Bioenergetic Limitations on Slow Microbial Growth in the Subsurface: What is the Burden of Maintenance on the Overall Energy Budget?

English Abstract:

In low energy environments such as the subsurface, the minimum energy required to maintain cellular integrity and function (maintenance energy) may represent a significant fraction of the total energy available to microbial communities. However, traditional kinetic and thermodynamic models incorporating key microbial processes are often developed using data collected in nutrient rich growth media. In this study, slow microbial growth in the subsurface was simulated using a flow through bioreactor system in experiments designed to determine the maintenance energy requirement of the model subsurface bacterium Shewanella oneidensis. An existing bioreactor system (Applikon EZ-control®, 2.4 L) was modified to include a biomass retention filtration unit (retentostat) resulting in biomass accumulation over time. An artificial low-nutrient groundwater medium was optimized for slow S. oneidensis growth and was supplied and removed from the reactor at flow rates on the order of 1 mL min^-1 with a dilution rate of 0.025 h^-1. The retentostat was run under electron donor limited conditions with nitrate, a common groundwater contaminant, supplied at 0.025 mM h^-1 and lactate supplied in excess at 0.125 mM h^-1. Respiratory ammonification of nitrate by S. oneidensis and cell growth was monitored over time (40 days) and compared to parallel incubations in batch reactors. Initial rates of ammonification were similar in the bioreactor and batch reactors, however, optical density and ATP measurements showed a slow yet increasing microbial growth over time (generation time = 17 days) in the retentostat. In contrast, cells in the batch reactors did not grow significantly and died within 2 weeks of inoculation. A maintenance energy demand was estimated (0.5 kJ C-mol biomass h^-1) by fitting the biomass data to the van Verseveld equation. The low maintenance energy demand of S. oneidensis as compared to typical maintenance energies reported in the literature (>10 kJ C-mol biomass h^-1) likely reflects its metabolic versatility and ability to persist in low energy environments. In addition to the retentostat results, we will also discuss our efforts to develop a kinetic and thermodynamic based biogeochemical model which explicitly accounts for energy used for both microbial maintenance and growth.