The Effect of Temperature and Fungal:Bacterial Ratio on Kombucha Culture Fermentation

Abstract: The fermented tea beverage kombucha is a traditional drink that has been consumed for thousands of years in Asia. Over the past decades, it has gained greatly in popularity amongst consumers and researchers also in the rest of the world. Kombucha is produced through fermentation of sweetened tea by a complex community of bacteria, mainly dominated by acetic acid bacteria, and a variety of yeast. During the fermentation process, bacteria and yeast interact through forming compounds and metabolites that stimulate the growth of the other part. The aim of this project was to investigate how temperature and the fungal:bacterial ratio affects the kombucha fermentation process. First, I attempted to construct a gradient of fungal:bacterial biomass between inoculums based on filtration and sedimentation. However, as the visual evaluation as well as the PCR results implied a weak effectiveness of the method, it was decided not to continue with this approach. Instead, fermentation vessels were places in different temperature zones around 15.1 °C, 19.8 °C, 24.0 °C, 35.9 °C, and allowed to ferment for 20 days. Previous research have shown contrasting results between yeast and bacteria on optimal temperature and temperature sensitivity. Thus, given that the two groups are competing for resources, temperature conditions which favor one of the groups more would indirectly disfavor the other and an alteration of the fungal:bacterial biomass might be achieved by the temperature difference. During the 20 day fermentation time, pH and brix was measured continuously every second day and samples were saved and frozen for later analysis including qPCR and sugar content. The pH followed an expected pattern throughout the fermentation processes such as faster decrease associated with higher temperature. In contrast, the brix results were unexpected, not changing as much as expected for the three lower temperatures and increasing significantly for the highest temperature. A decrease in sucrose content and increase in glucose and fructose content could be observed from the sugar analysis. Although few differences were significant, some tendencies could be observed when measuring the DNA amount and comparing amount of yeast and bacterial DNA between the different temperatures. Lower temperature seemed to favor the growth of both bacteria and yeast, although bacteria somewhat more. However, no evidence of a consistent effect of temperature on the fungal:bacterial ratio could be observed. Overall, the observable effects of temperature on fungal:bacterial ratio and kombucha fermentation dynamics in this project was not as significant as was hypothesized when planning this project but further research on the subject is suggested before final conclusions are drawn.