Sulphide tolerance and adaptation in the California killifish, Fundulus parvipinnis, a salt marsh resident

Hydrogen sulphide is a toxicant naturally produced in hypoxic marine sediments, hydrocarbon and brine seeps and hydrothermal vents. The California killifish, a salt marsh resident, is remarkably tolerant of sulphide. The 50% lethal concentration is 700 μM total sulphide in 96 h, and 5 mM in 8 h (determined in flow-through, oxygenated sea water). Killifish exposed to sulphide produce thiosulphate which accumulates in the blood. The cytochrome c oxidase (a major site of toxicity) of the killifish is 50% inhibited by <1 μM sulphide. Killifish liver mitochondria are poisoned by 50–75 μM sulphide but can oxidize 10–20 μM sulphide to thiosulphate. Sulphide causes sulphhaemoglobin formation (and impairment of oxygen transport) at 1–5 mM in vitro and to a small extent at 2 mM in vivo . Killifish blood neither catalyses sulphide oxidation significantly nor binds sulphide at environmental (low) sulphide concentrations. Exposure to 200 μM and 700 μM sulphide over several days causes significant increases in lactate concentrations, indicating shift to anaerobic glycolysis. However, individuals with the most lactate die. In terms of diffusible H₂S, the killifish can withstand concentrations two to three orders of magnitude greater than would poison cytochrome c oxidase. The high sulphide tolerance of the killifish, particularly of concentrations typical of salt marshes, can be explained chiefly by mitochondrial sulphide oxidation. Sulphide tolerance and mitochondrial sulphide oxidation in the killifish have a constitutive basis, i.e. do not diminish in fish held in the laboratory in sulphide-free water for 1–2 months, and are improved by prior acclimation.     

Biotechnological process for sulphide removal with sulphur reclamation

A new biotechnological process for sulphide removal is proposed. The process is based on the oxidation of sulphide into elemental sulphur, which can be removed by sedimentation. In this study it was found that elemental sulphur and sulphate are the main oxidation products of the biological sulphide oxidation. The settling characteristics become worse as the sulphide concentration increases, due to polysulphide formation. The start-up phase of this biological system is very short; Only four days are needed to reduce the sulphide concentration of 100 to 2 mg/l at a HRT (Residence time) of 22 minutes. Also some environmental factors were evaluated. The optimal pH is situated in the pH-range 8.0–8.5. Significantly lower conversion rates are found at pH = 6.5 to 7.5 and pH = 9.0, while at pH = 9.5 the sulphide oxidation capacity of the system detoriates. The process temperature was 20°C, although the optimal temperature is situated in the range 25–35°C. No substrate inhibition of sulphide was found at sulphide concentrations up to 100 mg/l.     

Oxidation of sulphide by cytochrome aa3

The effectiveness of H2S as an inhibitor of cytochrome c oxidase increase (Ki decreases) with sulphide concentration. A spectroscopic change in cytochrome aa3 is induced aerobically by sulphide at the same rate as that calculated for inhibition. The initial spectroscopic product is not inhibited, but an 'oxygenated' (oxyferri) form of the enzyme. Stoichiometric sulphide addition to cytochrome aa3 under anaerobic conditions produces another low-spin form of the enzyme; subsequent admission of oxygen gives rise to the 607 nm compound. At high enzyme levels sulphide itself acts as a substrate measured polarographically, with an oxygen uptake proportional to the amount of sulphide added. Binding of sulphide to ferric enzyme probably causes reduction at the oxygen-sensitive a3-Cu centre, which is followed aerobically by reoxidation to the oxyferri state via the 607 nm intermediate. A stable sulphide complex is formed only after the reduction of cytochrome a; but once formed this inhibited species is retained if cytochrome a is reoxidized.     

Optimization of sulphur production in a biotechnological sulphide-removing reactor

A new biotechnological process for sulphide removal is proposed. The principle of this process is that sulphide is converted into elemental sulphur, which can be removed by sedimentation. In this article, investigations on the optimization of the sulphur production are reported. It seems that less than 10% sulphate is produced at low oxygen concentration, when the sulphide concentration in the reactor exceeds 10 mg/L. At sulphide concentrations higher than 20 mg/L only 5% of the incoming sulphide is converted to sulphate even at high oxygen concentrations. An immobilized biomass on recticulated polyurethane produced more sulphate than a free cell suspension at the same oxygen and sulphide concentration.     

