Combination of natural antimicrobials for contamination control in ethanol production

  • Natalia Janaina Lago Maia
  • Jessica Audrey Feijó Corrêa
  • Rachel Tereza Rigotti
  • Anisio Antonio da Silva Junior
  • Fernando Bittencourt LucianoEmail author
Original Paper


Presence of bacterial contaminants at levels > 107 colony forming units per milliliter (CFU/mL) during ethanol production processes reduces the alcoholic fermentation yield by 30%. Antibiotics are currently used to control contamination, but their residues may be detected in yeast extract, restricting this by-product trade to several countries. Thus, the objective of this study was to assess antimicrobial activity of the natural compounds hops extract, 4-hydroxybenzoic acid, nisin Z, and lysozyme against Lactobacillus fermentum, Leuconostoc mesenteroides, and Saccharomyces cerevisiae, aiming development of a formula. Minimum Inhibitory Concentration of each antimicrobial was determined for bacteria and subsequently, nisin (30 mg/L) and hops extract (5 mg/L) were tested together, showing inhibitory effects combining doses of each antimicrobial that were equivalent to an eightfold reduction of their original Minimum Inhibitory Concentrations (3.75 and 0.625 mg/L, respectively), resulting in a FICIndex of 0.25. Thereon, a formula containing both compounds was developed and tested in fermentation assays, promoting reductions on bacterial population and no severe interferences in yeast viability or population even at extreme doses. Therefore, these compounds have great potential to successfully substitute conventional antibiotics in the ethanol industry.

Graphic abstract


Antimicrobial formula Hops extract Lactic-acid bacteria Nisin Sugarcane mills 



The authors would like to acknowledge Pontifícia Universidade Católica do Paraná for the financial support through scholarships provided for the students involved in this research.


