好氧厌氧氨氧化耦合颗粒污泥完全自营养脱氮机理与模拟优化
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摘要
好氧氨氧化与厌氧氨氧化耦合颗粒污泥完全自营养脱氮与传统的硝化反硝化过程相比可以减少60%以上的O_2消耗并且不消耗COD,能大幅减少废水生物脱氮过程的能量消耗和CO_2排放量。论文通过EGSB连续运行试验、SBR反应器间歇实验和分子生物学测试,以及完全自营养脱氮动力学模型研究与模拟优化等方法,研究好氧厌氧氨氧化耦合颗粒污泥完全自营养脱氮机理,优化颗粒污泥生物反应器的运行条件,研究添加微量NO_2条件下限制DO曝气方法强化生物反应器完全自营养脱氮特性等,论文得到如下主要研究结果:
①在EGSB反应器中接种厌氧颗粒污泥,采用间歇进水、间歇出水方式运行210天,成功启动了厌氧氨氧化反应器。在总氮容积负荷为0.11 kg/(m3·d)下,氨氮去除效率达75%,亚硝酸盐氮去除效率达85%,污泥颜色由原来的黑色渐渐变为棕色,形成新的厌氧氨氧化污泥颗粒粒径较小。氨氮、亚硝酸盐氮去除量和硝酸盐氮生成量的比例为1:1.1:0.18。在对厌氧氨氧化过程电子流分析基础上,建立了厌氧氨氧化细胞产率系数与NH_4~+、NO_2-去除量和NO3-生成量之间的计量学关系,估算得厌氧氨氧化菌产率系数为0.080mol CH2O0.5N0.15/mol NH4+。
②通过SBR反应器间歇实验,研究了基质浓度、pH值和温度对厌氧氨氧化活性的影响。结果表明可用Andrews方程描述氨氮和亚硝酸盐氮浓度对厌氧氨氧化动力学特性的影响,动力学分析表明理论最大氨氧化速率369.94 mg/(gMLSS·d)、氨氮半饱和系数值30.39mg/L、亚硝酸盐氮半饱和系数值25.54mg/L、氨氨抑制常数为148.44mg/L、亚硝酸盐对厌氧氨氧化的抑制常数为90.64 mg/L。当氨浓度为67.12 mg/L、亚硝酸盐氮浓度为48.11mg/L,表观最大厌氧氨氧化速率达93.53mg/(gMLSS·d)。pH值是影响厌氧氨氧化活性的重要因素,pH过高或过低都不利于细菌生长和反应进行,本研究中厌氧氨氧化菌的最适pH为8.04。20℃~ 30℃条件下,厌氧氨氧化反应速率与温度的关系可用修正的Arrhenius方程描述。
③使用电解质呼吸仪测定间歇实验中好氧氨氧化过程的累积耗氧量和耗氧速率,通过Mann-Kendell趋势检验方法确定好氧氨氧化菌进入内源呼吸的时刻以及内源呼吸过程中耗氧速率,计算氨氮氧化的耗氧量。基于好氧氨氧化过程中合成代谢和能量代谢耦合,测得好氧氨氧化污泥产率系数为0.199mgCOD/ mg NH4+-N。
④在EGSB反应器中同时接种好氧氨氧化污泥和厌氧氨氧化污泥进行完全自营养脱氮颗粒污泥的培养,pH控制在7.6～8.0,DO控制在0.6～0.8 mg/L,上升流速为4.2m/h。反应器经过120多天的运行,NH4+-N去除效率达75%,总氮去除效率为52%,总氮去除速率达0.101 kg/(m3·d),颗粒污泥粒径主要分布在0.5~1.0mm。
⑤在SBR间歇实验中,DO一定的条件下完全自营养脱氮速率在一定范围内随NH_4~+-N浓度而提高,NH_4~+-N浓度为150mg/L时未发现对完全自营养脱氮活性的显著抑制。DO对完全自营养脱氮过程的影响主要体现在两个方面:过高的DO会抑制厌氧氨氧化活性,提高亚硝酸盐氧化速率,导致NO_2-和NO_3~-在反应器内积累,降低总氮去除效率;DO过低时,好氧氨氧化过程中NO_2-生成速率较低,限制了厌氧氨氧化速率的提高,总氮去除速率较低,但反应器内NO_2-和NO_3~-积累较少,总氮去除效率较高。
⑥提取污泥中细菌总DNA并纯化,以好氧氨氧化菌AmoA基因为进化指标设计特异性引物AMo-F/AMo-R,对好氧氨氧化菌AmoA基因部分序列进行PCR扩增,扩增片段克隆到pMD19-T载体并测序,测得序列的系统发育分析表明污泥中的好氧氨氧化菌主要是亚硝化单胞菌属,与Nitrosomonas europaea具有95%～98%的同源性。根据厌氧氨氧化菌的16SrDNA序列中的特异序列片断设计特异引物Anammox1/ Anammox2,对厌氧氨氧化菌16SrDNA进行PCR扩增。扩增产物连接到pMD19-T载体,将载体转化到感受态细胞大肠杆菌JM109中,并对其16SrDNA基因进行测序。测序结果进行系统发育树分析,表明富集得到的厌氧氨氧化菌与Candidatus Anammoxoglobus propionicus进化关系比较接近。
⑦基于边界层假设模拟颗粒污泥与主体液相间传质过程,并将其与颗粒污泥内传质过程以及好氧氨氧化菌、厌氧氨氧化菌、亚硝酸盐氧化菌生长过程、内源呼吸过程相耦合,建立颗粒污泥完全自营养脱氮动力学模型。通过SBR反应器内间歇实验对模型进行验证,模型模拟结果与实测结果有较好的一致性。对间歇反应过程中完全自营养脱氮过程模拟结果分析表明,在一定NH4+-N浓度下,通过合理控制DO,可使总氮去除效率和去除速率同时达到较好水平。
