Thiobacillus sp. W5, the dominant autotroph oxidizing sulfide to sulfur in a reactor for aerobic treatment of sulfidic wastes

The floating filter technique was successfully adapted for the isolation of the dominant, chemolithoautotrophic, sulfide-oxidizing bacterium from a sulfur-producing reactor after conventional isolation techniques had failed. The inoculated polycarbonate filters, floating on mineral medium, were incubated under gaseous hydrogen sulfide at non-toxic levels. This technique gave 200-fold higher recoveries than conventional isolation techniques. Viable counts on the filters, making up 15% of the total count, appeared to be all of the same species. Chemostat cultures of the new isolate had a very high sulfur-forming capacity, converting almost all hydrogen sulfide in the medium to elemental sulfur under high sulfide loads (27.5 mmol l^-1 h^-1) and fully aerobic conditions. This behaviour closely resembled that of the microbial community in the sulfur-producing reactor. Moreover, similar protein patterns were obtained by electrophoresis of cell-free extracts from the isolate and the mixed culture. It has therefore been concluded that this isolate represents the dominant sulfide-oxidizing population in the reactor. The isolate has been shown to be a new Thiobacillus species, related to Thiobacillus neapolitanus. In view of the general confusion currently surrounding the taxonomy of the thiobacilli, a new species has not been formally created. Instead, the isolate has been given the working name Thiobacillus sp. W5.     

Removal of dimethyl sulfide,methyl mercaptan,and hydrogen sulfide by immobilized Thiobacillus thioparus TK-m

Cells of Thiobacillus thioparus TK-m were immobilized on cylindrical porous polypropylene pellets (5 mmφ × 5 mm) which were packed in an acrylic cylinder of 50 mm inner diameter up to the height of 800 mm. When a sulfur-containing malodorous gas was charged to this packed tower at the superficial velocity of 0.1 m/s, maximum loading capacity (mmol/l·d) for a malodorous gas to attain the removal rate of 95% or more was: 3.65 for dimethyl sulfide, 8.74 for methyl mercaptan, and 17.36 for hydrogen sulfide. At this time, the inlet concentration (μl/l) of the malodorous compound was: 7.44 for dimethyl sulfide, 17.8 for methyl mercaptan, and 35.4 for hydrogen sulfide. For every compound, higher loading resulted in greater removal quantities. The removal rate of dimethyl sulfide was not overly affected by the presence of a large amount of easily decomposable hydrogen sulfide.     

Flagella and Pili of Iron-Oxidizing Thiobacilli Isolated from a Uranium Mine in Northern Ontario, Canada

Five strains of Thiobacillus ferrooxidans, which included three recent isolates from a uranium mine, possessed flagella. Three of the strains had several pili per cell. The dimensions, fine structure, and orientation of the flagella were different. Both polar and peritrichous flagella were observed, indicating strain-dependent ultrastructural variation in acidophilic thiobacilli. Neither flagella nor pili were detected in eight other strains of T. ferrooxidans and two strains of Thiobacillus acidophilus by electron microscopy, although all of the cultures contained motile cells.     

