SBR和MBR反应器中好氧反硝化菌的筛选与分析

好氧反硝化菌是土壤、沉积物、活性污泥等介质中广泛存在的一类微生物。研究以相同种泥扩大培养的SBR(序批式活性污泥反应器)和MBR(膜生物反应器)中活性污泥为研究对象,采用平板分离法分别从运行6个月的SBR和MBR反应器中分离得到9株和11株好氧反硝化菌。经好氧反硝化性能测定发现,20株细菌中有16株总氮去除率在50%以上,且NO_2~--N累积量均在0.5 mg/L以下。经系统进化分析表明,在门分类级别中,采用同一种泥的2个反应器中好氧反硝化菌表现出极大的一致性,20株好氧反硝化菌全部属于变形菌门(Proteobacteria)。但是,进一步分析发现不同反应器中好氧反硝化菌表现出较大的属、种间的差异。其中,SBR反应器9株好氧反硝化菌分属于4个属,6个物种;而从MBR反应器获得的11株细菌分属于2个属,6个物种;仅1个物种(Pseudomonas toyotomiensis)在2个反应器均有发现。     

滇池可培养好氧反硝化细菌多样性及其脱氮特性

【目的】氮污染已成为当今水体污染的一个重要因素,为了解滇池可培养好氧反硝化细菌的多样性,获得高效好氧反硝化细菌资源,为污染水体或浅层地下水的生物修复提供材料。【方法】采用富集培养方法从滇池沉积物和水体样品中分离好氧反硝化细菌,对好氧反硝化细菌的16S r RNA基因序列进行系统发育分析,并筛选其中的高效好氧反硝化细菌。【结果】分离出260株好氧反硝化菌,经16S rRNA基因序列分析,260株菌分属于2门13科14属的59个种。假单胞菌属(Pseudomonas)为优势细菌属,其次是不动杆菌属(Acinetobacter)、气单胞菌属(Aeromonas)和代尔夫特菌属(Delftia)。筛选到12株高效好氧反硝化细菌菌株,其中8株属于假单胞菌(Pseudomonas spp.),4株为不动杆菌(Acinetobacter spp.)。定量分析发现菌株N15-6-1的反硝化效果较好。对菌株N15-6-1的脱氮条件优化结果显示,在以蔗糖为碳源,温度为30–35℃、C/N=12、静止培养时,反硝化能力较强,其在48 h内硝态氮的去除率达到98.81%,总氮的去除率达96.27%。【结论】滇池存在着较丰富的可培养好氧反硝化细菌,好氧反硝化细菌的分离丰富了好氧反硝化菌的种类,其中的高效脱氮菌株为污染水体或浅层地下水的生物修复提供了初步的候选菌株。     

好氧反硝化菌的研究进展

综述了好氧反硝化菌的种类和特性、好氧反硝化菌的反硝化作用机制和影响因素.好氧反硝化菌主要包括假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)、副球菌属(Para-coccus)和芽孢杆菌属(Bacillus)等,属好氧或兼性好氧异养微生物.好氧反硝化菌能在好氧条件下进行反硝化,其主要产物是N2O,并可将铵态氮直接转化成气态产物.催化好氧反硝化菌反硝化作用的硝酸盐还原酶是周质酶而不是膜结合酶.溶解氧和C/N往往是影响好氧反硝化菌反硝化作用的主要因素.介绍了间歇曝气法、选择性培养基法等好氧反硝化菌的主要分离筛选方法.概述了好氧反硝化菌在水产养殖、废水生物处理、降解有机污染物以及对土壤氮素损失的影响方面的研究进展.     

反应器进水管生物膜中喹啉降解菌的分离鉴定及降解特性

【背景】喹啉是一类高毒、致癌且难降解的含氮杂环化合物,本实验室建立了一个长期高效运行的反硝化喹啉降解生物反应器。【目的】从反应器进水管富集的生物膜中筛选有氧条件下降解喹啉的菌株。【方法】通过以喹啉为唯一碳源的培养基来富集、分离、纯化菌株;利用16S rRNA基因的序列分析鉴定分离株的系统发育地位;比较不同pH及温度条件下菌株的喹啉降解特性。【结果】经鉴定,4株分离物Q1、Q3、Q7和Q8分别属于Sphingobium、Massilia、Rhodococcus和Dyadobacter属。降解实验表明,以上菌株均能在48 h内实现50 mg/L喹啉的完全去除,但各自表现出不同的降解特性,其中Q1、Q3和Q8在降解过程中都检测到了喹啉降解产物2-羟基喹啉的积累。降解喹啉的Sphingobium、Massilia和Dyadobacter属菌株尚未见报道。【结论】从喹啉降解生物反应器的进水管内分离的4株喹啉降解菌可为设计处理含喹啉工业废水的反应器提供新菌种资源,对于完善喹啉生物降解机理研究具有实际意义。     

