Biodegradation of Anthracene Compound by Two Species of Filamentous Fungi

Isolation and identification fungi of Emericella nidulans and Aspergillus flavus from a pinkish and yellowish artificial clay, by using potato dextrose agar (PDA). Results revealed that E. nidulans was the best for degrading anthracene (92.3%) with maximum biomass production (3.7gm/l), compared to A. flavus with the rate of degradation (89%) and biomass production of (1.2gm/l), when methylene blue was used as redox indicator after incubating in a shaker incubator 120rpm at 30C o for 8days. Results indicated that E. nidulans has a high ability of anthracene degradation with the rate of (84%), while A. flavus showed the lower level with (77%) by using HPLC.


Introduction:
Polycyclic aromatic hydrocarbons (PAHs) are prevalent environmental pollutants. Uptake of anthracene via numerous routes in the body imposes toxicity to the skin, blood, intestine and the lymphatic system, thus leading to tumors, edema, itching and the upsurge of fluids in tissues (1,2).
Numerous procedures were utilized to expel PAHs, as physical treatment, chemicals and microbial degradation using bacteria, fungi, algae and yeasts (3,4).
Anthracene is a solidify PAH (C 14 H 10 ), comprising about three combined Benzene rings. It will be utilized to prepare the Red Dye Alizarin and other dyes. Anthracene may be colorless, but display a blue (400-500)nm peak fluorescence under ultraviolet light (5).
Many literatures show that filamentous fungi are playing an important role in removing of PAHs and even more effective than some bacteria. Increasing non-ligninolytic fungi, Penicillium simplicissmum and species belonged to the genera Trichoderma, Fusarium, Penicillium, Stachybotrys, Aspergillus, Cladosporium, Mortierella, Beauveria and Engyodontium, have been recently described as tolerant to pollutants such as PCBs and chlorobenzoic acids (6).

Purification of isolated fungi
Fungus isolates were purified by repeat culturing on plates containing PDA medium, till ensuring purity of fungal isolates. Then isolates subsequently cultured on PDA slants and allowed to grow for 5-7days stored at 4°C as stock cultures.

Identification of fungal isolates
A colony of fungal isolates were diagnosed due to their morphological characteristics by utilizing the lactophenol solution, then slides were recognized under the microscope.

Methylene blue (Redox indicator)
Erlenmeyer flasks (Volume 100ml) were used in this experiment containing a suitable amount of mineral salt medium preparing by Ameen et al., (2014) with some modification (7), containing 25µl of Tween 80, then volume flasks were completed to 25ml with the same media, (pH 7.0), 15µl of methylene blue as prepared by Wilson and Jones (8) and 100ppm anthracene were added after autoclaved media at 121 0 C for 15min. Flasks were inoculated with diameter 7mm of selected fungal isolates (One isolate for each flask). Flasks were incubated in a shaker incubator 120rpm at 30C o for Open Access 8days. All experiments were done in duplicate and control for each isolate. After the incubation period, growth culture was filtrated to separate biomass, followed by centrifugation at 10000rpm for 15min. Supernatant was analyzed by spectrophotometer at 609nm and the percentage of degradation was calculated by a formula as below (9).
Absorbance of control

Estimation of anthracene concentration by HPLC
After a period of incubation, the growth was harvested at 10000rpm for 20min and filtered through Whatman No. 1. Add 10ml of hexane to 25ml of supernatant, then mixing for 30min by using separate funnel, then 1ml from the upper phase (Hexane) was transferred to a sterile tube for HPLC analysis, which achieved with reverse-phase column C18 (Syknm Chromatography Products, Germany). Separation was accomplished by isocratic elution in (Acetonitrile: Water) (70:30), with a flow rate 1.0ml/min and UV absorbance detector set at 254nm.
Initial conc. of anthracene -Anthracene conc. after incubation Initial conc. of anthracene

Estimation of biomass
Twenty-five ml of growth media was centrifuged with speed 10000rpm at 4 0 C for 20min, then supernatant was discarded and biomass washed 2-3 times with distilled water, re-centrifuge under the same conditions. Dry weight was estimated after drying at 80 0 C for 24h (10].

