Nitrogen transforming bacteria within a full-scale partially saturated vertical subsurface flow constructed wetland treating urban wastewater.

The aim of this study was to characterize the nitrogen transforming bacterial communities within a partially saturated vertical subsurface flow constructed wetland (VF) treating urban wastewater in southern Brazil. The VF had a surface area of 3144m2, and was divided into four wetland cells, out of which two were operated while the other two rested, alternating cycles of 30days. The nitrifying and denitrifying bacterial communities were characterized in wetland cell 3 (764m2 surface area) over a period of 12months by using the FISH technique. Samples were collected monthly (from Feb 2014 to Feb 2015) from different layers within the vertical profile, during operation and rest periods, comprising a total of 6 sampling campaigns while the cell was in operation and another 6 when the cell was at rest. This wetland cell operated with an average organic loading rate (OLR) of 4gCODm-2d-1 and a hydraulic loading rate of 24.5mmd-1. The rest periods of the wetland cell presented influences on the abundance of ammonia-oxidizing bacteria (AOB) (8% and 3% for feed and rest periods, respectively), and nitrite-oxidizing bacteria (NOB) (5% and 2% for feed and rest periods, respectively). However, there was no influence of the rest periods on the denitrifying bacteria. AOB were only identified in the top layer (AOB ß-proteobacteria) in both operational and rest periods. On the other hand, the NOB (Nistrospirae and Nitrospina gracilis) were identified in feed periods just in the top layer and during rest periods just in the intermediate layer. The denitrifying bacteria (Pseudomonas spp. and Thiobacillus denitrificans) were identified from the intermediate layer downwards, and remained stable in both periods. Based on the identified bacterial dynamics, the partially saturated VF wetland operated under low OLR enabled favorable conditions for simultaneous nitrification and denitrification.

Dimethyl sulfide biofiltration using immobilized Hyphomicrobium VS and Thiobacillus thioparus TK-m in sugarcane bagasse.

Sugarcane bagasse was used as a carrier material of microorganisms in two different biofilters used to remove dimethyl sulfide (DMS) from a gas stream. The first biofilter was inoculated with Hyphomicrobium VS and the second with Thiobacillus thioparus Tk-m. During the operation of the biofilters the empty bed residence time (EBRT) was varied from 90 to 180 seconds and the inlet concentration of DMS from 12 to 50 ppmv. The inlet load was varied in the range of 0.62 to 5.2 (g DMS/m3 h). The maximum elimination capacity (EC) of the biofilter inoculated with Hyphomicrobium VS was 5 g DMS/m3 h; however, for the biofilter inoculated with T. thioparus Tk-m the maximum EC was 3.9 g DMS/m3 h. For T. thioparus TK-m the maximum removal efficiency (RE) obtained was 85.1 +/- 5.2% at 12 ppmv DMS inlet concentration, inlet load of 0.62 g DMS/m3 h and 180 s EBRT. The highest removal efficiency for Hyphomicrobium VS was 97.6 + 4.8% at 12 ppmv DMS inlet concentration, load of 0.62 g DMS/m3 h and 180 s EBRT.

Kinetics of the bio-oxidation of volatile reduced sulphur compounds in a biotrickling filter.

Mixtures of volatile reduced sulphur compounds (VRSCs) like hydrogen sulphide (H(2)S), methylmercaptan (MM), dimethyl sulphide (DMS) and dimethyl disulphide (DMDS) are found in gaseous emissions of several industrial activities creating nuisance in the surroundings. Hydrogen sulphide (H(2)S) decreases the removal efficiency of volatile reduced sulphur compounds (VRSCs) in biofilters but the kinetics of this effect is still unknown. Kinetic expressions that represent the rate of bio-oxidation of H(2)S, MM, DMS and DMDS are proposed. In order to observe and quantify this effect, equimolar mixtures of MM, DMS and DMDS were fed into a biotrickling filter inoculated with Thiobacillus thioparus at different H(2)S loads. Experimental results shown a good agreement with the simulations generated by the model considering the kinetic equations proposed. The estimated kinetic constants show that H(2)S and MM have a significant inhibitory effect on the bio-oxidation of DMS and DMDS, having the H(2)S the higher effect.

Oxidation of volatile reduced sulphur compounds in biotrickling filter inoculated with Thiobacillus thioparus

Reduced volatile sulphur compounds generate an impact on the environment, because of the bad smell and its low odour threshold. Compared with the existing physicochemical technologies for their elimination, biotrickling filters are an economically and environmentally sustainable alternative. Usually mixed cultures of microorganisms are used for inoculating biotrickling filters, in this case a pure culture of Thiobacillus thioparus is used for generating a biofilm, allowing to measure its capacity for the oxidation of four volatile reduced sulphur compounds: hydrogen sulphide, dimethyl sulphide, methyl mercaptan and dimethyl disulphide, using a residence time of 0.033 hrs. The viable cells of the biofilm were quantified by epifluorescence microscopy, staining the cells with ethidium bromide and acridine orange, polymerase chain reaction analysis in real time was used for testing the predominance of T. thiopharus in the biofilm. The microorganism was able to adhere and grow on the surface of rings made of polyethylene, with a viable population of 7•10(7) cell•ring-1, a 74 percent of total cells. The real time PCR showed a persistence of the population of T. thioparus for more than 300 days of operation, without being displaced by other microbial species. The maximum elimination capacities for each compound were 34.4; 21.8; 30.8 and 25.6 gS•m-3•h-1 for H2S, dimethylsulphide, dimethyldisulphide and methyl mercaptan, respectively. We conclude that it is possible to implement a biotrickling filter with the bacteria T. thioparus, which can oxidize volatile reduced sulphur compounds efficiently.

