【作者】 陈婷婷；

【导师】 郑平；

【作者基本信息】 浙江大学 ， 环境工程， 2013， 博士

【摘要】 随着经济的快速发展,废水排放量不断增加,其中,许多工业废水、农业废水和生活污水中均有含氮污染物。氮素污染已严重威胁到人类的生存环境和身心健康,氮素污染的治理迫在眉睫。在众多废水脱氮工艺中,厌氧氨氧化(anaerobic ammonium oxidation, ANAMMOX)工艺以其经济高效受到了环境工程界的广泛关注,但厌氧氨氧化菌(anaerobic ammonium oxidizing bacteria, AAOB)生长缓慢,严重制约了ANAMMOX工艺的工程应用。本课题以不同浓度的废水作为处理对象,考察了ANAMMOX-EGSB (expanded granular sludge bed)反应器的运行性能；从ANAMMOX富集培养物(以下简称“富集培养物”)的菌群组成和生长代谢切入,揭示了AAOB及其伴生菌的工作机制。主要研究结果如下：(1)考察了高、低基质浓度下ANAMMOX-EGSB反应器的运行性能。①考察了高基质浓度下ANAMMOX-EGSB反应器的运行性能。ANAMMOX-EGSB反应器适合处理高浓度含氨废水。当进水总氮浓度为1429.1mgN/L时,反应器容积负荷可达22.87kgN/(m3·d),容积去除负荷可达18.65kgN/(m3·d),且容积去除负荷仍呈上升趋势,反应器运行性能良好。反应器内富集培养物具有很高的比转化速率,对氨氮、亚硝氮、总氮的理论转化速率分别为381.2、304.7、731.7mgN/(gVSS·d);富集培养物具有很高的基质亲和力和很强的基质耐受性,对氨氮、亚硝氮、总氮的半饱和常数分别为36.75、0.657、29.26mgN/L,抑制常数分别为887.1、13942.1、1779.6mgN/L。②考察了低基质浓度下ANAMMOX-EGSB反应器的运行性能。ANAMMOX-EGSB反应器具有优良的达标性能。在出水达标(15mg/L)的情况下,反应器容积负荷为27.31kgN/(m3·d),容积去除负荷为25.86kgN/(m3·d),总氮转化率为94.68%。富集培养物具有很高的比转化速率,对氨氮、亚硝氮、总氮的最大比转化速率分别为907.13、841.76、1810.10mgN/(gVSS·d);富集培养物具有很高的基质亲和力,对氨氮、亚硝氮、总氮的半饱和常数分别为2.69、0.44、3.11mgN/L,基质亲和力显著高于同类研究报道值。(2)揭示了ANAMMOX富集培养物的微生物特性。①探明了不同容积负荷下ANAMMOX富集培养物的生长-代谢性能。研究发现,在富集培养物连续培养过程中,随着容积负荷的提高,富集培养物依次呈现饥饿、适宜和中毒状态。在饥饿、适宜和中毒期,生物量[以VSS (volatile suspended solid,挥发性悬浮固体)表征]的变化速率分别为-6.1×10-3gVSS/(gVSS·d)、1.45×10-2gVSS/(gVSS·d)和-5.78×10-2gVSS/(gVSS·d);用于提供维持能的基质消耗速率为0.05kgNH4+-N/(kgVSS·d)、0.07kgNO2-N/(kgVSS·d)、0.12kgN/(kgVSS·d);用于支持最大生长速率的基质消耗速率为0.21kgNH4+-N/(kgVSS·d)、0.24kgNO2--N/(kgVSS·d)、0.45kgN/(kgVSS·d)。在不同时期富集培养物的比氨氮转化活性、比亚硝氮转化活性、比氮转化活性分别为：饥饿期-0.03、0.04、0.07kgN/(kgVSS·d);适宜期—0.22、0.23、0.45kgN/(kgVSS·d);中毒期-0.08、0.10、0.18kgN/(kgVSS·d)。研究发现,在适宜期,富集培养物的VSS增长速率与氨氮、亚硝氮、总氮消耗速率以及硝氮生成速率都呈线性相关,相关系数分别为0.94、0.92、0.93、0.90。富集培养物得率为0.14gVSS/(gNH4+-N),0.12gVSS/(gNO2--N),0.70gVSS/(gNO3--N)。胞外多聚物(extracellular polymeric substances,EPS)得率为0.11gEPS/(gNH4+-N),0.09gEPS/(gNO2--N),0.55gEPS/(gNO3--N)。②揭示了高基质浓度对ANAMMOX富集培养物颗粒化的影响。氨氮、亚硝氮均为AAOB的基质,但超过一定浓度,氨氮、亚硝氮也会对AAOB产生抑制作用。高基质浓度(超过抑制浓度)会引起富集培养物(颗粒污泥)解体,其中以高浓度亚硝氮的抑制作用更大,对AAOB的抑制浓度阂为100mgN/L。颗粒污泥解体导致沉降性能变差,沉降速度由73.73m/h降低至16.49m/h,污泥被洗出反应器,最终引起反应器性能下降,反应器容积去除率由21.81kgN/(m3·d)降低至16.97kgN/(m3·d)。通过洗出反应器中剩余基质并重新从低浓度开始运行反应器,富集培养物可重新形成颗粒污泥,沉降速度重新升高至60.59m/h,反应器容积总氮去除率也恢复至22.51kgN/(m3·d),反应器性能得到恢复。③研究了高浓度低流量及高流量低浓度两种不同运行模式下两种富集培养物的菌群组成。研究发现,高浓度低流量(R1)及高流量低浓度(R2)两种不同运行模式下富集培养物的菌群组成各不相同。R1中富集培养物颜色偏白略带红色,内部菌体形态多样,微生物以球菌、丝状菌和杆菌为主,菌体细胞内含物形状多样且不规则；R2中富集培养物颜色鲜红,内部菌体形态相对统一,以球菌和杆菌为主,甚少观察到丝状菌,菌体细胞内含物形态相对一致但也不规则；运用PCR-DGGE手段分析表明,两种富集培养物的特征性条带差异显著,R2中富集培养物的条带明显多于R1中富集培养物的条带,即R2富集培养物中的微生物种群更丰富。两种富集培养物中的微生物数量差异显著。采用定量PCR对富集培养物中的细菌及AAOB进行定量分析,结果表明富集培养物(R2)中的细菌基因拷贝数与AAOB hzs基因拷贝数均大于富集培养物(R1)中的细菌基因拷贝数与AAOB hzs基因拷贝数,但是富集培养物(R1)中AAOB所占总细菌的比例(61.10士6.5%)高于富集培养物(R2)中AAOB所占的比例(34.27+3.7%)。从FISH结果可知,在两种富集培养物的所有AAOB中,Candidatus Kuenenia stuttgartiensis, Candidatus Brocadia anammoxidans为优势种。④分离研究了ANAMMOX富集培养物中的伴生菌。从富集培养物中分离获得了菌株ZU-H、ZU-I经形态观察、生理生化试验、(G+C)%(mol/mol)含量测定和16S rRNA系统发育分析,两者均归入Proteobacteria门、Burkholderiales目、Alcaligenaceae科,Castellaniella属,ZU-H与Castellaniella defragrans进化距离最近,ZU-I与Castellaniella denitrificans进化距离最近。试验证明,ZU-I具有显著的反硝化活性,无ANAMMOX活性。在反硝化培养基中,菌株ZU-I的反硝化速率为3.94x10-2mgC/(mg protein·h)和1.08×10-2mgN/(mg protein·h),反硝化消耗的COD/N为5.47:1。作为伴生菌,菌株ZU-I能形成荚膜,有助于ANAMMOX团聚体(如：颗粒污泥、生物膜)的形成。菌株ZU-I革兰氏阴性,异养菌,兼性厌氧,具有丰富的基质多样性。以分子生物学技术检测发现分离源中包含Burkholderiales目细菌,即菌株ZU-I能存在于ANAMMOX富集培养物中,并具有很强的有机物利用能力。更多还原

