Determination of metabolites involved in natural succinic acid production from glucose, glycerol and crude glycerin by HPLC methodology

  • L. Jaramillo
  • L.B. Pazutti
  • P.F. de Aguiar
  • V.S. Ferreira-Leitão
  • E.C. Sérvulo
Keywords: validation of analytical methodology, high added-value product, anaerobic fermentation, Actinobacillus succinogenes, Basfia succiniciproducens


Bio-succinic acid process involves complex biochemical pathways in which diverse metabolites may be cogenerated. Their identification and quantification allow an adequate monitoring and understanding of the bioprocess. In this work, a HPLC methodology for simultaneous determination of glucose, glycerol, ethanol and citric, pyruvic, succinic, lactic, formic, acetic and propionic acids was validated, presenting adequate selectivity and linearity. Matrix effect was observed for citric and lactic acids, glycerol and ethanol. The limits of detection and quantification ranged from 0.006 to 0.021 g.L-1 and 0.018 to 0.065 g.L-1, respectively. Recovery values were between 89 and 109% and variation coefficients were less than 2.3%. The intermediate precision was verified with short-term stability, after one freezing and thawing cycle and analysis by a second analyst. The analysis of fermentation samples showed that Actinobacillus succinogenes’s metabolism was carbon source dependent, while Basfia succiniciproducens presented similar metabolic behavior for the carbon sources evaluated, with less variety of generated products. Succinic acid represented around 37% and 25% of products originated by fermentation of glucose and glycerol sources, respectively, using A. succinogenes. Meanwhile, maximum succinic acid production was equivalent to 50% and 80% of metabolites produced in the fermentation of glucose and glycerol sources by B. succiniciproducens.


Abel, E. L., Digiovanni, J. (2015). Environmental carcinogenesis. In: The Molecular Basis of Cancer (e2, Elsevier Inc), Pp. 103-128. doi:10.1016/B978-1-4557-4066-6.00007-X

Ahn, J. H., Jang, Y.-S., Lee, S. Y. (2016). Production of succinic acid by metabolically engineered microorganisms. Current Opinion in Biotechnology 42, 54-66. doi:10.1016/j.copbio.2016.02.034

Anitha, M., Kamarudin, S. K., Kofli, N. T. (2016). The potential of glycerol as a value-added commodity. Chemical Engineering Journal 295, 119-130. doi:10.1016/j.cej.2016.03.012

ANVISA, Agência Nacional de Vigilância Sanitária. (2017). Guia para validação de métodos analíticos. RDC no 166/2017. Brasil.

AOAC International, Association of Official Analytical Chemists. (2016). Official Methods of Analysis, 20th edition. Appendix F, Guidelines for Standard Method Performance Requirements. ISBN 0-935584-87-0. Maryland, United States.

Ardi, M. S., Aroua, M. K., Hashim, N. A. (2015). Progress, prospect and challenges in glycerol purification process: A review. Renewable and Sustainable Energy Reviews 42, 1164-1173. doi:10.1016/j.rser.2014.10.091

Babajide, O. (2013). Sustaining Biodiesel Production via Value-Added Applications of Glycerol. Journal of Energy 2013, 1-7. doi:10.1155/2013/178356

Becker, J., Reinefeld, J., Stellmacher, R., Schafer, R., Lange, A., Meyer, H., Lalk, M., Zelder, O., Abendroth, G., Schroder, H., Haefner, S., Wittmann, C. (2013). Systems-wide analysis and engineering of metabolic pathway fluxes in bio-succinate producing Basfia succiniciproducens. Biotechnology and Bioengineering 110, 3013-3023. doi:10.1002/bit.24963

Biddy, M. J., Scarlata, C., Kinchin, C. (2016). Chemicals from Biomass: A Market Assessment of Bioproducts with Near-Term Potential. United States. doi:10.2172/1244312

Cao, Y., Zhang, R., Sun, C., Cheng, T., Liu, Y., Xian, M. (2013). Fermentative Succinate Production: An Emerging Technology to Replace the Traditional Petrochemical Processes. BioMed Research International 2013, 1-12. doi:10.1155/2013/723412

Carvalho, M., Matos, M., Roca, C., Reis, M. A. M. (2014). Succinic acid production from glycerol by Actinobacillus succinogenes using dimethylsulfoxide as electron acceptor. New Biotechnology 31, 133-139. doi:10.1016/j.nbt.2013.06.006

Cochran, W. G. (1941). The Distribution of the Largest of a Set of Estimated Variances as a Fraction of Their Total. Annals of Eugenics 11, 47-52. doi:10.1111/j.1469-1809.1941.tb02271.x