⑧基于颗粒污泥完全自营养脱氮动力学模型对EGSB反应器操作条件进行优化。结果表明较高的上升流速有利于提高EGSB的脱氮性能,当上升流速由1.1 m/h提高到4.9 m/h时,总氮去除速率提高约10.2%;合适的DO是提高总氮去除性能的关键,通过优化控制EGSB反应器内DO,总氮去除平均除率由52%提高到61%,平均去除速率由0.103 kg/(m~3·d)提高到0.114 kg/(m~3·d),与模型预测结果的误差均小于10%。
⑨采用SBR反应器间歇试验方法,研究微量NO_2氛围下颗粒污泥完全自营养脱氮动力学特性。无O_2时好氧氨氧化菌的NO_2型氨氧化可用Andrews方程描述,最大氨氮降解速率为10.46 mg/(g·h),NO_2半饱和系数和抑制数分别为2.09 mmol/m~3和10.62 mmol/m~3。存在O2时,NO/NO_2形成的NOx循环能强化好氧氨氧化过程,常规好氧氨氧化过程和NO_2强化好氧氨氧化过程同时发生,动力学特性分析表明,最大强化系数40.85,NO_2半饱和常数和抑制常数分别为1.32mmol/m~3和7.11 mmol/m~3。NO_2对厌氧氨氧化过程的强化采用基础速率系数修正的Andrews方程描述,最大强化系数43.5、NO_2半饱和常数和抑制常数分别为16.9 mmol/m~3、0.348 mmol/m~3,基础速率系数0.024。好氧氨氧化和厌氧氨氧化耦合有利于减小NOx对于NO_2型氨氧化过程的抑制。
⑩在对NO_2强化好氧、厌氧氨氧化动力学特性研究的基础上,建立NO_2强化完全自营养脱氮动力学模型,并对EGSB反应器内NO_2强化完全自营养脱氮过程进行模拟,根据模拟结果优化EGSB反应器操作运行模式,控制DO和NO_2分别为0.5～0.8mg/L和2.7～3.3 mmol/m3,总氮去除效率由26.86%～ 31.65%提高到58.83%～63.08%,总氮平均去除速率由0.113 kg/(m~3·d)提高到0.234kg/(m~3·d)。
Based on co-existing of aerobic and anaerobic ammonium oxidizing bacteria in granular sludge, completely autotrophic nitrogen removal process was achieved. Compared to traditional nitration-denitrification process, completely autotrophic nitrogen removal process technology could decrease O_2 consuming as much as 60% and hardly consumed COD, which could decrease the energy consuming and the release of CO2 in nitrogen removal process. Through EGSB (Expanded Granular Sludge Bed) reactor starting up, batch experiment in SBR, molecular biology ,and dynamic modeling optimization. The mechanism of coupling aerobic and anaerobic ammonium oxidizing process was investigated, and then operation mode was optimized. By adding trace NO_2, the enhancement of NO_2 on completely autotrophic nitrogen removal was studied and stimulated. The results were given as follows:
①The ANAMMOX bioreactor was successfully started-up with inoculating anaerobic granular sludge in an EGSB, by intermittent inflow and outflow operating mode for around 210 days. At the total nitrogen loading of 0.11 kg/(m~3·d), removal efficiency of NH_4~+-N and NO_2--N were 75% and 85% respectively. The sludge color changed from black to brown gradually, yet the ANAMMOX sludge granulation was not satisfied. The ratio of NH_4~+-N, NO-2-N removed and NO_3~--N produced was 1:1.1:0.18. Based on analyses of electron stream in ANAMMOX, the stoichiometrics formulae among cell yield, NH_4~+-N, NO-2-N removed and NO_3~--N produced were developed, the yield coefficients of ANAMMOX sludge was 0.080mol CH2O0.5N0.15/mol NH4+.