【Abstract】 With the rapid development of economy, large amount of wastewaters are emitted, in which a large number of industrial wastewater, agricultural wastewater and domestic sewage contain nitrogen. Nitrogen pollution has already seriously threated the living environment and the physical and mental health of human beings. It is urgent that nitrogenous pollutants should be removed from wastewaters. Due to the high efficiency and cost effectiveness, anaerobic ammonium oxidation (ANAMMOX) process has drawn much attention in the field of environmental engineering. However, the slow growth of anaerobic ammonium oxidizing bacteria (AAOB) severly restricted the full-scale application of ANAMMOX process. Using the wastewaters with different nitrogen concentrations as the treatment objects, this study revealed the performance of ANAMMOX-EGSB (expanded granular sludge bed) reactor. Commencing from the microbial consortium and the characteristics of growth and metabolism of ANAMMOX enrichment culture (hereafter referred as enrichment culture), this study revealed the working mechanisms of AAOB and associated bacteria. The main results are as follows:(1) The performances of ANAMMOX-EGSB reactors at high and low substrate concentrations were revealed.①The performance of ANAMMOX-EGSB reactor at high substrate concentrations was revealed. The ANAMMOX-EGSB reactor was suitable to treat strong ammonium-containing wastewaters. When the influent nitrogen concentrations were up to1429.1mgN/L, the nitrogen loading rate (NLR) and nitrogen removal rate (NRR) were as high as22.87kgN/(m3·d) and18.65kgN/(m3·d), respectively, moreover, the NRR was still on the rise. The enrichment culture in the reactor had high substrate conversion rates. The theoretical ammonium, nitrite and nitrogen specific conversion rates were381.2,304.7and731.7mgN/(gVSS·d), respectively. The enrichment culture possessed great substrate affinity and excellent substrate tolerance. The half-saturation constant (KS) values for ammonium, nitrite and nitrogen were36.75,0.657and29.26mg N/L and the inhibition constant (Ki) values for ammonium, nitrite and nitrogen were887.1,13942.1and1779.6mg N/L.