Study On The Effects Of Antibiotics On Bacteria In Activated Sludgeby Microfluidic Technology

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.