②The effecting of ammonium, nitrite, pH and temperatures on ANAMMOX were investigated by batch experiment in SBR. The results revealed that influence of ammonium and nitrite on anaerobic ammonium oxidation could be described by Andrews equation. Kinetic characteristics analyzing of anaerobic ammonium oxidation revealed that theoretical maximal rate of anaerobic ammonium oxidation was 369.94 mg(/gMLSS·d), half saturation constant for ammonium and nitrite were 30.39mg/L and 25.54mg/L respectively, and inhibition coefficient for ammonium and nitrite were 148.44mg/L and 90.64mg/L respectively. When the ammonium and nitrite concentration were 67.12 mg/L and 48.11 mg/L, the observed maximal rate of anaerobic ammonium oxidation was 93.53mg/(gMLSS·d). pH value, which is too high or too low, would inhibit bacteria activity, and the optimal pH value was 8.04 in ANAMMOX. The Influence of temperature to anaerobic ammonium oxidation could be described by revised Arrhenius equation between 20 and 30℃.
③Accumulated oxygen consuming and oxygen uptake rate (OUR) in aerobic ammonium oxidation process were measured by electrolytic respirometer in sequence experiment. With method of Mann-Kendell trend test, the time aerobic ammonium oxidizer got in endogenous respiration and the OUR in endogenous respiration were determined, and then the oxygen consuming in ammonium oxidizing was estimated. Based on the coupling of anabolism and energy metabolism in aerobic ammonium oxidation process, relationship between aerobic ammonium oxidizer growth and oxygen consuming in ammonium oxidizing process was established. Yield coefficients of aerobic ammonium oxidizer was determined, which was 0.199mgCOD/ mg NH4+-N.
④In a Expanded Granular Sludge Bed (EGSB)reactor, completely autotrophic nitrogen removal granular sludge was cultured inoculating simultaneously aerobic and anaerobic ammonium oxidation bacteria. The pH was controlled between 7.6 and 8.0, DO between 0.6 mg/L and 0.8 mg/L and the up-velocity 4.2m/h. At end, NH4+-N and TN average removal efficiency was 75% and 52% respectively, TN removal rate reached 0.101 kg/(m~3·d).
