We present a model for diauxic growth of denitrifying bacteria in which nitrate reductase synthesis kinetics dominate the overall growth kinetics. The model is based on the assumption of the existence of a nitrate respiration operon, thereby linking the rate of nitrate uptake to the activity of nitrate reductase. We show that this approach can model diauxic growth of Pseudomonas denitrificans by conducting experiments in which nitrate reductase activity was measured during both lag and ensuing exponential growth phases. We consistently observed the pattern of low nitrate reductase enzyme activity during the lag phase, increasing before the onset of growth. By fitting model parameters we were able to successfully match experimental data for growth, nitrate uptake, and enzyme activity level.
Microaerobic (oxygen limited) conditions are advantageous for several industrial applications since a majority of the carbon atoms can be directed for synthesis of desired products. Oxygen limited conditions, however, can result in high levels of undesirable by-products such as acetate, which subsequently can have an impact on biomass and product yields. The molecular mechanisms involved in acetate accumulation under oxygen limited conditions are not well understood. Our results indicate that a majority of the genetic modifications known to decrease acetate under aerobic conditions results in similar or even higher acetate under oxygen limitation. Deletion of arcA, whose gene product is a global transcriptional regulator, was the only modification among those evaluated that significantly decreased acetate under both transient and prolonged oxygen limitation. Transcriptome results indicate that the arcA deletion results in an increased expression of the operon involving acs and actP (whose gene products are involved in acetate assimilation and uptake respectively) and some genes in the TCA cycle, thereby promoting increased acetate assimilation. These results provide useful cues for strain design for improved manufacturing of biopharmaceuticals under oxygen limited conditions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:303-314, 2018.
Disposable technology is being used more each year in the biotechnology industry. Disposable bioreactors allow one to avoid expenses associated with cleaning, assembly and operations, as well as equipment validation. The WAVE bioreactor is well established for Chinese Hamster Ovary (CHO) production, however, it has not yet been thoroughly tested for E. coli production because of the high oxygen demand and temperature maintenance requirements of that platform. The objective of this study is to establish a robust process to generate inoculum for E. coli production fermentations in a WAVE bioreactor. We opted not to evaluate the WAVE system for production cultures because of the high cell densities required in our current E. coli production processes. Instead, the WAVE bioreactor 20/50 system was evaluated at laboratory scale (10-L) to generate inoculum with target optical densities (OD(550)) of 15 within 7-9 h (pre-established target for stainless steel fermentors). The maximum settings for rock rate (40 rpm) and angle (10.5) were used to maximize mass transfer. The gas feed was also supplemented with additional oxygen to meet the high respiratory demand of the culture. The results showed that the growth profiles for the inoculum cultures were similar to those obtained from conventional stainless steel fermentors. These inoculum cultures were subsequently inoculated into 10-L working volume stainless steel fermentors to evaluate the inocula performance of two different production systems during recombinant protein production. The results of these production cultures using WAVE inocula showed that the growth and recombinant protein production was comparable to the control data set. Furthermore, an economic analysis showed that the WAVE system would require less capital investment for installation and operating expenses would be less than traditional stainless steel systems.
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