铁矿物强化厌氧氨氧化效能及其微生物机制研究进展

自然界中的氮循环与铁循环相互交联,参与氮循环的厌氧氨氧化（anaerobic ammonium oxidation,anammox）菌的生长代谢及活性发挥也与铁元素紧密关联。自然界广泛存在的铁矿物因具有运行成本低廉、稳定性好、二次污染小等优势,在污水处理领域得到广泛应用。在厌氧氨氧化脱氮系统中引入适量铁矿物,不仅有助于促进anammox菌和铁还原菌的富集,提高功能基因丰度和相关酶活性,还可能通过影响污泥浓度、血红素c含量、胞外聚合物含量和颗粒化程度,改善污泥性能和提高厌氧氨氧化系统的稳定性。同时,铁矿物具有促进体系多种氮素转化途径（如anammox、铁自养反硝化、铁氨氧化、异化硝酸盐还原成铵和反硝化）相耦合的潜能,可以提高anammox污水处理系统的总氮去除率。本文基于铁矿物在促进污水生物脱氮方面的良好性能及其在anammox系统中的变化,从脱氮效能、污泥特性、微生物特征及酶活性等方面,系统综述了铁矿物对厌氧氨氧化系统的强化作用机制,并从anammox菌对铁矿物的利用及铁元素的摄取角度展望了后续的研究方向,以期为铁矿物强化厌氧氨氧化系统的实际应用提供理论和技术指导。     

厌氧氨氧化在污水处理中的研究进展

厌氧氨氧化(ANAMMOX)是指厌氧氨氧化细菌在厌氧条件下以亚硝酸盐为电子受体将氨氮氧化为氮气的过程。由于在节能降耗和环境友好上的独特优点,基于厌氧氨氧化原理的脱氮技术被公认是目前最具应用前景的生物脱氮技术,因此自发现以来一直是国内外研究的热点。综述近年有关厌氧氨氧化细菌、厌氧氨氧化机理、反应的影响因素及其在污水处理应用方面的研究进展,并展望厌氧氨氧化在污水处理领域的发展方向。     

污水脱氮功能微生物的组学研究进展

生物脱氮是污水处理厂的核心,掌握生物脱氮过程相关微生物代谢特性,对于探索微生物资源和提高污水处理厂脱氮性能具有重要意义。近年来,分子生物学方法不断发展和改进,已被广泛应用于揭示脱氮微生物群落多样性、组成结构和潜在功能等方面,大幅提升了研究者们对污水生物脱氮系统中微生物,尤其是不可培养微生物的代谢机理、抑制调控原理及新型生物脱氮工艺途径的认识。本文对流行的分子生物学方法（16S rRNA基因测序、实时荧光定量PCR技术、宏基因组学、宏转录组学、宏蛋白质组学和代谢组学）进行了介绍,综述了其在硝化细菌、反硝化细菌、完全氨氧化细菌、厌氧氨氧化细菌、厌氧铁氨氧化细菌、硫酸盐型厌氧氨氧化细菌及亚硝酸盐/硝酸盐型厌氧甲烷氧化微生物等方面的研究进展,阐明了这些氮素转化微生物在氮循环过程的代谢途径和酶促反应,并从标准测定方法构建、不同方法的联用及跨学科结合和检测方法的简易化这3个方面展望了分子生物学方法的技术突破及其在污水生物处理系统中的应用前景。本综述从系统角度全面认识脱氮微生物群落及其结构,为未来污水处理生物脱氮微生物的研究提供了新方向。     

单级自养脱氮系统中厌氧氨氧化菌的分子生物学鉴定

对具有厌氧氨氧化作用的细菌进行更深入的分析有助于了解该菌在生物脱氮过程的应用。对稳定运行、氨氮转化率及总氮去除率分别达到90%及80%左右的单级自养脱氮系统的底部取活性污泥,采用分子生物学方法提取活性污泥细菌总DNA,利用特异引物Pla46rc/Amx820对单级自养脱氮系统中的厌氧氨氧化菌16S rDNA基因进行PCR扩增。扩增产物经克隆、测序及BLAST分析,结果表明该单级自养脱氮系统中存在的厌氧氨氧化菌与Candidatus Kueneniastuttgartiensis和Candidatus Brocadia anammoxidans的16S rDNA序列同源性达99%,进化分析证明与Candidatus Kuenenia stuttgartiensis进化上较为接近。     