⑤Completely autotrophic nitrogen removal rate increased with the NH4+-N concentration in a certain range. In experiment, the NH4+-N concentration as high as 150mg/L would not inhibit nitrogen removal significantly. With much higher DO, TN removal efficiency decreased, which was caused by inhibition of anaerobic ammonium oxidation and increasing of nitrite oxidation rate. With much lower DO, TN removal rate was limited for lacking of NO_2-, and TN removal efficiency was higher because of less accumulation of NO_2- and NO3-.
⑥A pair of specific primers AMo-F/AMo-R based on AmoA (ammonia monooxygenase)gene of ammonium oxidation bacteria was designed to amplify and identify the ammonium oxidation bacteria from the completely autotrophic nitrogen removal granular sludge. After total community genomic DNA was harvested from the sludge, the gene AmoA was amplified by Polymerase Chain Reaction(PCR) method. Then the AmoA gene region was cloned into pMD19-T simple vector and sequenced.The results showed that the amplified DNA fragments were in high homology with AmoA of Nitrosomonas europaea. It indicated that the predominant population of ammonia oxidation bacteria in the sludge was Nitrosomonas.sp. ANAMMOX bacterium was identified by molecular biology. The partial 16SrDNA sequence of ANAMMOX bacterium was amplified by PCR with a pair of specific primers Anammox1/ Anammox2. The PCR product was cloned into the vector pMD19-T and was transfected into competent cells E.coil JM109, then 16SrDNA sequence was determined. Phylogenetic analysis indicated that the cultivated ANAMMOX bacterium closed to Candidatus Anammoxoglobus propionicus.
⑦Based on hypothesis of boundary layer, the transfer processes between granular sludge and bulk liquid was modified, which was coupled with substance transfer processes in granular sludge and aerobic ammonium oxidation, anaerobic ammonium oxidation, nitrite oxidation process, and completely autotrophic nitrogen removal model was found. The stimulation was in a good agreement with measuring results in batch experiment. In a certain NH4+-N concentration, TN removal rate and TN removal efficiency can simultaneously reach higher levels by controlling appropriate DO.
⑧The completely autotrophic nitrogen removal process was stimulated and optimized in EGSB reactor. The concentration of NH4+-N, NO_2--N, NO3--N along reactor in different up velocity revealed that appropriate up velocity had good effect on TN removal by mixture of sludge and water. When up velocity arose from 1.1 m/h to4.9 m/h, TN removal efficiency arose 10.2%. According to the stimulating of total nitrogen removal on different DO, the DO in EGSB was optimized ,TN removal rate and TN removal efficiency raised from 52%to 61% and 0.103 kg/(m~3·d) to 0.114 kg/(m~3·d).the error less than 10% comparing to simulation.
⑨The effect of trace NO_2 and kinetic characteristics for coupling of aerobic and anaerobic ammonium oxidation in granular sludge was investigated by batch experiment in SBR. Without O2, the NO_2 -dependent ammonia oxidation could be described by the Andrews model. The maximum ammonia oxidation rate was 10.46 mg/(g·h), the half saturate coefficient and inhibition coefficient of NO_2 were 2.09 mmol/m3 and 10.62mmol/m3 respectively. Under trace NO_2 atmosphere conditions, aerobic ammonium oxidation was enhanced by increasing the activity of O2 in NOx circulation. The maximum enhancing coefficient was 40.85, the half saturate coefficient and inhibition coefficient of NO_2 were 1.32 mmol/m3 and 7.11 mmol/m3 respectively. Anaerobic ammonium oxidation process could be enhanced by NO_2, which described by revised Andrews model. The maximum enhancing coefficient was 43.5, the half saturate coefficient and inhibition coefficient of NO_2 were 16.9 mmol/m3 and 0.348 mmol/m3 , the basic rate coefficient was 0.024. The toxic of NO_2 for aerobic ammonium oxidizer was decreased in coupling system of aerobic and anaerobic ammonium oxidation.
