Enhanced Arsenate Immobilization by Kaolinite via Heterogeneous Pathways during Ferrous Iron Oxidation.

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.

Fodinisporobacter ferrooxydans gen. nov., sp. nov.-A Spore-Forming Ferrous-Oxidizing Bacterium Isolated from a Polymetallic Mine.

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).

Microbial immobilisation and adaptation to Cu2+ enhances microbial Fe2+ oxidation for bioleaching of printed circuit boards in the presence of mixed metal ions.

A circular economy requires effective re-use of finite resources, such as metals from waste electrical and electronic equipment (WEEE). Bioleaching for extraction and recovery of base metals from printed circuit boards (PCBs) before recovering precious metals has potential to increase metal circularity. However, inhibition by base metals released from the PCBs and accumulated in PCB leachates on microbial Fe2+ oxidation, a critical bioleaching sub-process for Fe3+ regeneration, can limit this approach. Here, we explore the potential of microbial immobilisation on polyurethane foam (PUF) and adaptation to cupric ions to minimise inhibition by mixed metals released from PCBs, particularly zinc, nickel, and tin, and enhancing Fe2+ oxidation rates in PCB bioleaching systems. A mixed mesophilic culture dominant in Leptospirillum ferriphilum, Acidiplasma cupricumulans and Acidithiobacillus caldus was immobilised on PUF and adapted to 6 g/L Cu2+. Tolerance of Cu-adapted immobilised cells to the inhibitory metal ions Zn2+, Ni2+, and Sn2+, as individual (0-10 g/L) and mixed metal ions at concentrations typically leached from PCBs at solids loadings of 0-20% (mass/volume) was compared to that of non-adapted immobilised cells. Further, the impact of solutes from PCB leachates was evaluated. Inhibition by individual metal ions decreased in the order Sn2+ > Ni2+ > Zn2+. Inhibition of ferrous iron oxidation by mixed metal ions was synergistic with respect to individual metal ions. PCB leachates were more inhibitory than both mixed and individual metal ions even where metal concentration was low. Cu-adapted immobilised cells exhibited higher tolerance to increasing concentrations of inhibitory metal ions than non-adapted cells. These results are promising for the application of Cu-adapted cells in the bioleaching of PCBs and multi-metal concentrates.

Sequence similarity network and protein structure prediction offer insights into the evolution of microbial pathways for ferrous iron oxidation.

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.

Phosphate Recovery from Aqueous Solutions via Vivianite Crystallization: Interference of FeII Oxidation at Different DO Concentrations and pHs.

Vivianite (Fe3(PO4)2·8H2O) crystallization has attracted increasing attention as a promising approach for removing and recovering P from wastewaters. However, FeII 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 FeII 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 FeII 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 FeII 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 FeII oxidation and FeIII 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 biotransformation genes and As transportation in soil-rice system affected by iron-oxidizing strain (Ochrobactrum sp.).

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.

Simultaneous removal of nitrate and heavy metals in a continuous flow nitrate-dependent ferrous iron oxidation (NDFO) bioreactor.

Nitrogen and heavy metals can co-occur in various industrial wastewaters such as coke-oven wastewater. Removal of these contaminants is important, but cost-efficient removal technology is limited. In this study, we examined the usefulness of nitrate-dependent ferrous iron oxidation (NDFO) for the simultaneous removal of nitrate and heavy metals (iron and zinc), by using an NDFO strain Pseudogulbenkiania sp. NH8B. Based on the batch culture assays, nitrate, Fe, and Zn were successfully removed from a basal medium as well as coke-oven wastewater containing 5 mM nitrate, 10 mM Fe(II), and 10 mg/L Zn. Zinc in the water was most likely co-precipitated with Fe(III) oxides produced during the NDFO reaction. Simultaneous removal of nitrate, Fe, and Zn was also achieved in a continuous-flow reactor fed with a basal medium containing 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L Zn. However, when the reactor is fed with coke-oven wastewater supplemented with 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L ZnCl2, the reactor performance significantly decreased, most likely due to the inhibition of bacterial growth by thiocyanate or organic contaminants present in the coke-oven wastewater. Use of mixed culture of NDFO bacteria and thiocyanate/organic-degrading denitrifiers should help improve the reactor performance.

