一株产漆酶土壤真菌F-5的分离及土壤修复潜力

漆酶因可氧化许多种有机污染物,在土壤污染修复方面的应用潜力受到广泛重视。筛选具有较高漆酶活性的土壤真菌,可以为污染土壤修复提供生物资源。通过培养基中愈创木酚颜色反应,从土壤中筛选获得1株真菌菌株F-5。18S rRNA基因序列显示该菌株属于巨座壳科(Family Magnaporthaceae)。单因素试验和正交试验结果显示,蔗糖和蛋白胨分别是最有利于该菌产漆酶的碳源和氮源。在适当培养条件下,真菌F-5培养液酶活性可达4033U/L,表现出该菌具有较强的产漆酶能力。在多环芳烃(PAHs)污染土壤的生物修复中,真菌F-5可使土壤中苯并(a)芘、二苯并(a,h)蒽等高环、高毒性多环芳烃降解,并使土壤多环芳烃毒性当量大幅降低。因此,真菌F-5适合修复PAHs污染土壤。

Isolation and the remediation potential of a Laccase-producing Soil Fungus F-5

Policyclic aromatic hydrocarbons (PAHs) are a class of organic compounds containing two or more fused aromatic rings, and composed of only carbon and hydrogen atoms. They are persistent organic pollutants (POPs) that exist extensively in environment. The United States Environmental Protection Agency (USEPA) has classified 16 of the PAHs as priority pollutants based on toxicity, potential for human exposure, frequency of occurrence at hazardous waste sites, and the extent of information available. The environmental risk cannot be ignored due to the possible carcinogenic, teratogenic and mutagenic effects of some high-molecular-weight PAHs. PAHs are widespread in the soil in some economically well-developed areas of China, such as Bohai Bay Rim, the Yangtze River and Pearl River Delta regions. In some cases, the concentration of PAHs has already reached a very high level in the soil of these regions. Laccases (EC1.10.3.2) are copper-containing oxidase enzymes that are found in many plants, fungi, and microorganisms. Because laccase have the ability to oxidize many kinds of organic pollutants, application potential of laccase in remediating organic contaminated soil has drawn extensive attention. Fungus is an essential part of the soil ecosystem, which plays an important role in the detoxification and cleaning up of organic contaminated soil. Many studies have shown that PAHs can be degraded efficiently by white-rot fungi, which usually grows on dead trees. However, white-rot fungi is not suitable to grow in soil and have limited application in soil remediation. Fungi with laccase-producing activity can transform and degrade PAHs. Thus, isolating PAHs-degrading fungi can be changed into isolating fungi with laccase-producing activity, which can simplify isolation procedure and save isolation time and work capacity. Isolating soil Fungi with high laccase-producing activity is promising to provide biological source for remediating PAHs-contaminated soil. Based on the phenomenon that media containing guaiacol can turn red, potent PAHs-transforming fungi strains can be isolated from soil. In this study, one fungal strain, F-5, was isolated from an agricultural soil based on the color development of guaiacol in medium. Analysis of partial 18S rRNA gene sequences showed that strain F-5 is affiliate within the Family Magnaporthaceae. Sucrose and peptone are the optimal carbon and nitrogen sources for laccase production respectively,according to one-factor experimental results, as well as orthogonal test results. The laccase activity in medium reached to 4033U/L in suitable cultural condition, showing the Fungi's high ability to produce laccase. Low-molecular-weight PAHs are readily degraded by microoganisms. However, high-molecular-weight PAHs (four and more rings) are more persistent, in part because of their low bioavailability and their strong adsorption onto the soil organic matter. Our result showed that fungus strain F-5 can degradate a few high-molecular-weight PAHs with strong toxicity, such as benzo(a)pyrene and dibenzo(a,h)anthracene, in an aged PAHs-contaminated soil. In consequence, the toxic equivalence quantity of soil was reduced greatly. This finding suggested that the Fungus strain F-5 is suitable for remediating PAHs-contaminated soil. This research is a pilot study on the bioremediation of the PAHs contaminated soil. More work will be done in our lab to determine the applicability and practicality of utilizing this organism in remediating PAHs-contaminated field sites.