⑩.Based on kinetic analysis, completely autotrophic nitrogen removal model enhanced NO_2 by was found, and then completely autotrophic nitrogen removal process with NO_2 in EGSB was stimulated. According to stimulation, the operation was optimized, DO and NO_2 were controlled to 0.5~0.8mg/L and 2.7～3.3 mmol/m3 respectively, nitrogen removal efficiency increased from 26.86%～31.65% to 58.83%～63.08%, and removal rate increased from 0.113 kg/(m~3·d) to 0.234kg/(m~3·d).
①在EGSB反应器中接种厌氧颗粒污泥,采用间歇进水、间歇出水方式运行210天,成功启动了厌氧氨氧化反应器。在总氮容积负荷为0.11 kg/(m3·d)下,氨氮去除效率达75%,亚硝酸盐氮去除效率达85%,污泥颜色由原来的黑色渐渐变为棕色,形成新的厌氧氨氧化污泥颗粒粒径较小。氨氮、亚硝酸盐氮去除量和硝酸盐氮生成量的比例为1:1.1:0.18。在对厌氧氨氧化过程电子流分析基础上,建立了厌氧氨氧化细胞产率系数与NH_4~+、NO_2-去除量和NO3-生成量之间的计量学关系,估算得厌氧氨氧化菌产率系数为0.080mol CH2O0.5N0.15/mol NH4+。
②通过SBR反应器间歇实验,研究了基质浓度、pH值和温度对厌氧氨氧化活性的影响。结果表明可用Andrews方程描述氨氮和亚硝酸盐氮浓度对厌氧氨氧化动力学特性的影响,动力学分析表明理论最大氨氧化速率369.94 mg/(gMLSS·d)、氨氮半饱和系数值30.39mg/L、亚硝酸盐氮半饱和系数值25.54mg/L、氨氨抑制常数为148.44mg/L、亚硝酸盐对厌氧氨氧化的抑制常数为90.64 mg/L。当氨浓度为67.12 mg/L、亚硝酸盐氮浓度为48.11mg/L,表观最大厌氧氨氧化速率达93.53mg/(gMLSS·d)。pH值是影响厌氧氨氧化活性的重要因素,pH过高或过低都不利于细菌生长和反应进行,本研究中厌氧氨氧化菌的最适pH为8.04。20℃~ 30℃条件下,厌氧氨氧化反应速率与温度的关系可用修正的Arrhenius方程描述。
③使用电解质呼吸仪测定间歇实验中好氧氨氧化过程的累积耗氧量和耗氧速率,通过Mann-Kendell趋势检验方法确定好氧氨氧化菌进入内源呼吸的时刻以及内源呼吸过程中耗氧速率,计算氨氮氧化的耗氧量。基于好氧氨氧化过程中合成代谢和能量代谢耦合,测得好氧氨氧化污泥产率系数为0.199mgCOD/ mg NH4+-N。
④在EGSB反应器中同时接种好氧氨氧化污泥和厌氧氨氧化污泥进行完全自营养脱氮颗粒污泥的培养,pH控制在7.6～8.0,DO控制在0.6～0.8 mg/L,上升流速为4.2m/h。反应器经过120多天的运行,NH4+-N去除效率达75%,总氮去除效率为52%,总氮去除速率达0.101 kg/(m3·d),颗粒污泥粒径主要分布在0.5~1.0mm。
⑤在SBR间歇实验中,DO一定的条件下完全自营养脱氮速率在一定范围内随NH_4~+-N浓度而提高,NH_4~+-N浓度为150mg/L时未发现对完全自营养脱氮活性的显著抑制。DO对完全自营养脱氮过程的影响主要体现在两个方面:过高的DO会抑制厌氧氨氧化活性,提高亚硝酸盐氧化速率,导致NO_2-和NO_3~-在反应器内积累,降低总氮去除效率;DO过低时,好氧氨氧化过程中NO_2-生成速率较低,限制了厌氧氨氧化速率的提高,总氮去除速率较低,但反应器内NO_2-和NO_3~-积累较少,总氮去除效率较高。
⑥提取污泥中细菌总DNA并纯化,以好氧氨氧化菌AmoA基因为进化指标设计特异性引物AMo-F/AMo-R,对好氧氨氧化菌AmoA基因部分序列进行PCR扩增,扩增片段克隆到pMD19-T载体并测序,测得序列的系统发育分析表明污泥中的好氧氨氧化菌主要是亚硝化单胞菌属,与Nitrosomonas europaea具有95%～98%的同源性。根据厌氧氨氧化菌的16SrDNA序列中的特异序列片断设计特异引物Anammox1/ Anammox2,对厌氧氨氧化菌16SrDNA进行PCR扩增。扩增产物连接到pMD19-T载体,将载体转化到感受态细胞大肠杆菌JM109中,并对其16SrDNA基因进行测序。测序结果进行系统发育树分析,表明富集得到的厌氧氨氧化菌与Candidatus Anammoxoglobus propionicus进化关系比较接近。
⑦基于边界层假设模拟颗粒污泥与主体液相间传质过程,并将其与颗粒污泥内传质过程以及好氧氨氧化菌、厌氧氨氧化菌、亚硝酸盐氧化菌生长过程、内源呼吸过程相耦合,建立颗粒污泥完全自营养脱氮动力学模型。通过SBR反应器内间歇实验对模型进行验证,模型模拟结果与实测结果有较好的一致性。对间歇反应过程中完全自营养脱氮过程模拟结果分析表明,在一定NH4+-N浓度下,通过合理控制DO,可使总氮去除效率和去除速率同时达到较好水平。
⑧基于颗粒污泥完全自营养脱氮动力学模型对EGSB反应器操作条件进行优化。结果表明较高的上升流速有利于提高EGSB的脱氮性能,当上升流速由1.1 m/h提高到4.9 m/h时,总氮去除速率提高约10.2%;合适的DO是提高总氮去除性能的关键,通过优化控制EGSB反应器内DO,总氮去除平均除率由52%提高到61%,平均去除速率由0.103 kg/(m~3·d)提高到0.114 kg/(m~3·d),与模型预测结果的误差均小于10%。
