10:45 〜 12:15
[BBC02-P04] Cultivation and isolation of methane-oxidizing bacteria living inside tree stems
キーワード：メタン、メタン酸化細菌、森林、樹幹
Forest ecosystems have been generally recognized as a sink of atmospheric methane, but recent studies have shown that some trees emit significant amounts of methane through the stems. Our previous study also showed that tree stems of several native species in Japan emitted methane and methanogenic archaea inhabited inside the tree stems. However, the methane fluxes were not always correlated to the abundance of methanogenic archaea in the trees. These gaps may be attributed in part to the consumption of methane by methane-oxidizing bacteria (MOB) before releasing from the tree stems. Some molecular-based studies showed the presence of MOB on the bark of trees, but the cultivation and isolation of MOB have not been reported yet from trees, and no study has focused on MOB inside tree stemwood. In the present study, we aimed to cultivate and isolate MOB living inside tree stems and analyzed their phylogenetic, physiological, and genetic characteristics to show the inhabitance of MOB inside tree stems and reveal their features.
To cultivate and isolate MOB, eleven heartwood and nine sapwood samples were collected from seven tree species, Acer pictum (Itayakaede), Aesculus turbinate (Tochinoki), Cryptopmeria japonica (Sugi), Fraxinus mandchurica (Yachidamo), Magnolia obovate (Hoonoki), Populus tremula var. sieboldii (Yamanarashi), and Quercus crispula (Mizunara) in the Ashiu Experimental Forest of Kyoto University in July 2020 and September 2021. These samples were pulverized and incubated in a nitrate liquid mineral-salt (NMS) medium supplemented with about 12% (v/v) methane as the sole energy source to enrich MOB. A portion (1%[v/v]) of the cultures that showed methane consumption activities were transferred to a fresh medium to enrich MOB further. After several trials of the enrichment, the cultures were inoculated on an NMS plate medium with gellan gum, followed by subculturing of colonies formed on the plate to a new liquid medium. These steps were repeated until a pure culture of MOB was obtained. The physiological and phylogenetic characteristics of the isolated strain were analyzed.
Methane concentrations in the first cultures inoculated with a sapwood sample of A. pictum and a heartwood sample of C. japonica reduced to almost zero in the 1 and 4 months of incubation, respectively, indicating the growth of MOB in the cultures. The active growth of MOB was observed within a few days in the second and subsequent subcultures, ensuring that the MOB were successfully enriched in the cultures. After several trials of isolation, one strain, is20ea, has been finally isolated from the culture derived from a sapwood sample of A. pictum. Strain is20ea actively grew with methane within about two days. Cells of strain is20ea were motile rods with a single polar flagellum. Phylogenetic analysis of 16S rRNA genes showed that the strain is20ea was closely related to Methylosinus sporium 5T in Alphaproteobacteria with 99.7% identity. The comparison of whole genome sequences among the related species suggested that the strain is20ea represents a novel species in the genus Methylosinus.
In the present study, we cultivated and isolated for the first time a novel methane-oxidizing bacterium inside a tree. This finding suggests a possibility that MOB living in stemwood consume methane produced in tree stems.
To cultivate and isolate MOB, eleven heartwood and nine sapwood samples were collected from seven tree species, Acer pictum (Itayakaede), Aesculus turbinate (Tochinoki), Cryptopmeria japonica (Sugi), Fraxinus mandchurica (Yachidamo), Magnolia obovate (Hoonoki), Populus tremula var. sieboldii (Yamanarashi), and Quercus crispula (Mizunara) in the Ashiu Experimental Forest of Kyoto University in July 2020 and September 2021. These samples were pulverized and incubated in a nitrate liquid mineral-salt (NMS) medium supplemented with about 12% (v/v) methane as the sole energy source to enrich MOB. A portion (1%[v/v]) of the cultures that showed methane consumption activities were transferred to a fresh medium to enrich MOB further. After several trials of the enrichment, the cultures were inoculated on an NMS plate medium with gellan gum, followed by subculturing of colonies formed on the plate to a new liquid medium. These steps were repeated until a pure culture of MOB was obtained. The physiological and phylogenetic characteristics of the isolated strain were analyzed.
Methane concentrations in the first cultures inoculated with a sapwood sample of A. pictum and a heartwood sample of C. japonica reduced to almost zero in the 1 and 4 months of incubation, respectively, indicating the growth of MOB in the cultures. The active growth of MOB was observed within a few days in the second and subsequent subcultures, ensuring that the MOB were successfully enriched in the cultures. After several trials of isolation, one strain, is20ea, has been finally isolated from the culture derived from a sapwood sample of A. pictum. Strain is20ea actively grew with methane within about two days. Cells of strain is20ea were motile rods with a single polar flagellum. Phylogenetic analysis of 16S rRNA genes showed that the strain is20ea was closely related to Methylosinus sporium 5T in Alphaproteobacteria with 99.7% identity. The comparison of whole genome sequences among the related species suggested that the strain is20ea represents a novel species in the genus Methylosinus.
In the present study, we cultivated and isolated for the first time a novel methane-oxidizing bacterium inside a tree. This finding suggests a possibility that MOB living in stemwood consume methane produced in tree stems.