Lysosomes and sulfide-oxidizing bodies in the bacteriocytes of Lucina pectinata, a cytochemical and microanalysis approach

Lucina pectinata is a large tropical clam living deeply burrowed in the black, reducing mud of mangrove swamps. It is known to possess hemoglobin in the cytoplasmic areas of its bacteriocytes, which harbor sulfide-oxidizing bacteria. The bacteriocytes also possess lysosome-like microbodies containing either membrane whorls or electron-dense granules in which free heme compounds have been identified. The cytochemical detection of acid phosphatase and arylsulphatase through EDX (energy-dispersive X-ray) microanalysis strongly suggests that the bacteriocytes of L. pectinata contain, in fact, two different types of microbodies. Some of these (devoid of dense granules) possess a variable amount of lysosomal enzymes and occasionally a limited quantity of iron, which may result from a recycling process of hemoglobin. Their main function seems to be the digestion of a limited proportion of symbiotic bacteria. They represent genuine secondary lysosomes with a functionally acidic pH. The second type of microbodies is characterized by dense granules containing sulfur and iron hemes but no lysosomal enzymes. Their sulfide-oxidizing activity was substantiated by benzyl viologen assay, with Na₂S as a substrate. These microbodies appear to be similar to the sulfide-oxidizing bodies (SOBs) described in the bacteriocytes of other bivalve species with symbiotic thioautotrophic bacteria; however, their sulfide-oxidizing activity appears to be non-enzymatic. They are discrete organelles, characterized by a functionally basic pH and pseudoperoxidasic activity, and have been termed SOBs. Therefore, the bacteriocytes of L. pectinata possess at the same time functional lysosomes and functional SOBs. Received: 17 August 2000 / Accepted: 20 December 2000     

Contrasting effects of sulfide and thiosulfate on symbiotic CO2-assimilation of Phallodrilus leukodermatus (Annelida)

Experiments were conducted in order to specify the reductants responsible for the carbon dioxide fixation of the symbiotic sulfur bacteria in the gutless marine oligochaete Phallodrilus leukodermatus (Annelida) from shallow calcareous sediments in Bermuda. Carbon dioxide-uptake rates were suppressed by S⁼ and stimulated by S₂O₃ ⁼. Individuals which hosted bacteria containing reserve energy substances maintained a high short-term CO₂-uptake activity, while bacteria in worm homogenates and in worms treated with an antibiotic (Baypen) did not show any significant metabolic activity. Absolute uptake rates in P. leukodermatus were usually considerably higher than those reported for other animals harbouring prokaryotic sulfuroxidizing symbionts. Utilization of thiosulfate rather than sulfide is compatible with the preferred occurrence of the worms around the redox discontinuity layer and has been confirmed in other thiobiotic animals. Sulfur stored in the symbiotic bacteria appears to be oxidized to sulfate and be excreted when the worms are held under energy-limited conditions. The data emphasize the complexity of the possible metabolic pathways involved in the oxidation of reduced-sulfur compounds by bacterial symbionts in marine invertebrates.     

Sulfide tolerance and detoxification in shallow-water marine fishes

Hydrogen sulfide is a potent inhibitor of aerobic respiration. Sulfide is produced in sediments, and many species of fish live in association with the bottom. Tolerance tests, enzyme assays, and chromatography of sulfur compounds in thirteen species of shallow-water marine fishes (collected in San Diego, California, USA in 1987–1988) indicate adaptations to sulfide that vary with habitat and lifestyle. Tidal-marsh inhabitants, like Gillichthys mirabilis and Fundulus parvipinnis, have higher tolerance to sulfide (96 h LC₅₀ at 525 to 700 M) relative to outer-bay and open-coast inhabitants (surviving <12 h at much lower concentrations). The cytochrome c oxidase of all species shows high activity and susceptibility to sulfide poisoning, with 50% inhibition at 30 to 500 nM in various tissues. The two marsh species are able to survive at sulfide concentrations already inhibitory to their cytochrome c oxidase and fatal to other species. All species detoxify sulfide by oxidizing it to thiosulfate. All have sulfide-oxidizing activity in the blood, spleen, kidney, liver and gills, which correlates significantly with heme content. Thiosulfate appears in the tissues of sulfide-exposed fish and builds up to high concentrations (up to 2 mM) with stronger and longer exposure. Unexposed fish contain little or no thiosulfate. Sulfide is barely detectable in the tissues, even in high-sulfide exposure tests. We suggest that fish blood, in having high sulfide-oxidizing activity and no cytochrome c oxidase, can act as a short-term first line of defense against sulfide, and thus minimize the amount that reaches the vital organs. The results of this study indicate that sulfide is a significant environmental factor influencing the ecological distribution of marine fishes.     

