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.

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.

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.

[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.

Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2.

Genes encoding toxin-antitoxin proteins are frequently found on plasmids where they serve to stabilize the plasmid within a bacterial population. The toxin-antitoxin proteins do not increase the likelihood of a progeny cell receiving a plasmid but rather function as post-segregational killing mechanisms which decrease the proportion of cells that survive after losing the plasmid. These toxin-antitoxin couples therefore act as plasmid addiction systems. Several new proteic toxin-antitoxin systems have been identified and these systems appear to be ubiquitous on the chromosomes of bacteria and archaea. When placed on plasmids, these chromosomal systems also have the ability to stabilize plasmids and in at least one case, chromosomal- and plasmid-based toxin-antitoxin systems have been shown to interact. Recent findings regarding toxin-antitoxin systems and questions that have arisen as a result of these findings are reviewed.

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.

Effect of sulphur inoculated with Thiobacillus on soil salinity and growth of tropical tree legumes.

A greenhouse experiment was carried out with the objective of evaluating the effects of the elementary sulphur inoculated with Thiobacillus, compared with gypsum, in the amendment of a alluvial sodic saline soil from the Brazilian semiarid region, irrigated with saline water and grown with the tropical legumes leucena and mimosa. The treatments consisted of levels of sulphur (0; 300 and 600 kg/ha) and gypsum (1,200 and 2,400 kg/ha), irrigation using different waters containing the salts NaHCO3, MgCl2, CaCl2, NaCl and KCl, with different electrical conductivities (ECs: 0.2. 6.1 and 8.2 dS/m at 25 degrees C). Based on the results it appears that saline water increased exchangeable Na+, K+, Ca2+, Mg2+, and soil pH. Sulphur inoculated with Thiobacillus was more efficient than gypsum in the reduction of the exchangeable sodium of the soil and promoting leaching of salts, especially sodium. Sulphur inoculated with Thiobacillus reduced the EC of the soil saturation extract to levels below that adopted in soil classification of sodic or saline sodic. Leucena was more tolerant to salinity and mimosa more resistant to acidity promoted by sulphur inoculated with Thiobacillus.

Bioleaching of heavy metals from sediment: significance of pH.

Bioleaching process, which causes acidification and solubilization of heavy metals, is one of the promising methods for removing heavy metals from contaminated sediments. The solubilization of heavy metals from contaminated sediments is governed by the sediment pH. In the present study, the significance of pH in bioleaching of heavy metals from contaminated sediment was evaluated at different solid contents of sediments in a bench-scale reactor. Results showed that a temporal change of pH in the bioleaching process was effected by the buffering capacity of the sediment particulates. The variations of pH in this bioleaching process were calculated by a modified logistic model. It was observed that solubilization of heavy metals from sediments is highly pH-dependent. In addition, a non-linear equation for metal solubilization relating pH value in the bioleaching process was established. This allows an easier and faster estimate of metal solubilization by measuring pH in the bioleaching process.

Biotreatment of H2S- and NH3-containing waste gases by co-immobilized cells biofilter.

Gas mixture of H2S and NH3 in this study has been the focus in the research area concerning gases generated from the animal husbandry and the anaerobic wastewater lagoons used for their treatment. A specific microflora (mixture of Thiobacillus thioparus CH11 for H2S and Nitrosomonas europaea for NH3) was immobilized with Ca-alginate and packed inside a glass column to decompose H2S and NH3. The biofilter packed with co-immobilized cells was continuously supplied with H2S and NH3 gas mixtures of various ratios, and the removal efficiency, removal kinetics, and pressure drop in the biofilter was monitored. The results showed that the efficiency remained above 95% regardless of the ratios of H2S and NH3 used. The NH3 concentration has little effect on H2S removal efficiency, however, both high NH3 and H2S concentrations significantly suppress the NH3 removal. Through product analysis, we found that controlling the inlet ratio of the H2S/NH3 could prevent the biofilter from acidification, and, therefore, enhance the operational stability. Conclusions from bioaerosol analysis and pressure drop in the biofilter suggest that the immobilized cell technique creates less environmental impact and improves pure culture operational stability. The criteria for the biofilter operation to meet the current H2S and NH3 emission standards were also established. To reach Taiwan's current ambient air standards of H2S and NH3 (0.1 and 1 ppm, respectively), the maximum inlet concentrations should not exceed 58 ppm for H2S and 164 ppm for NH3, and the residence time be kept at 72 s.

Effect of substrate concentration on bioleaching of metal-contaminated sediment.

The remediation of metal-contaminated sediment was studied using the bioleaching process with a mixed culture of sulfur-oxidizing bacteria. The effects of substrate concentration (elemental sulfur) on sediment acidification, sulfur oxidation and metal solubilization from contaminated sediment during the bioleaching process were investigated with free-cell suspensions. Sulfur concentration greater than 0.5% (w/v) was found to be inhibitory to bacterial activity and metal solubilization from sediment. The sulfate production was well described by a substrate inhibition expression and Haldane's equation. In addition, an empirical equation related to sulfur concentration was also used to describe the metal solubilization in the bioleaching process.

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.

Carbon disulfide and hydrogen sulfide removal with a peat biofilter.

Simultaneous removal of H2S and CS2 was studied with a peat biofilter inoculated with a Thiobacillus strain that oxidizes both compounds in an acidic environment. Both sulfurous gases at concentrations below 600 mg S/m3 were efficiently removed, and the removal efficiencies were similar, 99%, with an empty bed retention time (EBRT) of more than 60 sec. Concentrations greater than 1300-5000 mg S/m3 caused overloading of the filter material, resulting in high H2SO4 production, accumulation of elemental sulfur, and reduced removal efficiency. The highest sulfur removal rate achieved was 4500 g-S/day/m3 filter material. These results indicate that peat is suitable as a biofilter material for the removal of a mixture of H2S and CS2 when concentrations of gases to be purified are low (less than 600 mg/m3), but it is still odorous and toxic to the environment and humans.