好氧反硝化菌的筛选及其脱氮除磷性质的研究

利用富集培养基, 从用生活污水驯化后的活性污泥中筛选得到一株具有好氧反硝化兼具除磷功能的细菌。通过形态学及生理生化指标鉴定其为假单胞菌属。利用此好氧反硝化菌处理模拟废水及生活废水, 通过监测总氮、无机磷及CODcr变化确定在C/N摩尔比为3:1、接种量为10%、pH 6.8、30°C条件下处理2 d, 该菌株脱氮、除磷及去除有机物的效果最佳, 活性污泥经此好氧反硝化菌强化后, 对生活废水的处理能力得到明显提升。     

异养硝化-好氧反硝化细菌的研究进展

异养硝化-好氧反硝化菌株的发现是对传统硝化反硝化的突破和发展。近年来由于其独特的生物学特性及其在污水处理中的巨大优势,受到众多学者的青睐。文章介绍了异养硝化-好氧反硝化菌的筛选,异养硝化-好氧反硝化代谢途径和异养硝化-好氧反硝化菌的影响因素,并总结了异养硝化-好氧反硝化菌在废水处理中的研究进展,最后展望未来研究的方向。     

洱海沉积物中反硝化细菌的分离与反硝化作用研究

自洱海十个点位的沉积物中富集筛选出101株反硝化细菌并从中筛选出1株较强反硝化能力的细菌,命名为EH314。该细菌接触酶(过氧化氢酶)试验、产硫化氢试验和淀粉水解均为阳性,葡萄糖氧化发酵实验结果为氧化菌,产脂酶(Tween 80)试验结果为阴性;初步鉴定该菌为产碱杆菌属细菌;对细菌反硝化能力进行测定发现,菌株EH314能有效地降解水体中的硝酸盐且反硝化可在有氧条件下进行。     

好氧条件下复合生物膜-活性污泥反应器中的反硝化菌群结构

以nirK和nirS基因为标记,利用PCR-DGGE技术研究了好氧条件下复合生物膜-活性污泥反应器中的反硝化群落结构。结果表明,样品中大部分nirK-反硝化菌都属于α-变形菌纲中的根瘤菌目Rhizobiales,而nirS-反硝化菌则与β-变形菌纲(包括红环菌目Rhodo-cyclales和伯克氏菌目Burkholderiales)及γ-变形菌纲(假单胞菌目Pseudomonadales)细菌相似。在nirK-及nirS-群落中均发现了未与已知菌群聚类的特殊序列。细菌的生长方式(生物膜或活性污泥)对反硝化群落结构有显著影响。这些结果将为生物膜-活性污泥复合工艺中的反硝化过程提供基础数据。     

北京典型景观水体好氧反硝化菌组成特征

好氧反硝化菌对环境水体氮素的循环起到非常重要的作用。对北京市6个典型景观水体中好氧反硝化菌进行富集培养和分离,并开展菌株的16S rRNA基因测序和组成特征分析。结果表明,从6个水体中共富集分离得到80株好氧反硝化菌,均为变形菌门 (Proteobacteria),聚类于其3个纲(α-Proteobacteria、β-Proteobacteria、γ-Proteobacteria),分属于9个属,31个物种。其中90%左右的菌株具有良好的好氧反硝化能力,是水体进行生物脱氮修复的重要微生物基础。在不同景观水体中,好氧反硝化菌表现出较为明显的分布差异性和性能差异性,除了普遍存在的假单胞菌属（Pseudomonas）和不动杆菌属（Acinetobacter）外,每个水体基本都有属于自己的特异菌属,其中重要的特异菌属包括Alishewanella、Delftia、Hydrogenophaga和Rheinheimera,这对水体修复具有指导意义。     

