水稻土中铁还原菌多样性

微生物介导的异化Fe(III) 还原是非硫厌氧环境中Fe(III) 还原生成Fe(II) 的主要途径,然而相关的铁还原菌还不是很清楚,特别是在水稻土中.本文采用富集培养的方法,以乙酸和氢气作为电子供体,水铁矿和针铁矿作为电子受体,通过末端限制性片段长度多态性（T-RFLP）技术和16S rRNA基因克隆测序相结合的分子生物学方法研究了水稻土中铁还原菌的多样性.结果表明：无论是以乙酸或氢气为电子供体,水铁矿或针铁矿为电子受体,地杆菌（Geobacter）和梭菌（Clostridiales）是富集到的主要微生物群落;乙酸为电子供体时,富集到的主要微生物群落还包括红环菌（Rhodocyclaceae）;因此,除地杆菌外,梭菌和红环菌很可能也是水稻土中重要的铁还原菌.     

铁还原菌降解石油烃的研究进展

铁还原菌是指能够利用细胞外Fe(III)作为末端电子受体,通过氧化有机物将Fe(III)还原为Fe(II)微生物的总称。铁还原作用广泛存在于土壤、河流、海洋、地表含水层以及高温高压的地下深部油藏。在厌氧或兼性厌氧条件下,Fe(III)还原耦合有机物的降解,对铁、碳元素的生物地球化学循环具有重要意义。本文介绍了铁还原菌的多样性和铁还原作用机理,综述了铁还原菌在石油烃降解方面的研究进展。此外,还总结了铁还原菌在生物修复中的潜在作用,并对未来的研究方向进行了展望。     

不同电子受体下铁还原细菌异化还原Fe(Ⅲ)性质及菌群特征

将渤海沉积物进行厌氧培养,富集异化Fe(Ⅲ)还原混合菌群。在不同电子受体下,分析铁还原菌群异化还原Fe(Ⅲ)性质。以柠檬酸铁和氢氧化铁为电子受体培养体系,在培养12 h时,累积Fe(Ⅱ)浓度分别为(100.67±0.75)和(53.24±3.63)mg·L~(-1);当培养60h时,累积Fe(Ⅱ)浓度达到(118.95±1.47)和(119.74±3.96)mg·L~(-1)。这表明可溶性与不可溶性电子受体能够显著影响细菌异化Fe(Ⅲ)还原过程,而对累积Fe(Ⅲ)还原量影响不明显。通过高通量测序技术,分析不同电子受体下的异化Fe(Ⅲ)还原混合菌群多样性与优势菌组成。菌群多样性分析表明,以柠檬酸铁和氢氧化铁为电子受体时,菌群多样性Shannon指数分别是3.40和3.11,较对照组(Shannon指数2.07)高,表明培养体系中加入Fe(Ⅲ)能显著提高铁还原混合菌群多样性。异化Fe(Ⅲ)还原混合菌群在不同电子受体下优势菌主要是Clostridium_sensu_stricto和Romboutsia,属于梭菌目Clostridiales,这表明梭菌是参与Fe(Ⅲ)还原的优势菌。     

碳源和淹水时间对水稻土微生物Fe(Ⅲ)还原能力的影响

以我国6个省的水稻土为供试样品,采用厌氧恒温培养方法,研究了分别以葡萄糖、丙酮酸盐、乳酸盐和乙酸盐为惟一碳源时不同淹水时间土壤微生物群落对Fe(Ⅲ)的还原能力.结果表明：不同淹水时间对Fe(Ⅲ)还原特征值V_max的影响显著,表现为淹水20 d > 30 d > 12 d > 1 d > 5 d,不同淹水时间下水稻土微生物群落结构不同是导致Fe(Ⅲ)还原能力不同的主要原因.不同碳源对微生物铁还原过程有显著影响,葡萄糖和丙酮酸盐在不同淹水时间中始终为优势碳源,其Fe(Ⅲ)还原率分别为88.1%～99.9%和58.0%～97.9%;不同土壤铁还原微生物群落对乳酸盐的利用差距较大,湖南和浙江水稻土在整个淹水周期中Fe(Ⅲ)还原率达到87.1%～100%,而其他土壤则表现为淹水前5 d为5.0%～49.4%,12 d后增加到52.2%～99.9%;乙酸盐处理在不同淹水时间中都表现为随时间推移Fe(Ⅲ)还原率逐渐增大的趋势,其中浙江水稻土的变化最大,在5.3%～75.8%.     

