An electrochemical sensing approach for scouting microbial chemolithotrophic metabolisms.

The present study was aimed to test an electrochemical sensing approach for the detection of an active chemolithotrophic metabolism (and therefore the presence of chemolithotrophic microorganisms) by using the corrosion of pyrite by Acidithiobacillus ferrooxidans as a model. Different electrochemical techniques were combined with adhesion studies and scanning electron microscopy (SEM). The experiments were performed in presence or absence of A. ferrooxidans and without or with ferrous iron in the culture medium (0 and 0.5 g L-1, respectively). Electrochemical parameters were in agreement with voltammetric studies and SEM showing that it is possible to distinguish between an abiotically-induced corrosion process (AIC) and a microbiologically-induced corrosion process (MIC). The results show that our approach not only allows the detection of chemolithotrophic activity of A. ferrooxidans but also can characterize the corrosion process. This may have different kind of applications, from those related to biomining to life searching missions in other planetary bodies.

The toxicity of graphene and its impacting on bioleaching of metal ions from sewages sludge by Acidithiobacillus sp.

The increasing production of graphene raised concerns about their releasing into sewage sludge, however, there is little information about graphene impacting on the growth of bacteria and hence their bioleaching of metal ions from sewages sludge. In this study, we reported that Acidithiobacillus sp., isolated from sewages, were used to bioleach Cu2+ and Zn2+ from sewages sludge in the presence of graphene. The negative effect on the growth of Acidithiobacillus sp. and dose-dependent were observed in presence of graphene, where the optical density (OD420) of the culture decreased from 0.163 to 0.045, while the bioleaching efficiency of Cu2+ (70%-16%) and Zn2+ (80%-48%) were also reduced when the graphene dose decreased from 50 mg L-1 to 1 mg L-1. Furthermore, scanning electron microscopy (SEM) and atomic force microscopy (AFM) confirmed that the direct contacts between graphene and cell at 1 mg L-1 graphene caused cell membrane disruption, while Acidithiobacillus sp. grew better by forming dense biofilms around the suspended graphene at a 50 mg L-1. LIVE/DEAD staining further demonstrated that almost no live cells were detected at 1 mg L-1 graphene. The toxicity of graphene could generally be explained by depending on the concentration of graphene. The new findings provide an insight into dose dependence, which impacted on the growth of Acidithiobacillus sp. and their bioleaching of metal ion from sludge.

Characterization of endogenous promoters for control of recombinant gene expression in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an important iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is used extensively in metal extraction and refining, and more recently in the bioproduction of chemicals. However, a lack of genetic tools has limited the further development of this organism for industrial bioprocesses. Using prior microarray studies that identified genes, which may express differentially in response to the availability of iron and sulfur, the cycA1 and tusA promoter sequences have been characterized for their ability to drive green fluorescent protein expression. The promoters exhibited opposite control behavior, where the cycA1 sequence was repressed and the tusA promoter was induced by the presence of sulfur in the growth medium. Sulfur was found to be the dominant signal. The sulfur IC50 for cycA1 was 0.56 mM (18 mg/L), whereas the sulfur EC50 of tusA was 2.5 mM (80 mg/L). Together these sequences provide two new tools to selectively induce or repress gene expression in A. ferrooxidans. Acidithiobacillus ferrooxidans is an important industrial organism; however, genetic tools for control of gene expression do not exist. Here, we report the identification of promoter sequences that allow for the development of control of gene expression for engineering this organism.

Immobilization of Acidithiobacillus ferrooxidans on cotton gauze for biological oxidation of ferrous ions in a batch bioreactor.

The ability of Acidithiobacillus ferrooxidans to oxidize ferrous iron has been extensively studied in bioleaching to recover metal resources. Although immobilization of A. ferrooxidans is of great importance to achieve high bioleaching performance in practical application, the reported approaches of immobilization of A. ferrooxidans are still limited. This paper is attempting to develop a novel method to immobilize A. ferrooxidans by a less-costly effective carrier from zeolite, activated carbon, and cotton gauze. The results showed that cotton gauze was the most suitable carrier to immobilize A. ferrooxidans cells in comparison with zeolite and activated carbon. Acidithiobacillus ferrooxidans immobilized on the cotton gauze by gravity dehydration could achieve an average ferrous iron oxidation rate of 0.73 g/(L·h). Furthermore, the ferrous iron oxidation ratio attained in the bioreactor under batch operation was maintained above 97.83%. All results indicated that cotton gauze could be an efficient carrier for immobilizing A. ferrooxidans cells for the biooxidation of ferrous ions.

