以亚硝氮为唯一氮源生长的海洋紫色硫细菌去除无机三态氮

【目的】揭示以亚硝氮为唯一氮源生长的海洋紫色硫细菌去除水体中无机三态氮的特征和规律。【方法】在光照厌氧环境下,以乙酸盐为唯一有机物,在分别以氨氮、亚硝态氮、硝态氮为唯一氮源和三氮共存的模拟水体中,采用Nessler’s试剂分光光度法、N-(1-萘基)-乙二胺分光光度法和紫外分光光度法分别测定水体中氨氮、亚硝态氮和硝态氮的含量,比浊法测定菌体生物量。【结果】随着时间的延长,海洋紫色硫细菌Marichromatium gracile YL28分别在氨氮、亚硝态氮和硝态氮为唯一氮源的水体中对三氮的去除量增加,生物量增大,水体pH升高,并逐渐趋于平衡;YL28对氨氮的最大去除量和最大耐受浓度分别为9.64 mmol/L和36.64 mmol/L,当氨氮浓度低于3.21 mmol/L时,去除率可达97.61%以上;与氨氮相比,以亚硝态氮和硝态氮为唯一氮源,菌体的生长速率、生物量和水体最终pH较低,但对亚硝态氮和硝态氮的去除速率和去除量仍然很高,当亚硝态氮和硝态氮浓度分别达13.50 mmol/L和22.90 mmol/L时,YL28仍能够完全去除。在三氮共存的水体中,YL28也能良好的去除无机三态氮,对亚硝态氮和硝态氮去除能力更强。【结论】在模拟水体中,海洋紫色硫细菌YL28能够分别以氨氮、亚硝态氮和硝态氮为唯一氮源生长,具有良好的耐受和去除无机三态氮的能力,尤其对亚硝态氮具有良好的去除能力。本研究为进一步开发高效脱氮,尤其是去除亚硝态氮的不产氧光合细菌水质调节剂奠定了基础,也为微生物制剂的合理应用提供参考。     

金属离子对粪产碱杆菌C16的脱氮和亚硝酸盐积累的影响

【目的】研究不同金属离子对异养氨氧化细菌C16的生长和脱氮性能影响,探讨适于C16生长和脱氮的金属离子及其浓度。【方法】实验选用Mg2+、Mn2+、Fe2+、Cu2+、Zn2+5种金属离子,对C16的生长﹑脱氮性能﹑亚硝酸盐氮积累以及相关酶活性进行研究。【结果】Mg2+明显促进C16的生长和NH4+-N氧化速率;较高浓度Mn2+使得C16无法生长;原培养基中缺少Fe2+会抑制C16的生长和NH4+-N氧化速率;在原培养基中加入0.1 mmol/L的Cu2+对C16的生长和脱氮具有一定的促进作用,Cu2+使得培养基中基本无NO2--N和NH2OH的积累;不同浓度的Zn2+对C16的生长和氨氮去除有抑制作用。酶活实验结果显示,0.1 mmol/L Mg2+促进了羟胺氧化还原酶(HAO)的活性;0.1 mmol/L Cu2+促进了硝酸盐还原酶(Nar)和亚硝酸盐还原酶(Nir)的活性。【结论】Mg2+是C16生长和脱氮过程中的一种重要金属离子;加入Cu2+可避免过量亚硝酸盐积累。     

厌氧氨氧化菌混培物生长及代谢动力学研究

研究了厌氧氨氧化菌混培物的动力学特性.测得细胞产率系数1.573mgVS(mmolNH4+)-1;细胞衰减常数0.052mgVS(g@VS@d)-1.厌氧氨氧化菌混培物的最大氨氧化速率1.320～2.761mmol(gVS@d)-1,最大亚硝酸盐转化(反硝化)速率14.497mmol(gVS@d)-1.厌氧氨氧化菌混培物利用氨的Km值1.801～4.215mmol@L-1,利用亚硝酸盐的Km值0.468mmol@L-1.氨自身的抑制常数38.018～98.465mmol@L,实际最大氨氧化速率的氨浓度16.656mmol@L-1.亚硝酸盐对厌氧氨氧化的抑制常数5.401～11.995mmol@L-1.厌氧氨氧化的最适pH7.605.厌氧氨氧化的最适温度30℃.Vmaxa、Kma、Kia和Kin的活化能依次为37.316、30.239、33.695和30.473kJ@mol-1.     

