Dumsday Geoff J, Ocal Gunseli, Bridger John S, Zachariou Michael
CSIRO Molecular and Health Technologies, Bayview Avenue, Clayton, Bag 10, Clayton South MDC, Victoria 3169, Australia.
Biotechnol Bioeng. 2009 Feb 15;102(3):673-83. doi: 10.1002/bit.22115.
Arising from the requirement for discovery of novel biocatalysts with unusual properties, a process was developed which uniquely combines aspects of continuous culture with the measurement of oxygen uptake. This adaptation of the chemostat can be used to facilitate the isolation of a number of microorganisms with desirable properties, particularly those with useful metabolic capabilities and/or enzymes. The technique was also used to provide feedback on the metabolic status of a microbial population and increase the feed flow rate (i.e., dilution rate) thereby enabling the isolation of microorganisms with enhanced 1,3-propanediol dehydrogenase activity. The use of oxygen uptake as an indicator of cellular activity enables indirect measurement of substrate utilization and provides a real-time online assessment of the status of microbial enrichment or evolutionary processes and provides an opportunity, through the use of feedback systems, to control these processes. To demonstrate the utility of the technique, oxygen uptake rate (OUR) was compared with a range of conventional analytical techniques that are typically used to monitor enrichment/evolutionary processes and showed good correlation. Further validation was demonstrated by monitoring a characterizable microbial population shift using OUR. The population change was confirmed using off-line analytical techniques that are traditionally used to determine microbial activity. OUR was then used to monitor the enrichment of microorganisms capable of using a solvent (1-methyl-2-pyrrolidinone) as the sole source of carbon for energy and biomass formation from a heterogeneous microbial population. After purification the microorganisms taken from the enrichment process were able to completely utilize 1 g L(-1) 1-methyl-2-pyrrolidinone within 24 h demonstrating that the technique had correctly indicated the enriched population was capable of growth on 1-methyl-2-pyrrolidinone. The technique improves on conventional microbial enrichment that utilizes continuous culture by providing a real-time assessment of the enrichment process and the opportunity to use the OUR output for automated control and variation of one or more growth parameters.
基于发现具有特殊性质的新型生物催化剂的需求，开发了一种将连续培养的各个方面与氧气摄取量测量独特结合的方法。这种恒化器的改进形式可用于促进分离出许多具有理想特性的微生物，特别是那些具有有用代谢能力和/或酶的微生物。该技术还用于提供有关微生物群体代谢状态的反馈，并提高进料流速（即稀释率），从而能够分离出具有增强的1,3 - 丙二醇脱氢酶活性的微生物。将氧气摄取作为细胞活性的指标，能够间接测量底物利用情况，并对微生物富集或进化过程的状态进行实时在线评估，还通过使用反馈系统提供了控制这些过程的机会。为了证明该技术的实用性，将氧气摄取率（OUR）与一系列通常用于监测富集/进化过程的传统分析技术进行了比较，结果显示出良好的相关性。通过使用OUR监测可表征的微生物群体变化，进一步证明了该技术的有效性。使用传统上用于确定微生物活性的离线分析技术确认了群体变化。然后，OUR被用于监测能够利用溶剂（1 - 甲基 - 2 - 吡咯烷酮）作为唯一碳源来生成能量和生物量的微生物从异质微生物群体中的富集情况。经过纯化后，从富集过程中获取的微生物能够在24小时内完全利用1 g L(-1)的1 - 甲基 - 2 - 吡咯烷酮，这表明该技术正确地表明富集的群体能够在1 - 甲基 - 2 - 吡咯烷酮上生长。该技术通过对富集过程进行实时评估，并提供利用OUR输出对一个或多个生长参数进行自动控制和变化的机会，对利用连续培养的传统微生物富集方法进行了改进。
