Characterization of an acetate-decarboxylating,non-hydrogen-oxidizing methane bacterium

A methanogenic bacterium, commonly seen in digested sludge and referred to as the fat rod or Methanobacterium soehngenii, has been enriched to a monoculture and is characterized. Cells are gramnegative, non-motile and appear as straight rods with flat ends. They form filaments which can grow to great lengths. The structure of the outer cell envelop is similar to Methanospirillum hungatii. The organism grows on a mineral salt medium with acetate as the only organic component. Acetate is the energy source, and methane is formed exclusively from the methyl group. Acetate and carbon dioxide act as sole carbon source and are assimilated in a molar ratio of about 1.9:1. The reducing equivalents necessary to build biomass from these two precursors are obtained from the total oxidation of some acetate. Hydrogen is not used for methane formation and is not needed for growth. Formate is cleaved into hydrogen and carbon dioxide. Coenzyme M was found to be present at levels of 0.35 nmol per mg of dry cells and F₄₂₀ amounted to 0.55 g per mg protein. The mean generation time was 9 days at 33°C.     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

Methanosphaera stadtmaniae gen. nov., sp. nov.: a species that forms methane by reducing methanol with hydrogen

Methanosphaera stadtmaniae is a non-motile, Gram-positive spherical-shaped organism that obtains energy for growth by using hydrogen to reduce methanol to methane. It does not produce methane from hydrogen and carbon dioxide, formate, acetate or methylamines and cannot grow with hydrogen and carbon monoxide, nitrate, fumarate, sulfate or choline. Its pH optimum is 6.5 to 6.9 and its temperature optimum is 36° to 40° C. It is not inhibited by bile salts, inhibitors of the synthesis of folic acid coenzymes, cephalothin or clindamycin but is inhibited by metronidazole, bacitracin, monensin, lasalocid, or bromoethanesulfonate. It requires acetate, carbon dioxide, isoleucine, ammonium, and thiamin for growth and biotin is stimulatory. It does not contain cytochromes and the mol % G+C of its DNA is 25.8. The composition of its cell wall and 16 S rRNA and its immunological fingerprint are consistent with characterization of the organism as a member of a new genus of the family Methanobacteriaceae. The habitat of the type strain is the human large intestine.     

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.     

Growth characteristics of an obligately psychrophilic Vibrio sp.

The growth characteristics of an obligately psychrophilic Vibrio sp. have been studied in a chemostat with glucose or lactose as the limiting substrate over a temperature range 0–23°C. Vibrio AF-1 has an optimum growth temperature of 15°C and maximum growth temperature which is dependent upon the carbon source. On glucose growth ceases at 20°C whereas on lactose growth continues to 23°C. Growth rate is also a function of the carbon source provided. When grown on glucose, fructose, sucrose, maltose and galactose _max values of 0.046 h^-1 at 15°C were recorded whereas on lactose, mannose, ribose and xylose _max values of 0.020 h^-1 were obtained. Substrate affinities (K _s ) for the 9 sugars also fall into 2 divisions as for _max and are temperature dependent. Those sugars which support a high growth rate have highest K _s values at 0°C whereas these which give a low growth rate show maximum affinities at 15°C. Vibrio AF-1 produces the maximum cell yield (0.6 g/g sugar consumed) at temperature <8°C irrespective of the carbon source utilised and correlated with maximum rates of sugar uptake and minimum O₂ consumption. Maintenance energy determination on glucose grown cells show that at 2° C 2% of the carbon input is used for maintenance whereas at 20°C the requirement increases to 10% of the carbon input.     

Isolation and characterization of a marine methanogenic bacterium from the biofilm of a shiphull in Los Angeles harbor

A marine mesophilic, irregular coccoid methanogen, which shows close resemblance toMethanococcus sp., was isolated from the biofilm of shiphulls docked in Los Angeles harbor. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8–7.0 and a temperature optimum of 37°C. Elevated levels of sodium chloride were required for optimum growth. Optimum levels of total sulfide and magnesium chloride for growth were 1.0mm and 10mm respectively. The isolate used ammonia as nitrogen source. The concentration of 30mm ammonium chloride supported maximum growth of the isolate.     

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.     

