1株滩涂沉积物反硝化细菌的鉴定及其性能研究

利用富集培养法从江苏沿海滩涂沉积物中分离筛选获得1株具有反硝化性能的细菌,命名为MD5。通过形态学观察、糖发酵及16S r DNA序列分析对其进行了分类鉴定、系统发育地位及反硝化性能等方面的研究。结果表明,该菌株能在厌氧条件下利用硝酸钠进行反硝化反应,在初始硝酸盐质量浓度为1 500 mg/L、30℃培养条件下,144 h后培养基中硝酸盐脱除率可达90.1%;在好氧培养条件下,该菌株可使能力测试管中硝酸盐质量浓度下降89.3%。菌株MD5的形态学、糖发酵结果与盐单胞菌一致,且基因序列与盐单胞属(Halomonas)细菌Halomonas sp.IW11-1(AB305262.1)的序列同源性达到99%,表明菌株MD5为1株盐单胞属细菌。本研究分离得到的菌株MD5在厌氧和好氧条件下均具有较强的反硝化能力,对海洋沉积物脱氮具有一定的意义。     

好氧反硝化细菌及其在微污染水源水修复中的应用研究进展

相比于氨氮,天然水体中的硝酸盐氮通常更稳定,导致更难将其从水中去除。由于好氧反硝化可以在有氧环境下进行反硝化作用去除硝酸盐氮,该过程对含有较高溶解氧的天然水体中硝酸盐氮处理有重要作用。本文综述了好氧反硝化菌的分离纯化现状、微生物代谢机制和环境影响因子,并介绍了功能菌群在微污染饮用水源水生物修复的应用研究进展。与一般的厌氧反硝化类似,好氧反硝化菌的种属分布较广,常见的如假单胞菌属(Pseudomoas)、产碱杆菌属(Alcaligenes)、副球菌属(Paracoccus)和芽孢杆菌属(Bacillus)等所属部分微生物均有好氧反硝化能力。大部分好氧反硝化菌株在最佳生长条件下(25–37℃、溶解氧浓度为3–5mg/L、pH为7–8、碳氮比为5–10)具有高效的脱氮效率。但目前好氧反硝化作用在微污染饮用水源水的生物修复方面的应用仍有着脱氮性能不稳定、菌剂流失等不足。此外,目前较少相关中试及实际工程应用的研究,需要进一步的深入探究。     

异养硝化-好氧反硝化菌脱氮相关酶系及其编码基因的研究进展

水体氮素污染日益严重,如何经济、高效地去除水体氮素已成为研究热点。近年来,研究人员已从不同环境中分离到许多同时具有异养硝化和好氧反硝化功能的菌株,此类菌生长迅速,可在好氧条件下同时实现硝化和反硝化的过程,并可用于脱除有机污染物,是一类应用潜力巨大的脱氮菌。目前,异养硝化-好氧反硝化菌的脱氮途径和机制主要是通过测定氮循环中间产物或终产物、测定相关酶活性、注释部分氮循环相关基因及参考自养硝化菌和缺氧反硝化菌的氮循环途径等进行研究,其完整的氮素转化途径和氮代谢机制还需要进一步明确。总结了目前异养硝化-好养反硝化菌的脱氮相关酶系及其编码基因的研究进展,以期为异养硝化-好氧反硝化菌的理论研究及其在污水脱氮处理上的应用提供参考。     

耐盐好氧反硝化菌A-13菌株的分离鉴定及其反硝化特性

[目的]筛选高效好氧脱氮的反硝化细菌,对菌株进行多项鉴定及条件优化,为后续富营养化人工湖水体治理提供理论依据.[方法]利用反硝化培养基分离筛选好氧反硝化细菌,通过形态、生理生化、16S rRNA基因序列分析、周质硝酸还原酶亚基基因( napA)同源性分析进行菌株鉴定;通过反硝化培养基,对菌株生长及反硝化的最适pH、温度、碳源、溶解氧、接种量等进行了考察.[结果]从福州市闽侯县上街镇高岐村某排污口分离出1株耐盐高效好氧反硝化细菌A-13,多项鉴定表明该菌株为Pseudomonas stutzeri,与Pseudomonas stutzeri DSM 50283亲缘关系最近.菌株生长及反硝化的最适pH为6.5,最适温度为33℃,最适碳源为丁二酸钠,最适摇床转速为150 r/min,最适接种量为5％.在此条件下,最大可去除NO3-浓度约为1900 mg/L.该菌能够在高盐培养基( 10％ NaCl)中良好生长.对人工废水的净化效果表明,该菌具有一定的工程应用价值.[结论]分离所得好氧反硝化细菌为Pseudomonas stutzeri,将其命名为P.stutzeri YHA-13.具备高耐盐性的好氧反硝化功能的P.stutzeri未见报道.这对含盐废水/富营养化水体的工程应用有一定的潜在价值.     

