【作者】 李俊；

【导师】 李爱民；

【作者基本信息】 南京大学 ， 环境工程（专业学位）， 2017， 硕士

【摘要】 制药、造纸、印染、食品加工等领域在生产过程中会产生大量含有高浓度硫酸盐的有机废水。如果这些废水进入水体,将会造成水体酸化,影响水生生物的正常生长;在厌氧环境中,硫酸盐会被还原产生H2S,腐蚀设备管道;此外,高H2S浓度的还会引起人神经中毒。因此,硫酸盐有机废水的处理研究具有重要的实际意义。厌氧生物法由于处理负荷高,占地面积小,能耗低等优点在高浓度硫酸盐有机废水的处理上得到了广泛的应用。但是当废水中硫酸盐含量较高时,会对厌氧过程产生很大影响。一方面,产甲烷菌(MPB)与硫酸盐还原菌(SRB)之间存在着基质竞争,竞争共同的底物(氢气和乙酸);另一方面,硫酸盐还原产物硫化物对MPB和SRB产生毒性抑制作用。因此,硫酸盐的存在不仅影响微生物的活性,严重时会导致厌氧处理系统的崩溃。两相厌氧工艺通过相的分离,使硫酸盐还原过程和产甲烷过程分别发生在两个单独反应器内,实现了硫酸盐还原过程和产甲烷过程的分离,减弱SRB与MPB的相互干扰。目前,关于两相厌氧工艺处理硫酸盐有机废水的研究多集中于硫酸盐浓度1000mg/L以内,而对于高浓度硫酸盐条件下(尤其当硫酸盐浓度超过10000 mg/L),两相厌氧反应器的处理效能、微生物群落结构与功能以及两者之间关联的研究较少。本论文采用第三代厌氧膨胀颗粒污泥床反应器(Expanded granular sludge bed reactor,EGSB)作为两相厌氧反应器的酸相反应器和甲烷相反应器,采用模拟废水,对酸相反应器进行动力学控制法和pH调控实现相的分离。在完成两相厌氧反应器启动和相的分离的基础上,研究硫酸盐浓度对酸相反应器的影响、高浓度硫酸盐条件下COD/SO42-对酸相反应器的影响以及甲烷相反应器对硫酸盐的耐受性。结合分子生物学技术,如Illumina Mi-seq高通量测序和实时定量PCR(Real time PCR,q-PCR),对两相厌氧系统各个阶段内微生物群落结构和功能基因的丰度进行解析,揭示硫酸盐废水厌氧处理过程的微生物作用机制,并在此基础上探索反应器运行状况和微生物群落结构之间的联系。本研究主要结论如下:(1)采用低负荷启动方式及pH调控和动力学控制法实现两相反应器的启动和相分离。结果表明,低负荷启动方式可以顺利完成两相厌氧反应器的启动,启动完成后,酸相反应器的COD去除率为21.87%,硫酸盐还原率为69.4%;甲烷相反应器COD去除率高达81.3%,硫酸盐还原率为81.4%。两相厌氧系统总的COD去除率为85.4%,硫酸盐还原率高达94.3%,两相厌氧反应器对有机物和硫酸盐均取得了良好的处理效果;通过pH调控和动力学控制法进行相的分离,酸相反应器酸化率超过50%,分相效果较为理想。(2)系统研究了进水硫酸盐浓度对酸相反应器运行的影响及微生物作用机制,结果表明,在COD/SO42-为3,HRT为20h的条件下,酸相反应器最高可以处理高达12000 mg/L的硫酸盐废水,对应硫酸盐负荷为14.4 g SO42-/(L·d),此时反应器可以保持50%左右的硫酸盐还原率和30%左右的COD去除率。高浓度硫酸盐条件下(12000 mg/L),出水VFA维持在10000 mg/L以上,出水硫化物维持在200 mg/L以上,VFA和硫化物毒性抑制作用可能是影响高浓度硫酸盐条件下酸相反应器正常运行的主要原因。硫酸盐浓度升高导致厌氧污泥中微生物群落多样性降低。优势菌门为厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。其中,Firmicutes的丰度受进水硫酸盐影响较大,当进水SO42-浓度由9000 mg/L上升至12000 mg/L时,其相对丰度由40%降至1%以下。由于很多Firmicutes的微生物可以参与VFA的降解过程,所以高浓度硫酸盐条件下VFA的积累可能是由于Firmicutes受到抑制所引起的。不同进水硫酸盐条件下发挥主要脱硫作用的SRB都是脱硫弧菌属(Desulfovibrio)。dsrA功能基因的绝对丰度远高于mcrA功能基因,说明酸相反应器有着较好的脱硫功能,但产甲烷功能很微弱。(3)研究了高浓度硫酸盐进水条件下COD/SO42-对酸相反应器运行的影响及其微生物作用机制。结果表明,高浓度硫酸盐条件下(SO42-=12000 mg/L),酸相反应器保持良好运行所需的最低COD/SO42-为2,此时酸相反应器可以维持40%以上的硫酸盐还原率。高COD/SO42-条件下,丁酸是最主要的VFA,其次为乙酸。随着COD/SO42-的降低,乙酸浓度上升,丁酸浓度下降,乙酸逐渐取代丁酸成为最主要的VFA。厌氧污泥中生物多样性指数均随着COD/SO42-的降低而降低。Firmicutes是酸相反应器内相对丰度最高的菌门,其相对丰度不受COD/SO42-改变而变化;Proteobacteria和 Thermotogae 受 COD/SO42-影响较大,Proteobacteria主要存在于COD/SO42-≤2的条件下,而Thermotogae主要存在于COD/SO42->2的条件下。Desulfovibrio、脱硫化曲菌属(Desulfocurvus)和脱硫代硫酸盐菌属(Dethiosulfovibrio)是酸相反应器主要的SRB,其中Desulfovibrio的相对丰度最高。Desulfovibrio、Desulfocurvus和Dethiosulfovibrio的相对丰度均随 COD/SO42-降低而降低。(4)研究了硫酸盐浓度对甲烷相反应器运行的影响及其微生物作用机制。结果表明,以乙酸为碳源,而甲烷相反应器最高可以耐受约50000 mg/L的硫酸盐。甲烷相反应器取得良好的脱硫效果(70%)需要保持进水COD/SO42-超过2.67;Proteobacteria、Firmicutes和Bacteroidetes是甲烷相反应器内主要的优势菌门,其相对丰度的总和在各个阶段下均占到了 70%左右。Desulfomicrobium是甲烷相反应器内相对丰度最高的SRB,也是高硫酸盐条件下甲烷相反应器中发挥脱硫作用的主要微生物。古细菌中,广古菌门(Euryarchaeota)是甲烷相反应器内主要的优势菌门,其相对丰度在各个阶段均达到97%以上;甲烷八叠球菌属(Methanosarcina)是甲烷相反应器内主要的优势菌属,当进水硫酸盐浓度在40000 mg/L以内,其相对丰度均在95%以上,而当SO42-浓度升至50000 mg/L时,Methanosarcina受到了高盐的抑制,相对丰度降至56.78%。50000 mg/L时硫酸盐可以有效抑制群落甲烷相的功能,但对群落脱硫功能没有明显的抑制。更多还原

【Abstract】 In the field of pharmaceutical,paper making,printing and dyeing,food processing,a large number of organic wastewater containing high concentration sulfate can be produced in the production process.If the wastewater was discharged into water body,the water will be acidified,affecting the normal growth of aquatic organisms.In the anaerobic environment,the sulfate will be degraded into S2-to produce H2S,which is one of the main causes of equipment pipeline corrosion.In addition,high concentration of H2S will also cause human nerve poisoning.Therefore,the study on treatment of sulfate organic wastewater has important practical significance.Anaerobic biological process has been widely used in the treatment of high concentration sulfate organic wastewater due to its high loading capacity,small footprint and low energy consumption.But