Acidithiobacillus ferrianus sp. nov.: an ancestral extremely acidophilic and facultatively anaerobic chemolithoautotroph.

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.

Complete Genome Sequence of Acidithiobacillus Ferrooxidans YNTRS-40, a Strain of the Ferrous Iron- and Sulfur-Oxidizing Acidophile.

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.

Immobilization of arsenic as scorodite by a thermoacidophilic mixed culture via As(III)-catalyzed oxidation with activated carbon.

In this study we describe the immobilization of arsenic as scorodite (FeAsO4.2H2O) 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.

Nitrate-Dependent Iron Oxidation: A Potential Mars Metabolism.

This work considers the hypothetical viability of microbial nitrate-dependent Fe2+ oxidation (NDFO) for supporting simple life in the context of the early Mars environment. This draws on knowledge built up over several decades of remote and in situ observation, as well as recent discoveries that have shaped current understanding of early Mars. Our current understanding is that certain early martian environments fulfill several of the key requirements for microbes with NDFO metabolism. First, abundant Fe2+ has been identified on Mars and provides evidence of an accessible electron donor; evidence of anoxia suggests that abiotic Fe2+ oxidation by molecular oxygen would not have interfered and competed with microbial iron metabolism in these environments. Second, nitrate, which can be used by some iron oxidizing microorganisms as an electron acceptor, has also been confirmed in modern aeolian and ancient sediment deposits on Mars. In addition to redox substrates, reservoirs of both organic and inorganic carbon are available for biosynthesis, and geochemical evidence suggests that lacustrine systems during the hydrologically active Noachian period (4.1-3.7 Ga) match the circumneutral pH requirements of nitrate-dependent iron-oxidizing microorganisms. As well as potentially acting as a primary producer in early martian lakes and fluvial systems, the light-independent nature of NDFO suggests that such microbes could have persisted in sub-surface aquifers long after the desiccation of the surface, provided that adequate carbon and nitrates sources were prevalent. Traces of NDFO microorganisms may be preserved in the rock record by biomineralization and cellular encrustation in zones of high Fe2+ concentrations. These processes could produce morphological biosignatures, preserve distinctive Fe-isotope variation patterns, and enhance preservation of biological organic compounds. Such biosignatures could be detectable by future missions to Mars with appropriate instrumentation.

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans.

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.

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.

Proteomic Analysis of Differential Protein Expression in Acidithiobacillus ferrooxidans Grown on Ferrous Iron or Elemental Sulfur.

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(2+)) or elemental sulfur (S°). Sixteen of these proteins were upregulated by growth on S° or downregulated by growth on Fe(2+), 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(2+) 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(2+) oxidation by this extreme acidophile.

Toward a mechanistic understanding of anaerobic nitrate-dependent iron oxidation: balancing electron uptake and detoxification.

The anaerobic oxidation of Fe(II) by subsurface microorganisms is an important part of biogeochemical cycling in the environment, but the biochemical mechanisms used to couple iron oxidation to nitrate respiration are not well understood. Based on our own work and the evidence available in the literature, we propose a mechanistic model for anaerobic nitrate-dependent iron oxidation. We suggest that anaerobic iron-oxidizing microorganisms likely exist along a continuum including: (1) bacteria that inadvertently oxidize Fe(II) by abiotic or biotic reactions with enzymes or chemical intermediates in their metabolic pathways (e.g., denitrification) and suffer from toxicity or energetic penalty, (2) Fe(II) tolerant bacteria that gain little or no growth benefit from iron oxidation but can manage the toxic reactions, and (3) bacteria that efficiently accept electrons from Fe(II) to gain a growth advantage while preventing or mitigating the toxic reactions. Predictions of the proposed model are highlighted and experimental approaches are discussed.