利用硝酸盐和亚硝酸盐同步富集厌氧甲烷氧化微生物的比较实验

An experimental comparison of simultaneous enrichment of anaerobic methane oxidizing microorganisms using nitrate and nitrite

摘要:

【背景】反硝化厌氧甲烷氧化(Denitrifying anaerobic methane oxidation，DAMO)是以硝酸盐或亚硝酸盐为电子受体以甲烷为电子供体的厌氧氧化过程，对认识全球碳氮循环、削减温室气体排放和开发废水脱氮新技术等方面具有重要意义。【目的】认识以硝酸盐和亚硝酸盐为电子受体的DAMO微生物富集过程和结果的差异性。【方法】在序批式反应器(Sequencing batch reaetor，SBR)内接种混合物、分别以硝酸盐和亚硝酸盐为电子受体连续培养800 d，定期检测反应器基质浓度变化、计算转化速率；利用16S rRNA基因系统发育分析研究功能微生物的多样性，利用实时荧光定量PCR技术定量测定功能微生物。【结果】以亚硝酸盐为电子受体的1、3号反应器富集到了DAMO细菌、没有检测到DAMO古菌；以硝酸盐为电子受体的2号反应器富集到了DAMO细菌和古菌的混合物；3个反应器的脱氮速率经过初始低速期、快速提升期，最终达到稳定，但2号快速提升期开始时间比1、3号晚了80 d左右，达到稳定的时间更长，稳定最大速率为1、3号的44.7%、40.3%。【结论】硝酸盐和亚硝酸盐对富集产物有决定性影响；以硝酸盐为电子受体富集得到的DAMO古菌和细菌协同体系可以长期稳定共存，DAMO古菌可能是协同体系中脱氮速率的限制性因素。

Abstract:

[Background] Denitrifying anaerobic methane oxidation (DAMO) is an anaerobic oxidation process, in which nitrate/nitrite works as the electron acceptor, while methane works as the electron donor. DAMO has great significance to understand the global carbon and nitrogen cycle, reduce greenhouse gas emissions and develop new technologies for nitrogen removal from wastewater. [Objective] Feeding nitrate or nitrite as the electron acceptor, we investigated the differences on the performance of the denitrifying anaerobic methane oxidation reactors. [Methods] The SBR reactors in which nitrate/nitrite works as the electron acceptor were inoculated sludge mixture and then consecutively long-term incubation for 800 days, meanwhile we measured the nitrite and nitrate concentration in the reactors and calculated the conversion rate regularly. The abundance and diversity of functional microorganisms were investigated by 16S rRNA gene phylogenetic analysis. Then we analyzed the amount of the functional microorganisms by real-time fluorescence quantitative PCR technique. [Results] The DAMO bacteria were detected in the No. 1 reactor and No. 3 reactor which were fed with nitrite as the electron acceptor but the DAMO archaea was not detected. However in the No. 2 reactor which was fed with nitrite as the electron acceptor, the DAMO archaea and bacteria were detected simultaneously. The nitrogen removal rate of the three reactors kept a low value at the very beginning, then experienced a fast lifting phase and maintained a steady value at last. The time when nitrogen removal rate of No. 2 reactor started to increase rapidly was about 80 days later than No. 1 and No. 3 reactor. Its stable maximum rate is only 44.7% of No. 1 reactor and 40.3% of No. 3 reactor, spending more time reaching the stability. [Conclusion] The different substrate fed leads to the different results of enrichment. The DAMO archaea and bacterial synergistic system, which is enriched with nitrate as the electron acceptor, can coexist for a long time. DAMO archaea may be a limiting factor in the nitrogen removal rate in the synergistic system.