Enumeration and Characterization of Iron(III)-Reducing Microbial Communities from Acidic Subsurface Sediments Contaminated with Uranium(VI)

Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml⁻¹ for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.     

异化铁还原细菌LQ25还原重金属Cr (VI)的特性研究

[背景] 异化铁还原细菌能够在还原Fe （III）的同时将毒性较大的Cr （VI）还原成毒性较小的Cr （III）,解决铬污染的问题。[目的] 基于丁酸梭菌（Clostridium butyricum） LQ25异化铁还原过程制备生物磁铁矿,开展异化铁还原细菌还原Cr （VI）的特性研究。[方法] 构建以氢氧化铁为电子受体和葡萄糖为电子供体的异化铁培养体系。菌株LQ25培养结束时制备生物磁铁矿。设置不同初始Cr （VI）浓度（5、10、15、25和30 mg/L）,分别测定菌株LQ25对Cr （VI）还原效率以及生物磁铁矿对Cr （VI）的还原效率。[结果] 菌株LQ25在设置的Cr （VI）浓度范围内都能良好生长。当Cr （VI）浓度为15 mg/L时,在异化铁培养条件下,菌株LQ25对Cr （VI）的还原率为63.45%±5.13%,生物磁铁矿对Cr （VI）的还原率为87.73%±9.12%,相比菌株还原Cr （VI）的效率提高38%。pH变化能影响生物磁铁矿对Cr （VI）的还原率,当pH 2.0时,生物磁铁矿对Cr （VI）的还原率最高,几乎达到100%。电子显微镜观察发现生物磁铁矿表面有许多孔隙,X-射线衍射图谱显示生物磁铁矿中Fe （II）的存在形式是Fe （OH）₂。[结论] 基于异化铁还原细菌制备生物磁铁矿可用于还原Cr （VI）,这是一种有效去除Cr （VI）的途径。     

Alkaline iron(III) reduction by a novel alkaliphilic,halotolerant, <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from salt flat sediments of Soap Lake

A halotolerant, alkaliphilic dissimilatory Fe(III)-reducing bacterium, strain SFB, was isolated from salt flat sediments collected from Soap Lake, WA. 16S ribosomal ribonucleic acid gene sequence analysis identified strain SFB as a novel Bacillus sp. most similar to Bacillus agaradhaerens (96.7% similarity). Strain SFB, a fermentative, facultative anaerobe, fermented various hexoses including glucose and fructose. The fructose fermentation products were lactate, acetate, and formate. Under fructose-fermenting conditions in a medium amended with Fe(III), Fe(II) accumulated concomitant with a stoichiometric decrease in lactate and an increase in acetate and CO₂. Strain SFB was also capable of respiratory Fe(III) reduction with some unidentified component(s) of Luria broth as an electron donor. In addition to Fe(III), strain SFB could also utilize nitrate, fumarate, or O₂ as alternative electron acceptors. Optimum growth was observed at 30°C and pH 9. Although the optimal salinity for growth was 0%, strain SFB could grow in a medium with up to 15% NaCl by mass. These studies describe a novel alkaliphilic, halotolerant organism capable of dissimilatory Fe(III) reduction under extreme conditions and demonstrate that Bacillus species can contribute to the microbial reduction of Fe(III) in environments at elevated pH and salinity, such as soda lakes.     