好氧反硝化微生物多样性及其反硝化功能初步研究

为研究污水厂/养殖池中好氧反硝化微生物的多样性及菌株反硝化能力,本研究采集了位于福建省厦门市和漳州市的污水处理厂、排污口、污水池、对虾养殖池的污水和污泥样品进行好氧反硝化微生物的富集、分离、鉴定和功能筛选。分别以NaNO3、NaNO2作为唯一氮源共分离纯化获得128株单菌。其中以NaNO3为唯一氮源分离得到63株,以NaNO2为唯一氮源分离得到65株。16SrRNA基因序列分析表明,128株单菌分属于γ-变形菌纲(Gammaproteobacteria,58.6%)、芽胞杆菌纲(Bacilli,6.4%)、放线菌纲(Actinobacteria,11.7%)、α-变形菌纲(Alphaproteobacteria,8.6%)、纤维菌纲(Cytophagia,2.3%)、鞘脂杆菌纲(Sphingobacteria,0.8%)和黄杆菌纲(Flavobacteria,1.6%)7个纲中的38个属。其中盐单胞菌属(Halomonas,29.7%)和芽胞杆菌属(Bacillus,12.5%)为优势菌属,并且广泛存在于各个样品中。反硝化功能初筛结果表明,35株菌能在72h内将20mg·L-1 NO-3-N/NO-2-N完全去除;复筛结果表明,21株菌能在72h内将100 mg·L-1 NO-3-N/NO-2-N完全去除,并且盐单胞菌属、卓贝尔氏菌属(Zobellella)、斯塔普氏菌属(Stappia)及节杆菌属(Arthrobactor)反硝化效果较好,其中斯塔普氏属是首次报道具有好氧反硝化功能。本研究结果表明,污水场/养殖池等环境中可培养反硝化细菌多样性丰富,同时高效反硝化菌的获得也为含氮废水的生物处理提供了良好的菌种资源。     

Anaerobic and aerobic degradation of pyridine by a newly isolated denitrifying bacterium.

New denitrifying bacteria that could degrade pyridine under both aerobic and anaerobic conditions were isolated from industrial wastewater. The successful enrichment and isolation of these strains required selenite as a trace element. These isolates appeared to be closely related to Azoarcus species according to the results of 16S rRNA sequence analysis. An isolated strain, pF6, metabolized pyridine through the same pathway under both aerobic and anaerobic conditions. Since pyridine induced NAD-linked glutarate-dialdehyde dehydrogenase and isocitratase activities, it is likely that the mechanism of pyridine degradation in strain pF6 involves N-C-2 ring cleavage. Strain pF6 could degrade pyridine in the presence of nitrate, nitrite, and nitrous oxide as electron acceptors. In a batch culture with 6 mM nitrate, degradation of pyridine and denitrification were not sensitively affected by the redox potential, which gradually decreased from 150 to -200 mV. In a batch culture with the nitrate concentration higher than 6 mM, nitrite transiently accumulated during denitrification significantly inhibited cell growth and pyridine degradation. Growth yield on pyridine decreased slightly under denitrifying conditions from that under aerobic conditions. Furthermore, when the pyridine concentration used was above 12 mM, the specific growth rate under denitrifying conditions was higher than that under aerobic conditions. Considering these characteristics, a newly isolated denitrifying bacterium, strain pF6, has advantages over strictly aerobic bacteria in field applications.     

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, γ-aminobutyrate/α-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of diverse halobenzoate-degrading denitrifying bacteria from soils and sediments

Denitrifying bacteria capable of degrading halobenzoates were isolated from various geographical and ecological sites. The strains were isolated after initial enrichment on one of the monofluoro-, monochloro-, or monobromo-benzoate isomers with nitrate as an electron acceptor, yielding a total of 33 strains isolated from the different halobenzoate-utilizing enrichment cultures. Each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor. The isolates obtained on 2-fluorobenzoate could use 2-fluorobenzoate under both aerobic and denitrifying conditions, but did not degrade other halobenzoates. In contrast, the 4-fluorobenzoate isolates degraded 4-fluorobenzoate under denitrifying conditions only, but utilized 2-fluorobenzoate under both aerobic and denitrifying conditions. The strains isolated on either 3-chlorobenzoate or 3-bromobenzoate could use 3-chlorobenzoate, 3-bromobenzoate, and 2- and 4-fluorobenzoates under denitrifying conditions. The isolates were identified and classified on the basis of 16S rRNA gene sequence analysis and their cellular fatty acid profiles. They were placed in nine genera belonging to either the alpha-, beta-, or gamma-branch of the Proteobacteria, namely, Acidovorax, Azoarcus, Bradyrhizobium, Ochrobactrum, Paracoccus, Pseudomonas, Mesorhizobium, Ensifer, and Thauera. These results indicate that the ability to utilize different halobenzoates under denitrifying conditions is ubiquitously distributed in the Proteobacteria and that these bacteria are widely distributed in soils and sediments.     

