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     

Oxidation of reduced sulphur compounds by intact cells of Thiobacillus acidophilus

Oxidation of reduced sulphur compounds by Thiobacillus acidophilus was studied with cell suspensions from heterotrophic and mixotrophic chemostat cultures. Maximum substrate-dependent oxygen uptake rates and affinities observed with cell suspensions from mixotrophic cultures were higher than with heterotrophically grown cells. ph Optima for oxidation of sulphur compounds fell within the pH range for growth (pH 2–5), except for sulphite oxidation (optimum at pH 5.5). During oxidation of sulphide by cell suspensions, intermediary sulphur was formed. Tetrathionate was formed as an intermediate during aerobic incubation with thiosulphate and trithionate. Whether or not sulphite is an inter-mediate during sulphur compound oxidation by T. acidophilus remains unclear. Experiments with anaerobic cell suspensions of T. acidophilus revealed that trithionate metabolism was initiated by a hydrolytic cleavage yielding thiosulphate and sulphate. A hydrolytic cleavage was also implicated in the metabolism of tetrathionate. After anaerobic incubation of T. acidophilus with tetrathionate, the substrate was completely converted to equimolar amounts of thiosulphate, sulphur and sulphate. Sulphide- and sulphite oxidation were partly inhibited by the protonophore uncouplers 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) and by the sulfhydryl-binding agent N-ethylmaleimide (NEM). Oxidation of elemental sulphur was completely inhibited by these compounds. Oxidation of thiosulphate, tetrathionate and trithionate was only slightly affected. The possible localization of the different enzyme systems involved in sulphur compound oxidation by T. acidophilus is discussed.     

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.     

A case study for late Archean and Proterozoic biogeochemical iron‐ and sulphur cycling in a modern habitat—the Arvadi Spring

As a consequence of Earth's surface oxygenation, ocean geochemistry changed from ferruginous (iron(II)‐rich) into more complex ferro‐euxinic (iron(II)‐sulphide‐rich) conditions during the Paleoproterozoic. This transition must have had profound implications for the Proterozoic microbial community that existed within the ocean water and bottom sediment; in particular, iron‐oxidizing bacteria likely had to compete with emerging sulphur‐metabolizers. However, the nature of their coexistence and interaction remains speculative. Here, we present geochemical and microbiological data from the Arvadi Spring in the eastern Swiss Alps, a modern model habitat for ferro‐euxinic transition zones in late Archean and Proterozoic oceans during high‐oxygen intervals, which enables us to reconstruct the microbial community structure in respective settings for this geological era. The spring water is oxygen‐saturated but still contains relatively elevated concentrations of dissolved iron(II) (17.2 ± 2.8 μM) and sulphide (2.5 ± 0.2 μM) with simultaneously high concentrations of sulphate (8.3 ± 0.04 mM). Solids consisting of quartz, calcite, dolomite and iron(III) oxyhydroxide minerals as well as sulphur‐containing particles, presumably elemental S⁰, cover the spring sediment. Cultivation‐based most probable number counts revealed microaerophilic iron(II)‐oxidizers and sulphide‐oxidizers to represent the largest fraction of iron‐ and sulphur‐metabolizers in the spring, coexisting with less abundant iron(III)‐reducers, sulphate‐reducers and phototrophic and nitrate‐reducing iron(II)‐oxidizers. 16S rRNA gene 454 pyrosequencing showed sulphide‐oxidizing Thiothrix species to be the dominating genus, supporting the results from our cultivation‐based assessment. Collectively, our results suggest that anaerobic and microaerophilic iron‐ and sulphur‐metabolizers could have coexisted in oxygenated ferro‐sulphidic transition zones of late Archean and Proterozoic oceans, where they would have sustained continuous cycling of iron and sulphur compounds.     

