Improved sulfur autotrophic denitrification using supplementary bovine serum albumin.

Excess nitrate presented in natural water body and drinking water has been a challenge for maintaining safe ecosystem and human health. Sulfur autotrophic denitrification is proved a feasible technology to remove nitrate from water environment. However, comparatively low rate of sulfur autotrophic denitrification needs to be addressed before wide application of this technology, which is a result of the low solubility of elemental sulfur. Therefore, this study employed bovine serum albumin (BSA) as a supplementary material to modify the elemental sulfur for improved sulfur autotrophic denitrification rate. Artificial biofilm of Thiobacillus denitrificans was prepared and employed in experiments. By testing different amount of BSA applied in both elemental sulfur and the biofilm, including 1 %, 2 % and 4 % mass ratios, it was found that larger employment of BSA had significant effect in increasing the denitrification rate. Particularly when 4 % BSA was added into elemental sulfur, the highest denitrification rate reached 26.8 mg-N/(L·d), 3.7 times of the control group. Meanwhile, the largest reaction rate constant was achieved, 4.13 mg0.5/(L0.5·d), 2.78 times of the control group. This effect was attributed to promoted conversion of elemental sulfur to polysulfide that was easily utilized by sulfur-oxidizing bacteria. A long-term operation (14 days) of packed bed reactor filled with sulfur particles and 1 % BSA delivered a much faster start-up than the control and outperformed it with better denitrification performance all-through the experiment. This result evidenced again that BSA could make a highly effective supplement in sulfur autotrophic denitrification.

Light-driven nitrous oxide production via autotrophic denitrification by self-photosensitized Thiobacillus denitrificans.

N2O (Nitrous oxide, a booster oxidant in rockets) has attracted increasing interest as a means of enhancing energy production, and it can be produced by nitrate (NO3-) reduction in NO3--loading wastewater. However, conventional denitrification processes are often limited by the lack of bioavailable electron donors. In this study, we innovatively propose a self-photosensitized nonphototrophic Thiobacillus denitrificans (T. denitrificans-CdS) that is capable of NO3- reduction and N2O production driven by light. The system converted >72.1 ±â€¯1.1% of the NO3--N input to N2ON, and the ratio of N2O-N in gaseous products was >96.4 ±â€¯0.4%. The relative transcript abundance of the genes encoding the denitrifying proteins in T. denitrificans-CdS after irradiation was significantly upregulated. The photoexcited electrons acted as the dominant electron sources for NO3- reduction by T. denitrificans-CdS. This study provides the first proof of concept for sustainable and low-cost autotrophic denitrification to generate N2O driven by light. The findings also have strong implications for sustainable environmental management because the sunlight-triggered denitrification reaction driven by nonphototrophic microorganisms may widely occur in nature, particularly in a semiconductive mineral-enriched aqueous environment.

Thermophilic prokaryotic communities inhabiting the biofilm and well water of a thermal karst system located in Budapest (Hungary).

In this study, scanning electron microscopy (SEM) and 16S rRNA gene-based phylogenetic approach were applied to reveal the morphological structure and genetic diversity of thermophilic prokaryotic communities of a thermal karst well located in Budapest (Hungary). Bacterial and archaeal diversity of the well water (73.7 °C) and the biofilm developed on the inner surface of an outflow pipeline of the well were studied by molecular cloning method. According to the SEM images calcium carbonate minerals serve as a surface for colonization of bacterial aggregates. The vast majority of the bacterial and archaeal clones showed the highest sequence similarities to chemolithoautotrophic species. The bacterial clone libraries were dominated by sulfur oxidizer Thiobacillus (Betaproteobacteria) in the water and Sulfurihydrogenibium (Aquificae) in the biofilm. A relatively high proportion of molecular clones represented genera Thermus and Bellilinea in the biofilm library. The most abundant phylotypes both in water and biofilm archaeal clone libraries were closely related to thermophilic ammonia oxidizer Nitrosocaldus and Nitrososphaera but phylotypes belonging to methanogens were also detected. The results show that in addition to the bacterial sulfur and hydrogen oxidation, mainly archaeal ammonia oxidation may play a decisive role in the studied thermal karst system.

