Phosphate-Catalyzed Hydrogen Peroxide Formation from Agar, Gellan, and κ-Carrageenan and Recovery of Microbial Cultivability via Catalase and Pyruvate

ABSTRACTPreviously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H2O2) is formed in the resulting medium (PT medium), and the colony count on the medium inoculated with environmental samples becomes much lower than that on a medium in which agar and phosphate are autoclaved separately (PS medium) (T. Tanaka et al., Appl Environ Microbiol 80:7659–7666, 2014,https://doi.org/10.1128/AEM.02741-14). However, the physicochemical mechanisms underlying this observation remain largely unknown. Here, we determined the factors affecting H2O2formation in agar. The H2O2formation was pH dependent: H2O2was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H2O2formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and κ-carrageenan) also produced H2O2after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H2O2scavengers, are effective in yielding high colony counts. The colony count on PT medium was only 5.7% of that on PS medium. Catalase treatment effectively restored the colony count of PT medium (to 106% of that on PS medium). In contrast, pyruvate was not as effective as catalase: the colony count on sodium pyruvate-supplemented PT medium was 58% of that on PS medium. Given that both catalase and pyruvate can remove H2O2from PT medium, these observations indicate that although H2O2is the main cause of reduced colony count on PT medium, other unknown growth-inhibiting substances that cannot be removed by pyruvate (but can be by catalase) may also be involved.IMPORTANCEThe majority of bacteria in natural environments are recalcitrant to laboratory culture techniques. Previously, we demonstrated that one reason for this is the formation of high H2O2levels in media prepared by autoclaving agar and phosphate buffer together (PT medium). In this study, we investigated the factors affecting H2O2formation from agar. H2O2formation is pH dependent, and ammonium ions promote this phosphate-catalyzed H2O2formation. Amendment of catalase or pyruvate, a well-known H2O2-scavenging agent, effectively eliminated H2O2. Yet results suggest that growth-inhibiting factor(s) that cannot be eliminated by pyruvate (but can be by catalase) are present in PT medium.