Initial stage of the biofilm formation on the NiTi and Ti6Al4V surface by the sulphur-oxidizing bacteria and sulphate-reducing bacteria.

The susceptibility to the fouling of the NiTi and Ti6Al4V alloys due to the adhesion of microorganisms and the biofilm formation is very significant, especially in the context of an inflammatory state induced by implants contaminated by bacteria, and the implants corrosion stimulated by bacteria. The aim of this work was to examine the differences between the sulphur-oxidizing bacteria (SOB) and sulphate-reducing bacteria (SRB) strains in their affinity for NiTi and Ti6Al4V alloys. The biofilms formed on alloy surfaces by the cells of five bacterial strains (aerobic SOB Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans, and anaerobic SRB Desulfovibrio desulfuricans-3 strains) were studied using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The protein concentrations in liquid media have also been analyzed. The results indicate that both alloys tested may be colonized by SOB and SRB strains. In the initial stage of the biofilm formation, the higher affinity of SRB to both the alloys has been documented. However, the SOB strains have indicated the higher (although differentiated) adaptability to changing environment as compared with SRB. Stimulation of the SRB growth on the alloys surface was observed during incubation in the liquid culture media supplemented with artificial saliva, especially of lower pH (imitated conditions under the inflammatory state, for example in the periodontitis course). The results point to the possible threat to the human health resulting from the contamination of the titanium implant alloys surface by the SOB (A. thiooxidans and A. ferrooxidans) and SRB (D. desulfuricans).

Chemical and surface analysis during evolution of arsenopyrite oxidation by Acidithiobacillus thiooxidans in the presence and absence of supplementary arsenic.

Bioleaching of arsenopyrite presents a great interest due to recovery of valuable metals and environmental issues. The current study aims to evaluate the arsenopyrite oxidation by Acidithiobacillus thiooxidans during 240h at different time intervals, in the presence and absence of supplementary arsenic. Chemical and electrochemical characterizations are carried out using Raman, AFM, SEM-EDS, Cyclic Voltammetry, EIS, electrophoretic and adhesion forces to comprehensively assess the surface behavior and biooxidation mechanism of this mineral. These analyses evidence the formation of pyrite-like secondary phase on abiotic control surfaces, which contrast with the formation of pyrite (FeS2)-like, orpiment (As2S3)-like and elementary sulfur and polysulfide (Sn(2-)/S(0)) phases found on biooxidized surfaces. Voltammetric results indicate a significant alteration of arsenopyrite due to (bio)oxidation. Resistive processes determined with EIS are associated with chemical and electrochemical reactions mediated by (bio)oxidation, resulting in the transformation of arsenopyrite surface and biofilm direct attachment. Charge transfer resistance is increased when (bio)oxidation is performed in the presence of supplementary arsenic, in comparison with lowered abiotic control resistances obtained in its absence; reinforcing the idea that more stable surface products are generated when As(V) is in the system. Biofilm structure is mainly comprised of micro-colonies, progressively enclosed in secondary compounds. A more compact biofilm structure with enhanced formation of secondary compounds is identified in the presence of supplementary arsenic, whereby variable arsenopyrite reactivity is linked and attributed to these secondary compounds, including Sn(2-)/S(0), pyrite-like and orpiment-like phases.

A new genome of Acidithiobacillus thiooxidans provides insights into adaptation to a bioleaching environment.

Acidithiobacillus thiooxidans is a sulfur oxidizing acidophilic bacterium found in many sulfur-rich environments. It is particularly interesting due to its role in bioleaching of sulphide minerals. In this work, we report the genome sequence of At. thiooxidans Licanantay, the first strain from a copper mine to be sequenced and currently used in bioleaching industrial processes. Through comparative genomic analysis with two other At. thiooxidans non-metal mining strains (ATCC 19377 and A01) we determined that these strains share a large core genome of 2109 coding sequences and a high average nucleotide identity over 98%. Nevertheless, the presence of 841 strain-specific genes (absent in other At. thiooxidans strains) suggests a particular adaptation of Licanantay to its specific biomining environment. Among this group, we highlight genes encoding for proteins involved in heavy metal tolerance, mineral cell attachment and cysteine biosynthesis. Several of these genes were located near genetic motility genes (e.g. transposases and integrases) in genomic regions of over 10 kbp absent in the other strains, suggesting the presence of genomic islands in the Licanantay genome probably produced by horizontal gene transfer in mining environments.