Effects of drainage and temperature on carbon balance of tussock tundra micrososms

We examined the importance of temperature (7°C or 15°C) and soil moisture regime (saturated or field capacity) on the carbon (C) balance of arctic tussock tundra microcosms (intact blocks of soil and vegetation) in growth chambers over an 81-day simulated growing season. We measured gaseous CO₂ exchanges, methane (CH₄) emissions, and dissolved C losses on intact blocks of tussock (Eriophorum vaginatum) and intertussock (moss-dominated). We hypothesized that under increased temperature and/or enhanced drainage, C losses from ecosystem respiration (CO₂ respired by plants and heterotrophs) would exceed gains from gross photosynthesis causing tussock tundra to become a net source of C to the atmosphere. The field capacity moisture regime caused a decrease in net CO₂ storage (NEP) in tussock tundra micrososms. This resulted from a stimulation of ecosystem respiration (probably mostly microbial) with enhanced drainage, rather than a decrease in gross photosynthesis. Elevated temperature alone had no effect on NEP because CO₂ losses from increased ecosystem respiration at elevated temperature were compensated by increased CO₂ uptake (gross photosynthesis). Although CO₂ losses from ecosystem respiration were primarily limited by drainage, CH₄ emissions, in contrast, were dependent on temperature. Furthermore, substantial dissolved C losses, especially organic C, and important microhabitat differences must be considered in estimating C balance for the tussock tundra system. As much as 20% of total C fixed in photosynthesis was lost as dissolved organic C. Tussocks stored 2x more C and emitted 5x more methane than intertussocks. In spite of the limitations of this microcosm experiment, this study has further elucidated the critical role of soil moisture regime and dissolved C losses in regulating net C balance of arctic tussock tundra.     

Carbon assimilation by the autotrophic thermophilic archaebacterium Thermoproteus neutrophilus

Growth of Thermoproteus neutrophilus at 85°C was studied using an improved mineral medium with CO₂, CO₂ plus acetate, CO₂ plus propionate, or CO₂ plus succinate as carbon sources; sulfur reduction with H₂ to H₂S was the sole source of energy. None of the carbon compounds added was oxidized to CO₂. The organism grew autotrophically with a generation time of 9–14 h, up to a cell density of 0.5 g dry weight per liter (2×10⁹ cells/ml). Propionate did not stimulate, succinate slightly stimulated the growth rate. Acetate, even at low concentrations (0.5 mM), stimulated the growth rate, the generation time being shortened to 3–4 h. Acetate provided 70% of the cell carbon, which shows that Thermoproteus neutrophilus is a facultative autotroph. The path of these carbon precursors into cell compounds was studied by ¹⁴C long-term labelling and investigation of enzyme activities. Propionate could not be used as a major carbon source and was incorporated only into isoleucine, probably via the citramalate pathway. Acetate was a preferred carbon source which suppressed autotrophic CO₂ fixation: acetate grown cells exhibited an incomplete citric acid cycle in which 2-oxoglutarate dehydrogenase was present, but fumarate reductase was repressed. The succinate incorporation pattern and enzyme pattern indicated that autotrophic CO₂ fixation proceeded via a yet to be defined reductive citric acid cycle.     

Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing,strictly anaerobic bacterium

A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H₂ as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H₂, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H₂. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160M), a dechlorination rate of about 50 nmol min^-1 (mg cell protein)^-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.Abbreviations PCE Perchloroethylene, tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon     

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and O-12, growing in the temperature range from 30 to 61°C with a optimum at 55°C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes and genes of membrane-bound methane monooxygenase, as well as on phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulosephosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55°C than in cells grown at 37°C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH₄ ⁺ assimilation occurred through reductive amination of -ketoglutarate and via the glutamate cycle. The relationship between the physiological and biochemical peculiarities of the isolates and their thermophilic nature is discussed.     

Methane production capacities of different rice soils derived from inherent and exogenous substrates

Methane production rates were determined at weekly intervals during anaerobic incubation of eleven Philippine rice soils. The average production rates at 25 °C varied in a large range from 0.03 to 13.6 g CH_{4 g(d.w. soil)} ^-1d^-1. The development of methane production rates derived from inherent substrate allowed a grouping of soils in three classes: those with instantaneous development, those with a delay of approximately two weeks, and those with a suppression of methane production of more than eight weeks. Incubation at 30 and 35 °C increased production capacities of all soils, but the grouping of soils was still maintained. The Arrhenius equation provided a good fit for temperature effects on methane production capacities except for those soils with suppressed production. Acetate amendment strongly enhanced methane production rates and disintegrated the grouping. However, the efficiencies in converting acetate to methane differed among soils. Depending on the soil, 16.5–66.7% of the added acetate was utilized within five weeks incubation at 25 °C.Correlation analyses of methane production (over eight weeks) and physico-chemical soil parameters yielded significant correlations for the concentrations of organic carbon (R² = 0.42) and organic nitrogen (R² = 0.52). Correlation indices could substantially be enhanced by using the enriched fraction of organic carbon (R² = 0.94) and organic nitrogen (R² = 0.77), i.e. the differential between topsoil and subsoil concentrations of the respective compounds. The enriched organic material in the topsoil corresponds to the biologically active fraction and thus represents a good indicator of methane production derived from inherent substrate. The best indicators of the conversion rate of acetate in different soils were pH-value (R² = 0.56) and organic carbon content (R² = 0.52).Apparently, soil properties affect methane production through various pathways. Inherent organic substrate represents a considerable source of methane in some soils and is negligible in others. Likewise, soils also differ regarding the response to exogenous substrate. Both mechanisms yield in a distinct spatial variability of methane production in rice soils.     

