Production of succinate from simply purified crude glycerol by engineered Escherichia coli using two-stage fermentation

Abstract

Background

Crude glycerol is a main by-product from biodiesel production, and efficient utilization of crude glycerol will bring significant economic and environmental benefits. However, the complex compositions of crude glycerol may impair the cellular growth and inhibit the crude glycerol consumption. Therefore, it is necessary to find a simple method to treat the crude glycerol and release the inhibition on cell metabolism.

Results

The simply purified crude glycerol by activated carbon can be used as the carbon source to produce succinate in two-stage fermentation by the engineered Escherichia coli strain, MLB (ldhA ⁻, pflB ⁻) expressing phosphoenolpyruvate carboxykinase. In the flask experiments, succinate production from crude glycerol without treatment was less than that from pure glycerol. However, in the experiments of 1.5-L bioreactor, little succinate was produced in crude glycerol. The simply purified crude glycerol was used as carbon source for succinate production, and the glycerol consumption and succinate production were enhanced greatly. The succinate produced from the simply purified crude glycerol reached 566.0 mM, which was about ten times higher as that of non-purified one (50.3 mM). The succinate yield of the anaerobic stage achieved 0.97 mol/mol, which was 97% of the theoretical yield.

Conclusion

The treatment of crude glycerol by activated carbon could effectively release the inhibition on the glycerol consumption and succinate production of the engineered E. coli strains, so that the fermentation result of the treated crude glycerol was similar as the pure glycerol. The results showed that the metabolically engineered E. coli strains have great potential to produce succinate from crude glycerol.

Keywords

Escherichia coli / Succinate / Crude glycerol / Activated carbon / Two-stage fermentation

References

[1] Ahn JH, Bang J, Kim WJ, Lee SY. Formic acid as a secondary substrate for succinic acid production by metabolically engineered Mannheimia succiniciproducens. Biotechnol Bioeng, 2017, 114: 2837-2847.

[2] Anand P, Saxena RK. A comparative study of solvent-assisted pretreatment of biodiesel derived crude glycerol on growth and 1,3-propanediol production from Citrobacter freundi. New Biotechnol, 2011, 29: 199-205.

[3] Balzer GJ, Thakker C, Bennett GN, San KY. Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase. Metab Eng, 2013, 20: 1-8.

[4] Blankschien MD, Clomburg JM, Gonzalez R. Metabolic engineering of Escherichia coli for the production of succinate from glycerol. Metab Eng, 2010, 12(5): 409-419.

[5] Booth IR (2005) Glycerol and methylglyoxal metabolism. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE, editors. Escherichia coli and Salmonella: Cellular and molecular biology (web edition). ASM Press, Washington, DC. http://www.ecosal.org

[6] Bretz K. Succinic acid production in fed-batch fermentation of Anaerobiospirillum succiniciproducens using glycerol as carbon source. Chem Eng Technol, 2015, 38: 1659-1664.

[7] Carvalho M, Roca C, Reis MA. Improving succinic acid production by Actinobacillus succinogenes from raw industrial carob pods. Bioresour Technol, 2016, 218: 491-497.

[8] Chen C, Ding S, Wang D, Li Z, Ye Q. Simultaneous saccharification and fermentation of cassava to succinic acid by Escherichia coli NZN111. Bioresour Technol, 2014, 163: 100-105.

[9] Chung SC, Park JS, Yun J, Park JH. Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum. Metab Eng, 2017, 40: 157-164.

[10] Clark DP. The fermentation pathways of Escherichia coli. FEMS Microbiol Rev, 1989, 63: 223-234.

[11] Dharmadi Y, Murarka A, Gonzalez R. Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioeng, 2006, 94: 821-829.

[12] Dobrowolski A, Mituła P, Rymowicz W, Mirończuk AM. Efficient conversion of crude glycerol from various industrial wastes into single cell oil by yeast Yarrowia lipolytica. Bioresour Technol, 2016, 207: 237-243.

[13] Gonzalez R, Murarka A, Dharmadi Y, Yazdani SS. A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. Metab Eng, 2008, 10: 234-245.

[14] Jian X, Li N, Zhang C, Hua Q. In silico profling of cell growth and succinate production in Escherichia coli NZN111. Bioresour Bioprocess, 2016, 3: 48.

[15] Jiang M, Liu SW, Ma JF, Chen KQ, Yu L, Yue FF, Xu B, Wei P. Effect of growth phase feeding strategies on succinate production by metabolically engineered Escherichia coli. Appl Environ Microb, 2010, 76: 1298-1300.

[16] Jiang M, Chen X, Liang L, Liu R, Wan Q, Wu M, Ouyang P. Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli. Enzyme Microb Technol, 2014, 56: 8-14.

[17] Lee JW, Yi J, Kim TY, Choi S, Ahn JH, Song H, Lee MH, Lee SY. Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens. Metab Eng, 2016, 38: 409-417.

[18] Li N, Zhang B, Chen T, Wang Z, Tang Y, Zhao X. Directed pathway evolution of the glyoxylate shunt in Escherichia coli for improved aerobic succinate production from glycerol. J Ind Microbiol Biotechnol, 2013, 40(12): 1461-1475.

[19] Li Q, Wu H, Li ZM, Ye Q. Enhanced succinate production from glycerol by engineered Escherichia coli strains. Bioresour Technol, 2016, 218: 217-223.