A conspicuous H<Subscript>2</Subscript>S-oxidizing microbial mat from a high-latitude Arctic fjord (Young Sound,NE Greenland)

A several centimeter thick, H₂S-oxidizing microbial mat was encountered in a high-latitude Arctic fjord. Microscopic investigations revealed that the mat contained numerous filamentous bacteria, and fluorescence in situ hybridization showed that the vast majority of these belonged to -Proteobacteria and that Thiothrix spp. was present. The mat grew on a layer of accumulated decaying macroalgae and was characterized by a complex three-dimensional structure consisting of numerous holes, voids and chimney-like structures. Microsensor measurements performed in situ and in a laboratory-established mesocosm showed a highly dynamic O₂, pH and H₂S distribution, especially around protruding structures of the mat. Detailed microsensor investigations documented that anoxic sulfidic water emerged trough the chimneys and indicated a microcirculation, replenishing the emerging water with oxygenated water in areas were the mat was absent. This governed an efficient advective exchange of solutes and presumably allowed the mat to grow to the extensive thickness of several centimeters.Communicated by M. Kühl, Helsingør     

Microbial activity, oxygen status and fermentation in the gut of the irregular sea urchin Echinocardium cordatum (Spatangoida: Echinodermata)

Microbial activity in the gut of the detrivore irregular sea urchin Echinocardium cordatum (Pennant) was studied by measurements of oxygen profiles, fermentation end products, C/N ratios, pH and concentrations of sulphide and sulphate in the gut segments, including the nodules of the intestinal caecum. The highest oxygen flux and consumption, and highest concentrations of short-chained fatty acids, and the lowest pH values occurred in the anterior stomach segments, including the anterior caecum. The C/N ratios indicate synthesis of microbial biomass in the caeca. The concentration of sulphate was high in the anterior stomach segments, whereas sulphide was only detectable in the nodules of the intestinal caecum. The anterior and intestinal caeca were the major sites for microbial activity, and oxidation of acetate and propionate produced in the two caeca corresponded to at least 9% of the total respiration of the sea urchin. Microbial fermentation, especially in the anterior caecum, seems to be important for the metabolism of E. cordatum, allowing the sea urchin to utilise refractile carbohydrates. The functions of the intestinal caecum are probably both residual fermentation and oxidation of accumulated sulphide. Received: 25 February 1998 / Accepted: 30 June 1998     

Elemental sulfur in the gills of three species of clams containing chemoautotrophic symbiotic bacteria: a possible inorganic energy storage compound

Sulfur content and fine structure were studied for tissues of three species of clams, Lucinoma annulata, Calyptogena elongata and Lucina floridana, which inhabit sulfide-rich environments and whose gills harbor symbiotic sulfur bacteria. Lucinoma annulata and C. elongata were dredged from the Santa Barbara basin, California, USA, at a depth of 480 to 490 m, and Lucina floridana were dug from below the roots of seagrasses in Saint Joseph Bay, Florida, at a depth of 0.25 to 2m. Foot tissue of Lucinoma annulata, without symbionts, had a total sulfur content of 1.4±0.1 (SD) mg 100 mg^-1 dry weight of tissue (%DW). The symbiont-containing gill tissue of different individuals of L. annulata varied in color from dark red to pale yellow, and the total sulfur content was 2.5±0.4% DW in red gills and was 5.6±3.3 % DW in the yellowest gills. Maintenance of L. annulata in the laboratory for 21 d in the absence of sulfide resulted in the loss from the gill of yellow deposits which were elemental sulfur in the form of liquid-crystalline sulfur globules rather than solid orthorhombic sulfur crystals. The foot tissue did not contain elemental sulfur. When examined by freeze-etch microscopy, sulfur globules were found only within bacteria and not in the animal host cytoplasm. Sulfur globules were confined to the periplasmic space of the bacteria. C. elongata and Lucina floridana resembled Lucinoma annulata in the physical form and distribution of elemental sulfur. The absence of elemental sulfur in the animal cytoplasm suggests that its formation from sulfide is not a detoxification scheme to protect animal tissue from sulfide toxicity. The sulfur deposits probably represent inorganic energy reserves that permit the symbiotic bacteria to function even during the temporary absence of external sulfide.     