Selection of sulphur sources for the growth of Butyribacterium methylotrophicum and Acetobacterium woodii

Sulphide and cysteine inhibited growth of batch cultures of Butyribacterium methylotrophicum at moderate concentrations (above 0.5 mM) during growth on glucose (10 mM). The ability of several sulphur sources to replace sulphide was tested in cultures of B. methylotrophicum or Acetobacterium woodii. With sulphite (1 mM), thiosulphate (0.5 mM), elemental sulphur, and dithionite (1 mM), but not sulphate (1 mM), cultures of both organisms grew and produced some sulphide. With elemental sulphur as the sulphur source, toxic levels of sulphide accumulated. Optimal levels for the cultivation of B. methylotrophicum with sulphite were 0.5–2.0 mM, but at higher concentrations the growth rate decreased rapidly, while with dithionite up to 4.0 mM the growth rate was relatively unaffected. In chemostat cultures of B. methylotrophicum with dithionite (1 mM) as the sulphur source and glucose as the limiting substrate, dilution rates up to 0.40 h^–1 were obtained. Thiosulphate could only be used in batch cultures in combination with the reductant titanium(III)nitriloacetate, but in continuous cultures the addition of the reductant to the reservoir was not necessary, because once growth had started enough sulphide was produced to keep the fermentor reduced. The maximum growth rate of B. methylotrophicum with thiosulphate in batch and continuous culture was 0.26 h^–1. Both thiosulphate and dithionite are more convenient sulphur sources than sulphide, but dithionite is more versatile because of its reductive properties and the faster growth it allows.Offprint requests to: T. A. Hansen     

Cysteine desulphurase plays an important role in environmental adaptation of the hyperthermophilic archaeon Thermococcus kodakarensis

The sulphur atoms of sulphur‐containing cofactors that are essential for numerous cellular functions in living organisms originate from L‐cysteine via cysteine desulphurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologues. The existence of CSD orthologues in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulphur‐trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulphur (S⁰)‐reducing hyperthermophilic archaeon, encoding Tk‐CSD, conferred a growth defect evident only in the absence of S⁰, and that growth can be restored by the addition of S⁰, but not sulphide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S⁰, but is necessary for iron‐sulphur cluster biosynthesis in the absence of S⁰. Recombinant form of Tk‐CSD expressed in Escherichia coli was obtained and it was found to catalyse the desulphuration of L‐cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD‐dependent iron‐sulphur cluster biogenesis, which allows them to thrive outside solfataric environments.     

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation.     

Chemolithoutotrophic growth of Thiothrix ramosa

Thiothrix has been shown for the first time to be able to grow chemolithoautotrophically with thiosulphate or carbon disulphide as sole energy substrate. Thiosulphate served as the growth-limiting substrate for Thiothrix ramosa in chemostat culture. Maximum growth yield (Y_max) from yields at growth rates between 0.029–0.075 h^-1 was 4.0 g protein/mol thiosulphate oxidized. The key enzyme of the Calvin cycle, ribulose 1,5-bisphosphate carboxylase, was present in these cells, as were rhodanese, adenylyl sulphate (APS) reductase and sulphur-oxidizing enzyme. Thiosulphate-grown cells oxidized thiosulphate, sulphide, tetrathionate and carbon disulphide. Oxidation kinetics for sulphide, thiosulphate and tetrathionate were biphasic: oxygen consumption during the fast first phase of oxidation indicated oxidation of sulphide, and the sulphane moieties of thiosulphate and tetrathionate, to elemental sulphur, before further oxidation to sulphate. Kinetic constants for these four substrates were determined. T. ramosa also grew mixotrophically in batch culture on lactate with a number of organic sulphur compounds: carbon disulphide, methanethiol and diethyl sulphide. Substituted thiophenes were also used as sole substrates. The metabolic versatility of T. ramosa is thus much greater than previously realised.     

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.     

