【作者】 陈丹；

【导师】 杨开；

【作者基本信息】 武汉大学 ， 市政工程， 2017， 博士

【摘要】 由于地表及地下水中超标的硝酸盐会对水环境和人类健康造成严重威胁,寻求高效、低耗、无二次污染的硝酸盐去除技术已成为当今水处理界的一个重要课题。而生物处理技术因具有高效低耗的特点,己成为最具优势的硝酸盐去除技术。虽然硝酸氮的生物处理技术已经得到了广泛的研究和论证,但是很多情况下硝酸氮污染的水体会同时面临着重金属和致毒有机物的威胁,目前,虽然部分研究开始涉足生物反应器对硝酸氮、重金属及有机物的同步去除,但是关于重金属和致毒有机物对反硝化的影响以及与硝酸氮同步降解的机理并没有系统的研究,本文尝试在反硝化的前提下,研究几种重金属及致毒有机物对生物自养反硝化系统反硝化效果、优势菌群、关键酶活性、反硝化功能基因、胞外聚合物等的影响,从宏观及微观层面揭示几种重金属及致毒有机物对反硝化过程的毒性效应,对生物自养反硝化的实际应用具有重要的意义。本文构建硫自养-电化学氢自养耦合反硝化系统,研究重金属离子Cr(VI)和V(V),致毒有机物苯酚(phenol)、对硝基酚(PNP)和氯霉素(CAP)对耦合系统反硝化过程的影响。结果表明当耦合反硝化系统初始Cr(VI)的浓度从零增加到150 mg/L时,耦合反硝化系统的脱氮效率不断降低。在100mg/L初始Cr(VI)的胁迫下,耦合反硝化系统对100 mg/L浓度以下的NO3--N均能够基本实现完全反硝化,但当初始N03--N的浓度为200 mg/L时,耦合系统的脱氮效果降低到81.69%。在100 mg/L初始Cr(VI)的胁迫下,耦合反硝化系统在pH为7时能取得最好的反硝化效果,硝酸氮的平均去除率达到93.01%。当初始Cr(VI)的浓度为150 mg/L时,耦合反硝化系统中的硝酸盐还原酶和亚硝酸盐还原酶的活性均出现了较大幅度的降低。100 mg/L以内的Cr(VI)对耦合反硝化系统生物膜的形成的影响较小,但是继续增加Cr(VI)的浓度到150 mg/L,耦合反硝化系统内的微生物受到明显影响,将分泌较少的EPS。耦合系统在Cr(VI)的胁迫环境下某些反硝化微生物会受到一定的毒性影响而使其减少或者消失。耦合系统能够取得较好的反硝化效果,主要依靠Proteobacteria、Firmicutes菌门和Betaproteobacteria、Clostridia菌纲的反硝化功能。当初始Cr(VI)的浓度从0增加到150mg/L,耦合反硝化系统中主要的反硝化功能基因nirS,nirK,narG,napA的拷贝数均呈现下降的趋势。Cr(VI)在耦合反应系统内被去除主要是通过生物还原过程变为Cr(III)与生物膜结合在一起,或最终随生物膜一起脱落并被排出反应器。当耦合反硝化系统初始V(V)的浓度从零增加到100 mg/L时,耦合反硝化系统的脱氮效率不断降低。在60 mg/L初始V(V)的胁迫下,当电流从50 mA增加到200 mA时,耦合反硝化系统的反硝化效果逐渐增加,但当电流增大到400 mA时,耦合系统反硝化的效果反而降低。在60 mg/L初始V(Ⅴ)的胁迫下,耦合反硝化系统在中性环境下脱氮效果最佳,酸性或碱性环境都会降低脱氮效果。当初始V(Ⅴ)的浓度大于60 mg/L时,硝酸盐还原酶活性和亚硝酸盐还原酶活性均显著地降低,这与明显降低的硝酸氮的去除率相对应。LDH释放率和ROS产率的分析结果表明了高于60 mg/L的V(Ⅴ)的胁迫环境会对硫自养-电化学氢自养耦合反硝化系统内的微生物产生损害。V(Ⅴ)的胁迫会使硫自养-电化学氢自养耦合反硝化系统内的微生物丰度、物种总数和微生物多样性都呈现下降的趋势。耦合反硝化系统中占主导地位的菌门和菌纲为Proteobacteria、Firmicutes菌门和Betaproteobacteria、Clostridia、Gammaproteobacteria、Alphaproteobacteria菌纲。不断增加的V(V)的浓度会对耦合反硝化系统中主要的反硝化功能基因的丰度产生抑制作用,从而导致耦合系统反硝化效果的不断降低。被去除的V(Ⅴ)在耦合反硝化系统中被生物还原的最终产物为V(Ⅳ)。当耦合反硝化系统初始苯酚的浓度从零增加到100 mg/L时,耦合反硝化系统的脱氮效率不断降低,100 mg/L的苯酚对耦合反硝化系统的抑制作用最为明显。在50 mg/L初始苯酚的胁迫下,随着初始硝酸氮的浓度的增加,耦合反硝化系统对硝酸氮的去除率逐渐降低。在50 mg/L初始苯酚的胁迫下,pH为7时硫自养-电化学氢自养耦合反硝化系统取得最佳的脱氮效果。随着初始苯酚的浓度的增加,硝酸盐还原酶活性和亚硝酸盐还原酶活性均呈现出降低的趋势,这与不断降低的反硝化效率相一致。随着初始苯酚浓度的增加,耦合反硝化系统分泌越来越少的EPS,从而影响耦合反硝化系统内生物膜的形成,降低脱氮效率。当初始苯酚浓度低于50 mg/L时,耦合反硝化系统内的微生物多样性在苯酚的刺激下表现出增加的趋势,但是当苯酚浓度增加到100 mg/L时,耦合反硝化系统内的微生物的多样性下降,100 mg/L的苯酚对耦合反硝化系统内的微生物产生了较为明显的负面影响。苯酚在耦合反硝化系统中被去除,主要是作为碳源参与异养反硝化过程,此时耦合系统中的反硝化过程是自养和异养结合的反硝化过程。当初始对硝基酚的浓度从零增加到100 mg/L,耦合反硝化系统的反硝化效率逐渐降低,但是降低的幅度相对较小。在60 mg/L的初始对硝基酚的胁迫环境下,随着初始硝酸氮的浓度不断增加,耦合反硝化系统对硝酸氮的去除率逐渐降低。在60 mg/L的初始对硝基酚的胁迫环境下,pH为7时硫自养-电化学氢自养耦合反硝化系统取得最佳的脱氮效果,当pH为酸性或碱性时,耦合反硝化系统内的微生物的活性会受到抑制,从而导致反硝化不完全,亚硝酸氮也出现了一些累积。100 mg/L以内的对硝基酚对耦合反硝化系统的硝酸盐还原酶活性和亚硝酸盐还原酶活性影响相对较小。乳酸脱氢酶的释放量和活性氧的产生量随着对硝基酚的浓度的增加升高的幅度并不是很大,说明100 mg/L以内的对硝基酚对耦合反硝化系统内的微生物的细胞膜的损害相对较小。当耦合反硝化系统在对硝基酚的胁迫环境下时,系统中最优势的Proteobacteria菌门占耦合反硝化系统总菌门的比例波动相对较小,这表明了对硝基酚对耦合反硝化系统的主导菌门影响较小,这与对硝基酚胁迫环境下耦合系统降低程度较小的反硝化效率是相一致的。对硝基酚在耦合反硝化系统中被去除主要是被反硝化微生物还原为对氨基酚。当初始氯霉素的浓度从零增加到150 mg/L,耦合反硝化系统的反硝化效率逐渐降低。在100 mg/L的氯霉素的环境下,随着初始硝酸氮的浓度的增加,有限的电子供体不能无限制的用于还原硝酸氮,所以硝酸氮的去除效果降低。在100 mg/L的氯霉素的胁迫环境下,随着电流从50 mA增加到200 mA,在氯霉素的胁迫环境下耦合反硝化系统对硝酸氮的去除率逐渐增加,但是当电流为400 mA时,耦合反硝化系统对硝酸氮的去除率反而降低为85.11%。耦合反硝化系统中初始氯霉素的浓度的不断增加,会使系统硝酸盐还原酶活性和亚硝酸盐还原酶活性都降低。随着氯霉素浓度的增加,耦合反硝化系统中乳酸脱氢酶的释放量和活性氧不断增加的趋势表明系统中的微生物受到的损害越来越严重,从而反硝化效果越来越差。氯霉素的环境会对耦合反硝化系统内的微生物物种产生较大的影响。随着氯霉素的浓度从零增加到150 mg/L,耦合反硝化系统中的反硝化功能基因nirS,nirK,narG和napA的拷贝数均呈现降低的趋势,表明氯霉素的胁迫环境会使系统中主要的反硝化功能基因的丰度降低。氯霉素在耦合反硝化系统中被降解可能是作为异养反硝化的碳源被利用。更多还原

