Research On Heavy Metal Resistance Mechanisms And Bioaccumulation Characteristics Of Fungus

Heavy metal and its compounds are widely used in many industries and a large quantity of containing heavy metals wastes are released into environment. Heavy metal has posed great threat on environmental safety and human health. The reduction and removal of heavy metal is the key for the detoxification of heavy metal. Many tolerance strains have been found to be able to bioaccumulation of heavy metal and bioremediation has been regarded as a promising approach for the treatment of heavy matal pollution. In this paper, different fungal strains were applied to the biosorption and bioaccumulation of heavy metals. The resistence mechanisms, bioaccumulation characteristics of heavy metal by growing cells, and the biosorption characteristics and mechanisms by living and dead cells were studied in detail.A novel fungus CTB430-1 with resistance to copper and zinc was isolated from soil at a copper and zinc Tailing of Linxiang in Yueyang city, Hunan province. The fungus was identified as Aspergillus flavus by analyzing the morphology and measuring the ITS sequence.The minimal inhibitory concentration of the strain CTB430-1 for Cu(II)and Zn(II) were 400 and 800mg/L, respectively. Compared with CTB430-1, the minimal inhibitory concentration of Aspergillus niger which was contrastive stain in our study were only 200 and 300 mg/L. So the tolerance of CTB430-1 was high. The maximum bioaccumulation capacities of Cu(II)and Zn(II) by CTB430-1 were 30.82 and 40.37 mg/g at initial concentrations of 200 and 250 mg/L, and that of A.niger were 8.89 and 22.80 mg/g at initial concentrations of 150 and 250 mg/L, respectively.The factors that affected the bioaccumulation efficiency by isolated strain CTB430-1 and contrastive stain A. niger, were thoroughly investigated, such as initial pH value of liquid medium, initial Cu(II)and Zn(II) ions concentration, co-ions in the liquid medium, spores inoculum amount and temperature.The results showed that the initial pH value of liquid medium had important effect on growth and bioaccumulation of CTB430-1 and A. niger, at low pH value(≤2.0), the growth was inhibited when the initial. With the pH value increased, the growth and bioaccumulation capacity by the two strains increased with increasing pH value in the range of 2.0-5.0 for Cu(II)and Zn(II), and the maximum growth and bioaccumulation capacity were hold at pH 5.0. The potential inhibition on growth of CTB430-1 and A. niger were increase by increasing intial concentration of Cu(II) and Zn(II) in range of 25-100mg/L, and the growing period were delay,all the stains reached the growth balance in 5d. The bioaccumulation amount of Cu(II) and Zn(II) ions increases by CTB430-1 and A. niger with the increasing initial metal ion concentration and contact time. The bioaccumulation equilibriums of Cu(II) and Zn(II) by CTB430-1 reached in 4d, which of A.niger reached in 5d.In the binary Cu(II)+Zn(II) system, the presence Cu(II)/Zn(II) ions induced to the inhibition in the growth of CTB430-1 and A. niger except that the concentration of (25+25) and (25+50)mg/L could promote little the growth of A. niger. When the concentration of Cu(II)/Zn(II) ions was high, the competition and bioaccumulation ability of Cu(II)/Zn(II) ions were strong. With the spores inoculum amount increased, the growth and bioaccumulation of CTB430-1 and A. niger were increase, and reached balance at 1mL of the inoculum amount. Maximum growth and bioaccumulation capacities were obtained at different temperatures in the range of 25-30℃and 30-35℃, respectively.The changes of micrographs obtained from scanning electron micrograph and transmission electron microscope of CTB430-1 and A. niger before and after Cu(II) and Zn (II) accumulation showed that all the stain suffered the heavy metal toxicity in the growing process, which could destroy the surface structure possibly or some heavy metal ions maybe were uptaked on the surface of mycelium that made the surface become blurry. The bioaccumulation included mostly extracellular and intracellular biosorption. The results of FTIR spectrum showed that the Cu(II) ions counld not destroy the structure of CTB430-1 in the process of bioaccumulation, hydroxyl was the primary activity groups in the biosorption or complexing/chelated Cu(II) ions. However, the biosorption of Cu(II) ions by A.niger were made metal-ligand complexes by the phosphorus as donor atoms which in polyoses groups.The resistant mechanism of Cu(II) and Zn(II) by CTB430-1 and A. niger were studied. It was observed that high concentration of Cu(II) and Zn(II) could restrict the growth of CTB430-1 and A. niger obviously, cause decrease the soluble protein content and increase in GSH levels, which could alleviate the oxidation stress of Cu(II) and Zn(II). The activities of SOD and CAT in cells of CTB430-1 and A. niger increased obviously with the concentration of Cu(II) and Zn(II) increasing, which was one of the attributes of two stains'resisting peroxidation. The enzyme activities of CTB430-1 increased highly, so the resistance to Cu(II) and Zn(II) of CTB430-1 were stronger than A.niger. The MDA levels enhanced in the cells of CTB430-1 and A. niger under the stress of Cu(II) and Zn(II), but the chang were prominent of all the stains under the stress of Cu(II). It showed that the mechanisms for Cu(II) to damage CTB430-1 and A. niger by in ducing ROS and lipid peroxidation.The effects of exogenous nitric oxide (NO) on antioxidation system of CTB430-1 and A. niger under the stress of Cu(II) were explored, the result indicated that the pretreatment of sodium nitroprusside (SNP), a NO donor, at 0.1nmol/L could increase the growth of two stains, and alleviate the reduction of soluble protein, induce the creation of GSH, and enhance the activities of SOD and CAT, all of that could enhance the capacity of antioxidation for CTB430-1 and A. niger. At the same time, the NO (0.1mmol/L SNP) reduced the toxicy of Cu(II) by decreasing the MDA amount in cells of CTB430-1 and A. niger. However, the alleviations of high NO concentration (0.3mmol/L SNP) were not obvious.The biosorption characteristics of Cd(II) and Zn(II) by living A. niger were investigated. The optimum adsorption pH value for Cd(II) and Zn(II) were 4.0 and 6.0. The best temperature and agitation rate were in the range of 25-30℃and 120r/min for all metal ions. Under the optimal conditions, the maximum uptake capacities of Cd(II) and Zn(II) ionsare 15.50 and 23.70 mg/g at initial concentrations of 75 and 150 mg/L, respectively. Biosorption equilibrium isestablished within 24h for Cd(II) and Zn(II) ions. The adsorption data provide an excellent fit to Langmuir isotherm model. The results of the kinetic studies show that the rate of adsorption follows the pseudo-second order kinetics.The isotherms, kinetics and thermodynamics of Cd(II), Zn(II) and Pb(II) biosorption by non-living Penicillium simplicissimum were also investigated in a batch system. The effects of pH, initial metal ions concentration, biomass dose, contact time, temperature and co-ions on the biosorption were studied. Adsorption data were well described by the Redlich-Peterson model. Chemical ion-exchange was found to be an important process based on free energy value from Dubini-Radushkevich isotherm for all metal ions. The results of the kinetic studies of all metal ions at different temperature showed that the rate of adsorption followed the pseudo second-order kinetics well. The thermodynamics constantsΔG o,ΔH oandΔS oof the adsorption process showed that biosorption of Cd(II), Zn(II) and Pb(II) ions on Penicillium simplicissimum were endothermic and spontaneous.