Insights to the effects of free cells on community structure of attached cells and chalcopyrite bioleaching during different stages.

The effects of free cells on community structure of attached cells and chalcopyrite bioleaching by Acidithiobacillus sp. during different stages were investigated. The attached cells of Acidithiobacillus thiooxidans owned the community advantage from 14thd to the end of bioprocess in the normal system. The community structure of attached cells was greatly influenced in the free cells-deficient systems. Compared to A. thiooxidans, the attached cells community of Acidithiobacillus ferrooxidans had a higher dependence on its free cells. Meanwhile, the analysis of key biochemical parameters revealed that the effects of free cells on chalcopyrite bioleaching in different stages were diverse, ranging from 32.8% to 64.3%. The bioleaching contribution of free cells of A. ferrooxidans in the stationary stage (8-14thd) was higher than those of A. thiooxidans, while the situation was gradually reversed in the jarosite passivation inhibited stage (26-40thd). These results may be useful in guiding chalcopyrite bioleaching.

Immobilization of Acidithiobacillus ferrooxidans on Cotton Gauze for the Bioleaching of Waste Printed Circuit Boards.

The bioleaching parameters of metal concentrates from waste printed circuit boards by Acidithiobacillus ferrooxidans immobilized on cotton gauze in a two-step reactor were investigated in this study. The results indicated that an average ferrous iron oxidation rate of 0.54 g/(L·h) and a ferrous iron oxidation ratio of 96.90 % were obtained after 12 h at aeration rate of 1 L/min in bio-oxidation reactor. After 96 h, the highest leaching efficiency of copper reached 91.68 % under the conditions of the content of the metal powder 12 g/L, the retention time 6 h, and the aeration rate 1 L/min. The bioleaching efficiency of copper could be above 91.12 % under repeated continuous batch operation. Meanwhile, 95.32 % of zinc, 90.32 % of magnesium, 86.31 % of aluminum, and 59.07 % of nickel were extracted after 96 h. All the findings suggested that the recovery of metal concentrates from waste printed circuit boards via immobilization of A. ferrooxidans on cotton gauze was feasible.

Insights into the relation between adhesion force and chalcopyrite-bioleaching by Acidithiobacillus ferrooxidans.

This paper presents a study on the relation between bacterial adhesion force and bioleaching rate of chalcopyrite, which sheds light on the influence of interfacial interaction on bioleaching behavior. In our research, Acidithiobacillus ferrooxidans (A. ferrooxidans) were adapted to grow with FeSO4 · 7H2O, element sulfur or chalcopyrite. Then, surface properties of Acidithiobacillus ferrooxidans and chalcopyrite were analyzed by contact angle, zeta potential and Fourier transform infrared spectroscopy (FTIR). Adhesion force between bacteria and chalcopyrite was measured by atomic force microscopy (AFM). Attachment and bioleaching behaviors were also monitored. The results showed that A. ferrooxidans adapted with chalcopyrite exhibited the strongest adhesion force to chalcopyrite and the highest bioleaching rate. Culture adapted with sulfur bacteria took second place and FeSO4 · 7H2O-adapted bacteria were the lowest. Bioleaching rate and bacterial attachment capacity were positively related to bacterial adhesion force, which is affected by the nature of energy source. According to this work, the attachment of bacteria to chalcopyrite surface is one of the most important aspects that influence the bioleaching process of chalcopyrite.

Community dynamics of attached and free cells and the effects of attached cells on chalcopyrite bioleaching by Acidithiobacillus sp.

The community dynamics of attached and free cells of Acidithiobacillus sp. were investigated and compared during chalcopyrite bioleaching process. In the mixed strains system, Acidithiobacillus ferrooxidans was the dominant species at the early stage while Acidithiobacillus thiooxidans owned competitive advantage from the middle stage to the end of bioprocess. Meanwhile, compared to A. ferrooxidans, more significant effects of attached cells on free biomass with A. thiooxidans were shown in either the pure or mixed strains systems. Moreover, the effects of attached cells on key chemical parameters were also studied in different adsorption-deficient systems. Consistently, the greatest reduction of key chemical ion was shown with A. thiooxidans and the loss of bioleaching efficiency was high to 50.5%. These results all demonstrated the bioleaching function of attached cells was more efficient than the free cells, especially with A. thiooxidans. These notable results would help us to further understand the chalcopyrite bioleaching.