An evaluation of the outer membrane charge and softness of Thiobacillus ferrooxidans by the Ohshima's electrophoretic model of a "soft" particle.

The surface charge of bacterial cells plays an important role in their interfacial physiology and adhesion to substrata mediated by the electrostatic double-layer interaction. The surface charge or potential of biological cells is generally calculated from the experimentally measurable electrophoretic velocity of these cells migrating in an external electric field, applying the well-known Smoluchowski equation which is valid for "hard" particles with a sharp interface. However, bacterial cells possessing a structured outer membrane of a finite thickness (dependent on the ionic strength and pH of the surrounding liquid medium) are expected to obey Ohshima's electrophoretic mobility equation derived recently for "soft" particles. The electrophoretic mobility of Thiobacillus ferrooxidans was measured here by the fully automated technique of electrophoretic light scattering, based on the proportionality between the mobility and the Doppler shift in the frequency of light scattered by electrophoresing cells. Agreement was obtained between the experimentally determined electrophoretic mobility expressed as a function of low ionic strength (60-6000 mumol/L) at different pH values and the best-fit theoretical predictions of the "soft" particle electrophoresis theory, which is better than in the case of applying the Smoluchowski formula. The best-fit surface-charge and softness parameters predict a rather rigid and low-charge outer membrane of the bacterium examined, as compared to the parameters obtained for other bacteria in media of high ionic strength.

Heavy metal removal from wastewater and leachate co-treatment sludge by sulfur oxidizing bacteria.

Heavy metal concentration in sludge is one of the major obstacles for the application of sludge on land. There are various methods for the removal of heavy metals in sludge. Using sulfur oxidizing bacteria for microbiological removal of heavy metals from sludges is an outstanding option because of high metal solubilization rates and the low cost. In this study, bioleaching by indigenous sulfur oxidizing bacteria was applied to sludges generated from the co-treatment of municipal wastewater and leachate for the removal of selected heavy metals. Sulfur oxidizing bacteria were acclimated to activated sludge. The effect of the high organic content of leachate on the bioleaching process was investigated in four sets of sludges having different concentrations of leachate. Sludges in Sets A, B, C and D were obtained from co-treatment of wastewater and 3%, 5%, 7% and 10% (v/v) leachate respectively. The highest Cr, Ni and Fe solubilization was obtained from Set A. Sulfur oxidizing bacteria were totally inhibited in Set D that received the highest volume of leachate.

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.

Autotrophic denitrification for combined hydrogen sulfide removal from biogas and post-denitrification.

In this paper we describe an alternative flow-chart for full treatment of wastewaters rich in organic substrates, ammonia (or organic nitrogen), and sulfate, such as those generated in fish cannery industries. Biogas generated during anaerobic pretreatment of these wastewaters is rich in hydrogen sulfide that needs to be removed to enable application of the biogas. Nitrogen elimination is traditionally achieved by subsequent nitrification and denitrification of the effluent of the anaerobic reactor. Alternatively, the hydrogen sulfide in the biogas can be applied as an electron donor in an autotrophic post-denitrification step. In order to study whether sufficient hydrogen sulfide containing biogas for denitrification was produced in the anaerobic reactor, the biogas composition as a function of the anaerobic reactor-pH was estimated based on a typical wastewater composition and chemical equilibrium equations. It is demonstrated that typical sulfate and nitrogen concentrations in fish cannery wastewater are highly appropriate for application of autotrophic post-denitrification. A literature review furthermore suggested that the kinetic parameters for autotrophic denitrification by Thiobacillus denitrificans represent no bottleneck for its application. Initial experimental studies in fixed-film reactors were conducted with sodium sulfide and nitrate as an electron donor-acceptor couple. The results revealed that only moderate volumetric treatment capacities (< 1 g-NO3- N l(-1) day(-1)) could be achieved. Mass balances suggested that incomplete sulfide oxidation to elemental sulfur occurred, limiting biomass retention and the treatment capacity of the reactor. Future research should clarify the questions concerning product formation from sulfide oxidation.

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.

Biological sulfide oxidation in a fluidized bed reactor.

Feasibility of a laboratory scale fluidized bed process for biological sulfide oxidation to elemental sulfur and the formation of well-settleable sulfur sludge is demonstrated. Sulfide oxidation strongly depends upon oxygen concentration, sulfide loading rate and upflow velocity. At reactor dissolved oxygen concentrations (DOr) higher than 0.1 mg l(-1), sulfate was the main product of sulfide oxidation Upon increasing the sulfide loading rate, the sulfate production rate decreased as sulfide oxidation to sulfur showed marked increase. Low formation of sulfate could mean that sulfide was inhibitory to sulfate producing bacteria or that conversion of sulfide to sulfur was more favorable than sulfate production. Sulfide conversions higher than 90% were obtained at sulfide loading rates of 0.13-1.6 kgS mr(-3) d(-1). At DOr less than 0.1 mg l(-1), sulfur was the major end product of the sulfide oxidation. Upflow velocity in the range of 16-26 m h(-1) and sulfide loading rate of 0.9-1.6 kgS mr(-3) d(-1) were necessary for generation of biogranules containing 65-76% of elemental sulfur. The elemental sulfur production of 76% was obtained at upflow velocity of 17 m h(-1) with sulfide loading rate up to 1.6 kgS mr(3)d(-1). Morphological examination of the biogranules showed elemental sulfur deposition in the sludge granule and outside the bacterial cells.