The use of oxygen uptake rate to monitor discovery of microbial and enzymatic biocatalysts.
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.
Dumsday
Geoff J
GJ
CSIRO Molecular and Health Technologies, Bayview Avenue, Clayton, Bag 10, Clayton South MDC, Victoria 3169, Australia. geoff.dumsday@csiro.au
Ocal
Gunseli
G
Bridger
John S
JS
Zachariou
Michael
M
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D019905:Benzyl Alcohol / Q000378:metabolism; D055162:Biocatalysis; D018533:Biomass; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D030541:Databases, Genetic; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D016680:Genome, Bacterial / Q000235:genetics; D020407:Internet; D010101:Oxygen Consumption; D011157:Population Dynamics; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D011760:Pyrrolidinones / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012722:Sewage / Q000382:microbiology
D011760:Pyrrolidinones; D012336:RNA, Ribosomal, 16S; D012722:Sewage; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C052370:1,3-propanediol dehydrogenase; C038678:N-methylpyrrolidone; D019905:Benzyl Alcohol