Effect of crude glycerol, 2,3-butanediol, and acetoin initial concentrations on Enterobacter aerogenes ATCC 13048 growth in batch runs


  • Bruna Campos de Souza
  • Stefanie Lemos Barbosa
  • Larissa Pereira Machado
  • Analia Borges Folle
  • Sabrina Carra
  • Eloane Malvessi




Crude Glycerol, 2,3-Butanediol, Acetoin, Product Inhibition, Enterobacter aerogenes


The 2,3-butanediol (BD) and acetoin production from several carbon sources and microorganisms has been extensively studied, mainly due to the possibility of replacing petroleum derivatives by the diol. Crude glycerol is highlighted among the substrates that can be used to produce 2,3-BD. However, this carbon source contains impurities that can affect microbial metabolism. Furthermore, there are scarce studies about the inhibitory effect of fermentation products, such as 2,3-BD and acetoin, on the process. The aim of this study was to evaluate the effect of crude glycerol, 2,3-BD and acetoin initial concentrations on Enterobacter aerogenes growth and metabolism. For this purpose, batch runs were performed using crude glycerol concentrations varying from 20 to 80 g.L-1. To evaluate the inhibitory effect of 2,3-BD plus acetoin, batch runs were performed using product addition from 0 to 40 g.L-1. During batch runs performed using increasing crude glycerol concentrations, the values of maximum specific growth rates were decreasing, evidencing the metabolism inhibition possibly due to the osmotic pressure and substrate composition. In all batch runs, acetic and lactic acids, and ethanol were identified as co-products. In the case of fermentation products, the 2,3-BD plus acetoin initial concentration of 20 g.L-1 was found inhibitory to E. aerogenes growth by a reduction of 45% in maximum specific growth rate. The data also proved the major influence of 2,3-BD rather than acetoin on the overall rate of fermentation, since acetoin concentrations of up to 3.5 g.L-1 did not influence the E. aerogenes growth. Furthermore, the strain ability to adapt to the 2,3-BD and acetoin initial concentrations was demonstrated, opening possibilities for new processes strategies.


Attarbachi T, Kingsley MD, Spallina V. New trends on crude glycerol purification: A review. Fuel, v. 340, p. 127485, 2023. DOI: 10.1016/j.fuel.2023.127485

Białkowska AM. Strategies for efficient and economical 2,3-butanediol production: new trends in this field. World J Microbiol Biotechnol, v. 32, p. 200, 2016. DOI: 10.1007/s11274-016-2161-x

Celińska E, Grajek W. Biotechnological production of 2,3-butanediol: Current state and prospects. Biotechnol Adv, v. 27 p. 715-725, 2009. DOI: 10.1016/j.biotechadv.2009.05.002

Converti A, Perego P. Use of carbon and energy balances in the study of the anaerobic metabolism of Enterobacter aerogenes at variable starting glucose concentrations. Appl Microbiol Biotechnol, v. 59, p. 303-309, 2002. DOI: 10.1007/s00253-002-1009-5

Cui Z, Wang Z, Zheng M, Chen T. Advances in biological production of acetoin: a comprehensive overview. Crit Rev Biotechnol, v. 42, p. 1135-1156, 2021. DOI: 10.1080/07388551.2021.1995319

Dobson R, Gray V, Rumbold K. Microbial utilization of crude glycerol for the production of value-added products. J Ind Microbiol Biotechnol, v. 39, p. 217-226, 2012. DOI: 10.1007/s10295-011-1038-0

Folle AB, Souza BC, Reginatto C et al. Medium composition and aeration to high (R,R)-2,3-butanediol and acetoin production by Paenibacillus polymyxa in fed-batch mode. Arch Microbiol, v. 205 p. 171, 2023. DOI: 10.1007/s00203-023-03521-z

Garg SKK, Jain A. Fermentative production of 2,3-butanediol: a review. Bioresour Technol, v. 51, p. 103-109, 1995. DOI: 10.1016/0960-8524(94)00136-O

Hazeena SH, Shurpali NJ, Siljanen H et al. Bioprocess development of 2,3-butanediol production using agro-industrial residues. Bioprocess Biosyst Eng, v. 45, p. 1527-1537, 2022. DOI: 10.1007/s00449-022-02761-5

Ji XJ, Huang H, Ouyang PK. Microbial 2,3-butanediol production: A state-of-the-art review. Biotechnol Adv, v. 29, p. 351-364, 2011. DOI: 10.1016/j.biotechadv.2011.01.007

Koutinas AA, Yepez B, Kopsahelis N et al. Techno-economic evaluation of a complete bioprocess for 2,3-butanediol production from renewable resources. Bioresour Technol, v. 204, p. 55-64, 2016. DOI: 10.1016/j.biortech.2015.12.005

Lee SJ, Choi HS, Kim CK et al. Process strategy for 2,3-butanediol production in fed-batch culture by acetate addition. J Ind Eng Chem, v. 56, p. 157-162, 2017. DOI: 10.1016/j.jiec.2017.07.008

Lee SJ, Kim SB, Kang SW et al. Effect of crude glycerol-derived inhibitors on ethanol production by Enterobacter aerogenes. Bioprocess Biosyst Eng, v. 35, p. 85-92, 2012. DOI: 10.1007/s00449-011-0607-y

