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

Abstract

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 0.8 µm, 0.6 µm and 0.2 µm 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.