Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O₂). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO₂ as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO₂. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Isotope dilution mass spectrometry for the quantification of sulfane sulfurs

Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that allows them to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H₂S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfur concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulfide product, PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a ¹³C₃-labeled phosphine sulfide, PS1, as the internal standard. The specificity and efficiency of the method were proven by model reactions. It was also applied to the measurement of sulfane sulfurs in mouse tissues including brain, kidney, lung, liver, heart, spleen, and blood.     

Determination of hydrogen sulfide and acid-labile sulfur in animal tissues by gas chromatography and ion chromatography

A sensitive and reliable method was developed for the determination of hydrogen sulfide and acid-labile sulfur (ALS) in animal tissues using gas chromatography with flame photometric detector (GC-FPD) and ion chromatography (IC). Hydrogen sulfide trapped in alkaline solution was determined by GC-FPD as hydrogen sulfide or by IC as sulfate after oxidation with hydrogen peroxide. Sodium sulfide used as a source of hydrogen sulfide was standardized by IC. Fresh rat liver and heart tissues contained 112.2±23.0 and 274.1±34.6 nmol/g of ALS respectively. Free hydrogen sulfide was not detected.     

Reduced sulfur and nitrogen compounds and molecular hydrogen as electron donors for anaerobic CO2 photoreduction in Anacystis nidulans

Photosynthesis by Anacystis nidulans was studied in presence of reduced sulfur or nitrogen compounds, or of hydrogen. O₂ evolution and CO₂ fixation were depressed by sulfide, sulfite, cysteine, thioglycollate, hydroxylamine and hydrazine. Sulfite, cysteine and hydrazine inhibited O₂ evolution much more strongly than CO₂ fixation, indicating ability to supply electrons for CO₂ photoreduction; DCMU suppressed these photoreductions. In contrast, some anoxygenic photosynthetic CO₂ fixation insensitive to DCMU was found with sulfide, thiosulfate and hydrogen. Emerson enhancement studies confirmed that sulfite, cysteine and hydrazine acted on photosystem II, while photoreduction supported by sulfide, thiosulfate and hydrogen needed photosystem I only.Sulfite was photooxidized to sulfate, sulfide to elemental sulfur, and thiosulfate to sulfate plus elemental sulfur; the sulfur accumulated inside the cells. Results on the stoichiometries of the photoreductions were consistent with the photooxidation products determined. Inhibitor studies suggested photosynthetic CO₂ fixation through the Calvin cycle.While photoreduction by all reductants used was found to be constitutive in Anacystis, the process was stimulated by anaerobic preincubation with the reductants only in the cases of hydrogen and thiosulfate; this adaptation was prevented by chloramphenicol and by O₂. Anaerobic photoautotrophic growth of Anacystis was, however, not observed; the increase in dry weight with H₂ and thiosulfate was not accompanied by cell multiplication or by an increase in chlorophyll content. Parallel short-term experiments with Chlorella did not reveal any constitutive photoreduction in this eukaryotic alga.Abbreviations CAP chloramphenicol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone - DCMU dichlorophenyl dimethyl urea - DSPD disalicylidenepropane diamine-(1,3) - EDAC 1-ethyl-3(3-dimethylaminopropyl-) carbodiimide     

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography,Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.     

Origin of the labile sulfide in the iron-sulfur proteins of Escherichiacoli

A method has been devised for measuring the abundance of sulfur-34 in the hydrogen sulfide released upon the acidification of $\text{Escherichia}\text{coli}$ cells. Evidence is presented, based on the rate at which the hydrogen sulfide is released from the cells as well as the total amount released, that this hydrogen sulfide originates from the iron-sulfur proteins present in the cells. The sulfur-34 abundance in this hydrogen sulfide which was isolated from cells grown with [sulfane-³⁴S]thiocystine, a compound which can differentially label $\text{in}\text{vivo}$ the sulfur-34 abundance of cysteine and hydrogen sulfide, shows cysteine sulfur and not hydrogen sulfide to be the origin of the sulfide sulfur of iron-sulfur proteins in aerobically grown $\text{E.}\text{coli}$     

Structure-function relationship in hemoproteins: The role of cytochrome c 3 in the reduction of colloidal sulfur by sulfate-reducing bacteria

Cytochromes c ₃ of different strains of sulfatereducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c ₃ is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c ₃. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c ₃ is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c ₃ (or another sulfur reductase) than lactate-sulfate-grown cells.     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Investigations on microbial sulfur respiration

