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A novel acidophilic, aerobic bacterium strain, MYW30-H2T, was isolated from a heap of polymetallic mine. Cells of strain MYW30-H2T were Gram-stain-positive, endospore-forming, motile, and rod-shaped. Strain MYW30-H2T grew at a temperature range of 30–45 °C (optimum 40 °C) and a pH range of 3.5–6.0 (optimum 4.0) in the presence of 0–0.5% (w/v) NaCl. Strain MYW30-H2T could grow heterotrophically on yeast extract and glucose, and grow mixotrophically using ferrous iron as an electron donor with yeast extract. Menaquinone-7 (MK-7) was the sole respiratory quinone of the strain. Iso-C15:0 and anteiso-C15:0 were the major cellular fatty acids. The 16S rRNA gene sequence analysis showed that MYW30-H2T was phylogenetically affiliated with the family Alicyclobacillaceae, and the sequence similarity with other Alicyclobacillaceae genera species was below 91.51%. The average amino acid identity value of the strain with its phylogenetically related species was 52.3–62.1%, which fell into the genus boundary range. The DNA G+C content of the strain was 44.2%. Based on physiological and phylogenetic analyses, strain MYW30-H2T represents a novel species of a new genus of the family Alicyclobacillaceae, for which the name Fodinisporobacter ferrooxydans gen. nov., sp. nov. is proposed. The type strain is MYW30-H2T (=CGMCC 1.17422T = KCTC 43278T). [ABSTRACT FROM AUTHOR]

A novel acidophilic, aerobic bacterium strain, MYW30-H2T, was isolated from a heap of polymetallic mine. Cells of strain MYW30-H2T were Gram-stain-positive, endospore-forming, motile, and rod-shaped. Strain MYW30-H2T grew at a temperature range of 30–45 °C (optimum 40 °C) and a pH range of 3.5–6.0 (optimum 4.0) in the presence of 0–0.5% (w/v) NaCl. Strain MYW30-H2T could grow heterotrophically on yeast extract and glucose, and grow mixotrophically using ferrous iron as an electron donor with yeast extract. Menaquinone-7 (MK-7) was the sole respiratory quinone of the strain. Iso-C15:0 and anteiso-C15:0 were the major cellular fatty acids. The 16S rRNA gene sequence analysis showed that MYW30-H2T was phylogenetically affiliated with the family Alicyclobacillaceae, and the sequence similarity with other Alicyclobacillaceae genera species was below 91.51%. The average amino acid identity value of the strain with its phylogenetically related species was 52.3–62.1%, which fell into the genus boundary range. The DNA G+C content of the strain was 44.2%. Based on physiological and phylogenetic analyses, strain MYW30-H2T represents a novel species of a new genus of the family Alicyclobacillaceae, for which the name Fodinisporobacter ferrooxydans gen. nov., sp. nov. is proposed. The type strain is MYW30-H2T (=CGMCC 1.17422T = KCTC 43278T).

ABSTRACT Dissimilatory ferrous iron [Fe(II)] oxidation is a well-established microbial energy generation strategy. This study aims to comprehensively investigate the distribution and evolution of recognized Fe(II) oxidation pathways through comparative analysis. Interestingly, we have discovered a wide range of taxonomic groups that harbor homologs to known Fe(II) oxidation proteins. The presence of these homologs among phylogenetically distant lineages and their frequent association with mobile genetic elements strongly suggest horizontal gene transfer events involving Fe(II) oxidation proteins, such as the rus operon of Acidithiobacillus and Cyc572 from Leptospirillum lineages belonging to classes Gammaproteobacteria and Betaproteobacteria often present at the hub positions of the protein sequence similarity networks from which homologs of other taxa are derived. In addition, RoseTTAFold predictions have provided valuable insights into the structural characteristics of previously unknown Fe(II) oxidation components. Despite having limited sequence identity, a significant number of acknowledged proteins involved in different Fe(II) oxidation pathways exhibit close structural similarities, including Cyc2 and Cyc572. Collectively, this study significantly enhances our understanding of the distribution and evolution of microbial ferrous iron oxidation pathways. IMPORTANCE Microbial Fe(II) oxidation is a crucial process that harnesses and converts the energy available in Fe, contributing significantly to global element cycling. However, there are still many aspects of this process that remain unexplored. In this study, we utilized a combination of comparative genomics, sequence similarity network analysis, and artificial intelligence-driven structure modeling methods to address the lack of structural information on Fe(II) oxidation proteins and offer a comprehensive perspective on the evolution of Fe(II) oxidation pathways. Our findings suggest that several microbial Fe(II) oxidation pathways currently known may have originated within classes Gammaproteobacteria and Betaproteobacteria.

