Affiliation:
1. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan
2. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
Abstract
ABSTRACT
Previously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H
2
O
2
) 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 H
2
O
2
formation in agar. The H
2
O
2
formation was pH dependent: H
2
O
2
was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H
2
O
2
formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and κ-carrageenan) also produced H
2
O
2
after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H
2
O
2
scavengers, 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 H
2
O
2
from PT medium, these observations indicate that although H
2
O
2
is 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.
IMPORTANCE
The 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 H
2
O
2
levels in media prepared by autoclaving agar and phosphate buffer together (PT medium). In this study, we investigated the factors affecting H
2
O
2
formation from agar. H
2
O
2
formation is pH dependent, and ammonium ions promote this phosphate-catalyzed H
2
O
2
formation. Amendment of catalase or pyruvate, a well-known H
2
O
2
-scavenging agent, effectively eliminated H
2
O
2
. Yet results suggest that growth-inhibiting factor(s) that cannot be eliminated by pyruvate (but can be by catalase) are present in PT medium.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
39 articles.
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