Biosci. Biotechnol. Biochem., 74 (11), 2316–2319, 2010 Note Mixed-Species Biofilm Formation by Direct Cell-Cell Contact between Brewing Yeasts and Lactic Acid Bacteria

y Soichi FURUKAWA,1; Kanako YOSHIDA,1 Hirokazu OGIHARA,1 y y Makari YAMASAKI,2; and Yasushi MORINAGA1;

1Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, , 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan 2Advanced Research Institute for the Sciences and Humanities, Nihon University, 12-5, 5-bancho, Chiyoda-ku, 102-8251, Japan

Received May 10, 2010; Accepted July 22, 2010; Online Publication, November 7, 2010 [doi:10.1271/bbb.100350]

Mixed-species biofilm was remarkably formed in a have high ability for mixed-species biofilm formation, in static co-culture of Lactobacillus plantarum ML11-11 actual traditional fermentation processes. and Saccharomyces cerevisiae Y11-43 isolated from We focused on the combinations of yeasts and LAB brewing samples of Fukuyama pot vinegar. Mixed- isolated from brewing samples of Fukuyama pot species biofilm is probably formed by direct cell-cell vinegar, one of the most primitive types of rice vinegar contact between ML11-11 and S. cerevisiae including in Japan.15–18) Fermentation of Fukuyama pot vinegar is Y11-43 and laboratory yeast strains. Scanning electron conducted in roughly capped large pots laid in the open- microscopic observation suggested that the mixed- air without any refined culture-manipulation. Recently, species biofilm had a thick, bi-layer structure. we investigated the microbiological and biochemical transition of the fermentative process of Fukuyama pot Key words: mixed-species biofilm; Lactobacillus plan- vinegar to determine the mechanism of the establish- tarum; Saccharomyces cerevisiae ment of acetic acid fermentation through diverse microbes.19) In Fukuyama pot vinegar, three reaction Some traditional fermentation processes start from steps, saccharification, alcohol fermentation, and acetic solid biomaterials e.g., rice in rice wine (sake), vegeta- acid fermentation, proceed sequentially and partly in bles in pickled foods such as sauerkraut and kimchi, parallel, suggesting the existence of indispensable cereals (soy bean and wheat) in soy sauce, and marine interactions. These might include mixed-species biofilm products in traditional Asian condiments such as nam formation among the microorganisms catalyzing the pla.1) We hypothesized that the formation of a biofilm, a reaction steps. microbial community on the solid-liquid interface,2–4) is We obtained yeasts isolates and LAB isolates from important in the traditional fermentation process. autumn-2006 samples of Fukuyama pot vinegar fermen- Coexistence or symbiosis of yeasts and lactic acid tation collected on the 5th and 11th brewing day. We bacteria (LAB) in traditional fermented foods, such as investigated mixed-species biofilm formation between sake, wine (malolactic fermentation by LAB), lambic the yeast and LAB. To prepare seed cultures, LAB were beer, whiskey, kefir, sourdough, and pickles is well grown in DeMan, Rogosa, Sharpe broth (MRS; Becton, known.1,5) Dickinson, Franklin Lakes, NJ) at 28 C for 24 h in Several investigators have also done pioneering work static aerobic culture. Yeasts were grown in YPD broth on mixed-species biofilm formation.6–13) Especially, (YPD; Becton, Dickinson) at 28 C for 24 h in static Hogan and Kolter reported initially on pathogenic aerobic culture. Cells were cultured to the stationary bacteria and yeast co-aggregative interaction.10,12) They phase. found that Pseudomonas aeruginosa forms a dense Biofilm formation and assay protocol was almost as in biofilm on Candida albicans filaments and kills the our previous study.20) To assay biofilm formation, fungus, and, in contrast, P. aeruginosa neither binds to stationary phase LAB or yeast cultures were inoculated nor kills yeast-form C. albicans. into fresh YPD at the dilution rate of 1:100 in These pioneering works on the mixed-species biofilm monoculture, while for co-culture, stationary phase prompted us to investigate the possibility that yeasts and cultures of the LAB and yeast were inoculated into LAB can form a mixed-species biofilm in co-culture. fresh YPD at a dilution rate of 1:200. A 96-well We have found positive combinations of yeasts and polystyrene micro-titer plate (#92696, TPP AG, Adolf LAB, in which LAB produced extracellular factors Ku¨hner, Switzerland) was used for biofilm formation. supporting the formation of yeast biofilm in a single After inoculation, both mono- and co-culture samples culture.14) These yeasts and LAB were arbitrarily were incubated at 30 C for 24 h. Quantification of selected laboratory and industrial strains. In this study, biofilm formation was done by the conventional titer we tried to identify LAB and yeast combinations that plate method.14,20,21)