Comparison and evaluation of immobilization methods for preparing bacterial probes using acidophilic bioleaching bacteria Acidithiobacillus thiooxidans for AFM studies.

We evaluated different strategies for constructing bacterial probes for atomic force microscopy studies of bioleaching Acidithiobacillus thiooxidans interacting with pyrite mineral surfaces. Of three available techniques, the bacterial colloidal probe technique is the most reliable and provides a versatile platform for quantifying true interactive forces between bioleaching microorganisms and mineral surfaces.

Evaluation of E. coli biofilm as a protective barrier against microbiologically influenced deterioration of concrete (MICD) under mesophilic temperatures.

In this study, Escherichia coli DH5α biofilm was evaluated for its potential to control and minimize microbiologically influenced concrete deterioration (MICD) under mesophilic temperatures (37 °C). Escherichia coli DH5α biofilm was first grown on Portland cement mortar disks for 8 days. Mortar disks were then exposed to two different types of sulfur oxidizing bacteria (SOB) (Thiobacillus neapolitanus and Thiobacillus thiooxidans), which use sulfur compounds as substrate and oxidize them to sulfate and sulfuric acid. The effectiveness of the biofilm against MICD was evaluated by measuring pH, sulfate, calcium concentrations in the reactors and surface analysis of the mortar samples using X-ray diffraction and visual inspection. Overall, the results indicate that the E. coli DH5α biofilm showed good protection against MICD induced by SOB at 37 °C.

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans.

Surfaces of massive chalcopyrite (CuFeS2) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S n(2-); elemental sulfur, S(0); and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S n(2-)) to active (containing increasing amounts of S(0)) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S n(2-)/S(0) speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S n(2-) and S n(2-)/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S(0)). Furthermore, it was observed that cells were partially covered by CuS and S(0) phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S n(2-)) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.

Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans.

Biofilms of Acidithiobacillus thiooxidans were grown on the surface of massive chalcopyrite electrodes (MCE) where different secondary sulfur phases were previously formed by potentiostatic oxidation of MCE at 0.780≤Ean≤0.965 V (electrooxidized MCE, eMCE). The formation of mainly S° and minor amounts of CuS and Sn²â» were detected on eMCEs. The eMCEs were incubated with A. thiooxidans cells for 1, 12, 24, 48, and 120 h in order to temporally monitor changes in eMCE's secondary phases, biofilm structure, and extracellular polymeric substance (EPS) composition (lipids, proteins, and polysaccharides) using microscopic, spectroscopic, electrochemical, and biochemical techniques. The results show significant cell attachments with stratified biofilm structure since the first hour of incubation and EPS composition changes, the most important being production after 48-120 h when the highest amount of lipids and proteins were registered. During 120 h, periodic oxidation/formation of S°/Sn²â» was recorded on biooxidized eMCEs, until a stable CuS composition was formed. In contrast, no evidence of CuS formation was observed on the eMCEs of the abiotic control, confirming that CuS formation results from microbial activity. The surface transformation of eMCE induces a structural transformation of the biofilm, evolving directly to a multilayered biofilm with more hydrophobic EPS and proteins after 120 h. Our results suggest that A. thiooxidans responded to the spatial and temporal distribution and chemical reactivity of the Sn²â»/S°/CuS phases throughout 120 h. These results suggested a strong correlation between surface speciation, hydrophobic domains in EPS, and biofilm organization during chalcopyrite biooxidation by A. thiooxidans.

Evolution of biofilms during the colonization process of pyrite by Acidithiobacillus thiooxidans.