海洋氮循环中细菌的厌氧氨氧化

细菌厌氧氨氧化过程是在一类特殊细菌的厌氧氨氧化体内完成的以氨作为电子供体硝酸盐作为电子受体的一种新型脱氮反应.厌氧氨氧化菌的发现,改变人们对传统氮的生物地球化学循环的认识:反硝化细菌并不是大气中氮气产生的唯一生物类群.而且越来越多的证据表明,细菌厌氧氨氧化与全球的氮物质循环密切相关,估计海洋细菌的厌氧氨氧化过程占到全球海洋氮气产生的一半左右.由于氮与碳的循环密切相关,因此可以推测,细菌的厌氧氨氧化会影响大气中的二氧化碳浓度,从而对全球气候变化产生重要影响.另外,由于细菌厌氧氨氧化菌实现了氨氮的短程转化,缩短了氮素的转化过程,因此为开发更节约能源、更符合可持续发展要求的废水脱氮新技术提供了生物学基础.     

厌氧氨氧化反应器启动过程的影响因素及微生物群落变化研究进展

自厌氧氨氧化反应发现以来,由于其具有低能耗、无需外加碳源等优点,已成为人们在污水生物脱氮研究与应用中的最新关注点。然而,由于极低的生长速率、极长的倍增时间以及严格的代谢条件等特点,限制了厌氧氨氧化菌的应用。综述了厌氧氨氧化菌富集培养过程中的影响因素,介绍了不同污泥来源的厌氧氨氧化优势菌属、分子鉴定方法,提供了部分用于厌氧氨氧化菌鉴定使用的引物序列和厌氧氨氧化菌最新发现的属与种。最后,对未来的研究方向提出一些建议思考,以期为厌氧氨氧化工艺在污水处理中的应用提供参考。     

厌氧氨氧化颗粒污泥聚集机制研究进展

厌氧氨氧化(anaerobic ammonium oxidation,anammox)工艺被认为是当前污水生物脱氮领域最经济的处理工艺,有利于实现污水处理厂的能源自给。厌氧氨氧化菌是该工艺的核心功能微生物。以厌氧氨氧化菌为主导微生物形成的厌氧氨氧化颗粒污泥具有沉速大、污泥持留能力强及对不利环境抵抗能力强等突出优势,是实现厌氧氨氧化工艺最有前景的污泥形态。本论文围绕厌氧氨氧化颗粒,介绍了厌氧氨氧化菌的特性、种类及代谢途径,综述了厌氧氨氧化颗粒污泥的形成假说及与厌氧氨氧化颗粒污泥聚集密切相关的胞外聚合物(extracellular polymeric substance,EPS)和群体感应研究现状,并对今后厌氧氨氧化颗粒的研究进行了展望,以期为后续厌氧氨氧化颗粒的研究及厌氧氨氧化颗粒工艺的优化提供参考。     

厌氧氨氧化菌驱动脱氮途径的研究进展

厌氧氨氧化菌(anaerobic ammonia-oxidizing bacteria, AnAOB)的代谢多样性,使得该菌群能够在海洋、湿地和陆地等不同的自然生态系统中广泛分布,甚至在一些极热和极寒环境中也检测到了该菌群的存在。本文回顾并总结了厌氧氨氧化菌在不同生态系统中的发现、分布及脱氮贡献等方面的研究,分析了厌氧氨氧化菌分布的主要环境影响因素。该综述将帮助我们更好地理解全球氮循环中厌氧氨氧化菌的实际角色和功能,并基于厌氧氨氧化(anaerobicammoniaoxidation,anammox)过程,探究能与其进行协作的新型生物脱氮工艺,以期为这些工艺的研发和推广提供生态学基础和新的思考,从而实现脱氮工艺的技术变革。     