Banded Iron Formations (BIFs) are ancient marine chemical sediments that contain various Fe-bearing minerals such as hematite (Fe 2 O 3), magnetite (Fe 3 O 4), siderite (FeCO 3) and a variety of FeII - /FeIII-silicates. The prevailing opinion is that primary Fe(III) (oxyhydr)oxides, such as ferrihydrite (simplified formula of Fe(OH) 3), were precipitated from the ocean's photic zone by marine plankton, and a fraction of these minerals was subsequently transformed into secondary magnetite and siderite by dissimilatory Fe(III)-reducing bacteria (DIRB). However, aside from broad estimates, it is currently unknown what fraction of the primary Fe(III) minerals was sedimented to the seafloor where it was eventually lithified, and what fraction was reduced by DIRB in the water column, thus forming a microbial Fe cycle in the water column. To test this, we conducted Fe cycling experiments with marine phototrophic Fe(II)-oxidizing bacteria and DIRB under conditions mimicking the Precambrian ocean water column with elevated Fe(II) and Si concentrations. We followed secondary mineral formation over three consecutive redox cycles (oxidation followed by reduction) over a time interval of up to 58 days to determine which mineral phases would ultimately have settled as BIF forming sediments. We used wet geochemical methods to follow Fe speciation, measured dissolved silica and volatile fatty acid (VFA) concentrations, determined cell-mineral associations using fluorescence and electron microscopy, and characterized the mineralogy of the precipitates using 57Fe-Moessbauer spectroscopy and X-ray diffraction (XRD). Our results showed that both the absence of silica and an increasing number of Fe cycles favored the formation of more crystalline minerals, such as goethite (α-FeOOH). However, in the presence of high concentrations of monomeric silica, as suggested for ancient oceans (2.2 mM), only short-range ordered (SRO) Fe(III) minerals such as ferrihydrite were observed. These did not transform into the more thermodynamically stable goethite during repeated Fe cycling. Interestingly, no magnetite formed in any of the setups. Instead, increasing Si concentrations favored the formation of increasing quantities of Fe(II) minerals. Microscopy revealed a tight association between microbial biomass and minerals formed. Dissolved silica analysis showed the removal of Si from solution congruent with Fe(II) oxidation and a release of Si during Fe(III) reduction. Together, these results suggest an important role of co-precipitated biomass as well as silica for secondary mineral formation by either constraining crystal growth and/or inhibiting Fe(II)-induced mineral transformation. Overall, our results imply that microbial Fe cycling during settling of primary ferrihydrite through the photic zone in a Precambrian ocean would have resulted in the partial transformation of ferrihydrite into secondary Fe(II) mineral phases in the water column. This would have resulted in the accumulation of mixtures of a ferrihydrite-silica composite and Fe(II) minerals in the initial BIF forming sediments. [ABSTRACT FROM AUTHOR]

ABSTRACT. The main aim of this study was to assess the effects of ferrous iron oxidation, hydrolysis and precipitation of the ferric iron on the mechanisms of arsenate removal from acid mine drainage. The study was realized at the pH range 2,0 – 6,5. H2O2 was the oxidant used in the chemical test. A mixed culture of mesophilic iron–oxidizing chemolithotrophic bacteria (Acidithiobacillus ferrooxidans, Leptospirillum ferroxidans) carried out the ferrous oxidation in the biological test. NaOH was used as an alkalizing agent which triggered the ferric iron hydrolysis and precipitation. The results showed that the total content of retained arsenic by ferric iron hydrous oxides and its speciation as geochemical fractions depended on the iron and phosphate concentrations in acid solution. The results showed that phosphate concentration lower than 6 mg/ L limited the ferrous iron oxidation rate. At the tested experimental conditions, arsenic sorption by formed ferric iron hydrous oxides was the main mechanism for its removal from acid mine drainage, as the ratio between iron and phosphate determined the proportions between the geochemical fractions of scavenged arsenic.

Clay minerals are ubiquitous in subsurface environments and have long been recognized as having a limited or negligible impact on the fate of arsenic (As) due to their negatively charged surfaces. Here, we demonstrate the significant role of kaolinite (Kln), a pervasive clay mineral, in enhancing As(V) immobilization during ferrous iron (Fe(II)) oxidation at near-neutral pH. Our results showed that Fe(II) oxidation alone was not capable of immobilizing As(V) at relatively low Fe/As molar ratios (≤2) due to the generation of Fe(III)-As(V) nanocolloids that could still migrate easily as truly dissolved As did. In the presence of kaolinite, dissolved As(V) was significantly immobilized on the kaolinite surfaces via forming Kln-Fe(III)-As(V) ternary precipitates, which had large sizes (at micrometer levels) to reduce the As mobility. The kaolinite-induced heterogeneous pathways for As(V) immobilization involved Fe(II) adsorption, heterogeneous oxidation of adsorbed Fe(II), and finally heterogeneous nucleation/precipitation of Fe(III)-As(V) phases on the edge surfaces of kaolinite. The surface precipitates were mixtures of amorphous basic Fe(III)-arsenate and As-rich hydrous ferric oxide. Our findings provide new insights into the role of clay minerals in As transformation, which is significant for the fate of As in natural and engineered systems.