We have applied epifluorescence principles, atomic force microscopy, and Raman studies to the analysis of the colonization process of pyrite (FeS(2)) by sulfuroxidizing bacteria Acidithiobacillus thiooxidans after 1, 15, 24, and 72 h. For the stages examined, we present results comprising the evolution of biofilms, speciation of S (n) (2-) /S(0) species, adhesion forces of attached cells, production and secretion of extracellular polymeric substances (EPS), and its biochemical composition. After 1 h, highly dispersed attached cells in the surface of the mineral were observed. The results suggest initial non-covalent, weak interactions (e.g., van der Waal's, hydrophobic interactions), mediating an irreversible binding mechanism to electrooxidized massive pyrite electrode (eMPE), wherein the initial production of EPS by individual cells is determinant. The mineral surface reached its maximum cell cover between 15 to 24 h. Longer biooxidation times resulted in the progressive biofilm reduction on the mineral surface. Quantification of attached cell adhesion forces indicated a strong initial mechanism (8.4 nN), whereas subsequent stages of mineral colonization indicated stability of biofilms and of the adhesion force to an average of 4.2 nN. A variable EPS (polysaccharides, lipids, and proteins) secretion at all stages was found; thus, different architectural conformation of the biofilms was observed during 120 h. The main EPS produced were lipopolysaccharides which may increase the hydrophobicity of A. thiooxidans biofilms. The highest amount of lipopolysaccharides occurred between 15-72 h. In contrast with abiotic surfaces, the progressive depletion of S (n) (2-) /S(0) was observed on biotic eMPE surfaces, indicating consumption of surface sulfur species. All observations indicated a dynamic biooxidation mechanism of pyrite by A. thiooxidans, where the biofilms stability and composition seems to occur independently from surface sulfur species depletion.

Enhancing sewage sludge dewaterability by bioleaching approach with comparison to other physical and chemical conditioning methods.

The sewage sludge conditioning process is critical to improve the sludge dewaterability prior to mechanical dewatering. Traditionally, sludge is conditioned by physical or chemical approaches, mostly with the addition of inorganic or organic chemicals. Here we report that bioleaching, an efficient and economical microbial method for the removal of sludge-borne heavy metals, also plays a significant role in enhancing sludge dewaterability. The effects of bioleaching and physical or chemical approaches on sludge dewaterability were compared. The conditioning result of bioleaching by Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans on sludge dewatering was investigated and compared with the effects of hydrothermal (121 degrees C for 2 hr), microwave (1050 W for 50 sec), ultrasonic (250 W for 2 min), and chemical conditioning (24% ferric chloride and 68% calcium oxide; dry basis). The results show that the specific resistance to filtration (SRF) or capillary suction time (CST) of sludge is decreased by 93.1% or 74.1%, respectively, after fresh sludge is conditioned by bioleaching, which is similar to chemical conditioning treatment with ferric chloride and calcium oxide but much more effective than other conditioning approaches including hydrothermal, microwave, and ultrasonic conditioning. Furthermore, after sludge dewatering, bioleached sludge filtrate contains the lowest concentrations of chroma (18 times), COD (542 mg/L), total N (TN, 300 mg/L), NH4(+)-N (208 mg/L), and total P (TP, 2 mg/L) while the hydrothermal process resulted in the highest concentration of chroma (660 times), COD (18,155 mg/L), TN (472 mg/L), NH4(+)-N (381 mg/L), and TP (191 mg/L) among these selected conditioning methods. Moreover, unlike chemical conditioning, sludge bioleaching does not result in a significant reduction of organic matter, TN, and TP in the resulting dewatered sludge cake. Therefore, considering sludge dewaterability and the chemical properties of sludge filtrate and resulting dewatered sludge cakes, bioleaching has potential as an approach for improving sludge dewaterability and reducing the cost of subsequent reutilization or disposal of dewatered sludge.

Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans.

Massive pyrite (FeS2) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S(n)²â», S°). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe(1-x )S2) to active sulfur species (e.g., Fe(1-x )S(2-y ), S°). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S(n)²â»/S° speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S(n)²â»/S° surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.

The inhibitory effect of antimicrobial zeolite on the biofilm of Acidithiobacillus thiooxidans.

