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.     

Physiology of sulfide in the rat colon: use of bismuth to assess colonic sulfide production.

Colonic bacteria produce hydrogen sulfide, a toxic compound postulated to play a pathogenetic role in ulcerative colitis. Colonic sulfide exposure has previously been assessed via measurements of fecal sulfide concentration. However, we found that <1% of fecal sulfide of rats was free, the remainder being bound in soluble and insoluble complexes. Thus fecal sulfide concentrations may reflect sulfide binding capacity rather than the toxic potential of feces. We utilized bismuth subnitrate to quantitate intracolonic sulfide release based on observations that bismuth 1) avidly binds sulfide; 2) quantitatively releases bound sulfide when acidified; and 3) does not influence fecal sulfide production by fecal homogenates. Rats ingesting bismuth subnitrate excreted 350 +/- 18 micromol/day of fecal sulfide compared with 9 +/- 1 micromol/day in control rats. Thus the colon normally absorbs approximately 340 micromol of sulfide daily, a quantity that would produce local and systemic injury if not efficiently detoxified by the colonic mucosa. Studies utilizing bismuth should help to clarify the factors influencing sulfide production in the human colon.     

Competition for Dimethyl Sulfide and Hydrogen Sulfide by Methylophaga sulfidovorans and Thiobacillus thioparus T5 in Continuous Cultures

Pure and mixed cultures of Methylophaga sulfidovorans and Thiobacillus thioparus T5 were grown in continuous cultures on either dimethyl sulfide, dimethyl sulfide and H(inf2)S, or H(inf2)S and methanol. In pure cultures, M. sulfidovorans showed a lower affinity for sulfide than T. thioparus T5. Mixed cultures, grown on dimethyl sulfide, showed coexistence of both species. M. sulfidovorans fully converted dimethyl sulfide to thiosulfate, which was subsequently further oxidized to sulfate by T. thioparus T5. Mixed cultures supplied with sulfide and methanol showed that nearly all the sulfide was used by T. thioparus T5, as expected on the basis of the affinities for sulfide. The sulfide in mixed cultures supplied with dimethyl sulfide and H(inf2)S, however, was used by both bacteria. This result may be explained by the fact that the H(inf2)S-oxidizing capacity of M. sulfidovorans remains fully induced by intracellular H(inf2)S originating from dimethyl sulfide metabolism.     

Modulation of sulfide oxidation and toxicity in rat mitochondria by dehydroascorbic acid

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H(2)S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid.     

Rapid hydrogen sulfide consumption by Tetrahymena pyriformis and its implications for the origin of mitochondria

Although sulfide is typically regarded as toxic to eukaryotic cells, it is avidly consumed by Tetrahymena pyriformis. That was observed only when the sulfide concentration was kept below 1 microM. Previously concentrations that were too high had been tested. A new device (Sulfidostat) was used to measure sulfide consumption in steady-state concentrations as low as 10(-12)M. The technique was validated non-biologically by slowly injecting AgNO(3) into buffer and using Ag(2)S precipitation to mimic sulfide consumption, confirming that rates of sulfide consumption could be measured independently of sulfide concentrations. With T. pyriformis, sulfide consumption was 0.25 micromol (gprotein)(-1)s(-1) in 0.5 microM sulfide. Sulfide consumption required O(2) and was inhibited by HCN or by too much sulfide. When cells were separated into fractions, sulfide consumption occurred in the particulate (mitochondrial) fraction. Unexpectedly, the soluble cytosolic fraction slowly produced sulfide even when aerated. The observations are consistent with the conjecture that mitochondria evolved from sulfidotrophic symbionts in a sulfidogenic host cell.     

Hydrogen sulfide consumption measured at low steady state concentrations using a sulfidostat

Although >10 microM hydrogen sulfide typically is toxic to eukaryotic cells, <1 microM sulfide is rapidly consumed and oxidized. To measure sulfide consumption in such low concentrations, we built a "Sulfidostat." The apparatus uses a sulfide-specific electrode to measure the concentration of free sulfide. The electrode is connected to a computer that controls a syringe pump. The pump injects Na(2)S solution into the sample chamber to maintain a constant concentration. Since the response of the electrode to low sulfide concentrations at neutral pH had not been previously validated, that was measured. Then using the Sulfidostat, the rate of sulfide consumption is the rate at which it is pumped into the sample to maintain a constant concentration. The protozoan Tetrahymena pyriformis was used to demonstrate the apparatus; maximum sulfide consumption occurred near 0.5 microM sulfide at a rate of 250 nmol (g protein)(-1) s(-1). That is higher than the rate calculated from the disappearance of sulfide following a bolus addition, a difference that can be explained by the slow response of the electrode and by reversible binding of sulfide by the cells. The Sulfidostat can measure sulfide consumption at concentrations lower than previously have been possible.     

