津田弯孢C12铅耐受性研究

Lead tolerance of Curvularia tsudae C12

【目的】测定津田弯孢C12(Curvularia tsudae C12)对重金属铅的耐受性,并分析相关的理化机制,为该菌在重金属环境污染治理中的应用提供理论基础,并丰富真菌重金属抗性机制理论。【方法】采用固、液两种培养方法,分别检测不同Pb^2+质量浓度(0,200,400,800,1 200,1 600,2 000 mg/L)胁迫下津田弯孢C12的生长状况,并依据菌丝体干质量计算铅对该菌的半效应浓度(EC50),明确该菌对铅的耐受性;测定不同Pb^2+质量浓度胁迫下津田弯孢C12培养液的pH值和有机酸含量、菌丝体中的谷胱甘肽(GSH)和丙二醛(MDA)含量,初步分析津田弯孢C12可能的铅耐受性机制。【结果】津田弯孢C12在所测Pb^2+质量浓度下均能生长,但相同Pb^2+质量浓度下液体培养的菌丝体干质量和EC50(990.8 mg/L)均显著高于固体培养(P<0.05);铅胁迫下(200~2 000 mg/L),津田弯孢C12菌落直径、菌丝体干质量和培养液pH值均显著(P<0.05)低于不加铅(0 mg/L)的对照,而培养液中4种有机酸含量和有机酸总量及菌丝体内GSH含量均高于不加铅的对照,且草酸和柠檬酸含量显著(P<0.05)高于酒石酸和苹果酸。随培养基中Pb^2+质量浓度的增加,菌落直径和菌丝体干质量呈逐渐降低的趋势;草酸含量先升高后稳定,在Pb^2+质量浓度800~2 000 mg/L时达到最大;酒石酸、苹果酸、柠檬酸的含量和有机酸总量都呈先升高后降低并趋于平稳,在Pb^2+质量浓度400~800 mg/L时达到最大值;培养液pH则呈先降低后升高并稳定的趋势,Pb^2+质量浓度为800 mg/L时培养液pH值最低;菌丝体内GSH含量逐渐上升,低Pb^2+质量浓度(200~1 200 mg/L)下显著低于(P<0.05)高Pb^2+质量浓度(1 600~2 000 mg/L),而MDA含量则是先增加后降低再升高,但各个Pb^2+质量浓度下均无显著变化(P>0.05)。【结论】津田弯孢C12对重金属铅具有较高的耐受性,其在液体振荡培养条件下的生长状况和铅耐受性均明显优于固体培养,改变形态以增加吸附表面积、分泌黑色素、积累GSH和有机酸,尤其是草酸和柠檬酸,可能是该菌减轻重金属铅毒害、具有较高铅耐受性的潜在机制。

【Objective】 To provide premise and theoretical basis for application of Curvularia tsudae C12 in treatment of heavy metal environmental pollution and enrich theory of fungal heavy metal resistance mechanism,the tolerance of Curvularia tsudae C12 to heavy metal lead was determined and the related physiochemical mechanism was analyzed in this study.【Method】 The growth statuses of C.tsudae C12 under different Pb^2+ concentrations(0,200,400,800,1 200,1 600 and 2 000 mg/L) were measured by both solid and liquid culture methods.The EC50 value of Pb^2+ was calculated according to hyphal dry weight,pH value and organic acids concentration in liquid culture medium.The contents of glutathione(GSH) and malondialdehyde(MDA) in liquid culture hyphae were also analyzed.【Result】 C. tsudae C12 could grow under all tested Pb^2+ concentrations,but the hyphal dry weights and Pb^2+ EC50 values(990.8 mg/L) in liquid culture condition were significantly higher(P<0.05) than those in solid culture condition at same concentration.Under Pb^2+ stress(200-2 000 mg/L),the colony diameter,hyphal dry weight and pH value of liquid culture medium were significantly(P<0.05) decreased compared to the control(0 mg/L),but the concentrations of organic acids in liquid culture medium and hyphal GSH were increased.The concentrations of oxalic acid and citric acid were significantly higher than tartaric acid and malic acid.With the increase of Pb^2+ concentration in culture media,the hyphal color and morphological characteristics of C. tsudae C12 were varied and the colony diameter and hyphal dry weight were decreased gradually.The oxalic acid concentration was increased firstly and reached the maximum value under 800-2 000 mg/L,but the concentrations of tartaric acid,malic acid,citric acid and total organic acids increased firstly and then decreased with maximum values under 400-800 mg/L.The pH value of liquid culture medium decreased firstly and then increased with the lowest value under 800 mg/L.The hyphal GSH content was increased gradually and significantly lower(P<0.05) under low Pb^2+ concentrations(200-1 200 mg/L) than under high Pb^2+ concentrations(1 600-2 000 mg/L).The hyphal MDA content was increased firstly,then decreased and increased again without significant changes(P>0.05) under all concentrations.【Conclusion】 C. tsudae C12 showed higher lead tolerance,especially under liquid culture condition.The morphological changes,secretion of melanin,and accumulation of hyphal GSH and organic acids,especially oxalic acid and citric acid,may be one of the potential mechanisms for C. tsudae C12 to reduce damage of lead.