②The performance of ANAMMOX-EGSB reactor at low substrate concentrations was revealed. The ANAMMOX-EGSB reactor had an excellent performance to meet the Chinese integrated wastewater discharge standard (GB8978-1996) in which the ammonium concentration should be lower than15mg/L. When the effluent ammonium concentrations was11.90mgN/L which was lower than15mgN/L, the NLR, NRR and nitrogen remove efficiency (NRE) were27.31kgN/(m3·d),25.86kgN/(m3·d) and94.68%, respectively. The enrichment culture had high specific substrate conversion rate (qmax) and great substrate affinity. The qmax for ammonium, nitrite and nitrogen were907.13,841.76and1810.10mg N/(g VSS-d), the KS for ammonium, nitrite and nitrogen were2.69,0.44and3.11mg N/L which were significantly stronger than those reported in the similar studies.(2) The microbial characteristics of ANAMMOX enrichment culture were investigated.①The growth and metabolism characteristics of ANAMMOX enrichment culture at different NLRs were explored. The results showed that the enrichment culture presented starved, suitable and inhibitory phase along with the increase of substrate supply. The change rates of biomass in the starved, suitable and inhibitory phase were-6.1×10-3gVSS/(gVSS·d),1.45×10-2gVSS/(gVSS·d) and-5.78×10-2gVSS/(gVSS·d), respectively. The substrate conversion rates for survival were0.05kgNH4+-N/(kgVSS·d),0.07kgNO2--N/(kgVSS·d) and0.12kgN/(kgVSS·d); the substrate conversion rates for maximum growth were0.21kgNH4+-N/(kgVSS·d),0.24kgNO2--N/(kgVSS·d) and0.45kgN/(kgVSS·d), respectively. In different phase, the specific ammonium, nitrite and nitrogen conversion rates were as follows:starved phase-0.03,0.04,0.07kgN/(kgVSS·d); suitable phase-0.22,0.23,0.45kgN/(kgVSS·d); inhibitory phase-0.08,0.10,0.18kgN/(kgVSS·d). In suitable phase, the VSS accumulation rate showed a linear correlation with the substrate (ammonium, nitrite and nitrogen) removal rate and product (nitrate) formation rate, the correlation coefficients were0.94,0.92,0.93and0.90, the yield of enrichment culture was0.14gVSS/(gNH4+-N),0.12gVSS/(gNO2--N) and0.70gVSS/(gNO3--N), the yield of EPS was0.11gEPS/(gNH4+-N),0.09gEPS/(gNO2--N) and0.55gEPS/(gNO3--N).②The effects of high substrate concentration on the granulation of ANAMMOX enrichment culture were revealed. The ammonium and nitrite are both substrates of AAOB, but they will become inhibitors when their concentrations exceed a certain level. The substrate concentrations exceeded the inhibitory threshold concentration could result in the physiological changes of AAOB which were an important factor for enrichment culture (granular sludge) dispersal. The inhibitory effects of nitrite were greater than those of ammonium, the inhibitory threshold concentration of nitrite to AAOB was around100mgN/L. The dispersal of granular sludge directly resulted in poor settleability (the settling velocity decreased form73.73m/h to16.49m/h), some sludge were washed-out which lead to the deterioration of reactor performance [the NRR decreased from21.81kgN/(mj-d) to16.97kgN/(m3·d)]. By rinsing out the residual substrate from the reactor and then re-running the reactor from low substrate concentrations, the enrichment culture could reform granular sludge (the settling velocity increased to60.59m/h) and reactor performance also could recover [the NRR went up to22.51kgN/(m3·d)].③The microbial consortia of two different enrichment cultures were studied. The two enrichment cultures were derived from two ANAMMOX-EGSB reactors which were operated with two different operation modes-high concentration with low flow rate (R1) and high flow rate with low concentration (R2). The microbial populations of enrichment cultures in R1and R2were different. The enrichment culture in R1was white and reddish, in which the cell morphology was various, the predominant bacteria were cocci, filamentous bacteria and bacilli, and the shape of cell inclusion was various and irregular. The enrichment culture in R2was red. Compared to the enrichment culture in R1, the cell morphology of enrichment culture in R2was more consistent, predominant bacteria were cocci and bacilli, rarely filamentous bacteria were observed, and shape of cell inclusion was consistent but irregular. The results from PCR-DGGE analysis showed that the fingerprints of the two enrichment cultures were significantly different. The bands of enrichment cultures in R2were more than the bands of enrichment culture in R1, which meant the microbial species of enrichment culture in R2were more than those of enrichment culture in R1. The number of bacteria in two enrichment cultures was obviously different. By using quantitative PCR to analyze the number of bacteria and AAOB in enrichment cultures, it was found that both of the bacterial gene copies and AAOB hzs gene copies in enrichment culture (R2) were more than those in enrichment culture (R1). However, the proportion of AAOB to the total bacteria in enrichment culture (R1)(61.10±6.5%) was higher than the proportion of AAOB to the total bacteria in enrichment culture (R2)(34.27±3.7%). The results from the fluorescence in situ hybridization (FISH) showed that, in the two enrichment cultures, the predominant AAOB were Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans.④Two associated bacteria in the ANAMMOX enrichment culture were isolated and studied. The strains ZU-H and ZU-I were isolated from the enrichment culture and were identified as Castellaniella defragrans and Castellaniella denitrificans in phylum Proteobacteria. Strain ZU-I had denitrification activity but no ANAMMOX activity. The denitrification rates of strain ZU-I were3.94×10-2mgC/(mg protein-h) and1.08×10-2mgN/(mg protein-h), the consumed COD/N in denitrification was5.47:1. As an associated bacterium, stain ZU-I was able to form capsule, which was helpful to the formation of ANAMMOX aggregates. Strain ZU-I was Gram-negative, heterotrophic, facultatively anaerobic and had rich diversity of carbon source. It was found from the study of microbial consortium that the isolation source contained the bacteria in Burkholderiales, which meant strain ZU-I was able to live in the enrichment culture and could use organic matters very well.更多还原