Identification of fungal isolates
Morphological characteristics of fungal colonies by using a dissecting microscope, were the initial identification according to their macroscopic and microscopic features. Results showed that most active isolates were identified as E. nidulans and A. flavus (11) [Fig. 1, 2 (A&B)].

Biodegradation of anthracene
The period of incubation was the most important factor that affects degradation processes (12) and the optimum condition for anthracene degradation (100ppm), by E. nidulans and A. flavus isolated from artificial clay, were incubated in a shaker incubator 120rpm at 30C o for 8days.
The result showed that increasing concentration of a water soluble synthetic compound (Tween 80) improved the biodegradation and bioremediation of toxic pollutants. Muthusamy et al., (13), just as (1, 2.5 and 5) gm/l in the media create gradual increasing in decane biodegradation (55.0, 74.5 and 79.3)%, respectively, at 20°C. Whereas 10gm/l of tween 80 caused decreases in biodegradation (57.2)% compared to 5gm/l (79.3)% at the same degree.
We can be inferred that low nutrients, low pH, accumulation of metabolites resulting from oxidation processes, lack oxygen and ventilation, were the reasons of decreasing anthracene degradation after 8days (14).
Ravelet et al., (15) recorded that Zygomycota Mucor racemosus was able to degrade more than 40% of anthracene after 8days of incubation. This result elucidates that biodegradation happened due to the higher surface area (Fungal mycelium), which increased both mechanical and enzymatic contacts with an insoluble substrate such as anthracene and invade a larger volume of soil (16). At the same time, Mohammed et al., (17) verified that degradation of anthracene by fungi are more practical than bacterial degradation.

A-Methylene blue method
The statistical results showed that E. nidulans was significantly the better isolate in degradation of anthracene compound by using methylene blue as a reduction agent to the residue of anthracene, with an OD 0.2 and rate 92.3%, compared to an OD of A. flavus 0.3 and rate of degradation was 89% (Fig. 3) ( Table 1).

Figure 3. Reduction of methylene blue (Redox indicator) by E. nidulans and A. flavus after 8days of incubation in shaker incubator 120rpm
at 30 0 C.
The ability of these isolates to induce the color change in the media is presumably due to a reduction of the indicator by oxidized products of hydrocarbon degradation or due to the fungal growth, which utilizes oxygen for their metabolism (9).

B-Estimation of biomass method
The results showed variation in the capacity of the fungal isolates in the consumption of anthracene in the liquid mineral salts media. It was found that the pink colony (E. nidulans) was the best isolate for anthracene degradation with the rate of 84% and yield maximum biomass 3.7gm/l, while the yield maximum biomass of yellow colony (A. flavus) was 1.2gm/l, with the rate of anthracene degradation reaching to 77% (Table 2). Results showed that there is a relationship between rates of anthracene consumption concentrations and biomass production. E. nidulans was the best isolate that decreases anthracene concentration and produces maximum biomass. Garapati and Mishra (18) were used Penicillium sp. and Aspergillus versicolor upon growth in liquid medium containing 500ppm of crude oil, they recorded that these isolates increase in biomass (2.3 and 2.56)gm/l, respectively, according to decrease in crude oil concentration with a rate of degradation (76.5 and 78.4)%, respectively.
Biomass of Stereum ostrea and Phanerochaete chrysosporium growing in liquid medium contains 1% anthracene under shaking conditions, were initially slow for 4days, then picked up and remained steady at 8days of incubation. Stereum ostrea produced maximum biomass of 1.89gm/flask (25ml) after 8days of incubation with anthracene degradation 62.3%, while the fungus Phanerochaete chrysosporium gave 1.78gm/flask (25ml) with anthracene degradation 56.6% over the same period of incubation (19).