y To whom correspondence should be addressed. Soichi FURUKAWA, Tel/Fax: +81-466-84-3973; E-mail: [email protected]; Makari YAMASAKI, E-mail: [email protected]; Yasushi MORINAGA, Tel/Fax: +81-466-84-3971; E-mail: [email protected] Mixed-Species Biofilm Formation by Yeasts and Lactic Acid Bacteria 2317 Table 1. Mixed-Species Biofilm Formation between Yeast and LAB Isolates from Fukuyama Pot Vinegar Fermentation Collected on the 5th and 11th Brewing Day

Yeast Y5-1 Y5-11 Y5-26 Y5-31 Y5-43 Yeast Y11-10 Y11-15 Y11-34 Y11-43 Y11-50 LAB single 1 1 1 1 1 LAB single 1 1 1 1 1 GML5-1 1 1 1 1 1 1 ML11-1 1 1 1 1 1 1 GML5-2 1 1 1 1 1 1 ML11-2 1 1 1 1 1 1 GML5-3 1 1 1 1 1 1 ML11-3 1 1 1 1 1 1 GML5-4 1 1 1 1 1 1 ML11-4 1 1 1 1 1 1 GML5-5 1 1 1 1 1 1 ML11-5 1 1 1 1 1 1 GML5-6 1 1 1 1 1 1 ML11-6 1 4 2 2 2 3 GML5-7 1 1 1 1 1 1 ML11-7 1 1 1 1 1 1 GML5-8 1 1 1 1 1 1 ML11-8 1 1 1 1 1 1 GML5-9 1 2 2 2 2 1 ML11-9 1 1 1 1 1 1 GML5-10 1 1 1 1 1 2 ML11-10 1 1 1 1 1 1 GML5-11 1 1 1 1 1 1 ML11-11 2 4 2 2 5 3 GML5-12 1 1 1 1 1 1 ML11-12 1 1 1 1 1 1 GML5-13 1 1 1 1 1 1 ML11-13 1 1 1 1 1 1 GML5-14 1 1 1 1 1 1 ML11-14 1 1 1 1 1 2 GML5-15 1 1 1 1 1 1 ML11-15 1 1 1 1 1 1 GML5-16 1 1 1 1 1 1 ML11-16 1 1 1 1 1 1 GML5-17 1 1 1 1 1 1 ML11-17 1 1 1 1 1 1 GML5-18 1 1 1 2 1 2 ML11-18 1 1 1 1 1 1 GML5-19 2 1 1 2 2 2 ML11-19 1 1 1 1 1 1 GML5-20 2 1 1 2 1 1 ML11-20 1 1 1 1 1 1 GML5-21 2 2 2 2 2 1 ML11-21 1 1 1 1 1 1 GML5-22 1 1 1 1 1 1 ML11-22 1 1 1 1 1 1 GML5-23 1 1 3 2 2 2 ML11-23 1 1 1 1 1 2 GML5-24 1 1 1 1 1 1 ML11-24 1 1 1 1 1 1 GML5-25 1 2 1 2 2 2 ML11-25 1 1 1 1 1 1 GML5-26 1 1 1 1 1 1 ML11-26 1 1 1 1 1 1 GML5-27 1 1 1 1 1 1 ML11-27 1 1 1 1 1 1 GML5-28 1 1 1 1 1 1 ML11-28 1 3 1 2 2 1 GML5-29 1 1 3 2 2 1 ML11-29 1 1 1 1 1 1 GML5-30 1 1 1 1 1 1 ML11-30 1 1 1 1 1 1 GML5-31 1 2 1 2 2 2 ML11-31 2 2 2 1 1 1 GML5-32 1 1 1 1 1 1 GML5-33 1 1 1 1 1 1 GML5-34 1 1 1 1 1 1 GML5-35 1 1 1 1 1 1 GML5-36 1 1 1 1 1 1 GML5-37 1 1 2 1 1 1 GML5-38 1 1 1 1 1 1 GML5-39 1 1 1 1 1 1 GML5-40 2 2 1 2 2 2 GML5-41 1 1 1 1 1 1 GML5-42 1 1 1 1 1 1 GML5-43 1 1 1 1 1 1 GML5-44 2 2 2 2 2 2 GML5-45 1 1 1 1 1 1

