| Along with the development of the economy,heavy metal pollution has become one of the most serious problems,and the presence of these metals even in traces is toxic.Among the various wastewater treatment techniques,biosorption of heavy metals is a promising alternative method due to its high selectivity,easy handling,lower operating costs,high efficiency,and regeneratability of biosorbents.Sclerotial differentiation plays a vital role in tolerating and resisting heavy metals.Strain G15,which was isolated from forest soil of Pangquangou National Nature Reserve,was able to form abundant black sclerotia,and showed very strong tolerance to copper and lead.The strain was identified as Aspergillus oryzae by morphological observation and the ITS sequence analysis method.The resistance mechanisms,bioaccumulation characteristics of copper and lead by growing cells,and the biosorption characteristics and mechanisms by mycelia and sclerotia of G15 were studied in detail.The main conclusions are as follows:1.Strain G15 was cultured on Czapek yeast extract agar?CYA?medium containing different concentration of copper and lead to investigate the heavy metal tolerance.G15 strain can grow on CYA containing 300 mg/l Cu???and Pb???,which indicated that this strain had a great potential application in the copper and lead pollution restoration.The mycelial density and diameters of colonies treated with copper ions were significantly decreased when compared to control.Cu treatment?200–300 mg/l?delayed sclerotial maturation by 46-53 h.The spore formation and mycelial growth were inhibited by Pb treatment.G15 adsorbed most Cu and Pb on the cell surface,about 20 to 40 percent of the heavy metals entered into the cell.The contents of copper and lead in sclerotia were relatively more than that in mycelia,which indicated sclerotial differentiation had a positive effect on the heavy metal accumulation.2.A.oryzae G15 was cultured on CYA containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance.The effects of heavy metals metallothionein?MT?,malondialdehyde?MDA?and antioxidant metabolism were evaluated.The results showed that MDA levels had a positive correlation with copper and lead concentration,which revealed the appearance of heavy metal-induced oxidative stress.In the case of mycelia,copper treatment elevated the MT levels significantly in a concentration dependent manner.Lead treatment with concentrations of 50 and 100 mg/l also significantly increased the MT contents.In the case of sclerotia,MT levels were unaffected by the copper and lead.The data demonstrated that MT were mostly activated early?stage of mycelia growth?to scavenge ROS and chelate metal ions.Superoxide dismutase?SOD?activity was significantly increased by Cu treatment.Cu???in different concentrations improved catalase?CAT?activity in different degrees,and 50 and 100 mg/l Cu???was the most effective one for mycelia and sclerotia,respectively.Induction of peroxidase?POD?was also detected at lower copper concentrations,while,Cu in the relatively high level caused loss in the activity.In the case of Pb treatment,a significant decline in SOD and CAT activity was observed.Pb treatment promoted the POD activity in mycelia,while it had no significant effect on the activity in sclerotia.In sum,Cu treatment enhanced the activities and reduced sclerotial biomass,which indicated that G15 strain was protected efficiently from the copper-induced damage.Pb treatment inhibited the activities of SOD and CAT and promoted the sclerotial differentiation,suggesting that the responses of G15 were insufficient to protect the cells against lead-induced damage.3.Mycelia and sclerotia of G15 were ground into powder and used as biosorbents.The biosorption characteristics for the biosorbents and the factors that affected the adsorption efficiency were investigated.The results showed the solution pH values had a great influence on the biosorption of Cu and Pb by mycelia and sclerotia.The maximum capacity for Cu???were hold at pH 5.0.The biosorption capacities for Pb???increased as the increases of pH value.More than 90% of the Pb???were adsorbed on mycelia and sclerotia at pH 6.0.Thus,pH value of 6.0 was considered to be the optimum pH.Adsorption temperature,contact time and adsorbent concentration had a few effects on the adsorption efficiency.The optimum conditions of the biosorption was adsorbent concentration = 2 g/l,temperature = 30 ? and contact time = 60 min.In addition,co-ions(Na+,K+,Mg2+,Ca2+)in solution led to the decrease in adsorption efficiency of biosorbents for Cu???and Pb???.However,low concentration of KCl enhanced the adsorption capacity of mycelia for Pb???.4.The comparative study of biosorption characteristics of mycelia and sclerotia of G15 was carried out.Equilibrium biosorption was modeled using Langmuir and Freundlich isotherm equations.The biosorption process obeyed the Langmuir adsorption isotherms,which indicated that mycelia and sclerotia of G15 would provide the mono-layer adsorption for Cu???and Pb???.The maximum biosorption capacities of sclerotia calculated from Langmuir isotherm were 73.53 and 123.46 mg/g for Cu???and Pb???,respectively.The maximum biosorption capacities of mycelia were 35.84 and 60.61 mg/g for Cu???and Pb???,respectively.The kinetics of the biosorption was better described by pseudo-second order kinetic model,indicating that the adsorption process could be divided into physical adsorption stage?external mass transfer?and chemical adsorption stage?intraparticle diffusion?.Desorption and regeneration studies using different eluants were also studied.The results showed that more than 90% of adsorbed Cu and Pb ions were desorbed by the mineral acids?HCl and HNO3?and EDTA solution,suggesting that most of the biosorbed metal ions were sticked to cell surface and the biosorbents could be regenerated and reused.5.The study on the biosorption mechanisms of the biosorbents was carried out through a variety of analytical methods,such as zeta potential,potentiometric titrator?PT?,scanning electron microscopy?SEM?,fourier transform infrared spectroscopy?FTIR?and X-ray photoelectron spectroscopy?XPS?.XPS analysis confirmed that there was surface biosorption of Cu???and Pb???on the adsorbents.SEM observation showed that copper and lead were toxic to cell of G15,and they caused obvious change in surface morphology of mycelia and sclerotia.PT,FTIR and XPS analyses indicated that carboxyl group was mainly responsible for the adsorption,the hydroxy and amido groups took minor responsibility.According to the XPS results,mycelia of G15 were not loaded with Cu and Pb showed 1.37% sodium,whereas,heavy metal loaded mycelia did not show the presence of sodium.The result revealed that ion-exchange with Na+ was a mechanism related with the adsorption on mycelia.The obtained results showed that adsorption capacity of mycelia and sclerotia of G15 depend mainly on the amount of adsorption sites.Sclerotia were superior biosorbent than mycelia,which could be ascribed to the higher amount of carboxyl sites exposed after differentiation.The amount of absorbed Pb???on mycelia was higher than that onsclerotia at pH 2.0 – 3.0.Thus,it could be speculated that the Pb???adsorption on mycelia at low pH value was mainly caused by ion exchange. |
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