Etude des eaux rouges dues à la prolifération des bactéries photosynthétiques sulfo-oxydantes dans l'étang du Prévost,lagune saumâtre méditerranéenne

Dystrophic crises or malaígues with red water, were observed in summer, in Mediterranean brackish lagoons near Montpellier, France. During July 1976, the photosynthetic sulfide-oxidizing bacteria causing this phenomenon were isolated from the Prévost lagoon. The most important genera isolated from this red water wereChromatium, Thiocystis andThiocapsa (Chromatiaceae): the predominant species wasThiocapsa roseopersicina. Water and sediment samples from the same lagoon were collected during the winter season (October 1976 and January 1977) and were enriched with organic matter. The samples were incubated in aquaria under artificial light (800 to 1000 lux) at room temperature (ca. 22°C). Eight to 10 days later, red water developed which was similar to that observedin situ in summer. These red waters develop under conditions of anaerobiosis and H₂S production. Such conditions are necessary for the growth of Chromatiaceae. Excessive deposists of organic matter in the lagoon water lead to increased oxygen consumption and overproduction of hydrogen sulfide, which can be oxidized by photosynthetic sulfideoxidizing bacteria. This bacteria bloom (causing the red water) removes the hydrogen sulfide, thus re-establishing aerobiotic conditions in the water of the Prévost lagoon.     

Response of hydrothermal vent vestimentiferan <Emphasis Type="Italic">Riftia pachyptila</Emphasis> to differences in habitat chemistry

Vestimentiferan tubeworms, which rely on intracellular sulfide-oxidizing autotrophic bacteria for organic carbon, flourish at deep-sea hydrothermal vents despite the erratic nature of their habitat. To assess the degree to which differences in habitat chemistry (sulfide, pH/CO₂) might impact host and symbiont metabolic activity, Riftia pachyptila tubeworms were collected from habitats with low (H₂S < 0.0001 mM) and high (up to 0.7 mM) sulfide concentrations. The elemental sulfur content of the symbiont-containing trophosome organ was lower in specimens collected from the low-sulfide site. Symbiont abundance, RubisCO activity, and trophosome carbon fixation rates were not significantly different for individuals collected from low- versus high-sulfide habitats. Carbonic anhydrase activities were higher in the anterior gas exchange organs of R. pachyptila from the low-sulfide habitat. Despite large differences in habitat chemistry, symbiont abundance and autotrophic potential were consistent, while the host appears to tailor carbonic anhydrase activity to environmental CO₂ availability.     

A brominated secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue

The dictyoceratid marine sponge Dysidea herbacea (Keller, 1889) is common in shallow waters of the tropical Pacific Ocean. Polybrominated biphenyl ethers such as 2-(2,4-dibromophenyl)-4,6-dibromophenol (1) are characteristic secondary metabolites of some specimens of this sponge and may represent as much as 12% of the dry weight. We have found 1 to be deposited as conspicuous crystals throughout the sponge tissue. The dominant prokaryotic endosymbiont in the mesohyl of the sponge is a filamentous cyanobacterium (Oscillatoria spongeliae), although a vacuole-containing, heterotrophic bacterium is also present. The cyanobacteria were separated from the sponge cells and heterotrophic bacteria by flow cytometry. Coupled gas chromatography—mass spectrometry and proton nuclear magnetic-resonance spectroscopy revealed that the major brominated Compound 1 isolated from the intact symbiotic association is found in the cyanobacteria and not in the sponge cells or heterotrophic bacteria. This suggests that the production of the compound is due to the cyanobacterium, and not to the sponge or symbiotic heterotrophic bacteria, as had been suggested earlier.     

