Enrichment Of Soil Iron-reducing Microbial Consortium And Its Effect In The PAHs Elimination Combined With Fenton Treatment

PAHs(polycyclic aromatic hydrocarbons)are a kind of aromatic hydrocarbons with two or more fused benzene rings,which are lipophilic,hydrophobic,and stable,and can remain for a long time in the soil,threatening humanity and environmental health.The Fenton reagent can effectively remove PAHs and other stable organic matters in the soil based on the principle of Fe(?)catalytic oxidation.It is one of the commonly used methods of PAHs elimination in the soil,but it is often restricted by the availability of PAHs,secondary pollution with iron,and destruction of soil ecology after applying.Iron is an abundant element in soil,and the Fe(?I)reduction process driven by iron-reducing microbes can be coupled with the metabolism of various organics such as PAHs or transformation of heavy metal ions valence.The iron-reducing process is likely to exert a crucial role in the anaerobic degradation of PAHs.The function of Fenton reagent and iron-reducing microbial consortia to the transformation for the iron valence is opposite,and then the combined application of these two is extremely probable to remove PAHs in the soil efficiently.At present,little research had reported the combined effect of them on the elimination of soil PAHs.Therefore,to enriching iron-reducing organism,and analyze the bacterial enrichment community structure,six soil samples with potential PAHs contamination were collected from Chongqing Fuling shale gas field,four and two of which were from the oil-based ash dump site(DC1,DC2,DC3,DC4)and the vicinity of abandoned gas wells(FS1,FS2),respectively.The basic properties and iron reducing potential(IRP)of the soil were measured to explore the relationship between the IRP and environmental factors.Thereafter,two representative PAHs(phenanthrene and pyrene)and microbial consortia with the highest IRP were chosen,and then the effect of the combined pattern of Fenton and iron-reducing microbes on the removal rate of PAHs was examined through an inner-room incubation experiment.The results of this study should provide a theoretical basis for the application of the chemical-biological joint pathway in the remediation of soil polluted with PAHs,and lay a foundation for the development of high efficiency and saving energy new strategies of the PAHs elimination in soil.The main findings are as follows:(1)The results of the iron-reducing bacterial community reveal that Firmicutes,Proteobacteria,and Bacteroidetes were the dominant bacterial phyla which the iron-reducing bacterial consortium enriched from the soil samples in the Fuling shale gas field.Higher relative abundance of some bacterial genera,such as Pseudomonas,norank Peptococcaceae,and Lentimicrobium is detected in the high IRP samples.The result of the principal coordinate analysis(PCo A)based on the OTU level indicated that there was a significant difference in the iron-reducing microbial community enriched using the low,medial,and high IRP samples(R²=0.25,p<0.01).Compared to microbial consortia in the low IRP samples,some OTUs belonging to the bacterial genera Acetoanaerobium,Proteiniphilum,Petrimonas,Tessaracoccus,and Exiguobacterium was significantly upregulated in the high IRP consortiums.Structural equation model derived from the IRP,soil properties,and microbial community characteristics demonstrated that iron-reducing bacterial community structure was the sole and key factor directly affecting soil IRP;nevertheless,soil ammonia and available phosphorus could indirectly affect soil IRP by their direct influence on soil microbial community structure.Moreover,soil ammonia also showed a sign of an indirect effect on soil IRP by its step-by-step impact on available phosphorus,microbial community,and IRP.In summary,the iron-reducing microbial consortia are more sensitive to environmental factors.The iron-reducing microbial community structure is a key factor that directly affects the IRP of the soil in this gas field.The bacterial that is significantly upregulated in high IRP samples play an important role in the ferrum reduction process.