【Abstract】 Because excessive amounts of nitrate in surface and ground waters pose serious threats to water environment and human health,exploring high-efficiency,low-consumption and non-secondary pollution methods for nitrate removal is becoming an important subject for water treatment.The biological treatment is regarded as the most dominant nitrate removal technology because of its high-efficiency and low-consumption features.Although biological nitrate removal method have been widely studied and demonstrated,the nitrate polluted waters may face threats from heavy metal ions and toxic organics.Nowadays,even though some researchers have focused on simultaneous removal of nitrate,heavy metals and organics,the effects caused by heavy metals and organics concerning denitrification process and the simultaneous degradation mechanisms are not systematically studied.This work aims to explore the effects of denitrification efficiency,dominant microbial community,key enzyme activity,denitrifying functional gene,and extracellular polymeric substance caused by heavy metals and organics concerning denitrification process,and reveal the toxic effects of heavy metals and organics onto denitrification from the macroscopic and microscopic aspects.This study will provide important supports for the practical application of denitrification process.This work develops a combined sulfur autotrophic and electrochemical hydrogen autotrophic denitrification system in order to investigate the effects of heavy metal ions Cr(Ⅵ)and V(Ⅴ),toxic organics phenol,p-nitrophenol and chloramphenicol onto denitrification process.Results demonstrate that nitrate removal efficiency decreases with initial Cr(Ⅵ)concentration increases from zero to 150 mg/L in the combined denitrification system.Under the stress condition of 100 mg/L Cr(Ⅵ),the combined system can achieve completed denitrification when the nitrate concentration is lower than 100 mg/L.However,nitrate removal efficiency decreases to 81.69%under 200 mg/L nitrate condition.Under the stress condition of 100 mg/L Cr(Ⅵ),the combined system achieves optimal denitrification efficiency at pH 7 and the highest nitrate removal efficiency reaches 93.01%.When the initial Cr(Ⅵ)concentration is 150 mg/L,the nitrate reductase activity and nitrite reductase activity both decrease obviously.The effects of biofilm formation are not obvious when the initial Cr(Ⅵ)concentration is lower than 100 mg/L,however,when the initial Cr(Ⅵ)concentration is further increased to 150 mg/L,the microorganisms are apparently influenced and then secrete fewer amounts of EPS.Under the stress condition of Cr(VI),some denitrifying microorganisms may decrease or disappear because of the toxic effects.The combined system can achieve satisfied denitrification efficiency,attributing to the