【作者】 王岩；

【导师】 邢薇；

【作者基本信息】 北京交通大学 ， 环境科学与工程， 2021， 硕士

【摘要】 地表水体的氮污染治理已成为国内外热点。针对有机碳不足的低碳氮比(COD/TN)水质特征,自养/异养耦合反硝化工艺已成为地表水氮污染修复的热门技术。然而在自养/异养耦合反硝化体系中,自养反硝化菌与异养反硝化菌并非各自独立完成反硝化作用,其耦合作用机制尚不清楚。本研究以硫铁矿自养/异养耦合反应器运行为基础,同时建立自养/异养耦合微生物个体生长模型(Newcastle University Frontiers in Engineering Biology,NUFEB),分别从宏观的工艺角度和微观的个体角度对耦合反硝化体系中自养反硝化菌与异养反硝化菌的耦合机制进行了探究,最后利用NUFEB模型对硫铁矿自养/异养耦合反硝化工艺参数进行了评价。主要结论如下:(1)在COD/TN=1的水质条件下,当在硫代硫酸盐自养反硝化体系中添加少量有机物构建硫铁矿自养/异养耦合反硝化体系时,TN去除率从11.47±0.02%提升至49.00±3.07%,证明了自养/异养耦合反硝化体系对于脱氮的提升效果,并且在耦合反硝化体系中异养反硝化菌占绝对优势。利用高通量测序和实时定量PCR技术对微生物的群落结构和功能基因表达情况进行分析,表明硫铁矿自养/异养耦合反硝化阶段的微生物主要优势菌为自养反硝化菌Thiobacillus(硫杆菌属)和异养反硝化菌Thauera(陶厄氏菌属),所占比例分别为8.21%和23.10%;且填料内侧的自养反硝化菌丰度大于异养反硝化菌。根据实时定量PCR结果显示,填料外侧的表征反硝化的nir S基因表达情况多于内侧;填料内侧表征自养反硝化菌固氮的cbb L基因表达情况大于外侧。结果表明在硫铁矿自养/异养耦合反硝化体系中,自养反硝化菌多生长于靠近硫铁矿的填料内侧;异养反硝化菌则多生长于填料外侧,二者在自养/异养耦合反硝化体系空间中存在物理水平上的协作关系。(2)以硫代硫酸盐为电子供体,建立了硫自养/异养耦合反硝化NUFEB模型,以序批式的方式运行,在三种进水氮浓度(45、100、300 mg N/L)下验证了耦合反硝化体系的工艺脱氮效果大于单独自养或单独异养反硝化体系,并且随着进水有机物浓度的增加,异养反硝化菌所占比例随之增加。分别从二维的浓度变化和三维的个体代谢生长角度探究了自养反硝化菌和异养反硝化菌在耦合反硝化体系中协作关系与竞争关系。得出结论:在硫代硫酸盐自养/异养耦合反硝化体系中,自养反硝化菌与异养反硝化菌呈现化学水平上的协作关系以及“良性”的竞争关系。序批式反应器中的自养反硝化菌与异养反硝化菌的生长周期交替,轮流承担硝酸盐的去除工作,从时间关系上呈现协作关系。在此过程中异养反硝化菌所占种群比例与脱氮贡献比例先增加后减少,并随着进水氮浓度和COD/TN的增加而增加;耦合菌群明显缩短了单独营养体系中反硝化菌的迟缓期,从而增强脱氮效果。异养反硝化菌在反硝化过程中产生大量碳酸氢盐,可以为自养反硝化菌提供无机碳源,同时促使异养反硝化反应朝着正向进行,呈现出化学水平上的协作关系。在竞争关系上,自养反硝化菌与异养细菌争夺电子受体与生长空间。其中异养反硝化菌的竞争能力和抗竞争能力明显好于自养反硝化菌。当异养反硝化菌入侵优势的自养反硝化菌时,自养反硝化菌总量减少,然而脱氮效果反而好于入侵前的单独自养反硝化体系,因此称之为“良性”竞争。从侧面也说明对于整体的耦合反硝化体系,自养反硝化菌和异养反硝化菌之间协作关系对工艺脱氮的正面促进效果大于竞争关系所带来的负面效果。(3)以硫铁矿为电子供体,建立了硫铁矿自养/异养耦合反硝化NUFEB模型,以连续流的方式运行,模拟受硝酸盐污染的地表水,在进水氮浓度45 mg N/L,COD浓度45 mg N/L,即COD/TN=1的水质条件下验证了耦合反硝化体系的工艺脱氮效果大于单独自养或单独异养反硝化体系。分别从工艺角度和微生物角度出发研究COD/TN和初始菌群比例两个因素对硫铁矿自养/异养耦合反硝化反应器连续流运行的影响。结果表明:随着COD/TN的增加,脱氮效果增加,但增加到一定量后,反硝化速率增幅减小。随着自养反硝化菌起始数量比例的增加,耦合反硝化体系的脱氮速率随之增加。在实际的反应器接种运行中,增加初始接种时的自养反硝化菌菌量,可以有效缩短自养反硝化菌的生长周期,从而增加脱氮效果。本研究结果表明,自养反硝化菌与异养反硝化菌在耦合反硝化体系中存在协作关系,分别体现于:1)物理水平上的填料内外侧生长空间利用;2)序批式反应器周期互补交错生长时段利用;3)化学水平上的碳酸氢盐的生产再利用。自养反硝化菌与异养反硝化菌在耦合反硝化体系中存在“良性”竞争关系,异养反硝化菌的竞争能力和抗入侵能力明显好于自养反硝化菌,竞争所产生的负面效果小于协作关系的正面效果。从整体上看,自养与异养反硝化菌在耦合反硝化体系中更多的体现协作关系。为实际的硫源自养/异养耦合反硝化在实际氮污染修复中的利用提供了技术支持和理论基础。图66幅,表18个,参考文献126篇更多还原

【Abstract】 Nitrogen pollution control of surface water has become a hot spot at home and abroad.According to the characteristics of low carbon to nitrogen ratio(COD/TN)water quality with insufficient organic carbon,autotrophic/heterotrophic coupled denitrification process has become a popular technology for surface water nitrogen remediation.However,in the autotrophic/heterotrophic coupled denitrification system,the autotrophic denitrification bacteria and heterotrophic denitrification bacteria do not complete the denitrification independently,and the coupling mechanism is still unclear.Based on the operation of an autotrophic/heterotrophic coupled reactor in pyrite,an individual growth model of autotrophic/heterotrophic coupled microorganisms was established(Newcastle University Frontiers in Engineering Biology,NUFEB).The coupling mechanism of autotrophic denitrification bacteria and heterotrophic denitrification bacteria in the coupled denitrification system was explored from the macro process point of view and the micro individual point of view.Finally,the NUFEB model was used to optimize the autotrophic/heterotrophic coupled denitrification process of pyrite.The main conclusions are as follows.