The reduction of elemental or sulfane sulfur to hydrogen sulfide by eubacteria was investigated. Spirillum 5175 had the most active sulfur oxidoreductase. It could be cultivated with fumarate (F), elemental sulfur (S) or nitrate (N) as electron acceptor. Maximum activity was found for Spirillum 5175_S but activity was also present in Spirillum 5175_F and Spirillum 5175_N, i.e. the sulfur oxidoreductase is a constitutive enzyme. It was localized in the membrane, and no activity was found in the cytoplasm in contrast to Desulfovibrio baculatus. Different procedures were applied for the measurement of the sulfur oxidoreductase activity. In the manometric assay hydrogenase was coupled to the sulfur oxidoreductase, and the uptake of dihydrogen was measured in the presence of elemental sulfur. Alternatively, H₂S was assayed directly or was trapped in 12% NaOH and determined by the methylene blue procedure. Using ³⁵S sulfur and ³⁵S-labelled compounds both the substrate and H₂S could be measured. A further increase in sensitivity was achieved using phenosafranin. It was reduced photochemically, and served as the electron donor to the sulfur oxidoreductase, i.e. no hydrogenase was required. This was an important result in view of the fact that not all sulfur-reducing bacteria contain hydrogenase. However, in those cases the hydrogenase isolated from Clostridium pasteurianum could be coupled to the sulfur oxidoreductase. Among the different forms of elemental sulfur Janek sulfur gave the best results in terms of activity and reproducibility. The reduction of elemental sulfur to hydrogen sulfide had a pH optimum at pH 8.7–8.9. There was always a lag-phase which was pH-dependent. During this period the turbidity of the solution changed. Addition of thiols, such as GSH, shortened the lag-phase and caused an increase in activity of the sulfur oxidoreductase. In the presence of p-chloromercuribenzenesulfonic acid the reaction rate decreased significantly. Comparable reaction rates and activity values of the sulfur oxidoreductase in Spirillum 5175_F were obtained with organic trisulfides, RS-S-SR. In contrast to elemental sulfur RS-S-SR are well-defined chemical compounds suitable for quantitative and mechanistic investigations. Labelling the central sulfur of RS-S-SR with ³⁵S gave a satisfactory recovery of the total radioactivity in form of (³⁵S) H₂S in our assay. Trisulfides were shown to be formed as reactive intermediates in bacteria. This process required the sulfur transferase rhodanese which was present in Spirillum 5175, or other sulfur-reducing eubacteria.Abbreviations EPR Electron Paramagnetic Resonance - A Absorbance - PCMS p-chloromercuribenzenesulfonic acid - Sp. 5175_F Splrillum 5175 grown with fumarate - Sp. 5175_S with sulfur - Sp. 5175_N with nitrate - SCE Standard Calomel Electrode     

Characterization of elemental sulfur in isolated intact spinach chloroplasts

Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of ³⁵SO₄²⁻ resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. We have identified this compound as the most stable form (S₈) of elemental sulfur (S⁰, valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S⁰) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S₈ under the experimental conditions used for analysis.     

Inhibition of microbiological sulfide oxidation by methanethiol and dimethyl polysulfides at natron-alkaline conditions

To avoid problems related to the discharge of sulfidic spent caustics, a biotechnological process is developed for the treatment of gases containing both hydrogen sulfide and methanethiol. The process operates at natron-alkaline conditions (>1 mol L⁻¹ of sodium- and potassium carbonates and a pH of 8.5–10) to enable the treatment of gases with a high partial CO₂ pressure. In the process, methanethiol reacts with biologically produced sulfur particles to form a complex mixture predominantly consisting of inorganic polysulfides, dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS). The effect of these organic sulfur compounds on the biological oxidation of sulfide to elemental sulfur was studied with natron-alkaliphilic bacteria belonging to the genus Thioalkalivibrio. Biological oxidation rates were reduced by 50% at 0.05 mM methanethiol, while for DMDS and DMTS, this was estimated to occur at 1.5 and 1.0 mM, respectively. The inhibiting effect of methanethiol on biological sulfide oxidation diminished due to its reaction with biologically produced sulfur particles. This reaction increases the feasibility of biotechnological treatment of gases containing both hydrogen sulfide and methanethiol at natron-alkaline conditions.     

A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulfite as reducing agent

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na₂S) was replaced by sodium sulfite (Na₂SO₃), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H₂S was simply added to the headspace of serum bottles by a syringe. The use of H₂S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably. Received: January 4, 2000 / Accepted: April 5, 2000     

Comparative study of using different materials as bacterial carriers to treat hydrogen sulfide

The use of support media for the immobilization of microorganisms is widely known to provide a surface for microbial growth and protect the microorganisms from inhibitory compounds. In this study, molecular sieve, granular porous carbon, and ferric oxide desulfurizer, immobilized with autotrophic bacteria capable of oxidizing ferrous iron to ferric iron, were developed to treat hydrogen sulfide (H₂S). Their corresponding bioreactors were referred to as BMS, BPC, and BFO, respectively. H₂S loading, gas retention time, hydrogen ion, and aluminous, ferric, and ferrous iron concentrations of recycling effluents were evaluated. Thermogravimetric analysis, Brauner-Emmett-Teller method, and scanning electron microscopy were used to characterize packing materials. Results showed that the elimination capacity was in the order of BFO > BPC > BMS. This study suggested that the material characteristics progressively influenced the deodorization capacities of bioreactors. H₂S was oxidized into elemental sulfur and oxidized sulfur species, according to differences of carriers. Furthermore, this study revealed the potential application of simultaneously treating of H₂S under extremely acidic conditions.     

