摘  要

生物被膜泛指物体表面相关的微生物群体以及微生物形成的絮凝体或凝聚体，近年来其用于微通道催化研究日趋成为微型生物催化的研究热点。然而，大多情况下，生物被膜在反应器内无序过度生长甚至无法控制，使物料传质受阻，而降低反应效率。由此，本文以重组大肠杆菌作为研究对象，在微通道反应器采用分段流形式培养生物菌膜，探讨微通道中形成生物菌膜的过程规律。

（1）对空气-水分段流的调控方法进行了优化。实验结果显示，分段流的最佳调控方法为：LB培养液-空气分段流，共同流速为45 μL/min，培养温度为32 ℃，培养时间为72 h。在此条件下，微管道中重组大肠杆菌的生长状况良好，OD590最大为1.729。

（2）研究了重组大肠杆菌的生长曲线。结果表明，将延滞期、指数生长期、稳定期和衰亡期不同时期的重组大肠杆菌分别通入微通道中进行培养，指数生长期的大肠杆菌在微通道中生长状况最好，其最快时间在微通道中形成稳定的生物被膜，时间为72 h，相对生长最慢延滞期的大肠杆菌时间缩短了1/3。

（3）考察了微通道中的温度和pH对重组大肠杆菌形成生物被膜的影响。结果表明，在微通道中重组大肠杆菌的最佳生长温度为32 ℃，最佳生长pH为7.0。在此最佳培养条件下，重组大肠杆菌形成生物被膜的状况最佳，OD590最大为1.319。

（4）考察了MW-CN、Fe2O3和Graphene三种不同纳米材料对生物被膜成膜特性的影响。碳纳米管、Fe2O3和石墨烯（10 mg/L）相比较空白抑制率分别为3.2%、5.3%和 15.7%。在相同浓度下，碳纳米管、Fe2O3和石墨烯纳米对生物菌膜的抑制率与对重组菌株比较分别下降19.4%、13.1%和12.1%。

综上所述，采用双相分段流方法在微通道反应器内培养大肠杆菌制备生物被膜且优化其条件，不仅可以解决在常规反应器内生物被膜生长不可控的问题，还可以为生物被膜催化提供一条新的思路。

关键词：生物被膜；微流控；分段流；大肠杆菌；重组菌株

Abstract

Biofilm is a ubiquitous microbial life form in nature consisting of cells embedded within a self-produced matrix of extracellular polymeric substances (EPS). In recent years, the research of biofilms catalysis has become a hotspot in microbial catalysis. However, the mass transfer and reaction efficiency in biofilm reactors are often limited due to excessive growth. Thus, with reorganizing the Escherichia coli as a research object, segmented flow strategy was used to culture E. coli for biofilm formation in microreactors and the discipline of biofilm development was explored. The main research contents are as follows:

(1) The regulation process parameters of segmented flow method were optimized. The experimental results showed that the optimum conditions were as follows: the optimal flow rate was 45 μL/min, the segmented flow was LB medium-air segmented flow, the culture temperature was 32 °C, and the culture time was 72 h. The method allowed the formation of robust biofilms under aqueous-air segmented flow conditions and the maximum value of OD590 was 1.729.

(2) The growth curve of recombinant E. coli was studied. The results showed that the growth cycle of recombinant E. coli was consisted of four periods: retardation period, exponential growth period, stable period and decay period. The biofilm was prepared in microchannels in four different periods. The results showed that recombinant E. coli in exponential growth period could stably form the biofilm on the surface of the microchannel in 72 h. Compared with recombinant E. coli in lag phase, the culture time reduced by 1/3.

(3) The effects of temperature and pH on the biofilm formation of recombinant E. coli were investigated. The results showed that the optimal temperature was 37 °C and the optimum pH was 7.0. Under the optimum conditions, the recombinant E. coli biofilm could form rapidly and steadily and the maximum value of OD590 was 1.319.