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     

Kinetic parameters of a mixed culture oxidizing sulfide and sulfur with oxygen

Kinetic parameters of biological sulfide oxidation are described. The influence of the sulfide loading rate on growth yield and specific oxidation rate were investigated with free-cell suspensions. It is concluded that at least two types of bacteria were present, namely, sulfate producers (type A) that grow at higher loading rates. Type A bacteria have a growth yield of 04 g dry S/mol S, while type B bacteria have a growth yield of 04 g dry S/mol S. Type A has a high affinity for sulfide and is inhibited by sulfide at sulfide concentrations exceeding 10 mg/L. Type B has a low affinity for sulfide and is not inhibited by sulfide, but by oxygen.     

Adaptation dynamics and evolutionary rescue under sulfide selection in cyanobacteria: a comparative study between Microcystis aeruginosa and Oscillatoria sp. (cyanobacteria)

Experimental evolution studies using cyanobacteria as model organisms are scarce despite the role of cyanobacteria in the evolution of photosynthesis. Three different experimental evolution approaches have been applied to shed light on the sulfide adaptation process, which played a key role in the evolution of this group. We used a Microcystis aeruginosa sulfide‐sensitive strain, unable to grow above ~0.1 mM, and an Oscillatoria sp. strain, isolated from a sulfureous spa (~0.2 mM total sulfide). First, performing a fluctuation analysis design using the spa waters as selective agent, we proved that M. aeruginosa was able to adapt to this sulfide level by rare spontaneous mutations. Second, applying a ratchet protocol, we tested if the limit of adaptation to sulfide of the two taxa was dependent on their initial sulfide tolerance, finding that M. aeruginosa adapted to 0.4 mM sulfide, and Oscillatoria sp. to ~2 mM sulfide, twice it highest tolerance level. Third, using an evolutionary rescue approach, we observed that both speed of exposure to increasing sulfide concentrations (deterioration rate) and populations’ genetic variation determined the survival of M. aeruginosa at lethal sulfide levels, with a higher dependence on genetic diversity. In conclusion, sulfide adaptation of sensitive cyanobacterial strains is possible by rare spontaneous mutations and the adaptation limits depend on the sulfide level present in strain’s original habitat. The high genetic diversity of a sulfide‐sensitive strain, even at fast environmental deterioration rates, could increase its possibility of survival even to a severe sulfide stress.     

Biosynthesis and urinary excretion of methyl sulfonium derivatives of the sulfur mustard analog, 2-chloroethyl ethyl sulfide, and other thioethers

Thioether methyltransferase was previously shown to catalyze the S-adenosylmethionine-dependent methylation of dimethyl selenide, dimethyl telluride, and various thioethers to produce the corresponding methyl onium ions. In this paper we show that the following thioethers are also substrates for this enzyme in vitro: 2-hydroxyethyl ethyl sulfide, 2-chloroethyl ethyl sulfide, thiodiglycol, t-butyl sulfide, and isopropyl sulfide. To demonstrate thioether methylation in vivo, mice were injected with [methyl-3H]methionine plus different thioethers, and extracts of lungs, livers, kidneys, and urine were analyzed by high-performance liquid chromatography for the presence of [3H]methyl sulfonium ions. The following thioethers were tested, and all were found to be methylated in vivo: dimethyl sulfide, diethyl sulfide, methyl n-propyl sulfide, tetrahydrothiophene, 2-(methylthio)ethylamine, 2-hydroxyethyl ethyl sulfide, and 2-chloroethyl ethyl sulfide. This supports our hypothesis that the physiological role of thioether methyltransferase is to methylate seleno-, telluro-, and thioethers to more water-soluble onium ions suitable for urinary excretion. Conversion of the mustard gas analog, 2-chloroethyl ethyl sulfide, to the methyl sulfonium derivative represents a newly discovered mechanism for biochemical detoxification of sulfur mustards, as this conversion blocks formation of the reactive episulfonium ion that is the ultimate alkylating agent for this class of compounds.     

包埋法固定化对硫氧化微生物菌群结构和功能的影响

【目的】为探讨包埋法固定化过程对硫氧化菌群硫化物去除能力及菌群微生物群落结构的影响,【方法】以聚乙烯醇-海藻酸钠-活性炭为载体,对硫氧化菌群进行了固定化,并采用富含硫化物的无机盐培养基,对比固定化与非固定化硫氧化菌群对硫化物的氧化去除能力。同时,利用PCR-DGGE技术,探讨硫氧化菌群在固定化前后以及在硫化物氧化去除过程中微生物群落结构变化。【结果】在对硫氧化菌群进行固定化之后,12 h之内对硫化物的最大去除能力从1000 mg/L下降为600 mg/L。硫氧化菌群的微生物群落结构发生了明显变化,但菌群中的硫氧化菌Catenococcus thiocycli未受影响,硫氧化菌Thioclava pacifica在菌群中的地位反而得到了强化。【结论】受制于底物在载体材料中的扩散迁移效率,硫氧化菌群对硫化物的氧化去除能力在固定化之后有所下降。由于不同微生物对固定化形成的微环境的适应能力以及对载体附着能力的不同,固定化对硫氧化菌群的微生物群落结构产生较大影响。     

