APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2007, p. 4354–4356 0099-2240/07/$08.00⫹0 doi:10.1128/AEM.00437-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 13
Dekkera bruxellensis and Lactobacillus vini Form a Stable Ethanol-Producing Consortium in a Commercial Alcohol Production Process䌤 Volkmar Passoth,* Johanna Blomqvist, and Johan Schnu ¨rer Department of Microbiology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7025, SE-750 07 Uppsala, Sweden Received 26 February 2007/Accepted 24 April 2007
relation had changed and the LAB were less than 10% of the total cell number (Table 1). However, these changes did not influence the ethanol concentration in the fermentor (B. Johansson, personal communication). The quantifications are based on CFU determinations on the selective media described above. It was not possible to perform microscopic counts because of the high number of particles in the industrial medium that were difficult to distinguish from microbial cells. From every sample, 20 yeast and 20 bacterial colonies were randomly chosen for PCR fingerprint analysis. For a comparison, we also analyzed 20 yeast and 20 LAB colonies from commercial baker’s yeast, which is used as the inoculum for the fermentation process (see above). Fingerprints were generated with genomic DNA as the template by using GTG5 primers (13). The reaction conditions used were 94°C for 2 min; 39 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 2 min; and 72°C for 5 min for yeasts and 95°C for 7 min; 29 cycles of 90°C for 30 s, 95°C for 1 min, 40°C for 1 min, and 65°C for 4 min; and 65°C for 16 min for LAB. In the fingerprints of the yeast isolates from the first sample from the production process, the band intensities showed slight differences from each other but the fingerprints were completely different from those of the isolates from baker’s yeast. These slight differences might indicate some intraspecific genetic variants, as all of the isolates were found to belong to the same species (see below). The LAB from the production process showed a uniform banding pattern. Among the isolates from baker’s yeast, four slightly different patterns were found, none of them similar to those of the fermentation isolates. We identified all of the yeast isolates and four LAB isolates from the first sampling by sequencing the D1/D2 and partial 16S rRNA gene regions by using the 16S-s primer for sequencing (3, 22). For the yeast isolates, a BLAST search yielded a more than 99% match to Dekkera bruxellensis. The sequences were then aligned with the D1/D2 regions of the type strain of D. bruxellensis (CBS 74, NRRL Y-12961) and other isolates designated D. bruxellensis. These alignments revealed 100% identity within the D1/D2 stretch that has been used for phylogenetic characterization of the genus Dekkera (corresponding to bp 83 to 653 of the homologous Saccharomyces cerevisiae gene) (5). Thus, we designated our isolates D. bruxellensis. For
We analyzed the population dynamics of yeasts and lactic acid bacteria (LAB) in a Swedish ethanol production plant. The production process runs as a continuous fermentation with recirculation of the yeasts. The substrate for fermentation is formed from wheat starch. The material is first liquefied with ␣-glucoamylase at 90°C and then further degraded by ␣-glucosidase at 60°C to release fermentable sugars. By this procedure, about 96% of the starch is degraded to fermentable sugar. The glucose concentration in the fermentor is always below 0.1 g/liter because of the substrate-limited continuouscultivation method (B. Johansson, personal communication). The process is started by mixing 1 ton of baker’s yeast (Ja¨stbolaget, Sollentuna, Sweden; cell viability, ⬎90%) with the substrate in the fermentor (fermentor size, about 100 m3). According to observations of the staff, it usually takes up to 3 weeks until stable fermentation is obtained; thereafter, the process can run stably for 2 or even more years. During the first 3 weeks, the process is prone to infections and stuck fermentation. After stabilization, the staff noticed a change in the cell shape of the production yeast, which was regarded by them either as a physiological adaptation to the harsh conditions in the fermentor (high cell density, sugar and oxygen limitation, a pH of about 3.5, and a temperature of 35°C) or as a selected genetic variant of the inoculated baker’s yeast. Our intention was to analyze this genetic variant but also to investigate the role of potentially contaminating LAB. Samples were taken in January, March, and July 2006 from the fermentor, which had been running stably since July 2005. Appropriate dilutions of the fermentation broth were spread onto plates selective for either yeasts (malt extract agar containing 20 g/liter malt extract and 0.1 g/liter chloramphenicol) or LAB (de Man-Rogosa-Sharp medium containing 0.1 g/liter Delvocid). Surprisingly, during the first two samplings the number of LAB was very high, constituting about 70% of the total cell number (yeast plus LAB). At the last sampling, this
* Corresponding author. Mailing address: Department of Microbiology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7025, SE-750 07 Uppsala, Sweden. Phone: 46 18 673380. Fax: 46 18 673392. E-mail:
[email protected]. 䌤 Published ahead of print on 4 May 2007. 4354
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The ethanol production process of a Swedish alcohol production plant was dominated by Dekkera bruxellensis and Lactobacillus vini, with a high number of lactic acid bacteria. The product quality, process productivity, and stability were high; thus, D. bruxellensis and L. vini can be regarded as commercial ethanol production organisms.