城市河道污水中好氧反硝化菌的分离鉴定与特性

采用富集培养和BTB(溴百里酚蓝)平板法从城市河道污水中筛选、分离获得了一株高效的好氧反硝化菌株ADZ1, 48 h内对硝酸盐的降解率为93.1%, 总氮的去除率为34.7%。16S rRNA测序及系统发育分析结果表明该菌株属于Pseudamonas sp., 经VITEK? 2系统鉴定为Pseudomonas putida。对该菌株的反硝化特性进行了研究, 结果表明, 该菌株以乙醇为最佳碳源, 在碳氮比达到12:1时, 对硝酸盐的去除率达到98%以上, 总氮去除率达到41.3%。该菌株对溶解氧、pH有着广泛的适应性, 菌体活力强, 有着良好的应用前景。     

一株耐盐的卓贝儿氏菌(Zobellella sp.)的分离鉴定及其硝酸盐还原能力

【背景】好氧反硝化是指在有氧条件下进行反硝化作用,使得硝化和反硝化过程能够在同一反应器中同时发生,是废水脱氮最具竞争力的技术。红树林湿地中蕴藏着丰富的微生物资源,分布着大量好氧反硝化微生物。【目的】了解耐盐微生物的脱氮机制,为含盐废水生物脱氮的工程实践提供理论依据,对一株分离于红树林湿地中的耐盐好氧细菌A63的硝酸盐异化还原能力进行分析。【方法】利用形态学特征及16S rRNA基因序列测定分析,对其种属进行了鉴定,采用单因子实验测定该菌在不同环境因子下的硝酸盐还原能力,并对其反硝化脱氮条件进行了优化。【结果】初步判定该菌株为卓贝儿氏菌(Zobellellasp.),其能在盐度0%-10%、pH5.0-10.0、温度20-40°C范围内进行反硝化脱氮和硝酸盐异化还原为氨(dissimilatorynitratereductiontoammonium,DNRA)作用。菌株A63最适生长碳源为柠檬酸钠(1.2 g/L),适宜脱氮盐度为3%、pH 7.0-7.5、温度30-35°C,且C/N为10。在最适脱氮条件下,该菌株12h内能将培养基中208.8mg/L硝态氮降至0,且仅有少量铵态氮生成,无亚硝态氮积累,脱氮率高达99%。此外,该菌株在高盐度、低C/N比、弱酸性和低温等不利生境中DNRA作用显著。【结论】细菌A63生长范围宽,脱氮效率显著,适用于海水养殖废水处理。研究为今后开发高效含盐废水生物脱氮工艺奠定了基础,对于加深氮素转化规律的认识、丰富生物脱氮理论有着重要意义。     

深海沉积物高效脱氮菌筛选分离及其脱氮特性研究

【背景】深海海域具有高压、低温、无光等环境条件,蕴含着丰富而独特的微生物资源。【目的】从深海沉积物中定向分离、筛选脱氮效率高的好氧脱氮菌株资源,并揭示其脱氮特性,为开发水体脱氮微生物技术提供物质基础。【方法】以东太平洋、南大西洋、西南印度洋共10个站位的深海沉积物为研究材料,在28°C下使用无机氮源连续进行两轮富集培养,然后定性筛选可以脱除氨氮、亚硝态氮和硝态氮的菌株,并通过形态学和16S rRNA基因序列分析进行初步分类鉴定;对优选得到的功能菌株,分别采用以氨氮、亚硝态氮、硝态氮为唯一氮源的培养基定量研究其生长和脱氮性能。【结果】从10份大洋深海沉积物样品中共分离得到49株好氧反硝化菌,其中3株在有氧条件下反硝化效率较高,分别命名为Pseudomonassp.G111、Pseudomonassp.G112和Dietziamaris W023a,其中菌株G111和G112与模式菌株博岑假单胞菌Pseudomonas bauzanensis BZ93T的16S rRNA基因序列相似度为99.2%,菌株W023a与模式菌株海洋迪茨氏菌DietziamarisATCC35013T的16SrRNA基因序列相似度为99.9%。菌株G111、G112和W023a培养48h后,对氨氮的脱除率分别为98.0%、85.2%和97.6%;对亚硝态氮的脱除率分别为71.9%、67.5%和34.7%;对硝态氮的脱除率分别为66.0%、52.6%和56.3%。菌株G111、G112和W023a均为异养硝化-好氧反硝化菌,可通过好氧反硝化作用将亚硝态氮和硝态氮还原为含氮气体,也可通过异养硝化-好氧反硝化作用将氨氮转化为含氮气体。【结论】从深海沉积物中分离筛选得到3株高效好氧反硝化菌,所获得的菌株在水体净化、污水处理、生态系统修复等领域具有应用潜力。     