Sulphide release from anoxic sediments in relation to iron availability and organic matter recalcitrance and its effects on inorganic phosphorus recycling

This research aims to analyse the sediment capacity to buffer free sulphide release in three coastal lagoons which differ in terms of eutrophication level, tide influence and primary producer communities. A preliminary estimate of soluble reactive phosphorus (SRP) regeneration coupled with sulphide fluxes is also made. Sediment profiles of ferrous and ferric iron and reduced sulphur pools were determined in three stations in the Bassin d'Arcachon (South West France), in one site in the Etang du Prévost lagoon (Southern France), and in three stations in the Sacca di Goro lagoon (Northern Italy). Laboratory experiments were also conducted by incubating sediment slurries. Slurries from the French lagoons were also enriched with about 2% d.w. of organic detritus obtained from the dominant macrophytes of each site, namely Zostera noltii and Ruppia cirrhosa (Bassin d'Arcachon), and Ulva rigida (Etang du Prévost). In the Sacca di Goro, slurry experiments were conducted at two sites with different salinity range, sediment composition and hydrodynamics.Field data showed that concentrations of available iron (Fe(II)+Fe(III)) ranged from a minimum of 28.5 µmol cm^–3 (Etang du Prévost) to a maximum of 275.7 µmol cm^–3 (Sacca di Goro). Moreover, in the French lagoons, acid volatile sulphide (AVS) accumulation in the superficial sediment was related to ferrous iron concentrations. Laboratory experiments showed that in spite of strong reducing conditions, sulphide and SRP release was weaker in iron-rich sediments and in those enriched with the most refractory organic matter. The highest fluxes were detected in sediment slurries from the Etang du Prévost, which had the lowest iron content, supplied by 2% of the labile detritus from Ulva rigida. In this case, SRP release was directly related to sulphide production.Two factors seem significant to evaluate the buffer capacity against free sulphide and SRP release from anoxic sediment: organic matter biodegradability, which forces sediment toward reducing conditions, and iron availability, which can affect sulphide mobility as well as the iron hydroxide-phosphate-sulphide system.     

The formation of iron plaques on roots and rhizomes of the seagrass Cymodocea serrulata (R. Brown) Ascherson with implications for sulphide intrusion

Despite the fact that iron plaque formation is ubiquitous in aquatic macrophytes and has been known for several decades, there are few reports of plaque occurrence in seagrasses to date. Herein we present the first microscopical observation and chemical quantification of iron (Fe) plaques on the shoots, rhizomes and roots of the seagrass Cymodocea serrulata (R. Brown) Ascherson collected from intertidal seagrass beds in Thailand. Plaques were observed on shoot bases, rhizomes and roots with the highest concentrations of iron in the plaques from the roots, reaching an average of 509 μmol gDW⁻¹. Interestingly, the most negative stable sulphur isotope (δ³⁴S) values, indicating H₂S intrusion into the plants occurred in the sampling site with the most intense root oxidizing capacity, as indicated by a greater Fe plaque formation. These apparently contradictory findings may be attributed to oxidizing capacity of root tips and root hairs sufficient to promote Fe(III) deposition in the rhizosphere, preceding deposition of plaques on the roots. While this rhizosphere oxidation may result in a more efficient sulphide detoxification during the day photosynthetic phase, root tips and hairs may serve as vulnerable sites for sulphide intrusion at night. The presence of Fe plaque on C. serrulata roots and rhizomes reveals the complexity of seagrass–sediment interactions and deserves further attention to understand if this is a local phenomenon or a newly discovered adaptive mechanism in seagrasses.     

Natural occurrence of microbial sulphur oxidation by long-range electron transport in the seafloor

