Sulfide Tolerance and Detoxification in Arenicola marina and Sipunculus nudus

SYNOPSIS. The lugworm Arenicola marina and the peanut worm Sipunculusnudus both inhabit intertidal flats. Sulfide concentrationsin the pore water of the sediment can be as high as 340 µMin the habitat of A. marina. Sulfide levels in the burrow waterand in the coelomic fluid increase during low tide. In contrast,S. nudus is only rarely exposed to sulfide. Neither A. marinanor S. nudus is able to exclude sulfide completely from itsbody. However, both worms accumulate only 15 to 40% respectivelyof the external sulfide concentration during hypoxic 1 mM sulfideincubations. During normoxia, sulfide accumulation is more thanten-fold lower than under hypoxic conditions in both species.When oxygen is available, sulfide is oxidized to thiosulfate.The oxidation rate is about 5 times higher in A. marina thanin S. nudus. A major component of sulfide oxidation is due tothe mitochondria of both species. Mitochondrial sulfide oxidationin the tissues of A. marina is insensitive to low cyanide andto high sulfide concentrations, but can be inhibited by salicylhydroxamicacid. The existence of an alternative terminal oxidase is suggested.When the sulfide level in the body of A. marina and S. nudusincreases above a toxic level, both species survive by switchingto an anaerobic metabolism.     

Cellular Energetics of Animals from High Sulfide Environments

SYNOPSIS.Mitochondrial ATP production is influenced by manyfactors, including the adenylate status of the cell, the supplyof reducing equivalents to the electron transport chain, thesupply of oxygen to cytochrome oxidase, and the demand for ATPto do cellular work. Hydrogen sulfide, which is naturally producedin marine sediments, is a poison of aerobic ATP production mainlybecause it inhibits cytochrome oxidase in the electron transportchain. However, most animals from high sulfide environmentsexhibit aerobic respiration, and may avoid sulfide poisoningwith detoxification reactions that may be useful sources ofenergy. Sulfide stimulates ADP phosphorylation in mitochondriaisolated from gills of Solemya reidi, a sulfide-oxidizing symbiont-harboringbivalve, and a P/O ratio near unity indicates that electronsfrom sulfide enter the electron transport chain at the levelof cytochrome c. Current investigations into the effects ofsulfide on oxygen consumption rate, ATP level, cytochrome reductionstate and ciliary beat frequency of symbiont-free gills of themussels Geukensia demissa and Mytilus edulis indicate that animalsfrom high sulfide environments may gain sufficient energy fromsulfide oxidation to support cellular work.     

Multiple Mechanisms Provide Tolerance to Environmental Sulfide in Urechis caupo

SYNOPSIS. During tidal exposure the echiuran worm Urechis caupoencounters toxic sulnde in its burrow microhabitat on the marinemudflat. Although sulnde freely penetrates the two respiratoryepithelia (the body wall and hindgut) and tissue cytochromeoxidases are extremely sulnde sensitive, the worm toleratessulnde concentrations which exceed environmental exposures.Sulnde tolerance results from a suite of sulnde detoxificationmechanisms that extend from the coelomic fluid to the epithelialsurfaces. The coelomic fluid is rich in hemoglobin and hematin,both of which bind sulfide and catalyze the oxidation of sulfideto thiosulfate in vitro. Peripheral defense mechanisms responsiblefor protection of aerobic epithelia from sulfide poisoning mayinclude mucus secretion, sulfide oxidation by symbiotic bacteriaand/or specialized organelles termed SOBs, and sloughing ofsulfide damaged cells. Thiosulfate, the principal detoxificationendproduct produced in vivo, is eliminated primarily by diffusionacross the hindgut, and sulfur metabolites may be sequesteredand eliminated by the anal sacs. Based on a hypothetical model,we conclude that sulfide tolerance in U. caupo is due primarilyto the sulfide oxidation activity of the coelomic fluid andthat the specialized integument and hindgut protect the metabolicallyactive, sulfide-exposed epithelial cells. These sulfide detoxificationmechanisms may allow populations of this filter-feeding wormto exploit nutrient-rich, sulfidic environments.     