biochemical reaction mechanisms in sulfur oxidation by chemosynthetic bacteria

Summary Aspects of the biochemistry of the oxidation of inorganic sulfur compounds are discussed in thiobacilli but chiefly inThiobacillus denitrificans. Almost all of the thiobacilli (e.g. T. denitrificans, T. neapolitanus, T. novellus, andThiobacillus A ₂) were capable of producing approximately 7.5 moles of sulfuric acid aerobically from 3.75 moles of thiosulfate per gram of cellular protein per hr. By far the most prolific producer of sulfuric acid (or sulfates) from the anaerobic thiosulfate oxidation with nitrates wasT. denitrificans which was capable of producing 15 moles of sulfates from 7.5 moles of thiosulfate with concomitant reduction of 12 moles of nitrate resulting in the evolution of 6 moles of nitrogen gas/g protein/hr. The oxidation of sulfide was mediated by the flavo-protein system and cytochromes ofb, c, o, anda-type. This process was sensitive to flavoprotein inhibitors, antimycin A, and cyanide. The aerobic thiosulfate oxidation on the other hand involved cytochromec : O₂ oxidoreductase region of the electron transport chain and was sensitive to cyanide only. The anaerobic oxidation of thiosulfate byT. denitrificans, however, was severely inhibited by the flavoprotein inhibitors because of the splitting of the thiosulfate molecule into the sulfide and sulfite moieties produced by the thiosulfate-reductase. Accumulation of tetrathionate and to a small extent trithionate and pentathionate occurred during anaerobic growth ofT. denitrificans. These polythionates were subsequently oxidized to sulfate with the concomitant reduction of nitrate to N₂. Intact cell suspensions catalyzed the complete oxidation of sulfide, thiosulfate, tetrathionate, and sulfite to sulfate with the stoichiometric reduction of nitrate, nitrite, nitric oxide, and nitrous oxide to nitrogen gas thus indicating that NO₂ ⁻, NO, and N₂O are the possible intermediates in the denitrification of nitrate. This process was mediated by the cytochrome electron transport chain and was sensitive to the electron transfer inhibitors. The oxidation of sulfite involved cytochrome-linked sulfite oxidase as well as the APS-reductase pathways. The latter was absent inT. novellus andThiobacillus A ₂. In all of the thiobacilli the inner as well as the outer sulfur atoms of thiosulfate were oxidized at approximately the same rate by intact cells. The sulfide oxidation occurred in two stages: (a) a cellular-membrane-associated initial and rapid oxidation reaction which was dependent upon sulfide concentration, and (b) a slower oxidation reaction stage catalyzed by the cellfree extracts, probably involving polysulfides. InT. novellus andT. neapolitanus the oxidation of inorganic sulfur compounds is coupled to energy generation through oxidative phosphorylation, however, the reduction of pyridine nucleotides by sulfur compounds involved an energy-linked reversal of electron transfer. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974. Summary already inserted on p. 189 of the present volume.     

Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as Thiobacillus ferrooxidans 13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection, Leptospirillum ferrooxidans 2705 and 2391 from the Deutsche Sammlung von Mikroorganismen, L. ferrooxidans BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of T. ferrooxidans AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.     

A new mechanism for the aerobic catabolism of dimethyl sulfide.

Aerobic degradation of dimethyl sulfide (DMS), previously described for thiobacilli and hyphomicrobia, involves catabolism to sulfide via methanethiol (CH3SH). Methyl groups are sequentially eliminated as HCHO by incorporation of O2 catalyzed by DMS monooxygenase and methanethiol oxidase. H2O2 formed during CH3SH oxidation is destroyed by catalase. We recently isolated Thiobacillus strain ASN-1, which grows either aerobically or anaerobically with denitrification on DMS. Comparative experiments with Thiobacillus thioparus T5, which grows only aerobically on DMS, indicate a novel mechanism for aerobic DMS catabolism by Thiobacillus strain ASN-1. Evidence that both organisms initially attacked the methyl group, rather than the sulfur atom, in DMS was their conversion of ethyl methyl sulfide to ethanethiol. HCHO transiently accumulated during the aerobic use of DMS by T. thioparus but not with Thiobacillus strain ASN-1. Catalase levels in cells grown aerobically on DMS were about 100-fold lower in Thiobacillus strain ASN-1 than in T. thioparus T5, suggesting the absence of H2O2 formation during DMS catabolism. Also, aerobic growth of T. thioparus T5 on DMS was blocked by the catalase inhibitor 3-amino-1,2,4-triazole whereas that of Thiobacillus strain ASN-1 was not. Methyl butyl ether, but not CHCl3, blocked DMS catabolism by T. thioparus T5, presumably by inhibiting DMS monooxygenase and perhaps methanethiol oxidase. In contrast, DMS metabolism by Thiobacillus strain ASN-1 was unaffected by methyl butyl ether but inhibited by CHCl3. DMS catabolism by Thiobacillus strain ASN-1 probably involves methyl transfer to a cobalamin carrier and subsequent oxidation as folate-bound intermediates.     