denitrification abilities of phyla Proteobacteria and Firmicutes and classes Betaproteobacteria and Clostridia.When the initial Cr(VI)concentration increases from zero to 150 mg/L,the copy numbers of denitrifying functional genes nirS,nirK,narG,and napA show declining trends.Cr(VI)is reduced to Cr(III)through biological reduction process and then combined with biofilms,or dropped off with biofilms,then removed from the reactor.When the initial V(V)concentration increased from 0 to 100 mg/L,the denitrification efficiency of the combined system presents decreased tendency.Under the stress condition of 60 mg/LV(V),the denitrification efficiency increases with current increases from 50 to 200 mA,However,the denitrification efficiency decreases when current increases to 400 mA.Under the stress condition of 60 mg/L V(V),the combined system achieve optimal nitrate removal efficiency under neutral condition,acidic or alkaline conditions cause decreased denitrification efficiencies.When the initial V(V)concentration is higher than 60 mg/L,the nitrate reductase activity and nitrite reductase activity both decrease obviously,which is in accordance with the decreased nitrate removal efficiency.The results of LDH release rate and ROS production rate demonstrate that the combined denitrifying microorganisms can be adversely affected by V(V)which concentration is higher than 60 mg/L.The stress of V(V)decreases the bacterial abundance,the total number of species,and microbial diversity.The dominant phyla and classes are Proteobacteria,Firmicutes and Betaproteobacteria,Clostridia,Gammaproteobacteria,Alphaproteobacteria,respectively.The main denitrifying functional genes are inhibited by the increasing V(V)concentration,so that the denitrification efficiency constantly decreases.V(V)is ultimately reduced to V(IV)through biological reduction process in the system.When initial phenol concentration increases from zero to 100 mg/L,denitrification efficiency shows decreased trend and the lowest denitrification efficiency achieves at 100 mg/L phenol.Under the stress condition of 50 mg/L phenol,nitrate removal efficiency gradually decreases with increasing initial nitrate concentration.Under the stress condition of 50 mg/L phenol,the combined system can achieve optimal denitrification efficiency at pH 7.With increasing initial phenol concentration,nitrate reductase activity and nitrite reductase activity both present decreased tendencies,which is in accordance with decreasing denitrification efficiency.With increasing initial phenol concentration,the combined system secretes declining EPS,and thus affecting the biofilm formation and decreasing nitrate removal efficiency.When initial phenol concentration is lower than 50 mg/L,the bacterial abundance shows increased tendency under the phenol stress condition.However,when initial phenol concentration increases to 100 mg/L,the community diversity shows decreased trend,demonstrating that 100 mg/L phenol causes negative effects onto microorganisms in the combined