Investigation On Electron Acceptors And Co-metabolic Effects Of Typical Soil Pahs Removal Under Denitrification

Among the environmental pollutants that disturb ecosystems and threaten human health,polycyclic aromatic hydrocarbons（PAHs）have attracted much attention because of their strong toxicity,refractory to be degraded,and bioaccumulation along the food chain.With the characteristics of high stability and low water solubility,PAHs can be easily accumulated in some micro-aerobic or anaerobic environments such as sediments,water,and soil,so anaerobic metabolism driven by microbes has been considered to be essential for the elimination of PAHs in these habitats.Although the anaerobic PAHs metabolism of microorganisms can occur in various reducing conditions supported by different electronic acceptors,denitrification with nitrate as an electron acceptor is thought to be the optimal pathway due to its high efficiency,low risk,and ubiquity.However,there are still some problems to be solved for the actual application of denitrification in the remediation of PAHs-contaminated soils.Firstly,the dosage-effect of electron acceptors is unsure;secondly,the interaction between denitrification and other reducing conditions is unclear;finally,the actual contaminated sites usually contain multiple PAHs,but the co-metabolism characteristics of multiple PAHs under denitrification are still unknown.Therefore,in this study,using the typical low-molecular-weight PAH（LMW-PAH:phenanthrene,PHE）and high-molecular-weight PAH（HMW-PAH:benzopyrene,BEN）as objects,through a soil incubation experiment with a gradient addition of nitrate,the dosage-effect of nitrate on soil PAHs removal and denitrification process was investigated.Then,an incubation experiment amended with nitrate（electron acceptor for denitrification）or sulfate（electron acceptor for sulfate reducing）was conducted to further investigate the effect of sulfate on the removal of soil BEN under denitrification.Finally,the co-metabolic effect of the PHE and BEN under denitrification in soil was investigated,and the possible co-metabolic mechanism of those PAHs under denitrification in soil was explored by Illumina Mi Seq sequencing.The main results are as follows:（1）To investigate the relationships between the electron acceptor（nitrate,NO₃^-）dosage,anaerobic removal of PAHs,and denitrification process in soil,the following soil microcosm-incubation experiment were carried out.Two typical PAHs treatment groups were set up:PHE 20 mg·kg^-1（P treatment group）and BEN 20 mg·kg^-1（B treatment group）;Six NO₃^-concentrations:single batch addition of（N）0,5,10,20,40mg·kg^-1 and multiple batch addition（NI）25 mg·kg^-1(5 mg·kg^-1each at 0,7,14,21 and28 days of incubation).A total of 12 treatments(P treatment group:PN₀,PN₅,PN₁₀,PN₂₀,PN₄₀,PNI;B treatment group:BN₀,BN₅,BN₁₀,BN₂₀,BN₄₀,BNI)were incubated at 25°C in the dark for 42 days.During the incubation,the dynamic changes in the generation rates of soil N₂O and CO₂ were monitored,and the content of soil inorganic nitrogen（NO₃^-,NO₂^-and NH₄⁺）were determined,while the soil PAHs contents and abundances of denitrification-related genes（cd₁-nitrite reductase gene,nir S;cooper-containing nitrite reductase gene,nir K）were detected at the end of incubation.The results indicated that nitrate addition within the range of 0～40 mg·kg^-1 promoted the anaerobic removal rate of PHE and BEN,and a more efficient promotion was found for the LMW-PAH（PHE）（the highest increase was 88.46%）,showing a significantly positive relationship with the dosage of nitrate（R²=0.739,P<0.01）.The anaerobic removal of HMW-PAH（BEN）was significantly promoted when the nitrate addition was higher than 20 mg·kg^-1（the highest increase was 15.93%）.Although nitrate amendment significantly promoted the generation rates of N₂O and CO₂ in each PAHs-contaminated soil,a stronger promotion was detected in the HMW-PAH（BEN）treatments.In addition,the BEN treatments had higher abundances of nir S and nir K than the PHE treatment,indicating a positive relationship between soil N₂O generation rate and denitrification gene abundance.Compared with the single-batch addition,the treatments with multiple-batch addition of nitrate did not promote the PAHs anaerobic removal rate but had higher abundances of denitrifier genes and CO₂ generation rate at the middle and late stages during the incubation.