Continuous microbial leaching of a pyritic concentrate by Leptospirillum-like bacteria

Summary Continuous leaching of a pyritic flotation concentrate by mixed cultures of acidophilic bacteria was studied in a laboratory scale airlift reactor. Enrichment cultures adapted to the flotation concentrate contained Thiobacillus ferrooxidans and Thiobacillus thiooxidans. During the late stationary growth phase of these thiobacilli growth of Leptospirillum-like bacteria was observed, too. In discontinuous cultivation no significant influence of Leptospirillum-like bacteria on leaching rates could be detected. During continuous leaching at pH 1.5 Leptospirillum-like bacteria displaced Thiobacillus ferrooxidans. The iron leaching rate achieved by Leptospirillum-rich cultures was found to be up to 3.9 times higher than that by Leptospirillum-free cultures.     

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.     

Sulfide oxidation under oxygen limitation by a thiobacillus thioparus isolated from a marine microbial mat

Abstract The colorless sulfur bacterium Thiobacillus thioparus T5, isolated from a marine microbial mat, was grown in continuous culture under conditions ranging from sulfide limitation to oxygen limitation. Under sulfide-limiting conditions, sulfide was virtually completely oxidized to sulfate. Under oxygen-limiting conditions, sulfide was partially oxidized to zerovalent sulfur (75%) and thiosulfate (17%). In addition, low concentrations of tetrathionate and polysulfide were detected. The finding of in vivo thiosulfate formation supports the discredited observations of thiosulfate formation in cell free extracts in the early sixties. In a microbial mat most sulfide oxidation was shown to take place under oxygen-limiting conditions. It is suggested that zerovalent sulfur formation by thiobacilli is a major process resulting in polysulfide accumulation. Implications for the competition between colorless sulfur bacteria and purple sulfur bacteria are discussed.     

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.     

Sulfide and ammonium oxidation, acetate mineralization by denitrification in a multipurpose UASB reactor

The physiological and kinetic behavior of a denitrifying granular sludge exposed to different sulfide loading rates (55-295 mg/L d) were evaluated in a UASB reactor fed with acetate, ammonium and nitrate. At any sulfide loading rates, the consumption efficiencies of sulfide, acetate and ammonium were above 95%, while nitrate consumption efficiencies were around 62-72%. At the highest sulfide loading rate the ammonium was used as electron donor for N₂ production. The increase of sulfide loading rate also affected the fate of sulfide oxidation, since elemental sulfur was the main end product instead of sulfate. However, the lithotrophic denitrifying kinetic was not affected. FISH oligonucleotide probes for Thiobacillus denitrificans, Thiomiscropira denitrificans, genus Paracoccus and Pseudomonas spp. were used to follow the microbial ecology. The results of this work have shown that four pollutants could simultaneously be removed, namely, sulfide, ammonium, acetate and nitrate under well defined denitrifying conditions.     

A new test procedure for biogenic sulfuric acid corrosion of concrete

A new test method is described for biogenic sulfuric acid corrosion of concrete, more specifically in sewer con-ditions.The aim of the new test method is the development of an accelerated and reproducible procedure formonitoring the resistance of different types of concrete with regard to biogenic sulfuric acid corrosion. This exper-imentalprocedure reflects worst case conditions by providing besides H₂S, also an enrichment of thiobacilli andbiologically produced sulfur. By simulating the cyclic processes occurring in sewer pipes, significant differencesbetween concrete mixtures could be detected after 51 days. Concrete modified by a styrene-acrylic ester polymerdemonstrated a higher resistance against biogenic sulfuric acid attack.     