异化铁还原细菌Clostridium sp.LQ25分离及其产氢与铁还原特性

[背景] 一些异化铁还原细菌兼具铁还原和发酵产氢能力,可作为发酵型异化铁还原细菌还原机制研究的对象。[目的] 筛选出一株发酵型异化铁还原细菌。在异化铁还原细菌培养体系中,设置不同电子供体并分析电子供体。[方法] 通过三层平板法从海洋沉积物中筛选纯菌株,基于16S rRNA基因序列进行菌株鉴定。通过测定细菌培养液Fe （II）浓度及发酵产氢量分析菌株异化铁还原和产氢性质。[结果] 菌株LQ25与Clostridium butyricum的16S rRNA基因序列相似性达到100%,结合电镜形态观察,菌株命名为Clostridium sp.LQ25。在氢氧化铁为电子受体培养条件下,菌株生长较对照组（未添加氢氧化铁）显著提高。菌株LQ25能够利用丙酮酸钠、葡萄糖和乳酸钠进行生长。丙酮酸钠为电子供体时,菌株LQ25细胞生长和异化铁还原效率最高,菌体蛋白质含量是（78.88±3.40） mg/L,累积产生Fe （II）浓度为（8.27±0.23） mg/L。以葡萄糖为电子供体时,菌株LQ25发酵产氢量最高,达（475.2±14.4） mL/L,相比对照组（未添加氢氧化铁）产氢量提高87.7%。[结论] 筛选到一株具有异化铁还原和发酵产氢能力的菌株Clostridium sp.LQ25,为探究发酵型异化铁还原细菌胞外电子传递机制提供了新的实验材料。     

Evidence for rapid microscale bacterial redox cycling of iron in circumneutral environments

The potential for microscale bacterial Fe redox cycling was investigated in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella alga strain BrY). The Fe(II)-oxidizing organism was isolated from freshwater wetland surface sediments which are characterized by steep gradients of dissolved O₂ and high concentrations of dissolved and solid-phase Fe(II) within mm of the sediment–water interface, and which support comparable numbers (10⁵–10⁶ mL⁻¹) of culturable Fe(II)-oxidizing and Fe(III)-reducing reducing. The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O₂ input from the atmosphere and measurable dissolved O₂ to a depth of 2 mm below the sand–water interface. In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(III)-reducing bacteria alone. Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the upper few mm of sand. Our results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe. This revised version was published online in August 2006 with corrections to the Cover Date.     

Electron shuttle-stimulated RDX mineralization and biological production of 4-nitro-2,4-diazabutanal (NDAB) in RDX-contaminated aquifer material

The potential for extracellular electron shuttles to stimulate RDX biodegradation was investigated with RDX-contaminated aquifer material. Electron shuttling compounds including anthraquinone-2,6-disulfonate (AQDS) and soluble humic substances stimulated RDX mineralization in aquifer sediment. RDX mass-loss was similar in electron shuttle amended and donor-alone treatments; however, the concentrations of nitroso metabolites, in particular TNX, and ring cleavage products (e.g., HCHO, MEDINA, NDAB, and NH₄ ⁺) were different in shuttle-amended incubations. Nitroso metabolites accumulated in the absence of electron shuttles (i.e., acetate alone). Most notably, 40–50% of [¹⁴C]-RDX was mineralized to ¹⁴CO₂ in shuttle-amended incubations. Mineralization in acetate amended or unamended incubations was less than 12% within the same time frame. The primary differences in the presence of electron shuttles were the increased production of NDAB and formaldehyde. NDAB did not further degrade, but formaldehyde was not present at final time points, suggesting that it was the mineralization precursor for Fe(III)-reducing microorganisms. RDX was reduced concurrently with Fe(III) reduction rather than nitrate or sulfate reduction. Amplified 16S rDNA restriction analysis (ARDRA) indicated that unique Fe(III)-reducing microbial communities (β- and γ-proteobacteria) predominated in shuttle-amended incubations. These results demonstrate that indigenous Fe(III)-reducing microorganisms in RDX-contaminated environments utilize extracellular electron shuttles to enhance RDX mineralization. Electron shuttle-mediated RDX mineralization may become an effective in situ option for contaminated environments.     