The endogenous production of hydrogen sulphide in intrauterine tissues

Background  

Hydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro.     

Sulphide utilization and injuries in hypoxic roots and rhizomes of common reed (Phragmites australis)

The presented investigations have been carried out in order to estimate toxic sulphide levels and to examine detoxification capabilities in roots and rhizomes of the common reed (Phragmites australis). Underground organs of common reed are sensitive towards sulphide above 1 mM applied exogenously under hypoxia. However, certain tolerance may be achieved by sulphide detoxification. Accumulated sulphide is partially used for the synthesis of non-toxic thiols, mainly glutathione. But the detoxification capacity of the underground organs is limited. Maximum concentrations of thiols are about 60 nmol/g^?1 fw in roots and 300 nmol/g^?1 fw in rhizomes. Energy metabolism is considerably affected by low sulphide concentrations of 1 mM for 4 days, and immediately disturbed by increased concentrations up to 6 mM sulphide. Adenylate energy charge, total adenylates, posthypoxic respiration, and fermentation capacity decrease significantly. Roots are more sensitive than rhizomes.     

Biochemical and biophysical studies on cytochrome c oxidase. XX. Reaction with sulphide.

1.Upon addition of sulphide to oxidized cytochrome c oxidase, a low-spin heme sulphide compound is formed with an EPR signal at gx = 2.54, gy = 2.23 and gz = 1.87. Concomitantly with the formation of this signal the EPR-detectable low-spin heme signal at g = 3 and the copper signal near g = 2 decrease in intensity, pointing to a partial reduction of the enzyme by sulphide. 2. The addition of sulphide to cytochrome c oxidase, previously reduced in the presence of azide or cyanide, brings about a disappearance of the azido-cytochrome c oxidase signal at gx = 2.9, gy = 2.2, and gz = 1.67 and a decrease of the signal at g = 3.6 of cyano-cytochrome c oxidase. Concomitantly the sulphide-induced EPR signal is formed. 3. These observations demonstrate that azide, cyanide and sulphide are competitive for an oxidized binding site on cytochrome c oxidase. Moreover, it is shown that the affinity of cyanide and sulphide for this site is greater than that of azide.     

The interaction of human neuroglobin with hydrogen sulphide

Neuroglobin has been identified to protect brain neurons from apoptotic stress. Hydrogen sulphide has a role in the brain as a neuromodulator, involving NMDA receptor activation. Here we report on studies of the in vitro interaction of ferric neuroglobin with hydrogen sulphide. Hydrogen sulphide binds very tightly to the heme group of neuroglobin in a biphasic reaction. The faster of the two reaction processes is concentration dependent whilst the slower process is not. The rate of hydrogen sulphide binding is pH sensitive and as the pH is reduced over the physiological range the rate of reaction increases by a factor of approximately 10. This change in reactivity appears to reflect the ionisation of the heme distal His ligand rather than a preference for the binding of H(2)S. We discuss the potential role of neuroglobin in the modulation of hydrogen sulphide sensitivity of neurons in the brain.     

Effect of organic substrates on biological sulphide oxidation

Summary Neither acetate nor higher fatty acids and glucose have a significant effect on the biotechnological process for sulphide removal at 20° C, in which sulphide is oxidized to sulphur using oxygen. The oxidation of acetate and propionate with oxygen is mainly dependent on the sulphide and oxygen concentrations in the reactor. The occurrence of Thiothrix filaments in sulphide-removing waste-water treatment systems has been investigated using a fixer-film upflow reactor. The influent of this reactor consisted of anaerobically treated paper-mill waste-water, with a sulphide concentration of 140 mg/1. It was found that sulphide loading rate is the decisive parameter as to whether or not Thiothrix will develop in a sulphide-removing reactor. Offprint requests to: C. J. N. Buisman     

Bacterial oxidation of sulphide under denitrifying conditions

Anoxic H2S oxidation under denitrifying conditions produced sulphur and sulphate in almost equal proportions by an isolated Thiobacillus denitrificans. Under nitrate reducing conditions the rate of sulphide oxidation was approximately 0.9 g sulphide/g biomass h. Nitrate was reduced to nitrite and accumulated during sulphide oxidation. Above 100 mg nitrite/l, the sulphide oxidation rate declined and at 500 mg/l it was totally arrested. The optimum pH for the anoxic sulphide oxidation was around 7.5. Concentrations of sulphate 1500 mg/l and acetate 400 mg/l had no effect on anoxic sulphide oxidation.     