Evaluation of a microbial method to reduce hydrogen sulfide levels in a porous rock biofilm

Summary The efficacy of nitrate addition, with and without inoculation with a sulfide-resistant strain ofThiobacillus denitrificans (strain F), in reducing sulfide levels in an experimental system using cores and subsurface formation water from a gas storage facility was examined. The addition of nitrate (40 mM) alone to the formation water injected into core systems operated at hydraulic retention times of 3.2 and 16.7 h resulted in lower effluent sulfide concentrations, from an influent concentration of about 170–190 M to an effluent concentration of 110 and 3 M, respectively. A reduction in effluent nitrate concentrations in both core systems indicated the presence of indigenous nitrate-using populations. After strain F was inoculated into the core system operated at the shorter retention time, the effluent sulfide concentration decreased from 110 to 16–25 M. The effluent sulfate concentration increased, and the effluent nitrate concentration decreased concomitant with the presence of high concentrations of denitrifying thiobacilli in the inoculated core system. The denitrifying thiobacilli detected after inoculation were presumed to be strain F since these organisms were not detected in this core system before inoculation, or in any of the samples from the uninoculated core system. These data suggest that the efficacy of the nitrate treatment may depend on the residence times of the liquids in the core system, and that inoculation with strain F was required to reduce sulfide levels to <20 M in the core system operated at a short hydraulic retention time.     

Identification of Thiobacilli by Replica Plating on Thallous Sulfide Paper

Colonies of Thiobacillus thioparus and T. thiooxidans grown on thiosulfate medium can be identified by replica plating on thallous sulfide paper.     

Reactivity versus flexibility in thiobacilli

The results of ecophysiological studies on obligately and facultatively chemolithotrophic thiobacilli performed over the past years clearly show that the two types of organisms occupy different ecological niches. Chemostat experiments with cultures of the obligate chemolithotroph Thiobacillus neapolitanus and the facultative chemolithotroph Thiobacillus A2 have been carried out to explain the competitiveness of T. neapolitanus under conditions of strongly fluctuating substrate supply. Thiobacillus neapolitanus appeared to be very resistant to starvation periods whereafter it could oxidize sulfide (or thiosulfate) almost instantaneously at the original rate. Under alternate supply of 4 h sulfide and 4 h sulfate (or acetate which does not support growth of the organism either) to a chemostat culture of T. neapolitanus (D=0.05 h^–1) the sulfide concentration in the growth vessel never reached levels higher than 4m. This strategy is aimed at maximal reactivity. In contrast to T. neapolitanus the facultative chemolithotroph T.A2 appeared to be very flexible with respect to its energy generation. Under alternate supply of 4 h sulfide and 4 h acetate (D=0.05 h^–1) T.A2 was able to grow continuously since it directed its metabolism to either heterotrophy or autotrophy by rapid induction-repression mechanisms. This flexible strategy seems to be incompatible with a reactive strategy within one organism, since the oxidation capacity for sulfide decreased during the acetate period resulting in accumulation of sulfide during the sulfide period. It is concluded that T.A2 needs a continuous supply of an inorganic and an organic substrate to thrive whereas T. neapolitanus needs only a continuous supply of a reduced inorganic sulfur source but also will persist in environments with interrupted addition of sulfide provided that the starvation period does not last too long.     

脱氮硫杆菌的脱硫特性及其处理恶臭物质硫化氢的应用

污水和污泥的处理过程中会产生大量的恶臭气体硫化氢(H2S)。脱氮硫杆菌是氧化H2S和其他硫化物的重要的脱硫工程菌。本文阐述了脱氮硫杆菌的生物学特性和氧化H2S的两种途径。分析了反应体系中的硫化物负荷、硝酸盐和亚硝酸盐的浓度、氧含量以及pH值等因素对氧化效果、反应速率、氧化途径及产物形式的影响。介绍了脱氮硫杆菌在恶臭污染治理中的应用及其在同步处理含氮含硫恶臭物质方面的发展趋势。     

An immobilized Thiobacillus thioparus biosensing system for monitoring sulfide hydrogen; optimized parameters in a bioreactor

