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...     

微生物介导铁还原耦合氨氧化过程的研究进展

铁的氧化还原过程可以显著影响环境中次生矿物的形成、养分转化和污染物的归趋。作为厌氧环境中新发现的铁循环过程,铁氨氧化过程对自然和农田生态系统中氨氧化的贡献可达10%以上,对环境保护和农业生产具有深远的意义。文章主要从发展历程、相关微生物、反应机制、影响因素和环境意义等方面综述了铁氨氧化过程。在此过程中,Acidimicrobiaceaesp.A6和异化铁还原菌(DIRB)是驱动铁氨氧化过程的关键微生物,环境pH、Fe(Ⅲ)的浓度和种类、碳源和Mn(Ⅳ)氧化物是重要环境影响因子。铁氨氧化过程可能由微生物独立驱动完成,也可能由微生物-化学耦合作用驱动完成。从环境意义看,铁氨氧化过程对减少温室气体排放、固定重金属等方面具有积极影响,但也会导致氮素流失等负面环境效应。后续的研究可以从纯化微生物、拓展研究方法等方面着手,进一步提升铁氨氧化过程的研究广度和深度。     

海水养殖生境中硫酸盐还原菌活性的抑制机制

【目的】海水养殖生境中的硫化物(H₂S)严重损害养殖生物健康,控制该条件下硫酸盐还原菌(sulfate-reducing bacteria,SRB)的代谢活性是有效抑制H₂S产生的重要途径。【方法】本研究利用稀释涂布-叠皿夹法对海水养殖生境底泥中SRB进行富集筛选,获得SRB菌株,通过投加硝酸盐对菌株产H₂S的活性进行抑制。【结果】获得的2株SRB Desulfovibrio sp.NY-1和Clostridium sp.NH-1,能够在35℃、pH为7.0及盐度为20–30 mg/L条件下,分别积累高达435和150 mg/L H₂S。硝酸盐不能有效抑制NY-1产H₂S的活性,基因调控作用以及缺乏将硝酸盐作为电子受体的酶体系是其不能被抑制的主要原因。硝酸盐对NH-1 H₂S产生活性有可逆性抑制,其具有硝酸盐异化还原成铵(dissimilatory nitrate reduction to ammonium,DNRA)的能力,优先利用硝酸盐作为电子受体。DNRA作用下的中间代谢产物亚硝酸盐是有效抑制菌株NH-1产H₂S活性的主要原因,其抑制机理主要为抑制菌株的生长繁殖。【结论】硝酸盐对不同SRB菌株具有不同的抑制机制和效果,在进行硫化物污染控制前需要对产生硫化物的SRB菌群进行分析判别。     

一株兼性氧化亚氮还原菌的还原N2O能力

【目的】从水稻土中分离筛选出一株兼性氧化亚氮还原菌,并探索其在不同条件下还原N_2O的能力,为减少温室气体N_2O的排放提供重要依据。【方法】通过微生物富集培养分离技术从水稻土中分离得到纯菌;利用nosZ基因和16S rRNA的测序分析鉴定菌株;通过测定菌株在不同条件下N_2O的还原量,分析该菌株还原N_2O的能力及调控因子。【结果】经鉴定,该菌株含有nos Z基因,属于假单胞菌属,在温度30°C、厌氧条件下还原N_2O速率高达0.0219μmol/min以上,改变不同温度和氧气浓度后其能力相对减弱,但仍具备较强的还原N_2O作用。【结论】从水稻土中分离筛选得到的兼性氧化亚氮还原菌为假单胞菌,它在不同环境条件下都具备较强的还原N_2O能力,该菌株可能为减少土壤N_2O排放提供新途径,对保障生态环境安全具有重要的应用价值。     