when the sulfate content in wastewater is high,it will have a great influence on the anaerobic process.On the one hand,methane-producing bacteria(MPB)and sulfate-reducing bacteria(SRB)will compete for common substrates(hydrogen and acetate).On the other hand,the products of sulfate reduction process,sulfide,will bring toxicity inhibition on MPB and SRB.Therefore,the presence of sulfate not only affects the activity of microorganisms,but also leads to the collapse of anaerobic treatment systems.In two phase anaerobic process,the sulfate reduction and methanogenesis occurred respectively in two reactors to achieve the separation of sulfate reduction process and methanogenesis process,so as to reduce the mutual interference between SRB and MPB,At present,the study about the treatment of high concentration sulfate organic wastewater by two phase anaerobic process mainly within sulfate concentration less than 1000 mg/L,while for the high concentration sulfate conditions(especially when the sulfate concentration is more than 10000 mg/L),there were few study about the association between the treatment performance of two-phase anaerobic reactor,the structure and function of microbial communities.This paper adopts the third generation anaerobic expanded granular sludge bed reactor(EGSB)as the two-phase anaerobic reactor acidogenic phase reator reactor and methane reactor and use simulated wastewater.Phase separation was achieved by kinetic control and pH control.After the start-up of two-phase anaerobic reactor,the effect of sulfate concentration on the acidogenic phase reator reactor,the effect of COD/SO42-on the acidogenic phase reator reactor in the presence of high concentration sulfate and the tolerance of the methanogenic phase reactor to sulfate were studied.Using molecular biology techniques,such as Illumina Mi-seq high-throughput sequencing and real-time quantitative PCR(q-PCR),to analyze the abundance of all stages of the two-phase anaerobic system within the microbial community structure and function of genes and to reveal the microbial mechanism of anaerobic treatment of sulfate wastewater,and explore the relationship between reactor operating conditions and microbial community structure.The main conclusions of this study are as follows:(1)The start-up and phase separation of the two phase reactor were carried out by means of low load start-up mode,pH control and dynamic control.The results show that the low load start-up method can successfully complete the start-up of the two-phase anaerobic reactor.After the start-up,the COD removal rate of the acidogenic phase reactor was 21.87%and the sulfate reduction rate was up to 69.4%.In methanogenic phase reactor,the COD removal rate was up to 81.3%and the sulfate reduction rate was 81.4%.The total COD removal rate was 85.4%in the two-phase anaerobic system and sulfate reduction rate is as high as 94.3%,the two-phase anaerobic reactor achieved high treatment efficiency of both organic matter and sulfate.The phase separation was carried out by pH control and kinetic control.The phase separation was ideal with the acidification rate of in the acidogenic phase reactor.