A microbial biosensing system for detection of hydrogen sulfide has been developed by using immobilized Thiobacillus thioparus TK-m in poly vinyl alcohol matrix, together with a dissolved oxygen sensor. Parameters of immobilization (poly vinyl alcohol concentration and amount of wet cell) were optimized by using statistical software. The obtained values for concentration of poly vinyl alcohol and wet cell weight were 11.3% (w/v) and 45 mg, respectively, where the response time of biosensor was 80 s. Calibration of oxygen concentration based on hydrogen sulfide concentration was investigated between 1 mg/L and 20 mg/L. The effect of pH and temperature were investigated in specific range of experimental conditions as well. Some parameters including operational stability and detection limit were studied in detail for characterization of biosensing system. In order to determine the operational stability, bio-sensing system at optimized working conditions was used to distinguish viability of microorganisms in polymer beads in period of time.     

Characterization of Thiobacillus species by gas-liquid chromatography of cellular fatty acids

Summary Fatty acids of 18 strains representing 10 species of Thiobacillus were extracted from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparison of the resulting chromatograms for the presence and relative amounts of major peaks allowed rapid differentiation between such closely related species as Thiobacillus neapolitanus and T. thioparus and of eight other species. Except for a feature common to all thiobacilli tested, T. thiooxidans, T. neapolitanus and T. thioparus each possessed a characteristic fatty acid methyl ester profile that was exhibited by all the strains of that species. Hence, the thiobacilli could be divided into three distinct groups. It was possible to use the gas-liquid chromatographic patterns of the cellular fatty acids for rapid identification or grouping of these microorganisms since the fatty acid composition of the genus Thiobacillus thus appeared to be of taxonomic significance.Non-standard abbreviations GLC Gas-liquid chromatography - FAME Fatty acid methyl ester(s)     

Phenol and sulfide oxidation in a denitrifying biofilm reactor and its microbial community analysis

A microbial consortium attached onto a polyethylene support was used to evaluate the simultaneous oxidation of sulfide and phenol by denitrification. The phenol, sulfide and nitrate loading rates applied to an inverse fluidized bed reactor were up to 168 mg phenol–C/(l d), 37 mg S^2?/(l d) and 168 mg NO₃^?–N/(l d), respectively. Under steady state operation the consumption efficiencies of phenol, sulfide and nitrate were 100%. The N₂ yield (g N₂/g NO₃^?–N) was 0.89. The phenol was mineralized resulting in a yield of 0.82 g bicarbonate–C/g phenol–C and sulfide was completely oxidized to sulfate with a yield of 0.99 g SO₄^2?–S/g S^2?. 16S rRNA gene-based microbial community analysis of the denitrifying biofilm showed the presence of Thauera aromatica, Thiobacillus denitrificans, Thiobacillus sajanensis and Thiobacillus sp. This is the first work reporting the simultaneous oxidation of sulfide and phenol in a denitrifying biofilm reactor.     

Isolation and Characterization of a Hydrogen Sulfide-Removing Bacterium,Pseudomonas sp. Strain DO-1

Five microbial strains that removed hydrogen sulfide (H₂S) or methylmercaptan (CH₃SH) gas were newly isolated from soil samples. Strain DO-1, one of the isolates, was identified as a member of Pseudomonas sp., and it’s immobilized cells removed 1 or 10 ppm of H₂S gas within 2 hours. When strain DO-1 was cultured aerobically in a flask containing nutrient broth medium, the deodorizing activity increased, depending on the growth of the culture, and the maximum activity was obtained after 48 hours. Even though the immobilized cells were stored at 4 or 25°C in sealed bottles for 6 months, the deodorizing activity remained. Throughout this study, strain DO-1 removed H₂ S gas without preliminary feeding or exposure to sulfur com-pounds as growth substrates or inducers. These characteristics are advantageous for the deodorization of the malodorous gases surrounding us in daily life.