乙腈是一种同化率较低的化学物质，是大多数污水处理厂中常见的污染物，因此，本研究将乙腈作为目标基质，尝试利用稳定性同位素示踪技术（Stable isotope probing，以下简称SIP）和磁性纳米颗粒分离技术（magnetic-nanoparticle mediated isolation，以下简称MMI）鉴别和分离活性污泥中的乙腈降解菌。SIP法是一种独立培养方法，能够在原位实现不能培养的功能微生物的鉴别，其在研究过程中需依赖于功能微生物将被15N同位素标记的基质（乙腈）同化为细胞内组分。MMI法是另一种不需基质标记的独立培养方法，借助磁性纳米颗粒的附着作用分离去除活性低且生长劣势的细菌，从而富集得到优势降解功能菌群。实验中设置了两个微生态系统以模拟不同的污水处理工艺，短期处理阶段模拟原位生物降解条件，一次性投加乙腈研究降解过程中的功能微生物；长期处理阶段模拟序批式培养条件，每隔一定时间加入乙腈，驯化培养功能微生物。得到的结论主要有： （1）在短期好氧处理阶段，乙腈在所有实验组中均发生降解且降解率差异不大。14N_SIP，15N_SIP与MMI体系中乙腈的降解率分别为55.4 %、55.0 %及53 %。在长期序批式处理阶段，乙腈降解率随着反应的进行不断增加，至第30天，14N_SIP，15N_SIP与MMI中乙腈的降解率均达到最高，分别为83.7 %、83.6 %及96.4 %，表明功能微生物在体系中实现了富集。 （2）SIP法在短期处理阶段筛选出的降解菌包括：Pseudomonadaceae， Gammaproteobacteria，Xanthomonadales与Chitinophagaceae。在长期处理阶段筛选出的降解菌包括：cvE6，norank_o_HTA4，Bdellovibrionaceae，Moraxellaceae，Rickettsiales incertae_sedis，SBR1093，Obscuribacterales，Rhodocyclaceae与Saccharibacteria。MMI法在短期处理阶段筛选出的降解菌包括：SC-I-84，Bradyrhizobiaceae，Rhodobacteraceae，ABS-19与Hyphomicrobiaceae。在长期处理阶段，MMI法与SIP法筛选出了相同的功能微生物。SIP法与MMI法在短期筛选出的降解菌存在显著差异，由于乙腈的同化率较低，使得目标基质很难完全地转化为实验中选择的同位素，因此SIP法对同化率低的污染物研究存在一定缺陷。而MMI法可以弥补这一不足，其不需底物标记或超高速离心，乙腈降解菌在MMI体系中的富集率显著高于SIP体系，因此MMI法是一种研究同化率低的污染物生物降解过程的有效方法。增加培养时间有助于功能微生物将更多同位素标记的乙腈转化为细胞组分，因此经过序批式培养后，SIP法与MMI法鉴别出了相同的乙腈降解菌。值得注意的是，由于外部培养条件的变化，短期和长期降解过程的乙腈降解菌群落存在一定差异。长期处理阶段中的功能微生物均未被报道过参与乙腈降解过程，对这些微生物的研究也很少，但序批式培养方法更接近实际生产中的污水处理过程，丰富了乙腈降解功能微生物的菌株资料。 （3）宏基因组测序对乙腈代谢路径研究的结果表明：乙腈在腈水解酶（EC 3.5.5.1）的作用下一步直接水解为氨，MMI体系中编码腈水解酶相关基因的丰度高于SIP体系。表明MMI法能够富集体系中的功能微生物从而提高乙腈的降解效率，是一种鉴别复杂环境中具有特定代谢功能的微生物的十分有前景的技术，也为今后污水处理厂乙腈降解提供了数据资料。 MMI法不需对基质进行标记，因此即使目标污染物的同化率较低也能实现对功能降解微生物的鉴别，本研究也首次利用MMI法在原位分离出活性乙腈降解菌。通过模拟不同的污水处理工艺，探讨了不同处理方式下乙腈降解菌群落结构的连续性，获得了更接近实际生产过程的功能微生物，丰富了乙腈降解菌的群落信息。为今后废水处理过程中乙腈生物降解的研究提供了新的方法和菌株资料，同时为选择合理的处理方法提供了新的思路。

Acetonitrile was used as the target low-assimilation chemical found in many wastewater treatment plants and attempted to identify and separate the active acetonitrile degraders in the activated sludge, via Stable isotope probing (SIP) and magnetic-nanoparticle mediated isolation (MMI). DNA-SIP is a cultivation-independent technique identifying functional-yet-uncultivable microbes in situ. SIP relies on the incorporation of stable isotopes into the intracellular components. MMI is another cultivation-independent approach without the requirement of substrate labeling, which using the adhesion of magnetic nanoparticles to remove the inert bacteria and the enrichment can get the advantage of degrading bacteria group. Two microcosms were set up to mimic different wastewater treatment process. The short-term treatment represented degradation process in situ within the aerobic reactor, in which the acetonitrile was added once to the microcom; the long-term treatment represented the sequencing batch reaction and acetonitrile was added regularly to domesticate cultured functional microorganisms. Conclusions are as follows: (1) Acetonitrile was degraded in all experimental groups in the short-term aerobic treatment stage and the degradation rate had no significant differences. The degradation rates of acetonitrile in 14N_SIP, 15N_SIP and MMI systems were 55.4 %, 55 % and 53 % respectively. The degradation rate increased with the reaction and reached the highest after 30 days, which was 83.7 %, 83.6 % and 96.4 % respectively, indicating that the functional microorganisms were enriched in the system. (2) The active acetonitrile degraders revealed by SIP were Pseudomonadaceae, Gammaproteobacteria, Xanthomonadales and Chitinophagaceae in short-term aerobic treatment. In long-term sequencing