D. BRUXELLENSIS AND L. VINI IN ALCOHOL PRODUCTION
VOL. 73, 2007
TABLE 1. Quantification of yeasts and LAB in the fermentor of the ethanol production process Concn (CFU/ml) of: Date
20 January 2006 7 March 2006 29 June 2006
Yeast/LAB ratio Yeast
LAB
2.1 ⫻ 107 1.7 ⫻ 107 1.7 ⫻ 107
7.6 ⫻ 107 6.0 ⫻ 107 1.2 ⫻ 106
0.28 0.28 14
TABLE 2. Identification of yeast and LAB isolates from the fermentor of the ethanol production process to the species level Sampling date and species
No. of isolates
20 January 2006 Dekkera bruxellensis ................................................................... 20a Lactobacillus vini ....................................................................... 20b 3 March 2006 Dekkera bruxellensis ................................................................... 20a Lactobacillus vini ....................................................................... 19b Lactobacillus fermentum............................................................ 1b 29 June 2006 Dekkera bruxellensis ................................................................... 20a Lactobacillus vini ....................................................................... 19b Lactobacillus panis..................................................................... 1b a b
Out of 20 investigated yeast colonies. Out of 20 investigated LAB colonies.
To identify potential inoculum sources for this consortium, we investigated the yeast and LAB diversity in baker’s yeast and in the process water and the saccharification line of the factory. In the baker’s yeast, all of the isolated yeast colonies belonged to S. cerevisiae while the isolated LAB were identified as Lactococcus lactis, Leuconostoc pseudomesenteroides, and Pediococcus sp. However, there were only very few LAB in the baker’s yeast (ratio of CFU to yeast cells, 9 䡠 ⫻ 10⫺4). In the process water, we found the yeast species S. cerevisiae, Kluyveromyces marxianus, and Pichia galeiformis and the LAB Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Weissella confusa, and Lactobacillus salivarius. In the saccharification line, we identified the yeasts Candida cf. sorbosivorans and Candida magnoliae and the LAB Lactobacillus parabuchneri and Lactobacillus casei. However, none of the strains investigated belonged to D. bruxellensis or L. vini. We also tried to amplify D. bruxellensis-specific sequences from template DNA isolated from the process water, the saccharification line, baker’s yeast, or the fermentor by using primers Brett1 and DB4, which are specific for Dekkera-Brettanomyces bruxellensis (7, 11). Whereas we obtained clear amplification with positive controls (amplification from any source with primers NL1 and NL4, which amplify the D1/D2 region of the yeast rRNA gene), we did not get any amplification with the D. bruxellensis-specific primers, except from DNA isolated from the fermentor broth. Thus, the substrate, process water, and inoculum are most probably not the source of the D. bruxellensis growth in the fermentor. The same is probably true for L. vini, but this hypothesis is only based on our cultivation-based assays and not on molecular identification. There is only limited information available about the genome of this species, and thus, no species-specific probes or primers have been designed for molecular identification (17). Thus, it is not possible to exclude the occurrence of low numbers of this LAB in the environments investigated. We can only hypothesize about the physiological basis of the outcompetition of S. cerevisiae by D. bruxellensis in the fermentation facility investigated in this study. D. bruxellensis has an ethanol tolerance similar to or even higher than that of S. cerevisiae (20). The yeast has been reported to produce a large amount of acetic acid under aerobic conditions (2). However,
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the LAB strains, about 870 bp of the 16S rRNA gene were sequenced for each strain. A BLAST search yielded more than 99% identity to a strain designated Lactobacillus mobilis (EMBL accession number AB242320). However, other strains that have been designated Lactobacillus vini (17) also matched our sequences by more than 99%. Multiple alignments of an 850-bp core region revealed an identity of 100% among our isolates; five L. vini strains, including type strain DSM20605; and the L. mobilis strain. L. mobilis is not an officially described species (http://www.ncbi.nlm.nih.gov/Taxonomy /Browser/wwwtax.cgi?id⫽357762), but regarding the high degree of identity of the 16S rRNA gene with the strains of L. vini, it seems likely that L. vini and L. mobilis are conspecific. We also tested the LAB isolates for physiological characteristics by using the API CH 50 test. The four isolates of L. vini from the fermentor showed a metabolic profile somewhat different from that of the type strain of this species (17). For example, the type strain was not able to ferment D-galactose, methyl-␣-Dmannoside, and D-tagatose, which the investigated fermentation isolates were able to ferment. However, a number of other L. vini strains investigated in the same article had the ability to ferment those carbohydrates, so there are intraspecific variations in metabolic capacity. We therefore designated all of our isolates L. vini. The other two samplings yielded similar results; i.e., all of the yeast isolates belonged to D. bruxellensis and the LAB population was dominated by L. vini (Table 2). The ratio of yeast and LAB cell numbers varied over the test period from about 3:10 in January and March to 14:1 in June (Table 1). This implies a biomass ratio of 6:1 to 15:1 for January and March and less than 1% LAB biomass for June, assuming a 20to 50-fold higher biomass per yeast cell compared to bacteria. Interestingly, the yeast cell number does not seem to be negatively influenced by the number of LAB, as it was highest in January, when the LAB number was also highest. Furthermore, no enhanced yeast cell number was observed in June, when the LAB numbers were lowest. In contrast, other LAB have been shown to negatively affect yeast growth in grain mash (21). To test whether the originally inoculated baker’s yeast was still present in the fermentation medium, we tried to identify S. cerevisiae by real-time PCR (9) with total genomic DNA isolated from the fermentation broth. However, no specific target amplification was found. In contrast, we observed clear amplification when using primers specific for D. bruxellensis (7). Thus, the D. bruxellensis-L. vini consortium had completely taken over the fermentation process. In spite of this, the production process was running continuously during the investigated time period and no problems regarding productivity, yield, or by-product formation were observed by the staff (B. Johansson, personal communication).