The inhibitory effect of antimicrobial zeolite coated concrete specimens (Z2) against Acidithiobacillus thiooxidans was studied by measuring biomass dry cell weight (DCW), biological sulphate generation, and oxygen uptake rates (OURs). Uncoated (UC), and blank zeolite coated without antimicrobial agent (ZC) concrete specimens were used as controls. The study was undertaken by exposing inoculated basal nutrient medium (BNM) to the various specimens. The coating material was prepared by mixing zeolite, epoxy and cure with ratios, by weight of 2:2:1. Concrete specimens were characterized before and after exposure to inoculated or sterile BNM by field emission-scanning electron microscopy (FE-SEM). Gypsum, which was absent in the other test concrete specimens, was detected in uncoated specimens exposed to the bacterium. In UC and ZC, the growth of the bacteria increased throughout the duration of the experiment. However, significant biomass inhibition was observed in experiments where Z2 was used. The overall biomass growth rate in suspension before the specimens were placed ranged from 3.18 to 3.5 mg DCW day(-1). After the bacterium was exposed to UC and ZC, growth continued with a corresponding value of 4 + or - 0.4 and 5.5 + or - 0.6 mg DCW day(-1), respectively. No biomass growth was observed upon exposure of the bacterium to Z2. Similarly, while biological sulphur oxidation rates in UC and ZC were 88 + or - 13 and 238 + or - 25 mg SO(4)(2-) day(-1), respectively, no sulphate production was observed in experiments where Z2 concrete specimens were used. Peak OURs for UC and ZC ranged from 2.6 to 5.2 mg l(-1) h(-1), and there was no oxygen uptake in those experiments where Z2 was used. The present study revealed that the antimicrobial zeolite inhibits the growth of both planktonic as well as biofilm populations of Acidithiobacillus thiooxidans.

[Dependence of the genotypic characteristics of Acidithiobacillus ferrooxidans on the physical, chemical, and electrophysical properties of pyrites].

Comparison of Acidithiobacillus ferrooxidans strains TFV-1 and TFBk with respect to their capacity to oxidize pyrite 1, with hole-type (p-type) conductivity, or pyrite 2, with an electron-type (n-type) conductivity, showed that, at a pulp density of 1%, both before and after its adaptation to the pyrites, strain TFBk, isolated from a substrate with a more complex mineral composition, grew faster and oxidized the pyrites of both conductivity types more efficiently than strain TFV-1, which was isolated from a mineralogically simple ore. At a pulp density of 3-5%, the oxidation of pyrite 1 by strain TFV-1 and both of the pyrites by strain TFBk began only after an artificial increase in Eh to 600 mV. If the pulp density was increased gradually, strain TFBk could oxidize the pyrites at its higher values than strain TFV-1, with the rate of pyrite 2 oxidation being higher than that of pyrite 1. During chemical oxidation of both of the pyrites, an increase was observed in the absolute values of the coefficients of thermoelectromotive force (KTEMF); during bacterial-chemical oxidation, the KTEMF of pyrite 1 changed insignificantly, whereas the KTEMF of pyrite 2 decreased.

Analysis of differential-expressed proteins of Acidithiobacillus ferrooxidans grown under phosphate starvation.

Acidithiobacillus ferrooxidans is one of the most important bacterium used in bioleaching, and can utilize Fe(2+) or sulphide as energy source. Growth curves for Acidithiobacillus ferrooxidans under phosphate starvation and normal condition have been tested, showing lag, logarithmic, stationary and aging phases as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for Acidithiobacillus ferrooxidans cultivated with normal cultivating condition and from 20 to 60 hrs for Acidithiobacillus ferrooxidans cultivated phosphate starvation. Differences of protein patterns of Acidithiobacillus ferrooxidans growing in case of normal or phosphate starvation were separately investigated after cultivation at 30(o)C by the analysis of two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization (MALDI)-Mass spectrometry. There were total 6 protein spots identified, which were Recombination protein recA, RNA helicase, AP2 domain-containing transcription factor, NADH dehydrogenase I chain D, Hyothetical protein PF1669, and Transaldolase STY3758. From the 6 identified protein spots, 3 proteins were found to be decreased in expression at the cultivating condition of phosphate starvation, while another three upregulated.

Microbial influenced degradation of solidified waste binder.

Ordinary cement pastes with water/cement (w/c) ratios of 0.2, 0.4 and 0.5 were used to examine the chemical and physical effects of microbial influenced degradation (MID). Samples were exposed to an active culture of Thiobacillus thiooxidans or to sterile media containing sulphuric acid using an intermittent immersion technique. Acid consumption and Ca, Al and Fe releases are presented for an exposure period of 90 days. Exposed samples were also sectioned and analysed by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX). All cement paste samples were subject to significant degradation in either sterile acid media or the T. thiooxidans lixiviant. Corrosion depths observed from SEM examination of exposed samples were affected by the w/c ratio of the cement. The extent and rate of degradation were not apparent from the calculated rate of hydrogen ion consumption, or the leaching rates of Ca, Al and Fe. It was not possible to distinguish differences in corrosion due to the chemical and microbial influenced degradation from the results obtained to date and further work is focusing on modified procedures to address this.