Light gray area show that the biofilm formation slightly increased in the mixed-culture. Concentrated gray area show that the biofilm formation highly increased in the mixed-culture. 1 to 5 show the level of the biofilm formation. The criteria of the biofilm formation level were light transmissibility (thickness) and coverage of the micro-titer plate well bottom of the biofilm. Level 5: micro-titer plate well bottom was not light transmissible and fully covered by biofilm. Level 4: micro-titer plate well bottom was not light transmissible but partly not covered by biofilm. Level 3: micro-titer plate well bottom was light transmissible but fully covered by biofilm. Level 2: micro-titer plate well bottom was light transmissible and partly covered by biofilm. Level 1: biofilm formation was not observed.

Ten yeasts and 124 LAB were isolated from samples strain identified as Saccharomyces cerevisiae Y11-43 of Fukuyama pot vinegar fermentation collected on the showed the most notable biofilm formation (Fig. 1). 5th and 11th brewing day, and mixed-species biofilm Both strains were identified by sequencing their 16S and formation between the yeasts and LAB was investigated 18S rDNA sequences. L. plantarum ML11-11 slightly (Table 1). The degree of biofilm formation was assayed formed a biofilm in single culture (thin mono-layer by naked eye observation. The criteria of the biofilm biofilm), but S. cerevisiae Y11-43 did not form a biofilm formation level were thickness (light transmissibility) in single culture, i.e., did not attach to polystyrene. and coverage area (biofilm coverage of the micro-titer Microorganisms belonging to the genera Saccharomyces plate well bottom) of the biofilm. Out of 380 combina- and Lactobacillus are commonly isolated from tradi- tions, there were 36 positive combinations between tional fermented foods,1) and these microorganisms have yeasts and LAB, and the combination of the strain been isolated from Fukuyama pot vinegar,14,15,18) but identified as Lactobacillus plantarum ML11-11 and the mixed-species biofilm formation by yeast and LAB 2318 S. FURUKAWA et al.

