Isolation and Screening of Microbial Isolates from Kombucha Culture for Bacterial Cellulose Production in Sugarcane Molasses Medium
Abstract
Kombucha tea is a traditional fermented beverage of Manchurian origins which is made of sugar and tea. The fermentation involves the application of a symbiotic consortium of bacteria and yeast (SCOBY) in which their metabolites provide health benefits for the consumer and subsequently allow the product to protect itself from contamination. Additionally, kombucha tea fermentation also produces a byproduct in the form of a pellicle composed of cellulose (Bacterial Cellulose, BC). Compared to plant cellulose, BC properties are more superior, which makes it industrially important. However, BC production at industrial scale has been faced with many challenges, including low yield and high fermentation medium cost. Many researchers have focused their studies on the use of alternative low cost media, such as molasses, which is a by-product of sugar refining process. To maximize the BC production in molasses medium, it is important to select the microbial strains that can grow and produce BC at high yield in molasses. This study aimed to isolate and characterize BC-producing bacteria and a dominant yeast from kombucha culture which had been previously adapted in molasses medium. The isolation of bacteria was performed using Nutrient Agar (NA) and Hestrin and Schramm (HS) supplemented with cycloheximide, while yeast was isolated using Potato Dextrose Agar (PDA) supplemented with chloramphenicol. The most dominant colonies were isolated and then subjected to microscopic observation for morphological analysis. The pure bacteria and yeast isolates were then identified by sequencing the 16S rRNA gene and D1/D2 region of the 26S rRNA, respectively. The bacteria isolates obtained were all from closely related genera: Komagataeibacter sp. DS1MA.62A, Komagataeibacter xylinus, Komagataeibacter saccharivorans, Komagataeibacter xylinus and Gluconacetobacter saccharivorans. The single isolated yeast was identified as Brettanomyces bruxellensis. This study helps to elucidate the BC-producing species which thrive in molasses medium for potential use in the BC production using molasses as alternative cheap carbon source. Also, the study revealed that the co-culture of Komagataeibacter sp. DS1MA.62A and B. bruxellensis could produce BC from molasses supplemented with caffeine and acetate buffer at an average yield of 27.7±1.83 g/L.
Keywords: Kombucha, SCOBY, bacterial cellulose, Acetobacter, Komagataeibacter, Brettanomyces, alternative medium, molasses, caffeine, acetate buffer.
References
[1] Jayabalan, R., Malbaša, R. V., Lončar, E. S., Vitas, J. S., & Sathishkumar, M. (2014). A review on kombucha tea-microbiology, composition, fermentation, beneficial effects, toxicity, and tea fungus. Comprehensive Reviews in Food Science and Food Safety, 13(4), 538–550. http://doi.org/10.1111/1541-4337.12073
[2] Chakravorty, S., Bhattacharya, S., Chatzinotas, A., Chakraborty, W., Bhattacharya, D., & Gachhui, R. (2016). Kombucha tea fermentation: Microbial and biochemical dynamics. International Journal of Food Microbiology, 220, 63–72.
[3] Chawla, P. R., Bajaj, I. B., Survase, S. A., & Singhal, R. S. (2009). Bacterial cellulose: Fermentative production and applications. Food Technology and Biotechnology, 47(2), 107–124.
[4] Huang, Y., Zhu, C., Yang, J., Nie, Y., Chen, C., & Sun, D. (2014). Recent advances in bacterial cellulose. Cellulose, 21(1), 1–30. http://doi.org/10.1007/s10570-013-0088-z
[5] Lee, K. Y., Buldum, G., Mantalaris, A., & Bismarck, A. (2014). More than meets the eye in bacterial cellulose: Biosynthesis, bioprocessing, and applications in advanced fiber composites. Macromolecular Bioscience, 14(1), 10–32. http://doi.org/10.1002/mabi.201300298
[6] Cakar, F, Özer, I., Aytekin, A.Ö., & Şahin, F. (2014). Improvement Production of Bacterial Cellulose by Semi-Continuous process in Molasses Medium. Carbohydrate Polymers 106, 7–13.
[7] Carreira, P., Mendes, J., A., Trovatti, E., Serafim, L., S., Freire, C., S., Silvestre, A., J., & Neto, C., P. (2011). Utilization of residues from agro-forest industries in the production of high value bacterial cellulose. Bioresource Technol, 102(15):7354–7360.
[8] Hong, F., Zhu, Y., Yang, G., & Yang, X. (2011). Wheat straw acid hydrolysate as a potential cost-effective feedstock for production of bacterial cellulose. J Chem Technol Biot, 86(5):675–680.
[9] Teoh, A. L., Heard, G., & Cox, J. (2004). Yeast ecology of Kombucha fermentation. International Journal of Food Microbiology, 95(2), 119–126. http://doi.org/10.1016/j.ijfoodmicro.2003.12.020
[10] Liu, C., Hsu, W., Lee, F., & Liao, C. (1996). The isolation and identification of microbes from a fermented tea beverage, Haipao, and their interactions during Haipao fermentation. Food Microbiology, 13(6), 407-415. doi:10.1006/fmic.1996.0047
[11] Naritomi, T., Kouda, T., Yano, H., & Yoshinaga, F. (1998). Effect of ethanol on bacterial cellulose production from fructose in continuous culture. Journal of Fermentation and Bioengineering, 85(6), 598-603. doi:10.1016/s0922-338x(98)80012-3
[12] Krystynowicz, A., Czaja, W., Wiktorowska-Jezierska, A., Gonçalves-Miśkiewicz, M., Turkiewicz, M., & Bielecki, S. (2002). Factors affecting the yield and properties of bacterial cellulose. Journal of Industrial Microbiology & Biotechnology, 29(4), 189-195. doi:10.1038/sj/jim/7000303
[13] Villarreal-Soto, S. A., Beaufort, S., Bouajila, J., Souchard, J., & Taillandier, P. (2018). Understanding Kombucha Tea Fermentation: A Review. Journal of Food Science, 83(3), 580-588. doi:10.1111/1750- 3841.14068
[14] Marsh, A. J., Osullivan, O., Hill, C., Ross, R. P., & Cotter, P. D. (2014). Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples. Food Microbiology, 38, 171-178. doi:10.1016/j.fm.2013.09.003
[15] Goh, W. N., Rosma, A., Kaur, B., Fazilah, A., Karim, A. A., & Bhat, R. (2012). Fermentation of black tea broth (kombucha): I. effects of sucrose concentration and fermentation time on the yield of microbial cellulose. International Food Research Journal, 19(1), 109–117. http://doi.org/10.1016/j.pec.2010.07.039
[16] Coton, M., Pawtowski, A., Taminiau, B., Deniel, F., Coulloumme-labarthe, L., Fall, A., … Coton, E. (2017). Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods. FEMS Microbiology Ecology, 95(3). https://doi.org/10.1093/femsec/fix048
[17] Zahan, K. A., Pa’e, N., & Muhamad, I. I. (2015). Monitoring the Effect of pH on Bacterial Cellulose Production and Acetobacter xylinum 0416 Growth in a Rotary Discs Reactor. Arabian Journal for Science and Engineering, 40(7), 1881–1885. http://doi.org/10.1007/s13369-015-1712-z