Linking TFT-LCD wastewater treatment performance to microbial population abundance of Hyphomicrobium and Thiobacillus spp.

This study investigated the linkage between performance of two full-scale membrane bioreactor (MBR) systems treating thin-film transistor liquid crystal display (TFT-LCD) wastewater and the population dynamics of dimethylsulfoxide (DMSO)/dimethylsulfide (DMS) degrading bacteria. High DMSO degradation efficiencies were achieved in both MBRs, while the levels of nitrification inhibition due to DMS production from DMSO degradation were different in the two MBRs. The results of real-time PCR targeting on DMSO/DMS degrading populations, including Hyphomicrobium and Thiobacillus spp., indicated that a higher DMSO oxidation efficiency occurred at a higher Hyphomicrobium spp. abundance in the systems, suggesting that Hyphomicrobium spp. may be more important for complete DMSO oxidation to sulfate compared with Thiobacillus spp. Furthermore, Thiobacillus spp. was more abundant during poor nitrification, while Hyphomicrobium spp. was more abundant during good nitrification. It is suggested that microbial population of DMSO/DMS degrading bacteria is closely linking to both DMSO/DMS degradation efficiency and nitrification performance.

Biological oxidation of hydrogen sulfide in mineral media using a biofilm airlift suspension reactor.

Hydrogen sulfide (H(2)S) removal in mineral media using Thiobacillus thioparus TK-1 in a biofilm airlift suspension reactor (BAS) was investigated to evaluate the relationship between biofilm formation and changes in inlet loading rates. Aqueous sodium sulfide was fed as the substrate into the continuous BAS-reactor. The reactor was operated at a constant temperature of 30 degrees C and a pH of 7, the optimal temperature and pH for biomass growth. The startup of the reactor was performed with basalt carrier material. Optimal treatment performance was obtained at a loading rate of 4.8 mol S(2-) m(-3) h(-1) at a conversion efficiency as high as 100%. The main product of H(2)S oxidation in the BAS-reactor was sulfate because of high oxygen concentrations in the airlift reactor. The maximum sulfide oxidation rate was 6.7 mol S(2-) m(-3) h(-1) at a hydraulic residence time of 3.3 h in the mineral medium. The data showed that the BAS-reactor with this microorganism can be used for sulfide removal from industrial effluent.

Direct current stimulation of Thiobacillus ferrooxidans bacterial metabolism in a bioelectrical reactor without cation-specific membrane.

A bioelectrical reactor without cation-specific membrane was designed to test effects of direct electrical current on growth of Thiobacillus ferrooxidans bacterium. The results indicated that the cell significantly enhanced the growth of T. ferrooxidans. At a current of 30 mA, the maximum cells density reached 1.39 x 10(9)cells/mL within 84 h, which was 10 times faster than under a conventional cultivation method, in which electrical current is not used. A lag phase during the growth of T. ferrooxidans was observed when direct electrical current was applied, and the lag phase became longer under higher current intensity.

Removal of hydrogen sulfide by immobilized Thiobacillus thioparus in a biotrickling filter packed with polyurethane foam.

In the work described here, a biotrickling filter with Thiobacillus thioparus (ATCC 23645) immobilized on polyurethane foam is proposed for the removal of hydrogen sulfide contained in air. The effect of surface velocity of the recirculation medium (5.9-1.2 m/h), sulfate concentration inhibition (3.0-10.7 g/L), pH (6.0-8.2), empty bed residence time (EBRT) (150-11 s) for constant loads of 11.5 and 2.9 g S/m(3)/h, and pressure drop of the system were investigated. The total amount of biomass immobilized on the carrier was 8.2+/-1.3x10(10) cells/g. The optimal values of the operating variables were: pH between 7.0 and 7.5, surface velocity of 5.9 m/h and sulfate concentration below 5 g/L. The critical EC value was 14.9 g S/m(3)/h (removal efficiency of 99.8%) and the EC(max) was 55.0 g S/m(3)/h (removal efficiency of 79.8%) for an EBRT of 150 s. For loads of 2.89+/-0.05 and 11.5+/-0.1 g S/m(3)/h, the removal efficiency was higher than 99% for an EBRT over 90 s.

