Adenylate energy charge (AEC) – computed from the ATP, ADP and AMP concentrations in a specimen – reflect the net physiological state of the microbial population in that specimen. Previous research has demonstrated that healthy microbial populations maintain AEC≥0.8. As populations are subjected to stresses, or – in closed systems – deplete the available nutrients, respond to the accumulation of toxic metabolites, or both, AEC decreases (often to <0.5). Aqueous-phase samples from a set of fuel–water microcosms were tested for cellular ATP (cATP) and AEC. This paper reports on the precision of the AEC test method and the relationship between cellular AEC and cATP bioburdens in the aqueous phase of fuel over aqueous-phase microcosms.