2.5

) A B C

590 2.0

1.5

1.0 CM enriched CM

0.5 Biofilm formation (A formation Biofilm

0.0 10 µm 10 µm Medium YPD YPD YPD Y-CM L-CM Co-CM Co-CM D Microbes Y L Co LYY L

10 µm Fig. 1. Effects of Conditioned Media (CMs) of L. plantarum ML11- 11, S. cerevisiae Y11-43 or Both on the Biofilm Formation of F L. plantarum ML11-11 and S. cerevisiae Y11-43. Gray bar, biofilm formation in YPD medium; white bar, biofilm formation in CM; black bar, biofilm formation in enriched CM. CM, conditioned media; the preparation method is described in the text. L, L. plantarum ML11-11; Y, S. cerevisiae Y11-43, and Co, µ 40 µm 5 µm co-culture. Enriched CM (pH 7.0), all ingredients of YPD added E 1 m to the above CM at final concentrations as for YPD, dissolved and filtrated. After filtration, the pH of the enriched CM was adjusted Fig. 2. Scanning Electron Microscopic Images of Biofilms Formed to 7.0. on Glass Plates. A, Attached single culture cells of L. plantarum ML11-11. B, Attached single culture cells of S. cerevisiae Y11-43. C, isolated from traditional fermented food had not been Mixed-species biofilm of ML11-11 and X2180-1A. D, Mixed- investigated so far. species biofilm of ML11-11 and Y11-43. E, Magnified picture of In our previous study,14) the yeast S. cerevisiae formed mixed-species biofilm of ML11-11 and Y11-43. F, Edge part of the biofilm in single culture in response to LAB, Lactoba- mixed-species biofilm of ML11-11 and S. cerevisiae BY4741. cillus casei, conditioned medium (CM). A conditioned medium (CM) was prepared from cultured broth (stat- tion was also investigated. Neither sex nor ploidy of the ically cultured at 30 C for 24 h) by removing cells by chromosome of yeast S. cerevisiae X2180 affected centrifugation (5;600 g for 20 min) and filtration with a mixed-species biofilm formation with L. plantarum 0.2-mm pore-size sterile filter (Toyo Roshi, Tokyo). The ML11-11 (data not shown). This indicates that mixed- effects of the addition of CMs on biofilm formation by species biofilm formation occurred independently of the S. cerevisiae Y11-43 were investigated. In contrast to sex or the ploidy of the chromosome. our previous study,14) no biofilm formation occurred in The structure of the mixed-species biofilm formed the single culture of S. cerevisiae Y11-43 induced by the by L. plantarum ML11-11 and S. cerevisiae Y11-43, addition of the CM prepared from the cultured broth of BY4741 or X2180-1A was observed by scanning L. plantarum ML11-11 (Fig. 1), suggesting a require- electron microscope (SEM). S. cerevisiae Y11-43, ment of direct cell-cell contact between Y11-43 cells and BY4741, and X2180-1A formed almost the same ML11-11 cells. In addition, the mixed-species biofilm structural mixed-species biofilm with L. plantarum formed in the mixed culture of L. plantarum ML11-11 ML11-11. For observation by SEM, biofilms were and S. cerevisiae Y11-43 was remarkably thicker (more formed on glass slides by culture in a petri dish than 10 times) than which we found in our previous containing YPD at 30 C for 24 h. SEM observation study.14) These results indicate mechanical differences was performed as described by Guzel-Seydim et al.14,23) between the previously found mixed-species biofilm Glass slides with biofilm were immersed in 0.1 M sodium formation and that found in the present study. Direct cell- phosphate buffer and fixed with 2% glutaraldehyde. The cell contact in the latter case probably contributes to samples were observed using a Keyence VE-8800 thick, strong biofilm formation. scanning electron microscope (Keyence, Osaka, Japan). Mixed-species biofilm formation between L. planta- In single culture, LAB cells attached to the glass surface rum ML11-11 and laboratory yeast strains was inves- and formed a thin biofilm (Fig. 2A), but yeast cells tigated. Laboratory yeast strains S. cerevisiae X2180 scarcely attached to the glass surface (Fig. 2B). These and BY4741 formed a biofilm with L. plantarum ML11- observational results correspond to the results of biofilm 11 in mixed culture as did S. cerevisiae Y11-43, and assay (Fig. 1). In mixed-culture, LAB and yeast cells these biofilm formation were not induced by the addition formed an extraordinarily thick biofilm (Fig. 2C and D). of the CM prepared from cultured broth of L. plantarum In the mixed-species biofilm, large yeast cells were ML11-11 (data not shown). The yeast strains used in this bound together by small LAB cells, which acted like study were S. cerevisiae BY4741 (MATa leu20 bridging clamps, that is, LAB and yeast showed direct ura30 his3-1 met150), S. cerevisiae X2180-1a cell-cell contact to form a co-aggregate (Fig. 2E and F). (X2180-1A) (MATa SUC2 mel gal2 CUP1), S. cerevi- The fringe of the biofilm also suggested that LAB cells siae X2180-1 (X2180-1B) (MAT SUC2 mel gal2 formed a basal layer in the mixed-species biofilm CUP1), and S. cerevisiae X2180-1a/1. (Fig. 2F). Thus SEM observation suggested that the Sexual agglutination of yeast is mediated by cell- mixed-species biofilm formed by L. plantarum ML11- surface glycoproteins.22) Hence the effect of sex of yeast 11 and yeast had a thick, bi-layer structure. on the mixed-species biofilm formation was investigated. The mixed-species biofilm-forming strains used in At the same time, the effect of the ploidy of the this study were isolated from samples of Fukuyama pot chromosome of yeast on mixed-species biofilm forma- vinegar collected on the 11th day, the critical time when Mixed-Species Biofilm Formation by Yeasts and Lactic Acid Bacteria 2319 ethanol fermentation reaches maximum and acetic acid 4) Kolter R and Greenberg EP, Nature, 441, 300–302 (2006). fermentation starts. This finding encouraged us to 5) Wood BJB, ‘‘Mixed Culture Fermentations,’’ eds. 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