Lennen RM, Lim HG, Jensen K et al. Laboratory evolution reveals general and specific tolerance mechanisms for commodity chemicals. Metab Eng, v. 76, p. 179-192, 2023. DOI: 10.1016/j.ymben.2023.01.012

Li H, Zhang G, Dang Y. Adaptive laboratory evolution of Klebsiella pneumoniae for improving 2,3-butanediol production. Bioengineered, v. 7, p. 432-438, 2016. DOI: 10.1080/21655979.2016.1199304

Luo Q, Wu J, Wu M. Enhanced acetoin production by Bacillus amyloliquefaciens through improved acetoin tolerance. Process Biochem, v. 49, p. 1223-1230, 2014. DOI: 10.1016/j.procbio.2014.05.005

Maina S, Prabhu AA, Vivek N et al. Prospects on bio-based 2,3-butanediol and acetoin production: Recent progress and advances. Biotechnol Adv, v. 54, p. 107783, 2022. DOI: 10.1016/j.biotechadv.2021.107783

Okonkwo CC, Ujor V, Ezeji TC. Investigation of relationship between 2, 3-butanediol toxicity and production during growth of Paenibacillus polymyxa. N Biotechnol, v. 34, p. 23-31, 2017. DOI: 10.1016/j.nbt.2016.10.006

Parate RD, Rode CV, Dharne MS. 2,3-Butanediol Production from Biodiesel Derived Glycerol. Curr Environ Eng, v. 5, p. 4-12, 2018a. DOI: 10.2174/2212717805666180112162517

Parate R, Mane R, Dharne M, Rode, C. Mixed bacterial culture mediated direct conversion of bio-glycerol to diols. Bioresour Technol, v. 250, p. 86-93, 2018b. DOI: 10.1016/j.biortech.2017.11.019

Perego P, Converti A, Del Borghi A, Canepa P. 2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues. Bioprocess Eng, v. 23, p. 613-620, 2000. DOI: 10.1007/s004490000210

Pirt SJ, Callow DS. Exocellular product formation by microorganisms in continuous culture I – Production of 2,3-butanediol by Aerobacter aerogenes in a single stage process. J Appl Bacteriol, v. 21, p. 188-205, 1958. DOI: 10.1111/j.1365-2672.1958.tb00134.x

Silveira MM, Berbert-Molina MA, Prata AMR, Schmidell W. Production of 2,3-butanediol from sucrose by Klebsiella pneumoniae NRRL B199 in batch and fed-batch reactors. Braz Arch Biol Technol, v. 41, p. 329-334, 1998. DOI: 10.1590/S1516-89131998000300009

Song CW, Park JM, Chung SC et al. Microbial production of 2,3-butanediol for industrial applications. J Ind Microbiol Biotechnol, v. 46, p. 1583-1601, 2019. DOI: 10.1007/s10295-019-02231-0

Souza BC. Bioprodução de 2,3-butanodiol em meio mineral contendo glicerol derivado da indústria de biodiesel. Dissertação (Mestrado), Universidade de Caxias do Sul, 126 f., 2018.

Souza BC, Bossardi FF, Furlan GR et al. Validated High-Performance Liquid Chromatographic (HPLC) Method for the Simultaneous Quantification of 2,3-Butanediol, Glycerol, Acetoin, Ethanol, and Phosphate in Microbial Cultivations. Anal Lett, v. 54, p. 2395-2410, 2021. DOI: 10.1080/00032719.2020.1869754

Tinôco D, Seldin L, Coutinho PLA, Freire DMG. Optimization of fermentation conditions as a metabolic strategy for the high-yield and high-selectivity bio-based 2,3-butanediol production. J Ind Eng Chem, v. 125, p. 345-359, 2023. DOI: 10.1016/j.jiec.2023.05.044

Xiao Z, Lu JR. Strategies for enhancing fermentative production of acetoin: A review. Biotechnol Adv, v. 32, p. 492-503, 2014.

Yang Z, Zhang Z. Recent advances on production of 2,3-butanediol using engineered microbes. Biotechnol Adv, v. 37, p. 569-578, 2019.

Zeng AP, Deckwer WD. A model for multiproduct-inhibited growth of Enterobacter aerogenes in 2,3-butanediol fermentation. Appl Microbiol Biotechnol, v. 35, p. 1-3, 1991.

Zeng AP, Biebl H, Deckwer WD. 2,3-Butanediol production by Enterobacter aerogenes in continuous culture: role of oxygen supply. Appl Microbiol Biotechnol, v. 33, p. 264-268, 1990a.

Zeng AP, Biebl H, Deckwer WD. Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture. Appl Microbiol Biotechnol, v. 33, p. 485-489, 1990b.




How to Cite

Souza, B. C. de, Barbosa, S. L., Machado, L. P., Folle, A. B., Carra, S., & Malvessi, E. (2024). Effect of crude glycerol, 2,3-butanediol, and acetoin initial concentrations on Enterobacter aerogenes ATCC 13048 growth in batch runs. Caderno Pedagógico, 21(5), e4094. https://doi.org/10.54033/cadpedv21n5-017