生物造粒流化床污水处理反应器中微生物的分布特征

对生物造粒流化床污水处理反应器10cm、60cm、110cm处好氧细菌总数以及反硝化菌、反硫化菌分别进行计数,同时,用光学显微镜、扫描电子显微镜以及石蜡切片技术对粒状污泥中细菌的分布情况进行研究。结果发现,流化床中好氧细菌非常丰富,在反应器10cm处,每克污泥微生物的数量可达1.6×10⁸个,说明好氧细菌在生物造粒流化床有机物生物降解中起主导作用;同时,流化床中也有一定数量的兼性厌氧菌存在,并且随着流化床床体的升高有增加的趋势,这与溶解氧(DO)随流化床床体高度的增加而迅速降低有关;随着回流比的增加,溶解氧增高,相应的好氧细菌有所增加而兼性厌氧菌减少;对于颗粒污泥,其表面和内部微生物分布数量有很大的差异。     

Aerobic and anaerobic growth of rifampin-resistant denitrifying bacteria in soil.

The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth followed by a gradual decline in numbers. After 28 days, both isolates maintained viable populations of between 10(4) and 10(5) CFU g dry soil-1 under both denitrifying and aerobic conditions. One of the isolates consistently grew better under denitrifying conditions, and the other isolate consistently grew better under aerobic conditions. The relative pattern of denitrifying versus aerobic growth for each organism was not affected by the addition of glucose. The growth yields of the two isolates varied with soil type, but the relative pattern of denitrifying versus aerobic growth was consistent in three soils with greatly different properties. Five of nine isolates introduced into Bruno soil at low population densities (approximately 10(5) CFU g dry soil-1) exhibited better growth after 2 days under denitrifying conditions. It was not possible to predict the prevalence of the denitrifying or aerobic mode of growth in nonsterile soil from the growth characteristics of the isolates in pure cultures or sterile soil.     

Aerobic and anaerobic growth of rifampin-resistant denitrifying bacteria in soil

The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth followed by a gradual decline in numbers. After 28 days, both isolates maintained viable populations of between 10(4) and 10(5) CFU g dry soil-1 under both denitrifying and aerobic conditions. One of the isolates consistently grew better under denitrifying conditions, and the other isolate consistently grew better under aerobic conditions. The relative pattern of denitrifying versus aerobic growth for each organism was not affected by the addition of glucose. The growth yields of the two isolates varied with soil type, but the relative pattern of denitrifying versus aerobic growth was consistent in three soils with greatly different properties. Five of nine isolates introduced into Bruno soil at low population densities (approximately 10(5) CFU g dry soil-1) exhibited better growth after 2 days under denitrifying conditions. It was not possible to predict the prevalence of the denitrifying or aerobic mode of growth in nonsterile soil from the growth characteristics of the isolates in pure cultures or sterile soil.     

好氧反硝化细菌碳氮代谢特点及途径的研究进展

好氧反硝化作用的发现打破了反硝化只能在严格厌氧条件下进行的传统认知,为生物脱氮提供了一条新的途径,已成为近些年的研究热点。碳源可为好氧反硝化过程提供能量和电子供体,其代谢难易程度直接影响着好氧反硝化细菌的脱氮效率,因此有必要明确碳源在好氧反硝化脱氮过程中的代谢机理。基于此,本文阐述了好氧反硝化细菌的种类及其对硝态氮与亚硝态氮的代谢途径;系统分析了不同好氧反硝化细菌对碳氮源代谢的差异与代谢机理;综合分析了碳代谢差异对好氧反硝化脱氮过程的影响,并对未来的研究方向进行了展望,旨在深入理解好氧反硝化细菌同时去除碳氮的机理,为提高废水生物脱氮除碳效率提供理论依据。     

Development and application of a novel and effective screening method for aerobic denitrifying bacteria

Herein we describe a novel and effective screening method for aerobic denitrifying bacteria. For this procedure, we utilized KCN to inhibit the electron transference from Cytaa3 to oxygen in the bacteria respiratory chain. We employed a 3-h aeration operation cycle and intermittent rotations. The resultant bacterial suspensions were plated on a KCN-screening medium and incubated aerobically. Single colonies were selected and incubated in an aerobic culture medium. Culture nitrate and nitrite levels were determined over time, and ultimately four bacterial strains that performed denitrifying under aerobic conditions were identified by this method. Of these, strain Y2-1-1 demonstrated the best aerobic denitrifying ability. In a 5-day test, the NO3--N of the aerobic culture medium was reduced from 282.0+/-8.3 mg L(-1) to 149.2+/-17.1 mg L(-1), with little nitrite or N2O production. The morphological, physiological and biochemical characteristics and the 16S rRNA gene sequence homology comparison data for this strain were consistent with the classification of the genus Pseudomonas. We named this strain Pseudomonas sp. Y2-1-1.