Development of a genetic system for the chemolithoautotrophic bacterium Thiobacillus denitrificans.

Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.

Evidence for the involvement of betaproteobacterial Thiobacilli in the nitrate-dependent oxidation of iron sulfide minerals.

The Thiobacilli are an important group of autotrophic bacteria occurring in nature linking the biogeochemical cycles of sulfur and nitrogen. Betaproteobacterial Thiobacilli are very likely candidates for mediating the process of nitrate-dependent anoxic iron sulfide mineral oxidation in freshwater wetlands. A Thiobacillus denitrificans-like bacterium was present in an enrichment on thiosulfate and nitrate, derived from an iron-sulfide- and nitrate-rich freshwater environment. Preliminary FISH analysis showed that the 16S rRNA gene-based bacterial probe mix showed great variation in intensity under different culture conditions. Furthermore, the widely applied 23S rRNA gene-based probe set BET42a/GAM42a incorrectly identified the T. denitrificans-like bacterium as a member of the Gammaproteobacteria. To circumvent these problems, the 23S rRNA genes of two T. denitrificans strains were partially sequenced and a new 23S rRNA gene-based probe (Betthio 1001) specific for betaproteobacterial Thiobacilli was designed. Use of this new probe Betthio 1001, combined with field measurements, indicates the involvement of Thiobacilli in the process of nitrate-dependent iron sulfide mineral oxidation.

[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.

An innovative process of simultaneous desulfurization and denitrification by Thiobacillus denitrificans.

Thiobacillus denitrificans, a species of autotrophic facultative anaerobic bacterium, was found to be capable of oxidizing sulfide into elemental sulfur when nitrate was adopted as its electron acceptor and carbon dioxide as its sole carbon source under anoxic conditions. In this way, sulfur was accumulated extracellularly and nitrate was converted into nitrogen gas. Based on these special physiological characteristics, an innovative process of simultaneous desulfurization and denitrification (SDD) was developed to obtain sulfur from sulfide. A strain of T. denitrificans, named D(4), was isolated and used as sulfur producers in this study. The stoichiometric equations of SDD by T. denitrificans were also derived. The key factors affecting this process were investigated through continuous-flow and batch tests. The experimental results indicated that both the sulfide concentration and the ratio of S2-/NO3- in the influent are key factors. Their suitable levels are suggested to be controlled less than 300 mg/L and 5/3-5/2, respectively, to achieve a high sulfur conversion degree.

[Adhesion and colonisation of the glass surface by Thiobacillus thioparus and Stenotrophomonas maltophilia and their biculture].

The thionic bacteria Thiobacillus thioparus and its natural sattelite Stenotrophomonas maltophilia have been isolated from the soil, adjacent to the surface of Kyiv underground tunnel. The sterile glass, was used as a model surface which imitates the hydrophilic model surface. Beijerinck nutrition media were inoculated by pure and mixed culture of T. thioparus. Some differences in adhesion by mono- and mixed cultures were shown. Hemolithotrophic and heterotrophic bacteria could be interrelated and this could influence the biofilm formation. The formation of biofilm of T. thioparus mixed culture occurs more actively in comparison with the pure culture.

Sulfur formation by steady-state continuous cultures of a sulfoxidizing consortium and Thiobacillus thioparus ATCC 23645.

