Comparison of soil microbiome by single cell technology, classical microscope methods and high-throughput MiSeq sequencing

Abstract:

[Objective] Characterize classical microscope counting methods, single cell technology and modern molecular methods in determining abundance and composition of total soil microbiome and aerobic methanotrophs in the process of methane oxidation. [Methods] Based on the extracted microbial cells and their DNA (Cell DNA), Single cell DNA, and soil microbiome DNA (Soil DNA), the conventional microscope counting methods and real time quantitive PCR (qPCR) were used to assess the abundance changes of soil microbiome and major aerobic methanotroph groups in aerobic methane oxidation. MiSeq sequencing of 16S rRNA and pmoA genes was used to compare microbial composition changes of soil microbiome and aerobic methanotrophs. [Results] Total microbial abundance in the studied paddy soil obtained by qPCR could be up to 3 orders of magnitude higher than that obtain by microscope counting methods. DAPI staining, CARD-FISH, qPCR of 16S rRNA gene in Cell DNA and Soil DNA showed total microbial abundance (cells/g) were (5.8-7.4)×10⁷, (1.7-1.9)×10⁷, (2.8-6.3)×10⁸, and (1.5-2.7)×10¹⁰, respectively. For methanotrophs, the result of qPCR of Soil DNA is 1.1×10⁷ cells/g which is 3 orders of magnitude higher than that obtained by microscope counting methods. However, all different methods showed that methanotroph abundance increased by 54-fold (CARD-FISH), 388-fold (qPCR of Soil DNA) and 40-fold (qPCR of Cell DNA), respectively, after the soil consumed high-concentration methane. Soil DNA (30 phyla) and Cell DNA (25 phyla) showed similar composition at phylum level by MiSeq sequencing analysis of 16S rRNA genes. More than 95% of the sequences derived from single cell DNA were affiliated to Proteobacteria, though 20 phyla were detected. Furthermore, a similar tendency was found by MiSeq sequencing analysis of pmoA genes in Soil DNA, Cell DNA and Single cell DNA. That is Methylobacter/Methylosarcina of γ-Proteobacteria becoming domaniant genera after high-concentration methane oxidation though, 7 pmoA genotypes existed in the original soil. [Conclusion] Total microbial abundance in the studied paddy soil obtained by microscope counting methods could be 1-3 orders of magnitude lower than that obtained by qPCR. qPCR of 16S rRNA gene in Soil DNA and Cell DNA revealed that the extracted microbial cells constituted only 2% of the total microbial cells in the studied paddy soil. Howerer, the extraction efficiency of methanotrophic cells could be up to 6%. MiSeq sequencing analysis indicated that Cell DNA could reflect the microbiome composition of the paddy soil at phylum level but not genus level as compared to that in Soil DNA, while the single cell sorting technique tended to select for Proteobacteria. These results showed that the resolution of traditional methods and molecular techniques are obviously different, but both methods can well characterize the microbial physiological and ecological processes of methane oxidation in paddy soil. The future study of soil microbiome should pay more attention to the inherent needs of the scientific issues, and to maximize the advantages of different technologies.