The High-throughput Absolute Abundance Quantification Of Soil Microbial Community And Its Applications

Microorganisms are the major players for the soil nutrient cycling,and we have much to learn about their abundant diversity of their species,genes and living environments.High-throughput sequencing is a widely used method to characterize soil microorganisms and it has become a powerful research tool that dramatically broadened our horizon of the microbial community.However,due to the limit of this technique,it can only be used to compare the species in the samples by relative abundance.The method can further bias the analysis and lead to opposite conclusions when there are large fluctuances for the total microbial absolute abundances.To make up for the deficiency of relative abundance in high-throughput sequencing analysis for microbial community,we developed two new methods,the high-throughput absolute abundance quantification(HAAQ)method,of individual soil bacteria and soil microbiota across the three domains in this study.The new methods allow to more objective comparisons between different samples and kingdoms,as well as more comprehensively describe soil microbial communities and understand their correlations.The main results were as below:(1)We constructed an internal standard strain(ISS)for the bacterial community from the Escherichia coli 0157:H7 strain.The internal standard strain was green fluorescent protein(GFP)marked,ampicillin resistant,and had a specific gene.It can be absolutely quantified by the methods of plate count,microscope count and quantitative PCR before and after its addition to the soil.Our results showed that a soil bacterial community and its dynamics can be better characterized by the HAAQ method when the ISS was quantified before the addition with optimal adding concentration of 105-107 cells g-1 and a 16S rRNA gene copy number adjustment was performed.In the test soils,the absolute abundance of the total bacteria was 2.10×109 ± 8.65×108 cells g-1.The soil bacterial absolute abundances at the genus level fitted well to the partial log-normal distribution function,and most genera concentrations were in the range of 103 5 to 106 5 cells g-1 in the test soils.The bacterial genera with absolute abundance below 103 cells g-1 were difficult to detect.Based on the fitted curves,it was estimated that 4.11-31.69%genera were undetected in the test soils.(2)The high-throughput absolute abundance quantification method for the bacterial community was applied to the purple soil and its parent material,as well as soils with or without azide sodium treatment to characterize the bacterial communities and dynamics.Among the samples,approximately 33.87%and 40.58%of the total genera showed opposite results(increase or decrease)between the absolute and relative abundances.More comprehensive and accurate descriptions of soil bacterial communities and their dynamics can be achieved by using both the relative and absolute abundances than by the relative abundance alone.Comparing with the soil parent material,the bacterial absolute abundance in the purple soil increased.Some of the genera belonging to Acidobacteria and Proteobacteria phyla increased both in relative and absolute abundances,which indicated their better abilities for adaption and decomposing minerals.The sodium azide showed good bacteriostatic effects on soil bacteria.In the sodium azide treated samples,the total absolute abundance of the bacteria decreased,and none of the genera significantly increased in the absolute abundance.The relative abundances of Firmicutes and Actinobacteria phyla increased because they were more resistant to this bacteriostat,while other bacteria decreased substantially.(3)A DNA fragment that contains the archaeal and bacterial 16S rRNA genes,the fungal internal transcribed spacer(ITS)gene and a specific gene was designed and constructed.It was artificially synthesized and inserted to the genome of Escherichia coli Dh5a by homologous recombination with the Lambda Red recombinase system.The constructed universal ISS Escherichia coli WYL for microbial communities can be employed independently or simultaneously as an ISS for high-throughput absolute abundance quantification of the prokaryotic(archaea and bacteria)and eukaryotic(fungi)communities.For the first time,the high-throughput absolute abundance quantification method of soil microbiota across three domains was established and proved to be valid.(4)By applying the high-throughput absolute abundance quantification method of soil microbiota across the three domains to the polycyclic aromatic hydrocarbons(PAHs)contaminated soils,different responses of microbes in the three domains were revealed.With the microbial absolute abundance at any taxon level,the contribution of response in each domain to the total microbiota was calculated and correlations among three domains were further analyzed.The archaea had the lowest microbial diversity and was most sensitive to the environmental changes.Because of its low total absolute abundance(107 copies g-1 and occupying just 1%to the whole microbiota),archaea change made little impact on the whole microbial community of soil microbiota across three domains.Bacteria were the major players in the whole microbial community because of their rich diversity and high absolute abundance(>109 copies g-1,which made up for 57.03-92.94%to the whole microbiota).The total absolute abundance(108 copies g-1)and biodiversity of fungi were between those of archaea and bacteria.The frequency distribution of archaeal absolute abundance at the genus level was discontinuous in the range of 103 1 to 106.5 copies g-1.The absolute abundance of most bacterial and fungal genera ranged from 104-106.5 copies g-1 and 103 5-106 copies g-1,respectively.Moreover,the absolute abundance at the genus level of the bacteria and fungi,as well as the microbiota,fitted well to the partial log-normal distributions.During the long-term contamination of PAHs,microbes in the three domains tended to be more cooperative(6635 edges)than competitive(1364 edges).The microbial community was dominated by the bacteria-bacteria and bacteria-ffungi correlations,and microbes were mainly clustered by their function of biodegradation.This research broke the bottleneck of analysis across microbial kingdoms in the high-throughput sequencing results through the achievement of high-throughput absolute abundance quantification method for the archaeal-bacterial-fungal communities.The new method allows for more comprehensive analysis of soil microbial communities of soil microbiota across the three domains and can potentially be applied to the study of quantitative microbial ecology.