system.Phenol is removed in the denitrification system due to participating heterotrophic denitrification process as carbon source.The denitrification process in this system contains autotrophic and heterotrophic process.When initial p-nitrophenol concentration increases from 0 to 100 mg/L,nitrate removal efficiency presents slightly decreased tendency.Under the stress condition of 60 mg/L p-nitrophenol,nitrate removal efficiency decreases with increasing initial nitrate concentration,and the combined system achieves optimal denitrification efficiency at pH 7.Under acidic or alkaline condition,the microbial activity is inhibited,so that denitrification is not completed and nitrite concentration is increased.The nitrate reductase activity and nitrite reductase activity are not obviously influenced when initial p-nitrophenol concentration is lower than 100 mg/L.LDH release rate and ROS production rate present slight increased trends,demonstrating that p-nitrophenol which concentration is lower than 100 mg/L brings relatively small damage to microbial cell membrane.Under the stress condition of p-nitrophenol,the percentage of the dominant phylum Proteobacteria presents relatively small variation,which demonstrates that the dominant phylum in the denitrification process is less affected by p-nitrophenol,which is also in accordance with the decreasing denitrification efficiency in the combined system under p-nitrophenol condition.p-Nitrophenol is mainly reduced to p-aminophenol in the system.When initial chloramphenicol concentration increases from 0 to 150 mg/L,nitrate removal efficiency presents decreased tendency.Under the stress condition of 100 mg/L chloramphenicol,the limited electron donor cannot support nitrate reduction so that nitrate removal efficiency decreases with increasing initial nitrate concentration.Under the stress condition of 100 mg/L chloramphenicol,nitrate removal efficiency shows increased trend with current increases from 50 to 200 mA.However,denitrification efficiency decreases to 85.11%when current increases to 400 mA.The nitrate reductase activity and nitrite reductase activity both decrease with increasing initial chloramphenicol concentration.The increased LDH release rate and ROS production rate demonstrates that the microbial community suffers more and more serious damage with increasing initial chloramphenicol concentration,so that denitrification efficiency is getting worse and worse.The chloramphenicol condition causes relatively great influence onto microorganisms in the combined system.When initial chloramphenicol concentration increases from 0 to 150 mg/L,the copy numbers of the main functional denitrification genes nirS,nirK,narG,and napA show decreased trends,demonstrating that the abundances of main functional denitrification genes decrease under chloramphenicol stress condition in the combined system.Chloramphenicol is removed in the denitrification system probably due to participating heterotrophic denitrification process as carbon source.更多还原