【作者】 李冰；

【导师】 施汉昌；

【作者基本信息】 清华大学 ， 环境科学与工程， 2014， 博士

【摘要】 抗生素的大量使用导致其在生活污水中广泛存在，会对污水处理系统中的微生物产生影响。在研究这种影响的特征时，人们往往遇到常规手段（如摇瓶或小试）费时费力且信息量较少的困境。微流控技术能实现并行测试、原位实时观察、单细胞追踪等功能，且样品量少，省时省力，有利于上述问题的研究。因此，本研究建立了适合研究环境微生物的微流控芯片系统，实现了单细胞跟踪、原位实时观测、梯度并行实验等功能；将其应用于抗生素对细菌的抑制、细菌耐药性及细菌对抗生素降解的研究，获得了比常规方法更多的信息（如细菌形态变化、细菌个体差异等），并发现了细菌在抗生素作用下的“新行为”。论文首先采用孔板实验方法，获取了13种抗生素对8种纯菌及活性污泥混合菌的生长抑制曲线，发现喹喏酮类对大部分细菌的抑制能力强，半数抑制浓度（IC50）低于1mg/L；磺胺类最弱，IC50大于100mg/L；活性污泥混合菌具有很强的耐药性。然后开展了微流控平台的研究，设计及优化了微流控双通道芯片，用于抗生素对细菌的抑制过程研究。采用大肠杆菌及阿莫西林验证了芯片的可靠性，实时观察了单个细菌的形态变化特征。芯片可快速培养氨氧化菌，比生长速率（1.2d-1）远高于摇瓶实验（0.088d-1），可在四天内获取阿莫西林对氨氧化菌的抑制曲线。第三，利用微流控芯片技术开展了细菌耐药性的研究。基于对芯片上单个大肠杆菌的数量及形态变化的分析，首次观察到细菌的“机会耐药性”行为。在IC50浓度附近，正常细菌由于邻近破裂细菌释放的细胞质的存在而快速生长，其生长速率与培养基条件下相近，表现出与“耐药性”相似的行为。氨氧化菌的耐药实验发现，氨氧化菌在抑制浓度的阿莫西林下暴露三天后仍有细菌存活，恢复培养基培养后能复苏生长。最后，采用微流控芯片研究了抗生素的降解特征。利用微流控单通道芯片作为“微反应器”，确认了硝化菌对抗生素降解的贡献，并观察到了纯种氨氧化菌降解抗生素的行为。活性污泥混合菌群可以完全降解低浓度阿莫西林（进水均值0.35mg/L），能够通过共基质代谢去除70%的四环素（进水均值0.8mg/L）；活性污泥中的硝化菌对阿莫西林的降解起到促进作用。纯种氨氧化菌可降解阿莫西林和四环素，降解率分别为70%及25%左右，具体的降解途径还需进一步研究。更多还原

【Abstract】 The overuse of antibotics leads to ubiquitous residual in domestic wastewater,which canhave an adverse effect on microbes involved in wastewater treatment systems.Flasks or reactors assays are often used to characterize these effects, however, theseassays are usually time consuming, labor intensive and require large amounts ofreagents. Moreover, these assays are, in general, end-point measurements, so it isdifficult to monitor the kinetics of bacterial growth in situ or to retrieve otherinformation such as bacterial morphological changes.Microfluidics offers some clearadvantages over these existing formats, providing the possibility to develop highthroughput, real-time, low sample consumption, and single cell traking assays.Therefore, wedeveloped a microfluidic system for environmental bacteria study,integrating singlecell tracking, insitu real-time bacterial quantity and morphologicrecording, concentrationgradients forming on a small chip. Then we applied thismicrofluidic system in the study ofbacterial inhibitory tests, bacterial resistance toantibiotic and biodegradationof antibiotic.First we obtained inhibitory curves ofthirteen antibiotics to eight bacterial speciesand bacterial consortia from two wastewater treatment plants through conventionalwell-plate methods, and identified quilolines as the strongest inhibitorsto bacteria(IC50<1mg/L) and sulfanilamide as the weakest (IC50>100mg/L). Furthermore,bacteria of activated sludge showed strong resistance to all antibiotics.Then we designed and optimized the microfluidic chip for bacterial growthinhibition test, with Escherichia coli and amoxicillin we quantitatively proved that theon-chip assay can get same results with conventional laboratory-based dilution methods.Compared with previous studies, our system enables long-term tracking ofmorphological dynamics of individual bacteria under a wide range of inhibitorconcentrations. Moreover, we succeeded to culture ammoium oxidizing bacteria (AOB)at rather higher specific growth rate (1.2d-1) on chip than inflask (0.088d-1), then wefinished the inhibitory test within four days.Thirdly, we used microfluidic technology to do the study of bacterial resistance toantibiotics. The growth rates together with morphological dynamics of individual cells had led to the discovery of a new form of persistence to amoxicillin. Normal cells thatare sensitive to amoxicillin gain persistence or recover from the killing process, if theyhave had an opportunity to live in the cytoplasm released from lysed cells close-by. Weterm this acquired persistence in normal growing cells ‘opportunistic persistence’.Theresistant behavior of AOB was observed when culturing them at inhibitoryconcentration of amoxicillin, some bacterial cells can survive and regrow whenamoxicillin was removed by broth.At last, we used microfluidic technology to study the degradation of antibiotics.The microfluidic system can be used as micro-reactor for the study of antibioticsdegradation. Through the chip experiments we identfied that nitrifiers could improve thedegradation of antibiotics, and then we acquiredproofs of that AOB might be able todegrade antibiotics. The bacterial consortium in activated sludge could eliminateamoxicllin completely when the influent concentration was0.35mg/L, while up to70%of tetracycline can be degraded with the existence of carbon sourcewhen the influentconcentration was0.8mg/L. Further experiments confirmed that AOB strain exhibits anability to degrade amxocillin and tetracycline, and the degradation rates were70%and25%, respectively. Nonetheless, the path of degradation needs further study.更多还原