水稻土中铁氧化物对产甲烷古菌群落结构的影响

产甲烷菌广泛分布在淹水水稻土等各种厌氧环境中,在全球气候变化、碳循环和能源等领域都发挥着重要的作用。研究发现,厌氧条件下,水稻土中铁氧化物的生物还原会抑制产甲烷菌的甲烷合成作用。然而,目前关于铁氧化物对产甲烷菌群落结构的影响报道较少。通过泥浆厌氧培养实验,向采集的水稻土中添加甲酸盐作为甲烷合成的底物(Control,CK处理),并设置添加水铁矿作为体系中唯一电子受体的处理组(Ferrihydrite,Fh处理)。培养结束后,与CK相比,添加水铁矿显著降低了古菌在总微生物群落中的占比,但对古菌群落的物种多样性和均一度没有显著影响;且两组处理中优势种均为操作分类单元(Operational taxonomic unit,OTU)2056和OTU 911(76%—80%)。这说明碳源相同时,产甲烷菌的群落结构不受铁氧化物的影响。本研究为探索土壤中微生物铁还原与碳循环耦合的分子机制奠定基础。     

海洋沉积物中铁还原细菌组成及异化铁还原与产氢性质分析

【背景】一些铁还原细菌具有异化铁还原与产氢的能力,该类细菌在环境污染修复的同时能够解决能源问题。【目的】从海洋沉积物中富集获得异化铁还原菌群,明确混合菌群组成、异化铁还原及产氢性质。获得海洋沉积物中异化铁还原混合菌群组成,分析菌群异化铁还原和产氢性质。【方法】利用高通量测序技术分析异化铁还原菌群的优势菌组成,在此基础上,分析异化铁还原混合菌群在不同电子供体培养条件下异化铁还原能力和产氢性质。【结果】高通量数据表明,在不溶性氢氧化铁为电子受体和葡萄糖为电子供体厌氧培养条件下,混合菌群的优势菌属主要是梭菌(Clostridium),属于发酵型异化铁还原细菌。混合菌群能够利用电子供体蔗糖、葡萄糖以及丙酮酸钠进行异化铁还原及发酵产氢。葡萄糖为电子供体时,菌群累积产生Fe(Ⅱ)浓度和产氢量最高,分别是59.34±6.73 mg/L和629.70±11.42 mL/L。【结论】异化铁还原混合菌群同时具有异化铁还原和产氢能力,拓宽了发酵型异化铁还原细菌的种质资源,探索异化铁还原细菌在生物能源方面的应用。     

淹水时间对水稻土中地杆菌科群落结构及丰度的影响

【目的】通过模拟水稻土淹水过程,探讨地杆菌科(Geobacteraceae)群落结构和相对丰度随淹水时间的动态变化特征,揭示其群落结构和相对丰度变化与微生物Fe(Ⅲ)还原的内在联系。【方法】提取水稻土淹水培养1 h、1 d、5 d、10 d、20 d和30 d后的微生物总DNA,构建地杆菌科16S rDNA克隆文库,采用PCR-RFLP方法分析地杆菌科的群落结构和多样性变化特征,通过Real-time PCR技术测定地杆菌科相对丰度的动态变化。采用厌氧泥浆培养方法,测定水稻土中Fe(Ⅱ)产生量变化。【结果】供试水稻土中,微生物Fe(Ⅲ)还原过程在淹水培养初期变化明显,培养20 d后达到稳定期,最大铁还原潜势为10.16 mg/g,最大反应速率为1.064 mg/(g.d),最大反应速率对应的时间为4.84 d。α多样性指数显示,水稻土中地杆菌科的多样性随淹水时间延长呈现波动性变化,淹水5 d和20 d处理出现2个峰值,而淹水10 d和30 d处理的多样性明显减小。β多样性指数表明淹水过程中群落结构存在明显差异。不同淹水时间共产生了10种地杆菌科优势类型,分别属于Clade 1和Clade 2。Real-time PCR结果表明,地杆菌科与总细菌16S rDNA丰度的比值在淹水培养1 d时最小(1.20%),而20 d时达到最大值(4.54%)。【结论】淹水培养的水稻土中,地杆菌科微生物的多样性和相对丰度的动态变化与微生物Fe(Ⅲ)还原过程密切相关。     

