Study On Mechaisms Of Filamentous Fungi Involved In Response To As(?) And Eu(?) Stress

Waste water containing heavy metal or radionuclide after emissions can lead to environmental pollution,and then enter the body through food chain transmission,thus causing serious harm to human health.Therefore,it is necessary to effectively treat heavy metal or radionuclide pollution.In recent years,the microbial remediation,as a highly-efficient and cost-effective wastewater treatment method,is drawing more attention.This paper begins with the isolation and screening of arsenic-resistant filamentous fungi and has a systematical study on the enrichment,interaction and tolerance mechanism of filamentous fungi on As(?)and Eu(III).The main research contents and results are as follows:(1)Screening of the resistance fungi to As(?).28 As(?)-resistant fungi were isolated from arsenic-contaminated soils.By morphological identification,11 filamentous fungi were found.Among them,TLWK-06 and TLWK-09 strains had a certain degree of growth under the stress of 5000 mg/L As(?).And the two fungi had a better removal effect on As(?)----the removal rate of As(?)is higher than 50% after three days.Using morphological and molecular biology to identify fungal strain,the result showed that the colony morphology and spore structure of TLWK-06 and TLWK-09 were the same as the characteristics of Mucor circinelloides and Aspergillus oryzae,respectively.The mapping results of BLAST alignment of ITS gene sequences on NCBI showed that the similarity of TLWK-06 and TLWK-09 with M.circinelloides and A.oryzae was 99%,respectively.(2)The characteristics of As(?)biosorption on A.oryzae TLWK-09 were studied.It was found that initial pH and ionic concentration had a significant effect on the removal of As(?)by A.oryzae TLWK-09.As(?)adsorption decreases with pH increasing.The adsorption ability of fungus A.oryzae TLWK-09 in As(?)solution increased with the increasing amount of mycelium,and adsorption rate tended to increase slowly when the concentration of mycelia exceeded 0.6 g/L.Biosorption data of As(?)were fitted to Langmuir model,and the maximum uptake capacity of A.oryzae TLWK-09 for As(?)was54.12 mg/g at pH = 6.5 and 301 K.Scanning electron microscopy and transmission electron microscopy showed that the surface of mycelium was obscure after enrichment,the texture of the cell wall became loose,and the thickness of the mycelium increased.There were sediment composed of fine particles in the surrounding and interior of the cell wall,indicating that A.oryzae TLWK-09 removed As(?)mainly by surface adsorption andin tracellular enrichment.The results of infrared spectra analysis showed that the main active groups of A.oryzae TLWK-09 for adsorbing or chelating As(?)probably were hydroxy,amido,carboxyl and phosphoryl.(3)The sequencing of the transcriptome of A.oryzae TLWK-09 before and after As(?)stress was carried out by RNA-seq sequencing technology.A total of 4911 genes were significantly differentially expressed,of which 2103 genes showed up-regulation comparing with the control group,while the expression of 2808 genes showed a decrease.The GO and Pathway enrichment analysis of differentially expressed genes revealed that these genes were mainly enriched in 7528 GO entries and 295 KEGG Pathways.Among them,glutathione metabolism and Metabolism of xenobiotics by cytochrome P450 may be important pathways to As(?)detoxification in A.oryzae TLWK-09.Finally,based on the functional annotation of the differentially expressed genes,the differential expression of genes related to binding and transport of metal ions,anti-oxidant,DNA repair,cell wall and methylation pathway under arsenic stress were analyzed.It was made preliminary clear that metallothionein(MT),YCF1 in ABC-transporters(ABC),antioxidant enzyme(SOD,POD and CAT),phytochelatins(PCs)and arsenite-S-adenosylmethyltransferase(ArsM)were abundantly expressed under arsenic stress,indicating that they played an important biological role in the arsenic resistance of A.oryzae TLWK-09.It provides reference for further study of these functional genes in the future.Eight differential genes related to arsenic transport and detoxification were selected and their expression was verified by fluorescence quantitative PCR.RT-qPCR test results and sequencing results were basically the same,indicating that the RNA-seq sequencing results can truly reflect the gene expression of the sequencing sample.(4)The Eu(III)biosorption characteristics of mutated Cladosporium sphaerospermum was studied.The mutated C.sphaerospermum with better adsorption of Eu(III)was also obtained by low temperature plasma.The maximum adsorption capacities of Eu(III)on mutated C.sphaerospermum was 278.8 mg/g,which was approximately three times than that of raw C.sphaerospermum.The adsorption of Eu(III)by C.sphaerospermum was mainly affected by the initial pH of the solution,the reaction time and the amount of mycelia added.The best adsorption and the largest amount of adsorption were obtained at pH = 6.5.After 24 hours that was to achieve the balance,and mutated C.sphaerospermum dosing at 0.5 g/L was more cost-effective.The reaction of the mutated C.sphaerospermum to absorb Eu(III)accords with the second-order kinetic model,indicating that its adsorption was dominated by chemisorption.The results of FTIR and XPS indicatedthat a large number of nitrogen-containing and oxygen-containing functional groups existed on the surface of mutated C.sphaerospermum,which could complex with more Eu(III)and thus had a stronger ability of removing Eu(III)from the solution.