亚硝氮对海洋着色菌亚硝氮和氨氮去除以及光合色素合成的影响

【目的】在以亚硝氮为唯一氮源和亚硝氮-氨氮共存体系中,考察和分析海洋着色菌(Marichromatium gracile) YL28菌株对水体亚硝氮的环境适应能力。【方法】采用分光光度法分析亚硝氮、氨氮去除效率以及亚硝氮对菌体生物量和色素含量的影响,采用薄层层析法分析亚硝氮对菌体光合色素组成的影响。【结果】YL28菌株能以亚硝氮为唯一氮源生长,主要积累2种细菌叶绿素(BChl)组分(BChl aTHGG和BChl ap)、1种细菌脱镁叶绿素(Bphe)和玫红品(Rhodopin)、螺菌黄质(Spirilloxanthin)、脱水紫菌红醇(Anhydrorhodovibrin)、番茄红素(Lycopene) 4种类胡萝卜素(Car);YL28生物量和对亚硝氮的去除效率随亚硝氮浓度升高而降低,完全去除亚硝氮的浓度可达200 mg/L以上;当亚硝氮浓度高于25 mg/L,单位质量菌体BChl a和Car总量降低,BChl a和Car合成的末端产物(BChl ap和Spirilloxanthin)以及Bphe相对含量升高,其它4种色素组分相对含量则降低,但Car与BChl a相对含量的比值未见明显变化。当亚硝氮-氨氮共存时,YL28菌株对亚硝氮的耐受能力和去除能力明显提高,完全去除亚硝氮的浓度可达300 mg/L以上;氨氮减缓了亚硝氮对光合色素合成的抑制作用,提高了菌体色素合成总量,各色素组分相对含量的变化与亚硝氮为唯一氮源时的变化规律一致。【结论】YL28菌株能高效去除亚硝氮,亚硝氮对菌株生长和光合色素的合成有抑制作用,但氨氮能明显提高YL28菌株对亚硝氮的适应能力。这为进一步开发高效脱除亚硝氮的APB水质调节剂奠定了基础。     

利用硝酸盐和亚硝酸盐同步富集厌氧甲烷氧化微生物的比较实验

【背景】反硝化厌氧甲烷氧化(Denitrifying anaerobic methane oxidation,DAMO)是以硝酸盐或亚硝酸盐为电子受体以甲烷为电子供体的厌氧氧化过程,对认识全球碳氮循环、削减温室气体排放和开发废水脱氮新技术等方面具有重要意义。【目的】认识以硝酸盐和亚硝酸盐为电子受体的DAMO微生物富集过程和结果的差异性。【方法】在序批式反应器(Sequencing batch reaetor,SBR)内接种混合物,分别以硝酸盐和亚硝酸盐为电子受体连续培养800 d,定期检测反应器基质浓度变化、计算转化速率;利用16S rRNA基因系统发育分析研究功能微生物的多样性,利用实时荧光定量PCR技术定量测定功能微生物。【结果】以亚硝酸盐为电子受体的1、3号反应器富集到了DAMO细菌,未检测到DAMO古菌;以硝酸盐为电子受体的2号反应器富集到了DAMO细菌和古菌的混合物;3个反应器的脱氮速率经过初始低速期、快速提升期,最终达到稳定,但2号快速提升期开始时间比1、3号晚了80 d左右,达到稳定的时间更长,稳定最大速率为1、3号的44.7%、40.3%。【结论】硝酸盐和亚硝酸盐对富集产物有决定性影响;以硝酸盐为电子受体富集得到的DAMO古菌和细菌协同体系可以长期稳定共存,DAMO古菌可能是协同体系中脱氮速率的限制性因素。     

生产性短程硝化-厌氧氨氧化装置处理制药废水的启动性能

为拓展新型生物脱氮技术的应用领域,研究了生产性短程硝化-厌氧氨氧化装置处理制药废水的启动性能。制药废水氨氮浓度为(430.40±55.43)mg/L时,氨氮去除率达(81.75±9.10)%,实现了短程硝化-厌氧氨氧化工艺对制药废水的生物脱氮。制药废水短程硝化系统的启动时间约为74 d,亚硝氮积累率达(52.11±9.13)%,证明了结合模拟废水和实际废水的"两步法"模式对短程硝化系统启动的适用性。制药废水厌氧氨氧化系统的启动时间约为145 d,最大容积氮去除速率达6.35 kg N/(m3·d),容积效能为传统硝化-反硝化工艺的数十倍,证明了结合菌种自繁和菌种流加的模式对厌氧氨氧化系统启动的适用性。     