短期淹水培养对水稻土中地杆菌和厌氧粘细菌丰度的影响

模拟水稻土淹水过程,采用Real-time PCR技术测度了不同水稻土中地杆菌(Geobacteraceae spp.)和厌氧粘细菌(Anaeromyxobacter spp.)在不同淹水时期16S rDNA拷贝数的变化,比较了地杆菌和厌氧粘细菌丰度与培养过程中微生物Fe (Ⅲ)还原的关系。结果表明,在4类稻作区采集的水稻土样品中,Fe (Ⅲ)还原潜势有明显的区别,表现出由北向南逐渐降低的趋势。从淹水12 h的地杆菌和厌氧粘细菌拷贝数变化看出,采自浙江和天津的水稻土样品对淹水过程具有高度敏感性,而采自吉林和广西的水稻土样品对淹水响应不敏感。在17 d的短期淹水培养中,地杆菌丰度明显高于厌氧粘细菌,表明地杆菌对水稻土中铁还原的贡献大于厌氧粘细菌。地杆菌和厌氧粘细菌拷贝数总体上表现出在11 d 时达到峰值,17 d时显著下降。吉林水稻土中地杆菌丰度在5 d时达到38.3%,与其最大铁还原速率到达时间(T_Vmax)为4.07 d相对应,表明地杆菌对其铁还原过程具有重要贡献。四川水稻土中地杆菌和厌氧粘细菌丰度均较低,暗示其他兼性铁还原菌对其铁还原的作用值得重视。     

初始pH值对碱性和酸性水稻土微生物铁还原过程的影响

酸碱度(pH值)是水稻土铁还原过程的重要影响因素之一。通过模拟水稻土淹水厌氧培养,以Al2(SO4)3和Na2CO3溶液分别调节碱性和酸性水稻土pH值至强酸性(pH值5.0)、酸性(pH值5.0—6.5)、中性(pH值6.5—7.5)、碱性(pH值7.5—8.5)、强碱性(pH值8.5),以此来研究5种初始pH值对水稻土泥浆铁还原过程的影响;通过微生物群落厌氧培养研究了2种水稻土菌悬液在6种pH值条件下的铁还原能力差异。结果表明,碱性水稻土铁还原潜势(a)、最大铁还原速率(V max)随初始pH值的降低而下降,而达到最大铁还原速率所需的时间(T Vmax)则延长。提高酸性水稻土初始pH值使铁还原V max增加而T Vmax缩短,但土壤中无定形氧化铁均能还原,初始pH值与V max具有显著正相关关系。碱性和酸性水稻土的土壤菌悬液在试验pH值范围内厌氧培养,其铁还原能力在培养初期差异不显著,但培养后期的差异明显,且最终都能把培养液中氧化铁完全还原。随着初始pH值升高T Vmax延长,V max则降低,且均显著负相关,但碱性水稻土微生物群落的V max在pH值6.00时最大。初始pH值和土壤类型对水稻土铁还原过程具有显著影响,且对土壤菌悬液微生物群的铁还原具有一定影响。     