Comparison on the removal of hydrogen sulfide in biotrickling filters inoculated with Thiobacillus thioparus and Acidithiobacillus thiooxidans

Emissions of hydrogen sulfide (H2S) by industrial activities is frequent cause of corrosion and unpleasant odours. Treatment of gaseous emissions contaminated with H2S by biotrickling filters inoculated with single cultures of sulfur oxidizer bacteria exhibit several advantages over physicochemical methods, such as shorter adaptation times and higher removal ability. Biofilms of Thiobacillus thioparus and Acidithiobacillus thiooxidans have proved to exhibit high removal capacities, yet no comparative studies between them have been reported. This article reports the efficiency of biotrickling filters inoculated with T. thioparus and A. thiooxidans under similar conditions excepting the pH, that was the optimal for the bacterial growth, for the removal of H2S. The support was selected by determining the respirometric coefficients of the biomass. The maximum removal capacity of the biofilter inoculated with T. thioparus, operating within the range of pH (5.5-7.0) was 14 gS m-3 h-1, lower the value obtained for the biotrickling filter inoculated with A. thiooxidans; 370 gS m-3 h-1. Therefore, it is concluded that acid biotrickling filter inoculated with A. thiooxidans constitute the best strategy to remove H2S, with the advantage that the system not require an exhaustive pH control of the liquid media.

The role of higher polythionates in the reduction of chromium(VI) by Acidithiobacillus and Thiobacillus cultures.

In this paper, we report the chromium(VI) reduction by filtrates of Acidithiobacillus and Thiobacillus cultures. Chromium(VI) reduction by filtrates of A. ferrooxidans cultures under acidic conditions was higher than that observed for A. thiooxidans. However, at pH close to 7, chromium(VI) reduction by filtrates of T. thioparus cultures was as high as that by filtrates of A. thiooxidans cultures and much higher than that observed for A. ferrooxidans cultures at the same pH. The capability of these cultures to reduce chromium(VI) was associated specifically with the fraction of cultures (cells, sulphur and associated sulphur compounds) retained by filtration through a 0.45mum filter. In the fraction that comes from A. thiooxidans culture, polythionates (S(x)O(6)(2-)) with 3-7 sulphur atoms were detected and identified (by HPLC with MS as detector). The model of vesicles containing polythionates, sulphur and water agrees with our results.

Novel electrochemical-enzymatic model which quantifies the effect of the solution Eh on the kinetics of ferrous iron oxidation with Acidithiobacillus ferrooxidans.

The influence of solution Eh on the rate of ferrous iron oxidation by Acidithiobacillus ferrooxidans is characterized. The experimental approach was based on the use of a two-chamber bioelectrochemical cell, which can determine the ferrous iron oxidation rate at controlled potential. Results enabled the formulation of a novel kinetic model, which incorporates the effect of solution Eh in an explicit form but still integrates the effect of ferrous iron concentration and ferric inhibition. The results showed that at Eh values below 650 mV (standard hydrogen electrode, SHE) the bacterial oxidative activity is mainly dependent on ferrous iron concentration. At Eh values between 650 and 820 mV (SHE) the oxidation rate is mainly controlled by ferric inhibition. Over 820 mV (SHE) the bacterial oxidative activity is strongly inhibited by the Eh increase, being completely inhibited at Eh = 840 mV (SHE).

Estimation of kinetic rates in batch Thiobacillus ferrooxidans cultures.

In this work, the key problem of estimation in bioprocesses when no structural model is available is dealt with. A nonlinear observer-based algorithm is developed in order to estimate kinetic rates in batch bioreactors. The algorithm uses the measurements of biomass concentration and either substrate concentration or redox potential to perform the estimation of the respective specific kinetic rates. For this purpose, a general mathematical model description of the process is provided. The estimation algorithm design is based on a nonlinear reduced-order observer. The observer performance is validated with experimental results on a Thiobacillus ferrooxidans batch culture.

Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation.

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.

IST1 insertional inactivation of the resB gene: implications for phenotypic switching in Thiobacillus ferrooxidans.