Coelho, E. M., da Silva Padilha, C. V., Miskinis, G. A., de Sá, A. G. B., Pereira, G. E., de Azevêdo, L. C., dos Santos Lima, M. (2018). Simultaneous analysis of sugars and organic acids in wine and grape juices by HPLC: Method validation and characterization of products from northeast Brazil. Journal of Food Composition and Analysis 66, 160-167. doi:10.1016/j.jfca.2017.12.017

Costa, M. P. da, Frasao, B. da S., Lima, B. R. C. da C., Rodrigues, B. L., Junior, C. A. C. (2016). Simultaneous analysis of carbohydrates and organic acids by HPLC-DAD-RI for monitoring goat’s milk yogurts fermentation. Talanta 152, 162-170. doi:10.1016/j.talanta.2016.01.061

De Barros, M., Freitas, S., Padilha, G.S., Alegre, R.M. (2013). Biotechnological Production of Succinic Acid by Actinobacillus Succinogenes Using Different Substrate. Chemical Engineering Transactions 32, 985-990. doi:10.3303/CET1332165

Dessie, W., Xin, F., Zhang, W., Jiang, Y., Wu, H., Ma, J., Jiang, M. (2018). Opportunities, challenges, and future perspectives of succinic acid production by Actinobacillus succinogenes. Applied Microbiology and Biotechnology. doi:10.1007/s00253-018-9379-5

De Sá, L. R. V., de Oliveira, M. A. L., Cammarota, M. C., Matos, A., Ferreira-Leitão, V. S. (2011). Simultaneous analysis of carbohydrates and volatile fatty acids by HPLC for monitoring fermentative biohydrogen production. International Journal of Hydrogen Energy 36, 15177-15186. doi:10.1016/j.ijhydene.2011.08.056

De Sena Aquino, A. C. M., Azevedo, M. S., Ribeiro, D. H. B., Costa, A. C. O., Amante, E. R. (2015). Validation of HPLC and CE methods for determination of organic acids in sour cassava starch wastewater. Food Chemistry 172, 725-730. doi:10.1016/j.foodchem.2014.09.142

Eyéghé-Bickong, H. A., Alexandersson, E. O., Gouws, L. M., Young, P. R., Vivier, M. A. (2012). Optimisation of an HPLC method for the simultaneous quantification of the major sugars and organic acids in grapevine berries. Journal of Chromatography B 885-886, 43-49. doi:10.1016/j.jchromb.2011.12.011

EURACHEM (2014). Eurachem Guide: The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics, e2. ISBN 978-91-87461-59-0

FDA, Food and Drug Administration. (1996). Guidance for Industry- Q2B Validation of Analytical Procedures: Methodology. United States.

Gargalo, C. L., Cheali, P., Posada, J. A., Carvalho, A., Gernaey, K. V., Sin, G. (2016). Assessing the environmental sustainability of early stage design for bioprocesses under uncertainties: An analysis of glycerol bioconversion. Journal of Cleaner Production 139, 1245-1260. doi:10.1016/j.jclepro.2016.08.156

Grubbs, F. E. (1969). Procedures for Detecting Outlying Observations in Samples. Technometrics 11, 1-21. doi:10.1080/00401706.1969.10490657

ICH. International Conference on Harmonisation. (2005). Validation of analytical procedures: text and methodology, in Q2(R1). Harmonised Tripartite Guidelines, London.

INMETRO, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial. (2016). DOQ-CGCRE-008- Orientação Sobre Validação De Métodos Analíticos. Brasil.

Ivanova-Petropulos, V., Tašev, K., Stefova, M. (2016). HPLC method validation and application for organic acid analysis in wine after solid-phase extraction. Macedonian Journal of Chemistry and Chemical Engineering 35, 225-233. doi:10.20450/mjcce.2016.1073

Jansen, M. L., van Gulik, W. M. (2014). Towards large scale fermentative production of succinic acid. Current Opinion in Biotechnology 30, 190-197. doi:10.1016/j.copbio.2014.07.003

Jiang, M., Ma, J., Wu, M., Liu, R., Liang, L., Xin, F., Dong, W. (2017). Progress of succinic acid production from renewable resources: Metabolic and fermentative strategies. Bioresource Technology 245, 1710-1717. doi:10.1016/j.biortech.2017.05.209

Kong, P. S., Aroua, M. K., Daud, W. M. A. W. (2016). Conversion of crude and pure glycerol into derivatives: A feasibility evaluation. Renewable and Sustainable Energy Reviews 63, 533-555. doi:10.1016/j.rser.2016.05.054

Luo, X., Ge, X., Cui, S., Li, Y. (2016). Value-added processing of crude glycerol into chemicals and polymers. Bioresource Technology 215, 144-154. doi:10.1016/j.biortech.2016.03.042