The elemental sulfur formation by the partial oxidation of thiosulfate by both a sulfoxidizing consortium and by Thiobacillus thioparus ATCC 23645 was studied under aerobic conditions in chemostat. Steady state was attained with essentially total conversion to sulfate when the dissolved oxygen concentration was 5 mgO2 l(-1) and below a dilution rate (D) of 3.0 d(-1)for the consortium and 0.9 d(-1) for T thioparus. The consortium formed elemental sulfur in steady state under oxygen limitation. Fifty percent of the theoretical elemental sulfur yield was obtained with a dissolved oxygen concentration of 0.2 mgO2 l(-1). Growth of T thioparus was negatively affected with a concentration below 1.9 mgO2 l(-1). Consortium yield from batch cultures was 2.1 g(-1) (protein) mol(-1) (thiosulfate), which was comparable with the values obtained in the chemostat at dilution rates of 0.4 d(-1) and 1.2 d(-1). The consortium showed a maximum degradation rate of 0.105 g(thiosulfate) g(-1) (protein) min(-1) and a saturation rate for S2O3(2-) of 1.9 mM.

Nitrate removal from saline water using autotrophic denitrification by the bacterium Thiobacillus denitrificans MP-1.

Autotrophic denitrification of synthetic wastewater by Thiobacillus denitrificans MP-1 isolated from mangrove sediment was investigated in both up-flow packed-bed reactors and fermentor. More than 97.5% and 90% of the nitrate in inflow was removed after 8.8 and 161 hours at 250 and 195 mg l(-1) for the packed-bed reactor and fermentor system, respectively. The nitrate was quickly denitrified at very low flow rates (0.11 m h(-1)) for the packed-bed reactors, but as the flow rate was greater than 0.13 m h(-1), the nitrate removal rate increased as the flow rate increased. In the static fermentor system, the denitrification can be described by a secondary reaction, but at a flow rate between 1.31 to 1.49 m h(-1), the reactor performance can be described using the zero-order reaction in the packed-bed reactor. As the speed increases, the zero-order reaction translates into half-order reaction as the penetration efficiency of nitrate decreases. The mass ratios between the nitrate removed and the sulfate produced were determined to be 6.81 and 9.32 in the reactor column and fermentor, respectively. The results of this study suggest that efficient removal of high concentrations of nitrate in water or wastewater can be achieved effectively using autotrophic bacteria immobilized on surfaces of sulphur granules in packed-bed reactor.

Removal of high NO3- concentrations in saline water through autotrophic denitrification by the bacterium Thiobacillus denitrificans strain MP.

Autotrophic denitrification by Thiobacillus denitrificans MP isolated from mangrove was investigated in both a sulphur-limestone column reactor and a fermenter. More than 97.5% of the nitrate (NO3-) in the 250 mg NO3(-)-N/L strong influent was removed after 14.3 hours in the column reactor. Influent NO3- was completely depleted in the lower part of the column as the hydraulic retention time increased and a slight pH drop was also observed along the reactor column due to the exhaustion of the buffering ability of the limestone. Trace amounts of oxygen present in the lower part of the reactor column resulted in the accumulation of nitrite and subsequent inhibition of further denitrification. The species composition of the bacterial community in the higher parts of the reactor column was morphologically more diverse than in the lower part. Denitrification by T. denitrificans MP reached an optimal level when the dissolved oxygen was maintained between 1.5-2% of saturation level in the automated fermenter. The stoichiometric ratios of deltaSO4(2-) produced/deltaNO3(-)-N removed were 6.81 and 9.32 in the reactor column and fermenter, respectively. This study suggests that efficient removal of high NO3- concentrations in water or wastewater can be achieved using autotrophic bacteria immobilized on surfaces of sulphur granules in the column system.

Development of supporting materials for microbial immobilization and iron oxidation.

We developed the microbial immobilization particle with curdlan and activated carbon, which has great adsorption capacity. The characteristics of porosity and mechanical strength of these supporting particles are dependent on manufacturing method. The supporting particle showed the best performance when the ratio of curdlan and activated carbon was 30 to 6 g/L. Brumauer-Emmett-Teller (specific surface area) and swelling capacity of the carrier were 52.63 m2/g and 17 (w/w), respectively. The immobilization characteristics of iron-oxidizing bacteria on supporting particles were observed using a scanning electron microscope. The concentration of microorganism on the surface of supporting particle was increased with reaction time. As the number of iron oxidation batch cycles increased, the iron oxidation rate increased.