Competition between Fe(III)-Reducing and Methanogenic Bacteria for Acetate in Iron-Rich Freshwater Sediments

The kinetics of acetate uptake and the depth distribution of [2-¹⁴C]acetate metabolism were examined in iron-rich sediments from a beaver impoundment in northcentral Alabama. The half-saturation constant (K_m) determined for acetate uptake in slurries of Fe(III)-reducing sediment (0.8 µM) was more than 10-fold lower than that measured in methanogenic slurries (12 µM) which supported comparable rates of bulk organic carbon metabolism and V_max values for acetate uptake. The endogenous acetate concentration (S _n) was also substantially lower (1.7 µM) in Fe(III)-reducing vs methanogenic (9.0 µM) slurries. The proportion of [2-¹⁴C]acetate converted to ¹⁴CH₄ increased with depth from ca 0.1 in the upper 0.5 cm to ca 0.8 below 2 cm and was inversely correlated (r² = 0.99) to a decline in amorphous Fe(III) oxide concentration. The results of the acetate uptake kinetics experiments suggest that differences in the affinity of Fe(III)-reducing bacteria vs methanogens for acetate can account for the preferential conversion of [2-¹⁴C]acetate to ¹⁴CO₂ in Fe(III) oxide-rich surface sediments, and that the downcore increase in conversion of [2-¹⁴C]acetate to ¹⁴CH₄ can be attributed to progressive liberation of methanogens from competition with Fe(III) reducers as Fe(III) oxides are depleted with depth.     

Eu3+ Sequestration by Biogenic Nano-Hydroxyapatite Synthesized at Neutral and Alkaline pH

Biogenic hydroxyapatite (bio-HA) has the potential for radionuclide capture and remediation of metal-contaminated environments. Biosynthesis of bio-HA was achieved via the phosphatase activity of a Serratia sp. supplemented with various concentrations of CaCl₂ and glycerol 2-phosphate (G2P) provided at pH 7.0 or 8.6. Presence of hydroxyapatite (HA) was confirmed in the samples by X-ray powder diffraction analysis. When provided with limiting (1 mM) G2P and excess (5 mM) Ca²⁺ at pH 8.6, monohydrocalcite was found. This, and bio-HA with less (1 mM) Ca²⁺ accumulated Eu(III) to ～31% and 20% of the biomineral mass, respectively, as compared to 50% of the mineral mass accumulated by commercial HA. Optimally, with bio-HA made at initial pH 7.0 from 2 mM Ca²⁺ and 5 mM G2P, Eu(III) accumulated to ～74% of the weight of bio-HA, which was equal to the mass of the HA mineral component of the biomaterial. The implications with respect to potential bio-HA-barrier development in situ or as a remediation strategy are discussed.     

Role of apoplast acidification by the H+ pump

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H⁺ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H⁺ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO₄). These results support the hypothesis that the apoplastic pH shift caused by H⁺ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H⁺ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H⁺ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H⁺ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H⁺ transport. Our results shed light on the way by which the strong buffering effect of HCO ₃ ^- in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.     

Microbial Community Analysis and Effects of Fe(II)/Mn(II) Ratio on Denitrification in the Mixotrophic Biological Reactor

In this study, the denitrification performance of the mixotrophic biological reactor was investigated under varying Fe(II)/Mn(II) molar ratio conditions. Results indicate that the optimal nitrate removal ratio occurred at an Fe(II)/Mn(II) molar ratio of 9:1, pH of 7, with an HRT of 10?h. When the reactor was performing under optimal conditions, the nitrate removal reached 100.00% at a rate of 0.116?mmol·L^?1·h^?1. The proportion of oxidized Fe(II) and Mn(II) reached 99.29% and 21.88%, respectively. High-throughput sequencing results show that Pseudomonas was the dominant species in the mixotrophic biological reactor. Furthermore, the relative abundance of Pseudomonas and denitrification performance was significantly influenced by variation in the Fe(II)/Mn(II) molar ratio.     

Anoxic oxidation of arsenite linked to chemolithotrophic denitrification in continuous bioreactors

