好氧反硝化生物脱氮技术的研究进展

好氧反硝化生物脱氮技术自提出以来,凭借能实现同步硝化反硝化、节省基建投资及运行费用等诸多优点,受到国内外环境领域学者的广泛关注。本文首先总结了近年来好氧反硝化菌种的筛选分离情况,以及环境因子对好氧反硝化菌脱氮效能的影响,包括溶解氧(dissolved oxygen,DO)、碳氮比(C/N)、温度等。然后深入探讨了好氧反硝化生物脱氮技术的原理,好氧反硝化过程中的关键功能基因及酶,同时介绍了分子生物技术在好氧反硝化研究过程中的应用,以及好氧反硝化生物脱氮技术在实际应用方面的研究现状。最后,基于目前的研究瓶颈问题,对未来好氧反硝化生物脱氮技术的研究方向提出了科学展望。     

一株好氧反硝化细菌的分离鉴定及反硝化特性研究

通过对采集水样进行富集培养,利用溴百里酚蓝( BTB)选择性培养基初筛和活性测定复筛得到一株好氧反硝化细菌N22’,发现该菌在好氧条件下能有效去除培养液中的NO3--N.硝酸盐氮初始浓度为125 mg/L,培养40h后硝酸盐氮去除率达86.39％;扫描电镜照片显示,菌株N22’为短杆菌,无鞭毛,大小约为(0.75-1.25)μm×(0.5-0.75) μm范围内,菌落表面呈乳白色.通过形态、生理生化特征及16S rRNA基因序列分析,初步判断菌株N22’为不动杆菌属Acinetobacter sp..反硝化性能测试结果表明,该菌反硝化作用的最适温度为25-30℃,pH值7.0.     

限氧自养硝化-反硝化生物脱氮新技术

限氧自养硝化—反硝化是部分硝化与厌氧氨氧化相耦联的生物脱氮反应过程,通过严格控制溶解氧在0．1～0．3mg·L^-1,实现硝化反应控制在亚硝酸阶段,然后以硝化阶段剩余的NH4^+作为电子供体,在厌氧条件下实现反硝化,该反应过程是完全的自养硝化—反硝化过程,具有能耗低、脱氮效率高、反应系统占地面积小等优点,适用于处理COD／NH4^+—N低的废水,是一种非常有应用前景的生物脱氮技术,文中详细介绍了限氧自养硝化—反硝化生物脱氮反应过程的研究进展,讨论了其微生物学机理及应用前景。     

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。     

好氧反硝化菌脱氮特性研究进展

好氧反硝化菌的发现,是对传统反硝化理论的丰富与突破. 由于其在脱氮方面的独特优势,已成为目前废水生物脱氮领域研究的热点. 好氧反硝化菌能够在有氧条件下,利用有机碳源生长的同时将含氮化合物反硝化生成N2等气态氮化物,多数还能同时进行异养硝化作用,将铵态氮直接转化为含氮气体. 本文从电子理论、反硝化酶系等方面对目前已分离出的一些好氧反硝化菌的脱氮特性及其脱氮机理进行探讨,分析了溶解氧、碳源类型及C/N等环境条件对其脱氮作用的影响,介绍了好氧反硝化菌的筛选方法及应用现状,对其应用前景和发展方向进行了展望.     

一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性

【目的】确定一株分离自海水的异养硝化-好氧反硝化菌的系统发育地位并探索其脱氮特性和机理,以期为解释异养硝化-好氧反硝化机理以及改进海水养殖及废水的生物脱氮工艺提供理论依据。【方法】通过形态观察、生理生化实验和16S rRNA基因序列分析,鉴定该菌株;通过测定菌株在不同无机氮源降解测试液中的生长和脱氮效率,分析其异养硝化和好氧反硝化性能。【结果】经鉴定该菌株属于盐单胞菌属(Halomonas);最适生长条件为盐度3%、pH 8.5、温度28℃、碳氮比10:1,在盐度为15%的培养液中仍能生长;可以同时去除氨氮、亚硝酸氮和硝酸氮,24 h时对NH4+-N、NO2--N、和NO3--N的去除率可分别达到98.29%、99.07%、96.48%,3种形态无机氮同时存在时,会优先利用NH4+-N,且总无机氮去除率较单一存在时更高,说明该菌株可实现同步硝化反硝化。【结论】该分离自海水的异养硝化-好氧反硝化菌属于盐单胞菌属(Halomonas),在高盐环境中仍能生长,同时具有高效的异养硝化和好氧反硝化能力,能够独立完成脱氮的全部过程。     

