Abstract
Microbial ecological studies have been remarkably promoted by the high-throughput sequencing approach with explosive information of taxonomy and relative abundance. However, relative abundance does not reflect the quantity of the microbial community and the inter-sample differences among taxa. In this study, we refined and applied an integrated high-throughput absolute abundance quantification (iHAAQ) method to better characterize soil quantitative bacterial community through combining the relative abundance (by high-throughput sequencing) and total bacterial quantities (by quantitative PCR). The proposed iHAAQ method was validated by an internal reference strain EDL933 and a laboratory strain WG5. Application of the iHAAQ method to a soil phenanthrene biodegradation study showed that for some bacterial taxa, the changes of relative and absolute abundances were coincident, while for others the changes were opposite. With the addition of a microbial activity inhibitor (NaN3), the absolute abundances of soil bacterial taxa, including several dominant genera of Bacillus, Flavobacterium, and Paenibacillus, decreased significantly, but their relative abundances increased after 28 days of incubation. We conclude that the iHAAQ method can offer more comprehensive information to reflect the dynamics of soil bacterial community with both relative and absolute abundances than the relative abundance from high-throughput sequencing alone.
Highlights
Bacteria are the essential components of soil ecosystem and play a vital role in the material cycle and energy flow in the Earth ecosystem (Whitman, Coleman & Wiebe, 1998)
The total bacterial quantities in Treatments from E9 to E4 ranged from 7.00 × 1010 to 3.53 × 109 and from 7.23 × 1010 to 5.82 × 109 copies (g dry wt soil)−1 for V3 and V4 regions, respectively
A standard curve of the FliC gene (fliC) gene in the range of 3.60×101 to 3.60 ×109 copies μL−1 was constructed for quantitative PCR (qPCR)
Summary
Bacteria are the essential components of soil ecosystem and play a vital role in the material cycle and energy flow in the Earth ecosystem (Whitman, Coleman & Wiebe, 1998). It was estimated that there are 4–6×1030 cells of prokaryotes on Earth and 2.6×1030 in soil, which is the most diverse and abundant cellular life form on Earth (Whitman, Coleman & Wiebe, 1998; Curtis & Sloan, 2005). Many other methods have been applied to explore the absolute or relative abundances of soil bacterial community. Brookes et al (1985) and Vance, Brookes & Jenkinson (1987) tried to estimate the total microbial abundance by measuring the microbial biomass-N (MBN) and -C (MBC) in soil. With the development of molecular biology and the more verified specific target genes, the quantitative PCR (qPCR) becomes a powerful and accurate technique to measure the total or specific microbial absolute abundance in soil (Philippot et al, 2009)
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