PRODUCTION OF HYDROGEN SULFIDE FROM SULFITE BY A COPPER-ADAPTED YEAST

Activity of hydrogen sulfide production from sulfite was studiedusing a copper-resistant yeast strain (R), its parent strain(P), and the culture of R in the medium without copper addition(R(0)). More hydrogen sulfide was produced under aerobic conditionthan under anaerobic condition. Sulfide producing activity wasin the order of R(0)>P>R under either condition. Stationaryphase cells produced more sulfide than logarithmic phase cellswhen cultured without copper, while the reverse was the casewith R, cultured in copper medium. Sulfide production was inhibitedby high concentrations of sulfite and by salicylaldoxime. Differencein the pathway from sulfite to sulfide was suggested betweenthe resistant strain (R and R(0)) and P in that the former wasmore sensitive to these inhibitors. ¹ Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

Metabolic Responses to Sulfur in Lucinid Bivalves

SYNOPSIS. Lucinid bivalves occur widely in habitats rangingfrom subtidal to deep ocean basins. The evidence reported todate supports the contention that all lucinids contain intracellular,sulfur-oxidizing bacterial symbionts which contribute substantiallyto the nutrition of the intact association. These burrowingbivalves are found in both high and low sulfide habitats. Examinationsof sulfur compounds in the hemolymph of lucinids reveal thatthiosulfate may be a key intermediate in the metabolism of sulfidein all members of this family. The presence of free sulfidein the hemolymph of both freshly collected and experimentalclams suggests that the total sulfide oxidation/detoxificationcapacity of the lucinids may be substantially lower than thatfound in other sulfur bacteria/invertebrate symbioses. Intracellulargranules catalyze the oxidation of sulfide in L. floridana,one apparent line of sulfide toxicity defense in this species.These electron-dense granules occur in high densities in thebacteriocytes (bacteria-containing gill cells) of all lucinids.Thiosulfate produced during this detoxification may be availablefor further oxidation by the abundant symbionts housed in thesesame cells. Hemolymph thiol and elementalsulfur levels in clamsfrom low sulfide habitats suggest that these animals have evolvedmechanisms for sulfur acquisition. In sulfur-free seawater,sulfur-starved Lucina floridana are infrequent, periodic ventilators,but in the presence of sulfur show a significant increase inventilation frequency. This periodic ventilation may be a traitof all lucinids that plays a substantial role in reducing metaboliccosts during low nutrient availability.     

Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron transfer in colonocytes

Sulfide (H₂S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20 µM. Sulfide oxidation has been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (< 1 µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.     

Sulfide oxidation coupled to ATP synthesis in chicken liver mitochondria

Chicken liver mitochondria consumed O2 at an accelerated rate when supplied with low concentrations of hydrogen sulfide. Maximum respiration occurred in 10 microM sulfide, and continued more slowly up to concentrations as high as 60 microM. Sulfide oxidation was coupled to adenosine triphosphate (ATP) synthesis, as shown by firefly luciferase luminescence and by measurement of the mitochondrial membrane electrochemical gradient. Synthesis of ATP required low, steady-state concentrations of sulfide (< 5 microM), which were maintained by use of a syringe pump. The ratio of consumed O2 to sulfide changed at low sulfide and O2 concentrations, indicating alternative metabolic reactions and products. In low concentrations of sulfide, presumably most similar to physiological, the O2/sulfide ratio was 0.75. This is the first report of sulfide oxidation linked to ATP synthesis in any organism not specifically adapted to a sulfide-rich environment. We suggest that this may be a widespread mitochondrial trait, and that it is consistent with the hypothesis that mitochondria originated from sulfide-oxidizing symbionts.     

