双阳极室甲烷微生物燃料电池同步脱氮除硫性能及微生物群落分析

【背景】甲烷厌氧氧化(anaerobic oxidation of methane, AOM)包含反硝化型甲烷厌氧氧化和硫酸盐还原型甲烷厌氧氧化。目前,人们向水体中排放过量的含氮及含硫污染物,引起了严重的环境污染和生态破坏。【目的】利用甲烷厌氧氧化微生物燃料电池(microbial fuel cell, MFC)研究同步脱氮除硫耦合反应机理及反应过程中微生物的多样性信息。【方法】构建了3个微生物燃料电池(N-S-MFC、N-MFC、S-MFC),以甲烷作为唯一碳源,探究其同步脱氮除硫性能,并采用16S rRNA基因高通量测序技术对微生物群落结构进行分析。【结果】N-S-MFC中硝酸盐和硫酸盐的去除率分别为90.91%和18.46%。阳极室中微生物的相对丰度提高,与反硝化及硫酸盐还原菌相关的微生物大量富集,如门水平上拟杆菌门(Bacteroidota)、厚壁菌门(Firmicutes)和脱硫杆菌门(Desulfobacterota),同时属水平上Methylobacterium＿Methylorubrum、Methylocaldum、Methylomonas等常见的甲烷氧化菌增多。【结论...     

基于甲烷氧化菌的脱氮技术研究进展

甲烷氧化菌利用甲烷作为唯一碳源和能源,在氧化甲烷的过程中能有效地实现脱氮,该过程分为好氧甲烷氧化耦合反硝化(aerobic methane oxidation coupled to denitrification,AME-D)和厌氧甲烷氧化耦合反硝化(anaerobic methane oxidation coupled to denitrification,ANME-D),在碳循环和氮循环的研究中具有重要意义。本文通过总结近年来有关甲烷氧化菌的分类与分布,阐述AME-D和ANME-D的基本原理、影响因素和应用情况,提出相应的研究方向,以期为甲烷氧化菌在污水脱氮中的应用提供参考。     

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。     

以N-甲基吡咯烷酮(NMP)为电子供体进行反硝化的可行性及实验研究

【目的】利用N-甲基吡咯烷酮(N-methylpyrrolidone, NMP)作为电子供体进行反硝化实验,以实现废水资源化。【方法】分别将NMP废水和葡萄糖作为电子供体加入到模拟的城市污水处理尾水中进行反硝化,比较2种电子供体去除硝酸盐的规律。同时考查NMP在反硝化过程中的氮素释放规律,并对所释放的氮素进行后续处理。最后再对它们作为电子供体时的反硝化污泥采用高通量测序,从微生物群落的角度分析NMP作为电子供体时其作用机理是否相同。【结果】当以NMP为电子供体时,硝酸盐氮的去除速率比葡萄糖为电子供体时要快67%。在8 h的反硝化结束后,剩余的硝酸盐氮、累积的亚硝酸盐氮和NMP本身所释放氨氮之和的总氮,与葡萄糖为电子供体时相近。【结论】NMP废水可以作为电子供体用于城镇污水处理厂的深度脱氮。对2种碳源所驯化的反硝化污泥样品进行高通量分析表明,NMP与葡萄糖作为电子供体用于反硝化反应时,相关的作用机理是不同的。该项研究结果对利用含氮杂环化合物作为电子供体进行反硝化具有重要的理论指导意义。     

Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶活性

【背景】异养硝化-好氧反硝化菌由于能够同时实现硝化反硝化作用而备受关注,但由于菌的种类不同,其脱氮途径不尽相同,研究菌株脱氮关键酶的种类及其活性可以推测菌株的脱氮途径,从而为菌株在生产上的应用提供技术支撑。【目的】研究Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶的活性,为菌株脱氮分子机理研究奠定基础。【方法】以柠檬酸钠为碳源,以硫酸铵、亚硝酸钠、硝酸钾为氮源,研究菌株AD-28的脱氮性能并检测其关键酶氨单加氧酶(AMO)、羟胺氧化还原酶(HAO)、亚硝酸盐还原酶(NIR)、硝酸盐还原酶(NAR)的酶活性。【结果】菌株AD-28培养24h的菌密度(OD600)可达1.971,对初始浓度为18.85mg/L的氨氮、26.13mg/L的硝酸盐氮、19.47mg/L的亚硝酸盐氮、66.11 mg/L的总氮去除率均达到96%以上;关键酶AMO、HAO、NIR和NAR的比活力分别为0.028、0.003、0.011、0.027 U/mg。【结论】Pseudomonas alcaliphila AD-28能同时进行异养硝化-好养反硝化作用,该菌在AMO作用下将NH4+-N氧化为羟胺,然后由HAO氧化为NO2--N,NO2--N和NO3--N在NIR、NAR等酶的催化作用下脱氮。     

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。     

氢自养微生物的驯化及其反硝化特性研究北大核心

【目的】本研究筛选出弱酸性环境下利用无机碳源进行高效脱氮的氢自养微生物,探究不同无机碳源对体系反硝化能力的影响,以及长期驯化过程中反应器内水质参数、微生物群落结构和脱氮周期变化规律。【方法】氢自养微生物的驯化采用一种成本低廉、气密性优良、可计算氢气利用率的序批式反应器,通过及时向装置内补充氢气、无机碳源、营养液和硝酸盐对微生物进行连续驯化。【结果】驯化的微生物利用NaHCO_(3)和CO_(2)作为混合无机碳源对硝酸盐的脱氮效果要优于单一使用NaHCO_(3);在环境温度为20℃,pH为6.3-7.0,硝态氮初始投加量为15 mg-N/L时,NO_(3)--N最高反应速率为1.374 mg-N/(L·h),氢气最高利用率为43.4%,脱氮周期为16 h,且脱氮过程中无亚硝酸盐积累;驯化得到的微生物主要为嗜酸菌属(Acidovorax),占比达84.4%。【结论】利用本研究的装置和驯化方法对土著微生物进行脱氮驯化是可行且高效的,可筛选出在弱酸性环境下利用无机碳源进行反硝化的氢自养微生物,为地下水中硝酸盐污染的生物修复提供理论依据,也为后续进一步研究弱酸性环境下氢自养微生物同时脱氮固铀奠定基础。     

生物电化学耦合厌氧氨氧化强化脱氮及其微生物群落特征

为探究生物电化学强化厌氧氨氧化(anaerobic ammonia oxidation,anammox)脱氮作用过程,采用双室微生物电解池(microbial electrolysis cell,MEC)富集电活性微生物,构建耦合厌氧氨氧化阴极的生物电化学系统。具体地,在外加0.2 V电压条件下改变不同总氮进水浓度于30°C进行暗培养批次实验研究,结合循环伏安法、电化学阻抗谱、高通量测序方法等多种表征手段研究了强化脱氮机理。结果表明,在初始总氮浓度分别为200、300和400 mg/L时对应获得了96.9%±0.3%、97.3%±0.4%和99.0%±0.3%的总氮去除率,且阴极电极生物膜表现出良好的电化学活性。高通量测序结果表明外加电压富集了除厌氧氨氧化菌以外的其他脱氮功能菌群:反硝化菌(Denitratisoma)、Limnobacter和氨氧化菌SM1A02和Anaerolineaceae、亚硝化菌(Nitrosomonas europaea)和硝化螺菌属(Nitrospira)等,这些具有电化学活性的微生物构成了体系的氨氧化胞外产电菌(ammonium oxidizing exoelectrogens,AOE)和反硝化电养菌(denitrifying electrotrophs,DNE),它们连同厌氧氨氧化菌Candidatus Brocadia构成了系统的脱氮微生物群落结构。AOE和DNE的种间直接电子传递作用协同厌氧氨氧化是强化系统总氮去除的关键原因。     

