engleski

Difficulties in enrichment and isolation of methane utilizing bacteria

The main objective was the isolation of methane utilizing bacteria from different environments (meadow soil, landfill leachate, eutrophicated sealake sediment and marine coastal sediments) in order to elucidate the importance of these bacteria in global methane cycling and pollutants transformation. Culture enrichment started by the injection of methane into original samples. In the case of soil sample, suspension in nitrate mineral salts medium was prepared. When the subsequent turbidity was observed (optical density determined at 600 nm), the enrichments were subcultured in sealed 120-ml serum bottles containing nitrate mineral salts (NMS) medium and ammonium nitrate mineral salts (ANMS) medium (1). For marine samples, these media were supplemented with 2.5 percent (w/v) sodium chloride. All enrichments and subcultures were incubated under the methane/air atmosphere either statically or by shaking at 30^oC. Although there were differences in growth rate between the enrichments originating from different environments, further subculturing suggested that methane utilizing bacteria were present in all the samples. The enrichments from marine environment showed better growth in ANMS medium while those originating from soil, landfill leachate and eutrophicated sealake revealed better and more stable growth characteristics when NMS medium was used. This suggested the presence of different methane utilizing bacteria in each enrichment. The study of the community structure of serially diluted enrichments grown on ANMS and NMS agarose plates under the methane/air atmosphere as well as on Nutrient agar and Marine agar showed that the enriched cultures consisted of six to eight different types of heterotrophs and one to two types of methanotrophs. Most of the heterotrophs grew well on ANMS and NMS agarose plates, forming colonies of tightly bound aggregates with the methane utilizing bacteria. Even when single isolated colonies appeared on these plates, we were so far unable to isolate methane utilizing bacteria in pure culture in most enrichments. This suggested that, based on the specific and mutually dependent relationships, methane utilizing bacteria grew tightly coupled with heterotrophs, which caused main difficulties in their isolation. The obtained results confirmed that the fraction of methane utilizing bacteria recovered from environmental samples is a small fraction of those present, and that there are difficulties in their cultivation, isolation and identification, especially from marine environment. (1) Whittenbury, T. W., K. C.Phillips, and J. G. Wilkinson. 1970. J. Gen. Microbiol. 61, 205-218

methane utilizing bacteria; isolation

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