厌氧氨氧化反应器的启动研究

目的：对UASB-生物膜反应器进行厌氧氨氧化反应的启动研究。方法：以自配含氨氮和亚硝氮的废水为进水,以氧化沟工艺城市污水处理厂回流污泥为接种污泥。结果：反应器内部菌群进行了竞争,在运行至第66d时氨氮、亚硝酸盐氮的去除率分别达到了60.4%、58.7%,同时有硝酸盐氮生成,表明厌氧氨氧化反应已经成为反应器内的主导反应。结论：厌氧氨氧化反应器实现了快速启动。     

异养硝化-好氧反硝化菌Acinetobacter johnsonii sp. N26的脱氮性能及代谢途径

【背景】水体中含氮物质的大量累积会造成水体富营养化、水生生物死亡等问题,严重威胁水生态环境,制约我国环境保护的持续发展。【目的】为去除生活污水中的含氮污染物,从羊粪堆肥中筛选出了一株具有异养硝化-好氧反硝化功能的细菌——约氏不动杆菌Acinetobacter johnsonii sp.N26,研究其脱氮性能和代谢途径。【方法】测定菌株N26在氨氮和硝态氮中的生长和脱氮曲线,通过单因素试验对其脱氮性能进行优化,通过氮平衡分析和功能基因鉴定研究其脱氮代谢途径。【结果】生长和脱氮曲线表明,菌株N26对初始浓度均为50 mg/L的氨氮和硝态氮的去除速度快、效率高,其中9 h内对氨氮的去除效率为95.5%,最大去除速率为5.330 mg/(L·h);15 h内对硝态氮的去除效率为93.6%,最大去除速率为3.147 mg/(L·h),且最终仅有少量硝酸盐、亚硝酸盐积累。脱氮性能优化结果表明,该菌株的最适氮源为氯化铵,最适碳源为丁二酸钠,最适温度为30℃,最适接种量为15%,最适p H值为8.0-9.0,最适碳氮比为15,最适转速为120 r/min,最适氮负荷≤300 mg/L (氨氮)。氮平衡...     

硝化基质和产物对发光细菌的急性毒性

摘要：【目的】对硝化基质和产物对硝化过程的影响进行初步研究。【方法】采用发光细菌法,在pH=7.0的条件下,测定了氨、羟胺、亚硝酸和硝酸对发光细菌的急性毒性（15min-半抑制浓度（the half inhibitory concentration,IC50））。【结果】单一物质的毒性试验结果表明,硝化基质和产物对发光细菌的毒性随浓度的升高而增大,且具有较好的线性关系;氨、羟胺、亚硝酸和硝酸的IC50分别为2180.2 mg/L、6.2740 mg/L、1207.2 mg/L和3140.3 mg/L;其毒性大小顺序为：羟胺 >亚硝酸 >氨 >硝酸。按等效浓度混合法测定硝化基质和产物的联合毒性,结果表明：氨与羟胺、氨与亚硝酸、羟胺与亚硝酸对发光细菌的联合毒性呈相加作用;氨与硝酸、羟胺与硝酸、亚硝酸与硝酸对发光细菌的联合毒性呈独立作用;氨、羟胺、亚硝酸、硝酸四元混合物的联合毒性也呈相加作用。【结论】根据硝化基质和产物对发光细菌和硝化细菌抑制浓度的相关性,可用发光细菌发光强度的变化指示硝化基质和产物的抑制作用。     

氧对膜生物反应器短程硝化的影响

为了研究膜生物反应器的短程硝化性能以及氧对短程硝化的影响,通过对比耗氧率和供氧率,提出了膜生物反应器短程硝化的控制优化建议。在膜生物反应器硝化过程中,DO小于1 mg/L开始出现亚硝氮积累;DO降到0.5 mg/L,出水氨氮浓度与亚硝氮浓度之比接近1∶1;DO调控在0.5-1 mg/L范围内,有利于前置硝化反应器与后续厌氧氨氧化反应器衔接。膜生物反应器中污泥浓度可达20 g/L,耗氧能力可达19.86 mg O2/(L·s),但最大供氧能力仅为0.369 mg O2/(L·s),供氧成为反应器运行的制约瓶颈,"低DO高流量"曝气是继续提高短程硝化效能的控制策略。     

Characteristics of nitrogenous substrate conversion by anammox enrichment

The characteristics of nitrogenous substrates conversion by anammox enrichment were investigated using batch experiments. The anammox enrichment was proved able to convert hydroxylamine to hydrazine, as well as convert hydrazine to ammonia anaerobically, with the average conversion rates of 0.207 and 0.031 mmol gVSS⁻¹ h⁻¹. It could convert hydroxylamine and nitrite simultaneously, with ammonia as an intermediate product. The maximum conversion rates of hydroxylamine and nitrite were 0.535 and 0.145 mmol gVSS⁻¹ h⁻¹, respectively. Their conversion rates were enhanced by 26.7% and 120.7%, respectively, by raising the ratio of hydroxylamine to nitrite from 1:1 to 2:1. The characteristics of nitrogenous substrate conversion by anammox enrichment could be explained using the speculative anammox pathway based on van de Graaf model.     