In Situ Determination of Sulfide Turnover Rates in a Meromictic Alpine Lake

A push-pull method, previously used in groundwater analyses, was successfully adapted for measuring sulfide turnover rates in situ at different depths in the meromictic Lake Cadagno. In the layer of phototrophic bacteria at about 12 m in depth net sulfide consumption was observed during the day, indicating active bacterial photosynthesis. During the night the sulfide turnover rates were positive, indicating a net sulfide production from the reduction of more-oxidized sulfur compounds. Because of lack of light, no photosynthesis takes place in the monimolimnion; thus, only sulfide formation is observed both during the day and the night. Sulfide turnover rates in the oxic mixolimnion were always positive as sulfide is spontaneously oxidized by oxygen and as the rates of sulfide oxidation depend on the oxygen concentrations present. Sulfide oxidation by chemolithotrophic bacteria may occur at the oxicline, but this cannot be distinguished from spontaneous chemical oxidation.     

Response of Sulfide:Quinone Oxidoreductase to Sulfide Exposure in the Echiuran Worm Urechis unicinctus

Sulfide is a natural, widely distributed, poisonous substance, and sulfide:quinone oxidoreductase (SQR) is responsible for the initial oxidation of sulfide in mitochondria. In this study, we examined the response of SQR to sulfide exposure (25, 50, and 150 μM) at mRNA, protein, and enzyme activity levels in the body wall and hindgut of the echiuran worm Urechis unicinctus, a benthic organism living in marine sediments. The results revealed SQR mRNA expression during sulfide exposure in the body wall and hindgut increased in a time- and concentration-dependent manner that increased significantly at 12 h and continuously increased with time. At the protein level, SQR expression in the two tissues showed a time-dependent relationship that increased significantly at 12 h in 50 μM sulfide and 6 h in 150 μM, and then continued to increase with time while no significant increase appeared after 25 μM sulfide exposure. SQR enzyme activity in both tissues increased significantly in a time-dependent manner after 50 μM sulfide exposure. We concluded that SQR expression could be induced by sulfide exposure and that the two tissues studied have dissimilar sulfide metabolic patterns. A U. unicinctus sulfide-induced detoxification mechanism was also discussed.     

In situ determination of sulfide turnover rates in a meromictic alpine lake

A push-pull method, previously used in groundwater analyses, was successfully adapted for measuring sulfide turnover rates in situ at different depths in the meromictic Lake Cadagno. In the layer of phototrophic bacteria at about 12 m in depth net sulfide consumption was observed during the day, indicating active bacterial photosynthesis. During the night the sulfide turnover rates were positive, indicating a net sulfide production from the reduction of more-oxidized sulfur compounds. Because of lack of light, no photosynthesis takes place in the monimolimnion; thus, only sulfide formation is observed both during the day and the night. Sulfide turnover rates in the oxic mixolimnion were always positive as sulfide is spontaneously oxidized by oxygen and as the rates of sulfide oxidation depend on the oxygen concentrations present. Sulfide oxidation by chemolithotrophic bacteria may occur at the oxicline, but this cannot be distinguished from spontaneous chemical oxidation.     

Morphological and Ecophysiological Adaptations of the Marine Oligochaete Tubificoides benedii to Sulfidic Sediments

SYNOPSIS. The marine oligochaete worm Tubificoides benedii inhabitscoastal tidal sediments in which sulfide can reach toxic concentrations.The role of external ironsulfide deposition in sulfide detoxificationis discussed together with a review of morphological and ecophysiologicaladaptations of T. benedii to sulfide. The body wall of T. benediiturns black in the presence of sulfide. Histochemical studiesand micro-X-rayanalyses provide evidence for the reaction ofiron in the mucus layer above the cuticle of the worm with environmentalsulfide to produce ironsulfide. The deposited ironsulfides areeither reoxidized or shed off through moulting, a process otherwiseunknown in oligochaetes. However, calculations on the diffusionrate of sulfide into T. benedii show that the deposition ofironsulfides do not play an important role in sulfide detoxification.The first and last few segments of T. benedii are not blackenedby sulfide and do not appear to precipitate sulfide. The diffusionrate of sulfide through these segments is so rapid that internalsulfide concentrations reach levels inhibitory to cytochromec oxidase, the key enzyme of aerobic respiration, within minutes.When internal sulfide concentrations increase to toxic levels,reliance on an anaerobic metabolism represents a successfulmechanism of sulfide tolerance in T. benedii. Metabolic adaptationsto hypoxia and sulfide include the maintenance of aerobic pathwaysdespite low oxygen or high sulfide concentrations and the abilityto gain energy through anaerobic pathways when oxygen and/orsulfide concentrations become limiting     

