Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli

Abstract

Industrial 20-m3-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated in a scale-down reactor with a controlled oxygen-limitation compartment. In this scaledown reactor similar results were obtained: i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid product formation (acetate, formate, D-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment. Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful for detecting local oxygen deficiency in large-scale E. coli processes.

Soft sensor control of metabolic fluxes in a recombinant Escherichia coli fed-batch cultivation producing green fluorescence protein

“…Other studies [25][26][27] suggest that metabolites resulting from mixed acid fermentation have a similar influence on an E. coli culture.…”

“…As host-vector has been chosen the expression of the Green Fluorescent Protein (GFP) in E. coli, since it represents a typical biopharmaceutical protein 3 production system [24]. A previous study of this system by us [11] is here extended to include control of glucose overflow and MAF metabolites [25][26][27]. In order to simplify the control only one variable is controlled, namely the sum of the detectable metabolites.…”

“…In order to simplify the control only one variable is controlled, namely the sum of the detectable metabolites. This is based on the reasoning that it is the total flux of the glycolytic pathway that should be modulated in order to stabilize the final product protein titer; an idea that previously has been proposed by other researchers [25,28].…”

Recombinant protein production in an Escherichia coli reduced genome strain

“…For the low and high fed-batch growth rates, the formate profiles were independent of the induction strength. These formate profiles indicate that mixed acid metabolism was not the source of the accumulated acetate (Bylund et al, 2000;Castan et al, 2002;Xu et al, 1999). Thus, the slightly higher acetate levels observed for E. coli MDS40 in the batch phase, where growth rates were higher, is likely due to overflow metabolism.…”

Complete reuse of raw fishmeal wastewater: Evidence from a field cultivation study and economic analysis

“…It has been known that the biological properties such as metabolic reactions of microorganisms are dependent upon scale-up [25]. In comparison to the lab-scale, the typical differences in large-scale are reduced biomass yield, increased by-products production and the presence of limiting substrate gradient as measured at different heights in the bioreactor [36,37]. Therefore, such quality of liquid fertilizer produced in large-scale was necessary to compare with those that were produced under different culture conditions.…”