Evaluation of microbial stability of simulated solid and liquid waste forms using a refined biofilm formation method.

A refined biofilm formation method was used to evaluate the stability of a simulated liquid waste form containing a simulated liquid waste (salts) and cement in three different proportions, and a simulated solid waste form containing a simulated solid waste (resin) and cement in three different proportions. The experimental samples of all the simulated liquid waste forms showed evidence of microbial growth on them after 3 days of evaluation as indicated by substantial increase in sulfate production, and exhibited considerable instability to microbial degradation as indicated by substantial leaching of calcium. The experimental samples of all the simulated solid waste forms showed evidence of inhibition of growth of Thiobacillus thiooxidans for about 18 days, after which the growth of the microbe became evident in two out of three. Within the growth inhibition period, the differences between experimental and control samples were minor. After the growth of T. thiooxidans became evident, comparatively higher degradations were observed for the experimental samples of the resin containing solid waste forms.

Denitrification of industrial wastewater with sulfur and limestone packed column.

An autotrophic denitrification system was developed for nitrate contaminated industrial wastewater whose C/N ratio was very low. The microbes containing Thiobacillus denitrificans as a dominant species were attached on the surface of granular elemental sulfur packed in a column. Elemental sulfur was used as an electron donor for autotrophic denitrification. The granules of limestone were mixed with the granular sulfur to moderate the decrease of alkalinity during autotrophic denitrification. The stoichiometry and basic kinetics of denitrification were studied in column runs. The effects of minerals such as phosphorus on treatment performance were clarified. The wastewater from steel production plants was treated by the present biofilm process. Low extent of nitrogen removal was caused by the lack of minerals.

Stability evaluation of a cement based waste form to microbially induced degradation.

In this study the current Nuclear Regulatory Commission (NRC) protocol is used to evaluate the stability of Tuskegee cement/cobalt chloride waste form in the presence of Thiobacillus thiooxidans (T. thiooxidans). A critical examination of this protocol and identified limitations are reported also. Tuskegee cement/cobalt chloride waste forms were shown to exhibit considerable instability to microbial degradation as indicated by significant physical deterioration, and increased leaching of calcium and cobalt on exposure to T. thiooxidans. The instability was aggravated with higher levels of cobalt chloride content of the waste forms. The degradative capability of T. thiooxidans closely followed its ability to significantly decrease the pH of its environment. Inherent limitations in the NRC protocol were observed which could lead to serious result interpretation errors. The use of a T. thiooxidans culture that is significantly lower in pH in comparison to the control medium could lead to an overestimation of the degradative effect of T. thiooxidans, while the use of a culture that is substrate limited could result in an underestimation of T. thiooxidans capability.

Bioleaching of metals from anaerobic sewage sludge: effects of total solids, leaching microorganisms, and energy source.

The effects of municipal sewage sludge solids concentration, leaching microorganisms (Thiobacillus thiooxidans or Thiobacillus ferrooxidans) and the addition of energy source (S0 or Fe(II)) on the bioleaching of metals from sewage sludge has been investigated under laboratory conditions using shake flasks. The results show that metal solubilization was better accomplished if additional energy source is supplemented to the microorganisms and that T. thiooxidans furnishes, in general, more adequate conditions for the bioleaching than T. ferrooxidans. At a total solids concentration of 70 g L-1 (originally present in the sludge) pH drop and ORP increase are attenuated, so metal solubilization is negatively affected. It was also demonstrated that if lead (Pb) solubilization is to be achieved, than a special combination of microorganism/energy source must be applied.

[Dissolution of alloy components from Ni/Cr base alloys by bacterial leaching]. / Die Auslösung von Legierungsbestandteilen aus Ni/Cr-Legierungen durch bakterielle Laugung.

The method of bacterial leaching of non precious dental alloys represents a useful completion of the method arsenal of biological investigations. In contrast to other well known methods it allows to point out small differences in corrosion resistance between alloys of the same type in a biologic system.