Geomicrobiology of Terrestrial Subsurface Fluids and Potential Applications in Biotechnology

Extremophiles have developed various mechanisms to tolerate harsh environmental conditions, and some of these adaptations may be exploited for applications in biotechnology. This study aims to elucidate the taxonomy and physiological characteristics of bacteria cultured from two different extreme ecosystems, and to assess their potential for use in different areas of biotechnology. Field sites chosen for this study include hydrothermal springs in Yellowstone National Park and serpentinizing springs in the Zambales Ophiolite, the Philippines. Generally, hydrothermal springs in Yellowstone are characterized by high temperatures (>60C) and acidity, while serpentinizing springs are characterized by moderate temperatures (30-40C) and alkalinity. Microorganisms inhabiting such environments may display adaptations to one or more physicochemical extremes. The first investigation involves microbial metal tolerance and transformation, and the second focuses on microbial cellulose degradation at different pH ranges. The goals of the first investigation were to culture and isolate metal-tolerant bacteria from environmental samples, evaluate the extent of tolerance exhibited by each, and attempt to elucidate the associated mechanisms of tolerance. Metals of interest included Cr, Cu, Co, Ni, and Zn. These metals are toxic at elevated concentrations and pose a significant environmental threat in areas affected by metal pollution. Bacterial tolerance was examined to concentrations of each metal ranging from 25 to 600 mg/L. Over 20 bacterial isolates were obtained from environmental samples, and most display tolerance to >200 mg/L of each metal, while some are capable of growth at up to 1000 mg/L of some metals. 16S rRNA gene analysis determined that most isolates belong to the genus Bacillus, with several members of Pseudomonas and Microbacterium identified as well. Tolerance mechanisms varied between isolates, and it was found that most strains likely utilized an efflux pump to regulate intracellular metal concentrations. Other observed mechanisms include the release of chelators and intracellular sequestration of metal in the cell membrane, two functions that are relevant in biotechnology. Results of this investigation may be relevant in developing metal remediation strategies or in the field of biomining. The second investigation focuses on the ability of microorganisms from these same environments to degrade cellulose, the most abundant organic compound on Earth. The goals of this investigation were to enrich for cellulolytic bacterial communities from each field site under their respective pH ranges – acidic for hydrothermal springs and alkaline for serpentinizing springs. Cellulose degradation at extreme pH and temperatures has implications in the conversion of this abundant renewable resource to biofuels and other useful chemicals like acetic acid and glucose. Results demonstrate that in both alkaline and acidic conditions, most cellulolytic organisms belong to the phylum Firmicutes, specifically the families Clostridiaceae and Bacillaceae. The results may support targeted culturing efforts for cellulolytic bacteria that are active at a specific pH range. Additionally, a cellulolytic consortium of Thermoanaerobacterium and Caldicellulosiruptor was observed to efficiently degrade paper substrate at low pH (~4.0).