Magnetic properties of Acidithiobacillus ferrooxidans.

Understanding the magnetic properties of magnetotactic bacteria (MTBs) is of great interest in fields of life sciences, geosciences, biomineralization, biomagnetism, and planetary sciences. Acidithiobacillus ferrooxidans (At. ferrooxidans), obtaining energy through the oxidation of ferrous iron and various reduced inorganic sulfur compounds, can synthesize intracellular magnetite magnetosomes. However, the magnetic properties of such microorganism remain unknown. Here we used transmission electronmicroscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) assay, vibrating sample magnetometer (VSM), magneto-thermogravimetric analysis (MTGA), and low temperature magnetometry to comprehensively investigate the magnetic characteristics of At. ferrooxidans. Results revealed that each cell contained only 1 to 3 magnetite magnetosomes, which were arranged irregularly. The magnetosomes were generally in a stable single-domain (SD) state, but superparamagnetic (SP) magnetite particles were also found. The calcined bacteria exhibited a ferromagnetic behavior with a Curie Temperature of 454 °C and a coercivity of 16.36 mT. Additionally, the low delta ratio (δFC/δZFC=1.27) indicated that there were no intact magnetosome chains in At. ferrooxidans. Our results provided the new insights on the biomineralization of bacterial magnetosomes and magnetic properties of At. ferrooxidans.

Attachment of Acidithiobacillus ferrooxidans onto different solid substrates and fitting through Langmuir and Freundlich equations.

Attachments of Acidithiobacillus ferrooxidans ATCC 23270 onto elemental sulfur, quartz and complex chalcopyrite were investigated by analysis of its extracellular polymeric substances as well as applying Langmuir and Freundlich equations. The two equations fitted the adsorption equilibrium data with significant correlation coefficient over 0.9. This indicated that bacterial attachment is complicated and involves Langmuir and Freundlich characterizations. Sulfur-grown cells showed the highest affinity for the three solid substrates. The investigated complex chalcopyrite possessed a higher maximum adsorption capacity for A. ferrooxidans than elemental sulfur or quartz. The Freundlich fitting parameters suggested that quartz had a weaker adsorption capacity and smaller adsorption areas than elemental sulfur or the complex chalcopyrite. It is not the content of total carbohydrates or proteins in EPS but their ratios that determine the affinity differences between cells and substrates.

Increases of ferrous iron oxidation activity and arsenic stressed cell growth by overexpression of Cyc2 in Acidithiobacillus ferrooxidans ATCC19859.

Acidithiobacillus ferrooxidans plays an important role in bioleaching in reproducing the mineral oxidant of ferric iron (Fe(3+) ) by oxidization of ferrous iron (Fe(2+) ). The high-molecular-weight c-type cytochrome Cyc2 that is located in the external membrane is postulated as the first electron carrier in the Fe(2+) oxidation respiratory pathway of A. ferrooxidans. To increase ferrous iron oxidation activity, a recombinant plasmid pTCYC2 containing cyc2 gene under the control of Ptac promoter was constructed and transferred into A. ferrooxidans ATCC19859. The transcriptional level of cyc2 gene was increased by 2.63-fold and Cyc2 protein expression was observed in the recombinant strain compared with the control. The ferrous iron oxidation activity and the arsenic stressed cell growth of the recombinant strain were also elevated.

Shotgun proteomics study of early biofilm formation process of Acidithiobacillus ferrooxidans ATCC 23270 on pyrite.

Acidithiobacillus ferrooxidans is a chemolithoautotrophic, mesophilic Gram-negative bacterium able to oxidize ferrous iron, sulfur, and metal sulfides. It forms monolayer biofilms where extracellular polymeric substances are essential for cell attachment and metal sulfide leaching. High-throughput proteomics has been applied to study the early process of biofilm formation on pyrite by At. ferrooxidans ATCC 23270. After 24 h contact with the mineral, planktonic and sessile (biofilm) cell subpopulations were separated and proteins extracted. In total, 1319 proteins were detected in both samples. Sixty-two of these were found to be increased in biofilms. Additionally, 25 proteins were found to be decreased in the biofilm cell subpopulation. Three transcriptional factors were found to be increased or decreased among both cell subpopulations, suggesting their potential involvement in the regulation of these processes. Although no significant differences were observed for the known proteins related to ferrous iron and sulfur oxidation pathways among both cell subpopulations, the results presented here show that the early steps of At. ferrooxidans biofilm formation consist of a set of metabolic adaptations following cell attachment to the mineral surface. Functions such as extracellular polymeric substances biosynthesis seem to be pivotal. This first high-throughput proteomic study may also contribute to the annotation of several unknown At. ferrooxidans proteins found.