一株异养硝化-反硝化不动杆菌的分离鉴定及脱氮活性

[目的]分离筛选并鉴定一株异养硝化-反硝化细菌,并探讨其在脱氮中的作用.[方法]富集培养分离筛选微生物,通过形态观察和生理生化特征及16S rDNA鉴定细菌,定时测定其OD600研究生长曲线,正交试验研究其脱氮影响因素和最佳条件,与污水处理厂活性污泥共同作用检验其脱氮活性.[结果]分离到一株异养硝化-反硝化细菌,鉴定结果表明是一株不动杆菌,命名为Acinetobacter sp.YF14,这是已知报道的第一株进行异养硝化和好氧反硝化的不动杆菌.该菌在12 h时进入对数期,22 h时进入稳定期,45 h以后进入衰亡期.该菌能进行异养硝化,3d后氨氮和总氮的去除率可以达到92％和91％,且无硝酸盐氮和亚硝酸盐氮积累.好氧条件下该菌能进行反硝化,在硝酸盐和亚硝盐培养基中均能将氮几乎完全去除.对该菌脱氮的影响程度大小依次为转速＞接种量＞碳源＞碳氮比＞ pH.当转速为160 r/min,碳源取葡萄糖,接种量1％,碳氮比为8∶1,pH为6.5时,脱氮效果最好.该菌株可以提高活性污泥对于生活污水总氮脱除率约30％.[结论]菌YF14可以明显加强活性污泥脱氮效果,显示了良好的应用前景.     

一株好氧反硝化菌的分离鉴定及其除氮特性

【目的】生物除氮中反硝化菌具有重要的作用,需氧反硝化菌研究较少,有着很好的应用潜力,本研究主要从环境样品中分离具有高效去除铵氮和亚硝酸盐氮活性的好氧反硝化菌,并对其分类及除氮特性进行研究。【方法】以高效去除铵氮、除亚硝酸盐氮和好氧反硝化能力为主要指标,从富营养化的池塘淤泥水和工厂污泥样品中进行菌株分离筛选。通过生理生化特点以及16S rRNA序列分析对活性最好的菌株进行初步鉴定。在好氧条件下,分别以NO-3-N、NH+4-N和NO-2-N作为唯一氮源,考察菌株的好氧反硝化特性、去除铵氮和亚硝酸盐氮特性,以及不同初始pH值、温度、碳源、摇床转速对该菌去除铵氮和亚硝酸盐氮特性的影响。【结果】得到的细菌中,以菌株C-4的活性最好,其16S rRNA序列与不动杆菌的同源性达99%,结合生理生化特点,初步确定菌株C-4属于不动杆菌属(Acinetobacter sp.)。以柠檬酸钠作为碳源,30℃、120 r/min振荡培养,种龄为18 h,用初始pH为8.5的200 mg/L NH +4-N培养基和初始pH为7.5的100 mg/L NO -2-N培养基进行测定,分别培养15 h与12 h,净除氮率分别达到65.8%和47.8%。【结论】从鱼塘水样中分离到一株好氧反硝化菌C-4,初步鉴定为不动杆菌属的一个种(Acinetobacter sp.),具有较高的反硝化特性和高效去除铵氮与亚硝酸盐氮的能力,在处理实际池塘污水时中,净除氮率可达73.04%以上。     