Textures and traction: how tube‐dwelling polychaetes get a leg up

By controlling the traction between its body and the tube wall, a tube‐dwelling polychaete can move efficiently from one end of its tube to the other, brace its body during normal functions (e.g., ventilation and feeding), and anchor within its tube avoiding removal by predators. To examine the potential physical interaction between worms and the tubes they live in, scanning electron microscopy was used to reveal and quantify the morphology of worm bodies and the tubes they produce for species representing 13 families of tube‐dwelling polychaetes. In the tubes of most species there were macroscopic or nearly macroscopic (~10 μm–1 mm) bumps or ridges that protruded slightly into the lumen of the tube; these could provide purchase as a worm moves or anchors. At this scale (~10 μm‐1 mm), the surfaces of the chaetal heads that interact with the tube wall were typically small enough to fit within spaces between these bumps (created by the inward projection of exogenous materials incorporated into the tube wall) or ridges (made by secretions on the interior surface of the tube). At a finer scale (0.01–10 μm), there was a second overlap in size, usually between the dentition on the surfaces of chaetae that interact with the tube walls and the texture provided by the secreted strands or microscopic inclusions of the inner linings. These linings had a surprising diversity of micro‐textures. The most common micro‐texture was a “fabric” of secreted threads, but there were also orderly micro‐ridges, wrinkles, and rugose surfaces provided by microorganisms incorporated into the inner tube lining. Understanding the fine structures of tubes in conjunction with the morphologies of the worms that build them gives insight into how tubes are constructed and how worms live within them.     

Relationship between the lifestyle of a Siboglinid (Pogonophoran) polychaete, Oligobrachia mashikoi, and the total sulfide and nitrogen levels in its habitat

A gutless polychaete of the family Siboglinidae, Oligobrachia mashikoi, known in the past as a beard worm of the group Pogonophora, inhabits Tsukumo Bay of the Noto Peninsula in the Sea of Japan. Photographs were taken of this polychaete projecting about one third of the length of its tentacles outside of its tube. The tube protruded several mm from the sea bottom. These are the first field photographs of beard worms. The trophosome of this beard worm harbors sulfur-oxidizing bacteria. In fact, the muddy sediment where this worm inhabits smells slightly of hydrogen sulfide. Total sulfide levels, which can be an indicator of the generation of hydrogen sulfide gas, were measured at 10 locations in the bay. Furthermore, at the location which this species inhabits, the total sulfide levels in the vertical direction were determined. In addition, the total nitrogen levels, which can indicate the quantity of organic substances, were measured. The sediment inhabited by this worm was determined to have total sulfide levels of 0.24-0.39 mg/g dry mud, measured in the form of acid-volatile sulfide-sulfur. The total nitrogen levels were 1.0-1.5 microg/mg dry mud. These values suggest that the bottom of Tsukumo Bay has not been deteriorated by eutrophication. The levels were, however, highest in the surface layer of the sediment. These results suggest that hydrogen sulfide is generated in the surface of the sediment by sulfate-reducing bacteria, and that O. mashikoi appears to able to live in an environment that contains a slight amount of sulfide.     

