Study On Bioremediation Of Polycyclic Aromatic Hydrocarbon Contaminated Soil Based On Fungus Immobilization

Polycyclic aromatic hydrocarbons(PAHs)are typical organic pollutantsin soils in China.Such compounds are potentially mutagenic and carcinogenic,which pose a great threat to human health and ecological environment.Fungi have shown great potential in remediation of PAHs contaminated soil due to their multiple oxidase systems.However,during the degradation and transformation of pollutants,the related mechanisms about how fungi interact with bacteria in soils are elusive and remain unknown.In addition,its large-scale application in in situ remediation of contaminated soils is limited by some problems such as weak competitiveness,difficult survival,and low effective concentration of inoculated exogenous fungi.Therefore,it is necessary to clarify the relationship between fungi and indigenous bacteria in the process of mycoremediation,deepen the understanding of the degradation and transformation mechanisms of organic pollutants by fungi,and simultaneously develop technologies and products to enhance the adaptability,competitiveness and degradation ability of fungi in soils.This work is of great significance for the practical application of mycoremediation in PAHs contaminated field sites.To solve the above mentioned problems,first of all,a new type of fungal immobilization technology was developed based on two aspects of culture medium and production process.The autochthonous fungi bioaugmentation agents were developed and inoculated into PAHs contaminated soils to systematically explore the effect and mechanism of bioremediation.Secondly,the diversity and structure of phenanthrene(PHE)degrading microbial communities during the process of mycoremediation was explored by DNA stable isotope probing.Our findings could provide a theoretical basis for a more comprehensive understanding of the process and mechanism of PAHs mycoremediation.The major accomplishments are as follows:(1)Totally 23 PAHs degrading fungi,numbered FLQ-1 to FLQ-23,were isolated from petroleum contaminated soil.They were assigned to Ascomycota,Zygomycota and Basidiomycota.Trichoderma longibrachiatum FLQ-4 and Rigidoporus vinctus FLQ-16 have the best performances of PAHs removal in liquid media with a PHE initial concentration of 50 mg/L,whose PHE removal efficiencies were 94.6% and96.3% respectively.For Benzo[a]pyrene(Ba P)with initial concentration of 20 mg/L,their corresponding removal efficiencies were 90.7% and 92.7%,respectively.The results of testing the distribution of PHE in the various components of liquid media showed that the PHE degradation by Trichoderma longibrachiatum FLQ-4 was mainly carried out in intracellular;while PHE metabolism of Rigidoporus vinctus FLQ-16 was mainly occurred in extracellular.Compared with Rigidoporus vinctus FLQ-16,Trichoderma longibrachiatum FLQ-4 has a wider suitable p H range and higher salt tolerance during growth,so strain FLQ-4 is more suitable as a contaminated soil remediation material.(2)Encapsulated fungi by Rigidoporus vinctus FLQ-16 was optimized in terms of substrate(culture medium)and manufacturing process.Adding the co-metabolism substrate ABTS to the media will help to improve the enzymatic activity and PHE removal ability of the encapsulated fungus.During the immobilization processes,the optimal concentrations of sodium alginate and calcium chloride were 3% and 4%,respectively.The inoculation amount and drying time only affected the growth rate of mycelium on the carrier,but had no significant effects on enzyme activity and PHE removal efficiency.Scanning electron microscopy(SEM)characterization of the encapsulated fungus showed that the calcium alginate gel layer could effectively isolate the substrate from the external region,without affecting the growth and migration of fungi.Moreover,the hypha contacted with the outside through hydrogel layer cannot destroy the gel layer structure.(3)Encapsulated fungi by Trichoderma longibrachiatum FLQ-4 was successfully proliferated in the contaminated soil and exhibited excellent PHE removal efficiency.After 30 days of mycoremediation,the removal rate of PHE in soil reached 76.3%.The results of amplicon sequencing technology showed that both biostimulated NSLS and bioaugmented NSTL treatments significantly increased the diversity of bacteria in the soil and the relative abundance of Proteobacteria was most significantly increased.Rhodanobacter and Pseudomonas which belong to the phylum of Gammaproteobacteria were the dominant bacterial genera in bioaugmentation and biostimulation treatments,respectively.We speculated that Gammaproteobacteria in the soil may participate in the degradation process of PAHs through co-metabolism with fungi.(4)DNA stable-isotope probing(DNA-SIP)was performed to identify indigenous PHE-degrading bacteria and determine their diversity during the fungal bioremediation process.The results showed that a total of 15 operational taxonomic units(OTUs)related to seven genera(Sphingomonas,Sphingobacterium,Acidovorax,Massilia,Flavobacterium,Cupriavidus,Aeromicrobium,and unclassified Chitinophagaceae)were enriched in the heavy DNA fractions,indicating their important roles in the PHE degradation in contaminated soil.Besides the enhancement of PHE removal efficiency,the number and diversity of indigenous PHE-degrading bacteria in soil bioaugmented with fungi were significantly increased.Moreover,fungal bioaugmentation could promote indigenous functional Proteobacteria to participate in PAHs degradation through co-metabolism,suggesting that PAHs biodegradation was attributable to cooperative metabolism by both fungi and indigenous bacteria.Additionally,Sphingomonas affiliated with the phylum Proteobacteria play acritical role in the process of PAHs remediation.This research developed a new type of immobilized fungi applied to the remediation of PAH contaminated soil,and explored the degradation and transformation mechanisms of PAHs during the fungal remediation process from the perspective of soil functional bacteria,to further utilize the synergistic metabolic mechanism between indigenous bacteria and fungi to regulate and strengthen the degradation function of indigenous bacteria,and provide a theoretical foundation for the development and improvement of the bioremediation technology for treatment of PAH contaminated soil.