The Diversity And Abundance Of PAHs Degradation Genes And Bacterial Community In Contaminated Soil And Rice

Polycyclic aromatic hydrocarbons(PAHs)are a type of persistent toxic organic pollutants of great concern globally.So far,microbial remediation is considered to be the most economical and effective method to control this category of pollutants.The existence of PAH-degrading genes is the genetic basis for microorganisms in metabolizing PAHs as carbon and energy sources.The quantitative analysis of PAH-degrading genes in the environment can assess the degree of PAH pollution,to a certain extent.Therefore,the diversity and abundance of PAH-degrading genes in both the microbial communities and in the environment have been explored to reveal the relationship between PAH-degrading genes,microbial communities,and pollutant PAHs.These investigations have vital theoretical significance in utilizing microorganisms to control PAHs pollution.Many scholars have studied the abundance of the PAH-degrading genes and the microbial community diversity in soil,sediment and seawater to elucidate for their distribution characteristics in the environment.However,the distribution characteristics of PAH-degrading genes and microbial communities in polluted plants,and how they respond to PAH-pollution stress have been rarely studied.In this dissertation,rice and its rhizosphere soil were collected from long-term PAHcontaminated farmland.The PAH contents,copy numbers of PAH-degrading genes,and the diversity of bacterial communities using high-throughput sequencing technologies were analyzed to explore the relationship between PAH content,PAH-degrading genes and bacterial communities in rice endospheric and rhizospheric soil.Based on the experimental results,a pot experiment was designed to study for the response of PAH-degrading genes and indigenous bacterial communities in soil and rice grains to PAH pollution stress and inoculation of exogenous PAH-degrading bacteria.The main research results are as follows:(1)The distribution characteristics of PAHs and its degradation genes in farmland soil and rice were revealed.The PAHs and PAH-degradation genes in rice grain mainly come from the soil,but there are significant differences between them in quantity with soil.The total content of 16 PAHs ranged from 514.27-2199.31 μg/kg in farmland soil and 80.56-195.97 μg/kg in rice grain.There PAHs were in the soil and the rice grain;however,the composition has distinct differences with them.The LMW-PAHs(low molecular weight PAHs,i.e.,2 and 3-rings)accounted for about 77.44%average in the soil,while in rice grains was 43.56%on average.It proved that PAHs in the rice grain was not absorbed from the soil entirely by the rice the root system.Similarly,phe,nahAc,phnAc,and nidA genes were detected in the soil and rice grain,with the phe gene having the highest copy number in the soil,with a copy number of 2.24E+08 copies/g·dws on average,while the copy number of phe gene on average was also the highest,with an average copy number of 3.59E+05 copies/g·dws.In the soil environment,there were significant correlations among PAHs content,PAH-degrading genes,and soil physicochemical properties.For example,HMW-PAHs(high molecular weight PAHs,i.e.,4 to 6-rings)in the soil had an extremely significant negative correlation with C/N in the soil,while C/N ratio(carbon to nitrogen ratio)had a significant positive correlation with nahAc genes in the soil.Meanwhile,the nahAc gene had a significant positive correlation with HMW-PAHs as FLA,PYR,BaA,CHR,BbF,BkF,BaP,DiahA,BghiP,and BPE.(2)Clarified the diversity and abundance of bacterial communities in farmland soil and rice grain with PAHs contaminated.The bacterial community diversity in rice grains was lower than that in soil samples,and the dispersion degee between samples was higher than that in soil samples,but the relative and absolute contents of PAHs degradation bacterial in rice grains were much higher than that in soil samples.The bacterial in PAH-contaminated farmland soil and rice grain were analyzed by sequencing,with results showing that there were 1118 OTUs(operational taxonomic units)in the soil and rice grain samples.Meanwhile,they accounted for 89.66%of the total OTUs in rice grain,and 14.4%of total OTUs in soil,which proved that the microfloral abundance in rice grain was much lower than that of the soil,with most of the microflora in the rice grain originated from the soil.Both alpha diversity and PCoA(principal coordinate analysis)analyses showed that the diversity of soil bacterial community was much greater than in the rice grain,and the dispersion degree between soil samples was less than that of the rice grain,while the species uniformity was much higher than that of rice grain.For example,the observed species index of the soil sample is 2559.9 on average,which is higher than the observed species index of the rice grain with 120.69 on average.The results of PCoA analysis showed that there were significant differences and significant relationship between soil samples and rice grain samples at the same time.PAH-degrading bacteria were detected in both soil and rice grain samples,but the total relative content was much higher in the rice grains than that of soil.The total relative content of PAHs degrading bacteria in soil was 1.07%,and that was 16.61%in rice grain.The absolute content of Pseudomonas sp.(a PAH-degrading bacterium)in rice grain was about 600 times than that in soil.(3)The analyzed effects of inoculating the functional degrading bacteria on PAH bioremediation and the abundance of PAH-degrading genes in soil and rice grain.The high PAH concentration inhibited the growth and metabolism of bacterial communities in the soil and rice,which promoted the enrichment of nidA gene.The inoculation of PAH-degrading bacteria could promote PAH bioremediation in both soil and rice grain,change the bacterial community structure and increase the proportion of PAHdegrading microbes.PAH concentrations can affect the bacterial community abundance in soil.For example,the 16S rRNA gene copy number was 6.62E+08 copies/g·dws in treatment of high PAH concentration,while the 16S rRNA gene copy number in treatment of low PAH concentration was 1.30E+09 copies/g·dws in the mature stage of rice.The high PAH concentration had a certain stress on genes which had a "survival of the fittest" screening effect on either nidA genes or bacterial containing the nidA genes.After inoculating PRd5,RS1 and RS2 suspensions,the PAH degradation rate in soil and rice grains increased at an average of 14.21%and 15.35%,respectively.Meanwhile,the average copies of 16S rRNA genes increased by 2.74%,and copies of nidA genes increased by 78.07%in the soil,while that of the rice grains increased by 183.45%and 394.56%.The increase in copy numbers of nidA genes was much greater than that of 16S rRNA genes,indicating that the proportion of bacterial having the nidA genes in the environment increased.