ANALYSIS OF THE METABOLIC EFFECTS OF ELECTRON ACCEPTOR VARIATION AND ANODE POTENTIAL IN GEOBACTER SULFURREDUCENS

Open Access
Author:
Rose, Nicholas Dean
Graduate Program:
Environmental Engineering
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
None
Committee Members:
  • John Michael Regan, Thesis Advisor
Keywords:
  • NADP
  • BES
  • MFC
  • MEC
  • Cofactors
  • ATP
  • ADP
  • AMP
  • Adenylate
  • NAD
  • AEC
  • nicotinamide adenine dinucleotide
  • Geobacter sulfurreducens
Abstract:
Geobacter sulfurreducens is considered a model organism for environmental iron reduction and electricity production inside bioelectrochemical systems (BESs), and is also important for the bioremediation of certain contaminants. Developing a better understanding of how the metabolic processes of this organism respond to electron acceptors could provide useful insights for improving BES performance and bioremediation techniques. Nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) are five important metabolic cofactors representing reduction potential and cellular energy. These cofactors were analyzed in G. sulfurreducens cultures using fumarate, Fe(III)-citrate, or anodes poised at varying redox potentials as the electron acceptor. In addition, reactors started with anode potentials poised at 210 mV vs Standard Hydrogen Electrode (SHE) and then switched to 110, 10, or −190 mV (versus SHE) or open circuit were compared to reactors started directly at 110, 10, or −190 mV (versus SHE). Results showed that cellular redox potential given by ratios of reduced to oxidized NAD and NADP were similar when growing under all anode potentials tested and with Fe(III)-citrate, which is also believed to be reduced extracellularly, as the electron acceptor with an average of 0.088 ±0.022 (NAD) and 0.268 ±0.098 (NADP) for all conditions. However, both ratios were significantly increased when fumarate was provided as the electron acceptor, with values of 0.331 ±0.094 (NAD) and 1.955 ±0.369 (NADP). Energy potential of the cell represented by adenylate energy charge (AEC), an aggregate ratio of ATP, ADP, and AMP that accounts for all phosphoanhydride bonds, was maintained near 0.47 under almost all conditions tested. The reactor started at 110 mV maintained an AEC of 0.63 ± 0.03, which Bonferroni-corrected t tests showed was statistically different from the AEC for the reactor started at −190 mV and fumarate reducing cultures (0.47 ± 0.003 and 0.047 ± 0.02, respectively). Anode-growing biofilms demonstrated a significantly higher ATP/ADP ratio relative to cells growing in suspended culture on fumarate or Fe(III)-citrate. Reactors started at 210 mV and then switched to different anode potentials showed similar NAD(P) ratios and AECs as reactors started at 110, 10, or −190 mV. These results indirectly show that metabolic mechanisms regulated by the ratios of reduced to oxidized NAD and NADP are altered significantly between G. sulfurreducens cultures growing with extracellular and intracellular electron acceptors but that energy potential represented by AEC is not significantly altered. However, the ATP/ADP ratio and specific cellular ATP concentration increased during biofilm growth, suggesting a need for increased levels of ATP to maintain the biofilm. Analysis of reactors with anode potentials posed at 210 mV that were put into open circuit for 15 to 17 hours shows that G. sulfurreducens maintains similar reduced to oxidized NAD and NADP ratios as well as AEC when electron acceptors are or are not available. Overall anode potential has little effect on these important metabolic ratios, and specific electron acceptors only appear to affect these ratios based on whether they are intra or extracellularly.