Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid     

Untersuchungen zur Photoautotrophie mit molekularem Wasserstoff bei neuisolierten schwefelfreien Purpurbakterien

Zusammenfassung In einer Mineralsalzlösung mit 0,1% NH₄Cl und 0,01% Hefeextrakt wurden aus Schlammproben unter einer Atmosphäre aus 90% H₂ und 10% CO₂ im Licht acht verschiedene Stämme von schwefelfreien Purpurbakterien angereichert und isoliert, die zum photoautotrophen Wachstum mit H₂-CO₂ befähigt sind. Die neuisolierten Stämme wurden aufgrund ihrer mikroskopischen Merkmale und ihrer Fähigkeit zur Verwertung verschiedener organischer C-Verbindungen bestimmt. Drei Rps. capsulata-Stämme benötigen Thiamin als Wachstumsfaktor, zwei R. rubrum-Stämme Biotin und ein Rps. gelatinosa-Stamm benötigt Thiamin und Nicotinsäure. Ein Rps. palustris-Stamm wächst langsam in einem Medium ohne Vitaminzusatz. Der Wachstumsfaktor-Bedarf eines zweiten Rps. palustris-Stammes kann nur durch Hefeextrakt gedeckt werden. Bei allen Stämmen wird das Wachstum durch kleine Mengen (0,01%) Casaminosäuren bzw. Hefeextrakt merklich beschleunigt. Die kürzesten Verdoppelungszeiten beim photoautotrophen Wachstum mit H₂ weisen die drei Rps. capsulata-Stämme auf (17 bis 23 h in einem Mineralmedium mit Thiamin, 10,5–11 h in einem Mineralmedium mit 0,01% Hefeextrakt). Die Hydrogenase-Aktivitäten der Stämme sind in starkem Maße abhängig von der Anzucht der Zellen. Die niedrigsten Aktivitäten werden nach photoheterotropher Anzucht in einem Medium mit NH₄Cl als N-Quelle gemessen, mittlere Aktivitäten nach photoheterotropher Anzucht in einem Medium mit Glutamat als N-Quelle und die höchsten Aktivitäten nach photoautotropher Anzucht mit H₂-CO₂. Die höchsten Raten der H₂-abhängigen CO₂-Fixierung werden ebenfalls nach photoautotropher Anzucht gemessen.

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Studies on the photoautotrophic growth of new isolated nonsulfur purple bacteria at the expense of molecular hydrogen

Summary By incubating samples of mud in a mineral salt medium containing 0.1% NH₄Cl and 0.01% yeast extract under an atmosphere of 90% H₂ and 10% CO₂ in the light, eight strains of nonsulfur purple bacteria capable of photoautotrophic growth at the expense of H₂-CO₂ were enriched and isolated. The bacteria were characterized by their microscopical features and their ability to use various organic compounds for growth. Three strains of Rps. capsulata need thiamine as a growth factor and two strains of R. rubrum biotin. The Rps. gelatinosa strain is dependent on thiamine and nicotinic acid. One of two Rps. palustris strains grows slowly in a medium completely free of growth factors, the other strain is dependent on the presence of 0.01% yeast extract. The growth of all strains is markedly stimulated by small amounts (0.01%) of amino acids or yeast extract. The three Rps. capsulata strains differ significantly from the other strains with regards to their fast photoautotrophic growth (doubling time: 17–23 h in a mineral salt medium with thiamine, 10.5–11 h in a medium with 0.01% yeast extract). The hydrogenase activities of all strains are strongly dependent on the culture conditions. The lowest activities are obtained after photoheterotrophic growth in a medium with NH₄Cl as the nitrogen source, moderate activities after photoheterotrophic growth in a medium with glutamate as the nitrogen source and maximum activities after photoautotrophic growth at the expense of H₂-CO₂. Maximum rates of CO₂-fixation are also obtained after photoautotrophic growth.

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Abkürzungen MB Methylenblau - PHBS Poly--hydroxybuttersäure - R. Rhodospirillum - Rps. Rhodopseudomonas Meinem Vater gewidmet.     

