Change in the microbial community of saline geothermal fluids amended with a scaling inhibitor: effects of heat extraction and nitrate dosage.

Geothermal plants are often affected by corrosion caused by microbial metabolites such as H2S. In the Bad Blumau (Austria) geothermal system, an increase in microbially produced H2S was observed in the hot (107 °C) and scaling inhibitor-amended saline fluids and in fluids that had cooled down (45 °C). Genetic fingerprinting and quantification revealed the dominance, increasing abundance and diversity of sulfate reducers such as Desulfotomaculum spp. that accompanied the cooling and processing of the geothermal fluids. In addition, a δ34S isotopic signature showed the microbial origin of the H2S that has been produced either chemolithotrophically or chemoorganotrophically. A nitrate addition test in a test pipe as a countermeasure against the microbial H2S formation caused a shift from a biocenosis dominated by bacteria of the phylum Firmicutes to a community of Firmicutes and Proteobacteria. Nitrate supported the growth of nitrate-reducing sulfur-oxidizing Thiobacillus thioparus, which incompletely reduced nitrate to nitrite. The addition of nitrate led to a change in the composition of the sulfate-reducing community. As a result, representatives of nitrate- and nitrite-reducing SRB, such as Desulfovibrio and Desulfonatronum, emerged as additional community members. The interaction of sulfate-reducing bacteria and nitrate-reducing sulfur-oxidizing bacteria (NR-SOB) led to the removal of H2S, but increased the corrosion rate in the test pipe.

Reclassification of Thiobacillus aquaesulis (Wood & Kelly, 1995) as Annwoodia aquaesulis gen. nov., comb. nov., transfer of Thiobacillus (Beijerinck, 1904) from the Hydrogenophilales to the Nitrosomonadales, proposal of Hydrogenophilalia class. nov. within the 'Proteobacteria', and four new families within the orders Nitrosomonadales and Rhodocyclales.

The genus Thiobacillus comprises four species with validly published names, of which Thiobacillus aquaesulis DSM 4255T (=ATCC 43788T) is the only species that can grow heterotrophically or mixotrophically - the rest being obligate autotrophs - and has a significant metabolic difference in not producing tetrathionate during the oxidation of thiosulfate during autotrophic growth. On the basis of this and differential chemotaxonomic properties and a 16S rRNA gene sequence similarity of 93.4 % to the type species Thiobacillus thioparus DSM 505T, we propose that it is moved to a novel genus, Annwoodia gen. nov., for which the type species is Annwoodia aquaesulis gen. nov., comb. nov. We confirm that the position of the genus Thiobacillus in the Betaproteobacteria falls within the Nitrosomonadales rather than the Hydrogenophilales as previously proposed. Within the Nitrosomonadales we propose the circumscription of genera to form the Thiobacilliaceae fam. nov. and the Sterolibacteriaceae fam. nov. We propose the merging of the family Methylophilaceae into the Nitrosomonadales, and that the Sulfuricellaceae be merged into the Gallionellaceae, leaving the orders Methylophilales and Sulfuricellales defunct. In the Rhodocyclales we propose the Azonexaceae fam. nov. and the Zoogloeaceae fam. nov. We also reject the Hydrogenophilales from the Betaproteobacteria on the basis of a very low 16S rRNA gene sequence similarity with the class-proper as well as physiological properties, forming the Hydrogenophilalia class. nov. in the 'Proteobacteria'. We provide emended descriptions of Thiobacillus, Hydrogenophilales, Hydrogenophilaceae, Nitrosomonadales, Gallionellaceae, Rhodocyclaceae and the Betaproteobacteria.

A Novel Uncultured Bacterium of the Family Gallionellaceae: Description and Genome Reconstruction Based on the Metagenomic Analysis of Microbial Community in Acid Mine Drainage.