(2)Opting for a PAH(phenanthrene)with low molecular weight as the object,we explored the effect of the combined pattern of Fenton and enriched iron-reducing consortium(obtained from part 1)on the soil phenanthrene elimination.Based on phenanthrene contaminated soil,3 groups of Fenton treatments were set up,which were without Fenton(F_N),Fenton treatment(pH 3)without adjustment(F_Y3),and with adjustment of pH to 7(F_Y7).In each group,3 sub-treatments of aerobic non-inoculation(O_YM_N),anaerobic non-inoculation(O_NM_N),and anaerobic inoculation iron-reducing microbial consortium(O_NM_Y)was set.After 28 days of dark incubation at 30±1?,the soil phenanthrene content was measured for the calculation of PHErr(Phenanthrene removal rate,PHErr).The results showed that in the soil containing 144.087 mg·kg^-1phenanthrene,the PHErr of the treatment(incubation without Fenton)was 39.49%,whereas the PHErr of the Fenton treated treatments showed an average PHErr of96.63%,which was significantly higher than that of the former(p<0.001).No significant difference was detected about the PHErr between the combined treatments at pH 3 and 7.The PHErr of O_NM_Nin F_Y3(97.11%)was significantly higher than that of O_NM_Yand O_YM_N;the PHErr values of O_NM_Yand O_NM_Yin F_Y7(about 97.01%)were significantly higher than that of O_NM_N,and both of them could remove 22.06%and21.93%of the hard-to-remove phenanthrene in O_NM_N,respectively.Subsequently,we extracted soil DNA from the samples in the F_NO_NM_N,F_NO_NM_Y,F_Y7O_NM_N,and F_Y7O_NM_Yafter the incubation,and the soil bacterial community was analyzed by Illumina Mi Seq sequencing.The result indicated that the bacterial diversity and abundance of F_Y7were significantly lower than those of F_Nand F_Y7O_NM_Y.Furthermore,adonis test showed that Fenton reagent and iron-reducing bacteria can significantly affect the soil bacterial community structure(R²=0.444,p<0.01;R²=0.154,p<0.05).Therefore,Fenton reagent could effectively remove soil phenanthrene,but it destroyed the soil microbial diversity and community.Inoculation of iron-reducing microbial consortium at pH 7 not only enhanced the remediation effect of soil phenanthrene pollution but also was significant for the restoration of soil microbial community structure.(3)Choosing a high molecular weight PAH(pyrene)as the object,we explored the effect of the combined pattern of Fenton and enriched iron-reducing consortium(same with part 2)on the soil pyrene elimination.Based on the pyrene contaminated soil,nine treatments and incubation conditions same with part 2 were conducted.The results showed that in the soil containing 133.47 mg·kg^-1pyrene,the PYRrr of the treatment(incubation without Fenton)was 27.74%,whereas the PYRrr of the Fenton treated treatments showed an average PYRrr of 99.17%,which was significantly higher than that of the former(p<0.001).At the same time,the PYRrr of combined treatments at pH 3(b F_Y3,99.82%)was significantly higher than at pH 7(b F_Y7,98.53%)(p<0.01).No significant difference was detected about the PYRrr among the combined treatments with aerobic non-inoculation(b O_YM_N),anaerobic non-inoculation(b O_NM_N),and anaerobic inoculation iron-reducing microbial consortium(b O_NM_Y)at pH 3.The PYRrr values were in the order of 99.89%(b O_NM_Y)>98.34%(b O_NM_N)>97.35%(b O_YM_N),and b O_NM_Ycould remove 93.6%of the hard-to-remove pyrene in b O_NM_Nat pH7.Subsequently,we extracted soil DNA from the samples in the non-Fenton anaerobic(b F_NO_NM_N),non-Fenton inoculation iron-reducing bacteria(b F_NO_NM_Y),b F_Y7O_NM_N,and b F_Y7O_NM_Yafter the incubation,and the soil bacterial community was analyzed by Illumina Mi Seq sequencing.The result was similar to part 2,which indicated that the bacterial diversity and abundance of b F_Y7were significantly lower than those of b F_Nand b F_Y7O_NM_Y.Furthermore,adonis test showed that Fenton reagent and iron-reducing bacteria can significantly affect the soil bacterial community structure(R²=0.447,p<0.01;R²=0.140,p<0.05).Therefore,Fenton reagent could effectively remove soil pyrene,but it destroyed the soil microbial diversity and community.Inoculation of iron-reducing microbial consortium at pH 7 not only enhanced the remediation effect of soil pyrene pollution but also was significant for the restoration of soil microbial community structure.