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This study was supported by the VL-Stiftelsen, Lidko ¨ping, Sweden. REFERENCES 1. Abbott, D. A., S. H. Hynes, and W. M. Ingledew. 2005. Growth rates of Dekkera/Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentations. Appl. Microbiol. Biotechnol. 66:641–647. 2. Aguilar Uscanga, M. G., M.-L. Delia, and P. Strehaiano. 2003. Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. Appl. Microbiol. Biotechnol. 61:157–162. 3. Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143–169. 4. Barnett, J. A., R. W. Payne, and D. Yarrow. 2000. Yeasts: characteristics and identification, 3rd ed. Cambridge University Press, Cambridge, United Kingdom. 5. Boekhout, T., C. P. Kurtzman, K. O’Donnell, and M. T. Smith. 1994. Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera), Dekkera (anamorph Brettanomyces), and Eeniella as inferred from partial 26S ribosomal DNA nucleotide sequences. Int. J. Syst. Bacteriol. 44:781–786. 6. Dato, M. C. F., J. M. Pizauro, and M. J. R. Mutton. 2005. Analysis of the secondary compounds produced by Saccharomyces cerevisiae and wild yeast strains during the production of “cachaca.” Braz. J. Microbiol. 36:70–74. 7. Delaherche, A., O. Claisse, and A. Lonvaud-Funel. 2004. Detection and quantification of Brettanomyces bruxellensis and ‘ropy’ Pediococcus damnosus strains in wine by real-time polymerase chain reaction. J. Appl. Microbiol. 97:910–915. 8. de Souza Liberal, A. T., A. C. M. Basilio, A. do Monte Resende, B. T. V. Brasileiro, E. A. da Silva-Filho, J. O. F. de Morais, D. A. Simo ˜es, and M. A. de Morais, Jr. 2007. Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation. J. Appl. Microbiol. 102:538–547. 9. Feng, X. M., V. Passoth, C. Eklund-Jonsson, M. L. Alminger, and J. Schnu ¨rer. 2007. Rhizopus oligosporus and yeast co-cultivation during barley tempeh fermentation—nutritional impact and real-time PCR quantification of fungal growth dynamics. Food Microbiol. 24:393–402. 10. Fugelsang, K. C., and B. W. Zoecklein. 2003. Population dynamics and effects of Brettanomyces bruxellensis strains on pinot noir (Vitis vinifera L.) wines. Am. J. Enol. Vitic. 54:294–300. 11. Ibeas, J. I., I. Lozano, F. Perdigones, and J. Jimenez. 1996. Detection of Dekkera-Brettanomyces strains in sherry by a nested PCR method. Appl. Environ. Microbiol. 62:998–1003. 12. Kurtzman, C. P., and J. W. Fell. 1998. The yeasts, a taxonomic study, 4th ed. Elsevier, Amsterdam, The Netherlands. 13. Lieckfeldt, E., W. Meyer, and T. Borner. 1993. Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting. J. Basic Microbiol. 33:413–425. 14. Meroth, C. B., W. P. Hammes, and C. Hertel. 2003. Identification and population dynamics of yeasts in sourdough fermentation processes by PCRdenaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 69:7453– 7461. 15. Pedersen, C., H. Jonsson, J. E. Lindberg, and S. Roos. 2004. Microbiological characterization of wet wheat distillers’ grain, with focus on isolation of lactobacilli with potential as probiotics. Appl. Environ. Microbiol. 70:1522– 1527. 16. Renouf, V., M. Falcou, C. Miot-Sertier, M. C. Perello, G. De Revel, and A. Lonvaud-Funel. 2006. Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking. J. Appl. Microbiol. 100:1208–1219. 17. Rodas, A. M., E. Chenoll, M. C. Macia ´n, S. Ferrer, I. Pardo, and R. Aznar. 2006. Lactobacillus vini sp. nov., a wine lactic acid bacterium homofermentative for pentoses. Int. J. Syst. Evol. Microbiol. 56:513–517. 18. Rodas, A. M., S. Ferrer, and I. Pardo. 2005. Polyphasic study of wine Lactobacillus strains: taxonomic implications. Int. J. Syst. Evol. Microbiol. 55:197–207. 19. Silva, L. R., P. B. Andrade, P. Valentao, R. M. Seabra, M. E. Trujillo, and E. Velazquez. 2005. Analysis of non-coloured phenolics in red wine: Effect of Dekkera bruxellensis yeast. Food Chem. 89:185–189. 20. Silva, P., H. Cardoso, and H. Geros. 2004. Studies on the wine spoilage capacity of Brettanomyces/Dekkera spp. Am. J. Enol. Vitic. 55:65–72. 21. Thomas, K. C., S. H. Hynes, and W. M. Ingledew. 2001. Effect of lactobacilli on yeast growth, viability and batch and semi-continuous alcoholic fermentation of corn mash. J. Appl. Microbiol. 90:819–828. 22. Valente, P., J. P. Ramos, and O. Leoncini. 1999. Sequencing as a tool in yeast molecular taxonomy. Can. J. Microbiol. 45:949–958. 23. van Beek, S., and F. G. Priest. 2002. Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study. Appl. Environ. Microbiol. 68:297–305. 24. Verachtert, H., and E. Dawoud. 1990. Yeast in mixed cultures. Louv. Brewing Lett. 3:15–40.