Drainage waters at the metal mining areas often have low pH and high content of dissolved metals due to oxidation of sulfide minerals. Extreme conditions limit microbial diversity in- such ecosystems. A drainage water microbial community (6.5'C, pH 2.65) in an open pit at the Sherlovaya Gora polymetallic open-cast mine (Transbaikal region, Eastern Siberia, Russia) was studied using metagenomic techniques. Metagenome sequencing provided information for taxonomic and functional characterization of the micro- bial community. The majority of microorganisms belonged to a single uncultured lineage representing a new Betaproteobacteria species of the genus Gallionella. While no.acidophiles are known among the cultured members of the family Gallionellaceae, similar 16S rRNA gene sequences were detected in acid mine drain- ages. Bacteria ofthe genera Thiobacillus, Acidobacterium, Acidisphaera, and Acidithiobacillus,-which are com- mon in acid mine drainage environments, were the minor components of the community. Metagenomic data were -used to determine the almost complete (-3.4 Mb) composite genome of the new bacterial. lineage desig- nated Candidatus Gallionella acididurans ShG14-8. Genome analysis revealed that Fe(II) oxidation probably involved the cytochromes localized on the outer membrane of the cell. The electron transport chain included NADH dehydrogenase, a cytochrome bc1 complex, an alternative complex III, and cytochrome oxidases of the bd, cbb3, and bo3 types. Oxidation of reduced sulfur compounds probably involved the Sox system, sul- fide-quinone oxidoreductase, adenyl sulfate reductase, and sulfate adenyltransferase. The genes required for autotrophic carbon assimilation via the Calvin cycle were present, while no pathway for nitrogen fixation was revealed. High numbers of RND metal transporters and P type ATPases were probably responsible for resis- tance to heavy metals. The new microorganism was an aerobic chemolithoautotroph of the group of psychrotolerant iron- and sulfur-oxidizing acidophiles of the family Gallionellaceae, which are common in acid mine drainages.

Aerobic and Anaerobic Thiosulfate Oxidation by a Cold-Adapted, Subglacial Chemoautotroph.

Geochemical data indicate that protons released during pyrite (FeS2) oxidation are important drivers of mineral weathering in oxic and anoxic zones of many aquatic environments, including those beneath glaciers. Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the metastable intermediate thiosulfate (S2O3 (2-)), which represents an electron donor capable of supporting microbial metabolism. Subglacial meltwaters sampled from Robertson Glacier (RG), Canada, over a seasonal melt cycle revealed concentrations of S2O3 (2-) that were typically below the limit of detection, despite the presence of available pyrite and concentrations of the FeS2 oxidation product sulfate (SO4 (2-)) several orders of magnitude higher than those of S2O3 (2-). Here we report on the physiological and genomic characterization of the chemolithoautotrophic facultative anaerobe Thiobacillus sp. strain RG5 isolated from the subglacial environment at RG. The RG5 genome encodes genes involved with pathways for the complete oxidation of S2O3 (2-), CO2 fixation, and aerobic and anaerobic respiration with nitrite or nitrate. Growth experiments indicated that the energy required to synthesize a cell under oxygen- or nitrate-reducing conditions with S2O3 (2-) as the electron donor was lower at 5.1°C than 14.4°C, indicating that this organism is cold adapted. RG sediment-associated transcripts of soxB, which encodes a component of the S2O3 (2-)-oxidizing complex, were closely affiliated with soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial environment at RG mediated in part by populations closely affiliated with RG5. The consumption of S2O3 (2-) by RG5-like populations may accelerate abiotic FeS2 oxidation, thereby enhancing mineral weathering in the subglacial environment.

Reclassification of 'Thiobacillus prosperus' Huber and Stetter 1989 as Acidihalobacter prosperus gen. nov., sp. nov., a member of the family Ectothiorhodospiraceae.

Analysis of phylogenomic metrics of a recently released draft genome sequence of the halotolerant, acidophile 'Thiobacillus prosperus' DSM 5130 indicates that it is not a member of the genus Thiobacillus within the class Betaproteobacteria as originally proposed. Based on data from 16S rRNA gene phylogeny, and analyses of multiprotein phylogeny and average nucleotide identity (ANI), we show that it belongs to a new genus within the family Ectothiorhodospiraceae, for which we propose the name Acidihalobacter gen. nov. In accordance, it is proposed that 'Thiobacillus prosperus' DSM 5130 be named Acidihalobacter prosperus gen. nov., sp. nov. DSM 5130T ( = JCM 30709T) and that it becomes the type strain of the type species of this genus.