Zn(II)对好氧反硝化菌Acinetobacter sp. JR-142的代谢活性影响

【目的】研究不同Zn(Ⅱ)浓度对好氧反硝化菌Acinetobacter sp.JR-142生理活性,尤其是反硝化代谢特性的影响。【方法】筛选一株好氧反硝化菌,优化了最佳活性条件;分析了不同Zn(Ⅱ)浓度对生长曲线和反硝化效率的影响以及对细胞形态的影响;明晰了不同Zn(Ⅱ)浓度条件下,细胞特征活性酶-硝酸盐还原酶和亚硝酸盐还原酶的活性变化情况,分析了不同活性同关键酶的编码基因napA和nirS的相对表达量之间的规律。【结果】获得一株具有好氧反硝化功能的菌株,命名为JR-142,经鉴定为不动杆菌Acinetobacter sp.。在以琥珀酸钠为碳源,C/N为6,pH为7.0,温度30℃,转速为180 r/min的条件下,好氧反硝化活性最高。结果表明当Zn(Ⅱ)浓度为3.25 mg/L时,对菌株的生长及好氧反硝化速率有促进作用;当Zn(Ⅱ)浓度为52 mg/L以上浓度时,菌株的生长及反硝化速率均受到抑制。酶活及关键基因napA、nirS的定量分析结果显示,对照组及JR+0.05处理组的硝酸盐还原酶NR、亚硝酸盐还原酶NiR活性均高于JR+0.8处理组,在24 h时,JR+0.05 Zn(Ⅱ)处理组中,细胞的关键好氧反硝化基因napA及nirS的相对表达量显著高于对照组,这进一步说明3.25 mg/L Zn(Ⅱ)可以促进好氧反硝化过程,而在24 h及32 h时对照组及JR+0.05处理组的基因相对表达量远高于JR+0.8处理组,也说明52 mg/L Zn(Ⅱ)则会对反应产生抑制。【结论】探究并系统分析了不同Zn(Ⅱ)浓度对不动杆菌Acinetobacter sp.JR-142生长繁殖以及和在重金属锌离子存在的情况下影响好氧反硝化生理活性的影响,为后续硝酸盐-重金属复合污染废水的生物处理技术提供了数据指导。     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review

Bioremediation of aromatic hydrocarbons in groundwater and sediments is often limited by dissolved oxygen. Many aromatic hydrocarbons degrade very slowly or not at all under anaerobic conditions. Nitrate is a good alternative electron acceptor to oxygen, and denitrifying bacteria are commonly found in the subsurface and in association with contaminated aquifer materials. Providing both nitrate and microaerophilic levels of oxygen may result in oxidation of the stable benzene rings in aromatic contaminants and allow for the intermediates of this oxidation to degrade via denitrification. The effects of using mixed electron acceptors on biodegradation of subsurface contaminants is unclear. Below some critical oxygen threshold, aerobic biodegradation is inhibited, however high levels of oxygen inhibit denitrification. The mechanisms which regulate electron transfer to oxygen and nitrate are complex. This review: 1) describes the factors which may affect the utilization of oxygen and nitrate as dual electron acceptors during biodegradation; 2) summarizes the incidence of dual use of nitrate and oxygen (aerobic denitrification); and 3) presents evidence of the effectiveness of bioremediation under mixed oxygen/nitrate conditions. Received 08 November 1995/ Accepted in revised form 09 June 1996     

Denitrification Potential, Root Biomass, and Organic Matter in Degraded and Restored Urban Riparian Zones

Hydrologic changes associated with urbanization often lead to lower water tables and drier, more aerobic soils in riparian zones. These changes reduce the potential for denitrification, an anaerobic microbial process that converts nitrate, a common water pollutant, into nitrogen gas. In addition to oxygen, denitrification is controlled by soil organic matter and nitrate. Geomorphic stream restorations are common in urban areas, but their effects on riparian soil conditions and denitrification have not been evaluated. We measured root biomass, soil organic matter, and denitrification potential (anaerobic slurry assay) at four depths in duplicate degraded, restored, and reference riparian zones in the Baltimore, Maryland, U.S.A., metropolitan area. There were three main findings in this study. First, although reference sites were wet and had high soil organic matter, they had low levels of nitrate relative to degraded and restored sites and therefore there were few differences in denitrification potential among sites. Evaluations of riparian restorations that have nitrate removal by denitrification as a goal should consider the complex controls of this process and how they vary between sites. Second, all variables declined markedly with depth in the soil. Restorations that increase riparian water tables will thus foster interaction of groundwater nitrate with near-surface soils with higher denitrification potential. Third, we observed strong positive relationships between root biomass and soil organic matter and between soil organic matter and denitrification potential, which suggest that establishment of deep-rooted vegetation may be particularly important for increasing the depth of the active denitrification zone in restored riparian zones.     

Continuous rhamnolipid production using denitrifying Pseudomonas aeruginosa cells in hollow‐fiber bioreactor

Rhamnolipids are high‐value effective biosurfactants produced by Pseudomonas aeruginosa. Large‐scale production of rhamnolipids is still challenging especially under free‐cell aerobic conditions in which the highly foaming nature of the culture broth reduces the productivity of the process. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. A coupled system using immobilized cells and nitrate instead of oxygen as electron acceptor taking advantage of the ability of P. aeruginosa to perform nitrate respiration was evaluated. This denitrification‐based immobilized approach based on a hollow‐fiber setup eliminated the transfer limitation problems and was found suitable for continuous rhamnolipid production in a period longer than 1,500 h. It completely eliminated the foaming difficulties related to aerobic systems with a comparable specific productivity of 0.017 g/(g dry cells)‐h and allowed easy recovery of rhamnolipids from the cell‐free medium. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013     