Immobilization of Acidithiobacillus ferrooxidans on sulfonated microporous poly(styrene-divinylbenzene) copolymer with granulated activated carbon and its use in bio-oxidation of ferrous iron.

The immobilization efficiencies of Acidithiobacillus ferrooxidans cells on different immobilization matrices were investigated for biooxidation of ferrous iron (Fe(2+)) to ferric iron (Fe(3+)). Six different matrices were used such as the polyurethane foam (PUF), granular activated carbon (GAC), raw poly(styrene-divinylbenzene) copolymer (rawSDVB), raw poly(styrene-divinylbenzene) copolymer with granular activated carbon (rawSDVB-GAC), sulfonated poly(styrene-divinylbenzene) copolymer (sulfSDVB) and sulfonated poly(styrene-divinylbenzene) copolymer with granular activated carbon (sulfSDVB-GAC). The sulfSDVB-GAC polymer showed the best performance for Fe(2+) biooxidation. It was used at packed-bed bioreactor and the kinetic parameters were obtained. The highest Fe(2+) biooxidation rate (R) was found to be 4.02 g/L h at the true dilution rate (Dt) of 2.47 1/h and hydraulic retention time (τ) of 0.4 h. The sulfSDVB-GAC polymer was used for the first time as immobilization material for A. ferrooxidans for Fe(2+) biooxidation.

Volatilization of metal mercury from Organomercurials by highly mercury-resistant Acidithiobacillus ferrooxidans MON-1.

The iron-oxidizing bacterium Acidithiobacillus ferrooxidans MON-1 is highly resistant not only to mercuric chloride (HgCl(2)) but also to organomercurials such as methylmercury chloride (MMC). We have found that cytochrome c oxidase, purified from strain MON-1, reduces Hg(2+) to volatilizable metal mercury (Hg(0)) with reduced mammalian cytochrome c or Fe(2+) as an electron donor. In this study we found that cytochrome c oxidase can volatilize Hg(0) from MMC as well as from Hg(2+) with reduced mammalian cytochrome c or c-type cytochrome purified from strain MON-1 as an electron donor. We also found that MMC-Hg(0) volatilization activity is present in the MON-1 plasma membrane but not in the cytosol. These activities were strongly inhibited by sodium cyanide (NaCN) and the antibody produced against purified MON-1 cytochrome c oxidase. This is the first report to indicate that cytochrome c oxidase is involved in the degradation of organomercurials in microorganisms.

Modeling and analysis of nanoscale interaction forces between Acidithiobacillus ferrooxidans and AFM tip.

The specificity exhibited by Acidithiobacillus ferrooxidans, a chemolithoautotrophic acidophile, in their attachment to different sulphide minerals has resulted in effective physical separation of these minerals. This can be explained in terms of surface forces of interaction between the cells and substrates using AFM. In the light of this, the present study reports interaction studies with A. ferrooxidans cells and an AFM silicon nitride tip. The aim of the present investigation is to probe the nanoscale interactions between A. ferrooxidans cells and silicon nitride tip of an AFM, model the approach and retraction forces, and elucidate the effects of pH, ionic strength and surface biopolymers on interfacial forces.

Enhanced biofiltration using cell attachment promotors.

H2S polluted airstreams were treated in two biotrickling filter columns packed with polyurethane (PU) foam cubes, one with cubes coated with a solution of 25 mg/L of polyethyleneimine (PEI, coated reactor) and the other containing just plain PU cubes (uncoated reactor) at empty bed residence times (EBRT) ranging from 6 to 60 s. and inlet H2S concentrations ranging from 30 to 235 ppm, (overall loads of up to 44 gH2S/m3bed/h), with overall removal efficiencies (RE) in the range of 90-100% over 125 days. The acclimatization characteristics of the coated reactor outperformed those of the uncoated one, and both the observed elimination capacity (EC) of 77 gH2S/m3bed/h and retention of volatile solids (VS) of 42 mgVS/cube were maxima in the coated reactor. Insights into the controlling removal mechanisms were also provided by means of dimensionless analysis of the experimental data. Denaturing gradient gel electrophoresis (DGGE) showed that the dominant surviving species in both units belonged to the genus Acidithiobacillus.