batch treatment, cvE6, norank_o_HTA4, Bdellovibrionaceae, Moraxellaceae, Rickettsiales incertae_sedis, SBR1093, Obscuribacterales, Rhodocyclaceae and Saccharibacteria were functional microorganisms in SIP. In MMI system, SC-I-84, Bradyrhizobiaceae, Rhodobacteraceae, ABS-19 and Hyphomicrobiaceae were revealed in short-term treatment. MMI and SIP revealed same acetonitrile-degraders in long-term treatment. In short-term process, it was difficult for the target substrate to be completely or significantly converted to the isotope selected for the experiment due to low assimilation rate of acetonitrile, which lead to significant stripping of the pollutants during the SIP process. Therefore, SIP method may not suitable for studying pollutants with low assimilation rates. The enrichment ratio of acetonitrile-degraders in MMI was significantly higher than SIP. MMI method does not require substrate labeling or ultracentrifugation, showing the advantages of MMI in identifying degraders for substrates with low assimilation rates. In long-term sequencing batch treatment, we found that longer cultivation helped more incorporation of stable isotope labeled acetonitrile into the cellular components of acetonitrile-degraders. After 30-day sequencing batch degradation, SIP and MMI revealed same acetonitrile-degraders. It is worth mentioning that different acetonitrile-degrader communities were found in short-term and long-term degradation, possibly attributing to the impacts of environmental conditions. Functional microorganisms screened out by the two methods have not been reported involved in the nitriles degradation process in the long-term process. However, studies on these strains were rare, but the stages of domestication and cultivation were closer to the actual conditions of the reactor in the production and living, which could truly reflect the actual role of functional bacteria in the degradation process and had more reference value. (3) Results of metagenomic on acetonitrile metabolic pathway showed that the key enzyme involved in acetonitrile degradation was nitrilase (EC 3.5.5.1) suggesting that acetonitrile was degraded by functional microorganisms in one-step direct hydrolysis in this study. The genes coding enzyme (3.5.5.1) involved in nitrile degradation was more abundant in MMI microcosms than SIP microcosms. MMI method is capable of enriching functional microorganisms and improving the degradation efficiency of acetonitrile. It is a very promising technology for identifying microorganisms with specific metabolic functions in a complex environment and provides data for the degradation of acetonitrile in a sewage treatment plant in the future. MMI method does not require the labeling of the substrate. Therefore, even if the assimilation rate of the target contaminant is low, the identification of functional microorganisms can be achieved. Our study isolated the active acetonitrile-degraders using MMI for the first time. By simulating different wastewater treatment processes, the succession of active acetonitrile-degraders between wastewater treatment strategies was explored. We obtained functional microorganisms that were closer to the actual production process and the community of acetonitrile-degrading bacteria was enriched. These findings provided new insights into the biological process related to acetonitrile metabolisms during wastewater treatment process and offered suggestive conclusions for appropriate treatment selection in wastewater management.