Recently, a novel mode of sulphur oxidation was described in marine sediments, in which sulphide oxidation in deeper anoxic layers was electrically coupled to oxygen reduction at the sediment surface. Subsequent experimental evidence identified that long filamentous bacteria belonging to the family Desulfobulbaceae likely mediated the electron transport across the centimetre-scale distances. Such long-range electron transfer challenges some long-held views in microbial ecology and could have profound implications for sulphur cycling in marine sediments. But, so far, this process of electrogenic sulphur oxidation has been documented only in laboratory experiments and so its imprint on the seafloor remains unknown. Here we show that the geochemical signature of electrogenic sulphur oxidation occurs in a variety of coastal sediment environments, including a salt marsh, a seasonally hypoxic basin, and a subtidal coastal mud plain. In all cases, electrogenic sulphur oxidation was detected together with an abundance of Desulfobulbaceae filaments. Complementary laboratory experiments in intertidal sands demonstrated that mechanical disturbance by bioturbating fauna destroys the electrogenic sulphur oxidation signal. A survey of published geochemical data and 16S rRNA gene sequences identified that electrogenic sulphide oxidation is likely present in a variety of marine sediments with high sulphide generation and restricted bioturbation, such as mangrove swamps, aquaculture areas, seasonally hypoxic basins, cold sulphide seeps and possibly hydrothermal vent environments. This study shows for the first time that electrogenic sulphur oxidation occurs in a wide range of marine sediments and that bioturbation may exert a dominant control on its natural distribution.     

Isolation of the sulphur reductase and reconstitution of the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes can grow at the expense of sulphur reduction by formate. The enzymes involved in the catalysis of this catabolic reaction have been investigated. From the results the following conclusions are drawn: 1. The enzyme isolated as a sulphide dehydrogenase from the cytoplasmic membrane of W. succinogenes is the functional sulphur reductase that operates in the electron transport from formate to sulphur. 2. The enzyme (M_r 200,000) consists essentially of one type of subunit with the M_r 85,000 and contains equal amounts of free iron and sulphide (120 mol/g protein), but no heme. It represents the first functional sulphur reductase ever isolated. 3. The electron transport chain catalyzing sulphur reduction by formate consists merely of formate dehydrogenase and sulphur reductase. A lipophilic quinone which mediates the transfer of electrons between enzymes in other chains, is apparently not involved. This is the first known example of a phosphorylative electron transport chain that operates without a quinone. 4. The same formate dehydrogenase appears to operate in the electron transport both with sulphur and with fumarate as the terminal electron acceptor in W. succinogenes.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DTT dithiothreitol - MK menaquinone (vitamin K₂) - PMSF phenylmethane sulfonylfluoride - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine - Tea triethanolamine - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonate Dedicated to Professor F. Schneider (Philipps-Universität Marburg) on the occasion of his 60th birthday     

Quantitative measurement of sulphur formation by steady-state and transient-state continuous cultures of autotrophic Thiobacillus species

 Sulphur formation by the obligately chemolithoautotrophic Thiobacillus o and Thiobacillus neapolitanus was studied in aerobic, substrate-limited continuous cultures. The performance of transient-state and steady-state cultures was compared using different methods for measuring sulphur production. Below a dilution rate (D) of 0.3 h^-1 (at 50% air saturation), sulphate-producing steady states were obtained, and cultures grown with sulphide or thiosulphate (at D=0.06 h^-1) showed similar characteristics (e.g. cell yields, oxidation capacities and CO₂-fixation capacities). Elemental sulphur was a major product above D=0.3 h^-1, but steady states were difficult to achieve, because of adherence of sulphur to the fermentor surfaces and the accumulation of sulphide. These problems could be circumvented using transient-state experiments of 1 h. It was then found that elemental sulphur was formed under oxygen limitation or at high substrate load. The rates of sulphur formation obtained by sulphur analysis agreed with the values calculated from stoichiometric balances. Sulphide and thiosulphate proved to be equivalent substrates for both Thiobacillus species during elemental sulphur formation under the conditions tested. It is concluded that transient-state cultures of thiobacilli, pregrown as sulphate-producing steady-state cultures, provide experimental conditions for the quantitative assessment of sulphur formation from (labile) sulphide and from thiosulphate. Received: 15 May 1995 / Received revision: 4 August 1995 / Accepted: 22 August 1995     

Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells ofChromatium okenii

Growth experiments and short term experiments in a stirred cuvette showed thatChromatium okenii strain Ostrau is not able to oxidize any reduced sulphur compounds except sulphide and elementary sulphur; thiosulphate, sulphite, and thioglycolate can not be utilized as reducing agents for photosynthesis. The cells are not able to use H₂; hydrogenase could not be demonstrated. In the dark, sulphide is formed from intracellular sulphur and the carbon content of the cells decreases. Growth and turnover of sulphur compounds was followed in the light in the presence and absence of acetate as a second carbon source. Sulphide oxidation depends on the presence of CO₂ and on light intensity, i.e. sulphur metabolism is governed by the photosynthetic activity of the cells.     