Rhodopseudomonas adriatica sp. nov., a new species of the Rhodospirillaceae,dependent on reduced sulfur compounds

A new purple nonsulfur bacterium was isolated from enrichment cultures of a sulfide-containing marine lagoon. The bacterium is similar to Rhodopseudomonas capsulata and is described as a new species of the genus Rhodopseudomonas: Rhodopseudomonas adriatica. Cells are non-motile, 0.5–0.8 m by 1.3–1.8 m, and multiply by binary fission. Intracytoplasmic membranes are of the vesicular type. The photosynthetic pigments are bacteriochlorophyll a and carotenoids of the spheroidene group. Growth is possible anaerobically in the light and at low pO₂ in the dark. Biotin and thiamine are required as growth factors. A wide variety of organic compounds, as well as sulfide and thiosulfate, are used as photosynthetic electron donors. Sulfide is oxidized to elemental sulfur, which is subsequently converted to sulfate, whereas thiosulfate oxidation occurs without measurable intermediate. Rhodopseudomonas adriatica is unable to assimilate sulfate, growth is only possible in the presence of a reduced sulfur compound.     

Progress toward a biomimetic leaf: 4,000 h of hydrogen production by coating‐stabilized nongrowing photosynthetic Rhodopseudomonas palustris

Intact cells are the most stable form of nature's photosynthetic machinery. Coating‐immobilized microbes have the potential to revolutionize the design of photoabsorbers for conversion of sunlight into fuels. Multi‐layer adhesive polymer coatings could spatially combine photoreactive bacteria and algae (complementary biological irradiance spectra) creating high surface area, thin, flexible structures optimized for light trapping, and production of hydrogen (H₂) from water, lignin, pollutants, or waste organics. We report a model coating system which produced 2.08 ± 0.01 mmol H₂ m^?2 h^?1 for 4,000 h with nongrowing Rhodopseudomonas palustris, a purple nonsulfur photosynthetic bacterium. This adhesive, flexible, nanoporous Rps. palustris latex coating produced 8.24 ± 0.03 mol H₂ m^?2 in an argon atmosphere when supplied with acetate and light. A simple low‐pressure hydrogen production and trapping system was tested using a 100 cm² coating. Rps. palustris CGA009 was combined in a bilayer coating with a carotenoid‐less mutant of Rps. palustris (CrtI^?) deficient in peripheral light harvesting (LH2) function. Cryogenic field emission gun scanning electron microscopy (cryo‐FEG‐SEM) and high‐pressure freezing were used to visualize the microstructure of hydrated coatings. A light interaction and reactivity model was evaluated to predict optimal coating thickness for light absorption using the Kubelka‐Munk theory (KMT) of reflectance and absorptance. A two‐flux model predicted light saturation thickness with good agreement to observed H₂ evolution rate. A combined materials and modeling approach could be used for guiding cellular engineering of light trapping and reactivity to enhance overall photosynthetic efficiency per meter square of sunlight incident on photocatalysts. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Oxidation of Inorganic Sulfur Compounds by Obligately Organotrophic Bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria which are most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Characterization of Rhodopseudomonas capsulata

Thirty-three strains of Rhodopseudomonas capsulata have been studied in order to develop a more comprehensive characterization of the species. On the basis of morphological, nutritional, physiological and other properties, the characteristics of an ideal biotype have been defined, which can be used to distinguish Rps. capsulata from similar purple bacteria. In this connection, two properties of Rps. capsulata are of particular note: a) sensitivity to penicillin G is 10³–10⁵ times greater than that shown by closely related species, and b) all strains examined are susceptible to lysis by one or more strains of host species-specific virulent bacteriophages. It appears that members of the species Rps. capsulata form a stringent taxonomic grouping.     