Metabolic and blood characteristics of the hydrothermal vent tube-worm Riftia pachyptila

Specimens of the hydrothermal vent pogonophoran Riftia pachyptila Jones were collected by submersible at a depth of 2 600 m at the 21°N hydrothermal vent site on the East Pacific Rise (20°50N, 109°06W) in April and May of 1982. The worms were maintained in pressurized aquaria for up to 45 d for metabolic studies. Consumption of O₂ was regulated down to low P_{O 2} (oxygen partial pressure) values; O₂ consumption rates were 0.63 and 1.12 mol g^-1 wet wt h^-1 at 2.5° and 8°C, respectively; such rates were comparable to those previously measured for other pogonophorans. Intact specimens of R. pachyptila (including bacterial symbionts) did not consume significant amounts of CH₄ from the environment. The respiratory quotients, in the absence of added sulfide, indicated that metabolism was mainly heterotrophic. High rates of uptake of dissolved amino acids were recorded for one specimen. The total [CO₂] in the vascular blood and the Hb-containing coelomic fluid were high. Under anaerobic conditions, there were equilibrium distributions of pH, total [CO₂] and sulfide concentrations between the vascular blood and the coelomic fluid, apparently because these metabolites were readily exchanged between the two compartments. The vascular blood bound neither CH₄ nor H₂. However, sulfide was reversibly bound by both the vascular blood and coelomic fluid; because this binding depended strongly on pH (with a maximum at about 7.5), HS^- was probably the molecular species bound. Under anaerobic, but not aerobic conditions, the trophosome bound substantial amount of sulfide; thus, the high concentrations of sulfide in the trophosome may have resulted mainly from sulfide bound to sulfide oxidases under anaerobic conditions. The coelomic fluid had a relatively low buffering capacity (2.2 mmol CO₂pH^-1).     

The color of the trophosome: elemental sulfur distribution in the endosymbionts of<Emphasis Type="Italic"> Riftia pachyptila</Emphasis> (Vestimentifera; Siboglinidae)

Riftia pachyptila Jones, 1981, lives in association with a chemoautotrophic, sulfide-oxidizing -Proteobacterium that occurs in a specialized organ, the trophosome. Ultrastructurally different bacterial subpopulations occur in different regions of the trophosome lobules (central rods, median small cocci, peripheral large cocci) and contain vesicles, which have been proposed to be sites of elemental sulfur storage. Differently colored trophosomes have been suggested to reflect different amounts of elemental sulfur in the tissue. In this study, the presence of elemental sulfur (S⁰) was confirmed in the vesicles of the symbionts of R. pachyptila by electron energy loss spectrography (EELS). The proportion of (two-dimensional) area occupied by sulfur vesicles in the cells was found to be strongly correlated with trophosome color, both in individuals with uniformly colored trophosomes and individuals that exhibited a gradual color change along the length of their trophosomes. Elemental sulfur content was highly variable between individuals from a single collection, suggesting a high degree of microhabitat heterogeneity within vestimentiferan aggregations.Communicated by O. Kinne, Oldendorf/Luhe     

Metabolic studies on thiobiotic free-living nematodes and their symbiotic microorganisms

The marine, free-living Stilbonematinae (Nematoda: Desmodoridae) are remarkable for the ectosymbiotic, prokaryotic microorganisms that populate their entire body surface. These nematodes occur in sulfidic sediments in the microoxic zone just above the sulfide maximum. Several facts point to a chemolithotrophic, sulfide oxidizing nature of the microorganisms. The oxygen uptake of three species was measured with and without their microbial coat using Cartesian and Gradient Diver microrespirometry in February 1989 at Carrie Bow Cay (Belize Barrier Reef). Symbiont-free stilbonematids exhibited constant and uniform oxygen uptake rates over several hours; rates which are significantly lower than those of oxyphilic nematodes. Freshly extracted stilbonematids, with intact bacterial coats, consumed significantly more oxygen than symbiont-free worms in the first 3 h of measurement. While the rates of aposymbiotic worms were more or less constant over time, the rates of symbiont-carrying worms exhibited a conspicuous drop during prolonged respiration. InStilbonema sp., symbiont carrying individuals kept under oxygenated conditions for more than 12 h had a respiration rate similar to those of aposymbiotic specimens. When such worms were re-incubated in sulfide-enriched seawater the respiration rate was significantly elevated. The possibility of recharging the oxygenated symbiosis system via sulfide-uptake is seen as an indication that storage of reduced sulfur compounds, or reserve substances synthetized in the presence of sulfide, play a decisive role in the metabolisms of the symbiotic bacteria. Migration of nematodes between sulfidic and oxidized sediment-layers are, most likely, the key to understanding the success of this nematode-bacteria symbiosis.Please address all correspondence and requests for reprints to Professor J. Ott     