Strain MG, isolated from an acidic pond sediment on the island of Milos (Greece), is proposed as a novel species of ferrous iron- and sulfur-oxidizing Acidithiobacillus. Currently, four of the eight validated species of this genus oxidize ferrous iron, and strain MG shares many key characteristics with these four, including the capacities for catalyzing the oxidative dissolution of pyrite and for anaerobic growth via ferric iron respiration. Strain MG also grows aerobically on hydrogen and anaerobically on hydrogen coupled to ferric iron reduction. While the 16S rRNA genes of the iron-oxidizing Acidi-thiobacillus species (and strain MG) are located in a distinct phylogenetic clade and are closely related (98–99% 16S rRNA gene identity), genomic relatedness indexes (ANI/dDDH) revealed strong genomic divergence between strain MG and all sequenced type strains of the taxon, and placed MG as the first cultured representative of an ancestral phylotype of iron oxidizing acidithiobacilli. Strain MG is proposed as a novel species, Acidithiobacillus ferrianus sp. nov. The type strain is MGT (= DSM 107098T = JCM 33084T). Similar strains have been found as isolates or indicated by cloned 16S rRNA genes from several mineral sulfide mine sites. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Under aerobic conditions, oxidation of As(III) to As(V) in aqueous solution is catalysed by granular activated carbon (GAC). • GAC-catalysed As(III) oxidation in the presence of a thermoacidophilic culture oxidizing Fe(II) led to scorodite formation. • The removal efficiency for As(III) with 0.9% w/v GAC and Fe(II) as electron donor exceeded 90%. Abstract In this study we describe the immobilization of arsenic as scorodite (FeAsO 4.2H 2 O) by a thermophilic iron-oxidizing mixed culture from an acidic sulfate medium containing 500 mg L-1 of Fe(II), 500 mg L-1 As(III) and granular activated carbon (GAC) as the main arsenite oxidant. This study shows that crystalline scorodite can only be precipitated in the presence of the ferrous iron-oxidizing mixed culture (pH 1.3 and 70 °C). The efficiency of arsenite oxidation was over 99% with a maximum specific oxidation rate of 280 mgAs(III) gGAC-1 day-1. Ferrous iron and arsenite were also oxidized in the absence of the mixed culture, however, no scorodite precipitated under these conditions; consequently, scorodite precipitation was biologically induced. The precipitated scorodite particles had a size between 0.5 and 10 μm with an average of 5 μm, resulting in low settling rates. Ion activity product calculations and observations by Scanning Electron Microscopy (SEM) indicated that microbial cells served as surface for heterogeneous nucleation. The potential of the thermophilic mixed culture for the scorodite formation explored in this study provides the basis of a new approach for the treatment of As(III) polluted streams. [ABSTRACT FROM AUTHOR]

Importance: Microbial Fe(II) oxidation is a crucial process that harnesses and converts the energy available in Fe, contributing significantly to global element cycling. However, there are still many aspects of this process that remain unexplored. In this study, we utilized a combination of comparative genomics, sequence similarity network analysis, and artificial intelligence-driven structure modeling methods to address the lack of structural information on Fe(II) oxidation proteins and offer a comprehensive perspective on the evolution of Fe(II) oxidation pathways. Our findings suggest that several microbial Fe(II) oxidation pathways currently known may have originated within classes Gammaproteobacteria and Betaproteobacteria ., Competing Interests: The authors declare no conflict of interest.

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans, grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans, it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans, the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the −35 (or −24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed.

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans, grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans, it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans, the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the -35 (or -24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA systemin the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed. [ABSTRACT FROM AUTHOR]

Laboratory and field experiments were used to measure the effects of several variables on the rate of heterogeneous oxidation in acid mine discharges (AMD). A wider range of variables were used compared to previously published studies, i.e., Fe(III) solid from 0.012 to 2.40 g L −1 , pH 5.35 to 6.81, DO 0.11–0.36 mM, temperature 10.1–22.7 °C, and Fe(II) total 0.04–1.0 mM. The heterogeneous rate exceeded the predicted homogeneous rate for slightly acidic pH values when Fe(III) solid was greater than about 0.02 g L −1 , with the cross-over concentration of Fe(III) solid a function of temperature. An equation for the heterogeneous oxidation rate was derived, d [ F e ( I I ) a q ] d t = − k h e t e r o [ F e ( I I ) a q ] [ F e ( I I I ) s o l i d ] { O 2 } { H + } n where n was 1.4 for pH from 6.0 to 6.8 and 2.0 for pH from 5.3 to 6.0. All previous studies reported n equal to 1.0. One earlier study reported that the rate of heterogeneous oxidation was constant for Fe(III) solid greater than 0.4 g L −1 . In our experiments the rate remained linear to at least 2.4 g L −1 Fe(III) solid . The predicted rate constants along with DO, pH, Fe(III) solid , and temperature can be used to predict the removal of Fe(II) in existing facilities and for design of new reactors. Application can result in design of smaller reactors for slightly acidic pH values and in production of re-usable Fe(III) oxide solids. [ABSTRACT FROM AUTHOR]

Vivianite (Fe 3 (PO 4 ) 2 ·8H 2 O) crystallization has attracted increasing attention as a promising approach for removing and recovering P from wastewaters. However, Fe II is susceptible to oxygen with its oxidation inevitably influencing the crystallization of vivianite. In this study, the profile of vivianite crystallization in the presence of dissolved oxygen (DO) was investigated at pHs 5-7 in a continuous stirred-tank reactor. It is found that the influence of DO on vivianite crystallization was highly pH-related. At pH 5, the low rate of Fe II oxidation at all of the investigated DO of 0-5 mg/L and the low degree of vivianite supersaturation resulted in slow crystallization with the product being highly crystalline vivianite, but the P removal efficiency was only 30-40%. The removal of P from the solution was substantially more effective (to >90%) in the DO-removed reactors at pH 6 and 7, whereas the efficiencies of P removal and especially recovery decreased by 10-20% when Fe II oxidation became more severe at DO concentrations >2.5 mg/L (except at pH 6 with 2.5 mg/L DO). The elevated degree of vivianite supersaturation and enhanced rate and extent of Fe II oxidation at the higher pHs led to decreases in the size and homogeneity of the products. At the same pH, amorphous ferric oxyhydroxide (AFO)─the product of Fe II oxidation and Fe III hydrolysis─interferes with vivianite crystallization with the induction of aggregation of crystal fines by AFO, leading to increases in the size of the obtained solids.