Thiobacillus ferroxidans ATCC 19859 undergoes rapid phenotypic switching between a wild-type state characterized by the ability to oxidize ferrous iron (FeII) and reduced sulfur compounds and a mutant state where it has lost the capacity to oxidize FeII but retains the ability to oxidize sulfur. The mutant has also gained the capacity to swarm. It is proposed that loss of FeII oxidation is due to the reversible transposition of the insertion sequence IST1 into resB encoding a putative cytochrome c-type biogenesis protein. Downstream from resB and co-transcribed with it is resC, encoding another putative cytochrome biogenesis protein. IST1 insertional inactivation of resB could result in the loss of activity of its target c-type cytochrome(s). This putative target cytochrome(s) is proposed to be essential for FeII oxidation but not for sulfur oxidation. Curiously, resB and resC pertain to the proposed system II cytochrome biogenesis pathway whereas gamma Proteobacteria, of which T. ferrooxidans is a member, normally use system I. This could represent an example of lateral gene transfer.

An analysis of a trickle-bed bioreactor: carbon disulfide removal.

An analysis of the local processes occurring in a trickle-bed bioreactor (TBB) with a first-order bioreaction shows that the identification of the TBB operating regime requires knowledge of the substrate concentration in the liquid phase. If the substrate liquid concentration is close to 0, the rate-controlling step is mass transfer at the gas-liquid interface; when it is close to the value in equilibrium with the gas phase, the controlling step is the phenomena occurring in the biofilm. CS2 removal rate data obtained in a TBB with a Thiobacilii consortia biofilm are analyzed to obtain the mass transfer and kinetic parameters, and to show that the bioreactor operates in a regime mainly controlled by mass transfer. A TBB model with two experimentally determined parameters is developed and used to show how the bioreactor size depends on the rate-limiting step, the absorption factor, the substrate fractional conversion, and on the gas and liquid contact pattern. Under certain conditions, the TBB size is independent of the flowing phases' contact pattern. The model effectively describes substrate gas and liquid concentration data for mass transfer and biodegradation rate controlled processes.

An evaluation into the potential of biological processing for the removal of metals from sewage sludges.

The use of sewage sludge in agricultural land as a means of sludge disposal and recycling has been shown to be economical and suitable because of the presence of nutrients such as nitrogen and phosphorus. However, municipal sludges often contain high quantities of toxic metals and other compounds that must be removed for its safe use in agricultural soils. The biological leaching of metals from sewage sludges has been shown to be a promising technique for metal detoxifying in such complex matrix. The process efficiency is dependent on several physico-chemical parameters, such as total solids concentration, metal forms, pH-ORP, and temperature. Scale-up of the process has not yet been defined and is still pursuing the correct operational design. Current research involving the bioleaching of metals from sewage sludge and its application to land, which affects soil physical properties, are presented and discussed.

Bio-dissolution of spent nickel-cadmium batteries using Thiobacillus ferrooxidans.

In this study, the production of sulphuric acid in bioreactors with Thiobacillus ferrooxidans attached on elemental sulphur was investigated. These bioreactors reached a maximum H+ productivity of 80 mmol kg-1 d-1 of support. This medium was used for the indirect dissolution of spent nickel-cadmium batteries recovering after 93 days 100% of cadmium, 96.5% of nickel and 95.0% of iron. Moreover, recoveries higher than 90.0% were reached when anodic and cathodic materials were directly added to Thiobacillus ferrooxidans cultures with sulphur as the sole energy source. The results presented show an economic and effective method which could be considered the first step to recycle spent and and discarded batteries preventing one of the many problems of environmental pollution.

Phosphorylation of GroEL, DnaK and other proteins from Thiobacillus ferrooxidans grown under different conditions.

The levels of phosphorylation of the chaperones DnaK and GroEL and other proteins varied when cells of Thiobacillus ferrooxidans were subjected to phosphate starvation. The phosphorylated amino acid of GroEL was found to be threonine. Our results show that not only heat shock, but also a nutrient starvation stress leads to phosphorylation of chaperones and, in addition, support the possible role of phosphorylation of these proteins in the sensing and regulation of stress responses in bacteria.

[Application of immunologic methods to the analysis of bio-leaching bacteria]. / Aplicación de métodos inmunológicos al análisis de bacterias biolixiviadoras.

Pure cultures of Thiobacillus ferrooxidans and mixed cultures of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans isolated from the Matahambre mine (Cuba) were used to fit immunodiffusion and immunoelectron microscopy to the study of iron oxidizing bacteria. The possibilities, advantages and limits of those techniques have been studied from both the identification and the serological characterization points of view. Finally, the efficiency of these methods was tested by applying them to the identification of microorganisms from acidic waters from the mine.

Thiobacillus ferrooxidans detection using immunoelectron microscopy.

A specific, fast and very sensitive immunoelectron microscopy method was developed to morphologically and serologically distinguish different cultures of iron oxidizers. Bacteria isolated from the acidic waters of "Matahambre" and "Mina Delita" mines (Cuba) were characterized. An antiserum specific to Thiobacillus ferrooxidans did not react with other bacteria also present in the acidic waters of mine drainage. Our results suggest the occurrence of some strains of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans in these waters.