Abstract
Industrial ethanol fermentation is subject to bacterial contamination that causes significant economic losses in ethanol fuel plants. Chronic contamination has been associated with biofilms that are normally more resistant to antimicrobials and cleaning efforts than planktonic cells. In this study, contaminant species of Lactobacillus isolated from biofilms (source of sessile cells) and wine (source of planktonic cells) from industrial and pilot-scale fermentations were compared regarding their ability to form biofilms and their sensitivity to different antimicrobials. Fifty lactobacilli were isolated and the most abundant species were Lactobacillus casei, Lactobacillus fermentum and Lactobacillus plantarum. The majority of the isolates (87.8%) were able to produce biofilms in pure culture. The capability to form biofilms and sensitivity to virginiamycin, monensin and beta-acids from hops, showed inter- and intra-specific variability. In the pilot-scale fermentation, Lactobacillus brevis, L. casei and the majority of L. plantarum isolates were less sensitive to beta-acids than their counterparts from wine; L. brevis isolates from biofilms were also less sensitive to monensin when compared to the wine isolates. Biofilm formation and sensitivity to beta-acids showed a positive and negative correlation for L. casei and L. plantarum, respectively.
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References
Al-Ahmad A, Ameen H, Pelz K, Karygianni L, Wittmer A, Anderson AC, Spitzmüller B, Hellwig E (2014) Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth. J Endod 40:223–230. https://doi.org/10.1016/j.joen.2013.07.023
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Amorim HV, Basso LC, Lopes ML (2004) Evolution of ethanol fermentation in Brazil. In: Bryce JH, Stewart GG (eds) Distilled spirits: tradition and innovation. Nottingham University Press, Nottingham, pp 143–146
Amorim HV, Basso LC, Lopes ML (2009) Sugar cane juice and molasses, beet molasses and sweet sorghum: composition and usage. In: Ingledew WM, Kelsall DR, Austin GD, Kluhspies C (eds) The alcohol textbook: a reference for the beverage, fuel and industrial alcohol industries. Nottingham University Press, Nottingham, pp 39–46
Amorim HV, Lopes ML, Oliveira JVC, Buckeridge MS, Goldman GH (2011) Scientific challenges of bioethanol production in Brazil. Appl Microbiol Biotechnol 91:1267–1275. https://doi.org/10.1007/s00253-011-3437-6
Basso TO, Gomes FS, Lopes ML, Amorim HV, Eggleston G, Basso LC (2014) Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation. Antonie Van Leeuwenhoek 105:169–177. https://doi.org/10.1007/s10482-013-0063-6
Bayrock DP, Ingledew WM (2004) Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity? J Ind Microbiol Biotechnol 31:362–368. https://doi.org/10.1007/s10295-004-0156-3
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. https://doi.org/10.1007/s10295-007-0250-4
Braga LPP, Alves RF, Dellias MTF, Navarrete AA, Basso TO, Tsai SM (2017) Vinasse fertirrigation alters soil resistome dynamic: an analysis based on metagenomic profiles. BioData Min 10:1–7. https://doi.org/10.1186/s13040-017-0138-4
Brody JR, Kern SE (2004) Sodium boric acid: a tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36:214–216
Carvalho-Netto OV, Carazzolle MF, Mofatto LS, Teixeira PJ, Noronha MF, Calderón LAL, Mieczkowski PA, Argueso JL, Pereira GA (2015) Saccharomyces cerevisiae transcriptional reprograming due to bacterial contamination during industrial scale bioethanol production. Microb Cell Fact 14:13. https://doi.org/10.1186/s12934-015-0196-6
Chang IS, Kim BH, Shin PK, Lee WK (1995) Bacterial contamination and its effects on ethanol fermentation. J Microbiol Biotechnol 5:309–314
Costerton JW (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322. https://doi.org/10.1126/science.284.5418.1318
Davey ME, O´Toole G (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64:847–867. https://doi.org/10.1128/MMBR.64.4.847-867.2000
de Oliva-Neto P, Yokoya F (1994) Evaluation of bacterial contamination in a fed-batch alcoholic fermentation process. World J Microbiol Biotechnol 10:697–699. https://doi.org/10.1007/BF00327963
Dong SJ, Lin XH, Li H (2015) Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation. Int J Biochem Cell Biol 68:33–41. https://doi.org/10.1016/j.biocel.2015.08.010
Eykelbosh AJ, Johnson MS, Couto EG (2015) Biochar decreases dissolved organic carbon but not nitrate leaching in relation to vinasse application in a Brazilian sugarcane soil. J Environ Manage 149:9–16. https://doi.org/10.1016/j.jenvman.2014.09.033
Fernandes EAN, Nepomuceno N, Trevizam AB, Amorim HV (1998) From potential to reality: yeasts derived from ethanol production for animal nutrition. J Radioanal Nucl Chem 234:113–118
G-Alegría E, López I, Ruiz JI, Sáenz J, Fernández E, Zarazaga M, Dizy M, Torres C, Ruiz-Larrea F (2004) High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol. FEMS Microbiol Lett 230:53–61. https://doi.org/10.1016/S0378-1097(03)00854-1
Gallo CR (1990) Determinação da microbiota bacteriana de mosto e de dornas de fermentação alcoólica. PhD Thesis, Universidade Estadual de Campinas, Campinas, SP, Brazil. http://repositorio.unicamp.br/jspui/handle/REPOSIP/255723