Sulfuriferula multivorans gen. nov., sp. nov., isolated from a freshwater lake, reclassification of 'Thiobacillus plumbophilus' as Sulfuriferula plumbophilus sp. nov., and description of Sulfuricellaceae fam. nov. and Sulfuricellales ord. nov.

A sulfur-oxidizing bacterium, strain TTN(T), was isolated from a Thioploca sample obtained from a freshwater lake in Japan. The isolate shared 97.1% 16S rRNA gene sequence similarity with an obligately aerobic chemolithoautotroph, 'Thiobacillus plumbophilus' Gro7(T). Cells were rods, motile, and Gram-stain-negative. The G+C content of the genomic DNA was approximately 66 mol%. Strain TTN(T) grew over a temperature range of 8-32 °C (optimum 22-25 °C), an NaCl concentration range of 0-133.3 mM (optimum 0-3.3 mM) and a pH range of 5.3-8.6 (optimum pH 6.4-7.0). Strain TTN(T) was facultatively anaerobic and could utilize nitrate as an electron acceptor. The isolate oxidized tetrathionate, thiosulfate and elemental sulfur as the sole energy sources for autotrophic growth, and could also grow heterotrophically on a number of organic substrates. Based on its phylogenetic and phenotypic properties, strain TTN(T) represents a novel species of a novel genus, for which the name Sulfuriferula multivorans gen. nov., sp. nov. is proposed. The type strain is TTN(T) ( =NBRC 110683(T) =DSM 29343(T)). Along with this, the reclassification of 'Thiobacillus plumbophilus' as Sulfuriferula plumbophilus sp. nov. (type strain Gro7(T) =NBRC 107929(T) =DSM 6690(T)) is proposed. Based on the data obtained in this study, we describe the designations Sulfuricellaceae fam. nov. and Sulfuricellales ord. nov.

Microbiology of healing mud (fango) from Roman thermae aquae iasae archaeological site (Varazdinske Toplice, Croatia).

We found well-preserved, rocky artefacts that had been buried in the healing mud (fango) for more than 1,500 years at the Roman archaeological site at Varazdinske Toplice. This Roman pool with fango sediments and artefacts is fed from hot sulphidic springs. The fango exhibited nearly neutral pH, a high level of organic C, an elevated concentration of heavy metals and a high total microbial biomass, greater than 10(8) cells per gram of dry weight. The dominant microbes, assessed by molecular profiling (denaturing gradient gel electrophoresis), were affiliated with Thiobacillus, Sulfuricurvum, Polaromonas, and Bdellovibrio. Polymerase chain reaction screening for microbial functional guilds revealed the presence of sulphur oxidizers and methanogens but no sulphate reducers. The dominance of four Proteobacterial classes (α-, ß-, δ- and ε-Proteobacteria) was confirmed by fluorescence in situ hybridisation; Actinobacteria were less abundant. Cultivable bacteria represented up to 23.4 % of the total bacterial counts when cultivation media was enriched with fango. These bacteria represented the genera Acinetobacter, Aeromonas, Arthrobacter, Comamonas, Ewingella, Flavobacterium, Pseudomonas, Rahnella and Staphylococcus. This study showed that the heterogeneous nature of fango at neutral pH created various microniches, which largely supported microbial life based on sulphur-driven, autotrophic denitrification.

Denitrification of groundwater using a sulfur-oxidizing autotrophic denitrifying anaerobic fluidized-bed MBR: performance and bacterial community structure.

