Enhanced Treatment Of Lead-contaminated Agricultural Wastes By White-rot Fungus And Its Resistance Analysis

China is a large agricultural country with abundant agricultural resources.The rapid development of industry and the widespread use of chemical fertilizers have led to the increasing pollution of heavy metals in agricultural wastes.The traditional composting method for agricultural wastes has the disadvantages of low utilization efficiency and long reaction time.The presence of heavy metals will affect the activity of microorganisms during composting,which results in a decreased composting efficiency and a low-quality compost due to an excessive toxicity of heavy metals.In order to solve the above problems,this paper systematically carried out the research on the application of white rot fungi to the treatment of lead?Pb?contaminated agricultural wastes,and clarified the application potential of white rot fungi in the treatment of Pb contaminated agricultural wastes,and initially revealed the mechanism in enhancing the degradation of agricultural waste and stabilizing the Pb by white rot fungus.Besides,in order to explore the intrinsic mechanism of white rot fungus‘s resistance to heavy metal,this paper studied the oxidative damage,physiological response and gene differential expression of Phanerochaete chrysosporium to Pb stress.The results will provide theoretical guidance for improving the microbial remediation on the treatment of heavy metal contaminated wastewater and soils.The specific research work and achievements of this paper include the following five parts:The first part described the effect of P.chrysosporium inoculation on microbial community and metal stabilization in Pb-contaminated agricultural waste composting.The results showed that Pb stress significantly affected the diversity and abundance of microbial species in the composting of agricultural wastes,and the inoculation of P.chrysosporium could promote the passivation and reduce the toxicity of Pb to microorganisms.Redundancy analysis indicated that C/N ratio,total organic matter,temperature and soluble-exchangeable Pb were the significant paramete rs to affect the bacterial community structure.Pearson correlation analysis showed that t otal organic carbon?TOC?and carbon/nitrogen?C/N?ratio significantly?P<0.05?influenced the distribution of Pb fractions.It is shown that the stabilization of Pb is carried out along with the degradation of agricultural wastes in the composting,and composting is a potential method for repairing heavy metal Pb pollution.The second part focused on the endanced degradation of Pb contaminated agricultural wastes by white rot fungus and the mechanism of stabilization.In this study,a solid state fermentation system was established for the use of agricultural waste straw straw as a single substrate and P.chrysosporium as inoculant.The changes of organic matter content,fungal biomass,lignin degrading enzyme activity and oxalic acid were investigated.The leaching toxicity and chemical transformation of Pb were also evaluated,and the correlation between oxalic acid content and Pb leaching toxicity was analyzed.The study found that 100²00 mg kg^–1 Pb significantly induced the degradation of organic matter,and 400 mg kg^–1 Pb had an inhibition.Similarly,100²00 mg kg^–1 Pb significantly improved the activity of Li P and Mn P in the early stage of fermentation,indirectly confirmed the important role of Li P and Mn P in the degradation of organic matter.In addition,Pb stress significantly inhibited the growth of fungi in the initial fermentation and had no obvious effect at the later stage,which indicated that P.chrysosporium had resistance to Pb and could alleviate its toxicity,and gradually adapted to Pb exposure.In addition,P.chrysosporium can induce high levels of oxalic acid,which can chelate Pb ions to form lead oxalate or more stable forms in the early stage of Pb esposure,and thus reduces the toxicity of Pb.The stability of Pb at the later stage may be related to the formation of humic substances.X-ray diffraction analysis confirmed the presence of lead oxalate and pyromorphite in the end of fermentation,indicating that inoculation of P.chrysosporium in the solid-state fermentation of Pb-containing agricultural waste is an effective approach for agricultural waste degradation and Pb stabilization.The third part emphasized the effect of Pb stress on the oxidative damage of P.chrysosporium.In this study,DNA damage of P.chrysosporium by Pb was assessed by single cell gel electrophoresis,and the effects of Pb stress on the membrane lipid peroxidation and intracellular reactive oxygen species?ROS?were studied.The results showed that Pb stress reduced the intracellular the content of O₂^?–,which may be due to the anti-oxidative defense mechanism of P.chrysosporium,such as the stimulation of superoxide dismutase?SOD?.In addition,short time?8 h?of Pb exposure induced the accumulation of intracellular H₂O₂ and?OH,and triggered the lipid peroxidation,and after 24 h of Pb stress,the ROS was scavenged in the fungus of all group except group of 400 mg L^–1 Pb.The results of comet assay showed that there was a significant dose-dependent relationship between Pb exposure and DNA damage of P.chrysosporium.Pearson correlation analysis indicated that when P.chrysosporium were subjected to Pb stress,the reactive oxygen species changed and resulted in oxidative stress to the cells,and thus caused the lipid peroxidation of the cytoplasmic membrane and DNA damage.The forth part investigated the physiological response mechanism of P.chrysosporium to Pb stress.In this study,the effects of heavy metal Pb stress on the growth,oxidative stress and antioxidants of P.chrysosporium were studied,and the adsorption and accumulation characteristics of P.chrysosporium for Pb,as well as the correlation between Pb accumulation and biomass,oxidation pressure and antioxidant,were also analyzed.The results showed that P.chrysosporium had a good tolerance to low concentrations(<200 mg L^–1)of Pb and was able to remove Pb ions from the solution effectively by extracellular adsorption and intracellular accumul ation.However,Pb stress induced the accumulation of intracellular H ₂O₂ in P.chrysosporium,and induced lipid peroxidation,which resulted in oxidative damage to P.chrysosporium.The up-regulation of SOD activity and the decrease of CAT and POD activity promoted the accumulation of H₂O₂,and the stimulation of GSH promoted the alleviation of Pb toxicity.The above results indicate that P.chrysosporium exhibits an outstanding accumulation of Pb and tolerance of Pb-induced oxidative stress by the effective antioxidant defense mechanism.The last part focused on identifying the Pb-responsive genes in Phanerochaete chrysosporium by c DNA-AFLP technique.The result found that a total of about 300differentially expressed transcript derived fragments?TDFs?were observed.After being harvested,cloned and sequenced,43 effective TDFs were obtained.Through BLASTX homology analysis and GO function annotation using Blast2GO software,23 sequences had high homology with known genes,and the other 20 TDFs did not show significant matche that may be some new Pb-responsive genes.GO function annotation indicated that the response genes were mainly involved in ion binding?23%?,energy metabolism?12%?and signal transduction?12%?in response to different time of Pb exposure and were mainly related to ion binding?30%?and energy metabolism?20%?in response to different dose of Pb.The results of q RT-PCR validated the expression profile of c DNA-AFLP.It was confirmed that c DNA-AFLP was an effective method for the study of differentially expressed genes with high repetition rate and low false-positive results.In this paper,the potential of white-rot fungus in enhanced the composting or solid-state fermentation of Pb contaminated agricultural wastes was clarified,and the mechanism involved in agricultural wastes degradation and Pb stabilization by white rot fungus was revealed.The results provide a way for the treatment of heavy metal contaminated agricultural wastes.In addition,this paper revealed the physiological response mechanism and potential responsive genes of P.chrysosporium in response to Pb stress,which can provide theoretical guidance for improving its ability to deal with heavy metals.