| E. coli cells produce acetic acid as an extracellular co-product of aerobic fermentation, and this exists as the ion acetate at the neutral pH used in E. coli fermentations. Acetate is undesirable because it retards growth even at concentrations as low as 0.5 g/L, and it inhibits protein formation. Moreover, acetate production represents a diversion of carbon that might otherwise have generated biomass or the protein product. In this study, high level of acetic acid was detected during fermentative production of L-Phe. In order to reduce acetic acid accumulation and improve L- Phenylalanine production, we optimized the initial glucose concentration and its exponential feeding strategy during L-Phe fermentation in 3 L fermentor. The results showed that glucose concentration had direct impact on specific growth, which was closely related with acetic acid accumulation. Not only would excessive acetic inhibit cell growth, but also exert negative effect on L-Phe production. Under high concentration of glucose, cell would exceed critical specific growth rate (0.3 h-1), causing remarkable acetic acid accumulation, and ultimately inhibiting L-Phe synthesis. Firstly, we optimized initial glucose concentration, the results indicated that initial glucose of 20 g/L could control specific growth rate under critical value of 0.3 h-1, leading to great reduction in acetic acid accumulation and significant improvement in L-Phe production. Real maximal specific growth rate of 0.29 h-1 was achieved when preset specific growth rate of 0.4 h-1 was employed. The combination of initial glucose concentration of 20 g/L and preset specific growth rate of 0.4 h-1 greatly promoted L-Phe synthesis, gaining ultimate L-Phe concentration of 48.45 g/L, which was 37% higher than previously reported value in our lab.Acetate formation is strongly affected by the composition of the culture medium. Previous reports showed that supplementing the medium with yeast extract, methionine or glycine can similarly reduce acetate formation and enhance protein production. The effects of 20 amino acid supplements on growth of E.coli WSH-Z06(pAP-B03) were tested. Only Gly, Pro, Met and Arg brought about enhancement of cell growth. In shaken flasks, adding 0.5 g/L of Gly, Pro, Met and Arg could respectively improved maximal specific growth rate to 0.50, 0.52, 0.53 and 0.56 h-1. However, further study found that only Met could relieve cell growth from inhibitory effects of acetic acid: addition of 0.5 g/L Met into medium led to 0.37 h-1 maximal specific growth rate in presence of 2.5 g/L acetic acid, which was equivalent to the value achieved in absence of external acetic acid. Addition of Gly, Pro and Arg could not mitigate the significant reduction in specific growth in presence of acetic acid from 0.5-2.5 g/L. Previous studies suggested that the methionine biosynthetic pathway in E. coli can be perturbed by excessive acetic acid, leading to a reduced growth rate, which is caused by partial methionine auxotrophy. In 3 L fermentor experiment, addition of 0.5 g/L Met at different points during E.coli fermentation for production of L-Phe exhibited remarkable effects on cell growth, acetic acid accumulation as well as L-Phe synthesis. The addition of 0.5 g/L Met at 8 h achieved L-Phe productivity of 1.06 g/L/h, maximal DCW of 17.52 g/L, maximal L-Phe concentration of 42.47 g/L in presence of maximal acetic acid accumulation of 2.58 g/L, which were respectively 89%, 12% and 29% higher than the control without Met addition. However, despite the significant enhancement of cell growth (maximal specific growth rate was 0.55 h-1 and maximal DCW was 18.36 g/L) when 0.5 g/L Met was added at 0 h, the ultimate L-Phe concentration was 36.34 g/L, in particular, L-Phe formation mainly focused in exponential phase. In general, Met addition could indeed enhance cell growth and L-Phe synthesis, possibly due to its reliving effects on acetic acid inhibition.It is well known that E.coli cells need proper pH for normal growth. The effects of changes in pH value on DCW and L-Phe production were investigated. The results of our study showed that the optimal pH for E.coli WSH-Z06(pAP-B03) growth was 7.0, under which 7.4 g/L maximal DCW could be achieved. However, the maximal DCW under pH of 6.0, 8.0 were respectively 3.20 and 4.55 g/L. Interestingly, in shaken flasks, 3.72 g/L and 4.25 g/L L-Phe were synthesized within 4 h after changing pH to 8.0 and 9.0, respectively, which were increased by 13% and 29% compared to those achieved at pH 7.0. At the same time, glucose consumption rates were respectively increased by 25% and 43%.pH impulse between 8.0 and 7.0 with interval time of 2 h could remarkably enhance L-Phe production in 3 L fermentor: the maximal L-Phe concentration of 48.35 g/L, 14.2% higher than the control, was achieved at 64 h. |
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