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In various fermentation products, cost of the carbon source is a significant contributor to the overall cost of the production. Molasses, a by‐product of the sugar factory, is one of the most common carbon sources. However, glycerol is becoming available and inexpensive as the sequence of the rapid growth of biodiesel industry. Generally, every ton of biodiesel produced, nearly a hundred kilograms of crude glycerol is also produced. In fact, pure glycerol has been used in some fermentation processes and additional cost is added to purify glycerol. Therefore, the application of pure glycerol as a carbon source is limited. Escherichai coli is a common microorganisms frequently used as a host in various industrial applications. Interestingly, E. coli is capable of utilizing a variety of carbon sources that are glucose, xylose and fatty acids including glycerol. L‐phenylalanine is the product from E. coli and it is a raw material of low‐caloric sweetener aspartame production. Due to the increasing demand for soft drinks and low‐caloric food, the commercial value of L‐phenylalanine has increased greatly over the past few years. This work presents the production of L‐phenylalanine from crude glycerol using recombinant E. coli BL21 (DE3). The experiment was divided into two parts. The first part was to optimize the medium formula of E. coli BL21 (DE3) for the biomass and L‐phenylalanine productions using Plackett‐Burman Design (P‐BD) and Central Composite Design (C‐CD). The condition of the first part cultivation was 200 rpm of shaking speed and 7.4 of initial pH at constant cultivation temperature of 37˚C. The results showed that, optimum medium formula for the biomass production were crude glycerol of 46.71 g/l, (NH4)2SO4 of 40.55 g/l, KH2PO4 of 1.742 g/l, K2HPO4 of 1.742 g/l, NaCl of 2.554 g/l, MgCl2 of 0.5800 g/l, CaCl2 of 0.0560 g/l, CoCl2 of 0.0036 g/l, ZnSO4 of 0.0113 g/l, CuSO4 of 0.0064 g/l, Na2MoO4 of 0.0069 g/l and yeast extract of 1.411 g/l. In addition, different medium formula was recommended for the L‐phenylalanine production which were crude glycerol of 25.27 g/l, (NH4)2SO4 of 11.53 g/l, KH2PO4 of 0.585 g/l, K2HPO4 of 0.585 g/l, NaCl of 0.859 g/l, MgCl2 of 0.195 g/l, CaCl2 of 0.0567 g/l, CoCl2 of 0.0036 g/l, ZnSO4 of 0.0115 g/l, CuSO4 of 0.0065 g/l, Na2MoO4 of 0.0069 g/l and yeast extract of 1.4263 g/l. In the second part, the fed‐batch cultivation of E. coli BL21 (DE3) was performed. The optimum medium for the biomass production was applied during 0–16 h of cultivation.
After 16th h, the comparison of two different feedings, continuous and pulse feeding operations was carried out. For both feeding the investigation of single substrate feed of crude glycerol and double substrate feeds of crude glycerol and ammonia solution were prepared. The optimum condition of the second part cultivation was 400 rpm of agitation speed, and 6.0 l/min (during 0–16 h and 56–72 h) and 4.0 l/min (during 16–56 h) of aeration rates. The initial volume was 1.3 l, while cultivation temperature and pH were kept constant at 37°C and 7.4. It is obvious that the pulse feeding of crude glycerol and ammonia solution gave the highest biomass and L‐phenylalanine production of 22.71 and 0.781 g/l.