(1)Under the water quality condition of COD/TN=1 and HRT=48h,the TN removal rate increased from 11.47±0.02% to 49.00±3.07% when pyrite autotrophic/heterotrophic coupled denitrification system was constructed by adding a small amount of organic matter to the thiosulfate autotrophic denitrification system.It has been proved that the autotrophic/heterotrophic coupled denitrification system can improve the denitrification efficiency,and the heterotrophic denitrification bacteria are more dominant in the coupled denitrification system.High-throughput sequencing and real-time quantitative PCR were used to analyze the microbial community structure and functional gene expression.The results showed that the main dominant microorganisms in the autotrophic/heterotrophic coupled denitrification stage of pyrite were autotrophic denitrification bacteria Thiobacillus and heterotrophic denitrification bacteria Thauera.The proportions were 8.21% and 23.10%,respectively.The abundance of autotrophic denitrifying bacteria inside the fillers was higher than that of heterotrophic denitrifying bacteria.According to the real-time quantitative PCR results,the expression of denitrifying nir S gene in the outer fillers was more than that in the inner fillers.The expression of cbb L gene in autotrophic denitrifying bacteria for nitrogen fixation was greater on the inside than on the outside.The results show that in the pyrite autotrophic/heterotrophic coupled denitrification system,autotrophic denitrification bacteria mostly grow near the inner fillers of pyrite.Heterotrophic denitrification bacteria mostly grew on the outer side of the filler,and there was a cooperative relationship between them in the space of autotrophic/heterotrophic coupled denitrification system at the physical level.(2)With thiosulfate as electron donor,a sulfur autotrophic/heterotrophic coupled denitrification NUFEB model was established,which was run in a sequence batch mode.It was verified that the denitrification efficiency of the coupled denitrification system was better than that of the autotrophic or heterotrophic denitrification systems alone at three influent nitrogen concentrations(45,100,300 mg N/L).The proportion of heterotrophic denitrifying bacteria increased with the increase of the concentration of organics in the influent water.The cooperation and competition between autotrophic denitrifiers and heterotrophic denitrifiers in the coupled denitrification system were investigated from the perspectives of two-dimensional concentration change and three-dimensional individual metabolic growth.It is concluded that the autotrophic denitrification bacteria and the heterotrophic denitrification bacteria in the thiosulfate autotrophic/heterotrophic coupled denitrification system show a cooperative relationship and a "benign" competition relationship at the chemical level.The autotrophic denitrifying bacteria and heterotrophic denitrifying bacteria in the sequencing batch reactor alternate their growth cycles and take turns to undertake the removal of nitrate,presenting a cooperative relationship in terms of time relationship.In this process,the proportion of heterotrophic denitrifying bacteria in the population and the contribution proportion of denitrification increased first and then decreased,and increased with the increase of influent nitrogen concentration and COD/TN.The coupled bacteria significantly shortened the delay period of denitrifying bacteria in the single nutrient system,thus enhancing the denitrifying effect.Heterotrophic denitrification bacteria produce a large amount of bicarbonate in the process of denitrification,which can provide inorganic carbon source for autotrophic denitrification bacteria,and promote the heterotrophic denitrification reaction in a