[20] Li J, Li Y, Cui Z, Liang Q, Qi QS. Enhancement of succinate yield by manipulating NADH/NAD⁺ ratio and ATP generation. Appl Microbiol Biotechnol, 2017, 101: 3153-3161.

[21] Li Q, Huang B, Wu H, Li Z, Ye Q. Efficient anaerobic production of succinate from glycerol in engineered Escherichia coli by using dual carbon sources and limiting oxygen supply in preceding aerobic culture. Bioresour Technol, 2017, 231: 75-84.

[22] Lin EC. Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol, 1976, 30: 535-578.

[23] Luo X, Ge X, Cui S, Li Y. Value-added processing of crude glycerol into chemicals and polymers. Bioresour Technol, 2016, 215: 144-154.

[24] Manosak R, Limpattayanate S, Hunsom M. Sequential-refining of crude glycerol derived from waste used-oil methyl ester plant via a combined process of chemical and adsorption. Fuel Process Technol, 2011, 92: 92-99.

[25] Meng J, Wang B, Liu D, Chen T, Wang ZW, Zhao XM. High-yield anaerobic succinate production by strategically regulating multiple metabolic pathways based on stoichiometric maximum in Escherichia coli. Microb Cell Fact, 2016, 15: 141.

[26] Mienda BS, Shamsir MS, Illias RM. Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol. J Biomol Struct Dyn, 2016, 34: 1705-1716.

[27] Okino S, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H. An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain. Appl Microbiol Biotechnol, 2008, 81: 459-464.

[28] Pott RW, Howe CJ, Dennis JS. The purification of crude glycerol derived from biodiesel manufacture and its use as a substrate by Rhodopseudomonas palustris to produce hydrogen. Bioresour Technol, 2014, 152: 464-470.

[29] Sadhukhan S, Villa R, Sarkar U. Microbial production of succinic acid using crude and purified glycerol from a Crotalaria juncea based biorefinery. Biotechnol Rep, 2016, 10: 84-93.

[30] Thakker C, San KY, Bennett GN. Production of succinic acid by engineered E. coli strains using soybean carbohydrates as feedstock under aerobic fermentation conditions. Bioresour Technol, 2013, 130: 398-405.

[31] Vivek N, Sindhu R, Madhavan A, Anju AJ, Castro E, Faraco V, Pandey A, Binod P. Recent advances in the production of value added chemicals and lipids utilizing biodiesel industry generated crude glycerol as a substrate—metabolic aspects, challenges and possibilities: an overview. Bioresour Technol, 2017, 239: 507-517.

[32] Werpy T, Petersen G (2004) Top value added chemicals from biomass, vol 1. Results of screening for potential candidates from sugars and synthesis gas. National Renewable Energy Laboratory, Golden, CO. DOE/GO-102004-1992

[33] Wu H, Li ZM, Zhou L, Ye Q. Improved succinic acid production in the anaerobic culture of an Escherichia coli pflB ldhA double mutant as a result of enhanced anaplerotic activities in the preceding aerobic culture. Appl Environ Microbiol, 2007, 73: 7837-7843.

[34] Wu H, Li Q, Li ZM, Ye Q. Succinic acid production and CO₂ fixation using a metabolically engineered Escherichia coli in a bioreactor equipped with a self-inducing agitator. Bioresour Technol, 2012, 107: 376-384.

[35] Wu H, Karanjikar M, San KY. Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol. Metab Eng, 2014, 25: 82-91.

[36] Yang F, Hanna MA, Sun R. Value-added uses for crude glycerol—a byproduct of biodiesel production. Biotechnol Biofuels, 2012, 5: 13.

[37] Zeikus JG, Jain MK, Elankovan P. Biotechnology of succinic acid production and markets for derived industrial products. Appl Microbiol Biotechnol, 1999, 51: 545-552.

[38] Zhang XL, Jantama K, Moore JC, Jarboe LR, Shanmugam KT, Ingram LO. Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli. Proc Natl Acad Sci USA, 2009, 106: 20180-20185.

[39] Zhang X, Shanmugam KT, Ingram LO. Fermentation of glycerol to succinate by engineered strains of Escherichia coli. Appl Environ Microbiol, 2010, 76: 2397-2401.

[40] Zhou YQ, Nie K, Zhang X, Liu SH, Wang M, Deng L, Wang F, Tan TW. Production of fumaric acid from biodiesel-derived crude glycerol by Rhizopus arrhizus. Bioresour Technol, 2014, 163: 48-53.

[41] Zhu J, Thakker C, San KY, Bennett G. Effect of culture operating conditions on succinate production in a multiphase fed-batch bioreactor using an engineered Escherichia coli strain. Appl Microbiol Biotechnol, 2011, 92: 499-508.

[42] Zhu X, Tan Z, Xu H, Chen J, Tang J, Zhang XL. Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. Metab Eng, 2014, 24: 87-96.

[43] Zou W, Zhu LW, Li HM, Tang YJ. Significance of CO₂ donor on the production of succinic acid by Actinobacillus succinogenes ATCC 55618. Microb Cell Fact, 2011, 10: 87.

Funding

the National Natural Science Foundation of China(21776083)

the National Key Research and Development Program of China(2017YFB0039302)

the National High Technology Research and Development Program of China (2011AA02A203)

the Fok Ying-Tong Education Foundation, China(161017)