Arsenic (As) biotransformation in soil affects As biogeochemical cycling and is associated with As accumulation in rice. After inoculation with 1% iron-oxidizing bacteria (FeOB) in paddy soil, As speciation, As biotransformation genes in soil, As/Fe in Fe plaques, and As accumulation in rice were characterized. Compared with the control, the available As concentrations in soils decreased while amorphous and poorly crystalline Fe–Al oxidized As and crystalline Fe–Al oxidized As fractions increased of F (FeOB) and RF (rice and FeOB) treatments. Fe concentrations increased and positively correlated with As concentrations in Fe plaques on the rice root surface (***P < 0.001). Compared with R (rice), Monomethyl As (MMA), dimethyl As (DMA), arsenate (As(V)), and arsenite (As(III)) concentrations in rice plants showed a downwards trend of RF treatment. The As concentration in grains was below the National Standard for Food Safety (GB 2762-2017). A total of 16 As biotransformation genes in rhizosphere soils of different treatments (CK, F, R and RF were quantified by high-throughput qPCR (HT-qPCR). Compared with the control, the As(V) reduction and As transport genes abundance in other treatments increased respectively by 54.54%–69.17% and 54.63%–73.71%; the As(III) oxidation and As (de) methylation genes did not change significantly; however, several As(III) oxidation genes (aoxA, aoxB, aoxS, and arsH) increased. These results revealed that FeOB could reduce, transport As, and maybe also oxidize As. In addition, As(III) oxidation gene (aoxC) in rhizosphere soil was more abundant than in non-rhizosphere soil. It indicated that radial oxygen loss (ROL) promoted As(III) oxidation in rhizosphere soils. The results provide evidence for As biotransformation by ROL and FeOB in soil-rice system. ROL affects As oxidation and immobilization, and FeOB affects As reduction, transportation and may also affect As oxidation. [Display omitted] • FeOB increased the proportion of crystalline Fe–Al oxidized As in soil. • FeOB decreased As availability and accumulation in soil-rice system. • As biotransformation genes were quantified by HT-qPCR. • Several As(III) oxidation genes (aoxA, aoxB, aoxS and arsH) abundance increased with FeOB. • As(V) reduction and As transport genes abundance in soil-rice system increased with FeOB. [ABSTRACT FROM AUTHOR]

Recent understanding of microbial retention within heap bioleaching systems has highlighted the importance of quantifying microbial growth and activity in both the bulk flowing solution and in the ore-associated phases. Typically, industrial heap bioleaching operations report variations in process conditions such as inoculum preparation and concentration and elevated copper concentrations in the recycled irrigation solution. In this paper, a mini-column reactor system containing pre-constructed and agglomerated, low-grade ore samples representing grab samples from a larger heap, were used to investigate the effect of a selection of physico-chemical and operating conditions on microbial growth, colonisation and substrate utilisation kinetics, considering both the planktonic and sessile populations of Acidithiobacillus ferrooxidans . The factors studied included inoculum size, inoculum cultivation conditions, availability of ferrous iron in the bulk flowing solution and copper concentration in the bulk flowing solution. The microbial population in the interstitial phase, i.e. associated with, but not bound to, the ore, remained the most abundant within the heap under all physico-chemical conditions considered. A comparison of the tests with different inoculum sizes found that a smaller inoculum size resulted in an increased delay in microbial growth and ferrous iron oxidation, but similar apparent maximum specific growth rates and iron oxidation rates. In contrast to the microbial culture grown on pyrite, a delay in microbial activity was observed for the culture grown on ferrous iron. However, greater microbial cell densities were reached, in the interstitial and attached phases compared with the pyrite-grown culture. The introduction of 6 g L − 1 cupric ions into the feed solution containing 0.2 g L − 1 ferric iron resulted in decreased microbial growth rate in the interstitial phase but not in the attached phase. Where the pyrite culture was pre-exposed to cupric ion, the microbial growth rate in the interstitial and attached phases was significantly enhanced. Nevertheless, the presence of cupric ion in the irrigation solution resulted in a decrease in microbial ferrous iron oxidation rate, irrespective of pre-culture conditioning. This study emphasises the important role played by the stagnant interstitial phase during the colonisation of a low-grade heap, particularly under adverse conditions for microbial growth and activity. It also highlights the role of inoculum culture conditions on the potential trade-off between increased heap colonisation and increased lag periods in microbial activity during heap start-up. [ABSTRACT FROM AUTHOR]

This study examined the kinetics of phosphate re- moval during Fe(II) oxidation, and the quantitative relationship between phosphate removal and Fe(II) oxidation in synthetic wastewater. The phosphate removal rate increased with the increase of DO and Fe(II)0 but firstly increased and then decreased with pH. The reaction kinetics of phosphate removal as a function of DO, Fe(II)0 and pH all followed pseudo-first-order kinetics. For the tested DO conditions, quantitative relationship between kp and kFe was described by the equation: kp 0.6024 kFe+0.0004. For the tested Fe(II)0 conditions, the evolution of kp versus kFe was expressed by two equations: kp = 1.9573 kFe - 0.0233 (Fe(II)0 from 10 to 30 mg/L) and kp =13.252 kFe - 0.2215 (Fe(II)o from 30 to 45 mg/L). For the tested pH conditions, relationship between kp and kFe followed two equations: k= 0.5911 kFe 0.0001 (pH from 6.0 to 8.0) and kp = 0.2274 kFe + 0.4401 (pH from 8.0 to 9.0). The precipitates formed at higher DO tended to be more compact, and featured by relatively bigger size, thus it resulted in better solid-liquid separation. [ABSTRACT FROM AUTHOR]