Oxygen dependent sulfide detoxification in the lugwormArenicola marina

The lugwormArenicola marina L. oxidizes entering sulfide to thiosulfate. After 8 h of normoxic incubations with sulfide concentrations of 0.2 to 1.0 mmoll^-1 thiosulfate in the coelomic fluid amounted up to about 4 mmoll^-1 whereas sulfite concentrations were 100-fold lower and no accumulation of sulfate in the coelomic fluid was found. The sulfide oxidation was highly oxygen dependent. An increase of oxygen partial pressure ( ) in the medium was followed by enhanced thiosulfate production and by a decrease of sulfide concentration in the coelomic fluid. Under normoxia, the sulfide oxidation rate was sufficient to compensate the influx of sulfide into the coelomic fluid when the sulfide concentration in the medium was below 0.33 mmoll^-1. When external sulfide was raised beyond this level, sulfide up to 5 moll^-1 in the coelomic fluid appeared. Succinate in the body wall tissue was low as long as no sulfide appeared in the coelomic fluid, indicating the maintenance of an aerobic metabolism. The oxidation of sulfide to thiosulfate was localized in the mitochondria of the body wall tissue. The oxygen consumption of mitochondria was stimulated by the addition of sulfide. The mitochondrial sulfide oxidation rate depended on the amount of mitochondrial protein and followed a Michaelis-Menten kinetic. An apparentK _m of 0.68±0.29 moll^-1 and aV _max of 41.9±22.3 nmol min^-1 mg^-1 protein was calculated. Sulfide was stoichiometrically oxidized to thiosulfate with 1 mol sulfide consuming 1 mol oxygen. Sulfide oxidation was not inhibited by sulfide concentrations as high as 100 moll^-1. At low concentrations of cyanide or azide, when respiration without sulfide was already inhibited, sulfide oxidation could still be stimulated, tentatively indicating the existence of an alternative terminal oxidase. Specimens examined in the present study were collected near St. Pol de Leon, France, from 1989 to 1992.     

Effects of sulfide exposure history and hemolymph thiosulfate on oxygen-consumption rates and regulation in the hydrothermal vent crab Bythograea thermydron

The hydrothermal vent crab Bythograea thermydron is exposed to high environmental concentrations of sulfide and low levels of oxygen for extended periods of time. It has previously been shown that hydrogen sulfide is oxidized to the relatively non-toxic thiosulfate (S₂O ₃ ^2– ), which accumulates in the hemolymph. Hemolymph thiosulfate levels in freshly captured crabs vary significantly among crabs from different hydrothermal vent sites as well as between crabs from different microhabitats within the same site. Hemolymph thiosulfate concentrations were not significantly different between crabs captured at the same site 6 mo apart. Hemolymph thiosulfate concentrations ranged from 66 mol 1^–1 in a crab captured at a site with relatively low sulfide venting, to 3206 mol 1^–1 in an individual that was netted from an active smoker vent with much higher sulfide exposure. The differences in hemolymph thiosulfate between sites and the stability of hemolymph thiosulfate in crabs captured at the same site at different times suggest that sulfide exposure is significantly different between sites and that this exposure may not vary significantly over the course of a few months. B. thermydron experimentally exposed to sulfide had high levels of thiosulfate in their hemolymph and increased abilities to regulate oxygen consumption in conditions of low oxygen. This enhancement of regulatory abilities suggests that the previously demonstrated increased hemocyaninoxygen (Hc–O₂) affinity due to elevated thiosulfate may be adaptive in vivo. Average oxygen-consumption rates were much higher in crabs experimentally exposed to sulfide than in unexposed crabs. Crabs injected with isosmotic thiosulfate did not have increased oxygen-consumption rates as did the sulfide-exposed individuals, but did show a similar reduction in P _c (the critical partial pressure of oxygen at which crabs can no longer regulate oxygen consumption). This suggests that it is the sulfide exposure and/or detoxification rather than the elimination of thiosulfate that causes the increase in metabolic rate. Thiosulfate diffuses into dead crabs and into live crabs exposed to 15 mmol S₂O ₃ ^2- l^–1, indicating substantial permeability, and yet live crabs are able to eliminate thiosulfate when incubated in sea water containing 1.5 mmol S₂O ₃ ^2- l^–1, suggesting a process that has an active component.     