全程硝化菌微生物学特性及在水处理领域的应用潜力

全程硝化菌是近期微生物氮循环领域的重大发现之一,引发了对其全球分布、系统发育特征和生理生化特性的广泛关注。本文综述了全程硝化菌在土壤、地表水、废水处理系统等生境的分布规律及影响因子;并从底物亲和力、代谢多样性等方面阐述了其与传统硝化微生物间的竞争互作和生态位分离机制;基于上述特征提出全程硝化菌在水处理领域中的应用前景,可能与其他脱氮微生物如反硝化菌、厌氧氨氧化菌和厌氧甲烷氧化菌等耦合实现在低氨氮、低溶解氧条件下的污水深度脱氮,从而节省能耗并降低温室气体排放。未来研究应继续深入研究全程硝化菌的生理生化特性,评价其生态功能和对氮素地球化学循环的贡献,并探索其在生物水处理等领域的应用潜力。     

OLAND生物脱氮系统中硝化菌群16S rDNA的DGGE分析

为了考察生物脱氮系统中硝化菌群（氨氧化菌和亚硝酸氧化菌）的种群多样性及硝化菌群随溶解氧降低的种群变化规律,并建立一套行之有效的用于自养生物脱氮系统中功能微生物菌群的快速分子检测技术,采用DGGE（变性梯度凝胶电泳）分子检测技术对硝化菌群的16SrDNA的特异性PCR扩增产物进行了分析,结果表明：OLAND生物脱氮系统中氨氧化菌和亚硝酸氧化菌随溶解氧的降低表现出了不同的种群变化规律,氨氧化菌种群多样性受溶解氧的影响非常大,而非亚硝酸氧化菌的种群多样性比较单一,且不受溶解氧的影响。结合FISH（全细胞荧光原位杂交）分析结果表明,在OLAND限氧稳定运行后期,亚硝化单胞菌属（Nitrosomonas）是主要的氨氧化菌,占OLAND限氧亚硝化阶段反应器中总细菌数的72.5%左右。     

The anammoxosome: an intracytoplasmic compartment in anammox bacteria

Anammox bacteria belong to the phylum Planctomycetes and perform anaerobic ammonium oxidation (anammox); they oxidize ammonium with nitrite as the electron acceptor to yield dinitrogen gas. The anammox reaction takes place inside the anammoxosome: an intracytoplasmic compartment bounded by a single ladderane lipid-containing membrane. The anammox bacteria, first found in a wastewater treatment plant in The Netherlands, have the potential to remove ammonium from wastewater without the addition of organic carbon. Very recently anammox bacteria were also discovered in the Black Sea where they are responsible for 30-50% of the nitrogen consumption. This review will introduce different forms of intracytoplasmic membrane systems found in prokaryotes and discuss the compartmentalization in anammox bacteria and its possible functional relation to catabolism and energy transduction.     

Candidatus 'Brocadia fulgida': an autofluorescent anaerobic ammonium oxidizing bacterium

Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus 'Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus 'Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus 'Brocadia fulgida' did not incorporate acetate directly into their biomass. Candidatus 'Brocadia fulgida' exhibited the common characteristics of anammox bacteria: the presence of an anammoxosome and ladderane lipids and the production of hydrazine in the presence of hydroxylamine. Interestingly, the biofilm aggregates of this species showed strong autofluorescence. It is the only known anammox species exhibiting this feature. The autofluorescent extracellular polymeric substance had two excitation (352 and 442 nm) and two emission (464 and 521 nm) maxima.     

厌氧氨氧化菌的研究进展

近年来,有关厌氧氨氧化过程这一特殊的生化机制以及微生物类群的研究引起了人们的极大关注,尤其是这类微生物的生态生境可能比人们预想的范围更加广泛,因而在自然界N循环中可能具有重要意义。对这类菌结构特征、系统发育地位以及厌氧氨氧化小体和厌氧氨氧化机制的更深入认识将大大促进它们在污水处理工程中的应用。综述了近年来有关厌氧氨氧化菌的生理特性、生化机制、结构特点、生态生境以及工程应用等方面的最新进展。     

Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite to nitrogen gas in the absence of oxygen. These microorganisms form a significant sink for fixed nitrogen in the oceans and the anammox process is applied as a cost‐effective and environment‐friendly nitrogen removal system from wastewater. Anammox bacteria have a compartmentalized cell plan that consists of three separate compartments. Here we report the fractionation of the anammox bacterium Kuenenia stuttgartiensis in order to isolate and analyze the innermost cell compartment called the anammoxosome. The subcellular fractions were microscopically characterized and all membranes in the anammox cell were shown to contain ladderane lipids which are unique for anammox bacteria. Proteome analyses and activity assays with the isolated anammoxosomes showed that these organelles harbor the energy metabolism in anammox cells. Together the experimental data provide the first thorough characterization of a respiratory cell organelle from a bacterium and demonstrate the essential role of the anammoxosome in the production of a major portion of the nitrogen gas in our atmosphere.     

Ladderane lipid distribution in four genera of anammox bacteria

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C(18) and C(20) ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C(20) alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of "Candidatus Kuenenia stuttgartiensis", suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C(27) hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.     

Biochemistry and molecular biology of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N₂ gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.     

Ladderane phospholipids in anammox bacteria comprise phosphocholine and phosphoethanolamine headgroups

Anammox bacteria present in wastewater treatment systems and marine environments are capable of anaerobically oxidizing ammonium to dinitrogen gas. This anammox metabolism takes place in the anammoxosome which membrane is composed of lipids with peculiar staircase-like 'ladderane' hydrocarbon chains that comprise three or four linearly concatenated cyclobutane structures. Here, we applied high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to elucidate the full identity of these ladderane lipids. This revealed a wide variety of ladderane lipid species with either a phosphocholine or phosphoethanolamine polar headgroup attached to the glycerol backbone. In addition, in silico analysis of genome data gained insight into the machinery for the biosynthesis of the phosphocholine and phosphoethanolamine phospholipids in anammox bacteria.     

厌氧氨氧化菌的物种多样性与生态分布

厌氧氨氧化是微生物和环境领域的重大发现,厌氧氨氧化可同时去除氨氮和亚硝氮,在环境工程上具有重大开发价值.由于厌氧氨氧化菌生长极慢,倍增时间长达11 d以上,严重制约了该反应的开发进程,因此,对厌氧氨氧化菌的研究具有重要意义.厌氧氨氧化菌种类丰富,除了人们最早认识的浮霉状菌外,还有硝化细菌和反硝化细菌,这些菌群生态分布广泛,为开辟新的厌氧氨氧化菌种资源创造了条件.硝化细菌和反硝化细菌兼有厌氧氨氧化能力,其代谢多样性为加速厌氧氨氧化反应器的启动提供了依据.厌氧消化污泥可呈现硫酸盐型厌氧氨氧化活性,可为新型生物脱氮工艺的研发奠定基础.探明厌氧氨氧化菌种资源及其生态分布,将有利于厌氧氨氧化的开发应用.     

厌氧铵氧化过程中关键酶及相关分子标记在生态学研究中的应用进展

厌氧铵氧化是由微生物介导的氮素循环过程中的重要途径之一。近20年来,通过对厌氧铵氧化细菌生态学、基因组学和生理代谢特性的探索,人们对其微生物学机制已经有了较多的认识:厌氧铵氧化细菌通过亚硝酸盐还原酶将亚硝酸根离子还原为一氧化氮,进而与铵离子结合在联氨合成酶的作用下生成联氨,最后通过联氨氧化酶的催化产生终产物氮气。同时,对参与这些过程的关键酶及其功能基因的认识有助于选择新的分子标记,从而为研究厌氧铵氧化细菌的多样性和分子生态学特征提供新的工具,以弥补16S rRNA基因特异性相对较低且难以与生态功能关联等方面的不足。对目前已知的参与厌氧铵氧化过程的3种关键酶的研究历程和现状进行了评述,并总结了利用3种功能基因进行厌氧铵氧化细菌生态学研究的最新进展。     

Identification of key proteins involved in the anammox reaction

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N₂. Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N₂ production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N₂ and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.