硝酸盐诱导的GARP类转录因子AhNIGT1.2调控花生结瘤的功能分析

花生是重要的经济及油料作物,富含丰富的蛋白质和油脂等,在我国广泛种植。花生能与根瘤菌共生形成根瘤,根瘤的固氮酶能将空气中N₂转化为自身可吸收利用的氨态氮。解析结瘤固氮对避免化肥过度施用、发展可持续农业具有积极意义。本研究通过生物信息学分析鉴定到一个主要在花生根瘤中表达的NIGT家族成员AhNIGT1.2。进一步的时空表达分析表明AhNIGT1.2在根瘤中高表达,且显著响应高氮、低氮、高磷、低磷和根瘤菌处理,组化染色表明该基因主要在发育的根瘤中表达,并在成熟根瘤中和花生根系连接处表达。亚细胞定位表明35S::AhNIGT1.2-GFP融合蛋白定位于烟草表皮细胞的细胞核中。进一步的功能研究发现AhNIGT1.2-OE显著提高了花生的结瘤数量,AhNIGT1.2-RNAi显著抑制花生根瘤数量,表明AhNIGT1.2正向调控花生结瘤。对AhNIGT1.2-OE根中硝酸盐转运和信号通路中的基因表达进行分析发现,NRT1.2、NRT2.4、NLP1和NLP7的表达量显著降低,表明AhNIGT1.2可能通过影响硝酸盐转运和NLP1相关基因的表达进而调控花生的结瘤。对转化的AhNIGT1.2-OE和对照根进行转录组分析,发现过表达AhNIGT1.2会显著富集硝酸盐响应、结瘤因子信号通路、四萜类生物合成酶和类胡萝卜素生物合成等途径的差异表达基因。综上所述,AhNIGT1.2可能通过影响硝酸盐转运、硝酸盐响应等通路调控花生的结瘤过程。本研究对解析氮磷营养调控豆科植物结瘤固氮的分子机制和创制在高氮环境高效结瘤固氮的豆科作物具有重要意义。     

浅表层水稻土N2O消耗能力及其与N2O还原微生物的耦合关系

土壤不仅能够产生、排放温室气体N_2O,还具有截留、吸收、转化N_2O的能力。土壤消耗N_2O已经成为很重要的一种降低大气N_2O浓度的途径,但目前关于土壤N_2O消耗过程及其微生物调控机制的系统研究较为缺乏。试验以浅表层水稻土柱(0—5 cm)为研究对象,通过外源添加N_2O气体研究N_2O迁移通过淹水土柱的动态过程,以及N_2O消耗能力与氧化亚氮还原酶基因丰度变化和其他土壤养分含量变化的联系,揭示浅表层水稻土N_2O消纳量与N_2O还原微生物之间的耦合关系。结果显示,淹水厌氧条件下5 cm土壤深度外源添加的N_2O迁移通过浅表层土柱后,仅有7.17—9.80%部分逸散出土表,表明0—5 cm淹水水稻土层具有极强的N_2O截留能力(90%以上)而减少N_2O净排放量。排放出土表的N_2O也可被淹水土柱继续吸收消耗,且吸收转化速率随N_2O浓度增加而大幅提高,最高可达到3896.75μg N m~(-2) h~(-1)。与此同时,土壤DOC含量大量消耗,含nosZⅠ基因的反硝化微生物数量显著增长(P0.01),而nosZⅡ基因丰度的无显著变化。说明高浓度N_2O添加能够促进淹水土壤N_2O吸收消耗能力,此刺激作用可能主要由含nosZⅠ基因的N_2O还原微生物进行调控。浅表层土壤强大的N_2O吸收消耗功能可进一步深入系统研究,为实践温室气体减排提供理论基础。     

微生物厌氧氨氧化耦合铁还原的研究进展及展望

厌氧氨氧化耦合铁还原[ammonium oxidation coupled to Fe(Ⅲ) reduction, Feammox]作为一种连接氮循环和铁循环之间的氮代谢途径,在自然界中氨氮转化过程中起到了重要作用。系统研究Feammox驱动的氮铁的生物地球化学耦合过程及其受控因素,有助于深入理解地球元素循环的微生物机制,也有助于揭示Feammox在缺氧地质历史时期对古海洋氮库演变和含铁矿物形成过程中的作用。本文从Feammox发展历史、相关微生物、影响因素和潜在地质意义等方面综述了Feammox的研究过程和研究内容,并对Feammox的未来研究方向提出展望。     