In this study, the anoxic oxidation of arsenite (As(III)) linked to chemolithotrophic denitrification was shown to be feasible in continuous bioreactors. Biological oxidation of As(III) was stable over prolonged periods of operation ranging up to 3 years in continuous denitrifying bioreactors with granular biofilms. As(III) was removed with a high conversion efficiency (>92%) to arsenate (As(V)) in periods with high volumetric loadings (e.g., 3.5–5.1 mmol As L day^?1). The maximum specific activity of sampled granular sludge from the bioreactors was 0.98 ± 0.04 mmol As(V) formed g^?1 VSS day^?1 when determined at an initial concentration of 0.5 mM As(III). The microbial population adapted to high influent concentrations of As(III) up to 5.2 mM. However, the As(III) oxidation process was severely inhibited when 7.6–8.1 mM As(III) was fed. Activity was restored upon lowering the As(III) concentration to 3.8 mM. Several experimental strategies were utilized to demonstrate a dependence of the nitrate removal on As(III) oxidation as well as a dependence of the As(III) removal on nitrate reduction. The molar stoichiometric ratio of As(V) formed to nitrate removed (corrected for endogenous denitrification) in the bioreactors approximated 2.5, indicating complete denitrification was occurring. As(III) oxidation was also shown to be linked to the complete denitrification of NO to N₂ gas by demonstrating a significantly enhanced production of N₂ beyond the background endogenous production in a batch bioassay spiked with 3.5 mM As(III). The N₂ production also corresponded closely to the expected stoichiometry of 2.5 mol As(III) mol^?1 N₂–N for complete denitrification. Biotechnol. Bioeng. 2010;105: 909–917. © 2009 Wiley Periodicals, Inc.     

Aluminium toxicity and binding to Escherichia coli

The toxicity and binding of aluminium to Escherichia coli has been studied. Inhibition of growth by aluminium nitrate was markedly dependent on pH; growth in medium buffered to pH 5.4 was more sensitive to 0.9 mM or 2.25 mM aluminium than was growth at pH 6.6–6.8. In medium buffered with 2-(N-morpholino)ethanesulphonic acid (MES), aluminium toxicity was enhanced by omission of iron from the medium or by use of exponential phase starter cultures. Analysis of bound aluminium by atomic absorption spectroscopy showed that aluminium was bound intracellularly at one type of site with a K _m of 0.4 mM and a capacity of 0.13 mol (g dry wt)^-1. In contrast, binding of aluminium at the cell surface occurred at two or more sites with evidence of cooperativity. Addition of aluminium nitrate to a weakly buffered cell suspension caused acidification of the medium attributable to displacement of protons from cell surfaces by metal cations. It is concluded that aluminium toxicity is related to pH-dependent speciation [with Al(H₂O) ₆ ³⁺ probably being the active species] and chelation of aluminium in the medium. Aluminium transport to intracellular binding sites may involve Fe(III) transport pathways.     

Physiological and Genomic Characterization of a Nitrate-Reducing Fe(II)-Oxidizing Bacterium Isolated from Paddy Soil

In this study, a neutrophilic, heterotrophic bacterium (strain Paddy-2) that is capable of ferrous iron [Fe(II)] oxidation coupled with nitrate (NO₃^?) reduction (NRFO) under anoxic conditions was isolated from paddy soil. The molecular identification by 16S rRNA gene sequencing identified the strain as Cupriavidus metallidurans. Strain Paddy-2 reduced 97.7% of NO₃^?and oxidized 89.7% of Fe(II) over 6?days with initial NaNO₃ and FeCl₂ concentrations of 9.37?mM and 4.72?mM, respectively. Acetate (5?mM) was also supplied as a carbon source and an alternative electron donor. A poorly crystalline Fe(III) mineral was the main component observed after 15?days of growth in culture, whereas lepidocrocite was detected in the X-ray diffraction spectrum after 3?months of culture. The homologous genes in electron transfer during Fe(II) oxidation (cyc1, cymA, FoxY, FoxZ, and mtoD) were also identified in the genomes of strain Paddy-2 and other reported NRFO bacteria. These genes encoding c-Cyts may play a role in electron transfer during the process of NRFO. These results provide evidence for the potential of NO₃^? to affect Fe(II) oxidation and biomineralization in bacterium from anoxic paddy soil.     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Reductive Dechlorination of Trichloroethylene (TCE) in Competition with Fe and Mn Oxides—Observed Dynamics in H2-dependent Terminal Electron Accepting Processes