Seasonal changes in the structure of the anoxygenic photosynthetic bacterial community in Lake Shunet,Khakassia

Seasonal studies of the anoxygenic phototrophic bacterial community of the water column of the saline eutrophic meromictic Lake Shunet (Khakassia) were performed in 2002 (June) and 2003 (February–March and August). From the redox zone down, the lake water was of dark green color. Green sulfur bacteria predominated in every season. The maximum number of green sulfur bacteria was 10⁷ cells/ml in summer and 10⁶ cells/ml in winter. A multi-syringe stratification sampler was applied for the study of the fine vertical distribution of phototrophs in August 2003; the sampling was performed every 5 cm. A 5-cm-thick pink-colored water layer inhabited by purple sulfur bacteria was shown to be located above the layer of green bacteria. The species composition and ratio of purple bacterial species depended on the sampling depth and on the season. In summer, the number of purple sulfur bacteria in the layer of pink water was 1.6 × 10⁸ cells/ml. Their number in winter was 3 × 10⁵ cells/ml. In the upper oxygen-containing layer of the chemocline the cells of purple nonsulfur bacteria were detected in summer. The maximum number of nonsulfur purple bacteria, 5 × 10² cells/ml, was recorded in August 2003. According to the results of the phylogenetic analysis of pure cultures of the isolated phototrophic bacteria, which were based on 16S rDNA sequencing, green sulfur bacteria were close to Prosthecochloris vibrioformis, purple sulfur bacteria, to Thiocapsa and Halochromatium species, and purple nonsulfur bacteria, to Rhodovulum euryhalinum and Pinkicyclus mahoneyensis.     

Chemoautotrophic growth of Rhodopseudomonas species with hydrogen and chemotrophic utilization of methanol and formate

A chemolithoautotrophic type of metabolism, which was hitherto unknown for purple nonsulfur bacteria, was demonstrated by growth experiments using Rhodopseudomonas capsulata Kb1 and Rhodopseudomonas acidophila 10050. These strains were able to grow in a mineral medium in the dark at the expense of H₂, O₂, and CO₂. A minimum doubling time of 9 h was obtained for R. capsulata under an atmosphere containing less than 15% oxygen; higher oxygen concentrations suppressed autotrophic but not chemoorganotrophic growth. Oxygen sensitivity of chemoautotrophically growing cells of R. acidophila was even more pronounced, whereas cells growing chemotrophically on methanol almost tolerated the oxygen concentration of air. Highest oxygen sensitivity of growth of R. acidophila was observed with formate as substrate. The growth yield of cultures grown semiaerobically in the dark on methanol was 0.23 g dry cell material per g methanol consumed.     

Reduced inorganic sulfur oxidation supports autotrophic and mixotrophic growth of Magnetospirillum strain J10 and Magnetospirillum gryphiswaldense

Magnetotactic bacteria are present at the oxic–anoxic transition zone where opposing gradients of oxygen and reduced sulfur and iron exist. Growth of non‐magnetotactic lithoautotrophic Magnetospirillum strain J10 and its close relative magnetotactic Magnetospirillum gryphiswaldense was characterized in microaerobic continuous culture. Both strains were able to grow in mixotrophic (acetate + sulfide) and autotrophic (sulfide or thiosulfate) conditions. Autotrophically growing cells completely converted sulfide or thiosulfate to sulfate and produced 7.5 g dry weight per mol substrate at a maximum observed growth rate of 0.09 h^?1 for strain J10 and 0.07 h^?1 for M. gryphiswaldense. The respiratory activity for acetate was repressed in autotrophic and also in mixotrophic cultures, suggesting acetate was used as C‐source in the latter. We have estimated the proportions of substrate used for assimilatory processes and evaluated the biomass yields per mol dissimilated substrate. The yield for lithoheterotrophic growth using acetate as the C‐source was approximately twice the autotrophic growth yield and very similar to the heterotrophic yield, showing the importance of reduced sulfur compounds for growth. In the draft genome sequence of M. gryphiswaldense homologues of genes encoding a partial sulfur‐oxidizing (Sox) enzyme system and reverse dissimilatory sulfite reductase (Dsr) were identified, which may be involved in the oxidation of sulfide and thiosulfate. Magnetospirillum gryphiswaldense is the first freshwater magnetotactic species for which autotrophic growth is shown.     