Acidithiobacillus ferrooxidans plays an important role in bioleaching in reproducing the mineral oxidant of ferric iron ( Fe3+) by oxidization of ferrous iron ( Fe2+). The high-molecular-weight c-type cytochrome Cyc2 that is located in the external membrane is postulated as the first electron carrier in the Fe2+ oxidation respiratory pathway of A. ferrooxidans. To increase ferrous iron oxidation activity, a recombinant plasmid p TCYC2 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. [ABSTRACT FROM AUTHOR]

Abstract: A novel Acidithiobacillus ferrooxidans strain QXS-1 was isolated from the acid mine drainage of Qixiashan Pb–Zn–Ag mine area in China. The isolate QXS-1 is a motile, rod-shaped Gram-negative bacterium with an optimum growth at 30°C and pH 2.0. The phylogenetic analysis has demonstrated that QXS-1 is a new strain of A. ferrooxidans. The strain QXS-1 exhibited an unusual trait on the utilization of ferrous iron and sulfur as energy sources. The sulfur-domesticated QXS-1 cells only utilized sulfur as energy source for growth when both ferrous iron and sulfur are available, whereas ferrous-domesticated QXS-1 cells prefer to simultaneously utilize both ferrous iron and sulfur as energy source for growth in the presence of both substrates. The QXS-1 cells accumulated intracellular hydrophilic sulfur during growth. Based on the ultrastructure study, we deduced that the accumulated sulfur in QXS-1 exists in both octasulfur ring form and polysulfide form. The strain QXS-1 exhibited remarkable marmatite-bioleaching capacity. It is able to extract 6585mg/L zinc from marmatite within 18days in shake flask. [Copyright &y& Elsevier]

In contrast to iron-oxidizing Acidithiobacillus ferrooxidans, A. ferrooxidans from a stationary phase elemental sulfur-oxidizing culture exhibited a lag phase in pyrite oxidation, which is similar to its behaviour during ferrous iron oxidation. The ability of elemental sulfur-oxidizing A. ferrooxidans to immediately oxidize ferrous iron or pyrite without a lag phase was only observed in bacteria obtained from growing cultures with elemental sulfur. However, these cultures that shifted to ferrous iron oxidation showed a low rate of ferrous iron oxidation while no growth was observed. Two-dimensional gel electrophoresis was used for a quantitative proteomic analysis of the adaptation process when bacteria were switched from elemental sulfur to ferrous iron. A comparison of total cell lysates revealed 39 proteins whose increase or decrease in abundance was related to this phenotypic switching. However, only a few proteins were closely related to iron and sulfur metabolism. Reverse-transcription quantitative PCR was used to further characterize the bacterial adaptation process. The expression profiles of selected genes primarily involved in the ferrous iron oxidation indicated that phenotypic switching is a complex process that includes the activation of genes encoding a membrane protein, maturation proteins, electron transport proteins and their regulators. [ABSTRACT FROM AUTHOR]

In this study, a proteomic analysis of Acidithiobacillus ferrooxidans by two-dimensional electrophoresis identified 24 proteins that were differentially expressed when the cells were grown on ferrous iron (Fe) or elemental sulfur (S°). Sixteen of these proteins were upregulated by growth on S° or downregulated by growth on Fe, including four proteins involved in disulfide bond reduction such as pyridine nucleotide-disulfide oxidoreductase, heterodisulfide reductase subunit B, thioredoxin-disulfide reductase, and cysteine desulfurase IscS, and three proteins involved in saccharide metabolism. A total of eight proteins were upregulated by growth on Fe or downregulated by S°. Northern blots further confirmed the differences in transcription for these differentially expressed proteins. We functionally characterized cysteine desulfurase IscS, and found that its overexpression in E. coli promoted the growth of the cells in LB containing 2.5 % sodium thiosulfate. Our results provide new insights into the molecular basis for S° and Fe oxidation by this extreme acidophile. [ABSTRACT FROM AUTHOR]

Neutrophilic Fe-oxidizing bacteria precipitate positively charged Fe minerals that are expected to sorb to the negatively charged cell surface, leading to encrustation and thus limiting the cells' accessibility to substrates and nutrients. However, electron-microscopy analysis of phototrophic iron-oxidizing Thiodictyon sp. strain F4 cells showed no encrustation, but mineral precipitation at a distance from the cell surface. In situ fluorescence microscopy analysis of F4 cells using a pH-sensitive fluorescent dye revealed a low cell surface pH (6.0 ± 0.1) in contrast to the bulk pH (6.6 ± 0.1). Biogeochemical modeling showed that the pH difference reduces Fe sorption and Fe precipitation rates at the cell surface, therefore directing mineral formation away from the cells. The results from this study therefore suggest that the establishment of a low cell surface pH could provide a mechanism for photoferrotrophs to successfully prevent Fe mineral precipitation on the cell surface. [ABSTRACT FROM AUTHOR]