The determination of hydrogen (H₂) concentration together with the products of microbial reduction reactions in a trichloroethylene dechlorinating system is conducted to delineate the ongoing predominant terminal electron accepting processes (TEAP). Formate was used as electron donor and synthetic Fe minerals or environmental samples were used as the substrate. Iron(III) and Mn(IV) reduction limited microbial dechlorination by the mixed anaerobic culture by decreasing the level of H₂ in the system. The H₂ measurements indicated that the H₂ concentration at which different TEAPs occur can overlap and thus these TEAPs can therefore occur concurrently rather than exclusively. Difference in Fe(III) bioavailability and hence, Fe(III) reduction partially explain this wide range. The distinction between dechlorination and other microbial reduction processes based on H₂ threshold values is not feasible under such conditions, though there appears to be a relation between the rates of H₂ consuming process and the observed H₂ level.     

Cooperation of Three Denitrifying Bacteria in Nitrate Removal of Acidic Nitrate- and Uranium-Contaminated Groundwater

In uranium-contaminated aquifers co-contaminated with nitrate, denitrifiers play a critical role in bioremediation. Six strains of denitrifying bacteria belonging to Rhizobium, Pseudomonas, and Castellaniella were isolated from the Oak Ridge Integrated Field Research Challenge Site (OR-IFRC), where biostimulation of acidic (pH 3.5–6.5), nitrate-contaminated (up to 140 mM) groundwater occurred. Three isolates were characterized in regards to nitrite tolerance, denitrification kinetic parameters, and growth on different denitrification intermediates. Kinetic and growth experiments showed that Pseudomonas str. GN33#1 reduced NO^? ₃ most rapidly (V_max = 15.8 μmol e^?·min^?1·mg protein^?1) and had the fastest generation time (gt) on NO^? ₃ (2.6 h). Castellaniella str. 4.5A2 was the most low pH and NO^? ₂ tolerant and grew rapidly on NO^? ₂ (gt = 4.0 h). Rhizobium str. GN32#2 was also tolerant of low pH values and reduced NO^? ₂ rapidly (V_max = 10.6 μmol e^?·min^?1·mg protein^?1) but was far less NO^? ₂ tolerant than Castellaniella str. 4.5A2. Growth of and denitrification by these three strains incubated together and individually were measured in OR-IFRC groundwater at pHs 5 and 7 to determine whether they cooperate or compete during denitrification. Mixed assemblages reduced NO^? ₃ more rapidly and more completely than any individual isolate over the course of the experiment. The results described in this article demonstrate 1) that this synthetic assemblage comprised of three physiologically distinct denitrifying bacterial isolates cooperate to achieve more complete levels of denitrification and 2) the importance of pH- and nitrite-tolerant bacteria such as Castellaniella str. 4.5A2 in minimizing NO^? ₂ accumulation in high-NO^? ₃ groundwater during bioremediation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.     

微生物介导硝酸盐还原耦合亚铁氧化过程的动力学及其影响因素

【目的】探究不同菌浓度和亚铁浓度条件下,Acidovorax sp. strain BoFeN1介导的厌氧亚铁氧化耦合硝酸盐还原过程的动力学和次生矿物。【方法】构建包含菌BoFeN1、硝酸盐、亚铁的厌氧培养体系,测试硝酸根、亚硝酸根、乙酸根、亚铁等浓度,并收集次生矿物,采用XRD、SEM进行矿物种类和形貌表征。【结果】在微生物介导硝酸盐还原耦合亚铁氧化的体系中,高菌浓度促进硝酸盐还原,对亚铁氧化也有一定促进作用;高浓度亚铁在低菌浓度下氧化反应速率和程度降低,但是在高菌浓度下无明显影响;亚铁浓度越高次生矿物结晶度越高,但对硝酸盐还原具有一定抑制作用。在微生物介导亚硝酸盐还原耦合亚铁氧化的体系中,高的菌浓度和亚铁浓度都会促进亚硝酸盐还原,但亚铁氧化的次生矿物会对亚硝酸盐的微生物还原产生较强的抑制作用,次生矿物的种类和结晶度主要受亚铁浓度影响。【结论】硝酸盐还原主要是生物反硝化作用,亚硝酸盐还原包含生物反硝化和化学反硝化两部分,在硝酸盐体系中亚铁氧化与次生矿物生成是受生物和化学反硝化作用的共同影响,但亚硝酸盐体系中亚铁氧化与次生矿物生成主要是受化学反硝化作用影响。该研究可为深入理解厌氧微生物介导铁氮耦合反应机制提供基础数据和理论支撑。     

pH Gradient-Induced Heterogeneity of Fe(III)-Reducing Microorganisms in Coal Mining-Associated Lake Sediments

