Lawrence Berkeley National Laboratory

Background: Extremophiles are key for remediation of groundwater contaminants and for biofuels development. Standard fermenters are not equipped for anaerobic growth conditions, nor can the stainless steel withstand the reactivity of metal-reducing organisms and their metabolic by-products, such as hydrogen sulfide. We have developed methods to grow sulfate-reducing bacteria (Desulfovibrio vulgaris Hildenborough, DvH) under various conditions for detailed physiological and molecular analyses, using custom-built fermenters. Methods: Five-liter-volume fermenters have PEEK headplates and agitators. During culture growth, optical density and redox potential are continuously measured, and discrete samples for substrate utilization and metabolite concentrations, phospholipid fatty acid (PLFA), cell counts, total cell proteins and whole cell protein analysis are taken. DvH is grown in batch and continuous flow modes using defined lactate-sulfate media. DvH batch cultures have also been grown under stressed conditions, including pyruvate fermentation. Results: The growth rate constant for DvH in the 5 L fermenter is 0.11 1/hr. At the onset of deceleration phase, all lactate (60 mM) is depleted, and 30 mM of the initial 50 mM sulfate is utilized. The dilution rates to maintain the culture at mid-log and late-log phase during continuous flow are 0.15 and 0.13 1/hr, respectively. Cell densities and total proteins at late mid-log phase range from 5-10 x 108 cells/mL, and 80 to 120 ?mu g/mL, respectively. PLFA profiles for DvH are sensitive to the growth conditions and growth phase, while preliminary results show consistent whole-cell protein patterns when visualized using SDS-PAGE. Conclusion: Detailed characterization during DvH growth provides insights into physiological changes during different growth phases and has resulted in the development of protocols for controlled and reproducible growth conditions for DvH. These protocols are essential in evaluating effects of stressors on growth, which may influence efficiency during subsurface remediation of heavy metals and radionuclides.