The worm re-turns: hiding behavior of a tube-dwelling marine polychaete, Serpula vermicularis

The polychaete worm Serpula vermicularis (Serpulidae) filterfeeds at the mouth of its calcareous tube, but retreats intothe tube when startled by mechanical stimuli likely to be associatedwith predators. While in its tube, a worm is safe but cannotfeed. Thus, hiding has a lost-opportunity cost. We show thatthis cost can be substantial, given that food in the naturalhabitat appears in pulses, and good feeding conditions may notlast long or recur frequently. We expect that a worm's hidingtime will be sensitive to the lost-opportunity cost, and wepresent data from a series of experiments that support thisprediction. The worms seem able to track relatively short-termchanges in food availability, and some evidence suggests thatthey assess food availability on a relative basis, comparingcurrent feeding conditions to those recently experienced. Hidingand other types of cryptic behavior are common antipredatortactics, and animals may commonly adjust the durations of suchbehaviors to current benefits and costs (including lost opportunity),as they perceive them     

Is succession in wet calcareous dune slacks affected by free sulfide?

Abstract. Consequences of sulfide toxicity on succession in wet calcareous dune slacks were investigated. Sulfide may exert an inhibitory effect on dune slack plants, but several pioneer species exhibit ROL (Radial Oxygen Loss) and thereby protect themselves against free sulfide. Under oxic conditions free sulfide will be oxiginated to harmless sulfate. However, successive species when not capable of ROL may be sensitive to free sulfide and cannot invade the area. Therefore, the occurrence of free sulfide may have a stabilizing effect on the pioneer vegetation. Data on the vertical distribution of oxygen, redox and sulfide were collected in mesocosms with Littorella uniflora or Carex nigra, with and without microbial mats and compared to control mesocosms. Also, in situ data were collected in a dune slack on the Frisian Island of Texel. In the mesocosms, free sulfide was detected only at nighttime in C. nigra populated mesocosms and in unvegetated units, but not in L. uniflora vegetated mesocosms. In the field, sulfide and redox profiles showed distinct differences between the groundwater exfiltration and infiltration site of the dune slack. At the exfiltration site, sulfide was only occasionally found; in contrast, measurable amounts of free sulfide were regularly found at the infiltration site of the slack. Since Phragmites australis dominates in the infiltration site of the slack, the results suggest that free sulfide accelerate the succession, rather than slowing it down by the exclusion of some plant species.     

一株分离自网箱养殖区沉积物的硫氧化菌B1-1的鉴定及其硫氧化特性

【背景】海水网箱养殖是一种高密度、高投饵的人工养殖方式,其产生的硫化物污染严重影响了海水养殖业的经济效益及可持续发展。【目的】鉴定从网箱养殖区沉积物中分离得到的硫氧化菌B1-1并研究其硫氧化特性,为利用生物技术修复网箱养殖环境提供理论依据。【方法】通过形态观察、生理生化特征及16S rRNA基因序列分析对硫氧化菌B1-1进行鉴定;通过摇床培养对该菌株氧化硫代硫酸盐的最适培养条件(pH、温度、底物浓度、外加碳源、外加氮源、金属离子)进行研究。【结果】该菌株被鉴定为HalothiobacillushydrothermalisB1-1。菌株B1-1生物氧化硫代硫酸盐的最适pH为8.0,最适生长温度为30°C,最适底物浓度为5.0g/L;外加碳源对该菌株氧化能力无显著影响,外加铵盐(硫酸铵或草酸铵)可以将延滞期缩短至12 h,添加Mg~(2+)可以显著提高菌株的氧化能力,氧化率达100%。【结论】Halothiobacillus hydrothermalisB1-1耐盐性较强,对硫代硫酸盐氧化率高,可作为修复受硫化物污染网箱养殖环境的潜在菌株资源。     

Light-Dependent Sulfide Oxidation in the Anoxic Zone of the Chesapeake Bay Can Be Explained by Small Populations of Phototrophic Bacteria