微生物介导硝酸盐还原耦合亚铁氧化过程的动力学及其影响因素

【目的】探究不同菌浓度和亚铁浓度条件下,Acidovorax sp. strain BoFeN1介导的厌氧亚铁氧化耦合硝酸盐还原过程的动力学和次生矿物。【方法】构建包含菌BoFeN1、硝酸盐、亚铁的厌氧培养体系,测试硝酸根、亚硝酸根、乙酸根、亚铁等浓度,并收集次生矿物,采用XRD、SEM进行矿物种类和形貌表征。【结果】在微生物介导硝酸盐还原耦合亚铁氧化的体系中,高菌浓度促进硝酸盐还原,对亚铁氧化也有一定促进作用;高浓度亚铁在低菌浓度下氧化反应速率和程度降低,但是在高菌浓度下无明显影响;亚铁浓度越高次生矿物结晶度越高,但对硝酸盐还原具有一定抑制作用。在微生物介导亚硝酸盐还原耦合亚铁氧化的体系中,高的菌浓度和亚铁浓度都会促进亚硝酸盐还原,但亚铁氧化的次生矿物会对亚硝酸盐的微生物还原产生较强的抑制作用,次生矿物的种类和结晶度主要受亚铁浓度影响。【结论】硝酸盐还原主要是生物反硝化作用,亚硝酸盐还原包含生物反硝化和化学反硝化两部分,在硝酸盐体系中亚铁氧化与次生矿物生成是受生物和化学反硝化作用的共同影响,但亚硝酸盐体系中亚铁氧化与次生矿物生成主要是受化学反硝化作用影响。该研究可为深入理解厌氧微生物介导铁氮耦合反应机制提供基础数据和理论支撑。     

好氧反硝化细菌SY-D-22的分离、优化及脱氮机理

【背景】在处理含硝酸盐氮的废水中,常见微生物不能同时高效去除硝酸盐氮和总氮,导致处理废水时往往使用多种微生物复合菌剂或者使用复杂的工艺。【目的】高效、安全地去除水中的硝酸盐氮和总氮。方法】富集筛选出一株新型高效好氧反硝化细菌,对其进行16S rRNA基因鉴定。利用响应面法对其影响脱氮因素进行优化并探索其最佳脱氮条件。研究其对含硝酸盐氮废水的反硝化能力及脱氮特性。【结果】从活性污泥中筛选获得一株新型高效好氧反硝化细菌SY-D-22,经鉴定为葡萄球菌属(Staphylococcus)。响应面法优化其最佳反硝化条件为：pH 8.18,C/N为13.39,温度31.43°C,摇床转速130 r/min。当以最佳碳源柠檬酸钠为唯一碳源时,对于100 mg/L浓度的NO₃^--N去除率可达100%,同时对于总氮(total nitrogen,TN)的去除率为95.34%,具有高效脱氮能力。【结论】从活性污泥中筛选出一株新型好氧反硝化细菌Staphylococcus SY-D-22,通过响应面法条件优化,菌株的硝酸盐氮去除率达到100.00%,总氮去除率达到...     

The bioenergetics of denitrification

In anaerobically grown Paracoccus denitrificans the dissimilatory nitrate reductase is linked to the respiratory chain at the level of cytochromes b. Electron transport to nitrite and nitrous oxide involves c-type cytochromes. During electron transport from NADH to nitrate one phosphorylation site is passed, whereas two sites are passed during electron transport from NADH to oxygen, nitrite and nitrous oxide. The presentation of a respiratory chain as a linear array of electron carriers gives a misleading picture of the efficiency of energy conservation since the location of the reductases is not taken into account. For the reduction of nitrite and nitrous oxide, protons are utilized from the periplasmic space, whereas for the reduction of oxygen and nitrate, protons are utilized from the cytoplasmic side of the inner membrane. Evidence for two transport systems for nitrate was obtained. One is driven by the proton motive force; this system is used to initiate nitrate reduction. The second system is a nitrate-nitrite antiport system. A scheme for proton translocation and electron transport to nitrate, nitrite, nitrous oxide and oxygen is presented. The number of charges translocated across the membrane during flow of two electrons from NADH is the same for all nitrogenous oxides and is 67-71% of that during electron transfer to oxygen via cytochrome o. These findings are in accordance with growth yield studies. YMAX electron values determined in chemostat cultures for growth with various substrates and hydrogen acceptors are proportional to the number of charges translocated to these hydrogen acceptors during electron transport.     