This paper investigates a novel sulfur-oxidizing autotrophic denitrifying anaerobic fluidized bed membrane bioreactor (AnFB-MBR) that has the potential to overcome the limitations of conventional sulfur-oxidizing autotrophic denitrification systems. The AnFB-MBR produced consistent high-quality product water when fed by a synthetic groundwater with NO3 (-)-N ranging 25-80 mg/L and operated at hydraulic retention times of 0.5-5.0 h. A nitrate removal rate of up to 4.0 g NO3 (-)-N/Lreactord was attained by the bioreactor, which exceeded any reported removal capacity. The flux of AnFB-MBR was maintained in the range of 1.5-15 L m(-2) h(-1). Successful membrane cleaning was practiced with cleaning cycles of 35-81 days, which had no obvious effect on the AnFB-MBR performance. The (15) N-tracer analyses elucidated that nitrogen was converted into (15) N2-N and (15) N-biomass accounting for 88.1-93.1 % and 6.4-11.6 % of the total nitrogen produced, respectively. Only 0.3-0.5 % of removed nitrogen was in form of (15)N2O-N in sulfur-oxidizing autotrophic denitrification process, reducing potential risks of a significant amount of N2O emissions. The sulfur-oxidizing autotrophic denitrifying bacterial consortium was composed mainly of bacteria from Proteobacteria, Chlorobi, and Chloroflexi phyla, with genera Thiobacillus, Sulfurimonas, and Ignavibacteriales dominating the consortium. The pyrosequencing assays also suggested that the stable microbial communities corresponded to the elevated performance of the AnFB-MBR. Overall, this research described relatively high nitrate removal, acceptable flux, indicating future potential for the technology in practice.

Potentialities of coupling biological processes (biotrickler/biofilter) for the degradation of a mixture of sulphur compounds.

This study deals with the potential of biological processes combining a biotrickler and a biofilter to treat a mixture of sulphur-reduced compounds including dimethyl sulphide (DMS), dimethyl disulphide (DMDS) and hydrogen sulphide (H2S). As a reference, duplicated biofilters were implemented, and operating conditions were similar for all bioprocesses. The first step of this work was to determine the efficiency removal level achieved for each compound of the mixture and in a second step, to assess the longitudinal distribution of biodegradation activities and evaluate the total bacteria, Hyphomicrobium sp. and Thiobacillus thioparus densities along the bed height. A complete removal of hydrogen sulphide is reached at the start of the experiment within the first stage (biotrickler) of the coupling. This study highlighted that the coupling of a biotrickling filter and a biofilter is an interesting way to improve both removal efficiency levels (15-20% more) and kinetics of recalcitrant sulphur compounds such as DMS and DMDS. The total cell densities remained similar (around 1 × 10(10) 16S recombinant DNA (rDNA) copies g dry packing material) for duplicated biofilters and the biofilter below the biotrickling filter. The relative abundances of Hyphomicrobium sp. and T. thioparus have been estimated to an average of 10 ± 7.0 and 0.23 ± 0.07%, respectively, for all biofilters. Further investigation should allow achieving complete removal of DMS by starting the organic sulphur compound degradation within the first stage and surveying microbial community structure colonizing this complex system.

Phylogenetic assessment of culture collection strains of Thiobacillus thioparus, and definitive 16S rRNA gene sequences for T. thioparus, T. denitrificans, and Halothiobacillus neapolitanus.

The 16S rRNA gene sequences of 12 strains of Thiobacillus thioparus held by different culture collections have been compared. A definitive sequence for the reference type strain (Starkey; ATCC 8158(T)) was obtained. The sequences for four examples of the Starkey type strain were essentially identical, confirming their sustained identity after passage through different laboratories. One strain (NCIMB 8454) was reassigned as a strain of Halothiobacillus neapolitanus, and a second (NCIMB 8349) was a species of Thermithiobacillus. These two strains have been renamed in their catalog by the National Collection of Industrial and Marine Bacteria. The 16S rRNA gene sequence of the type strain of Halothiobacillus neapolitanus (NCIMB 8539(T)) was determined and used to confirm the identity of other culture collection strains of this species. The reference sequences for the type strains of Thiobacillus thioparus and Halothiobacillus neapolitanus have been added to the online List of Prokaryotic Names with Standing in Nomenclature. Comparison of the 16S rRNA gene sequences available for strains of Thiobacillus denitrificans indicated that the sequence for the type strain (NCIMB 9548(T)) should always be used as the reference sequence for new and existing isolates.

