Effect Of Nitrogen On PAH Degradation And Degrading Genes In Soils And Vegetables

Polycyclic aromatic hydrocarbons（PAHs）are a class of persistent organic pollutants that are widely present in the environment.Currently,soils in China are severely contaminated with PAHs,which can be taken up by crops and then distributed throughout the food chain,which seriously threatens the safety of the ecosystem and human health.The research focus in recent years has shifted to how to effectively remove PAHs from the soil.Microbial remediation is a high-efficiency,low-cost,low-pollution remediation technology that is widely used in contaminated land remediation to reduce the risk of PAHs pollution in crops.Nitrogen is used in agriculture as an essential nutrient for plant growth;however,nitrogen is also an electron acceptor and nutrient,which can stimulate the growth of degrading bacteria and accelerate the rate of PAH degradation.The abundance of PAHdegrading genes is often related to the ability of microorganisms to degrade PAHs,with the soil microbial community structure and diversity being the key factors affecting the microbial remediation.Therefore,studying the structure and diversity of microbial communities and PAHs-degrading genes under the condition of nitrogen addition,revealing the relationship between nitrogen and PAHs-degrading genes,microbial community,and PAHs degradation,which can provide a vital theoretical basis for using microorganisms to reduce PAH contamination of soil and crops.However,the respond of PAH-degrading genes and microbial communities to nitrogen have been rarely studied.In this dissertation,long-term contaminated soils containing PAHs were collected,and nitrate and ammonium nitrogen were added in the soil to explore the effect of nitrogen on PAH degradation,PAH-degrading genes and the microbial community in complex PAHcontaminated soils.Based on the experimental results,nitrogen was added to soil-Chinese cabbage system to explore the effect of nitrogen on PAH degradation and PAH-degrading genes in both soil and Chinese cabbage.The main research results are as follows:1.Elucidating the response of PAH degradation and PAH-degrading genes abundance in soils to nitrogen.40,80,160mg·kg-1 of NO3-or NH4+ were added to PAH-contaminated soil,and the soil was cultured,and then the PAHs content and the abundance of degrading genes were determined during the culture.The results showed that NO3-and NH4+at different concentrations（40-160 mg·kg-1）could promote the degradation of PAHs in soil.The content of PAHs in the nitrogen-treated group decreased by 23.23%～34.21%at 32 days compared with the control group without nitrogen,and the content of PAHs in the 80 mg·kg-1 NH4+treated group was the least.qPCR results showed that the addition of NO3-（40-160 mg·kg-1）and NH4+（40,80 mg·kg-1）increased the number of bacteria in the soil.Similarly,the 16S rRNA gene abundance of the nitrogen-treated group was significantly increased by 24.29%～33.64%at 32 days compared with the control group without nitrogen.Also,the addition of nitrogen promoted the enrichment of PAHs-degrading genes,and the abundance of nidA,nahAc and phe genes increased by 250.9%,2549.7%and 134.4%,respectively,compared with the control group without nitrogen.The correlation analysis found that the nitrogen residues had a significant positive correlation with the PAHs residues,and the nitrogen residues and PAHs residues had a significant negative correlation with 16S rRNA,nidA and nahAc genes in soil.The above results prove that the addition of nitrogen promoted the reproduction of microorganisms carrying PAH-degrading genes,enhanced the biodegradation of PAHs in the soil,and thus reduced PAHs residue in the soil.2.Addition of different forms of nitrogen changed the diversity and structure of microbial community in PAH-contaminated soils.Illumina MiSeq platform was used for 16S high-throughput sequencing analysis of soil microbial community,and the diversity and structural composition of soil microbial community were studied.The Alpha diversity index showed that the Chao1 index and Shannon index of the microbial community in the soil were significantly higher than the control group without nitrogen on the 8th day,and the two indexes of the 40 mg·kg-1 NH4+treated group were the highest.The results of PCoA analysis showed that adding different concentrations of nitrogen can change the community structure of the treatment group,and the difference was larger at 32 days.These results proved that the addition of nitrogen increased the abundance and diversity of soil microbial communities in the early stage of cultivation,and also changed the structure of the microbial community.The species analysis found that the Proteobacteria that produce PAHs degrading bacteria account for more than 60%of the total soil microorganisms,and was the dominant phylum in the community.Ten species of PAHs degrading bacteria that have been confirmed to carry nidA and nahAc genes,including Sphingomonas,Sphingobium and Mycobacterium were detected in the soil.The addition of nitrogen（40,160 mg·kg-1 NO3-and 80,160 mg·kg-1 NH4+）promoted the growth of PAHs degrading bacteria in the soil,the relative abundance of the degrading bacteria on the 8th day was higher than that of the control group without nitrogen,and the relative abundance was highest in the 160 mg·kg-1 NO3-.treated groups.3.Revealed the effects of nitrogen on PAH degradation,gene abundance and distribution in soils and crops.50,100 and 200 mg·kg-1 NO3-or NH4+ were added to PAHs contaminated soil and Chinese cabbage（Brassica chinensis L.）were planted,and then the PAHs content and the abundance of degrading genes in soil and plants were determined during the culture.The results showed that the addition of NO3-and NH4+（50-200 mg·kg-1）could effectively reduce the content of PAHs in soil and Chinese cabbage,and the content decreased with the increase of the nitrogen concentration.The 200 mg·kg-1 treated-group had the least content of PAHs in soil and Chinese cabbage,and the PAHs content in the soil decreased by 21.39%,the Chinese cabbage shoot and root decreased by 36.53%and 25.79%after 60 days.The abundance of 16S rRNA gene showed that the number of bacteria in soil was greater than the number of plant endophytes.The addition of nitrogen（50-200 mg·kg-1）promoted the increase in the number of bacteria in the soil.The promoting effect of 200 mg·kg-1 NO3-was the largest,which increases by 29.29%compared with the control group without nitrogen,while nitrogen had no obvious promotion effect on the number of endophytes bacteria in Chinese cabbage.The relationship between the nidA,nahAc and phe gene abundance of different parts were as follows:soil>Chinese cabbage root>shoot.The addition of nitrogen increased the abundance of the three degrading genes in different parts,the average abundance of the three genes in the soil increased by 54.16%,35.16%,and 92.12%,the shoots and roots increased by 51.36%,29.08%,25.62%and 62.03%,41.41%,29.09%,respectively.The above results indicated that nitrogen addition could increase the proportion of microorganisms carrying PAH-degrading genes in both soil and plants,and promote the biodegradation of PAHs in soil and plants.