[Characteristics of the restriction profile of chromosomal DNA in strains of Acidithiobacillus ferroxidans, adapted to various oxidation substrates]. / Kharakteristika restriktsionnykh profilei khromosomnoi DNK u shtammov Acidithiobacillus ferrooxidans, adaptirovannykh k raznym substratam okisleniia.

Restriction profiles of chromosomal DNA were studied in different Acidithiobacillus ferrooxidans strains grown on medium with Fe2+ and further adapted to another oxidation substrate (S0, FeS2, or sulfide ore concentrates). The restriction endonuclease XbaI digested the chromosomal DNA from different strains into different numbers of fragments of various sizes. Adaptation of two strains (TFBk and TFN-d) to new oxidation substrates resulted in structural changes in XbaI-restriction patterns of their chromosomal DNA. Such changes in the DNA restriction patterns occurred in strain TFBk after the adaptation to precyanidated gravitational pyrite-arsenopyrite concentrate (no. 1) from the Nezhdaninskoe deposit or to copper-containing ore from the Udokanskoe deposit and also in strain TFN-d adapted to untreated pyrite-arsenopyrite concentrate (no. 2) from the Nezhdaninskoe deposit. No changes in the number or size of the XbaI-restriction patterns of chromosomal DNA were revealed in either strain TFBk cultivated on media with pyrite from the Angren and Tulun deposits or in strains TFN-d and TFO grown on media with S0 and pyrite. Neither were changes observed in the XbaI-restriction patterns of the DNA from strain TFV-1, isolated from the copper ore of the Volkovskoe deposit, when Fe2+ was substituted with alternative substrates--S0, pyrite or concentrate no. 2 from the ore of Nezhdaninskoe deposit. In strain TFO, no differences in the XbaI-restriction patterns of the chromosomal DNA were revealed between the culture grown on medium containing concentrate no. 2 or the concentrate of surface-lying ore from Olimpiadinskoe deposit and the culture grown on medium with Fe2+. When strain TFO was cultivated on the ore concentrate from deeper horizons of the Olimpiadinskoe deposit, which are characterized by lower oxidation degree and high antimony content, mutant TFO-2 differing from the parent strain in the chromosomal DNA structure was isolated. The correlation between the lability of chromosomal DNA structure in A. ferrooxidans strains and the physical and chemical peculiarities of the isolation substrate and habitat is discussed.

Biological leaching of Mn, Al, Zn, Cu and Ti in an anaerobic sewage sludge effectuated by Thiobacillus ferrooxidans and its effect on metal partitioning.

The chemical fractionation and bioleaching of Mn, Al, Zn, Cu and Ti in municipal sewage sludge were investigated using Thiobacillus ferrooxidans as leaching microorganism. As a result of the bacterial activity, ORP increase and pH reduction were observed. Metal solubilization was accomplished only in experimental systems supplemented with energy source (Fe(II)). The solubilization efficiency approached approximately 80% for Mn and Zn, 24% for Cu, 10% for Al and 0.2% for Ti. The chemical fractionation of Mn, Al, Zn, Cu and Ti was investigated using a five-step sequential extraction procedure employing KNO3, KF, Na4P2O7, EDTA and HNO3. The results show that the bioleaching process affected the partitioning of Mn and Zn, increasing its percentage of elution in the KNO3 fraction while reducing it in the KF, Na4P2O7 and EDTA fractions. No significant effect was detected on the partitioning of Cu and Al. However, quantitatively the metals Mn, Zn, Cu and Al were extracted with higher efficiency after the bacterial activity. Titanium was unaffected by the bioleaching process in both qualitative and quantitative aspects.