Towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods

The archaea that can be readily cultivated in the laboratory are only a small fraction of the total diversity that exists in nature.Although molecular ecology methods,such as metagenomic sequencing,can provide valuable information independent of cell cultivation,it is only through cultivation-based experiments that they may be fully characterized,both for their physiological and ecological properties.Here,we report our efforts towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods.Initially,cells were retrieved from the sediment samples through a cell extraction procedure and the sediment-free mixed cells were then divided into different size-range fractions by successive filtration through 0.8µm,0.6µm and 0.2µm membranes.Archaeal 16S rRNA gene analyses indicated noticeable retention of different archaeal groups in different fractions.For each fraction,supplementation with a variety of defined substrates(e.g.,methane,sulfate,and lignin)and stepwise dilutions led to highly active enrichment cultures of several archaeal groups with Bathyarchaeota most prominently enriched.Finally,using a roll-bottle technique,three co-cultures consisting of Bathyarchaeota(subgroup-8)and a bacterial species affiliated with either Pseudomonas or Glutamicibacter were obtained.Our results demonstrate that a combination of cell extraction,size fractionation,and roll-bottle isolation methods could be a useful protocol for the successful enrichment and isolation of numerous slow-growing archaeal groups from marine sediments.

Elevated and atmospheric-level methane consumption by soil methanotrophs of three grasslands in China

Background:Methane(CH4)oxidation driven by soil aerobic methanotrophs demonstrates the capacity of grassland as a CH4 sink.Methods:In this study,we compared the oxidation characteristics of atmospheric-level and elevated concentration(10%)CH4 in a typical grassland(steppe)on the Loess Plateau,an alpine meadow(meadow)on the Qinghai-Tibet Plateau,and an inland arid-area artificial grassland(pasture)in northwest China and investigated the communities of active methanotrophs and their contribution to CH4 oxidation using DNA-based stable-isotope probing and Illumina Miseq sequencing.Results:The results showed that the oxidation of atmospheric CH4 only occurred in steppe and meadow soils where the USCγgroup of methanotrophs was numerically dominant in the methanotroph community.Pasture soils,with their very low relative abundance of USCγ,did not show atmospheric CH4 oxidation.However,a DNA-stable isotope probing experiment with 10%CH4 indicated that conventional CH4 oxidizers(Methylocaldum and Methylocystis)rather than USCγcommunities assimilated significant amounts of 13CH4 for growth.Conclusions:The CH4 oxidation mechanisms in the three experimental grassland soils varied significantly.The USCγgroup may be obligate oligotrophic microorganisms or their growth requires specific unknown conditions.