Phenanthrene removal from soil by a strain of Aspergillus niger expressing mnp1 gene from Phanerochaete chrysosporiuin

The polycyclic aromatic hydrocarbons (PAHs) are primarily biodegraded by enzymatic systems fungi and bacteria. The PAH removal by ligninolytic fungi is done mainly by peroxidases; therefore, this activity should get an increment in the degradative abilities of toxic compounds, by filamentous fungi. Solid culture is an alternative for biorremediation of contaminated soils. The peroxidases production in solid culture is higher than in submerged culture, causing discharges removal rates; however, the production of these enzymes in ligninolytic fungi is limited by several factors, such as, the quick enzymatic inactivation, nutrients limitation and the short half life of these fungi in this system. In solid culture, agricultural residuals are used and blended with the contaminated soil. They serve as a support for flingal growth, it can be used as alternative carbon source due to the high quantity of sugars that contains and, also, as texturizer (because it confers to the system certain porosity characteristics for a good oxygen transfer). The use of other nonligninolytic fungi with capacities of removing toxic compounds in solid culture, can be good for the heterologous expression of a peroxidase (manganese peroxidase, MnP) of Phanerochaete chiysosporiuni and to increase the toxic (PAHs) removal capacity. The general objective of this work consists on using non-ligninolytic strains fungi, isolated from sugarcane bagasse, for the increment in the production of MnP and to show its effect in the removal of Phe in solid culture. In this work a strain fungi was isolated, which was identified by the sequence of the fragments ITS (Internal Transcription Spacer) as: Aspergillus niger SCB2, since it was able to tolerate (800 ppm) and to remove 50% of the initial Phe. This strain was cotransformed with two plasmids (pDLAM with a resistance gene to hygromicina B, HB and pTAAMnP1, with the gene nmpl encoding a MnP from P. chysosporium). Transformants were selected by resistance to HB and for the decoloration of the Poly R478. The recombinant A. niger SCB2-T3 strain developed activity of MnP and was able to remove 95% of the initial Phe (400 ppm) in solid culture; using sugarcane bagasse with contaminated soil with Phe after 17 d to laboratory scale, while the wild strain removed 73% under the same conditions in solid culture.