Engineering the active site of ascorbate peroxidase.

The oxidation of a number of thioethers, namely methyl phenyl sulphide (1), ethyl phenyl sulphide (2), isopropyl phenyl sulphide (3), n-propyl phenyl sulphide (4), p-chlorophenyl methyl sulphide (5), p-nitrophenyl methyl sulphide (6) and methyl naphthalene sulphide (7), by recombinant pea cytosolic ascorbate peroxidase (rAPX) and a site-directed variant of rAPX in which the distal tryptophan 41 residue has been replaced with an alanine (W41A) has been examined. The electronic spectrum (pH 7.0, mu = 0.10 M, 25.0 degrees C) for the ferric derivative of W41A (lambda(max)/nm = 411, 534, 560, 632) is indicative of an increased quantity of 6-coordinate, high-spin and/or 6-coordinate, low-spin haem compared to rAPX. Steady state oxidation of sulphides 1-4 and 7, gave values for kcat that are approximately 10-fold and 100-fold, respectively, higher for W41A than for rAPX. For rAPX, essentially racemic mixtures of R- and S-sulphoxides were obtained for all sulphides. With the exception of sulphide 7, the W41A variant shows substantial enhancements in enantioselectivity, with R : S ratios varying between R : S = 63 : 37 (sulphides 1 and 4) and R : S = 85 : 15 (sulphide 6). Incubation of sulphide 2 with rAPX or W41A and [(18)O] H(2)O(2) shows 95% (rAPX) and 96% (W41A) transfer of labelled oxygen to the substrate. Structure-based modelling techniques have provided a fully quantitative rationalization of all the experimentally determined R : S ratios and have indicated that reorientation of the sidechain of Arg38, such that access to the haem is much less restricted, is influential in controlling the stereoselectivity for both rAPX and W41A.     

Sulphide intrusion in eelgrass (Zostera marina L.)

Sudden events of seagrass die‐off have been suggested to be induced by invasion of the phytotoxin sulphide under environmental stress generating low oxygen supply in seagrass tissues. Laboratory experiments were conducted with eelgrass (Zostera marina L.) to measure intra‐plant changes in oxygen and sulphide by means of microelectrodes at different oxygen concentrations in the water column. The objectives were to examine whether sulphide intrusion into seagrass tissues can be induced, to determine the role of plant oxygen status for sulphide intrusion and to determine how fast internal sulphide pools are depleted after internal oxygen supplies have been restored. Under conditions with oxygen partial pressures (pO₂) above 7.4 kPa (> 35% of air saturation) within eelgrass rhizomes or meristematic tissues no intrusion of sulphide occurred in spite of high sediment concentrations of gaseous sulphide (> 1000 µm ). Lack of sulphide intrusion at high internal pO₂ suggested that oxygen release from the roots ensured complete re‐oxidation of sulphide in the rhizosphere. Under oxygen stress, however, the experiments clearly demonstrated intrusion of sulphide in eelgrass rhizomes and meristematic tissues. Rates of sulphide intrusion were controlled by internal pO₂, which in turn was controlled by water column oxygen concentrations. Maximum internal sulphide content reached 325 µm which by far exceeded the 1–10 µm known to inhibit mitochondrial activity in eukaryotic cells. Sulphide and low levels of oxygen could coexist in the eelgrass tissues reflecting fast internal transport of sulphide and slow rates of sulphide re‐oxidation. Upon re‐establishment of high internal oxygen concentrations the depletion of the sulphide pool was slow (half‐life = 20–30 min) indicating, that sulphide re‐oxidation within the eelgrass tissue was not bacterially or enzymatically facilitated but occurred by simple chemical oxidation. The results of this study are consistent with the proposed detrimental role of sulphide intrusion in events of sudden seagrass die‐off.     