好氧反硝化菌

<正>养殖水体氮素污染问题是目前困扰我国水产养殖业可持续发展的一大难题。生物脱氮技术被认为是目前最具发展前景的水体脱氮技术,其效果的优劣与所采用菌株的特性密切相关。传统反硝化细菌仅能在厌氧及低氧条件下发挥脱氮作用,与养殖水体的高溶氧环境矛盾,而好氧反硝化细菌则可在高溶氧环境中发挥脱氮作用,显著提高生物脱氮技术在养殖水体中的应用效果,实现养殖水体的绿色、零污染脱氮。因而,对好氧反硝化细菌开展高效选育方法的研究,找到可适应养殖水体水环境的微生物菌株具有重要的理论价值和经济价值。     

脱氮硫杆菌特异引物/探针的设计和评价

自脱氮硫杆菌(Thiobacillus denitrificans)16S rRNA基因V3可变区中发现一条27 bp的特异序列, 以该序列为反向引物, 对高效同步脱硫反硝化系统污泥DNA进行了温度梯度PCR扩增和基因文库构建, 结果证实了该引物的高度专一性。应用该探针在去离子甲酰胺和NaCl的浓度分别为35%和100 mmol/L, 杂交/洗脱温度为48°C条件下对污泥样品杂交得到较好的阳性结果, 软件分析表明脱氮硫杆菌在污泥中约占15%。脱氮硫杆菌专一性引物/探针的提出, 将为不同生态环境中该种微生物的时空分布、结构动态以及实时定量等研究提供分子生物学工具。     

极端条件下异养硝化-好氧反硝化菌脱氮的研究进展

异养硝化-好氧反硝化(HN-AD)是对传统自养硝化异养反硝化理论的丰富与突破。HN-AD菌在好氧条件下可快速实现氨氮、硝态氮(NO_3~–-N)、亚硝态氮(NO_2~–-N)三氮同步脱除。它们不仅具有分布范围广、适应能力强、代谢通路特殊等特点,而且还具有世代时间短、脱氮速率快、高活性持久等独特优势,在高盐、低温、高氨氮等极端条件表现出了巨大的脱氮潜力,因此在废水生物脱氮领域受到广泛关注。文中在介绍HN-AD菌属类别及代谢机理的基础上,重点总结了在高盐、低温、高氨氮等极端条件下进行氨氮脱除的HN-AD种属,系统分析了它们在极端条件下的脱氮特性及潜力,并简述了HN-AD菌在极端条件下的工艺应用研究进展,最后展望了HN-AD脱氮技术的应用前景和研究方向。     

微生物甲烷氧化反硝化耦合反应研究进展

甲烷氧化反硝化耦合过程是连接碳循环和氮循环的重要桥梁.该过程的深入研究有助于完善人们对全球碳氮生物化学循环的认识.甲烷作为反硝化外加气体碳源,既能调控大气甲烷平衡,有效减缓由甲烷引起的温室效应,又能降低反硝化工艺中因投入外加碳源带来的成本.因此近年来甲烷氧化反硝化耦合反应及其机理研究倍受关注.本文主要讨论了好氧和厌氧两种类型的甲烷氧化反硝化过程,重点对其微生物耦合反应机理及其影响因素进行了综述,同时指出了其工程化应用存在的问题,并对其应用前景提出展望.
     

好氧反硝化微生物学机理与应用研究进展

近年来,关于好氧反硝化过程的研究主要集中在三个方面:分别是好氧反硝化菌株的分离和脱氮性能表征,好氧反硝化微生物的应用潜力分析,以及好氧反硝化过程的机理研究。好氧反硝化菌株分布范围广泛,可从多种环境中分离得到,种属以Pseudomonas sp.、Alcaligenes sp.和Paracoccus sp.为主。好氧反硝化菌株及菌群在实验室条件下表现出优良的耐冷、耐盐特性,并具有可降解毒性有机物及N_2O减排的潜力。关于好氧反硝化过程的机理研究表明,虽然硝酸盐作为电子受体的竞争力比氧气弱,但反硝化作为辅助电子传递途径,可提高产能效率,防止NAD(P)H的过量积累。因此,硝酸盐可与氧气同时参与微生物的新陈代谢,即发生好氧反硝化现象。未来除了继续分离更新更好的好氧反硝化菌株外,应加强对好氧反硝化机理及实际生物强化方面的研究。     

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH₄-based MBfR (<1.0 g N/m²-day) is about one order of magnitude smaller than that in the H₂-based MBfR (1.1–6.7 g N/m²-day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH₄-based MBfR is limited to <1.7 g N/m²-day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K₂) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (k_max2), with k_max2 being more important. Model simulations show that increasing k_max2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (D_m) could bring the nitrate-removal flux to ≥4.0 g N/m²-day, which is close to the nitrate-removal flux for the H₂-based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when D_m and k_max2 increase together.     

