogen, acetate and ethanol. The bacterium produced carboxymethylcellulase and xylanase, especially when growing on xylan. Growth was optimal between 25°C and 40°C and between pH 6.5 and 7.5. The guanine plus cytosine content of the DNA was 34.8±0.8%. The isolate was identified as a member of the genus Bacteroides, and a new species is proposed: Bacteroides xylanolyticus (xylan dissolving). The type strain of B. xylanolyticus is strain X5-1 (DSM 3808).     

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.     

Halonatronum saccharophilum gen. nov. sp. nov.: A New Haloalkaliphilic Bacterium of the Order Haloanaerobiales from Lake Magadi

A new alkaliphilic and moderately halophilic chemoorganotrophic anaerobic bacterium (strain Z-7986), which is spore-forming, rod-shaped, and has a gram-negative cell wall pattern, was isolated from the coastal lagoon mud of the highly mineralized Lake Magadi (Kenya). The organism is an obligatorily carbonate- and sodium chloride-dependent motile peritrichously flagellated rod that grows within a 3–17% NaCl concentration range (with an optimum at 7–12% NaCl) and within a pH range of 7.7–10.3 (with an optimum at pH values of 8–8.5). It is a moderate thermophile with a broad temperature optimum at 36–55°C; maximum growth temperature is 60°C. The bacterium catabolizes glucose, fructose, sucrose, maltose, starch, glycogen, N-acetyl-D-glucosamine, and, to a slight degree, peptone and yeast extract. Its anabolism requires yeast extract or casamino acids. Glucose fermentation yields formate, acetate, ethanol, H₂, and CO₂. The bacterium is sulfide-tolerant and capable of the nonspecific reduction of S⁰ to H₂S. The G+C content of the DNA is 34.4 mol %. The analysis of the 16S rRNA sequence revealed that strain Z-7986 belongs to the order Haloanaerobiales and represents a new genus in the family Halobacteroidaceae. We suggest the name Halonatronum saccharophilum gen. nov. sp. nov. The type strain of this species is Z-7986^T (= DSM13868, = Uniqem*211).     

Isolation and characterization of a thermophilic Methanobacterium able to use formate,the strain FTF

A thermophilic anaerobic which produced methane from formate and H₂ and CO₂ was isolated from a bench-scale digester treating a mixture of solid wastes at 55°C, after enrichment cultures on sodium acetate. The cells were slightly crooked rods occurring singly or in filaments. The bacterium was not motile, and stained Gram positive. Colonies appearing after 1 week of incubation were white with filamentous edges and 1 mm in diameter. The organism used H₂:CO₂ or formate as an energy source. Yeast extract was not required but stimulated growth significantly. Casamino acids were stimulatory and could serve as a nitrogen source. Cysteine was used as a sulfur source. The optimum pH for growth was 7.5. Growth occurred from 35 to 70°C with an optimum at 55°C. The deoxyribonucleic acid base composition was 49.2 mol% guanine plus cytosine. Though this isolate conforms to Methanobacterium thermoformicium, its proper assignment awaits further studies. It has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 3012.This work was supported in part by the Conseil Régional Nord/Pas-de-Calais     

Pelczaria aurantia gen. nov., sp. nov., a newly described organge-colored bacterium

An organism from a goldfish aquarium, isolated on barbital medium, was found to be a Grampositive coccus which divided in alternating planes, often appearing as a doublet or as a tetrad with adjacent sides flattened. It grew well, although slowly, on rich solid medium (LB agar) and in liquid brain-heart infusion at room temperature (ca. 22°C); growth was slower and less extensive at 30°C or 37°C. No growth was seen at 4–5°C or at 42°C. It withstands brief exposure to ultraviolet radiation. Its growth was inhibited by low levels (0.1 unit/ml) of penicillin but was unaffected by levels of acetazolamide in excess of 1 mg/ml, indicating that it lacks carbonic anhydrase. Acid was not produced from glucose, maltose, mannose, lactose, or sucrose and only weakly, if at all, from fructose. Its DNA has a G+C mol percent of 59 measured chromatographically and neither the DNA nor rRNA from the organism hybridized with DNA from any organism that seemed related on morphological or other bases. Thin-layer chromatography of chloroform: methanol extracts of the organism show that it contains phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl glycerol. Cell-wall preparations contain glutamic acid, serine, histidine, lysine, and alanine in the ratio of 1:1:1:1:8. Colonies were red-orange in color due, in larger measure, to a carotenoid tentatively identified as rhodopin. The organism was named Pelczaria aurantia.This paper is submitted in honor of Miss Dorothy Schoknecht of Kalispell, Montana, teacher prima inter pares     

Methanogenium,a new genus of marine methanogenic bacteria,and characterization ofMethanogenium cariaci sp. nov. andMethanogenium marisnigri sp. nov.