Abstract: A few researchers have reported on work concerning bioleaching of heavy-metal-contaminated soil using Acidithiobacillus ferrooxidans, since this acidophile is sensitive to dissolved low molecular weight (LMW) organic acids. Iron oxidation by A. ferrooxidans R2 as well as growth on ferrous iron was inhibited by a variety of dissolved LMW organic acids. Growth experiments with ferrous iron as an oxidant showed that the inhibition capability sequence was formic acid>acetic acid>propionic acid>oxalic acid>malic acid>citric acid. The concentrations that R2 might tolerate were formic acid 0.1mmolL−1 (2mmolkg−1 soil), acetic and propionic acids 0.4mmolL−1 (8mmolkg−1 soil), oxalic acid 2.0mmolL−1 (40mmolkg−1 soil), malic acid 20mmolL−1 (400mmolkg−1 soil), citric acid 40mmolL−1 (800mmolkg−1 soil), respectively. Although R2 was sensitive to organic acids, the concentrations of LMW organic acids in the contaminated soils were rather lower than the tolerable levels. Hence, it is feasible that R2 might be used for bioleaching of soils contaminated with metals or metals coupled with organic compounds because of the higher concentrations of LMW organic acids to which R2 is tolerant. [Copyright &y& Elsevier]

Laboratory simulation of clogging in the Lixi tailings dam (Shaanxi Province, China) is urgently required because clogging is an important factor affecting the dam stability. This work firstly presents the results of ferrous iron oxidation experiments using buffer solution. The results indicate that the ferrous iron oxidation follows first order kinetics, and the oxidation process is strongly dependent on pH, a higher pH resulting in a higher oxidation rate. Furthermore, when the pH exceeds 7.0, the oxidation rate constant increases significantly. Secondly, a column experiment was carried out under the conditions of the pH ranging from 6.8 to 7.5 and the natural oxygen supply. Ferrous iron oxidation and precipitation were found to reach equilibrium under these conditions. After 23 days, the column experiment was stopped when the clogging materials blocked the column outlet. The clogging materials were found to be a mixture of ferric hydroxide and its converted products, and these existed in amorphous form with a loose cluster microstructure according to the results of XRD and SEM. [ABSTRACT FROM AUTHOR]

Abstract: The oxidation of Fe(II) in acidic mine waters emerging from the portals of abandoned mines (Lomero, Esperanza, Tharsis and Poderosa) of the Iberian Pyrite Belt (IPB) massive sulphide province, has been investigated. Field and laboratory studies have been performed in mine effluents with different iron concentrations (290 to 1940 mg/L Fe(II)) and pH (1.9–3.1) to calculate: (1) the reaction rates at which dissolved Fe(II) is oxidised to Fe(III) in the mine portals, (2) the hourly variations of these oxidation rates, and (3) the rate at which Fe(III) is subsequently hydrolyzed and precipitated as schwertmannite. The calculated field rates, between 5.5×10−6 and 4×10−7 mol L−1 s−1, are characteristic of bacterial oxidation. A marked difference has been recognized between the different mine sites depending on the biomass density. The oxidation rates measured in the mine portals with dense biofilms of acidic slime streamers (Tharsis and Esperanza), are notably faster than those measured in the mine sites where streamers are not present (Lomero mine portal). Among the sites with the highest density of biofilms, pH also appears to control the oxidation rate, with the effluents of lowest pH (Poderosa mine, pH 1.9) showing slower rates than those with higher pH (Tharsis and Esperanza, pH 2.7–3.0). The oxidation kinetics of Fe(II) appears to be zero-order and highly dependent on the water temperature (T). Consequently, iron-oxidation rates vary significantly along the day, being minimum at the early morning and sunset (with T ∼15–25 °C), and maximum at midday (with T ∼25–35 °C). Laboratory oxidation studies performed with samples of these mine effluents have confirmed the influence of T, pH, dissolved oxygen content and biofilm presence/absence on the oxidation rate of Fe(II). Precipitation of Fe(III) takes place at pH 2.7–3.1 and rates ranging from 1.7×10−6 to 10−7 mol L−1 s−1, also showing a daily cycle. [Copyright &y& Elsevier]

Abstract: The fate of arsenic discharged from contaminated ground water to a small, shallow lake at a hazardous waste site was examined to understand the role of iron (hydr)oxide precipitation–dissolution processes within the water column. Field and laboratory observations indicate that arsenic solubility was controlled, in part, by the extent of ferrous iron oxidation–precipitation and arsenic sorption occurring near the lake chemocline. Laboratory experiments were conducted using site-derived water to assess the impact of these coupled processes on the removal of dissolved arsenic from the water column. The measured concentration of organic carbon from epilimnetic and hypolimnetic water sampled from the lake was approximately 1.3 mM and 17.0 mM, respectively. Experiments conducted with these samples along with synthetic controls containing no organic carbon demonstrated that observed rates of formation and crystallinity of the precipitated iron (hydr)oxide were dependent on the concentration of organic carbon in the lake water. Increasing dissolved organic matter concentration did not significantly interfere with ferrous iron oxidation, but inhibited iron (hydr)oxide precipitation and subsequent sorption of arsenic. For experiments using water sampled from the lake hypolimnion there was a strong relationship between the fraction of precipitated iron and the fraction of sorbed arsenic. Laboratory- and field-derived iron (hydr)oxide precipitates were characterized to evaluate mineralogy and arsenic distribution. In-situ suspended solids and precipitates formed in laboratory experiments using hypolimnetic water were identified as poorly crystalline 2-line ferrihydrite. These solids were readily dissolved in the presence of dithionite indicating that elevated dissolved iron and arsenic observed in the hypolimnion resulted, in part, from in-situ reductive dissolution of settling 2-line ferrihydrite near the sediment–water interface. These observations support the contention that the levels of dissolved arsenic observed in the shallow lake can be attributed to ground-water discharge and internal recycling of arsenic within the water column. The efficiency of the process resulting in iron (hydr)oxide precipitation and arsenic sorption limits the downgradient export of arsenic derived from ground-water discharge. [Copyright &y& Elsevier]