PCR-DGGE对长江河口八种野生鱼类肠道菌群多样性的比较研究

目的分析长江河口捕获的8种野生鱼类的肠道菌群多样性的差异并观察这种差异与食性的联系。方法采用PCR-DGGE(denaturing gradient gel electrophoresis)技术,DGGE图谱用PCA(principal component analy-sis)方法进行分析。结果建立了长江口8种鱼野生条件下肠道菌群的DGGE指纹图谱,观察到它们在野生条件下的肠道菌群的差异。其中,营底栖生活的舌鰕虎鱼的肠道菌群和其他7种野生鱼有着明显的差异,其他7种鱼的肠道菌群多样性的差异与它们的食性差异相关。结论PCR-DGGE技术是一种能够快速有效地分析研究鱼类肠道菌群结构的技术。8种野生鱼的肠道菌群的结构有明显的差别,并且食性差异大的鱼类之间肠道菌群差异也     

大肠杆菌16S rRNA的核酶设计和初步应用

针对细菌rRNA研发抑制细菌增殖的新型抗菌素是抗生素研究领域的新课题。细菌rRNA与基因mRNA一样自然形成折叠卷曲高级结构,其结构上可以结合反义核酸的位点即靶点,靶点的阐明是设计有效反义核酸、核酶（Ribozyme）和脱氧核酶（DNAzyme）的关键。MAST方法固定16S rRNA,将其与寡核苷酸文库杂交筛选出靶点,获得了大肠杆菌16S rRNA的6个反义核酸结合靶点,并鉴定5个靶点有效,其中1个为高效。5个靶点的反义核酸能在通透性大肠杆菌菌株培养中不同程度地抑制其生长,针对高效靶点的核酶在转化大肠杆菌中表达而抑制其生长。     

Archaeal rRNA diversity and methane production in deep boreal peat

Northern peatlands play a major role in the global carbon cycle as sinks for CO₂ and as sources of CH₄. These diverse ecosystems develop through accumulation of partially decomposed plant material as peat. With increasing depth, peat becomes more and more recalcitrant due to its longer exposure to decomposing processes. Compared with surface peat, deeper peat sediments remain microbiologically poorly described. We detected active archaeal communities even in the deep bottom layers (−220/−280 cm) of two Finnish fen-type peatlands by 16S rRNA-based terminal restriction fragment length polymorphism analysis. In the sediments of the northern study site, all detected archaea were methanogens with Rice Cluster II (RC-II) and Methanosaetaceae as major groups. In southern peatland, Crenarchaeota of a rare unidentified cluster were present together with mainly RC-II methanogens. RNA profiles showed a larger archaeal diversity than DNA-based community profiles, suggesting that small but active populations were better visualized with rRNA. In addition, potential methane production measurements indicated methanogenic activity throughout the vertical peat profiles.     

空气环境中几种细菌检测与16S rRNA测序鉴定

采集空气环境微生物,根据菌落和革兰氏染色特征,分离5株细菌。提取分离株DNA,采用设计的16SrRNA引物扩增DNA片段;纯化PCR产物,进行测序反应并再纯化,上机读序。拼接与校对DNA序列,在NCBI网站进行Blast,鉴定细菌型别。5株菌株分别是表皮葡萄球菌、头状葡萄球菌、施氏假单胞菌、溶血葡萄球菌和大肠杆菌;其中溶血葡萄球菌是致病菌,其他4株是条件致病菌。这提示,应警惕空气环境中致病菌的感染。     

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

Molecular characterization of a microbial consortium involved in methane oxidation coupled to denitrification under micro‐aerobic conditions

Methane can be used as an alternative carbon source in biological denitrification because it is nontoxic, widely available and relatively inexpensive. A microbial consortium involved in methane oxidation coupled to denitrification (MOD) was enriched with nitrite and nitrate as electron acceptors under micro‐aerobic conditions. The 16S rRNA gene combined with pmoA phylogeny of methanotrophs and nirK phylogeny of denitrifiers were analysed to reveal the dominant microbial populations and functional microorganisms. Real‐time quantitative polymerase chain reaction results showed high numbers of methanotrophs and denitrifiers in the enriched consortium. The 16S rRNA gene clone library revealed that Methylococcaceae and Methylophilaceae were the dominant populations in the MOD ecosystem. Phylogenetic analyses of pmoA gene clone libraries indicated that all methanotrophs belonged to Methylococcaceae, a type I methanotroph employing the ribulose monophosphate pathway for methane oxidation. Methylotrophic denitrifiers of the Methylophilaceae that can utilize organic intermediates (i.e. formaldehyde, citrate and acetate) released from the methanotrophs played a vital role in aerobic denitrification. This study is the first report to confirm micro‐aerobic denitrification and to make phylogenetic and functional assignments for some members of the microbial assemblages involved in MOD.