A new genus of marine methanogenic bacteria and two species within this genus are described.Methanogenium is the proposed genus andMethanogenium cariaci the type species. Cells of the type species are Gram-negative, peritrichously flagellated, irregular cocci with a periodic wall surface pattern. Colonies formed by these bacteria are yellow, circular and umbonate with entire edges. The DNA base composition is 52 mol% guanine plus cytosine. Formate or hydrogen and carbon dioxide serve as substrates for growth. Cells ofMethanogenium marisnigri are of similar shape but smaller diameter thanM. cariaci. The colonies ofM. marisnigri are convex, and the DNA base composition is 61 mol % G+C. Formate or hydrogen and carbon dioxide are growth substrates. Sodium chloride is required for growth of both methanogens.Abbreviations SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2 ethanesulfonic acid) - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid     

Thermoanaerobium brockii gen. nov. and sp. nov., a new chemoorganotrophic,caldoactive, anaerobic bacterium

The isolation of a new anaerobic thermophilic bacterium, Thermoanaerobium brockii, from volcanic features is described. Successful enrichment required a complex medium containing glucose or other fermentable sugars and incubation temperatures of 55–80° C. Strains of T. brockii were gram positive, rods of uneven length that existed singly, in pairs, chains or filaments. Electron micrographs of thin sections of cell revealed a monolayered cell wall and a constrictive or pinching off cell division process. The organism was nonsporeforming, obligately anaerobic and chemoorganotrophic. The optimal temperature for growth was 65–70° C, the maxium was below 85° C and the minimum above 35° C. The doubling time at the optimal temperature for growth was about 1 h. The DNA base composition of three strains of T. brockii varied from 30.0–31.4 mol % guanosine plus cytosine. Fermentable carbohydrates included glucose, sucrose, maltose, lactose, cellobiose and insoluble starch. The fermentation products of cells grown on glucose were ethanol, lactic acid, acetic acid, hydrogen and carbon dioxide. Growth of all strains tested was inhibited by fairly low concentrations of cycloserine, penicillin, streptomycin, tetracycline and chloramphenicol. The possible ecological, evolutionary, and industrial significance, and taxonomic relationships of Thermoanaerobium are discussed.Abbreviations TYEG complex medium containing mineral salts, 0.3% yeast extract, 1.0% tryptone and 0.5% glucose - O.D. optical density - G+C guanosine plus cytosine     

Methane formation from fructose by syntrophic associations of Acetobacterium woodii and different strains of methanogens

When Acetobacterium woodii was co-cultured in continuous or in stationary culture with Methanobacterium strain AZ, fructose instead of being converted to 3 mol of acetate was converted to 2 mol of acetate and 1 mol each of carbon dioxide and methane, showing that interspecies hydrogen transfer occurred. In continous culture the organisms formed a close physical association in clumps; the doubling time for each organism was 6h at 33°C. Methane mainly was derived from carbon positions 3 and 4 of the sugar, but other carbons also yielded methane; this was shown to be due to carbon dioxide-acetate exchange reactions by A. woodii in a manner similar to that carried out by Clostridium thermoaceticum. Four other methanogens, Methanobacterium M.o.H. and M.o.H. G, Methanobacterium formicicum, and Methanosarcina barkeri (not acetate-adapted) also produced similar results, when co-cultured with A. woodii.     

Evidence for the presence of the glyoxylate cycle in Chloroflexus

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were present in cell-free extracts of the phototrophic, green, thermophilic bacterium Chloroflexus aurantiacus grown with acetate as the sole organic carbon source.The optimum temperature of these enzymes was 40° C, and their specific activities were high enough to account for the observed growth rate. Lower levels of the enzymes were found in extracts from cells grown on a complete medium.Itaconate was shown to inhibit isocitrate lyase from C. aurantiacus 96% at a concentration of 0.25 mM and also had a profound effect on the growth of the organism on acetate, 0.25 mM inhibiting completely. Itaconate also inhibited the growth when added to the complex medium, but in this case much higher concentrations were required.