Sulfide assessment in bioreactors with gas replacement

Many biological processes have utilized the addition of sulfide constituents, such as sodium sulfide or cysteine-sulfide, to affect the redox potential, remove residual oxygen, and/or provide a source of sulfur for metabolism. However, the effects of sulfide constituents and associated sulfide concentrations on growth and product formation of cellular systems have shown considerable variance. In this work, models were developed that explained sulfide loss in bottle studies (batch reactors) and continuously gas-purged reactors. Since sulfide in liquid can be converted to volatile hydrogen sulfide (H₂S), mass transfer plays a key role for sulfide loss in continuous reactors, whereas equilibrium is critical for sulfide loss in batch reactors. Models of sulfide can be used to understand the fate of sulfide during an experiment and to design experiments to maintain constant sulfide levels for providing greater clarity when interpreting experimental results. Cellular experiments for ethanol/acetic acid formation from syngas were carried out to demonstrate the maintenance of constant sulfide levels of 0–1.9 mM throughout the experiment. Results showed that cell growth was slightly affected by the sulfide concentration, ethanol production was favored at higher sulfide concentrations, and acetic acid production was favored at lower sulfide concentrations.     

Studies on the Degradation Mechanisms of Proteins and their Derivatives in the Foods

The volatile sulfur components produced by boiling soybean meal hydrolyzates (AMINOSAN-EKI) have been identified as dimethyl sulfide and hydrogen sulfide. No mercaptan or disulfides were detected.

The main precursor of dimethyl sulfide is supposed to be methionine methylsulfonium compound derived from methionine and pectin substances (–COOCH₃) during the hydrolysis of soybean meal by hydrochloric acid.     

Bioconversion of hydrogen sulfide by free and immobilized cells ofChlorobium thiosulfatophilum

Summary The transformation of hydrogen sulfide into elementary sulfur and sulfate was investigated in a photo-bioreactor using autotropic bacteriaChlorobium thiosulfatophilum. The accumulations of sulfur and sulfate in the reactor were found to be dependent on the light energy and the feed rate of H₂S. The optimum operation lines were established to limit sulfide or sulfate. Immobilization of the whole cells in strontium-alginate matrix enhanced the conversion more than with the free cells.     

Isolation and characterization of strains CVO and FWKO B, two novel nitrate-reducing, sulfide-oxidizing bacteria isolated from oil field brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O(2)). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO(2) as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO(2). Both strains grow at temperatures between 5 and 40 degrees C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Elemental sulfur as an intermediate of sulfide oxidation with oxygen byDesulfobulbus propionicus

The sulfate-reducing bacteriumDesulfobulbus propionicus oxidized sulfide, elemental sulfur, and sulfite to sulfate with oxygen as electron acceptor. Thiosulfate was reduced and disproportionated exclusively under anoxic conditions. When small pulses of oxygen were added to washed cells in sulfide-containing assays, up to 3 sulfide molecules per O₂ disappeared transiently. After complete oxygen consumption, part of the sulfide reappeared. The intermediate formed was identified as elemental sulfur by chemical analysis and turbidity measurements. When excess sulfide was present, sulfur dissolved as polysulfide. This process was faster in the presence of cells than in their absence. The formation of sulfide after complete oxygen consumption was due to a disproportionation of elemental sulfur (or polysulfide) to sulfide and sulfate. The uncoupler tetrachlorosalicylanilide (TCS) and the electron transport inhibitor myxothiazol inhibited sulfide oxidation to sulfate and caused accumulation of sulfur. In the presence of the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), sulfite and thiosulfate were formed. During sulfur oxidation at low oxygen concentrations, intermediary formation of sulfide was observed, indicating disproportionation of sulfur also under these conditions. It is concluded that sulfide oxidation inD. propionicus proceeds via oxidation to elemental sulfur, followed by sulfur disproportionation to sulfide and sulfate. Dedicated to Prof. Dr. Dr. h.c. Norbert Pfennig on the occasion of his 70th birthday     

Polysulfides as Intermediates in the Oxidation of Sulfide to Sulfate by Beggiatoa spp.

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S⁰ under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S₈) and inorganic polysulfides (S_n²⁻). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that S_n²⁻ species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS⁻ as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S⁰ deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.     

Analytical measurement of discrete hydrogen sulfide pools in biological specimens

Hydrogen sulfide (H₂S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H₂S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H₂S. The complexity of analytical H₂S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H₂S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H₂S. Acid-labile H₂S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1 mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H₂S. Volatilized H₂S is then trapped in 100 mM Tris–HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H₂S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H₂S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H₂S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens.