【作者】 朱刚利；

【导师】 胡勇有；

【作者基本信息】 华南理工大学 ， 环境工程， 2010， 博士

【摘要】 厌氧氨氧化微生物只在高细胞浓度下才具有活性,生长缓慢且较难培养。如何获得足够的生物量并使之保持在反应器中不流失,便成为ANAMMOX应用的技术瓶颈之所在。微生物包埋固定后,可以防止微生物流失,增加其局部浓度,进而实现反应器快速启动并提高活性和耐冲击性。目前,国内外对固定化条件下的厌氧氨氧化缺乏系统的研究报道,鉴于固定化技术对于解决厌氧氨氧化生物量维持的可行性,有必要对固定化厌氧氨氧化工艺进行深入的研究。为此,本研究在富集厌氧氨氧化污泥的基础上,筛选了最佳固定化载体,优化了最佳固定化条件,并且探明了固定化小球的理化性质,明确了环境条件对固定化厌氧氨氧化的影响及其生物学特性。同时采用ASBR反应器,实现了厌氧氨氧化反应器的快速启动,探讨了固定化厌氧氨氧化工艺的耐冲击性能和有机环境下的运行特性。首先采用ASBR作为反应器,以好氧硝化污泥为种泥,成功启动厌氧氨氧化反应器,达到了富集厌氧氨氧化混培物的目的。反应器运行到60d时,反应器内总氮负荷已经达到0.47 kg/m³.d,此时氨氮和亚硝酸氮的去除率都已达到80%,反应器已经表现出良好的厌氧氨氧化性能,经过126d的培养,反应器稳定运行在厌氧氨氧化状态时,反应器的总氮容积负荷（以N计）最高可达到0.53 kg/m³.d,总氮去除率平均为83%。利用扫描电镜和透射电镜观察显示有典型厌氧氨氧化菌形态的细菌存在。研究采用包埋法固定化微生物的方法,选择四种材料（海藻酸钠SA、聚乙烯醇PVA、羧甲基纤维素钠CMC、聚乙烯醇与海藻酸钠混合液PVA-SA）作为固定化载体,通过对不同载体的固定化操作、固定化小球的厌氧氨氧化活性、理化性质,包埋材料成本等因素的分析比较,选择出PVA-SA作为固定化包埋材料,制作出了PVA-SA固定化厌氧氨氧化小球,并优化了固定化条件。对PVA-SA固定化厌氧氨氧化小球的理化性质的分析测定表明,PVA-SA固定化厌氧氨氧化小球有良好的机械强度与表面特性,有较好的扩散性能。通过扫描电镜、透射电镜观察,直观描述了小球的构造和表面特性。HRT影响实验得出,固定化厌氧氨氧化反应受到HRT的影响明显大,应不短于24h才能维持反应器内部厌氧氨氧化性能。温度在25⁴0℃范围内,进水pH6⁸之间时,固定化厌氧氨氧化反应器基本能保持高效稳定的运行,固定化之后,厌氧氨氧化微生物对温度和pH的耐受范围有所扩大。采用特异性引物成功从固定化小球内微生物总DNA中扩增出厌氧氨氧化特异性16SrDNA,证明了厌氧氨氧化微生物在固定化小球内部事实存在,测序结果固定化小球内部厌氧氨氧化微生物（EU661861）属于Planctomycetales属,其16SrDNA序列与其它的已发现的厌氧氨氧化菌差异较大,基本可以断定实验中起厌氧氨氧化作用的优势种群是一种尚未报道的的新型厌氧氨氧化菌。实验证明厌氧氨氧化的反应速率与生物量的增长呈直线关系。利用细菌的比生长速率（μ）与比基质利用速率（ U）之间的关系,可以求得固定化厌氧氨氧化生物产率系数（Y）=0.0645mgVS （mgNH₄⁺）^-1;细胞衰减常数（b）=0. 0452mgVS（gVS·d）^-1 ,其数学表达式为μ= 0.0645U - 0.0452（r2=0.9641）。采用ASBR反应器和固定化厌氧氨氧化小球,实现了厌氧氨氧化反应器的快速启动,总氮容积负荷从0.355 kgN/m^-3d^-1提高到0.63 kgN/m^-3d^-1,氨氮与亚硝氮去除率始终维持在90%左右,具有高稳定性。基质浓度对厌氧氨氧化反应的影响主要体现在亚硝氮浓度,反应器在受到2倍和3倍进水基质负荷的冲击后,能够在5-8天恢复性能。进水pH冲击实验证明固定化厌氧氨氧化反应器具备更高的抗碱性冲击性能,而酸性冲击对其影响甚大。在以有机物进水运行期间,有机物严重的影响了厌氧氨氧化反应的性能,但反应器可以较快的恢复性能。研究结果充分证明的固定化厌氧氨氧化工艺的可行性和有效性,具有良好的应用前景。研究丰富了厌氧氨氧化研究内容,为基于固定化技术的厌氧氨氧化工艺开发奠定了理论基础。更多还原

【Abstract】 Anaerobic ammonium oxidation （ANAMMOX） has been put forward recently as a promising alternative to treat ammonium rich wastewaters. However, the ANAMMOX bacteria have a low growth rate and the biomass yield is low. For these reason, maintaining a sufficient amount of ANAMMOX sludge in a reactor is the main factor in the development of a stable and high-rate ANAMMOX system. Fortunately, immobilization is an efficient method to prevent biomass from being washed out and to promote hyper-concentrated cultures. Compared with natural microorganism technology, immobilization of microorganism technology has the advantages such as high cell density, strong endurance of toxicity, easy separation of produce, low operating expenses, simple maintenance management and little residual sludge etc. In order to promote the application of ANAMMOX process, it was necessary to research immobilized ANAMMOX process.In this research, the ANAMMOX sludge was cultured by the anaerobic sequencing batch reactor （ASBR）. The best entrapped support was obtained by comparing immobilization operation, nitrogen removal ratio and cost of entrapped materials etc. and the best operating conditions was demonstrated by orthogonal experiment. The functional microbial group was identified by making specific 16srDNA clone library. In addition, the effect of hydraulic retention time （HRT）, pH, and temperature on immobilized beads were investigated. Then, with immobilized ANAMMOX beads as seed, the start-up, operation, impact factors of ANAMMOX ASBR were investigated in detail.ANAMMOX ASBR was successfully started up within 126d, with aerobic nitrifying sludge as inoculum. When the ANAMMOX ASBR was run at stable state, the volumetric load rate was 0.53 kg（TN）/m³.d, and the average removal efficiency of total nitrogen was 83%. SEM and TEM showed that the typical ANAMMOX bacteria exist in the sludge.Four materials, sodium carboxymethylcellulose （Na-CMC）, sodium alginate （SA）, polyvinyl alcohol （PVA）, and mixed liquor of polyvinyl alcohol and sodium alginate （PVA-SA）, were prepared as carriers for entrapping ANAMMOX sludge. The best entrapment support PVA-SA was selected by comparing ANAMMOX efficiency, the physical characters, immobilization operation, and costs of entrapment materials and so on. Moreover, PVA-SA gel entrapment orthogonal test was performed, consequently, the PVA-SA immobilized live ANAMMOX beads were prepared.The physicochemical characteristics of PVA-SA immobilized ANAMMOX beads were investigated. The results indicated that the PVA-SA immobilized ANAMMOX beads had preferable mechanical strength, surface structure and diffusibility. By means of scanning electron microscopy, transmission electron microscopy and digital photography, the surface structure of PVA-SA immobilized bead is loose and finely porous which facilitates diffusion of the nitrogen,and the ANAMMOX bacteria were confirmed in gel beads. Then, the effect of hydraulic retention time （HRT）, pH, and temperature on immobilized biomass were investigated. The effect of pH and temperature on the ANAMMOX process was evidently weakened in PVA-SA immobilized gel beads and the effect of HRT on the ANAMMOX reaction was reversely significant, which could be the key parameter of the immobilized ANAMMOX system. Stable operation could be achieved in an ASBR, which proved that gel immobilization was an excellent method to maintain the ANAMMOX biomass.PCR amplification, cloning, and phylogenetic analysis of the 16S rRNA genes were performed to identify the ANAMMOX bacteria immobilized in the gel bead culture. The results demonstrated that there are one ANAMMOX bacterial species existing in the gel beads （GenBank accession numbers EU661861）. Phylogenetic analysis based on 16S rRNA sequences revealed that these bacteria were groups of distantly relative microorganisms from the Planctomycete community, had a common node with the recently described anaerobic ammonia-oxidizing group KSU-1（AB057453） of the Planctomycetales. The test results indicated that the efficiency of ANAMMOX has linear relationship with the growth of biomass, it showed the sufficient ANAMMOX biomass maintain in reactor is necessary. The kinetic characteristics of anaerobic ammonia oxidation was studied, The cell yield was 0.0645mgVS （mgNH₄⁺）^-1 and the cell decay coefficient was 0.0452mgVS （g VS·d）^-1, the Mathematical expression wasμ= 0.0645U - 0.0452 （r2=0.9641）.Finally, the ASBR was performed using ANAMMOX bacteria entrapped in a gel carrier. There appeared to be no lag phase in activity of the ANAMMOX bacteria, because the seed sludge was ANAMMOX enrichment, nitrogen removal quickly increased, and the removal ratios of ammonium and nitrite by gel beads were 81.3% and 76.5% until day 10, respectively. Although nitrogen loading increased in the influent, nitrite and ammonium concentration in the effluent decreased to below 15 mg/L and 10 mg/L after 30 day, the nitrogen conversion rate continuously increased and reached an average of 0.58 kg N/m³ per day after day 60. The effect of pH and substrate shocks on the performance of ANAMMOX reactor was investigated. Whether the reactor was affected by substrate or pH shocks, the performance was considerably affected. Comparing with substrate shocks, the reactor was more sensitive to pH shocks. The performance was able to recover from the disturbances at all the tested shocks within 5-11 days. The recovering velocity was relatively stable, which did not increase with the intensity of the shocks. But the reactor was easier to recover from high pH shocks than low high pH shocks. The organic carbon substrate test demonstrated that the organic matter could inhibit the ANAMMOX, under organic carbon condition, the heterotrophic denitrification was enhanced, and the nitrate nitrogen produced from ANAMMOX reaction could be removed.From the above results, it is believed that gel entrapment was supposed to be a highly effective technique for immobilized ANAMMOX bacteria. The immobilized ANAMMOX can bring great economic benefits due to easy separation and maintenance of the ANAMMOX bacteria in the reactor. The results proved the feasibility and good prospect of immobilized ANAMMOX, and laid a theoretical foundation for ANAMMOX process based on immobilization technique.更多还原