positive direction,showing a chemical level of cooperation.In terms of competition,autotrophic denitrifying bacteria and heterotrophic bacteria compete for electron acceptor and growth space.The competitive ability and anti-competitive ability of heterotrophic denitrifying bacteria were obviously better than that of autotrophic denitrifying bacteria.When the heterotrophic denitrifying bacteria invaded the dominant autotrophic denitrifying bacteria,the total amount of autotrophic denitrifying bacteria decreased,but the denitrification effect was better than that of the single autotrophic denitrifying system before invasion,so it was called "benign" competition.It is also indicated from the side that for the whole coupling denitrification system,the positive promoting effect of the cooperative relationship between autotrophic denitrification bacteria and heterotrophic denitrification bacteria on the process denitrification is greater than the negative hindrance effect caused by competition.(3)With pyrite as electron donor,the autotrophic/heterotrophic coupling denitrification NUFEB model of pyrite was established,which was operated in a continuous flow mode to simulate the surface water polluted by nitrate.The influent nitrogen concentration was 45 mg N /L,COD concentration was 45 mg N /L,under the water quality condition of COD/TN=1,it is verified that the process denitrification efficiency of coupled denitrification system is better than that of autotrophic or heterotrophic denitrification system alone.The effects of COD/TN and initial flora ratio on the continuous flow operation of the autotrophic/heterotrophic coupled denitrification reactor of pyrite were studied from the perspective of process and microorganism.The results showed that the denitrification efficiency increased with the increase of COD/TN,but the rate of denitrification decreased after the increase to a certain amount.The denitrification rate of the coupled denitrification system increased with the increase of the initial proportion of autotrophic denitrification bacteria,but the increasing trend gradually weakened after exceeding a certain amount.In the actual reactor inoculation operation,increasing the amount of autotrophic denitrifying bacteria at the initial inoculation can effectively shorten the growth cycle of autotrophic denitrifying bacteria,so as to increase the denitrifying effect.The results showed that there was a cooperative relationship between autotrophic denitrifiers and heterotrophic denitrifiers in the coupled denitrification system,which was reflected in the following aspects: 1)the utilization of the growth space inside and outside the filler at the physical level;2)Utilization of the cycle complementary staggered growth period of the sequential batch reactor;3)The chemical level of bicarbonate production is utilized.Autotrophic denitrifying bacteria and heterotrophic denitrifying bacteria in coupling denitrification system there is a "good" competition relationship,heterotrophic denitrifying bacteria competition ability and the ability to resist competition is much better than autotrophic denitrification bacteria,competition generated by the negative effect is less than the positive effect of collaboration,overall,autotrophic and heterotrophic denitrifying bacteria in coupling denitrification system more reflects collaboration.It provides technical support and theoretical basis for the application of coupled denitrification of sulfur from trophic/heterotrophic sources in the actual nitrogen pollution remediation.更多还原