This study was funded by Sugar Cane Ltd. (Brazil). Additionally, this work was performed at Pontificia Universidade Católica do Paraná (Brazil), which also granted scholarship to the students involved in the research. Luciano, Maia and Rigotti hold a patent at INPI - Instituto Nacional da Propriedade Industrial (Brazil) on the developed formulation described in the present work. Register Number: BR1020160260205, deposited in 11/2016. Also, we point that Silva Junior is vinculated to Sugar Cane Inc., funding source of this research.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no potential conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Adeboye PT, Bettiga M, Olsson L (2014) The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates. AMB Express 4:46. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ali SS, Nugent B, Mullins E, Doohan FM (2013) Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose. PLoS ONE. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Araújo LF, Junqueira OM, Lopes EL, Araújo CSS, Ortolan JH, Laurentiz AC (2006) Utilização da levedura desidratada (Saccharomyces cerevisiae) para leitões na fase inicial. Ciência Rural 36:1576–1581. CrossRefGoogle Scholar
  4. Balasundram N, Sundram K, Samman S (2006) Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem 99:191–203. CrossRefGoogle Scholar
  5. Bischoff KM, Skinner-Nemec KA, Leathers TD (2007) Antimicrobial susceptibility of Lactobacillus species isolated from commercial ethanol plants. J Ind Microbiol Biotechnol 34:739–744. CrossRefPubMedGoogle Scholar
  6. Bordignon-Júnior SE, Miyaoka MF, Costa L, Benavente AT, Couto GH, Soccol CR (2012) Inibição do crescimento de bactérias Gram-negativas em microdiluição por tratamento com nisina e EDTA. J Biotechnol Biodivers 3:127–135CrossRefGoogle Scholar
  7. Chum HL, Warner E, Seabra JEA, Macedo IC (2014) A comparison of commercial ethanol production systems from Brazilian sugarcane and US corn. Biofuels Bioprod Biorefining 8:205–223. CrossRefGoogle Scholar
  8. Chung W, Hancock REW (2000) Action of lysozyme and nisin mixtures against lactic acid bacteria. Int J Food Microbiol 60:25–32. CrossRefGoogle Scholar
  9. Clinical and Laboratory Standards Institution (2017) CLSI standard M100. In: Performance standards for antimicrobial susceptibility testing, 27th edn, p 224Google Scholar
  10. Clinical and Laboratory Standards Institution (2018) CLSI standard M07. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 11th edn, pp 15–52.Google Scholar
  11. Cotter PD, Hill C, Ross PR (2005) Bacteriocins: developing innate immunity for food. Nat Rev Microbiol 3:777–788. CrossRefPubMedGoogle Scholar
  12. Cueva C, Moreno-Arribas MV, Martín-Álvarez PJ, Bills G, Vicente MF, Basilio A, Rivas CL, Requena T, Rodríguez JM, Bartolomé B (2010) Antimicrobial activity of phenolic acids against commensal, probiotic and pathogenic bacteria. Res Microbiol 161:372–382. CrossRefPubMedGoogle Scholar
  13. Cunha AF, Missawa SK, Gomes LH, Reis SF, Pereira GAG (2006) Control by sugar of Saccharomyces cerevisiae flocculation for industrial ethanol production. FEMS Yeast Res 6:280–287. CrossRefPubMedGoogle Scholar
  14. Companhia Nacional de Abastecimento - CONAB (2016) Acompanhamento da safra brasileira de cana-de-açúcar: terceiro levantamento - safra 2016/2017. Cia Nac Abast - CONAB 3:174Google Scholar
  15. European Union (2003) Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003. Off J Eur Union 4:L 268/29-42.
  16. Ghorbani F, Younesi H, Esmaeili Sari A, Najafpour G (2011) Cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by Saccharomyces cerevisiae. Renew Energy 36:503–509. CrossRefGoogle Scholar
  17. Jouany J-P, Medina B, Bertin G, Julliand V (2009) Effect of live yeast culture supplementation on hindgut microbial communities and their polysaccharidase and glycoside hydrolase activities in horses fed a high-fiber or high-starch diet. J Anim Sci 87:2844–2852. CrossRefPubMedGoogle Scholar
  18. Kaklamanos G, Vincent U, Von Holst C (2013) Multi-residue method for the detection of veterinary drugs in distillers grains by liquid chromatography-orbitrap high resolution mass spectrometry. J Chromatogr A 1322:38–48. CrossRefPubMedGoogle Scholar
  19. Leathers TD, Bischoff KM (2011) Biofilm formation by strains of Leuconostoc citreum and L. mesenteroides. Biotechnol Lett 33:517–523. CrossRefPubMedGoogle Scholar
  20. Leite IR, Faria JR, Marquez LDS, Reis MHM, de Resende MM, Ribeiro EJ, Cardoso VL (2013) Evaluation of hop extract as a natural antibacterial agent in contaminated fuel ethanol fermentations. Fuel Process Technol 106:611–618. CrossRefGoogle Scholar