硝酸盐型厌氧铁氧化菌的种类、分布和特性

硝酸盐型厌氧铁氧化(NAFO)是指微生物在厌氧条件下利用硝酸盐或亚硝酸盐作为电子受体,将低价铁(二价铁或零价铁)氧化为高价铁(三价铁)的过程。具有NAFO代谢能力的微生物称为硝酸盐型厌氧铁氧化菌(NAFOM)。NAFO是微生物领域的重大发现,也是环境领域开发新型脱氮技术和地学领域研究铁、氮循环的理论依据。整理文献报道的NAFOM资料,分析NAFOM系统发育性状,探讨典型NAFOM的生态分布及其营养、代谢特性,以期为NAFOM菌种资源的开发、地球铁素和氮素循环的研究、NAFO过程的优化提供借鉴。     

重金属胁迫对Pseudomonas alcaligenes LH7抗生素抗性的影响

从广州某养猪场废水处理系统中筛选出1株优势菌Pseudomonas alcaligenes LH7。为了研究重金属胁迫对细菌抗生素抗性响应的影响,采用琼脂稀释法和K-B纸片扩散法,测定了重金属(Cu2+、Zn2+、Cr6+)的最小抑制浓度(MIC),及不同重金属种类和浓度胁迫下,四种抗生素(红霉素、阿莫西林、头孢拉定、四环素)的抑菌圈直径。结果表明:菌体对Cu2+、Zn2+、Cr6+的MIC分别为125、125、100 mg/L,并且具有四环素、阿莫西林、红霉素和头孢拉定多重抗性。重金属与抗生素之间的交互作用对细菌的抗性有显著影响(P0.05)。重金属和抗生素间的交互作用随重金属种类和浓度的不同而改变,可分为三类:低浓度重金属与抗生素共存时表现为协同抗性,高浓度时则表现为协同杀菌,如Cr6+或Zn2+与红霉素,Cu2+与头孢拉定;低浓度重金属与抗生素共存时表现为协同杀菌,高浓度时则表现为协同抗性,如Cr6+或Zn2+与阿莫西林;只与共存重金属种类相关的抗性组合有Cu2+与四环素或阿莫西林或红霉素,Cr6+与头孢拉定。环境中重金属离子的共存将改变抗生素污染物的生态危害和环境行为,并最终影响对应的污染防治技术的开发和应用。     

三峡库区水体中硫氧化菌的群落多样性

【目的】探究三峡库区干流和支流水体硫氧化菌群落组成和多样性及其与水体理化性质的相关性。【方法】于2017年3月在三峡库区支流和干流共选取9个采样点,测量各采样点水体理化参数,针对水体样品的sox B基因进行系统发育分析、聚类分析和多样性分析,并分析水体理化性质与硫氧化菌群落组成和多样性的相关性。【结果】克隆文库分析显示,本研究的三峡库区水体样品中硫氧化菌分属于α-Proteobacteria和β-Proteobacteria纲,其中β-Proteobacteria的硫氧化菌是群落构成主体,在干流和支流的采样点中相对丰度均高于95.6%。聚类分析和主坐标分析(PCo A)显示,各支流采样点之间以及支流样点和其对应干流样点之间的硫氧化菌群落构成差异较大,而干流采样点间硫氧化菌群落构成差异较小。Mantel检验结果显示,硫氧化菌群落组成与水体的温度、盐度、溶氧量、藻含量和p H等理化参数呈显著(P0.05)相关。Pearson相关性分析显示硫氧化菌的群落多样性与藻含量和DIC呈现显著负相关。【结论】三峡库区支流和干流水体中硫氧化菌主要由β-Proteobacteria纲构成,库区干流水体中硫氧化菌群落构成较为相似,而不同支流水体中的硫氧化菌群落构成差异较明显,并且支流和对应干流交汇处的水体中硫氧化菌群落组成差异明显,水体理化性质是导致这一差异的重要原因之一。     

Enumeration of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the root zone of wetland plants: Implications for a rhizosphere iron cycle