Growth of Rhodopseudomonas capsulata under anaerobic dark conditions with dimethyl sulfoxide

The photosynthetic bacterium, Rhodopseudomonas capsulata, could be cultured anaerobically in the absence of light on a synthetic medium with glucose as the carbon source only when dimethyl sulfoxide (DMSO) was added. The extent of growth was proportional to both DMSO and glucose concentrations. Optimal growth was achieved with 20 mm DMSO and 0.25% glucose. Under the best conditions, cells divided with a doubling time of 12 h. Pyruvate also supported the anaerobic dark growth of R. capsulata when DMSO was present. R. capsulata, R. sphaeroides, and R. palustris strains were all able to grow under anaerobic dark conditions with DMSO. Experiments using [¹⁴C]DMSO showed that more than 95% of the ¹⁴C was converted by cultures of R. capsulata to a volatile compound, identified as dimethyl sulfide (DMS) by gas chromatography, thus demonstrating that DMSO was being reduced to DMS during growth. These results indicate that R. capsulata requires a terminal electron acceptor for anaerobic dark growth and that DMSO can serve that function.     

Selective enrichment of green sulfur bacteria in the presence of 4-aminobenzenesulfonate (sulfanilate)

A novel selective enrichment method is described for phototrophic green sulfur bacteria even in the presence of purple sulfur and purple nonsulfur bacteria using sulfanilate, which was discovered during efforts to selectively isolate sulfanilate-metabolizing anoxygenic phototrophic bacteria from marine habitats. Samples for these experiments were obtained from beaches, saltpans, subsurface mangrove soils, fish and prawn aquaculture ponds and backwaters of the East and West coasts of India. Photoorganoheterotrophic and photolithoautotrophic enrichments in the absence of sulfanilate predominantly yielded purple bacterial enrichments. In contrast, photolithoautotrophic enrichments in the presence of sulfanilate yielded green-colored enrichments from the same samples. Whole cell absorption spectra of the enrichment cultures revealed the presence of bacteriochlorophyll c and thus green phototrophic bacteria. Microscopic observation demonstrated the presence of sulfur globules outside the bacterial cells and the presence of non-motile cells, some of which had prosthecae. 16S rDNA sequences obtained from green sulfur bacterial strains isolated from enrichment cultures confirmed the presence of representatives of the green sulfur bacterial genera Prosthecochloris and Chlorobaculum. The selective pressure of sulfanilate exerted through inhibition of phototrophic purple sulfur bacteria was demonstrated by inhibition studies using the purple sulfur bacteria Marichromatium indicum JA100 and Marichromatium sp. JA120 (JCM 13533) and the green sulfur bacterium Prosthecochloris sp. JAGS6 (JCM 13299).     

Coexistence of organisms competing for the same substrate: An example among the purple sulfur bacteria

The purpose of this study was to find a possible explanation for the coexistence of large and small purple sulfur bacteria in natural habitats. Experiments were carried out withChromatium vinosum SMG 185 andChromatium weissei SMG 171, grown in both batch and continuous cultures. The data may be summarized as follows: (a) In continuous light, with sulfide as growth rate-limiting substrate, the specific growth rate ofChr. vinosum exceeds that ofChr. weissei regardless of the sulfide concentration employed. Consequently,Chr. weissei is unable to compete successfully and is washed out in continuous cultures. (b) With intermittant light-dark illumination, the organisms showed balanced coexistence when grown in continuous cultures. The “steady-state” abundance ofChr. vinosum was found to be positively related to the length of the light period, and that ofChr. weissei to the length of the dark period. (c) Sulfide added during darkness is rapidly oxidized on subsequent illumination, resulting in the intracellular storage of reserve substances, which are later utilized for growth. The rate of sulfide oxidation/mg cell N/hr was found to be over twice as high inChr. weissei as inChr. vinosum. The observed coexistence may be explained as follows. In the light, with both strains growing, most of the sulfide will be oxidized byChr. vinosum [see (a)]. In the dark, sulfide accumulates. On illumination, the greater part of the accumulated sulfide will be oxidized byChr. weissei [see (c)]. A changed light-dark regimen should then have the effect as observed [see (b)]. These observations suggest that intermittant illumination may, at least in part explain the observed coexistence of both types of purple sulfur bacteria in nature.     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.     