一株嗜水气单胞菌HS01的偶氮还原脱色特性

[目的]研究嗜水气单胞菌HS01的偶氮染料还原脱色特性.[方法]建立HS01/偶氮染料/电子供体序批式厌氧反应体系,研究Fe(Ⅲ)/腐殖质还原菌HS01以偶氮染料为电子受体的厌氧呼吸特性及影响因素;并构建HS01/偶氮染料/电子供体/铁氧化物体系,探讨铁氧化物对HS01偶氮还原的影响.[结果]HS01可将金橙Ⅰ迅速还原,菌体增殖;柠檬酸、丙三醇、蔗糖和葡萄糖体系中,16h金橙Ⅰ的脱色率分别达87％、85％、88％、90％;不同pH和金橙Ⅰ初始浓度条件下的脱色率不同;在反应体系中加入α-FeOOH,脱色率从90％增加至95％,Fe(Ⅱ)生成量与无染料对照体系相当.[结论]HS01能以葡萄糖为电子供体,金橙Ⅰ为唯一电子受体,进行厌氧呼吸;蔗糖、柠檬酸、丙三醇也可作为有效的电子供体,脱色率依次递减;甲酸、乙酸、乳酸、乙醇及丙酸不能作为HS01厌氧呼吸的电子供体.金橙Ⅰ脱色的最佳pH范围为6.0-8.0;高浓度(2.0 mmol/L)金橙Ⅰ负荷下,HS01仍保持高脱色率(＞85％).在HS01/α-FeOOH/金橙Ⅰ体系中,异化铁还原作用与偶氮呼吸作用同时发生,异化铁还原能促进偶氮脱色,而脱色对Fe(Ⅲ)还原没有明显影响.这可为铁/腐殖质还原菌在环境修复和废水处理等领域的应用提供研究积累.     

Phylogenetic diversity of dissimilatory Fe(III)-reducing bacteria in paddy soil

Microorganism-mediated dissimilatory Fe (III) reduction is recognized as the dominant mechanism for Fe(III) reduction to Fe(II) in non-sulfidogenic anaerobic environments, but the microorganisms involved, especially in paddy soil, are still poorly understood. In this paper, an enrichment culture was conducted to study the phylogenetic diversity of Fe (III)-reducing bacteria in paddy soil, with acetate or hydrogen as the electron donor and with ferrihydrite or goethite as the electron acceptor, and by the methods of terminal-restriction fragment length polymorphism (T-RFLP) technology and 16S rRNA genes cloning and sequencing. No matter what the electron donor and electron acceptor were supplemented, the most abundant microorganisms were Geobacter and Clostridiales, and Rhodocyclaceae were also abundant, when acetate was supplemented as electron donor, which suggested that besides Geobacter, Clostridiales and Rhodocyclaceae could be also the important Fe(III)-reducing bacteria in paddy soil.     