Lakes formed because of coal mining are characterized by low pH and high concentrations of Fe(II) and sulfate. The anoxic sediment is often separated into an upper acidic zone (pH 3; zone I) with large amounts of reactive iron and a deeper slightly acidic zone (pH 5.5; zone III) with smaller amounts of iron. In this study, the impact of pH on the Fe(III)-reducing activities in both of these sediment zones was investigated, and molecular analyses that elucidated the sediment microbial diversity were performed. Fe(II) was formed in zone I and III sediment microcosms at rates that were approximately 710 and 895 nmol cm⁻³ day⁻¹, respectively. A shift to pH 5.3 conditions increased Fe(II) formation in zone I by a factor of 2. A shift to pH 3 conditions inhibited Fe(II) formation in zone III. Clone libraries revealed that the majority of the clones from both zones (approximately 44%) belonged to the Acidobacteria phylum. Since moderately acidophilic Acidobacteria species have the ability to oxidize Fe(II) and since Acidobacterium capsulatum reduced Fe oxides at pHs ranging from 2 to 5, this group appeared to be involved in the cycling of iron. PCR products specific for species related to Acidiphilium revealed that there were higher numbers of phylotypes related to cultured Acidiphilium or Acidisphaera species in zone III than in zone I. From the PCR products obtained for bioleaching-associated bacteria, only one phylotype with a level of similarity to Acidithiobacillus ferrooxidans of 99% was obtained. Using primer sets specific for Geobacteraceae, PCR products were obtained in higher DNA dilutions from zone III than from zone I. Phylogenetic analysis of clone libraries obtained from Fe(III)-reducing enrichment cultures grown at pH 5.5 revealed that the majority of clones were closely related to members of the Betaproteobacteria, primarily species of Thiomonas. Our results demonstrated that the upper acidic sediment was inhabited by acidophiles or moderate acidophiles which can also reduce Fe(III) under slightly acidic conditions. The majority of Fe(III) reducers inhabiting the slightly acidic sediment had only minor capacities to be active under acidic conditions.     

Enumeration and characterization of iron(III)-reducing microbial communities from acidic subsurface sediments contaminated with uranium(VI)

Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml(-1) for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.     

Resource effects on denitrification are mediated by community composition in tidal freshwater wetlands soils

Accurate prediction of denitrification rates remains difficult, potentially owing to complex uncharacterized interactions between resource conditions and denitrifier communities. To better understand how the availability of organic matter (OM) and nitrate (NO₃^–), two of the resources most fundamental to denitrifiers, affect these populations and their activity, we performed an in situ resource manipulation in tidal freshwater wetland soils. Soils were augmented with OM to double ambient concentrations, using either compost or plant litter, and fertilized with KNO₃ at two levels (low: ～ 5 mg l^–1 NO₃^––N and high: ～ 50 mg l^–1 NO₃^––N) in a full factorial design. Community composition of nirS‐denitrifers (assessed using terminal restriction fragment length polymorphism) was interactively regulated by both NO₃^– concentration and OM type, and the associated shifts in community composition were relatively consistent across sampling dates (6, 9 and 12 months of incubation). Denitrification potential (pDNF) rates were also strongly affected by NO₃^– fertilization and increased by ～ 10–100‐fold. Path analysis revealed that the influence of resource availability on pDNF rates was largely mediated through changes in nirS‐denitrifier community composition. These results suggest that a greater understanding of denitrifier community ecology may enable more accurate prediction of denitrification rates.