Enhanced Remediation Of Copper Contaminated Soil By Trichoderma Reesei FS10-c

Copper is an essential trace element that is vital to the health of all living things. But excess copper accumulation in plants, animals and human being will disturb their normal physiological processes, lead to stunted growth or even death in plants, and worse enough, risk factors of animals and our human beings. Therefore, remediation of copper contamination is of great importance at present. Trichoderma sp. can be easily screened out by isolation and culturing methods to cost-effectively obtained their pure culture. In addition, Trichoderma sp. has larger production of biomass, and can grow well in low pH besides its capacity of heavy metal resistance or promoting plant growth. Therefore, it could be as a candidate with its great advantage for remediation of copper contamination. A copper resistant Trichoderma reesei strain FS10-C was studied in the present study, to investigate its effects and mechanisms in bioremediation of copper contaminated soil. The important research scope and results are summarized as follows:1) Trichoderma reesei FS10-C was evaluated for its environmental adaptability through verifying of the spore germination, hyphal growth and sporulation in diverse conditions of temperature, humidity, pH value and light. In addition, bio-safety assessment was conducted to evaluate the perspective application of Trichoderma reesei FS10-C in remediation of copper pollution. The results showed that spore germination and hyphal growth of Trichoderma reesei FS10-C were possible at temperature of10-40℃and in relative humidity of60-100%, and sporulation occurred favorably at20-35℃. Trichoderma reesei FS10-C grew vigorously in acid-biased environment (pH4-7) due to copper contamination. Light stimulated sporulation sporulation of Trichoderma reesei FS10-C, but had little effect on spore germination and hyphal growth. No toxicity effects of skin irritation, skin toxicity, eye irritation and hemolysis were observed in both Trichoderma reesei FS10-C and its metabolites. These findings show that Trichoderma reesei FS10-C has wider environmental suitability, yet no health risk factors for humans and animals. It suggested that Trichoderma reesei FS10-C is a safe candidate for bio-remediation of copper contamination.2) Shake Flask tests were performed to observe variations of Trichoderma reesei FS10-C under copper stress, such as growth and development while copper accumulation, morphology and element of adult cell while copper absorption, and physiological-biochemical characteristic. Besides, capacity of siderophore production was verified to study responses and mechanisms of copper resistance of Trichoderma reesei FS10-C. The results showed that (1) the spores of Trichoderma reesei FS10-C were well grown and developed in aqueous solutions with Cu(II) concentration below600mg/L. Copper accumulation rates of Trichoderma reesei FS10-C under100,200and400mg/L Cu(II) concentrations were50.6%,30.3%and4.7%, respectively. The SEM showed no morphological variation in Trichoderma reesei FS10-C at100mg/L Cu(II) and mostly complete cell structure at400mg/L Cu(II). EDX results showed that weight percentage of copper in Trichoderma reesei FS10-C was18.50%and30.40%at100and400mg/L Cu(II), respectively, and biosorption capacity of mycelium was higher than that of spore;(2) mechanisms of copper resistant and removal effect of Trichoderma reesei FS10-C were related to increase of soluble protein and GSH contents, siderophore production, nitrogen-containing group and phospholipid of cells. It suggested that Trichoderma reesei FS10-C is a fungal strain with higher copper resistance, and it can remove copper by bioaccumulation and biosorption by cell composition and physiological and biochemical regulating mechanisms.3) Spores of Trichoderma reesei FS10-C were made into Trichoderma sp. preparation by solid-state fermentation with condition screened by orthogonal tests. And a pot experiment of Elsholtzia splendens in soils spiked with copper was carried out to investigate effects of the preparation on growth and Cu accumulation of the plant and on soil quantity. The results indicated that (1) spores of Trichoderma reesei FS10-C can grow and develop in alfalfa meal as solid matrix and made into Trichoderma sp. preparation with high spore density;(2)the plant biomass of Elsholtzia splendens showed an increasing trend in soils with all spiked Cu concentrations after applying of the preparation, except for shoot dry weight in soils with spiked Cu levels of400mg/kg. The preparation increased NH4OAc-extractable Cu in all Cu contaminated soils significantly in comparison with that in the control (P＜0.05), and also led to an increasing trend in Cu accumulations of all plants, except for that in shoot in soils with external Cu level of100mg/kg. Total Cu accumulations were significantly enhanced in soils with external Cu levels of100and400mg/kg (P＜0.05);(3)the Trichoderma sp. preparation improved soil quantity by enhancing available P content, activity of dehydrogenase, urease and phosphatase, microbial biomass C content and pH value of Cu contaminated soil. The findings suggested that this Trichoderma reesei FS10-C preparation could enhance Cu accumulation by promoting growth of Elsholtzia splendens and increasing Cu phytoavailability in soil. It can be both bioremediation agent and microbial fertilizer for soil, and could be a potential agent for bioremediation of copper contaminated soil.4) The mechanisms of augmentation effect of Trichoderma reesei FS10-C on remediation of Cu contaminated soil with Elsholtzia splendens were investigated, such as plant growth promoting effect on Elsholtzia splendens, biological characteristics of plant growth promoting, enhancement of Cu resistance of Elsholtzia splendens and enhancement Cu. availability of soil.The results showed that (1) Trichoderma reesei FS10-C was able to produce ACC deaminase, siderophore, and have the ability of phosphate solubilization;(2) Trichoderma reesei FS10-C had significantly promoted seed germination of Elsholtzia splendens and root development of the seedling in condition of copper stress free;(3)even in environment of copper stress, Trichoderma reesei FS10-C were capable of promoting seed germination and enhancing biomass, improving root activity, decreasing MDA content and increasing POD, SOD and CAT activities of Elsholtzia splendens(P<0.05).(4) Trichoderma reesei FS10-C had reduced pH value of the culture media during its development process. The fermentation broth also had higher ability of extracting Cu from soils. It proved that Trichoderma reesei FS10-C may release certain acidic materials that were able to accelerate extraction of Cu from soils to eventually enhance Cu availability of soil. These findings suggested that Trichoderma reesei FS10-C could promote growth and development of the plant in Cu contaminates soil due to its biological activity of plant growth promoting and capacity of copper resistance enhancement, and it may enhance Cu. availability of soils due to producing some acid materials.In summary, copper resistant Trichoderma reesei FS10-C can be a potential candidate for bioremediation of copper pollution. Trichoderma reesei FS10-C preparation could play safe and effective role in phytoremediation of Cu contaminated soil.