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5–76.0% and 13.2–19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m³_biogas/kgCOD_i) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO₂ was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 ± 0.006 g_DM/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%.     

Isolation and characterization of a strictly xylan-degrading Dictyoglomus from a man-made,thermophilic anaerobic environment

A thermophilic, strictly anaerobic eubacterium which utilized an unusually limited range of substrates was isolated from a sludge and pulp sample from a paperpulp cooling tank at a paper-board factory in Finland. The organism grew only with beech wood or oat spelt xylan; no growth occurred with soluble sugars, other polysaccharides, peptone, or yeast extract. The organism was rod-shaped, long (up to 20 m), thin (0.3 m), gramnegative, and in late-exponential and stationary phase cultures formed ball of yarn like structures; endospores were not observed and the organism was not motile. The organism grew fastest (=0.08 – 0.09 h^-1) at 65 to 75°C and pH 6.5 to 8.4, with a maximum growth temperature between 75 and 80°C and an upper pH limit near 9. During growth on beech xylan the isolate produced only acetate, H₂, and CO₂ as fermentation products. The guanine + cytosine (G+C) content of the isolates cellular DNA was 34%. The unusual morphology of the isolate is characteristic of the genus Dictyoglomus, and the limited substrate range, higher G+C ratio, and different fermentation products indicated that the isolate was a new species in that genus.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Purification and properties of an extra cellular xylanase enzyme ofClostridium strain SAIV

An extracellular xylanase enzyme fraction A from a mesophilicClostridium strain SAIV was purified by ammonium sulfate precipitation, Sephadex G-50 gel filtration and DEAE-Sephadex A-50 ion exchange. The xylanase exhibited a molecular weight of 30,000 and it was stable upto 55° C with an optimum temperature of 50° C. It was most stable between pH 5–7, with an optimum pH of around 6. The Km value was 7.0 mg·xylan ml^-1 and V_max was 36 mol·xylose liberated mg^-1 min^-1. Carboxymethyl cellulose, filter paper cellulose and 4-p-nitrophenyl -D-xylopyranoside were not hydrolysed. The specific activity of xylanase fraction A (9.8 U mg^-1) is 2–10 fold higher than the specific activity of xylanase in other mesophilic, xylanolytic, obligate anaerobic bacteria. A minor fraction of xylanase activity designated as xylanase B was also obtained supporting the view that the multiplicity of xylanases is common in microorganisms.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Isolation and characterization of Methanocorpusculum parvum,gen. nov., spec. nov., a new tungsten requiring,coccoid methanogen

A new mesophilic, monotrichously flagellated methane-producing coccus of 1m in diameter was isolated from an anaerobic sour whey digester, originally inoculated with sewage sludge. Growth and methane production were observed with H₂/CO₂, formate and — less effectively — with 2-propanol/CO₂. The isolate grew at temperatures between 15° C and 45° C with the optimum at around 37° C. Acetate, yeast extract and tungstate were required in the medium. Clarified rumen fluid stimulated growth.The DNA of the new methanogen has a G+C content of 48.5 mol%. Comparative 16 S rRNA oligonucleotide cataloguing allows to define the new isolate as a member of a new genus of the order Methanomicrobiales. Further evidence for this is provided by the antigenic crossreactivity with anti-S probes and by metabolic features.Because of its small size the new methanogen is named Methanocorpusculum parvum.This work was supported by a grant of the Deutsche Forschungsgemeinschaft DFG to J. W. and E. S. Immunologic studies were supported in part by grants No. DE-FGO2-84 R 13197 from the U.S. Department of Energy, and No. 261.81/82 from the North Atlantic Treaty Organization (NATO)     

