title: Recovery of soil unicellular eukaryotes: an efficiency and activity analysis on the single cell level.
abstract: Eukaryotic unicellular organisms are an important part of the soil microbial community, but they are often neglected in soil functional microbial diversity analysis, principally due to the absence of specific investigation methods in the special soil environment. In this study we used a method based on high-density centrifugation to specifically isolate intact algal and yeast cells, with the aim to analyze them with flow cytometry and sort them for further molecular analysis such as deep sequencing. Recovery efficiency was tested at low abundance levels that fit those in natural environments (10(4) to 10(6) cells per g soil). Five algae and five yeast morphospecies isolated from soil were used for the testing. Recovery efficiency was between 1.5 to 43.16% and 2 to 30.2%, respectively, and was dependent on soil type for three of the algae. Control treatments without soil showed that the majority of cells were lost due to the method itself (58% and 55.8% respectively). However, the cell extraction technique did not much compromise cell vitality because a fluorescein di-acetate assay indicated high viability percentages (73.3% and 97.2% of cells, respectively). The low abundant algae and yeast morphospecies recovered from soil were cytometrically analyzed and sorted. Following, their DNA was isolated and amplified using specific primers. The developed workflow enables isolation and enrichment of intact autotrophic and heterotrophic soil unicellular eukaryotes from natural environments for subsequent application of deep sequencing technologies.
authors.affiliation: Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany; UFZ - Helmholtz Center for Environmental Research, Department of Soil Ecology, Theodor-Lieser-Str. 4, 06120 Halle/Saale, Germany. Electronic address: guillaume.lentendu@ufz.de.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lentendu
authors.forename: Thomas
authors.initials: T
authors.lastname: Hübschmann
authors.forename: Susann
authors.initials: S
authors.lastname: Müller
authors.forename: Susanne
authors.initials: S
authors.lastname: Dunker
authors.forename: François
authors.initials: F
authors.lastname: Buscot
authors.forename: Christian
authors.initials: C
authors.lastname: Wilhelm
mesh_terms: D002470:Cell Survival; D002498:Centrifugation; D000460:Chlorophyta / Q000145:classification / Q000235:genetics; D005434:Flow Cytometry; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D010274:Parasitology / Q000379:methods*; D012987:Soil / Q000469:parasitology*; D058009:Stramenopiles / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D012987:Soil
keywords: Cell recovery; Cell sorting; Flow cytometry; Low abundant microorganisms; Soil unicellular eukaryotes