Key Physiology of Anaerobic Ammonium Oxidation

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43°C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1.4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.     

Key physiology of anaerobic ammonium oxidation.

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43 degrees C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1. 4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.     

ANAMMOX process start up and stabilization with an anaerobic seed in Anaerobic Membrane Bioreactor (AnMBR)

ANaerobic AMMonium OXidation (ANAMMOX) process, an advanced biological nitrogen removal alternative to traditional nitrification--denitrification removes ammonia using nitrite as the electron acceptor without oxygen. The feasibility of enriching anammox bacteria from anaerobic seed culture to start up an Anaerobic Membrane Bioreactor (AnMBR) for N-removal is reported in this paper. The Anammox activity was established in the AnMBR with anaerobic digester seed culture from a Sewage Treatment Plant in batch mode with recirculation followed by semi continuous process and continuous modes of operation. The AnMBR performance under varying Nitrogen Loading Rates (NLR) and HRTs is reported for a year, in terms of nitrogen transformations to ammoniacal nitrogen, nitrite and nitrate along with hydrazine and hydroxylamine. Interestingly ANAMMOX process was evident from simultaneous Amm-N and nitrite reduction, consistent nitrate production, hydrazine and hydroxylamine presence, notable organic load reduction and bicarbonate consumption.     

Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium-oxidizing bacteria

Bacterial aerobic ammonium oxidation and anaerobic ammonium oxidation (anammox) are important processes in the global nitrogen cycle. Key enzymes in both processes are the octahaem cytochrome c (OCC) proteins, hydroxylamine oxidoreductase (HAO) of aerobic ammonium-oxidizing bacteria (AOB), which catalyses the oxidation of hydroxylamine to nitrite, and hydrazine oxidoreductase (HZO) of anammox bacteria, which converts hydrazine to N(2). While the genomes of AOB encode up to three nearly identical copies of hao operons, genome analysis of Candidatus'Kuenenia stuttgartiensis' showed eight highly divergent octahaem protein coding regions as possible candidates for the HZO. Based on their phylogenetic relationship and biochemical characteristics, the sequences of these eight gene products grouped in three clusters. Degenerate primers were designed on the basis of available gene sequences with the aim to detect hao and hzo genes in various ecosystems. The hao primer pairs amplified gene fragments from 738 to 1172 bp and the hzo primer pairs amplified gene fragments from 289 to 876 bp in length, when tested on genomic DNA isolated from a variety of AOB and anammox bacteria. A selection of these primer pairs was also used successfully to amplify and analyse the hao and hzo genes in community DNA isolated from different ecosystems harbouring both AOB and anammox bacteria. We propose that OCC protein-encoding genes are suitable targets for molecular ecological studies on both aerobic and anaerobic ammonium-oxidizing bacteria.     

Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate

Anaerobic ammonium oxidation with nitrite to N2 (anammox) is a recently discovered microbial reaction with interesting potential for nitrogen removal from wastewater. We enriched an anammox culture from a rotating disk contactor (near K?lliken, Switzerland) that was used to treat ammonium-rich leachate with low organic carbon content. This enrichment led to a relative population size of 88% anammox bacteria. The microorganism carrying out the anammox reaction was identified by analysis of the 16S rDNA sequence and by fluorescence in situ hybridization (FISH) with 16S-rRNA-targeting probes. The percentage sequence identity between the 16S rDNA sequences of the K?lliken anammox organism and the archetype anammox strain Candidatus Brocadia anammoxidans was 90.9%, but between 98.5 and 98.9% with Candidatus Kuenenia stuttgartiensis, an organism identified in biofilms by molecular methods. The K?lliken culture catalyzed the anaerobic oxidation of ammonium with nitrite in a manner seemingly identical to that of Candidatus B. anammoxidans, but exhibited higher tolerance to phosphate (up to 20 mM) and to nitrite (up to 13 mM) and was active at lower cell densities. Anammox activity was observed only between pH 6.5 and 9, with an optimum at pH 8 and a temperature optimum at 37 degrees C. Hydroxylamine and hydrazine, which are intermediates of the anammox reaction of Candidatus B. anammoxidans, were utilized by the K?lliken organisms, and approximately 15% of the nitrite utilized during autotrophic growth was converted to nitrate. Electron microscopy showed a protein-rich region in the center of the cells surrounded by a doughnut-shaped region containing ribosomes and DNA. This doughnut-shape region was observed with FISH as having a higher fluorescence intensity. Similar to Candidatus B. anammoxidans, the K?lliken anammox organism typically formed homogenous clusters containing up to several hundred cells within an extracellular matrix.     

