Enrichment culture and identification of endophytic methanotrophs isolated from peatland plants

Zofia Stępniewska,Magdalena Małysza,Weronika Goraj,Natalia Łopacka,Agnieszka Kuźniar

doi:10.1007/s12223-017-0508-9

Zofia Stępniewska, Magdalena Małysza + Show 3 more

Open Access

https://doi.org/10.1007/s12223-017-0508-9

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Abstract

Aerobic methane-oxidizing bacteria (MOB) are an environmentally significant group of microorganisms due to their role in the global carbon cycle. Research conducted over the past few decades has increased the interest in discovering novel genera of methane-degrading bacteria, which efficiently utilize methane and decrease the global warming effect. Moreover, methanotrophs have more promising applications in environmental bioengineering, biotechnology, and pharmacy. The investigations were undertaken to recognize the variety of endophytic methanotrophic bacteria associated with Carex nigra, Vaccinium oxycoccus, and Eriophorum vaginatum originating from Moszne peatland (East Poland). Methanotrophic bacteria were isolated from plants by adding sterile fragments of different parts of plants (roots and stems) to agar mineral medium (nitrate mineral salts (NMS)) and incubated at different methane values (1–20% CH4). Single colonies were streaked on new NMS agar media and, after incubation, transferred to liquid NMS medium. Bacterial growth dynamics in the culture solution was studied by optical density—OD600 and methane consumption. Changes in the methane concentration during incubation were controlled by the gas chromatography technique. Characterization of methanotrophs was made by fluorescence in situ hybridization (FISH) with Mg705 and Mg84 for type I methanotrophs and Ma450 for type II methanotrophs. Identification of endophytes was performed after 16S ribosomal RNA (rRNA) and mmoX gene amplification. Our study confirmed the presence of both types of methanotrophic bacteria (types I and II) with the predominance of type I methanotrophs. Among cultivable methanotrophs, there were different strains of the genus Methylomonas and Methylosinus. Furthermore, we determined the potential of the examined bacteria for methane oxidation, which ranged from 0.463 ± 0.067 to 5.928 ± 0.169 μmol/L CH4/mL/day.

Highlights

Cultivation of methanotrophs was started in 1906 by Söhngen, who isolated Bacillus methanicus (Söhngen 1906)
Wetlands are the main natural source of methane. This emission is the result of the balance between methanogenesis and methanotrophic processes and is actively affected by the composition of wetland plants, which can influence CH4 production, consumption, and transport in the soil. Studies on this phenomenon indicated a significant role of methanotrophic bacteria in CH4 emissions, both the free-living microorganisms in the rhizosphere attached to the root surface in the form of a biofilm and those living inside host tissues and colonizing parts of plants (Raghoebarsing et al 2005; Liebner et al 2011; Stępniewska et al 2013; Stępniewska and Kuźniar 2013; Putkinen et al 2014)
Cultivations of all methane-oxidizing bacteria (MOB) consortia were characterized by the dynamics of the gaseous phase

Summary

Introduction

Cultivation of methanotrophs was started in 1906 by Söhngen, who isolated Bacillus methanicus ( known as Methylomonas methanica) (Söhngen 1906). The crucial studies on cultivation of methanotrophs were performed by Whittenbury et al (1970), who isolated more than 100 strains of these bacteria from various environments. Methanotrophic bacteria were isolated from plants C. nigra (C), Vaccinium oxycoccus (V), and E. vaginatum (E) by adding sterile fragments of each part of plant: roots (R) and stems (S) to agar mineral medium (NMS) (Whittenbury et al 1970) and incubated in methane atmosphere 10% (v/v) CH4.

Results

Conclusion