(2)The effect of influent sulfate concentration on the operation of acidogenic phase reactor and microbial mechanism were studied.The results show that when the COD/SO42-and HRT were fixed at 3 and 20 h,the acidogenic phase reactor can handle up to 12000 mg/L sulfate wastewater with about 50%of the sulfate reduction rate and 30%of the COD removal efficiency.Under the condition of high concentration sulfate(12000 mg/L),the effluent VFA and sulfide were maintained above 10000 mg/L and 200 mg/L,the toxicity inhibition of VFA and sulfide may be the main factors that affect the normal operation of acidogenic phase reactor under the condition of high concentration sulfate.The increase of sulfate concentration resulted in the decrease of microbial community diversity in anaerobic sludge.The dominant bacteria were Firmicutes,Proteobacteria and Bacteroides on phylum level.Among them,the abundance of Firmicutes was greatly affected by influent sulfate.When the influent SO42-concentration increased from 9000 mg/L to 12000 mg/L,the relative abundance decreased from 40%to below 1%.As many microorganisms of Firmicutes can participate in the degradation of VFA,the accumulation of VFA in the condition of high concentration sulfate may be due to the inhibition of Firmicutes by high sulfate.The microorganism that played the role of desulfurization under different influent sulfate is Desulfovibrio.The absolute abundance of dsrA gene was much higher than that of mcrA functional gene,which indicated that the acidogenic phase reator reactor had better desulfurization function,but the methanogenic function was weak.(3)The effect of COD/SO42-on the operation of acidogenic phase reactor and microbial mechanism were studied.The results show that under the condition of high concentration of sulfate(SO42-=12000 mg/L),the minimum COD/SO42-need for the acidogenic phase reactor to keep running properly was 2 and at this time the acidogenic phase reactor could maintain the sulfate removal efficiency of more than 40%.Under the condition of high COD/SO42-,butyric acid were the most predominant VFA,followed by acetic acid.With the decrease of COD/SO42-,the concentration of acetic acid increased,but that of butyric acid decreased,acetic acid gradually replaced butyric acid as the most predominant VFA.The decrease of COD/SO42-resulted in the decrease of microbial community diversity in anaerobic sludge.Firmicutes was the most dominant bacteria in acidogenic phase reactor and its relative abundance didn’t change with COD/SO42-.Proteobacteria and Thermotogae was affected by COD/SO42-,Proteobacteria mainly existed when COD/SO42-was less than or equal to 2,while Thermotogae mainly existed in the condition of COD/SO42->2.Desulfovibrio,Dethiosulfovibrio and Desulfocurvus were the main SRB in the acidogenic phase reactor,and their relative abundance decreased with the decrease of COD/SO42-.(4)The effect of sulfate concentration on the operation of methanogenic phase reactor and the microbial mechanism were studied.The results show that the methanogenic phase reactor can tolerate up to 50000 mg/L sulfate with acetic acid as carbon source.It is necessary to maintain COD/SO42-over 2.67 for the methanogenic phase reactor to achieve high sulfate removal efficiency(70%).Proteobacteria,Firmicutes and Bacteroidetes were the dominant bacteria in the methanogenesis phase reactor on phylum level and their total relative abundance in each stage was accounted for about 70%.Desulfomicrobium was the most dominant bacteria in the methanogenic phase reactor under the condition of high sulfate.Among archaea,Euryarchaeota was the dominant bacteria on phylum level,its relative abundance in each stage were more than 97%.Methanosarcina was the dominant bacteria on genus level,when the influent sulfate concentration was less than 40000 mg/L,its relative abundance was above 95%,but when the concentration of SO42-was increased to 50000 mg/L,Methanosarcina was inhibited by the high salinity and it’s relative abundance decreased to 56.78%.50000 mg/L sulfate can effectively inhibit the desulfurization function of community in methanogenesis phase reactor,but there was no obvious inhibition on methanogenic function of the community.更多还原