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     

Desulphuring of natural gas and petroleum oil by autotrophic Thiobacillus ferrooxidans

Hydrogen sulphide is a common toxic contaminant in natural gas and oil. In this study, the strictly autotrophic bacterium Thiobacillus ferrooxidans , which oxidizes reduced sulphur compounds, was used to desulphur petroleum oil and gas. The reaction was carried out in a closed vessel containing substrate mixed with a bacterial suspension. The significance of the hydrogen sulphide oxidizing activity of T. ferrooxidans is discussed.     

Corrosion of Pembina crude oil pipeline

Summary Corrosion failure of the Pembina pipeline system of North Central Alberta, Canada, was frequent and was associated with constant bacterial load and sulphide in the crude oil and produced water. The bacterial load included a variety of anaerobic and aerobic/facultative bacteria which acted in concert to produce sulphide, giving rise to a cascade of sulphide generation.A total of 256 isolates from the crude oil were tested for ability to reduce oxidized sulphur compounds to sulphide. Five groups of bacteria, (A-E), based on this ability to reduce sulphur compounds, existed in the crude oil system. Group A reduced sulphur compounds with oxidation states +6; and lower, Group B reduced oxidation state +4 and below; Group C, oxidation states +2 and lower. Group D reduced only oxidation state 0 (elemental Sulphur), while Group E could reduce no sulphur compound to sulphide. It was found that a ceiling on the reductive capability of each bacterial group was set by the oxidation state of the sulphur compounds. The result is a synergistic relationship whereby intermediate products of reductive activities of each group form the substrate for subsequent action by other groups until sulphide is produced.     

Conversion of hydrogen sulphide by acidophilic bacteria

Conversion of hydrogen sulphide (H₂S) by the bacterium Thiobacillus thiooxidans to sulphur or sulphate was demonstrated in a continuous column contacter using a countercurrent flow of gas and liquid medium. The initial conversion to sulphur was much faster than subsequent oxidation to sulphate, allowing for removal of elemental sulphur. The rate of H₂S removal increased with available surface area in the column bed and with time. The number of bacteria in the column increased very slowly with time, placing great importance on the initial concentration of bacteria in the column. Correspondence to: H. M. Lizama     

Microbial treatment of a synthetic sour brine

Summary The ability of the chemoautotroph and facultative anaerobeThiobacillus denitrificans to deodorize and detoxify an oil-field-produced water containing sulphides was evaluated under simulated field conditions. A sulphide-tolerant strain ofT. denitrificans was used to remove inorganic sulphide from a synthetic sour brine containing 4000 mg L^–1 total dissolved solids (TDS) and 100 mg L^–1 sulphide. The sour brine was treated continuously in a rectangular plugflow reactor which approximated the scaled dimensions of an existing field detention pond. The head space of the reactor was purged with N₂ in order to capture H₂S off-gases in a zinc acetate trap. Brine was fed to the reactor continuously for 90 days at rates corresponding to residence times of 0.17–6 days. Temperature and pH ranged from 22 to 40.5°C and 7.5 to 8.8, respectively. The start-up biomass concentration was approximately 100 mg L^–1 (by dry weight). No. additionalT. denitrificans biomass was added to the reactor after start-up. At residence times of 0.3 days and greater inorganic sulphide was undetectable in the effuent. No H₂S was detected in the outlet gas or the zinc acetate trap. Approximately 80% of the sulphide feed was oxidized to sulphate and removed from the reactor in the liquid effluent. The remainder was partially oxidized to elemental sulphur which was retained in the reactor. It is suggested that oxidation of inorganic sulphides byT. denitrificans represents a viable process concept for the treatment of sour water co-produced with oil and gas.