⑨采用SBR反应器间歇试验方法,研究微量NO_2氛围下颗粒污泥完全自营养脱氮动力学特性。无O_2时好氧氨氧化菌的NO_2型氨氧化可用Andrews方程描述,最大氨氮降解速率为10.46 mg/(g·h),NO_2半饱和系数和抑制数分别为2.09 mmol/m~3和10.62 mmol/m~3。存在O2时,NO/NO_2形成的NOx循环能强化好氧氨氧化过程,常规好氧氨氧化过程和NO_2强化好氧氨氧化过程同时发生,动力学特性分析表明,最大强化系数40.85,NO_2半饱和常数和抑制常数分别为1.32mmol/m~3和7.11 mmol/m~3。NO_2对厌氧氨氧化过程的强化采用基础速率系数修正的Andrews方程描述,最大强化系数43.5、NO_2半饱和常数和抑制常数分别为16.9 mmol/m~3、0.348 mmol/m~3,基础速率系数0.024。好氧氨氧化和厌氧氨氧化耦合有利于减小NOx对于NO_2型氨氧化过程的抑制。
⑩在对NO_2强化好氧、厌氧氨氧化动力学特性研究的基础上,建立NO_2强化完全自营养脱氮动力学模型,并对EGSB反应器内NO_2强化完全自营养脱氮过程进行模拟,根据模拟结果优化EGSB反应器操作运行模式,控制DO和NO_2分别为0.5～0.8mg/L和2.7～3.3 mmol/m3,总氮去除效率由26.86%～ 31.65%提高到58.83%～63.08%,总氮平均去除速率由0.113 kg/(m~3·d)提高到0.234kg/(m~3·d)。
Based on co-existing of aerobic and anaerobic ammonium oxidizing bacteria in granular sludge, completely autotrophic nitrogen removal process was achieved. Compared to traditional nitration-denitrification process, completely autotrophic nitrogen removal process technology could decrease O_2 consuming as much as 60% and hardly consumed COD, which could decrease the energy consuming and the release of CO2 in nitrogen removal process. Through EGSB (Expanded Granular Sludge Bed) reactor starting up, batch experiment in SBR, molecular biology ,and dynamic modeling optimization. The mechanism of coupling aerobic and anaerobic ammonium oxidizing process was investigated, and then operation mode was optimized. By adding trace NO_2, the enhancement of NO_2 on completely autotrophic nitrogen removal was studied and stimulated. The results were given as follows:
①The ANAMMOX bioreactor was successfully started-up with inoculating anaerobic granular sludge in an EGSB, by intermittent inflow and outflow operating mode for around 210 days. At the total nitrogen loading of 0.11 kg/(m~3·d), removal efficiency of NH_4~+-N and NO_2--N were 75% and 85% respectively. The sludge color changed from black to brown gradually, yet the ANAMMOX sludge granulation was not satisfied. The ratio of NH_4~+-N, NO-2-N removed and NO_3~--N produced was 1:1.1:0.18. Based on analyses of electron stream in ANAMMOX, the stoichiometrics formulae among cell yield, NH_4~+-N, NO-2-N removed and NO_3~--N produced were developed, the yield coefficients of ANAMMOX sludge was 0.080mol CH2O0.5N0.15/mol NH4+.
②The effecting of ammonium, nitrite, pH and temperatures on ANAMMOX were investigated by batch experiment in SBR. The results revealed that influence of ammonium and nitrite on anaerobic ammonium oxidation could be described by Andrews equation. Kinetic characteristics analyzing of anaerobic ammonium oxidation revealed that theoretical maximal rate of anaerobic ammonium oxidation was 369.94 mg(/gMLSS·d), half saturation constant for ammonium and nitrite were 30.39mg/L and 25.54mg/L respectively, and inhibition coefficient for ammonium and nitrite were 148.44mg/L and 90.64mg/L respectively. When the ammonium and nitrite concentration were 67.12 mg/L and 48.11 mg/L, the observed maximal rate of anaerobic ammonium oxidation was 93.53mg/(gMLSS·d). pH value, which is too high or too low, would inhibit bacteria activity, and the optimal pH value was 8.04 in ANAMMOX. The Influence of temperature to anaerobic ammonium oxidation could be described by revised Arrhenius equation between 20 and 30℃.
③Accumulated oxygen consuming and oxygen uptake rate (OUR) in aerobic ammonium oxidation process were measured by electrolytic respirometer in sequence experiment. With method of Mann-Kendell trend test, the time aerobic ammonium oxidizer got in endogenous respiration and the OUR in endogenous respiration were determined, and then the oxygen consuming in ammonium oxidizing was estimated. Based on the coupling of anabolism and energy metabolism in aerobic ammonium oxidation process, relationship between aerobic ammonium oxidizer growth and oxygen consuming in ammonium oxidizing process was established. Yield coefficients of aerobic ammonium oxidizer was determined, which was 0.199mgCOD/ mg NH4+-N.
④In a Expanded Granular Sludge Bed (EGSB)reactor, completely autotrophic nitrogen removal granular sludge was cultured inoculating simultaneously aerobic and anaerobic ammonium oxidation bacteria. The pH was controlled between 7.6 and 8.0, DO between 0.6 mg/L and 0.8 mg/L and the up-velocity 4.2m/h. At end, NH4+-N and TN average removal efficiency was 75% and 52% respectively, TN removal rate reached 0.101 kg/(m~3·d).
⑤Completely autotrophic nitrogen removal rate increased with the NH4+-N concentration in a certain range. In experiment, the NH4+-N concentration as high as 150mg/L would not inhibit nitrogen removal significantly. With much higher DO, TN removal efficiency decreased, which was caused by inhibition of anaerobic ammonium oxidation and increasing of nitrite oxidation rate. With much lower DO, TN removal rate was limited for lacking of NO_2-, and TN removal efficiency was higher because of less accumulation of NO_2- and NO3-.
⑥A pair of specific primers AMo-F/AMo-R based on AmoA (ammonia monooxygenase)gene of ammonium oxidation bacteria was designed to amplify and identify the ammonium oxidation bacteria from the completely autotrophic nitrogen removal granular sludge. After total community genomic DNA was harvested from the sludge, the gene AmoA was amplified by Polymerase Chain Reaction(PCR) method. Then the AmoA gene region was cloned into pMD19-T simple vector and sequenced.The results showed that the amplified DNA fragments were in high homology with AmoA of Nitrosomonas europaea. It indicated that the predominant population of ammonia oxidation bacteria in the sludge was Nitrosomonas.sp. ANAMMOX bacterium was identified by molecular biology. The partial 16SrDNA sequence of ANAMMOX bacterium was amplified by PCR with a pair of specific primers Anammox1/ Anammox2. The PCR product was cloned into the vector pMD19-T and was transfected into competent cells E.coil JM109, then 16SrDNA sequence was determined. Phylogenetic analysis indicated that the cultivated ANAMMOX bacterium closed to Candidatus Anammoxoglobus propionicus.
⑦Based on hypothesis of boundary layer, the transfer processes between granular sludge and bulk liquid was modified, which was coupled with substance transfer processes in granular sludge and aerobic ammonium oxidation, anaerobic ammonium oxidation, nitrite oxidation process, and completely autotrophic nitrogen removal model was found. The stimulation was in a good agreement with measuring results in batch experiment. In a certain NH4+-N concentration, TN removal rate and TN removal efficiency can simultaneously reach higher levels by controlling appropriate DO.
⑧The completely autotrophic nitrogen removal process was stimulated and optimized in EGSB reactor. The concentration of NH4+-N, NO_2--N, NO3--N along reactor in different up velocity revealed that appropriate up velocity had good effect on TN removal by mixture of sludge and water. When up velocity arose from 1.1 m/h to4.9 m/h, TN removal efficiency arose 10.2%. According to the stimulating of total nitrogen removal on different DO, the DO in EGSB was optimized ,TN removal rate and TN removal efficiency raised from 52%to 61% and 0.103 kg/(m~3·d) to 0.114 kg/(m~3·d).the error less than 10% comparing to simulation.
⑨The effect of trace NO_2 and kinetic characteristics for coupling of aerobic and anaerobic ammonium oxidation in granular sludge was investigated by batch experiment in SBR. Without O2, the NO_2 -dependent ammonia oxidation could be described by the Andrews model. The maximum ammonia oxidation rate was 10.46 mg/(g·h), the half saturate coefficient and inhibition coefficient of NO_2 were 2.09 mmol/m3 and 10.62mmol/m3 respectively. Under trace NO_2 atmosphere conditions, aerobic ammonium oxidation was enhanced by increasing the activity of O2 in NOx circulation. The maximum enhancing coefficient was 40.85, the half saturate coefficient and inhibition coefficient of NO_2 were 1.32 mmol/m3 and 7.11 mmol/m3 respectively. Anaerobic ammonium oxidation process could be enhanced by NO_2, which described by revised Andrews model. The maximum enhancing coefficient was 43.5, the half saturate coefficient and inhibition coefficient of NO_2 were 16.9 mmol/m3 and 0.348 mmol/m3 , the basic rate coefficient was 0.024. The toxic of NO_2 for aerobic ammonium oxidizer was decreased in coupling system of aerobic and anaerobic ammonium oxidation.
⑩.Based on kinetic analysis, completely autotrophic nitrogen removal model enhanced NO_2 by was found, and then completely autotrophic nitrogen removal process with NO_2 in EGSB was stimulated. According to stimulation, the operation was optimized, DO and NO_2 were controlled to 0.5~0.8mg/L and 2.7～3.3 mmol/m3 respectively, nitrogen removal efficiency increased from 26.86%～31.65% to 58.83%～63.08%, and removal rate increased from 0.113 kg/(m~3·d) to 0.234kg/(m~3·d).
引文
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