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.

The Polar Fox Lagoon in Siberia harbours a community of Bathyarchaeota possessing the potential for peptide fermentation and acetogenesis

“…Definitive answers to these questions can only be obtained through physiological experiments and/or integrated multi-(meta)omics approaches, both of which require additional sampling campaigns. Future experiments can take advantage of recent progress in the enrichment of Bathyarchaeota reported by Hu et al ( 2021 ). Additionally, it will be of great interest to follow the community composition in the Polar Fox Lagoon sediments as it transitions into being fully connected to Tiksi bay, with the accompanying changes in salinity and water chemistry, and to unravel the influence of changes in environmental conditions on the different Bathyarchaeotal subgroups.…”

“…1). Recently, enrichment cultures using these compounds as substrates have supported the genome-based results from metagenomic studies (Hu et al 2021;Yu et al 2018). We mined the PFL Bathyarchaeotal MAGs for carbohydrate-active enzymes (CAZymes) using the dbCAN2 server (Yin et al 2012;Zhang et al 2018), including annotation of putative CAZyme gene clusters with CGC-finder (CAZyme Gene Cluster).…”

“…These archaea are implicated in the global carbon cycle and may have a large impact on carbon dioxide and methane emissions into the atmosphere. Unfortunately, no pure cultures of Bathyarchaeota currently exist so that we have an incomplete understanding of their metabolic potential, however, very recently, new enrichment strategies have been explored, resulting in several co-cultures of a Bathyarchaeon with a bacterial partner (Hu et al 2021 ). Several previous studies have set out to create a classification of Bathyarchaeal lineages (Fillol et al 2016 ; Kubo et al 2012 ), which were unified into a set of 25 distinct lineages in a review by Zhou et al ( 2018 ).…”

Marine microbial bioprospecting: Exploitation of marine biodiversity towards biotechnological applications—a review

“…Mainly, two strategies have been developed and used for the marine microbial bioprospecting such as (i) nutrient enrichment of the sample prior to culture then isolation, and (ii) isolation of single‐cell followed by nutrient enrichment and culture. Generally, as summarised in Figure 2, bioprospecting starts with collecting the environmental sample, then enriching the sample with nutrients to enhance the growth of microbes, and finally comes the step of isolating the cultured microorganisms [62]. Such simple methodologies provide qualitative and quantitative data of high sensitivity and reliability on the microbial species [63].…”

Draft Genome Sequences of Four Bacterial Strains Isolated from Sediment of the South China Sea

“…Marine ecosystems are a vast habitat for diverse metabolically active groups of microbes, including sulfur-oxidizing and polysaccharide-degrading bacteria ( 1 , 2 ). In the context of screening and isolating novel and uncultured metabolically active bacterial strains from marine settings in our laboratory ( 3 – 5 ), four strains have been isolated from sediment samples of the South China Sea (global positioning system [GPS] coordinates, 115.830°E, 18.813°N). The sediment cores were collected using a multicorer, sliced into 2-cm pieces onboard as soon as possible, placed into Nasco sampling bags, and preserved at 4°C.…”