Iron plaque occurs on the roots of most wetland and submersed aquatic plant species and is a large pool of oxidized Fe(III) in some environments. Because plaque formation in wetlands with circumneutral pH has been largely assumed to be an abiotic process, no systematic effort has been made to describe plaque-associated microbial communities or their role in plaque deposition. We hypothesized that Fe(II)-oxidizing bacteria (FeOB) and Fe(III)-reducing bacteria (FeRB) are abundant in the rhizosphere of wetland plants across a wide range of biogeochemical environments. In a survey of 13 wetland and aquatic habitats in the Mid-Atlantic region, FeOB were present in the rhizosphere of 92% of the plant specimens collected (n = 37), representing 25 plant species. In a subsequent study at six of these sites, bacterial abundances were determined in the rhizosphere and bulk soil using the most probable number technique. The soil had significantly more total bacteria than the roots on a dry mass basis (1.4 × 10⁹ cells/g soil vs. 8.6 × 10⁷ cells/g root; p < 0.05). The absolute abundance of aerobic, lithotrophic FeOB was higher in the soil than in the rhizosphere (3.7 × 10⁶/g soil vs. 5.9 × 10⁵/g root; p < 0.05), but there was no statistical difference between these habitats in terms of relative abundance (1% of the total cell number). In the rhizosphere, FeRB accounted for an average of 12% of all bacterial cells while in the soil they accounted for < 1% of the total bacteria. We concluded that FeOB are ubiquitous and abundant in wetland ecosystems, and that FeRB are dominant members of the rhizosphere microbial community. These observations provide a strong rationale for quantifying the contribution of FeOB to rhizosphere Fe(II) oxidation rates, and investigating the combined role of FeOB and FeRB in a rhizosphere iron cycle.     

Cryopreservation of anoxygenic phototrophic Fe(II)-oxidizing bacteria

Preservation and storage of microbial stock cultures is desirable since the risk of contamination or loss of living cultures is immanent while over long periods mutations accumulate. Generally, it is rather difficult to preserve photosynthetic bacteria due to their sensitive photosynthetic membranes [1]. Phototrophic Fe(II)-oxidizing bacteria face an additional challenge; since they are exposed to light and Fe(II) during growth, they have to cope with radicals from Fenton reactions of Fe(II)-species, light and water. Therefore, phototrophic Fe(II)-oxidizing strains are thought to be especially susceptible to genetic modifications. Here, we provide a simple and fast protocol using glycerol as cryo-protectant to cryopreserve three strains of anoxygenic phototrophic Fe(II)-oxidizing bacteria from different taxa: α-proteobacteria, γ-proteobacteria and chloroflexi. All three strains investigated could be revived after 17 months at −72 °C. This suggests that a long-term storage of phototrophic Fe(II)-oxidizing strains is possible.     

Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317

Pseudomonas stutzeri strain 1317 was found to grow on various fatty acids, alcohols, diols, as well as glucose and gluconate for the synthesis of polyhydroxyalkanoates (PHA) with various monomer units. The PHA monomer structures were dependent on the type of fatty acids and alcohols, as well as the diols in the culture media. Only even number monomers, such as 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD), were accumulated when even numbered fatty acids, alcohols, glucose and gluconate, as well as diol were used as carbon sources. Odd numbered fatty acids and odd numbered alcohols led to the formation of odd numbered monomers, such as 3-hydroxyvalerate (HV), 3-hydroxyheptanoate (HHp), 3-hydroxynonanoate (HN) and 3-hydroxyundecanoate (HU). The strain tolerated up to 1.5% of ethanol and made 8.3% of PHA when growth was conducted in 1.2% of ethanol. PHA formed up to 77% of cell dry weight when the strain was grown in tridecanoate. PHA synthesis was highly dependent on the nitrogen source. A depletion in nitrogen supply immediately resulted in PHA accumulation in cells grown in the glucose mineral medium.     

Natural transformation of Pseudomonas stutzeri by plasmids that contain cloned fragments of chromosomal deoxyribonucleic acid

Natural transformation of plasmids by Pseudomonas stutzeri was found to depend on their bearing inserts of chromosomal DNA. A set of plasmids derived from the nonconjugative broad host range plasmid pSa151 was constructed by integrating various chromosomal DNA fragments into the single EcoR1 site of pSa151. Selection for the kanamycin resistance determined by pSa151 demonstrated that the derivative plasmids were taken into the cells by natural transformation and stably maintained; each could be reisolated unchanged. Thirty-two different derivative plasmids, 14 to 31 kbase pairs in size, all transformed. The frequency of transformation increased with the size of the chromosomal insert over a twenty fold range. These results suggest that the mechanism of transformation of plasmids by the Gram-negative P. stutzeri is similar to those proposed to operate in Gram-positive bacteria.Dedicated to Prof. Dr. H.-G. Schlegel on the occasion of his 60 th birthday     

