Adenosine triphosphate (ATP) measurement by bioluminescence is used in microbial adsorption studies for which colony-forming units (CFU) may underestimate true microbial numbers. We observed that citric acid profoundly affected ATP measurement by luciferin-luciferase assay, and we assessed the effect of citric acid on ATP detection in water and buffer systems in the pH range 4.9 to 7.4. Bioluminescence depended on the buffer system, increasing as pH increased in each buffer system and decreasing as the buffer system changed from Na-phosphate to citrate-phosphate. Citrate-phosphate buffer decreased bioluminescence by 82% relative to water at the same pH. Consequences of poor buffer selection are potentially reduced luciferase activity in the bioassay system and underestimation of microbial numbers.