Microbial Metabolic Diversity Study of the Kuantzuling Mud Hot Spring in the Southwestern Taiwan

Organic carbon, sulfate, methane, and hydrogen are available for microorganisms to carry on diverse metabolisms in the Kuantzuling mud hot spring, southwestern Taiwan. On the basis of bioenergetic evaluations and environmental DNA analyses, previous studies have inferred diverse metabolic capabilities, including methanogenesis, sulfate reduction, fermentation, aerobic heterotrophy and methanotrophy. However, active metabolisms have never been confirmed by cultivation-based analysis. Due to the temperature fluctuation of the Kuantzuling mud spring, this study performed a set of enrichment experiments at temperatures ranging from 25oC to 80oC to understand the activity and interaction among microorganisms at various temperatures. Pure stains were also isolated along with their physiological tests to reveal their possible roles in this terrestrial hot spring ecosystem. According to the geochemical and molecular data, nine types of media were designed to enrich different kinds of metabolisms in the slurry. Positive enrichments were obtained in all types of media, but not at all investigated temperatures. Methanogens using acetate, methanol, and hydrogen and carbon dioxide, sulfate reducers, thiosulfate reducers, fermenters, aerobic heterotrophs could be enriched at temperatures higher than 50oC and even 80oC. Methanogen using methylamine and aerobic methanotroph can only be enriched at temperatures lower than 50 oC. This result is generally consistent with previous energetic evaluation and molecular analysis. It also inferred that microbial assemblages possessing diverse metabolisms were either competitive or collaborative to each other for degradation of organic carbon or carbon cycling. Two strains were isolated from aerobic heterotrophic media. The 16S rDNA gene sequence of one strain exhibited a very close affiliation (at a similarity of 99%) with Meiothermus ruber strain SPS242 and that of the other showed an affiliation to that of Rhodobacter vinaykumarii JA123 at the similarity of 95%. The former grew at the pH values between 5 and 9, at the temperatures ranging from 20 to 70oC with the optimal growth temperature at 60oC, while the later can grow at the pH values between 6 and 9, at the temperatures ranging from 20 to 60oC with the optimal growth temperature at 50oC. The Kuantzuling mud spring harbors diverse microorganisms. Such a wide range of physiological capability might represent an unstable ecosystem constantly exposed to the substantial environmental fluctuations, such as temperature, oxygen content and fluid source.