铁氧化菌耐砷机制及其砷污染修复应用的研究进展

砷污染作为全球性环境问题已经引起了人们的高度重视。无机砷化合物可与铁氢氧化物络合通过共沉淀作用去除。因此,利用具有砷耐性的铁氧化菌氧化环境中的铁元素去除砷化合物具有潜在的应用前景。目前已有利用铁氧化菌去除环境中砷污染物的报道。用于砷污染修复的铁氧化菌必须有一定的砷耐性才能在含砷环境中行使功能。微生物是否具有砷耐性往往取决于基因,并且不同的菌株具有不同的生理特征,适宜不同砷污染环境的修复。本文通过对8株代表性的铁氧化菌砷耐性基因的总结,阐述其耐砷机制、研究概况及应用前景,以期为铁氧化菌用于除砷新技术的开发提供参考。     

Silver resistance in Pseudomonas stutzeri

Silver resistance was studied in a silver-resistant Pseudomonas stutzeri AG259 strain and compared to a silver-sensitive P. stutzeri JM303 strain. Silver resistance was not due to silver complexation to intracellular polyphosphate or the presence of low molecular weight metal-binding protein(s). Both the silver-resistant and silver-sensitive P. stutzeri strains produced H₂S, with the silver-resistant AG259 strain producing lower amounts of H₂S than the silver-sensitive JM303 strain. However, intracellular acid-labile sulfide levels were generally higher in the silver-resistant P. stutzeri AG259 strain. Silver resistance may be due to formation of silver-sulfide complexes in the silver-resistant P. stutzeri AG259 strain.     

嗜酸氧化亚铁硫杆菌亚铁氧化酶基因分子多态性研究

嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans, A.f）属于化能自养、革兰氏阴性细菌,好氧嗜酸,以氧化亚铁离子或还原态硫化物（S0&S2-）获得能量生长。在其Fe2+氧化系统中,亚铁氧化酶起重要作用。对不同硫化矿区分离得到的5株A.f进行生长动力学和对亚铁氧化活性的对比研究,结果显示不同菌株之间存在表型差异。提取A.f菌株的基因组DNA,设计引物,对亚铁氧化酶基因进行PCR扩增,同时对扩增产物直接进行测序,并同GenBank的参考序列进行比对分析,发现序列相似性在99%～97%之间,分析编码区域发现在第187～195位可能存在一个高变异区：在第187～189位,菌株YTW编码的氨基酸Thr→Pro;而在第193～195位,菌株TK是Met→Asn; 菌株BY则是Met→Ile。另外在位点第219位,所有菌株都是T→C,但编码的氨基酸未发生变化,属于同义密码子。对于编码区上游序列,比对后没有差异;而对于编码区下游序列,经比对发现存在一些差异位点。     

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。     

Detection and characterization of natural transformation in the marine bacterium Pseudomonas stutzeri strain ZoBell

Soil isolates of Pseudomonas stutzeri have been shown previously to acquire genes by natural transformation. In this study a marine isolate, Pseudomonas stutzeri strain ZoBell, formerly Pseudomonas perfectomarina, was also shown to transform naturally. Transformation was detected by the Juni plate method and frequencies of transformation were determined by filter transformation procedures. Maximum frequencies of transformation were detected for three independent antibiotic resistance loci. Transformation frequencies were on the order of 4×10^-5 transformants per recipient, a frequency over 100 times that of spontancous antibiotic resistance. Transfer of antibiotic resistance was inhibited by DNase I digestion. Marine isolates achieved maximum competence 14 h after transfer of exponential cultures to filters on solid media, although lower levels of competence were detected immediately following filter immobilization. Like soil isolates, P. stutzeri strain ZoBell is capable of cell contact transformation, but unlike soil isolates where transformation frequencies are greater for cell contact transformation as compared to transformation with purified DNA, the maximum frequency of transformation achieved by cell contact in the marine strain was approximately 10-fold less than transformation frequencies with purified DNA. These studies establish the first marine model for the study of natural transformation.This paper is dedicated to John L. Ingraham, Professor Emeritus of Microbiology at the University of California, Davis. Professor Ingraham was the first person to recognize natural transformation in Pseudomonas stutzeri and has continued to contribute to our understanding of the process over the past eight years. This understanding of the genetics of P. stutzeri is only one of the many areas of microbiology to which Professor Ingraham has contributed in his exceptional career