  21. Limayem A, Hanning IB, Muthaiyan A, Illeghems K, Kim J-W, Crandall PG, O’Bryan C, Ricke SC (2011) Alternative antimicrobial compounds to control potential Lactobacillus contamination in bioethanol fermentations. J Environ Sci Health B 46:709–714. CrossRefPubMedGoogle Scholar
  22. Lucena BT, dos Santos BM, Moreira JL, Moreira APB, Nunes AC, Azevedo V, Miyoshi A, Thompson FL, de Morais M (2010) Diversity of lactic acid bacteria of the bioethanol process. BMC Microbiol 10:298. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ludwig KM, Oliva-Neto P, de Angelis DF (2001) Quantificação da floculação de Saccharomyces cerevisiae por bactérias contaminantes da fermentação alcoólica. Ciência e Tecnol Aliment 21:63–66. CrossRefGoogle Scholar
  24. Mckenzie HA, White FH (1991) Lysozyme and α-lactalbumin: structure, function, and interrelationships. In: Anfinsen CB, Richards FM, Edsall JT, Eisenberg DSBT-A in PC (eds) Advances in protein chemistry. Academic Press, pp 173–315Google Scholar
  25. Meira NVB, Holley RA, Bordin K et al (2017) Combination of essential oil compounds and phenolic acids against Escherichia coli O157:H7 in vitro and in dry-fermented sausage production. Int J Food Microbiol 260:59–64. CrossRefPubMedGoogle Scholar
  26. Murphree CA, Heist EP, Moe LA (2014) Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States. Curr Microbiol 69:277–285. CrossRefPubMedGoogle Scholar
  27. Muthaiyan A, Limayem A, Ricke SC (2011) Antimicrobial strategies for limiting bacterial contaminants in fuel bioethanol fermentations. Prog Energy Combust Sci 37:351–370. CrossRefGoogle Scholar
  28. Narendranath NV, Thomas KC, Ingledew WM (2000) Urea hydrogen peroxide reduces the numbers of Lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation. Appl Environ Microbiol 66:4187–4192. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Natarajan P, Katta S, Andrei I, Babu Rao Ambati V, Leonida M, Haas GJ (2008) Positive antibacterial co-action between hop (Humulus lupulus) constituents and selected antibiotics. Phytomedicine 15:194–201. CrossRefPubMedGoogle Scholar
  30. Neris DJG, Bordignon-Júnior SE, Baratto CM, Gelinski JMLN (2013) Nisin in the biopreservation of Bordô (Ives) and Niágara table wines from Santa Catarina, Brazil. J Biotechnol Biodivers 4:176–183CrossRefGoogle Scholar
  31. Palaniappan K, Holley RA (2010) Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria. Int J Food Microbiol 140:164–168. CrossRefPubMedGoogle Scholar
  32. Paulus Compart DM, Carlson AM, Crawford GI, Fink RC, Diez-Gonzalez F, Dicostanzo A, Shurson GC (2013) Presence and biological activity of antibiotics used in fuel ethanol and corn co-product production. J Anim Sci 91:2395–2404. CrossRefGoogle Scholar
  33. Prado JL, Venturini Filho WG (2014) Uso de antibióticos convencionais e antimicrobianos a base de lúpulo no controle da infecção bacteriana em fermentação alcoólica. Rev Energ na Agric 29:45Google Scholar
  34. Puupponen-Pimiä R, Nohynek L, Meier C, Kähkönen M, Heinonen M, Hopia A, Oksman-Caldentey KM (2001) Antimicrobial properties of phenolic compounds from berries. J Appl Microbiol 90:494–507. CrossRefPubMedGoogle Scholar
  35. Radler F (1990) Possible use of nisin in winemaking. I. Action of nisin against lactic acid bacteria and wine yeast in solid and liquid media. Am J Enol Vitic 41:1–6Google Scholar
  36. Rigotti RT, Corrêa JAF, Maia NJL, Cesaro G, Rosa EAR, Macedo REFD, Luciano FB (2017) Combination of natural antimicrobials and sodium dodecyl sulfate for disruption of biofilms formed by contaminant bacteria isolated from sugarcane mills. Innov Food Sci Emerg Technol. CrossRefGoogle Scholar
  37. Rückle L, Senn T (2006) Hop acids can efficiently replace antibiotics in ethanol production. Int Sugar J 108:139–147Google Scholar
  38. Sánchez-Maldonado AF, Schieber A, Gänzle MG (2011) Structure-function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria. J Appl Microbiol 111:1176–1184. CrossRefPubMedGoogle Scholar
  39. Silva-Filho EA, Santos SKB, Resende AM, Morais JOF, Morais MA Jr, Simões DA (2005) Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting. Antonie Van Leeuwenhoek 88:13–23. CrossRefPubMedGoogle Scholar
  40. Suzuki K, Sami M, Kadokura H, Nakajima H, Kitamoto K (2002) Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis. Int J Food Microbiol 76:223–230. CrossRefPubMedGoogle Scholar
  41. Tiukova I, Eberhard T, Passoth V (2014) Interaction of Lactobacillus vini with the ethanol-producing yeast Dekkera bruxellensis and Saccharomyces cerevisiae. Biotechnol Appl Biochem 61:40–44. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.School of Life SciencesPontifícia Universidade Católica do ParanáCuritibaBrazil
  2. 2.Sugar Cane Representações Comerciais LtdaCampinasBrazil

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