Thiobacillus thiophilus sp. nov., a chemolithoautotrophic, thiosulfate-oxidizing bacterium isolated from contaminated aquifer sediments.

Strain D24TN(T) was enriched and isolated from sediment collected from a tar oil-contaminated aquifer at a former gasworks site located in Duesseldorf-Flingern, Germany. Cells of strain D24TN(T) were rod-shaped, non-spore-forming and stained Gram-negative. Thiosulfate was used as an electron donor. The organism was obligately chemolithoautotrophic and facultatively anaerobic, and grew with either oxygen or nitrate as electron acceptor. Growth was observed at pH values between 6.3 and 8.7 and at temperatures of -2 to 30 degrees C; optimum growth occurred at pH 7.5-8.3 and 25-30 degrees C. The DNA G+C content was 61.5 mol%. On the basis of the 16S rRNA gene sequence analysis, strain D24TN(T) clustered in the Betaproteobacteria and was most closely related to Thiobacillus denitrificans (97.6 %) and Thiobacillus thioparus (97.5 %). Based on the phenotypic, chemotaxonomic and phylogenetic data, strain D24TN(T) represents a novel species of the genus Thiobacillus, for which the name Thiobacillus thiophilus sp. nov. is proposed. The type strain is D24TN(T) (=DSM 19892(T)=JCM 15047(T)).

Molecular analysis of the distribution and phylogeny of dissimilatory adenosine-5'-phosphosulfate reductase-encoding genes (aprBA) among sulfur-oxidizing prokaryotes.

Dissimilatory adenosine-5'-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo- and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92-93 % (2.1-2.3 kb) of the APS reductase locus aprBA. PCR-based screening of 116 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo- and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBA-based tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta- and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent 'authentic' and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo- and chemotrophic SOP is discussed.

Succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems.

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O3(2-)). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.

[Thiobacillus sajanensis sp. nov., a new obligately autotrophic sulfur-oxidizing bacterium isolated from Khoito-Gol hydrogen-sulfide springs, Buryatia].

Four strains of rod-shaped gram-negative sulfur-oxidizing bacteria were isolated from Khoito-Gol hydrogen-sulfide springs in the eastern Sayan Mountains (Buryatia). The cells of the new isolates were motile by means of a single polar flagellum. The strains were obligately chemolithoautotrophic aerobes that oxidized thiosulfate (with the production of sulfur and sulfates) and hydrogen sulfide. They grew in a pH range of 6.8-9.5, with an optimum at pH 9.3 and in a temperature range of 5-39 degrees C, with an optimum at 28-32 degrees C. The cells contained ubiquinone Q-8. The DNA G+C content of the new strains was 62.3-64.2 mol %. According to the results of analysis of their 16S rRNA genes, the isolates belong to the genus Thiobacillus within the subclass Betaproteobacteria. However, the similarity level of nucleotide sequences of the 16S rRNA genes was insufficient to assign the isolates to known species of this genus. The affiliation to the genus Thiobacillus was confirmed by DNA-DNA hybridization of the isolates with the type strain of the type species of the genus Thiobacillus, T. thioparus DSM 505T (= ATCC 8158T). Despite the phenotypic similarity, the hybridization level was as low as 21-29%. In addition, considerable differences were revealed in the structure of the genes encoding RuBPC, the key enzyme of autotrophic CO2 assimilation, between the known Thiobacillus species and the new isolates. Based on molecular-biological features and certain phenotypic distinctions, the new isolates were assigned to a new Thiobacillus species, T. sajanensis sp. nov., with the type strain 4HGT (= VKM B-2365T).

Isolation, characterization, and ecology of cold-active, chemolithotrophic, sulfur-oxidizing bacteria from perennially ice-covered Lake Fryxell, Antarctica.

