Preface

The optimization of feeding process is one of the most important research directions for fermentation process optimization, and a sustained and stable feeding rate often has a significant impact on the synthesis of the final product and the metabolic direction of the strain. The synthesis of most products requires strict control of the sugar concentration in the culture medium during the feeding process. Traditional sugar control mainly relies on operators constantly sampling and manually adjusting the feeding speed, which has poor timeliness and control effect. Fermentation engineers are always looking for more stable and precise ways to control sugar. This case takes the tryptophan producing strain E. coli TM01 as the starting strain, and uses a Sartorius (7 L) parallel bioreactor to maintain glucose concentration in the culture medium through two different methods: manual sugar control and automatic detection and sugar control strategies using a biological culture process online detector (BODS). The tryptophan production is used as an indicator to compare the differences in different sugar control modes.

Figure 1: Online detection instrument (BODS) for the biological cultivation process of Tianmu Biotechnology

Experimental Procedure

Experimental strain: E. coli WT01.

Seed liquid culture medium: peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, glucose 20 g/L;

Fermentation broth medium: glucose 25 g/L, yeast extract 7 g/L, citric acid monohydrate 1.1 g/L, ammonium sulfate 7.5 g/L, potassium dihydrogen phosphate 2 g/L, dipotassium dihydrogen phosphate trihydrate 3 g/L, magnesium sulfate heptahydrate 1 g/L, iron sulfate heptahydrate 0.1 g/L, vitamin B1 0.1 g/L, glycerol 10 g/L, ascorbic acid 0.45 g/L, shake flask fermentation with an additional 5 g/L of calcium carbonate added;

Fermentation culture in a fermenter: After activating the strain in a glycerol tube, transfer it to seed culture medium at 37 ℃ for 12 hours, and then transfer it to the fermentation medium at a 5% inoculation rate. The fermentation process is carried out in a Sartorius 7L fermenter with a ventilation rate of 1vvm. The dissolved oxygen is controlled at 30% through the correlation of dissolved oxygen and stirring speed, and the pH is maintained at 6.9 by adding ammonia water. After culturing at 37 ℃ to OD600 to around 10, cool down to 30 ℃ and induce with IPTG. After the glucose in the culture medium is consumed during the fermentation process, a 500 g/L glucose solution is added to control the residual sugar concentration at 2 g/L.

The manual feeding group takes samples every hour to detect the residual sugar concentration in the culture medium, and manually adjusts the feeding strategy based on the measured concentration. The automatic feeding group uses Tianmu Biotechnology's biological cultivation process online detection system (BODS) to real-time detect the residual sugar concentration in the culture medium, and the instrument automatically adjusts the sugar feeding strategy. The concentration of tryptophan in the fermentation broth is detected and compared by the liquid phase.

experimental results

As shown in Figure 2, in manual feeding mode, due to the inability to observe the growth of bacterial cells in real time, the residual sugar concentration in the culture medium fluctuates greatly. Moreover, due to the fatigue of night operators, the maximum error can even reach a difference of+10 g/L. The fluctuation of residual sugar concentration directly affects the biomass and tryptophan production of bacterial cells, and the highest tryptophan production in the manually controlled sugar group is only about 0.3 g/L. Compared with it, the intelligent associated feeding of BODS eliminates the errors caused by human operation, and can monitor the growth of bacterial cells in the fermentation broth in real-time around the clock, detect the residual sugar concentration in the culture medium (set to automatically sample and detect once every 1 hour in the case), and provide feedback to the system. Then, according to the set value, the feeding strategy is adjusted in real time to maintain the sugar stable at the set value (2 ± 0.2g/L). The better sugar control strategy also brings a significant increase in tryptophan production. Compared with the manual feeding mode, the intelligent associated feeding mode of BODS increases tryptophan production by about 71.4%.

Figure 2 Comparison of manual feeding and intelligent associated feeding fermentation results

Conclusion

This case study takes the tryptophan producing engineering strain as the starting strain and investigates the effects of different feeding strategies on the tryptophan production of the strain. The experimental results have shown that more stable sugar control methods often lead to a significant increase in yield. Compared with the manual feeding mode, the BODS intelligent correlation system stabilizes the residual sugar level in the culture medium at the set value (2 ± 0.2g/L) through automatic sampling detection, monitoring, feedback adjustment, and self-learning feeding strategies without the need for operators. It also increases the tryptophan production of the bacterial strain by about 71.4%, indicating the important role of BODS in fermentation process optimization. In addition, the control of residual sugar concentration in industrial production is also a key parameter that directly affects the quality of enterprise products. The real-time detection capability of BODS also helps production workshop personnel to control changes in the fermentation process in real time, adjust process parameters in a timely manner, and help enterprises better complete the scale-up production of products.

Since its establishment, Tianmu Biotechnology has adhered to the principle of customer first and is committed to providing highly specialized strain optimization solutions, providing more reliable choices for fermentation process optimization.