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this cannot play a role in this fermentation because the fermentation runs under severe oxygen limitation. Consequently, no or only a very small amount of acetic acid was found in the production process (B. Johansson, personal. communication). In a recent study, it has been demonstrated that S. cerevisiae outcompeted D. bruxellensis because of its higher growth rate (1). Growth rate should not play a role in the process since the yeast is recirculated. However, it has been shown that D. bruxellensis is more competitive than S. cerevisiae in wine fermentations at low substrate concentrations (16). Moreover, a recent study showed that contaminating D. bruxellensis increased at the expense of S. cerevisiae in industrial fermentations running in a mode similar to that investigated in our study (i.e., with yeast recirculation) (8). D. bruxellensis (anamorph Brettanomyces bruxellensis) is a yeast that occurs in different fermented foods (4, 12). The yeast has frequently been found in contaminated red wine, where it can produce 4-ethylphenol, which gives the wine an off flavor (10, 19). However, the yeast is commercially used for production of the alcoholic beverage lambic beer (24) and belongs to the natural microflora of sourdough (14). It has even been shown in a model fermentation that the yeast can produce ethanol and by-products (aldehydes and others) in accordance with the rules of the Brazilian legislation for cachaca, a national distilled beverage (6). D. bruxellensis obviously did not impair the production process, and we conclude that it is not a contaminant in this fermentation but instead the production organism. Taking into account the reports from the production staff about the changing cell shapes of the production yeast during the first 3 weeks, it seems likely that this yeast has been the production organism for several years. The ethanol company has no plans to try to change the D. bruxellensis-L. vini consortium back to S. cerevisiae, since there were no complains about the efficiency and productivity of the process. Not much is known about L. vini or L. mobilis, the dominating LAB in the fermentation. L. vini strains have been isolated from wine fermentation (18), whereas the one strain designated L. mobilis has been isolated from lactic acidcontaining beverages (http://www.ncbi.nlm.nih.gov/Taxonomy /Browser/wwwtax.cgi?id⫽357762). Thus, this is the first report of the occurrence of this bacterium in an industrial, grainbased ethanol production process. The interaction of yeasts and LAB in the production of strong alcoholic beverages has been investigated in malt whisky distilleries (23). In this study, L. fermentum was detected among other LAB, while we found it to be a minor organism in the ethanol fermentation process (Table 2). In whisky production, LAB are responsible for the formation of lactic and acetic acids during the middle and late phases of the fermentation process (23). The other minor lactic acid bacterium, Lactobacillus panis, has earlier been detected in wet wheat distiller’s grain (15) and may thus belong to the normal flora of ethanol production processes. There is very limited data available about the physiology of L. vini, and we can only speculate about the role of this bacterium in the fermentation process. However, as it apparently forms a stable community with D. bruxellensis, it might function to stabilize the population and the ethanol production in the fermentor.
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