Factors that influence microbial communities in spontaneously fermented wine, beer, and kombucha: insights from a metabarcoding perspective : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

It is well established that the world contains a vast diversity of microbes, many of which have been unexplored and even as yet uncultivated. Advances in molecular biology techniques such as metabarcoding have helped in improving our insights into this complex microbial world, in every aspect of human civilization, including food and beverage production. The importance of microbial diversity in the processing of wine and other fermented products in general cannot be overemphasized. In wine for example, grape health and the diversity of microorganisms introduced into the winery depends on the number of microbes who are able to form ecological niches in the vine and soil of the vineyard. With extensive knowledge of microbiome diversity and its interactions or shifts within fermentation processes, this could lead to an improved understanding of this activity. With next generation Sequencing (NGS) techniques, it is now feasible to accurately detect and quantify the relative abundance of microbes found in and around these products. Culture based methods previously used in microbial analysis neglects the non-culturable microbial fraction, an advantage NGS methods possesses over culture based techniques. Performing complex multifactorial analyses is now possible by coupling metabarcoding with bioinformatics tools. This is fast becoming the “go to” strategy in monitoring, traceability of products and diagnostic purposes. If properly harnessed, metabarcoding opens up the possibility of adequately monitoring and describing the microbial population during fermentation and how their profile changes with time. The aim of this research was to employ metabarcoding in exploring the microbiomes associated with spontaneously fermented wine and other foods/products such as kombucha and lambic styled beer in New Zealand. These studies included investigating the influences that the environment and/or climate play in shaping the microbiome of these products. Where applicable, the complex relationship between the microorganisms and the organoleptic and chemical properties of the products was also probed. The first experiment in this work explored the impact of differing environmental systems on the microbial communities utilized by an organic winemaker in producing wines. Results indicated that T. delbrueckii and Fructobacillus could be affected by environmental conditions as they were detected in one system but not the other. Together, significant differences were reported between the fungal (RANOSIM = 0.603, p = 0.0001) and bacterial (RANOSIM = 0.5814, p = 0.0001) diversity found in both systems. Assessment of these microbial differences were done to see if they brought about detectable sensory or chemical variations in the resulting wines. Certain volatile compounds were only found in wines from one of the environmental system but not the other, indicating that significantly different bacterial and/or fungal species could have major roles in synthesising these compounds. Specifically, it can be inferred that Gluconobacter may play a role in the taste (bitterness) and ‘mouth feel’ (astringency/tannin) attributes of Pinot noir wines. To gain insights into how the microbial communities associated with kombucha production might differ based on scale (large commercial scale as compared to a small scale production), another experiment was carried out. For the first time in kombucha literature, results uncovered indicated that the fungal and bacterial diversity associated with the commercial fermentation process had a higher diversity as compared to small scale kombucha production. Interestingly, the core fungal microbiome found in the SCOBY and tea phase of both the commercial and small-scale kombucha types were similar. Using linear modelling unravelled how Komagataeibacter might play a key role and shape the pH of kombucha. To understand if the microbial communities detected at the end of spontaneous beer fermentation varies across vintages, a beer experiment was conducted. Results from this experiment revealed how vintage has a huge influence in shaping the detected microbial populations. Climatic factors such as humidity and maximum temperature were identified as variables that may enhance the relative abundance of Penicillium and Hanseniaspora in beer microbiota. When the microbiome of all three fermented food/products were compared, in comparison to the bacterial communities, similarities can be seen with the fungal populations. Spontaneously fermented wine and kombucha fermentation had considerable amounts of Hanseniaspora, Metschnikowia and Saccharomyces. Beer and kombucha fermentation had substantial Brettanomyces and Saccharomyces proportions. Saccharomyces was the only genus present in significant amounts in all three foods/products. For bacteria, spontaneously fermented Pinot noir wine was dominated by Tatumella (2021 vintage) and Lactococcus (2018 vintage). Komagataeibacter and Gluconobacter were abundant in the home and commercial kombucha brew respectively. Lambic styled beer saw Pediococcus as the most dominant bacteria. Summarily, this research unravelled the complex microbiome of spontaneous Pinot noir wine fermentation, kombucha fermentation and lambic beer brewing. Description of how microbial populations can be affected by environmental and/or climatic influences were done. Where applicable, how these microbial differences could result in sensory and/or chemical variations in the resultant product were assessed. This study has described how metabarcoding can be reliable in assessing the microbiome of fermented food/products.