Energieabhängige 63Ni-Aufnahme bei Alcaligenes eutrophus Stamm H 1 und H 16

Zusammenfassung Die Kinetik der Aufnahme von ⁶³Ni wurde an zwei Stämmen von Alcaligenes eutrophus untersucht, die Nickelionen für das chemolithotrophe Wachstum benötigen. Mit Kohlendioxid als einziger Kohlenstoffquelle wird das Wachstum durch niedrige Konzentrationen von Nickel gefördert, wobei das Optimum der Wachstumsförderung bei 0,3 M Nickel lag. Höhere Nickelkonzentrationen wirkten hemmend. Das heterotrophe Wachstum mit Fructose wurde durch Nickelionen nicht gefördert. — Übertragen in Phosphatpuffer, der von Schwermetallionen befreit worden war, zeigten autotroph gewachsene Zellen eine rasche Aufnahme von ⁶³Ni, sofern Wasserstoff, Sauerstoff und Kohlendioxid zugegen waren. Dabei wurde Nickel innerhalb von 60 min aus dem umgebenden Medium bis zur 280 fachen Konzentration in den Zellen angehäuft. Die beobachtete Ni-Aufnahme zeigte ein Temperaturoptimum bei etwa 29° C und wurde durch Hemmstoffe wie Arsenit, Jodacetat, Methylenblau, Natriumazid und Natriumcyanid stark beeinträchtigt. Andere Schwermetallionen (Zn, Co, Mn und Cu) verminderten die Nickelaufnahme nur geringfügig. Durch ⁵⁸NiCl₂ und Toluol wurde der Efflux von ⁶³Ni aus den Zellen gefördert. Die Beobachtungen lassen darauf schließen, daß Nickelionen durch einen energieabhängigen Prozeß in chemolithotroph gewachsenen Zellen dieser Stämme angehäuft werden.

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Energy-dependent ⁶³Ni-uptake by Alcaligenes eutrophus strains H 1 and H 16

Kinetic studies of the uptake of ⁶³Ni were undertaken with two strains of Alcaligenes eutrophus, known to require nickel ions for chemolithotrophic growth. Using carbon dioxide as sole carbon source, growth is stimulated by low concentrations of nickel with optimum concentration for growth stimulation at about 0.3 M nickel. Higher nickel concentrations were inhibitory. Heterotrophic growth on fructose was not stimulated by nickel ions.-Upon transfer into phosphate buffer freed of heavy metal ions, autotrophically grown cells exhibited rapid uptake of ⁶³Ni which was dependent upon th presence of hydrogen, oxygen and carbon dioxide. Within 60 min nickel was accumulated from the medium, reaching 280-fold concentration in the cells. The observed uptake exhibited a temperature optimum at about 29° C and was markedly inhibited by metabolic inhibitors such as arsenite, iodoacetate, methylene-blue, sodium azide and sodium cyanide. Other heavy metal ions (Zn, Co, Mn and Cu) only slightly inhibited ⁶³Ni-uptake. The efflux of ⁶³Ni from the cells was stimulated by ⁵⁸NiCl₂ and by toluene. These data indicate that nickel ions are accumulated by an energy dependent mechanism in chemolithotrophically grown cells of these strains.

     

Upper temperature limits for growth and diazotrophy in the thermophilic cyanobacterium HTF Chlorogloeopsis

The effect of temperature and oxygen on diazotrophic growth of the thermophilic cyanobacterium HTF (High Temperature Form) Chlorogloeopsis was investigated using cells grown in light-limited continuous culture at a dilution rate of 0.02 h^-1. Diazotrophy was more sensitive to elevated temperatures than growth with combined nitrogen. The maximum temperature for growth of cultures gassed with CO₂-enriched air was more than 55 °C but less than 60 °C with N₂ as the sole nitrogen source, but between 60°C and 65°C when nitrate was present in the medium. The effect of temperature on nitrogenase activity, photosynthesis and respiration in the dark was determined using cells grown at 55°C. Maximal rates of all three processes were observed at 55°C and rates at 60°C during shortterm incubations were not less than 75% of the maximum. However, nitrogenase activity at 60°C was unstable and decayed at a rate of 2.2 h^-1 under air and at 0.3 h^-1 under argon. Photosynthesis and respiration were more stable at 60°C than anoxic nitrogen fixation. The upper temperature limits for diazotrophic growth thus seem to be set by the stability of nitrogenase.Abbreviations chl chlorophyll a - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - Taps N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid