新型MBR-PBBR组合系统捷径生物脱氮研究
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摘要
我国氮素污染所引起的水体富营养化问题日益突出,导致湖泊“水华”和近海“赤潮”频繁发生,严重危害了水体生态系统和人类健康,因此国家制定了越来越严格的氮素排放标准。传统的生物脱氮技术在氮素污染控制方面发挥了重要作用,但对高氮低碳废水而言,传统技术总氮去除率低、能耗高。针对此类废水探索高效、低能耗的脱氮技术成为当今研究的热点和难点。近年来提出的短程硝化反硝化工艺缩短了生物脱氮的途径,对高氮低碳废水的处理表现出很大的经济优越性,可降低能耗、节省碳源、减少污泥产量和缩小反应器容积等。
本研究针对短程硝化反硝化捷径生物脱氮难以稳定运行的问题,引入生物强化技术,通过将短程硝化功能菌和亚硝酸型反硝化功能菌接种至膜生物反应器(MBR)和填料床生物膜反应器(PBBR)中,构建了新型的MBR-PBBR捷径生物脱氮工艺。在工艺成功启动后,探讨工艺参数和不同生态因子对该工艺捷径生物脱氮性能和稳定性的影响,并进一步研究其微生物作用机理,为该工艺的实际应用提供理论依据和技术指导。
首先,采用系列稀释法筛选驯化得到高效短程硝化功能菌和亚硝酸型反硝化功能菌。结果表明短程硝化功能菌细胞呈球状,主要是由Nitrosomonas属的氨氧化菌组成,其最佳氨氧化条件是:NH_4~+-N 400 mg/L,pH 8.5,温度35℃,Alk/N 8.33;亚硝酸型反硝化功能菌细胞呈杆状,为兼氧菌,其最适生长和降解条件是:柠檬酸三钠为碳源,TOC/N=4,温度30℃,pH 9.0。
其次,通过将短程硝化功能菌和亚硝酸型反硝化功能菌接种至MBR和PBBR中,构建了两级MBR-PBBR新型捷径生物脱氮工艺,并实现了该工艺的快速启动。研究发现,启动期氮的脱除全部通过捷径途径完成。随后,MBR-PBBR工艺在曝气速率为0.4L/min和操作温度为30℃时,实现了105天稳定的捷径生物脱氮。但随水力停留时间的降低,MBR中氨氧化活性降低,氨氧化率由95%下降至60%,导致工艺总氮去除率降低。FISH杂交和MPN计数结果表明,MBR中氨氧化菌为绝对优势菌,第105 d氨氧化菌数量为3×10~8MPN/mL,存在极少量的亚硝酸氧化菌(4.5×10~3MPN/mL)。
再次,分别探讨了溶解氧、运行温度和外加有机碳源对MBR-PBBR捷径生物脱氮工艺稳定性的影响。研究表明,低溶解氧(0.8～1.2 mg/L)下,该工艺氮的脱除全部以捷径途径完成,但低溶解氧影响了氨氧化效率导致工艺总氮去除率降低至80%;高溶解氧(5～6 mg/L)刺激了MBR中少量亚硝酸氧化菌生长(从4.5×10~3MPN/mL增加到2×10~5MPN/mL),导致亚氮积累不稳定,工艺中只有70%的氮以捷径途径去除。升高温度可显著提高MBR-PBBR工艺捷径生物脱氮效率和稳定性,当温度从20℃升高到35℃,MBR的氨氧化率从45%升高至90%,亚氮积累率由55%～80%的波动逐渐稳定在85%。少量的外加有机碳源(TOC/N＜0.2)可促进氨氧化反应,MBR中氨氧化率从无有机碳源时的60%升高至75%;当进水TOC/N≥0.2时,氨氧化反应受到抑制,氨氧化率迅速下降至25%,原因是有机碳源刺激了MBR中异养菌的大量繁殖(11.5×10~8CFU/mL),使氨氧化菌对溶解氧和氨氮的竞争处于弱势地位:有机碳源的加入使该工艺96%以上氮的脱除以捷径途径完成,主要是由于亚硝酸氧化菌很难竞争到溶解氧来氧化NO_2~--N,其数量在TOC/N=0.3时降低到1.0×10~3MPN/mL。
最后,采用现代分子生态学技术中的变性梯度凝胶电泳(DOGE)和荧光原位杂交(FISH),跟踪监测MBR在不同时期微生物结构及动态变化规律。结果发现,生物强化的MBR-PBBR工艺能实现相对稳定和高效的捷径生物脱氮,主要归功于MBR中的功能微生物Nitrosomonas eutropha在运行过程中始终保持优势地位,占全菌数的比例大于54%;然而,当外加有机碳源较高(TOC/N≥0.2)时,可动摇氨氧化菌的优势地位,N.europha占全菌数的比例在TOC/N=0.3时下降至31%。生物强化的MBR随时间的运行,微生物群落多样性总体呈升高趋势,最终形成以N.europha为主的β-Proteobacteria、CFB种群和α-、γ-Proteobacteria三大功能微生物菌群间相互协调的物质代谢和能量传递有序的“生物链”。
Since eutrophication caused by nitrogen pollution,leading to "algae blooms" in lakes and "red tide" along coasts,is becoming a serious issue in China,an ever stricter nitrogen discharge standard has been established to protect ecosystems and human health. Conventional biological nitrogen removal technology has been widely applied,however,it has low TN removal rate and high energy consumption for the treatment of high-strength nitrogen wastewater with low COD/N.Therefore,current researches are ongoing to explore novel nitrogen removal technologies with high efficiency and low energy consumption.The partial nitrification and denitrification process being developed over recent years shows many economic advantages for the treatment of high-strength nitrogen wastewater with low COD/N, such as short reaction routes,low energy consumption,saving carbon resource,low sludge production and smaller reactor volume,etc.
In this study,the performance of a novel two-stage MBR-PBBR(membrane bioreactorpacked bed biofilm reactor) combined system for shortcut biological nitrogen removal is investigated to improve its stability.The MBR and PBBR in the process are seeded with two kinds of functional microorganisms for partial nitrification and denitrification via nitrite.After fast start-up of the MBR-PBBR process,the effects of different ecological factors on the performance of MBR-PBBR combined system for shortcut biological nitrogen removal are studied to evaluate the feasibility of this novel process.The microbial mechanism is further explored to provide theoretical and technical guidelines for the application of this novel process.
Firstly,the functional microorganisms for partial nitrification and denitrification via nitrite are obtained by the extinction dilution method.The results indicate that the functional microorganism for partial nitrification with spherical cell morphology is mainly composed of ammonia-oxidizing bacteria(AOB) belonging to Nitrosomonas,whose optimal conditions for ammonium oxidation are as follows:NH_4~+-N 400 mg/L,pH 8.5,temperature 35℃,Alk/N 8.33.The functional microorganism for denitrification via nitrite with rod-shaped cell morphology belongs to facultative bacteria,whose optimal conditions for nitrite reduction are as follows:sodium citrate as carbon source,TOC/N=4,temperature 30℃,pH 9.0.
Secondly,the novel two-stage MBR-PBBR combined system,seeded with two functional microorganisms,is constructed for shortcut biological nitrogen removal.The MBR-PBBR process is started up very fast and almost all nitrogen is removed via nitrite pathway during this period.Subsequently,stable shortcut biological nitrogen removal for a long period of 105 days is found in MBR-PBBR process with 0.4 L/min aerated rate and 30℃of operating temperature.However,as the decrease of HRT,ammonium oxidation rate in MBR decreases from 95%to 60%leading to the decrease of TN removal rate.The results from fluorescent in situ hybridization(FISH) and the most-probable-number(MPN) counts show that AOB(3×10~8 MPN/mL) is dominated in MBR with less nitrite oxidizing bacteria (NOB,4.5×10~3 MPN/mL) on day 105.
Thirdly,the effects of three important ecological factors,i.e.the dissolved oxygen(DO), operating temperature and additional organic carbon,on the stability of shortcut nitrogen removal performance of the MBR-PBBR process are further studied The results show that low DO(0.8～1.2 mg/L) has negative effect on ammonium oxidation rate in MBR resulting in the decrease of TN removal rate from 95%to 80%.High DO(5～6 mg/L) contributes to the instability of partial nitrification,and only 70%of nitrogen is removed by nitrite pathway due to the growth of few NOB in MBR(from 4.5×10~3 MPN/mL to 2×10~5 MPN/mL) under sufficient DO conditions.The increase of operational temperature significantly improves the nitrogen removal rate and stability of MBR-PBBR process.As increasing the operating temperature from 20℃to 35℃,the ammonium oxidation rate and nitrite accumulation rate in MBR are improved from 45%to 90%and 55～80%to 85%,respectively.Small amount of additional organic carbon(TOC/N<0.2) can accelerate the ammonium oxidation rate from 60%to 75%in MBR.However,large amount of additional organic carbon concentration (TOC/N≥0.2) inhibits ammonium oxidation reaction,leading to sudden drawdown in ammonium oxidation rate to 25%.It is mainly due to the fact that the heterotrophs with rapid growth(11.5×10~8 CFU/mL) could overeompete AOB for DO and NH_4~+-N.96%of nitrogen is removed by nitrite pathway along with the addition of organic carbon.Because NOB is less competitive for enough DO for the nitrite oxidation,the number of NOB decreases to 1.0×10~3 MPN/mL at TOC/N=0.3.
Finally,the denaturing gradient gel eletrophoresis(DGGE) and FISH in molecular ecology technology are performed to analyze microbial community structure and their variations in MBR during different periods.The results indicate that the high efficiency and stable performance of shortcut nitrogen removal in MBR-PBBR process is attributed to the predominance of functional microbiology Nitrosomonas eutropha during the period of operation,which occupies over 54%of total bacteria.However,large amount of organic carbon can destroy the predominance of N.eutropha,whose proportion decreased to 31%at TOC/N=0.3.The diversity of microbial community in MBR increases along with operating time.The microbial community is mainly composed of three groups:β-subclass of Proteobacteria dominated by N.europha,CFB andα-,γ- subclass of Proteobacteria.The three groups form an orderly eco-chain for material metabolism and energy transfer by cooperating and coordinating with each other during the degradation of ammonium and organic matters.
本研究针对短程硝化反硝化捷径生物脱氮难以稳定运行的问题,引入生物强化技术,通过将短程硝化功能菌和亚硝酸型反硝化功能菌接种至膜生物反应器(MBR)和填料床生物膜反应器(PBBR)中,构建了新型的MBR-PBBR捷径生物脱氮工艺。在工艺成功启动后,探讨工艺参数和不同生态因子对该工艺捷径生物脱氮性能和稳定性的影响,并进一步研究其微生物作用机理,为该工艺的实际应用提供理论依据和技术指导。
首先,采用系列稀释法筛选驯化得到高效短程硝化功能菌和亚硝酸型反硝化功能菌。结果表明短程硝化功能菌细胞呈球状,主要是由Nitrosomonas属的氨氧化菌组成,其最佳氨氧化条件是:NH_4~+-N 400 mg/L,pH 8.5,温度35℃,Alk/N 8.33;亚硝酸型反硝化功能菌细胞呈杆状,为兼氧菌,其最适生长和降解条件是:柠檬酸三钠为碳源,TOC/N=4,温度30℃,pH 9.0。
其次,通过将短程硝化功能菌和亚硝酸型反硝化功能菌接种至MBR和PBBR中,构建了两级MBR-PBBR新型捷径生物脱氮工艺,并实现了该工艺的快速启动。研究发现,启动期氮的脱除全部通过捷径途径完成。随后,MBR-PBBR工艺在曝气速率为0.4L/min和操作温度为30℃时,实现了105天稳定的捷径生物脱氮。但随水力停留时间的降低,MBR中氨氧化活性降低,氨氧化率由95%下降至60%,导致工艺总氮去除率降低。FISH杂交和MPN计数结果表明,MBR中氨氧化菌为绝对优势菌,第105 d氨氧化菌数量为3×10~8MPN/mL,存在极少量的亚硝酸氧化菌(4.5×10~3MPN/mL)。
再次,分别探讨了溶解氧、运行温度和外加有机碳源对MBR-PBBR捷径生物脱氮工艺稳定性的影响。研究表明,低溶解氧(0.8～1.2 mg/L)下,该工艺氮的脱除全部以捷径途径完成,但低溶解氧影响了氨氧化效率导致工艺总氮去除率降低至80%;高溶解氧(5～6 mg/L)刺激了MBR中少量亚硝酸氧化菌生长(从4.5×10~3MPN/mL增加到2×10~5MPN/mL),导致亚氮积累不稳定,工艺中只有70%的氮以捷径途径去除。升高温度可显著提高MBR-PBBR工艺捷径生物脱氮效率和稳定性,当温度从20℃升高到35℃,MBR的氨氧化率从45%升高至90%,亚氮积累率由55%～80%的波动逐渐稳定在85%。少量的外加有机碳源(TOC/N＜0.2)可促进氨氧化反应,MBR中氨氧化率从无有机碳源时的60%升高至75%;当进水TOC/N≥0.2时,氨氧化反应受到抑制,氨氧化率迅速下降至25%,原因是有机碳源刺激了MBR中异养菌的大量繁殖(11.5×10~8CFU/mL),使氨氧化菌对溶解氧和氨氮的竞争处于弱势地位:有机碳源的加入使该工艺96%以上氮的脱除以捷径途径完成,主要是由于亚硝酸氧化菌很难竞争到溶解氧来氧化NO_2~--N,其数量在TOC/N=0.3时降低到1.0×10~3MPN/mL。
最后,采用现代分子生态学技术中的变性梯度凝胶电泳(DOGE)和荧光原位杂交(FISH),跟踪监测MBR在不同时期微生物结构及动态变化规律。结果发现,生物强化的MBR-PBBR工艺能实现相对稳定和高效的捷径生物脱氮,主要归功于MBR中的功能微生物Nitrosomonas eutropha在运行过程中始终保持优势地位,占全菌数的比例大于54%;然而,当外加有机碳源较高(TOC/N≥0.2)时,可动摇氨氧化菌的优势地位,N.europha占全菌数的比例在TOC/N=0.3时下降至31%。生物强化的MBR随时间的运行,微生物群落多样性总体呈升高趋势,最终形成以N.europha为主的β-Proteobacteria、CFB种群和α-、γ-Proteobacteria三大功能微生物菌群间相互协调的物质代谢和能量传递有序的“生物链”。
Since eutrophication caused by nitrogen pollution,leading to "algae blooms" in lakes and "red tide" along coasts,is becoming a serious issue in China,an ever stricter nitrogen discharge standard has been established to protect ecosystems and human health. Conventional biological nitrogen removal technology has been widely applied,however,it has low TN removal rate and high energy consumption for the treatment of high-strength nitrogen wastewater with low COD/N.Therefore,current researches are ongoing to explore novel nitrogen removal technologies with high efficiency and low energy consumption.The partial nitrification and denitrification process being developed over recent years shows many economic advantages for the treatment of high-strength nitrogen wastewater with low COD/N, such as short reaction routes,low energy consumption,saving carbon resource,low sludge production and smaller reactor volume,etc.
In this study,the performance of a novel two-stage MBR-PBBR(membrane bioreactorpacked bed biofilm reactor) combined system for shortcut biological nitrogen removal is investigated to improve its stability.The MBR and PBBR in the process are seeded with two kinds of functional microorganisms for partial nitrification and denitrification via nitrite.After fast start-up of the MBR-PBBR process,the effects of different ecological factors on the performance of MBR-PBBR combined system for shortcut biological nitrogen removal are studied to evaluate the feasibility of this novel process.The microbial mechanism is further explored to provide theoretical and technical guidelines for the application of this novel process.
Firstly,the functional microorganisms for partial nitrification and denitrification via nitrite are obtained by the extinction dilution method.The results indicate that the functional microorganism for partial nitrification with spherical cell morphology is mainly composed of ammonia-oxidizing bacteria(AOB) belonging to Nitrosomonas,whose optimal conditions for ammonium oxidation are as follows:NH_4~+-N 400 mg/L,pH 8.5,temperature 35℃,Alk/N 8.33.The functional microorganism for denitrification via nitrite with rod-shaped cell morphology belongs to facultative bacteria,whose optimal conditions for nitrite reduction are as follows:sodium citrate as carbon source,TOC/N=4,temperature 30℃,pH 9.0.
Secondly,the novel two-stage MBR-PBBR combined system,seeded with two functional microorganisms,is constructed for shortcut biological nitrogen removal.The MBR-PBBR process is started up very fast and almost all nitrogen is removed via nitrite pathway during this period.Subsequently,stable shortcut biological nitrogen removal for a long period of 105 days is found in MBR-PBBR process with 0.4 L/min aerated rate and 30℃of operating temperature.However,as the decrease of HRT,ammonium oxidation rate in MBR decreases from 95%to 60%leading to the decrease of TN removal rate.The results from fluorescent in situ hybridization(FISH) and the most-probable-number(MPN) counts show that AOB(3×10~8 MPN/mL) is dominated in MBR with less nitrite oxidizing bacteria (NOB,4.5×10~3 MPN/mL) on day 105.
Thirdly,the effects of three important ecological factors,i.e.the dissolved oxygen(DO), operating temperature and additional organic carbon,on the stability of shortcut nitrogen removal performance of the MBR-PBBR process are further studied The results show that low DO(0.8～1.2 mg/L) has negative effect on ammonium oxidation rate in MBR resulting in the decrease of TN removal rate from 95%to 80%.High DO(5～6 mg/L) contributes to the instability of partial nitrification,and only 70%of nitrogen is removed by nitrite pathway due to the growth of few NOB in MBR(from 4.5×10~3 MPN/mL to 2×10~5 MPN/mL) under sufficient DO conditions.The increase of operational temperature significantly improves the nitrogen removal rate and stability of MBR-PBBR process.As increasing the operating temperature from 20℃to 35℃,the ammonium oxidation rate and nitrite accumulation rate in MBR are improved from 45%to 90%and 55～80%to 85%,respectively.Small amount of additional organic carbon(TOC/N<0.2) can accelerate the ammonium oxidation rate from 60%to 75%in MBR.However,large amount of additional organic carbon concentration (TOC/N≥0.2) inhibits ammonium oxidation reaction,leading to sudden drawdown in ammonium oxidation rate to 25%.It is mainly due to the fact that the heterotrophs with rapid growth(11.5×10~8 CFU/mL) could overeompete AOB for DO and NH_4~+-N.96%of nitrogen is removed by nitrite pathway along with the addition of organic carbon.Because NOB is less competitive for enough DO for the nitrite oxidation,the number of NOB decreases to 1.0×10~3 MPN/mL at TOC/N=0.3.
Finally,the denaturing gradient gel eletrophoresis(DGGE) and FISH in molecular ecology technology are performed to analyze microbial community structure and their variations in MBR during different periods.The results indicate that the high efficiency and stable performance of shortcut nitrogen removal in MBR-PBBR process is attributed to the predominance of functional microbiology Nitrosomonas eutropha during the period of operation,which occupies over 54%of total bacteria.However,large amount of organic carbon can destroy the predominance of N.eutropha,whose proportion decreased to 31%at TOC/N=0.3.The diversity of microbial community in MBR increases along with operating time.The microbial community is mainly composed of three groups:β-subclass of Proteobacteria dominated by N.europha,CFB andα-,γ- subclass of Proteobacteria.The three groups form an orderly eco-chain for material metabolism and energy transfer by cooperating and coordinating with each other during the degradation of ammonium and organic matters.
引文
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