Bacterial sulfate reduction within reduced microniches of oxidized marine sediments

Bacterial sulfate reduction was demonstrated in the oxidized surface layers of a coastal marine sediment using a radiotracer technique. The obligate anaerobic process takes place within reduced sediment pellets of 50 to 200 m diameter. The H₂S produced diffuses out into the interstitial solution and is oxidized before any detectable accumulation takes place. This microniche structure explains the presence of sulfate-reducing (Desulfovibrio spp.) and sulfide oxidizing (Beggiatoa spp.) bacteria and of ferrous sulfide and pyrite in the oxidized sediment. Sulfate reduction was also demonstrated within detrital particles experimentally decomposed in oxic seawater or sediment. The limiting conditions for the maintenance of a reduced microniche within an oxic environment is discussed in terms of a theoretical model.     

Role of glutamine synthetase in ammonia assimilation by symbiotic marine dinoflagellates (zooxanthellae)

The dinoflagellate symbionts (zooxanthellae) present in many reef corals aid in the survival of the symbiotic unit in nitrogen deficient tropical waters by providing additional routes of nitrogen uptake and metabolism. The enzymatic pathway of ammonia assimilation from seawater and the re-assimilation of coral ammonium waste by zooxanthellae was studied by examining the affinity of glutamine synthetase for one of its substrates, ammonia. Glutamine synthetase activity was measured in dinoflagellates of the species Symbiodinium microadriaticum found in symbiotic association with various marine coelenterates. Michaelis-Menten kinetics for the substrate ammonia were determined for freshly isolated dinoflagellates from Condylactis gigantea (apparent NH₃ K_m=33 M) and for cultured dinoflagellates from Zoanthus sociatus (apparent NH₃ K_m=60 M). On the basis of the low apparent K_ms for NH₃, it appears that ammonia assimilation by these symbiotic dinoflagellates occurs via the glutamine synthetase/glutamate synthase pathway. Additionally, the uptake of exogenous ammonium by an intact coelenterate-dinoflagellate symbiosis was strongly inhibited by 0.5 mM methionine sulfoximine, and inhibitor of glutamine synthetase.     

Stromatolitoid microbial nodules from Bermuda —a special micro habitat for meiofauna

In Bermuda, stromatolitoid microbial nodules are found in dense groups in seagrass beds and on subtidal sandy or rocky bottoms. They develop between January and the beginning of August and may reach 15 cm in diameter and 6 cm in height. Nodules grow on top of the sediment surface and consist of convex interwoven mats of the cyanobacterium Phormidium corium and fine sediment particles trapped in these mats. Nodules were studied in the field and laboratory (in 1989–1992) with respect to their structure, microbial community, sediment chemistry and associated meiofauna. Vertical profiles taken with microelectrodes showed steep gradients of oxygen and sulfide. While free oxygen was only detected in the upper 2 mm, the sulfide concentrations increased with depth and reached maximal 1250 moll^-1 inside the nodules. On the basis of microscopic observations and sediment chemistry, vertical sections through a nodule reveal four distinct layers: (A) an oxic green surface layer of growing cyanobacteria mats, (B) an anoxic yellow-white laminated layer of sediment particles, mucus and unpigmented cyanobacteria sheaths, (C) an anoxic sulfidic (sulfide <50 moll^-1) interface where colour changes from yellow to gray and (D) an anoxic sulfidic (sulfide can increase to 1250 moll^-1) gray core composed of sediment and decomposing cyanobacteria. The following meiofauna taxa were found in the nodules: Ciliata, Turbellaria, Gnathostomulida, Gastrotricha, Nematoda, Kinorhyncha, Polychaeta, Ostracoda and Harpacticoida. The distribution of the meiofauna was significantly different (p<0.05) within the four layers. The highest density of individuals was found in the sulfidic interface and core. Nematodes represented the dominant group in general. Thiobiotic organisms, such as the Gnathostomulida, Solenofilomorphidae, and Stilbonematinae were primarily found in the anoxic sulfidic layers of the nodules.     

