Stephen J. Molyneaux

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

ABSTRACT A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

Stable isotope studies of the carbon, nitrogen and sulfur cycles in the Black Sea and the Cariaco Trench

Abstract Samples for stable isotope studies of possible chemosynthesis in anoxic basins were collected in 1986 in the Cariaco Trench and May 1988 in the Black Sea. POM (particulate organic matter) collected at oxic/anoxic interfaces in the water column showed no distinctive carbon or nitrogen isotopic compositions that could be associated with chemosynthetic bacteria. Carbon and nitrogen isotopic compositions at POM concentration maxima near the top of the sulfide zone were −23%o and 4.5%o, respectively, in both the Black Sea and the Cariaco Trench. Measurements of dissolved inorganic carbon (DIC) in the Black Sea indicated that carbon respired during decomposition at depth had an isotopic composition of −23%o and was isotopically similar to phytoplankton, with no distinctive component that could be attributed to chemosynthetic carbon. These results indicate that either the biomass of chemosynthetic bacteria in the oxic/anoxic interface zones is low relative to sinking phytoplankton or that chemoautotrophic bacteria have isotopic compositions similar to those of phytoplankton. In the uppermost 50 m of sulfidic waters in the Black Sea, sulfide isotopic compositions changed significantly in a region of sulfide consumption, increasing up to 5%o vs deep-water background values of −40.5%o. These increases in sulfide isotopic compositions may be due to sulfide oxidation mediated by MnO 2 or oxygen, but are not consistent with sulfide oxidation by photosynthetic bacteria. Growth experiments with sulfate-reducing bacteria suggested that part of the increase in sulfide isotopic compositions could be due to rapid rates of sulfate reduction in the oxic/anoxic interface regions.

Chemosynthetic microbial activity at Mid‐Atlantic Ridge hydrothermal vent sites

Chemosynthetic production of microbial biomass, determined by 14CO2 fixation and enzymatic (RuBisCo) activity, at the Mid-Atlantic Ridge (MAR) 23° and 26°N vent sites was found in various niches: warm water emissions, loosely rock-attached flocculent material, dense morphologically diverse bacterial mats covering the surfaces of polymetal sulfide deposits, and filamentous microbes on the carapaces of shrimp (Rimicaris exoculata). The bacterial mats on polymetal sulfide surfaces contained unicellular and filamentous bacteria which appeared to use as their chemolithotrophic electron or energy source either dissolved reduced minerals from vent emissions, mainly sulfur compounds, or solid metal sulfide deposits, mainly pyrite. Moderately thermophilic Chemosynthetic activity was observed in carbon dioxide fixation experiments and in enrichments, but no thermophilic aerobic sulfur oxidizers could be isolated. Both obligate and facultative chemoautotrophs growing at mesophilic temperatures were isolated from all chemosynthetically active surface scrapings. The obligate autotrophs could oxidize sterilized MAR natural sulfide deposits as well as technical pyrite at near neutral pH, in addition to dissolved reduced sulfur compounds. While the grazing by shrimp on the surface mats of MAR metal sulfide deposits was observed and deemed important, the animals’ primary occurrence in dense swarms near vent emissions suggests that they were feeding at these sites, where conditions for Chemosynthetic growth of their filamentous microbial epiflora were optimal. The data show that the transformation of geothermal energy at the massive polymetal sulfide deposits of the MAR is based on the lithoautotrophic oxidation of soluble sulfides and pyrites into microbial biomass.

Sulfide Ameliorates Metal Toxicity for Deep-Sea Hydrothermal Vent Archaea

ABSTRACT The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.

Chemoautotrophic sulfur-oxidizing bacteria from the Black Sea

In contrast to earlier attempts, we were able to isolate nine strains of obligately chemoautolithotropic, sulfur-oxidizing bacteria from three offshore stations in the western basin of the Black Sea (R.V. Knorr Cruise 134-9, Black Sea leg 2). The isolates grew with doubling times averaging 1.3 h over a pH range of 6.5–9.0 in artificial sea water containing thiosulfate. They also oxidized hydrogen sulfide, elemental sulfur and tetrathionate. Although acid producing, growth of the isolates was neutrophilic (optimum pH 7.5). Nitrate or manganese and iron oxides were not utilized as alternate electron acceptors. If acetate was present, not more than 10% of the incorporated carbon was mixotrophically obtained from the organic source. With a DNA base composition range of 37–40 mol % G+C, the new isolates appear to belong to the genus Thiomicrospira (36–44 mol % G+C) rather than Thiobacillus (55–68mol % G+C). Experimental studies on the potential sulfide oxidation by the new isolates under in situ conditions suggest that, above a certain density of active cells (ca 104 ml−1 ), biological sulfide oxidation appears to be able to compete successfully with its spontaneous chemical oxidation.

Effects of Dissolved Sulfide, pH, and Temperature on Growth and Survival of Marine Hyperthermophilic Archaea

ABSTRACT The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments.

Survival and growth of two heterotrophic hydrothermal vent archaea, Pyrococcus strain GB-D and Thermococcus fumicolans , under low pH and high sulfide concentrations in combination with high temperature and pressure regimes

Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature, and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic, heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation. A moderate pH of 5.5–7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors, survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature.