Bioreactor Cultivation of Bacillus Methanolicus Expressing Green Fluorescent Protein

Bioreactors are used to cultivate microbial organisms as biocatalysts for the large scale production of proteins, specialty chemicals, and commodity chemicals, such as amino acids. Bacillus methanolicus is a thermotolerant organism that shows optimal growth at 50-53ºC using methanol as substrate, which offers potential cost savings in feed and cooling utility. Glucose feed limitation below the critical amount required to sustain the maximum growth rate in the exponential phase has previously been shown to improve peak cell biomass and protein production in Escherichia coli. In this study, a feed control program was tested to manage carbon substrate-limited conditions to improve peak cell biomass in B. methanolicus MGA3 with plasmid pNW33N-mdh-GFPuv (pNmG) expressing green fluorescent protein (GFP), with pNW33N (non-GFP expressing), and wild-type B. methanolicus MGA3. Assessments with GFP production could not be made due to issues with protein detection at room temperature incubation. A chemostat was run at dilutions between 0.04 and 0.12 h-1to determine growth yields for the feeding program, and the results showed a biomass yield from substrate (YMx/s) of 0.335 ± 0.029 g biomass/g substrate and oxygen consumption (YO2/X) between 72 120 g O2/g biomass. Indeed, limiting the carbon resulted in higher biomass yield from substrate as compared with feeding excess carbon