Microbial sulfide oxidation in aquatic environments is an important ecosystem process, as sulfide is potently toxic to aerobic organisms. Sulfide oxidation in anoxic waters can prevent the efflux of sulfide to aerobic water masses, thus mitigating toxicity. The contribution of phototrophic sulfide-oxidizing bacteria to anaerobic sulfide oxidation in the Chesapeake Bay and the redox chemistry of the stratified water column were investigated in the summers of 2011 to 2014. In 2011 and 2013, phototrophic sulfide-oxidizing bacteria closely related to Prosthecochloris species of the phylum Chlorobi were cultivated from waters sampled at and below the oxic-anoxic interface, where measured light penetration was sufficient to support populations of low-light-adapted photosynthetic bacteria. In 2012, 2013, and 2014, light-dependent sulfide loss was observed in freshly collected water column samples. In these samples, extremely low light levels caused 2- to 10-fold increases in the sulfide uptake rate over the sulfide uptake rate under dark conditions. An enrichment, CB11, dominated by Prosthecochloris species, oxidized sulfide with a K_s value of 11 μM and a V_max value of 51 μM min⁻¹ (mg protein⁻¹). Using these kinetic values with in situ sulfide concentrations and light fluxes, we calculated that a small population of Chlorobi similar to those in enrichment CB11 can account for the observed anaerobic light-dependent sulfide consumption activity in natural water samples. We conclude that Chlorobi play a far larger role in the Chesapeake Bay than currently appreciated. This result has potential implications for coastal anoxic waters and expanding oxygen-minimum zones as they begin to impinge on the photic zone.     

Polyethylene glycol improves the in vitro growth of Brassica pollen tubes without loss in germination

The culture media for the in vitro pollen assay of Brassicaspecies have so far shown good percentage germination, but limitedpollen tube growth. It is reported here that by lowering sucroseconcentration from 20% to 5 or 10 % and by adding polyethyleneglycol 4000 in the culture medium, high percentage germinationand high tube growth (10 times that in the standard media) canbe achieved in three species of cultivated Brassica. This improvedmedium should be useful in fundamental and applied studies onthe pollen biology of Brassica species. Key words: Brassica, pollen, pollen germination, pollen tube growth, polyethylene glycol     

Dissolved methane oxidation and competition for oxygen in down-flow hanging sponge reactor for post-treatment of anaerobic wastewater treatment

Post-treatment of anaerobic wastewater was undertaken to biologically oxidize dissolved methane, with the aim of preventing methane emission. The performance of dissolved methane oxidation and competition for oxygen among methane, ammonium, organic matter, and sulfide oxidizing bacteria were investigated using a lab-scale closed-type down-flow hanging sponge (DHS) reactor. Under the oxygen abundant condition of a hydraulic retention time of 2h and volumetric air supply rate of 12.95m(3)-airm(-3)day(-1), greater than 90% oxidation of dissolved methane, ammonium, sulfide, and organic matter was achieved. With reduction in the air supply rate, ammonium oxidation first ceased, after which methane oxidation deteriorated. Sulfide oxidation was disrupted in the final step, indicating that COD and sulfide oxidation occurred prior to methane oxidation. A microbial community analysis revealed that peculiar methanotrophic communities dominating the Methylocaldum species were formed in the DHS reactor operation.     

Sulfide utilization by the photosynthetic bacterium Chlorobium phaeobacteroides

Sulfide and sulfur are used by the photosynthetic bacterium Chlorobium phaeobacteroides as electron donors. Sulfide and sulfur consumption was found to be affected by sulfide concentration in the medium. Raising the sulfide concentration from 0.28 mM to 5.05 mM caused an increase in the amount of S⁼ utilized per growth unit from 0.58 mM to 2.32 mM. This increase in sulfide utilization was not reflected in a higher photosynthetic activity. Sulfide and sulfur consumption was also influenced by light intensity, with higher light intensity sulfide consumption was increased. In Lake Kinneret, Chlorobium phaeobacteroides did not bloom in the thermocline layer until sulfide concentrations reached 0.03–0.06 mM.