（2）Based on the result of the previous part that discussed the dosage-effect of nitrate on the PAHs removal and denitrification process in soil,here,with BEN as the object,a soil incubation experiment with single electron acceptor（nitrate or sulfate）,mixed electron acceptors（nitrate and sulfate）,or sulfate reduction inhibitor was conducted to investigate the effect of sulfate-reducing process on the removal of soil BEN under denitrification and discover corresponding microbial mechanism.The experiment design was briefly described as follows.Four treatments including without electron acceptors（CK）,containing 20 mg·kg^-1 sulfate（S）,containing 40 mg·kg^-1nitrate（N）,and containing nitrate and sulfate（NS）were constructed,and then a corresponding comparative treatment by addition of sulfate reduction inhibitor（sodium molybdate,20 m M,denoted as M）was set up for each treatment above;Finally,a total of 8 treatments（CK,S,N,NS,and MCK,MS,MN,MNS）were conducted and were incubated at 25°C in the dark and anaerobic condition for 28 days.During this period,the dynamic changes in the generation rates of soil N₂O and CO₂ were monitored,and the contents of soil inorganic nitrogen（NO₃^-,NO₂^-and NH₄⁺）and sulfate(SO₄^2-)were determined.After the incubation,the soil BEN contents were detected and the bacterial community structure of each soil sample was measured by Illumina Mi Seq sequencing.The results indicated that compared with CK,both mono-nitrate（N）and mixed nitrate-sulfate treatment（NS）significantly promoted the removal of soil BEN,while there was no significant difference between mono-sulfate treatment（S）and CK.In addition,there was no significant difference in the removal rate of BEN between each treatment and its corresponding sulfate reduction inhibitor added treatment.The generation rates of soil N₂O under nitrate-containing treatments（N,MN and NS,MNS）were significantly higher than those without nitrate（CK,MCK and S,MS）,and the mixed electron acceptors treatment（NS,MNS）had the highest values.Furthermore,there was no significant difference in the generation rate of soil N₂O in each treatment compared with its corresponding sulfate reduction inhibitor addition treatment.The CO₂ generation rates of nitrate,sulfate and mixed addition treatment（N,MN,S,MS and NS,MNS）were significantly higher than those of CK and MCK treatment without electron acceptors,with an order of NS,MNS>N,MN>S,MS>CK,MCK.The addition of electron acceptors or sulfate reduction inhibitor had no significant effect on theα-diversity of soil bacteria,but the addition of sulfate reduction inhibitor significantly changed the community structure of soil bacteria.The above results indicated that mono-sulfate addition failed to promote the removal of soil BEN.Although the BEN removal rate of the mixed electron acceptors treatment（NS）was significantly higher than that of mono-nitrate treatment（N）,no significant difference was detected between the NS treatment and its corresponding sulfate reduction inhibitor treatment（MNS）.Thus,the sulfate-reduction process may not directly participate in the metabolism of soil BEN,but the presence of sulfate might facilitate the removal of soil BEN under denitrification.（3）Co-metabolism is an effective way to remove complex organic pollutants in the environment,but the co-metabolism of PAHs under soil denitrification is rarely studied.In this section,the HMW-BEN and LMW-PHE were used as the target pollutant and co-metabolic matrix,respectively,to investigate the co-metabolism effect of PAHs under soil denitrification condition.Here,two treatment groups with the low(20 mg·kg^-1,B_L)and high(50 mg·kg^-1,B_H)BEN concentration were set up,and then three sub-treatments with the PHE concentration of 0 mg·kg^-1（P₀）,5 mg·kg^-1（P₅）and 10 mg·kg^-1(P₁₀)were set up in each treatment group.Thus,a total of 6 treatments(B_L treatment group:B_LP₀,B_LP₅,B_LP₁₀;B_H treatment group:B_HP₀,B_HP₅,B_HP₁₀)were conducted and were incubated under denitrification condition(anaerobic,40 mg·kg^-1 NO₃^-)at 25°C in the dark for 28 days.During this period,the dynamic changes in the generation rates of soil N₂O and CO₂ were monitored,and the content of soil inorganic nitrogen（NO₃^-,NO₂^-,and NH₄⁺）were determined.After the incubation,the soil PAHs contents were detected and the bacterial community structure of each soil sample from the B_H group was measured by Illumina Mi Seq sequencing.The results indicated that there was no significant difference in the removal rate of soil BEN（target PAH）among the treatments(B_LP₀,B_LP₅,and B_LP₁₀)in the B_L（low BEN concentration）group,while the BEN removal rates of B_HP₁₀ and B_HP₅ were significantly higher than that of B_HP₀ in the B_H（high BEN concentration）group.In both treatment groups,the PHE（co-metabolic matrix）removal rates of the treatments with low concentration of PHE（B_LP₅ and B_HP₅）were significantly higher than those of treatments with high concentration PHE(B_LP₁₀and B_HP₁₀).There was no significant difference in the soil N₂O generation rate among all treatments during the incubation,whereas the co-metabolic treatments(B_LP₅,B_LP₁₀and B_HP₅,B_HP₁₀)of the two groups all showed higher CO₂ generation rates than those of the single PAH treatments（B_LP₀and B_HP₀）in the late stage of incubation.In addition,the results of Illumina Mi Seq sequencing indicated that the co-metabolism of PHE-BEN under soil denitrification significantly reduced the bacterialα-diversity and changed the bacterial community structure,and the soil bacterial community structure was significantly correlated with the removal rate of BEN（R²=0837,P=0.002）.In summary,co-metabolism of HMW and LMW PAHs can occur in soil under denitrification,and the promoting effect might be closely related to the dose ratio of HMW to LMW PAHs,which should be explained by the shift of soil bacterial community composition induced by the co-metabolic matrix（PHE）.