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

 Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity. Received: 23 May 1995/Received revision: 10 October 1995/Accepted: 16 October 1995     

Suboxic zone in the Black Sea: real fact or analytical artefact

A reevaluation of the presence of a suboxic zone above the sulphide onset and the fine structure of the oxic/anoxic interface in the Black Sea is discussed. The results qualify this phenomenon as an analytical artefact and that the methods used for dissolved oxygen (DO) profiling are not applicable in the situation. Former and recent studies have established the stability of the oxic/anoxic interface structure with two principal components: the low DO gradient zone situated at the base of oxycline, and of the C-layer located beneath the sulphide onset boundary, where DO and sulphide coexist. The existence of the C-layer does not permit a suboxic zone void of perceptible DO and sulphide.     

Survivorship of juvenile surf clams Donax serra (Bivalvia, Donacidae) exposed to severe hypoxia and hydrogen sulphide

Toxic “sulphide eruptions” sporadically occur in the highly productive inshore regions of the central Namibian Benguela upwelling system. The surf clam Donax serra (Röding, 1798) dominates the intertidal and upper subtidal of large exposed sandy beaches of southern Africa and its recruitment seems to be affected by sulphide events. The reaction of juvenile surf clams to low oxygen concentrations and sulphide occurrence (0.1 mmol l⁻¹) was examined by in vitro exposure experiments in a gas-tight continuous flow system. After 2 h of hypoxic- and hypoxic/sulphidic conditions, clams moved to the sediment surface, aiding their passive transport to areas with more favourable conditions. The clams showed a high sulphide detoxification capacity by oxidising the penetrating hydrogen sulphide to non-toxic thiosulphate. Moreover, juvenile D. serra switched to anaerobic energy production, indicated by the significant accumulation of succinate and, to some extent, alanine. Test animals were not able to reduce their energy requirements enough to withstand long periods of exposure, leading to a median survival time (LT₅₀) of 80 h under hypoxic sulphide incubation. In conclusion, natural “sulphide eruptions”, especially those with a large spatial and temporal extension, have to be considered as an important factor for D. serra recruitment failures. Hydrogen sulphide is assumed to be a potential community structuring factor.     

Effect of hydrogen sulphide on liver somatic index and Fulton's condition factor in Mystus nemurus

The growth rate and liver somatic index were significantly ( P <0.05) lower in Mystus nemurus exposed to hydrogen sulphide compared to controls. These differences increased with corresponding increases in hydrogen sulphide concentrations. No significant differences ( P >0.05) in Fulton's condition factor were detected between the exposed fish and the controls. The results revealed that liver somatic index is a more sensitive indicator of hydrogen sulphide toxicity compared to Fulton's condition factor.     

Does sulphate enrichment promote the expansion of Typha domingensis (cattail) in the Florida Everglades?1

1. The expansion of Typha domingensis into areas once dominated by Cladium jamaicense in the Florida Everglades has been attributed to altered hydrology and phosphorus enrichment, although increased concentrations of sulphate and phosphorus often coincide. The potential importance of hydrogen sulphide produced from sulphate in the expansion of Typha has received little attention. The present study aimed to quantify the comparative growth and photosynthetic responses of Cladium and Typha to sulphate/sulphide. 2. Laboratory experiments showed that Cladium is less tolerant of sulphide than Typha. Cladium was adversely affected at sulphide concentrations of approximately 0.22 mm , while Typha continued to grow well and appeared healthy up to 0.69 mm sulphide. 3. Experiments in field mesocosms provided strong support for species‐specific differences in physiology and growth. Regardless of interstitial sulphide concentrations attained, Typha grew faster and had a higher photosynthetic capacity than Cladium. However, sulphide concentrations in the mesocosms reached only 0.18 mm which, based on the hydroponic study, was insufficient to affect the growth or photosynthetic responses of either species. Nevertheless, the upper range of sulphide (0.25–0.375 mm ) in Everglades’ soil is high enough, based on our results, to impact Cladium but not Typha. 4. This research supports the hypothesis that sulphide accumulation could affect plant species differentially and modify species composition. Consequently, the role of sulphate loading should be considered, in conjunction with hydroperiod, phosphorus availability and disturbances, in developing future management plans for the Everglades.     