Influence of Biogenic Fe(II) on Bacterial Crystalline Fe(III) Oxide Reduction

Bacterial crystalline Fe(III) oxide reduction has the potential to significantly influence the biogeochemistry of anaerobic sedimentary environments where crystalline Fe(III) oxides are abundant relative to poorly crystalline (amorphous) phases. A review of published data on solid-phase Fe(III) abundance and speciation indicates that crystalline Fe(III) oxides are frequently 2- to S 10-fold more abundant than amorphous Fe(III) oxides in shallow subsurface sediments not yet subjected to microbial Fe(III) oxide reduction activity. Incubation experiments with coastal plain aquifer sediments demonstrated that crystalline Fe(III) oxide reduction can contribute substantially to Fe(II) production in the presence of added electron donors and nutrients. Controls on crystalline Fe(III) oxide reduction are therefore an important consideration in relation to the biogeochemical impacts of bacterial Fe(III) oxide reduction in subsurface environments. In this paper, the influence of biogenic Fe(II) on bacterial reduction of crystalline Fe(III) oxides is reviewed and analyzed in light of new experiments conducted with the acetate-oxidizing, Fe(III)-reducing bacterium (FeRB) Geobacter metallireducens . Previous experiments with Shewanella algae strain BrY indicated that adsorption and/or surface precipitation of Fe(II) on Fe(III) oxide and FeRB cell surfaces is primarily responsible for cessation of goethite ( f -FeOOH) reduction activity after only a relatively small fraction (generally < 10%) of the oxide is reduced. Similar conclusions are drawn from analogous studies with G. metallireducens . Although accumulation of aqueous Fe(II) has the potential to impose thermodynamic constraints on the extent of crystalline Fe(III) oxide reduction, our data on bacterial goethite reduction suggest that this phenomenon cannot universally explain the low microbial reducibility of this mineral. Experiments examining the influence of exogenous Fe(II) (20 mM FeCl 2 ) on soluble Fe(III)-citrate reduction by G. metallireducens and S. algae showed that high concentrations of Fe(II) did not inhibit Fe(III)-citrate reduction by freshly grown cells, which indicates that surface-bound Fe(II) does not inhibit Fe(III) reduction through a classical end-product enzyme inhibition mechanism. However, prolonged exposure of G. metallireducens and S. algae cells to high concentrations of soluble Fe(II) did cause inhibition of soluble Fe(III) reduction. These findings, together with recent documentation of the formation of Fe(II) surface precipitates on FeRB in Fe(III)-citrate medium, provide further evidence for the impact of Fe(II) sorption by FeRB on enzymatic Fe(III) reduction. Two different, but not mutually exclusive, mechanisms whereby accumulation of Fe(II) coatings on Fe(III) oxide and FeRB surfaces may lead to inhibition of enzymatic Fe(III) oxide reduction activity (in the absence of soluble electron shuttles and/or Fe(III) chelators) are identified and discussed in relation to recent experimental work and theoretical considerations.     

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.     

Fe(III)-enhanced anaerobic transformation of 2,4-dichlorophenoxyacetic acid by an iron-reducing bacterium Comamonas koreensis CY01

This work studied the ability of Comamonas koreensis CY01 to reduce Fe(III) (hydr)oxides by coupling the oxidation of electron donors and the enhanced biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the presence of Fe(III) (hydr)oxides. The experimental results suggested that strain CY01 can utilize ferrihydrite, goethite, lepidocrocite or hematite as the terminal electron acceptor and citrate, glycerol, glucose or sucrose as the electron donor. Strain CY01 could transform 2,4-D to 4-chlorophenol through reductive side-chain removal and dechlorination. Under the anaerobic conditions, Fe(III) reduction and 2,4-D biodegradation by strain CY01 occurred simultaneously. The presence of Fe(III) (hydr)oxides would significantly enhance 2,4-D biodegradation, probably due to the fact that the reactive mineral-bound Fe(II) species generated from Fe(III) reduction can abiotically reduce 2,4-D. This is the first report of a strain of C. koreensis capable of reducing Fe(III) (hydr)oxides and 2,4-D, which extends the diversity of iron-reducing bacteria associated with dechlorination.     

Carbohydrate oxidation coupled to Fe(III) reduction, a novel form of anaerobic metabolism

An isolate, designated GC-29, that could incompletely oxidize glucose to acetate and carbon dioxide with Fe(III) serving as the electron acceptor was recovered from freshwater sediments of the Potomac River, Maryland. This metabolism yielded energy to support cell growth. Strain GC-29 is a facultatively anaerobic, gram-negative motile rod which, in addition to glucose, also used sucrose, lactate, pyruvate, yeast extract, casamino acids or H2 as alternative electron donors for Fe(III) reduction. Stain GC-29 could reduce NO3(-), Mn(IV), U(VI), fumarate, malate, S2O3(2-), and colloidal S0 as well as the humics analog, 2,6-anthraquinone disulfonate. Analysis of the almost complete 16S rRNA sequence indicated that strain GC-29 belongs in the Shewanella genus in the epsilon subdivision of the Proteobacteria. The name Shewanella saccharophilia is proposed. Shewanella saccharophilia differs from previously described fermentative microorganisms that metabolize glucose with the reduction of Fe(III) because it transfers significantly more electron equivalents to Fe(III); acetate and carbon dioxide are the only products of glucose metabolism; energy is conserved from Fe(III) reduction; and glucose is not metabolized in the absence of Fe(III). The metabolism of organisms like S. saccharophilia may account for the fact that glucose is metabolized primarily to acetate and carbon dioxide in a variety of sediments in which Fe(III) reduction is the terminal electron accepting process.     