The membrane bioreactor: a novel tool to grow anammox bacteria as free cells

In a membrane bioreactor (MBR), fast growth of anammox bacteria was achieved with a sludge residence time (SRT) of 12 days. This relatively short SRT resulted in a--for anammox bacteria--unprecedented purity of the enrichment of 97.6%. The absence of a selective pressure for settling, and dedicated cultivation conditions led to growth in suspension as free cells and the complete absence of flocs or granules. Fast growth, low levels of calcium and magnesium, and possibly the presence of yeast extract and a low shear stress are critical for the obtainment of a completely suspended culture consisting of free anammox cells. During cultivation, a population shift was observed from Candidatus "Brocadia" to Candidatus "Kuenenia stuttgartiensis." It is hypothesized that the reason for this shift is the higher affinity for nitrite of "Kuenenia." The production of anammox bacteria in suspension with high purity and productivity makes the MBR a promising tool for the cultivation and study of anammox bacteria.     

厌氧氨氧化菌的中心代谢研究进展

摘要: 厌氧氨氧化是以NH +4为电子供体,以NO－2为电子受体产生N2的生物反应。厌氧氨氧化菌是厌氧氨氧化过程的执行者,在废水生物脱氮和地球氮素循环中扮演着重要角色。研究厌氧氨氧化菌的代谢特性,将有助于理解厌氧氨氧化过程,开发厌氧氨氧化工艺。厌氧氨氧化菌是化能自养型细菌,以CO2或HCO－3为碳源,并通过偶联NH+4氧化和NO －2还原的生物反应获得能量。在NH+4/NO－2的生物氧化还原反应过程中,检出了中间产物N2H4,但未检出其他中间产物(如NH2OH、NO)。此外,由基因组信息推断,厌氧氨氧化菌     

New anaerobic, ammonium-oxidizing community enriched from peat soil

Anaerobic ammonium-oxidizing (anammox) bacteria have been recognized as an important sink for fixed nitrogen and are detected in many natural environments. However, their presence in terrestrial ecosystems has long been overlooked, and their contribution to the nitrogen cycling in natural and agricultural soils is currently unknown. Here we describe the enrichment and characterization of anammox bacteria from a nitrogen-loaded peat soil. After 8 months of incubation with the natural surface water of the sampling site and increasing ammonium and nitrite concentrations, anammox cells constituted 40 to 50% of the enrichment culture. The two dominant anammox phylotypes were affiliated with "Candidatus Jettenia asiatica" and "Candidatus Brocadia fulgida." The enrichment culture converted NH(4)(+) and NO(2)(-) to N(2) with the previously reported stoichiometry (1:1.27) and had a maximum specific anaerobic ammonium oxidation rate of 0.94 mmol NH(4)(+)·g (dry weight)(-1)·h(-1) at pH 7.1 and 32°C. The diagnostic anammox-specific lipids were detected at a concentration of 650 ng·g (dry weight)(-1), and pentyl-[3]-ladderane was the most abundant ladderane lipid.     

Propionate oxidation by and methanol inhibition of anaerobic ammonium-oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway and a cost-effective way to remove ammonium from wastewater. Anammox bacteria have been described as obligate chemolithoautotrophs. However, many chemolithoautotrophs (i.e., nitrifiers) can use organic compounds as a supplementary carbon source. In this study, the effect of organic compounds on anammox bacteria was investigated. It was shown that alcohols inhibited anammox bacteria, while organic acids were converted by them. Methanol was the most potent inhibitor, leading to complete and irreversible loss of activity at concentrations as low as 0.5 mM. Of the organic acids acetate and propionate, propionate was consumed at a higher rate (0.8 nmol min(-1) mg of protein(-1)) by Percoll-purified anammox cells. Glucose, formate, and alanine had no effect on the anammox process. It was shown that propionate was oxidized mainly to CO(2), with nitrate and/or nitrite as the electron acceptor. The anammox bacteria carried out propionate oxidation simultaneously with anaerobic ammonium oxidation. In an anammox enrichment culture fed with propionate for 150 days, the relative amounts of anammox cells and denitrifiers did not change significantly over time, indicating that anammox bacteria could compete successfully with heterotrophic denitrifiers for propionate. In conclusion, this study shows that anammox bacteria have a more versatile metabolism than previously assumed.