Sulphur metabolism in Thiorhodaceae. II. stoichiometric relationship of CO2 fixation to oxidation of hydrogen sulphide and intracellular sulphur inChromatium okenii

Stoichiometry of sulphide and intracellular sulphur oxidation in connection with CO₂ fixation was studied inChromatium okenii. The equipment used was a special stirred cuvette with a rapid-sampling arrangement, which allowed short-time experiments with illuminated bacterial suspensions under anaerobic conditions. Turnover of the sulphur compounds is controlled by a linear CO₂ fixation rate which amounts to 0.069µmoles of CO₂/min mg of cell protein at light saturation. Van Niel's equations for bacterial photosynthesis could be confirmed for short periods under the condition that sulphate is produced during increase of intracellular sulphur; i.e., oxidation of sulphide and of intracellular sulphur do not occur consecutively but simultaneously. The full oxidation rate of intracellular sulphur starts after complete consumption of sulphide. The time during which sulphide is oxidized to intracellular sulphur amounts to 1/3–1/4 of the time necessary for the complete quantitative oxidation of the sulphide to sulphate.     

Prevention of sulphide accumulation and phosphate mobilization by the addition of iron(II) chloride to a reduced sediment: an enclosure experiment

1. In an enclosure experiment carried out in a ditch receiving sulphate-enriched seepage water, iron(II) chloride was added to the sediment. In the sediment pore water of the iron-treated enclosures sulphide levels decreased to very low values (<1 μmol 1^?1) immediately after the iron addition while in the control enclosures sulphide reached values up to 500 μmoll^?1. 2. The sulphide levels in the sediment pore water were also strongly correlated with temperature. In summer, phosphate mobilization was observed in the non-treated enclosures while in the iron-treated enclosures phosphate levels remained low. 3. Total phosphate levels increased greatly in the water layer of the non-treated enclosures, coincident with an algal bloom and increased turbidity. It is suggested that phosphate mobilization in summer is caused by the reduction of iron(III) phosphate complexes and in this high sulphate water body probably also by the reduction of iron(III) by sulphide and the consequential precipitation of iron(II) sulphide. 4. Iron addition appeared to prevent sulphide toxicity in Potamogeton acutifolius Link which was planted in the enclosures immediately after iron(II) addition. In the non-iron-treated enclosures P. acutifolius plants decayed within a few weeks probably as a result of sulphide toxicity.     

Dynamics of corrosion rates associated with nitrite or nitrate mediated control of souring under biological conditions simulating an oil reservoir

Representative microbial cultures from an oil reservoir and electrochemical techniques including potentiodynamic scan and linear polarization were used to investigate the time dependent corrosion rate associated with control of biogenic sulphide production through addition of nitrite, nitrate and a combination of nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB) and nitrate. The addition of nitrate alone did not prevent the biogenic production of sulphide but the produced sulphide was eventually oxidized and removed from the system. The addition of nitrate and NR-SOB had a similar effect on oxidation and removal of sulphide present in the system. However, as the addition of nitrate and NR-SOB was performed towards the end of sulphide production phase, the assessment of immediate impact was not possible. The addition of nitrite inhibited the biogenic production of sulphide immediately and led to removal of sulphide through nitrite mediated chemical oxidation of sulphide. The real time corrosion rate measurement revealed that in all three cases an acceleration in the corrosion rate occurred during the oxidation and removal of sulphide. Amendments of nitrate and NR-SOB or nitrate alone both gave rise to localized corrosion in the form of pits, with the maximum observed corrosion rates of 0.72 and 1.4 mm year⁻¹, respectively. The addition of nitrite also accelerated the corrosion rate but the maximum corrosion rate observed following nitrite addition was 0.3 mm year⁻¹. Furthermore, in the presence of nitrite the extent of pitting was not as high as those observed with other control methods.