Poly aromatic hydrocarbons degradation by lignin peroxidase produced from local isolate Aspergillus terreus SG777

Lignin peroxidase produced from A. terreus SG777 precipitated with 80% ammonium sulfate saturation then desalting by dialysis against Tris buffer solution and partially purified by ion exchange chromatography using DEAEcellulose.The specific activity reached 56.67U/mg with purification fold was 5.4 times and 13.7% recovery. The optimum pH for activity and stability was (3). The maximum activity was observed at 40°C and the enzyme maintained the activity when it was incubated at (25-35) ̊C. Degradation of poly aromatic hydrocarbons when incubated with 65.51 U/ml lignin peroxidase at (40°C) for 4 hr compared with the Refinery treatment. Results showed (97.54%) removal efficiency of Di benzo (a, h) anthrancene by lignin peroxidase, while only (54.37%) was treated by Refinery treatment. Flourene and anthrancene degradation (95.98%) and (83.51%) respectively, while the flourene and anthrancene degradation treated by Refinery treatment only 84.51% and (46.83%) respectively. The aim of this study was degradation of poly aromatic hydrocarbons by using partial purification lignin peroxidase. Keyword Environment and Safety in Oil Industries, Aspergillus, lignin peroxidase, poly aromatic


Introduction
A chief hazard for the environment and in many cases its poly cyclic aromatic hydrocarbons; they comprise risk to human and animal health. Poly cyclic aromatic hydrocarbons have caused a significant concern due to their toxicity and carcinogenicity. Therefore the increased consumption of fossil fuels, their incidence in the environment has gradually increased since last (10 -150) years [1].
Poly cyclic aromatic hydrocarbons anthropogenic source are becoming more significant with increasing industrial processes like petroleum and carbon manufactures, wood treatment plants, and many others activities in which burning of fuels or organic materials is included. The maximum PAHs levels have been so far detected in petroleum gasification sites [2].
The oxidation of PAHs is catalyzed by lignin peroxidase which have an important role in the first attack on high molecular weight PAHs, contains at least four benzene rings in soil [3].

E 145
One of the most important ligninolytic enzymes is lignin peroxidase glycosylated, heme containing enzymes which functionally need H 2 O 2 for the oxidation of lignin associated aromatic structures [4]. Numerous reports have exposed that LiP from Phanerochaete chrysosporium is directly implicated in the degradation of different xenobiotic compounds and dyes [5].
The aim of this study to degradation poly aromatic hydrocarbons by lignin peroxidase which produce from locale isolate Aspergillus terreus SG777 with high efficient and short time compared when we use the microorganism itself.

Lignocellulosic Substrate
Lignocellulosic agro-industrial wastes such as Banana stalks and Corn stover were obtained from some farms in Baghdad. Another substrate such as Wheat and Rice were obtained from market respectively and Bush horse which contains (Soybean, Corn, kernels and Barley) obtained from Equestrian Club in Jadriya -Baghdad. The substrates were crushed into small pieces, oven dried (50 o C), and ground to 40 mm mesh particle size and stored in air-tight plastic jars.

Inoculum preparation
A pure culture of the indigenous strain A.terreus SG-777 which produces all extracellular ligninolytic enzymes, isolated from horse manure was used for the present study [6]. Inoculums were prepared by growing the fungus in Kirk's basal salt medium [7]. Hundred ml of media it was added in Erlenmeyer flasks (500ml) .The medium was supplemented with Millipore filtered (0.22μm) glucose (0.5%). Spores of A.terreus S.G-777 added to the media and incubation at (30 °C) for (5 to 10) days to obtain homogenous spore suspensions of fungi (1x106 spores / ml) to use as inoculum [8].

Enzyme production
kirk´s basal medium modified by using (5g) from Bush horses consist of (Soybean , Corn, kernels and Barley) as a substrate instead of glucose , the substrate moistened by Kirk's basal salt medium . All the flasks were autoclaved and inoculated with (4ml) (1x10 5 spore/ml) homogenous suspension of strain A.terreus SG-777. The inoculated flasks were incubated at (30°C) for 10 days.
Culture flasks were harvested by (100ml) of (100mM) sodium succinate buffer, the flasks were shaken (120 rpm) for 30min. The mixture was filtered through a Watman No.1 filter paper and then centrifuged at (3000 xg) for (10min). The clear supernatants were keep at (4°C) to determine the activities of LiP, MnP and lacc [9].
Lignin peroxidase enzyme activity was expressed as U/ml .An international unit IU (or U) is defined as the amount of enzyme, which catalyzed the transformation of 1 micromole of substrate per minute under standard conditions. This was calculated using the formula [11]:

Protein concentration
The protein concentration was estimated according to Lowry method [12].

Optimization of lignin peroxidase enzyme production
To further enhance the lignin peroxidase enzyme production capability of fungi, different nutritional substrate and different physical parameters (incubation time, moisture level, inoculums size, pH and temperature) were optimized according method [13].

Purification of lignin peroxidase
Under optimum condition the Lip which produce from selected isolate A. terreus SG-777 was purification by following steps were carried out.
2-Desalting by dialysis against Tris buffer solution. processing unit and the second sample after mechanical, physical chemical and biological treatment).
Extraction of PAHs from water:The extraction of PAHs was done according to [14] as following: Five hindered milliliters from the first sample (before entering the processing unit ) was taken in separator funnel and extracted by equal volume from n-hexane by vigorous shaking for 3 min. The organic phase was approved through anhydrous sodium sulphate to eliminate traces of water contents.
The extract volume were concentrated by evaporated at room temperature overnight to near dryness , then added 1 ml acetonitrile as another solvent . Sample was analytic by using High

Purification of lignin peroxidase
Lignin peroxidase from A. terreus SG 777 produced at optimum conditions, supernatant was collected from the medium, which gave specific activity 10.45 U/mg and concentrated by salt using

Conclusion
x Lignin peroxidase was extracted and concentrated by ammonium sulfite, then purified by ion exchange using DEAEcellulose (Column chromatography).
x Optimum temperature for LiP activity was at 40°C, and the enzyme was stabile between 25 -35°C after 30 min . Whereas the optimum pH for Lip activity and stability was 3 at 40°C.
x Purified LiP was more efficient for dyes removal than crude LiP or H 2 O 2 .
x Purified LiP exhibited higher removal efficiency for poly aromatic hydrocarbons in comparison with to treatment in AL-Dorah Refinery.