Abstract: Laboratory experiments were carried out to acquire more insight and understanding of the phenomena associated with the in-situ arsenic remediation. Visualization techniques are the most informative for the detection of Fe(II) while flowing in soils. Green Rust (GR) was considered as representative of in-situ iron precipitates. In a visualization flat cell, the change in color of GR to orange, due to oxidation, was monitored by a digital camera and the images were analyzed giving the spatial and temporal distribution of Fe(II). Moreover, both oxygen and pH changes in time were recorded in two sections along the flow direction in the cell. The measured and calculated concentration profiles were compared and the actual reaction rates were predicted. The reaction rate constants measured in this study, under flowing conditions, are in a good agreement with the values obtained from batch experiments reported in the literature. [Copyright &y& Elsevier]

Abstract: The influences of H+, Fe2+, Fe3+ and Cu2+ ion concentrations and their different combinations on the iron oxidation by an enrichment culture dominated by Leptospirillum ferriphilum were studied in batch experiments and continuous-flow fluidized-bed reactors. The iron oxidation rate did not significantly vary in the pH range of 0.9–1.5 and was only partially inhibited at pH 0.7. Ferric and ferrous iron at 5 and 24gL−1, respectively, had little or no effect on iron oxidation by the enrichment culture and the iron oxidation rate slightly decreased at higher concentrations. At a loading rate of 3g Fe2+ L−1 h−1, 2gL−1 Cu2+ did not affect the iron oxidation rate, whereas at a loading rate of 10g Fe2+ L−1 h−1 it reduced the rate by 69%. The maximum iron oxidation rate was 10g Fe2+ L−1 h−1 at pH 0.9 and in the presence of 21gL−1 Fe2+ and 2gL−1 Cu2+. [Copyright &y& Elsevier]

Abstract: Microbial oxidation of ferrous iron may be available alternative method of producing ferric sulphate, which is a reagent used for removal of H2S from biogas. For practical use of this process, this study evaluated some of the most efficient bioreactor systems for continuous ferrous iron oxidation by iron-oxidizing bacteria. Performances of various bioreactor systems were compared based on the ferrous iron oxidation efficiency according to the stepwise increase of hydraulic loading rates and ease of operation. A submerged-membrane bioreactor and immobilized bioreactor systems were used to increase the cell concentration. In the immobilized bioreactor system, various support media such as a granular activated carbon, polyurethane foam and honeycomb-type ceramics were tested with respect to the cell immobilizing performance. To improve the stability of biofilm, airlift-type immobilized bioreactor system was also developed. In this study, both the membrane bioreactor system and the immobilized bioreactor system using polyurethane foam achieved very good performance of biological ferrous iron oxidation. However, in consideration of economical and operational aspects, the immobilized bioreactor using polyurethane foam is the most practical and efficient system for indirect H2S removal using iron-oxidizing bacteria. [Copyright &y& Elsevier]

The oxidation of ferrous iron by Acidithiobacillus ferrooxidans in the pH range 2.5–7.0 was characterized. In order to measure the rate of bacterial oxidation of ferrous iron with A. ferrooxidans in this high pH range, a novel experimental methodology was developed. The results showed that the inhibition of ferrous iron oxidation activity by A. ferrooxidans observed at pH values above 3.0 is partially linked to the formation of ferric iron precipitates, which apparently hinder transport processes on the cell surface. By carefully controlling the amount of iron precipitates formed during the oxidation of ferrous iron in this pH range, enhanced bacterial activity was obtained. [Copyright &y& Elsevier]

A modeling approach of the bacterial oxidation of ferrous iron in batch culture is presented. It is based on the spatial interpolation of experimental data known as kriging interpolation. It is shown that, using the proposed method, the prediction error between model and true system variables is optimal in the sense of minimum variance. The results obtained improve the prediction error associated to the use of the current “knowledge-based” models. Results on modeling the kinetics of the bacterium Acidithiobacillus ferrooxidans, previously called Thiobacillus ferrooxidans, growing in ferrous iron as energy source in a batch airlift reactor are presented. The prediction error bound associated with the model is deduced theoretically and calculated for the application case. [Copyright &y& Elsevier]