Distribution of Purple Photosynthetic Bacteria in Wetland and Woodland Habitats of Central and Northern Minnesota

Enrichment cultures for purple nonsulfur and sulfur photosynthetic bacteria were prepared from soil samples collected in central and northern Minnesota. The purple nonsulfur bacteria were found in most wetland soils sampled but were uncommon in woodland and grassland soils. The pH range of the soils in which these bacteria occurred was 3.8 to 7.8, and the oxidation-reduction potential (E(h)) range was +510 to -65 mV. Soils with a pH below 5.0 or an E(h) above +370 mV had few purple nonsulfur bacteria (<10/g of soil). Rhodopseudomonas viridis, a photosynthetic bacterium containing bacteriochlorophyll b, and the purple sulfur bacteria were common only in low-acidity wetland soils that were usually being reduced.     

Anoxygenic phototrophic bacteria from gypsum karst lakes of Lithuania

The spatial distribution and composition of anoxygenic phototrophic bacteria in the enriched bacterial communities from different depths of karst lakes Kirkilai and Ramunelis was studied using spectrophotometric analysis, as well as microbiological and molecular methods. In Lake Kirkilai, the highest bacterial abundance was measured in the metalimnion and near the bottom (up to 10.7 × 10⁶ cell/mL); in Lake Ramunelis it was in the anoxic hypolimnion (up to 22.4 × 10⁶ cell/mL). Increased water mineralization (0.5–1.2 g/L) with the domination of SO ₄ ^2? and Ca²⁺ ions created favorable conditions for the development of sulfate-reducing bacteria; hydrogen sulfide produced as a result of their life activity facilitated the development of sulfur-oxidizing bacteria. The pigment analysis of phototrophic green and purple sulfur bacteria showed the domination of green sulfur bacteria in the enrichment culture. The results of phylogenetic analysis showed that Chlorobium limicola dominated in the enrichment culture for the green sulfur bacteria, whereas purple nonsulfur bacteria of the genus Rhodopseudomonas dominated in the enrichment culture for the purple sulfur bacteria.     

Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

The sulfide affinity of phototrophic bacteria in relation to the location of elemental sulfur

Seventeen strains of phototrophic bacteria (4 strains of Chromatium spp., 2 strains of Thiocapsa sp., 4 strains of Ectothiorhodospira spp., 2 strains of Rhodopseudomonas sp., and 5 strains of Chlorobium spp.) have been grown in sulfide-limited continuous cultures to assess the affinity for sulfide. It was found that the affinity (calculated as the initial slope of the specific growth rate versus the concentration of sulfide) is higher in those phototrophic bacteria that deposit elemental sulfur outside the cells, than in those bacteria that store the sulfur inside the cells. A hypothesis is presented to explain this correlation.Dedicated to Prof. Dr. Hans G. Schlegel on the occasion of his 60th birthday     

Oxidation of inorganic sulfur compounds by obligatory organotrophic bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some of denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N₂, no nitrogenase activity could be detected unless -ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH ₄ ⁺ . In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C₂H₂ reduced · min^-1 · mg^-1 dry weight and 50 nmol methylene blue reduced · min^-1 · mg^-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H₂ produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H₂ stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O₂ in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O₂), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.     

Continuous culture of Thiorhodaceae

Summary In sulfide limited continuous culture of a marine isolate of Chromatium vinosum, sulfide was undetectable in steady states below dilution rates of 0.06h^-1, that is 1/2 of the maximum specific growth rate. In the same range, sulfur is assumed to attain the role of the growth rate limiting substrate. Furthermore, it could be shown that the rate of sulfur oxidation is a function of the surface area of the sulfur globules rather than of the sulfur concentration. In completely filled chemostats, steady states were obtainable only at dilution rates not exceeding 0.09 h^-1. In the presence of a nitrogen flushed gas phase, steady states were obtained at dilution rates approaching the maximum specific growth rate (0.12h^-1). This phenomenon is ascribed to the particular sulfide tolerance of our strain of Chromatium vinosum. The saturation constant and the inhibition constant (lowest, respectively highest total sulfide concentration at which the specific growth rate is equal to one-half of the maximum specific growth rate in the absence of inhibition) were 0.007 mM and 0.85 mM, respectively.The ecological significance of the data is discussed.Contribution No. 2406 from the Woods Hole Oceanographic Institution.