Moldoveanu, S. C., David, V. (2013). Basic Information about HPLC. In: Essentials in Modern HPLC Separations (Elsevier eds.). doi:10.1016/b978-0-12-385013-3.00001-x

OECD/FAO Food and Agriculture Organization of the United Nations, (2018), Agricultural Outlook 2018-2027. Paris/ Rome. doi: 10.1787/agr_outlook-2018-en

Pateraki, C., Patsalou, M., Vlysidis, A., Kopsahelis, N., Webb, C., Koutinas, A. A., Koutinas, M. (2016). Actinobacillus succinogenes : Advances on succinic acid production and prospects for development of integrated biorefineries. Biochemical Engineering Journal 112, 285-303. doi:10.1016/j.bej.2016.04.005

Petrova, O. E., Sauer, K. (2017). High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP. Methods in Molecular Biology, 33-43. doi:10.1007/978-1-4939-7240-1_4

Quispe, C. A. G., Coronado, C. J. R., Carvalho Jr., J. A. (2013). Glycerol: Production, consumption, prices, characterization and new trends in combustion. Renewable and Sustainable Energy Reviews 27, 475-493. doi:10.1016/j.rser.2013.06.017

Qureshi, M. S., Bhongale, S. S., Thorave, A. K. (2011). Determination of organic acid impurities in lactic acid obtained by fermentation of sugarcane juice. Journal of Chromatography A 1218, 7147-7157. doi:10.1016/j.chroma.2011.08.025

Samul, D., Leja, K., Grajek, W. (2013). Impurities of crude glycerol and their effect on metabolite production. Annals of Microbiology 64, 891-898. doi:10.1007/s13213-013-0767-x

Schindler, B. D., Joshi, R. V., Vieille, C. (2014). Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production. Journal of Industrial Microbiology & Biotechnology 41, 1339-1352. doi:10.1007/s10295-014-1480-x

Scholten, E., Dägele, D. (2008). Succinic acid production by a newly isolated bacterium. Biotechnology Letters 30, 2143-2146. doi:10.1007/s10529-008-9806-2

Sivasankaran, C., Ramanujam, P. K., Balasubramanian, B., Mani, J. (2016). Recent progress on transforming crude glycerol into high value chemicals: a critical review. Biofuels, 1-6. doi:10.1080/17597269.2016.1174018

Song, H., Lee, S. Y. (2006). Production of succinic acid by bacterial fermentation. Enzyme and Microbial Technology 39, 352-361. doi:10.1016/j.enzmictec.2005.11.043

Tan, H. W., Abdul Aziz, A. R., Aroua, M. K. (2013). Glycerol production and its applications as a raw material: A review. Renewable and Sustainable Energy Reviews 27, 118-127. doi:10.1016/j.rser.2013.06.035

Thompson, M., Ellison, S. L. R., Wood, R. (2002). Harmonized guidelines for single-laboratory validation of methods of analysis (IUPAC Technical Report). Pure and Applied Chemistry 74, 835-855. doi:10.1351/pac200274050835

Westbrook, A. W., Miscevic, D., Kilpatrick, S., Bruder, M. R., Moo-Young, M., Perry Chou, C. (2018). Strain engineering for microbial production of value-added chemicals and fuels from glycerol. Biotechnology Advances. doi:10.1016/j.biotechadv.2018.10.006

Zaky, A. S., Pensupa, N., Andrade-Eiroa, Á., Tucker, G. A., Du, C. (2017). A new HPLC method for simultaneously measuring chloride, sugars, organic acids and alcohols in food samples. Journal of Food Composition and Analysis 56, 25-33. doi:10.1016/j.jfca.2016.12.010

Zavarize, K., Menten, J., Pereira, R., Freitas, L., Romano, G., Bernardino, M., Rosa, A. (2014). Metabolizable energy of different glycerine sources derived from national biodiesel production for broilers. Revista Brasileira de Ciência Avícola 16, 411-416. doi:10.1590/1516-635x1604411-416

Zhang, A., Fang, Y. L., Meng, J. F., Wang, H., Chen, S. X., Zhang, Z. W. (2011). Analysis of low molecular weight organic acids in several complex liquid biological systems via HPLC with switching detection wavelength. Journal of Food Composition and Analysis 24, 449-455. doi:10.1016/j.jfca.2010.09.017
How to Cite
Jaramillo, L., Pazutti, L., de Aguiar, P., Ferreira-Leitão, V., & Sérvulo, E. (2019). Determination of metabolites involved in natural succinic acid production from glucose, glycerol and crude glycerin by HPLC methodology. Revista Mexicana De Ingeniería Química, 19(2), 653-667.