This study used bacterial cellulose (BC) as novel support material for Acidithiobacillus ferrooxidans immobilization, with the objective of improving the performance of bioleaching and increasing its sustainability. The BC was synthesized in the laboratory, which allowed selecting the desired size of this highly porous and mechanically resistant material. After bacterial immobilization, the biologically active material (BAM) was used to assess the effect that several operating parameters had on the process (shaking mode and speed, the nutrient medium volume-to-exposed surface area ratio –NMV:ESA-, and Fe2+ concentration). The influence of dissolved copper and BAM storage was also determined. The results showed that the NMV:ESA ratio conditioned Fe2+ bio-oxidation time: after 33 h, 60% and 90% of the initial Fe2+ in the samples was converted, with a 1:0.1 and 1:0.6 ratio, respectively. In addition, Fe3+ productivity increased 16% when raising the initial Fe2+ content from 6 to 9 g L−1. A considerable increase in productivity (from 190 to 223 mg L−1 h−1) was also recorded when the shaking speed was increased from 130 to 170 rpm. BAM successfully adapted to the presence of dissolved copper and oxidized Fe2+ in the presence of up to 30 g Cu2+ L−1. Finally, the integrity of the material was not affected by the demanding operating conditions, and bacterial activity was successfully recovered after storage at 4 °C and 22 °C. In sum, biocellulose has proven to be a suitable candidate for A. ferrooxidans immobilization, which could contribute to improving the efficiency of bioleaching. [Display omitted] • Bacterial cellulose is a new suitable support for A. ferrooxidans immobilization. • The optimum operating conditions for reducing bio-oxidation time were established. • Fe3+ productivity of 223 mg L−1 h−1 was achieved at selected operating conditions. • The immobilized bacteria were resistant to copper concentrations up to 30 g L−1. • The use of bacterial cellulose enhanced the sustainability of bioleaching process. [ABSTRACT FROM AUTHOR]

[Display omitted] • Double rice conversion to maize-rice (MR) increased Fe-SOC content. • Ferrous iron oxidation and low phenol oxidase activity stabilized Fe-SOC under MR. • Iron gate theory dominated in maize season whereas enzyme latch in rice in MR. • Tradeoffs between enzyme latch and iron gate preserve Fe-SOC in MR system. To assess the impact of the incorporation of maize into double-rice (RR) on soil organic carbon (SOC) sequestration, a two-year experiment was conducted and changes in iron-bound SOC (Fe-SOC), as a proxy for stable carbon pool, were measured. The introduction of a maize-rice (MR) rotation enhanced Fe-SOC by 12–17 % in the maize season, and it stayed 13–36 % higher during rice cultivation compared to RR. Following the "iron gate" theory, Fe oxidation in the maize season was the leading mechanism for improving Fe-SOC content. However, "enzyme latch", i.e. the suppression of Fe-SOC loss by decreased phenol oxidase activity, was a dominant factor controlling the increase of Fe-SOC in the rice season of MR. Thus, the introduction of maize into the paddy field increased Fe-SOC, which was caused by combination of "iron gate" mechanism in maize season and "enzyme latch" in rice season. [ABSTRACT FROM AUTHOR]

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. 'Ferric iron reductase' activity of 9 nmol Fe mg protein min could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification). [ABSTRACT FROM AUTHOR]

Acidithiobacillus ferrooxidans YNTRS-40 (A. ferrooxidans) is a chemolithoautotrophic aerobic bacterium isolated from Tengchong hot springs, Yunnan Province, China, with a broad growth pH range of 1.0–4.5. This study reports the genome sequence of this strain and the information of genes related to the adaptation of diverse stresses and the oxidation of ferrous iron and sulfur. Results showed that YNTRS-40 possesses chromosomal DNA (3,209,933-bp) and plasmid DNA (47,104-bp). The complete genome of 3,257,037-bp consists of 3,349 CDS genes comprising 6 rRNAs, 52 tRNAs, and 6 ncRNAs. There are many encoded genes associated with diverse stresses adaptation and ferrous iron and sulfur oxidation such as rus operon, res operon, petI, petII, sqr, doxDA, cydAB, and cyoABCD. This work will provide essential information for further application of A. ferrooxidans YNTRS-40 in industry. [ABSTRACT FROM AUTHOR]

• A rapid method to determine the biomass of A. ferrooxidans in sludge was developed. • A quantitative relation between iron oxidation rate and CFU counts was established. • The basal specific rate of iron oxidation by A. ferrooxidans was 10−9 mg/h/CFU. • The rate increased by 10−10 mg/h/CFU per hour when ferrous iron is available. • The method reduces the time required for biomass determination from 12 d to ∼3 h. Overlay plate counting is the traditional way to determine the biomass of viable Acidithiobacillus ferrooxidans in iron-based bioleaching matrix of sewage sludge. However, the time required is 12 days at a minimum. Here, a rapid method based on a quantitative relationship between the extant rate of iron bio-oxidation by Acidithiobacillus ferrooxidans and the CFU counts was developed. The method reduces the time to ∼3 h. In the new method, the biomass is calculated by dividing the extant iron bio-oxidation rate by the specific rate of iron oxidation by Acidithiobacillus ferrooxidans. The extant rate of iron bio-oxidation was determined in a 2-h iron oxidation test. The effects of environmental factors such as pH on iron oxidation in all tests were the same. The basal specific rate of iron oxidation by Acidithiobacillus ferrooxidans was 10−9 mg/h/CFU. During bioleaching, the specific rate increased by 10−10 mg/h/CFU when the net contact time between Acidithiobacillus ferrooxidans and available ferrous iron increased by one hour. Therefore, according to the law, the biomass can be rapidly determined by the extant rate and the net contact time. [ABSTRACT FROM AUTHOR]