Structural basis of denitrification

Denitrification represents an important part of the biogeochemical cycle of the essential element nitrogen. It constitutes the predominant pathway of the reductive dissimilation of nitrate in the environment. Via four enzymatic reactions, nitrate is transformed stepwise to nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O), to finally yield dinitrogen gas (N2). All steps within this metabolic pathway are catalyzed by complex multi-site metalloenzymes with unique spectroscopic and structural features. In recent years, high-resolution crystal structures have become available for these enzymes with the exception of the structure for NO reductase.     

Using the combined bioelectrochemical and sulfur autotrophic denitrification system for groundwater denitrification

A combined bioelectrochemical and sulfur autotrophic denitrification system (CBSAD) was evaluated to treat a groundwater with nitrate contamination (20.9-22.0mgNO(3)(-)-N/L). The reactor was operated continuously for several months with groundwater to maximize treatment efficiency under different hydraulic retention times (HRT) and electric currents. The denitrification rate of sulfur autotrophic part followed a half-order kinetics model. Moreover, the removal efficiency of bioelectrochemical part depended on the electric current. The reactor could be operated efficiently at the HRT ranged from 4.2 to 2.1h (corresponding nitrogen volume-loading rates varied from 0.12 to 0.24 kg N/m(3)d; and optimum current ranged from 30 to 1000 mA), and the NO(3)(-)-N removal rate ranged from 95% to 100% without NO(3)(-)-N accumulation. The pH of effluent was satisfactorily adjusted by bioelectrochemical part, and the sulfate concentration of effluent was lower than 250 mg/L, meeting the drinking water standard of China EPA.     

Monitoring denitrification by pH-Stat titration

An improved pH-stat titrimetric procedure was developed, validated, and extensively applied to monitor biological heterotrophic denitrification in a lab-scale sequencing batch reactor (SBR). So far, titrimetric procedures were not successful in monitoring denitrification processes in full-scale wastewater (WW) treatment plants, mainly because the stoichiometric ratio between proton production and nitrate reduction is highly variable due to variability of both biomass and influent WW characteristics. In this article, a new titration procedure is proposed where a simple calibration step is performed before each experimental test. This procedure allows for the assessment of (i) nitrate content in a sample of mixed liquor; (ii) the maximum denitrification rate of sludge when fed on acetate; and (iii) the denitrification potential (DNP) of different substrates. As for (i), validation by comparison with spectrophotometric measures indicated an average discrepancy of less than 3% on more than 40 samples; as for (ii) and (iii) collected values compared well with literature data. The titrimetric method proposed here is also capable of assessing the biomass anoxic yield in a very simple way, since it does not require any analytical nitrate determination. According to the results of this experimentation, titrimetry appeared to be a simple, inexpensive, and powerful tool for monitoring and operating denitrification processes in WW treatment plants.     

Anoxic treatment wetlands for denitrification

Anoxic subsurface flow (SSF) constructed wetlands were evaluated for denitrification using nitrified wastewater. The treatment wetlands utilized a readily available organic woodchip-media packing to create the anoxic conditions. After 2 years in operation, nitrate removal was found to be best described by first-order kinetics. Removal rate constants at 20 °C (k₂₀) were determined to be 1.41–1.30 d^?1, with temperature coefficients (θ) of 1.10 and 1.17, for planted and unplanted experimental woodchip-media SSF wetlands, respectively. First-order removal rate constants decreased as length of operation increased; however, a longer-term study is needed to establish the steady-state values. The hydraulic conductivity in the planted woodchip-media SSF wetlands, 0.13–0.15 m/s, was similar to that measured in an unplanted gravel-media SSF control system.