Effect of sulfide on growth of marine bacteria

Severe hypoxia leads to excess production of hydrogen sulfide in marine environments. In this study, we examined the effect of sulfide on growth of four facultative anaerobic marine bacteria in minimal media under anaerobic conditions. The Gram-negative chemolithoautotrophic Marinobacter sp. tolerated sulfide concentrations up to 0.60 mM, with doubling and lag times increasing as a function of increasing sulfide concentration but with no change in maximum culture yields; growth did not occur at 1.2 mM sulfide. Similar results were obtained for the metabolically diverse Gram-negative denitrifying Pseudomonas stutzeri, except that growth occurred at 1.2 mM and culture yields at 0.60 and 1.2 mM sulfide were approximately 10-fold lower than at sulfide concentrations between 0 and 0.30 mM. Increases in doubling and lag times accompanied by an overall 10-fold decrease in maximum culture yields were found for the Gram-negative chemoheterotrophic Vibrio sp. at all sulfide concentrations tested. In contrast, growth of a Gram-positive chemoheterotrophic Bacillus sp. was resistant to all sulfide concentrations tested (0.15–1.2 mM). Our results highlight the variable responses of marine bacteria to sulfide and provide some insight into shifts that may occur in microbial community structure and diversity as a consequence of changes in sulfide levels that are the result of hypoxia.     

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.     

Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values

Hydrogen sulfide is gaining acceptance as an endogenously produced modulator of tissue function. The present paradigm of H(2)S (diprotonated, gaseous form of hydrogen sulfide) as a tissue messenger consists of H(2)S being released from the desulfhydration of l-cysteine at a rate sufficient to maintain whole tissue hydrogen sulfide concentrations of 30 microM to >100 microM, and these tissue concentrations serve a messenger function. Utilizing physiological concentrations of l-cysteine and aerobic conditions, we found that catabolism of hydrogen sulfide by mouse liver and brain homogenates exceeded the rate of enzymatic release of this compound such that measureable hydrogen sulfide release was less with tissue-containing vs. tissue-free buffers. Analyses of the gas space over rapidly homogenized mouse brain and liver indicated that in situ tissue hydrogen sulfide concentrations were only about 15 nM. Human alveolar air measurements indicated negligible free H(2)S concentrations in blood. We conclude rapid tissue catabolism of hydrogen sulfide maintains whole tissue brain and liver concentrations of free hydrogen sulfide that are three orders of magnitude less than conventionally accepted values and only 1/5,000 of the hydrogen sulfide concentration (100 microM) required to alter cellular function in vitro. For hydrogen sulfide to serve as an endogenously produced messenger, tissue production and catabolism must result in intracellular microenvironments with a sufficiently high hydrogen sulfide concentration to activate a local signaling mechanism, while whole tissue concentrations remain very low.     

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.     

Chemical aspects of hydrogen sulfide measurements in physiological samples

Background

Owing to recent discoveries of many hydrogen sulfide-mediated physiological processes, sulfide biology is in the focus of scientific research. However, the promiscuous chemical properties of sulfide pose complications for biological studies, which led to accumulation of controversial observations in the literature.

Scope of review

We intend to provide an overview of fundamental thermodynamic and kinetic features of sulfide redox- and coordination-chemical reactions and protonation equilibria in relation to its biological functions. In light of these chemical properties we review the strengths and limitations of the most commonly used sulfide detection methods and recently developed fluorescent probes. We also give a personal perspective on blood and tissue sulfide measurements based on proposed biomolecule–sulfide interactions and point out important chemical aspects of handling sulfide reagent solutions.

Major conclusions

The diverse chemistries of sulfide detection methods resulted in orders of magnitude differences in measured physiological sulfide levels. Investigations that were aimed to dissect the underlying molecular reasons responsible for these controversies made the important recognition that there are large sulfide reserves in biological systems. These sulfide pools are tightly regulated in a dynamic manner and they are likely to play a major role in regulation of endogenous-sulfide-mediated biological functions and avoiding toxic side effects.

General significance

Working with sulfide is challenging, because it requires considerable amounts of chemical knowledge to adequately handle reagent sulfide solutions and interpret biological observations. Therefore, we propose that a rigorous chemical approach could aid the reconciliation of the increasing number of controversies in sulfide biology. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

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.     

Removal of methanethiol, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide from contaminated air by Thiobacillus thioparus TK-m

Methanethiol, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide were efficiently removed from contaminated air by Thiobacillus thioparus TK-m and oxidized to sulfate stoichiometrically. More than 99.99% of dimethyl sulfide was removed when the load was less than 4.0 g of dimethyl sulfide per g (dry cell weight) per day.     

Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene

The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.