Soft-particle model analysis of effect of LPS on electrophoretic softness of Acidithiobacillus ferrooxidans grown in presence of different metal ions.

The effect of surface lipopolysaccharides (LPS) on the electrophoretic softness and fixed charge density in the ion-penetrable layer of Acidithiobacillus ferrooxidans cells grown in presence of copper or arsenic ions have been discussed. The electrophoretic mobility data were analyzed using the soft-particle electrophoresis theory. Cell surface potentials of all the strains based on soft-particle theory were lower than those estimated using the conventional Smoluchowski theory. Exposure to metal ions increased the surface electrophoretic softness with decrease in the fixed charge density. Effect of cell surface lipopolysaccharides on the model parameters are investigated and discussed.

Influence of chloride and sulfate on formation of akaganéite and schwertmannite through ferrous biooxidation by Acidithiobacillus ferrooxidans cells.

Iron (oxyhydr)oxides play important roles in the fixation of toxic elements and also in the distribution of nutrients for plants in soils. Akaganéite and schwertmannite, as the iron oxyhydroxides having an analogous tunnel structure, have been widely recognized in Fe-rich environments. The objective of this study was to examine the formation of akaganéite/ schwertmannite via biooxidation of 0.1 M of ferrous solution containing only Cl-, SO4(2-) or both the anions with a Cl-/SO4(2-) mole ratio of 1, 3, 6, and 10 by chloride-acclimated Acidithiobacillus ferrooxidans cells. Results showed that ferrous iron in chloride/sulfate-containing solutions could be easily biooxidized to ferric iron, and subsequent Fe(III)-hydrolysis/precipitation could result in the formation of large quantity of akaganéite/schwertmannite precipitates. The resulting precipitates were identified to be the pure akaganéite (Fe8O8(OH)7.1(Cl)0.9, the pure schwertmannite (Fe8O8(OH)4.42(SO4)1.79, and the main schwertmannite phase (Fe8O8(OH)(8-2x)(SO4)x, with 1.09 < or = x < or = 1.73), respectively, under different Cl-/SO4(2-) mole ratio conditions. Obviously, sulfate inhibited drastically the bioformation of akaganéite but facilitated schwertmannite phase occurrence in the ferrous solution containing both sulfate and chloride. However, the presence of chloride ion in initial ferrous solution containing sulfate and Acidithiobacillus ferrooxidans cells would affect the morphology and other characteristics of schwertmannite generated.

[Study on immobilization of Acidithiobacillus ferrooxidans using PVA-Ca(NO3)2 method].

A new cell immobilization technique utilizing the complex of PVA solution and sodium alginate solution as entrapment medium is reported. The mixture of Acidithiobacillus ferrooxidans suspension and the entrapment complex were extruded into the solution of Ca(NO3)2 (1% - 5%) to form beads, then the beads were frozen at -20 degrees C for 1d and thawed at room temperature. This method can simultaneously eliminate the agglomeration of PVA beads and the biological toxicity of H3 BO31. A maximum oxidation rate of 2.45g Fe2+ /(L x h) was achieved in batch cultures by this immobilized cells. In addition, the operation of this new technique is very simple, and the gelation solution (calcium nitrate) used is of low toxicity and very cheap. Moreover, the mechanical strength and the oxidation activity of the beads obtained by this technique are better than those obtained by the other methods. So its application on an industrial scale would be more practicable.

Electrochemistry of marmatite-carbon paste electrode in the presence of bacterial strains.

The electrochemical behaviors of a marmatite-carbon paste electrode with the chemical leaching of Fe3+ ions, or the microbial leaching using Acidithiobacillus ferrooxidans, were compared. The cyclic voltammograms of the electrode in the presence and absence of bacterial strains showed that the leaching process of marmatite was carried out by the different reactions occurring in the interface of the marmatite electrode-leach liquid. The polarization currents of the electrode under the differently applied potentials suggested that the microbial leaching of marmatite could be accelerated by the applied potential. The SEM observations indicated that the corrosion pits formed in the electrode surface were similar to the attached bacterial cells in shape and size, other than that by the chemical leaching of Fe3+ ions. The contact leaching of the attached cells on the mineral substrate played an important role on the dissolution of marmatite in addition to the chemical leaching of Fe3+ ions.