Reduction of diverse electron acceptors by Aeromonas hydrophila

Aeromonas hydrophila ATCC 7966 grew anaerobically on glycerol with nitrate, fumarate, Fe(III), Co(III), or Se(VI) as the sole terminal electron acceptor, but did not ferment glycerol. Final cell yields were directly proportional to the amount of terminal electron acceptor provided. Twenty-four estuarine mesophilic aeromonads were isolated; all reduced nitrate, Fe(III), or Co(III), and five strains reduced Se(VI). Dissimilatory Fe(III) reduction by A. hydrophila may involve cytochromes. Difference spectra obtained with whole cells showed absorption maxima at wavelengths characteristic of c-type cytochromes (419, 522, and 553 nm). Hydrogen-reduced cytochromes within intact cells were oxidized by the addition of Fe(III) or nitrate. Studies with respiratory inhibitors yielded results consistent with a respiratory chain involving succinate (flavin-containing) dehydrogenase, quinones and cytochromes, and a single Fe(III) reductase. Neither anaerobic respiration nor dissimilatory metal reduction by members of the genus Aeromonas have been reported previously. Received: 24 June 1997 / Accepted: 20 October 1997     

Microbial Communities Involved in Fe Reduction and Mobility During Soil Organic Matter (SOM) Mineralization in Two Contrasted Paddy Soils

Lowland rice fields of West Africa (Ivory Coast) and South Asia (Thailand) are affected by ferrous toxicity or salinity, respectively, and their soil waters contain large amounts of ferrous iron, depending on reducing irrigation condition and suggesting occurrence of bacterial reducing processes. To determine the involvement, dynamic and activities of bacterial communities in Fe(III) reduction and mobilization during anaerobic degradation and mineralization of soil organic matter (SOM), different experiments and analyses have been performed. Results demonstrated that the utilization of SOM as sole carbon, nutrient and energy sources favored the presence of large bacterial communities: facultative anaerobic and anaerobic bacteria, Fe(III)-reducing bacteria (FeRB) (fermentative and Fe respiring), sulfate reducing bacteria (SRB) which are involved in carbon, nitrogen, iron and sulfur cycling. The larger functional diversity is observed in the Ivory Coast paddy soils containing larger amounts of organic matter and sulfur compounds. These communities contained complementary populations (chemoorganotrophic, chemolitotrophic, aerobic, facultative anaerobic and anaerobic) that can be active at different steps of iron solubilization with simultaneous organic matter mineralization. Our results indicate that the pH controlled by bacterial activity, the nature much more than the content of organic matter, and consequently the structure and activity of bacterial communities influence significantly the availability and dynamic of iron in paddy fields which affect the soil quality.     

Review: microbial mechanisms of accessing insoluble Fe(III) as an energy source

Of all the terminal electron acceptors, Fe(III) is the most naturally abundant in many subsurface environments. Fe(III)-reducing microorganisms are phylogenetically diverse and have been isolated from a variety of sources. Unlike most electron acceptors, Fe(III) has a very low solubility and is usually present as insoluble oxides at neutral pH. The mechanisms by which microorganisms access and reduce insoluble Fe(III) are poorly understood. Initially, it was considered that microorganisms could only reduce insoluble Fe(III) through direct contact with the oxide. However, recent studies indicate that extracellular electron shuttling or Fe(III)-chelating compounds may alleviate the need for cell–oxide contact. These include microbially secreted compounds or exogenous electron shuttling agents, mainly from humic substances. Electron shuttling via humic substances is likely a significant process for Fe(III) reduction in subsurface environments. This paper reviews the various mechanisms by which Fe(III) reduction may be occurring in pure culture and in soils and sediments.