Gold RS, Meagher MM, Hutkins R, Conway T (1992) Ethanol tolerance and carbohydrate metabolism in lactobacilli. J Ind Microbiol 10:45–54. https://doi.org/10.1007/BF01583633
Hudzicki J (2013) Kirby-Bauer disk diffusion susceptibility test protocol. Am Soc Microbiol 1–23. http://www.asmscience.org/content/education/protocol/protocol.3189
Johnson JL (1994) Similarity analysis of rRNAs. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 683–700
Leathers TD, Bischoff KM, Rich JO, Price NPJ, Manitchotpisit P, Nunnally MS, Anderson AM (2014) Inhibitors of biofilm formation by biofuel fermentation contaminants. Bioresour Technol 169:45–51. https://doi.org/10.1016/j.biortech.2014.06.065
Li YH, Tian XL (2012) Quorum sensing and bacterial social interactions in biofilms. Sensors 22:2519–2538
Lucena BTL, dos Santos BM, Moreira JL, Moreira APB, Nunes AC, Azevedo V, Miyoshi A, Thompson FL, de Morais MA (2010) Diversity of lactic acid bacteria of the bioethanol process. BMC Microbiol 10:298. https://doi.org/10.1186/1471-2180-10-298
Ludwig KM, Oliva-Neto P, Angelis DF (2001) Quantificação da floculação de Saccharomyces cerevisiae por bactérias contaminantes da fermentação alcoólica. Ciência e Tecnol Aliment 21:63–66. https://doi.org/10.1590/S0101-20612001000100014
Lushia W, Heist P (2005) Antibiotic resistant bacteria in fuel ethanol fermentations. Ethanol Producer Magazine, pp 80–82
McLean RJC (2002) An overview of biofilm molecular ecology. In: McLean RJC, Decho AW (eds) Molecular ecology of biofilms. Horizon Scientific Press, Norfolk, pp 1–21
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. https://doi.org/10.1007/s00284-014-0583-y
Muthaiyan A, Ricke SC (2010) Current perspectives on detection of microbial contamination in bioethanol fermentors. Bioresour Technol 101:5033–5042. https://doi.org/10.1016/j.biortech.2009.11.005
Muthaiyan A, Limayem A, Ricke SC (2011) Antimicrobial strategies for limiting bacterial contaminants in fuel bioethanol fermentations. Prog Energy Combust Sci 37:351–370. https://doi.org/10.1016/j.pecs.2010.06.005
Narendranath NV, Hynes SH, Thomas KC, Ingledew WM (1997) Effects of lactobacilli on yeast-catalyzed ethanol fermentations. Appl Environ Microbiol 63:4158–4163
Rich JO, Leathers TD, Nunnally MS, Bischoff KM (2011) Rapid evaluation of the antibiotic susceptibility of fuel ethanol contaminant biofilms. Bioresour Technol 102:1124–1130. https://doi.org/10.1016/j.biortech.2010.08.118
Rich JO, Leathers TD, Bischoff KM, Anderson AM, Nunnally MS (2015) Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation. Bioresour Technol 196:347–354. https://doi.org/10.1016/j.biortech.2015.07.071
Salminen S, von Wright A, Morelli L, Marteau P, Brassart D, de Vos WM, Fondén R, Saxelin M, Collins K, Mogensen G (1998) Demonstration of safety of probiotics—a review. Int J Food Microbiol 44:93–106
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Schell DJ, Dowe N, Ibsen KN, Riley CJ, Ruth MF, Lumpkin RE (2007) Contaminant occurrence, identification and control in a pilot-scale corn fiber to ethanol conversion process. Bioresour Technol 98:2942–2948. https://doi.org/10.1016/j.biortech.2006.10.002
Skinner KA, Leathers TD (2004) Bacterial contaminants of fuel ethanol production. J Ind Microbiol Biotechnol 31:401–408. https://doi.org/10.1007/s10295-004-0159-0
Skinner-Nemec KA, Nichols NN, Leathers TD (2007) Biofilm formation by bacterial contaminants of fuel ethanol production. Biotechnol Lett 29:379–383. https://doi.org/10.1007/s10529-006-9250-0
Souza MAC, Mutton MJR (2004) Floculação de leveduras por Lactobacillus fermentum em processos industriais de fermentação alcoólica avaliada por técnica fotométrica. Ciência e Agrotecnologia 28:893–898. https://doi.org/10.1590/S1413-70542004000400023
Stewart PS (2002) Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol 292:107–113. https://doi.org/10.1078/1438-4221-00196
Stirling D (2003) DNA extraction from fungi, yeast and bacteria. In: Bartlett JMS, Stirling D (eds) Methods in molecular biology: PCR protocols, 2nd edn. Humana Press, Totowa, pp 53–54
Suzuki K, Iijima K, Sakamoto K, Sami M, Yamashita H (2006) A review of hop resistance in beer spoilage Lactic Acid Bacteria. J Inst Brew 112:173–191. https://doi.org/10.1002/j.2050-0416.2006.tb00247.x
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. Microbiology 173:697–703
Wright ES, Yilmaz LS, Noguera DR (2012) DECIPHER, a search-based approach to chimera identification for 16S rRNA sequences. Appl Environ Microbiol 78:717–725. https://doi.org/10.1128/AEM.06516-11
Yang L, Liu Y, Wu H, Song Z, Høiby N, Molin S, Givskov M (2012) Combating biofilms. FEMS Immunol Med Microbiol 65:146–157. https://doi.org/10.1111/j.1574-695X.2011.00858.x
Acknowledgements
This work was supported by the Brazilian funding agency “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq) [Grant Numbers 158347/2010-2, 153037/2012-1].
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Dellias, M.d., Borges, C.D., Lopes, M.L. et al. Biofilm formation and antimicrobial sensitivity of lactobacilli contaminants from sugarcane-based fuel ethanol fermentation. Antonie van Leeuwenhoek 111, 1631–1644 (2018). https://doi.org/10.1007/s10482-018-1050-8
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DOI: https://doi.org/10.1007/s10482-018-1050-8