Effect of oxygen on denitrification in continuous chemostat culture withComamonas sp SGLY2

Continuous cultures ofComamonas sp SGLY2 were grown anaerobically prior to establishing steady states at different oxygen flow rates. At a low oxygen transfer rate, no dissolved oxygen accumulated in the medium and all nitrate was reduced to dinitrogen. Concurrently with the increase of dissolved oxygen concentration in the liquid phase, the rate of denitrification decreased. However, at a dissolved oxygen concentration near saturation (33 mg L^–1), a part of the electron flow always diverted to nitrate with production of dinitrogen: the aerobic denitrification rate was equivalent to 35% of that calculated under anaerobic conditions. These experiments reflected the co-utilization of oxygen and N-oxides and the production of dinitrogen, up to saturated conditions, which implied synthesis and activity of the four denitrifying enzymes under various aeration conditions.     

Aerobic denitrification: a controversy revived

During studies on the denitrifying mixotroph, Thiosphaera pantotropha, it has been found that this organism is capable of simultaneously utilizing nitrate and oxygen as terminal electron acceptors in respiration. This phenomenon, termed aerobic denitrification, has been found in cultures maintained at dissolved oxygen concentrations up to 90% of air saturation.The evidence for aerobic denitrification was obtained from a number of independant experiments. Denitrifying enzymes were present even in organisms growing aerobically without nitrate. Aerobic yields on acetate were higher (8.1 g protein/mol) without than with (6.0 g protein/mol) nitrate, while the anaerobic yield with nitrate was even lower (4 g protein/mol). The maximum specific growth rate of Tsa. pantotropha was higher (0.34 h^-1) in the presence of both oxygen (>80% air saturation) and nitrate than in similar cultures not supplied with nitrate (0.27 h^-1), indicating that the rate of electron transport to oxygen was limiting. This was confirmed by oxygen uptake experiments which showed that although the rate of respiration on acetate was not affected by nitrate, the total oxygen uptake was reduced in its presence. The original oxygen uptake could be restored by the addition of denitrification inhibitors.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

细菌好氧反硝化研究进展

阐述了好氧反硝化细菌的种类及有关性质 ,并从电子理论、氧的浓度、反硝化酶系等方面对细菌好氧反硝化的作用机制进行了探讨 ,对细菌好氧反硝化的研究概况、进展及其研究意义和目前存在的问题作了较为详细的介绍。     

Soil and sediment bacteria capable of aerobic nitrate respiration.

Several laboratory strains of gram-negative bacteria are known to be able to respire nitrate in the presence of oxygen, although the physiological advantage gained from this process is not entirely clear. The contribution that aerobic nitrate respiration makes to the environmental nitrogen cycle has not been studied. As a first step in addressing this question, a strategy which allows for the isolation of organisms capable of reducing nitrate to nitrite following aerobic growth has been developed. Twenty-nine such strains have been isolated from three soils and a freshwater sediment and shown to comprise members of three genera (Pseudomonas, Aeromonas, and Moraxella). All of these strains expressed a nitrate reductase with an active site located in the periplasmic compartment. Twenty-two of the strains showed significant rates of nitrate respiration in the presence of oxygen when assayed with physiological electron donors. Also isolated was one member of the gram-positive genus Arthrobacter, which was likewise able to respire nitrate in the presence of oxygen but appeared to express a different type of nitrate reductase. In the four environments studied, culturable bacteria capable of aerobic nitrate respiration were isolated in significant numbers (10(4) to 10(7) per g of soil or sediment) and in three cases were as abundant as, or more abundant than, culturable bacteria capable of denitrification. Thus, it seems likely that the corespiration of nitrate and oxygen may indeed make a significant contribution to the flux of nitrate to nitrite in the environment.     

关于好氧反硝化菌筛选方法的研究

采用污泥驯化手段富集好氧反硝化细菌,将得到的驯化污泥分离纯化,共得到105株菌。用测TN的方法对所筛菌株进行初筛,得到25株对TN去除率达到50％以上的菌株。用氮元素轨迹跟踪测定法复筛,证实这25株菌都可以在好氧条件下进行硝酸盐呼吸,其中24株菌的反硝化过程为：NO3^-N→NO2^-N→N2,研究中还发现在反硝化过程中硝酸盐和亚硝酸盐不存在明显竞争被利用的作用。同时还提出了可能实现短程同步硝化反硝化以及在反馈作用的调节下,加快硝化反应速度的观点。