The bacterial symbiont from the hydrothermal vent tubewormRiftia pachyptila is a sulfide specialist

We have investigated the metabolic adaptations of the chemolithotrophic bacterial symbionts ofRiftia pachyptila. Specimens of the tubeworm were collected by submersible from depths of 2600 m at 13°N on the East Pacific Rise in 1987, and 2450 m at the Galápagos Rift in 1988. Isolated bacteria utilize sulfide, but not thiosulfate or sulfite, as their sole reduced-sulfur energy source. The bacteria rapidly oxidize a wide range of sulfide concentrations (5µM to 2 mM), with maximal respiration rates at concentrations >1 mM, and unlike many sulfur-oxidizing bacteria, show no inhibition in oxygen consumption at sulfide concentrations up to 2 mM. Incubations of freshly homogenized trophosome tissue or isolated bacteria with sodium [³⁵S] sulfide and subsequent analysis of sulfur products by high-performance liquid chromatography and flow-through scintillation counting showed that sulfide disappeared almost completely within 1 min. Both soluble and insoluble products of sulfide oxidation were produced. The soluble fraction contained sulfate and polysulfides, with no thiosulfate produced. However, the majority of the radioactivity was in the water-insoluble fraction, mostly as elemental sulfur. Whole-worm experiments under pressure showed a rapid removal of³⁵S-sulfide from the incubation water, with sulfide, sulfate, and polysulfides appearing in the blood within 4 h. There was no utilization of thiosulfate by the whole worms, freshly homogenized trophosome tissue, or isolated bacteria.     

The methane mussel: roles of symbiont and host in the metabolic utilization of methane

Methane mussels (Bathymodiolus sp., undescribed; personal communication by R. Turner to CRF) were collected in September 1989 and April 1990 from offshore Louisiana in the Gulf of Mexico. These mussels contain endosymbiotic methane-oxidizing bacteria and are capable of utilizing environmental methane as a source of energy and carbon. Oxygen consumption, methane consumption, and carbon dioxide production were measured in mussels with intact symbionts, functionally aposymbiotic mussels, and separated symbiont preparations under controlled oxygen and methane conditions, in order to study the roles of the symbionts and the hosts in methane utilization. The association was found to be very efficient in fixing methane carbon (only 30% of CH₄ consumed is released as CO₂), and to be capable of maximal rates of net carbon uptake of nearly 5 mol g^-1 h^-1. Rates of oxygen and methane consumption were dependent upon oxygen and methane concentrations. Maximal consumption rates were measured at 250 to 300 M O₂ and 200 to 300 M CH₄, under which conditions, oxygen consumption by the gill tissues (containing symbionts) had increased more than 50-fold over rates measured in the absence of methane. A model is proposed for the functioning of the intact association in situ, which shows the symbiosis to be capable of achieving growth rates (net carbon assimilation) in the range of 0.003 to 0.50% per day depending upon oxygen and methane concentrations. Under the conditions measured in the seep environment (200 M O₂, 60 M CH₄), a mussel consuming methane at rates found to be typical (4 to 5 mol g^-1 h^-1) should have a net carbon assimilation rate of about 0.1% per day. We suggest that the effectiveness of this symbiosis arises through integration of the morphological and physiological characteristics inherent to each of the symbiotic partners, rather than from extensive specialization exhibited by other deep-sea chemotrophic associations.