Novel strains of obligately chemolithoautotrophic, sulfur-oxidizing bacteria have been isolated from various depths of Lake Fryxell, Antarctica. Physiological, morphological, and phylogenetic analyses showed these strains to be related to mesophilic Thiobacillus species, such as T. thioparus. However, the psychrotolerant Antarctic isolates showed an adaptation to cold temperatures and thus should be active in the nearly freezing waters of the lake. Enumeration by most-probable-number analysis in an oxic, thiosulfate-containing medium revealed that the sulfur-oxidizing chemolithotroph population peaks precisely at the oxycline (9.5 m), although viable cells exist well into the anoxic, sulfidic waters of the lake. The sulfur-oxidizing bacteria described here likely play a key role in the biogeochemical cycling of carbon and sulfur in Lake Fryxell.

[Studies of polymorphisms of Thiobacillus Ferrooxidans using RAPD].

Random amplified polymorphic DNA(RAPD) was used in analyzing the polymorphisms of Thiobacillus ferrooxidans from seven different places. Of the 20 primers, four could generate reproducible RAPD profiles, and each one produced 1 approximately 9 bands. The similarity coefficients obtained from profiles generated by four primers among Thiobacillus Ferrooxidans were about 44% approximately 83%.

Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of 13C-labelled fatty acids.

A method for the detection of physiologically active autotrophic bacteria in complex microbial communities was developed based on labelling with the stable isotope 13C. Labelling of autotrophic nitrifying, sulphur-oxidizing and iron-oxidizing populations was performed in situ by incubation with NaH[13C]O3. Incorporated label into fatty acid methyl esters (FAMEs) was detected and quantified using gas chromatography-mass spectrometry in single ion monitoring mode. Before the analyses of different environmental samples, the protocol was evaluated in pure culture experiments. In different environmental samples a selective labelling of fatty acids demonstrated which microbial taxa were responsible for the respective chemolithoautotrophic activity. The most strongly labelled fatty acids of a sample from a sulphide treating biofilter from an animal rendering plant were cis-7-hexadecenoic acid (16:1 cis7) and 11-methyl hexadecanoic acid (16:0 11methyl), which are as-yet not known for any sulphide-oxidizing autotroph. The fatty acid labelling pattern of an experimental biotrickling filter sample supplied with dimethyl disulphide clearly indicated the presence and activity of sulphide-oxidizing bacteria of the genus Thiobacillus. For a third environmental sample from an acid mining lake sediment, the assignment of autotrophic activity to bacteria of the genus Leptospirillum but not to Acidithiobacillus could be made by this method, as the fatty acid patterns of these bacteria show clear differences.

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1.

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.

Mediation of arsenic oxidation by Thiomonas sp. in acid-mine drainage (Carnoulès, France).

AIMS: To isolate, identify, and characterize heterotrophic bacteria in acid-mine drainage that mediate oxidation of As(III). METHODS AND RESULTS: Samples of acid-mine drainage were collected over a period of 14 months. Heterotrophic and non-obligatory acidophilic bacteria in the samples were cultured on a solid medium (pH 7.0-7.2), and three strains were isolated. The three different strains belong to the genus Thiomonas, and have more than 99% homology with the group Ynys1. Culturing in mineral media demonstrated that the isolated strains used thiosulphate as an energy source, and oxidized iron in the presence of thiosulphate. However, none of the strains were able to oxidize arsenic in the presence of thiosulphate, nor could they use iron or arsenic alone as an energy source. In vitro experiments demonstrated that two of the Thiomonas strains were able to oxidize more than 90% of the As(III) present in the acid-mine drainage, whereas no abiotic oxidation of arsenic occurred. CONCLUSIONS: Two strains of newly identified Thiomonas sp. found in acid-mine drainage are capable of oxidizing arsenic. SIGNIFICANCE AND IMPACT OF STUDY: These results represent the first reported oxidation of arsenic by Thiomonas sp. Biologically mediated oxidation and subsequent immobilization of arsenic is of great interest for the remediation of contaminated mine sites.