Bioremediation & Biodegradation Biodegradation of Natural Rubber Latex of Calotropis procera by Two Endophytic Fungal Species

In this study eight species related to Aspergillus (3 species), Fusarium (1), Penicillium (1), Emericella (1), Nigrospora (1) and trichoderma (1) were isolated from leaves and latex of Calotropis procera . Only P. chrysogenium and A. niger were able to grow on natural rubber but other species were not. The degrading ability of ( Penicillium chrysogenum and Aspergillus niger ), isolated from latex of calotropis procera was assessed. The degradation of rubber latex was determined by measuring the increase in protein content of the fungus (mg/g dry wt), reduction in molecular weight (g/mol) and inherent viscosity (dl/g) of the latex. Moreover, the degradation was also confirmed by observing the growth of these species strain using scanning electron microscopy.


Sampling, fractionation and preparation of natural rubber latex
The latex of Calotropis procera plant was obtained by taping method. Fresh latex was centrifuged at 17,000 rpm for 20 min at 4°C in SR4000 Prolabo centrifuge (France). latex was separated into three layers: a sticky top layer containing Natural Rubber; clear middle fraction of serum, and a small quantity of lipids at the bottom of the tube (Figure 1).
The natural rubber fraction was separated and washed three times by deionized water to remove impurities and dried at 30°C for 24 h, A known weight (20 mg) of the prepared natural rubber was dissolved in 2 ml of tetrahydrofuran (THF), then its molecular weight was determined by gel permeation chromatography (GPC) [9,13].
Isolation of endophytic fungi from C. procera leaves and latex, calculation of colonizing frequency and Identification of endophytic fungi were done by the current authors according to these papers [13,21,22].
Methods used for measuring the capabilities of endophytic fungi to degrade pure natural rubber Growing of isolates on solid media coated with natural rubber film for 15 days of incubation in tetrahydrofuran (THF) (20 mg/2 ml THF), and spotted on to agar media (each 50 µl/colony), then left in an oven at 30°C until THF is evaporated and the NR became white in appearance. Fungi were inoculated with tooth pick on the top of pure natural rubber (NR) spot, incubated for 15 days at 28°C. The diameters of colonies were measured, then fungi which grow well in solid media Growing of in liquid media for measuring biodegradation ability after 30 days. At the end of incubation period the rubber pieces and microorganisms were separated by centrifuging at 10000 rpm for 15 min, microorganisms (bottom layer) were subjected for protein determination while natural rubber pieces (top layer) were collected, washed with deionized water, and dried at 30°C and then subjected for analytical methods (molecular weight and viscosity) as well as SEM photography has been described before [13,22].

Protein determination
The total protein pellet was determined according to the method adopted by Lowry et al. [27].

Molecular weight determination by Gel Permeation Chromatography (GPC)
A known weight of natural rubber was dissolved in 2 ml THF and passed through a 0.45 μm-pore size filter. Gel permeation chromatogram of the the model Agilent technologies 1100, Germany (present in National Research Center, Doki, Cairo) was used. THF was used as the eluent with a flow rate 1 mL / min. Polymethylmethacrylate and polystyrene standards were used to calibrate the columns. The refractive index detector 10 4 A°, Colum PL gel Micrometer 100; 10000; 100000. The GPC apparatus was run under the following conditions: flow rate=2.000 ml/min., injection volume=100.000 μl, sample concentration=1.000 g/l. The values of molecular weight were calculated by means of a computer program [13,22].

Scanning Electron Microscopy (SEM)
The morphological changes during growth of the organism on natural rubber was also studied by scanning electron microscopy. The inoculated samples as well as the control were fixed overnight in glutaraldehyde 5%, then dried at 50°C. The samples were mounted on metal stubs and coated with gold and palladium (Jeol JFC1100E Iosputtering Device). Micrographs were taken with a Joel JSM-4500 LV electron microscope operating at 15 KV in Assiut University, electron Microscopy unit [13,22].

Isolation and identification of fungi
Six hundred and seventy-five segments of leaf and 15 latex sample of C. procera were processed for the isolation of endophytic fungi. Eight species related to 6 genera were isolated and these were Aspergillus niger, A. fumigatus, A. flavus, Emericella nidulans, Fusarium oxysporum, Nigrospora oryzae, Penicillium chrysogenum and Trichoderma harzianum. Isolation of endophytic fungi from C. procera leaves and latex, calculation of colonizing frequency and Identification of endophytic fungi were done by the current authors (data not shown).

Growth of mesophilic endophytic fungi on solid media coated with natural rubber film after 15 days of incubation
Eight isolates (representative for the species isolated) were tested for their ability of growing on sugar-free Czapek agar coated with thin film of pure natural rubber (3 replicate agar plates for each isolate).

Biodegradation abilities of Penicillium chrysogenum and Aspergillus niger grown in liquid media after 15 and 30 days of incubation
Fungal protein, viscosity and molecular weight of P. chrysogenum: Protein content of P. chrysogenum grown on natural rubber increased significantly from 2.71 mg/g mycelial dry weight after 15 days to 5.23 mg/g after 30 days of incubation in liquid Cz medium. On the other hand rubber viscosity decreased significantly from 6.0 dl/g after 15 days to 5.5 dl/g after 30 days of incubation.
Molecular weight of natural rubber (that was previously inoculated with P. chrysogenum), decreased from 8.08 × 10 4 (in control medium) to 4.88 × 10 4 g/mol after 30 days (degradation rate of P. chrysogenum after 30 days=40.0% comparable with the control (Table 1; Figure 3).

Fungal protein, rubber viscosity and molecular weight of A. niger:
Protein content for A. niger grown on natural rubber increased significantly from 1.71 mg/g mycelial dry weight after 15 days to 4.22 mg/g after 30 days. On the other hand rubber viscosity decreased significantly from 6.2 dl/g after 15 days to 5.9 dl/g after 30 days of incubation.

Molecular weight of separated natural rubber inoculated with
A. niger, decreased from 8.08 × 10 4 to 5.82 × 10 4 g/mol after 30 days (degradation rate of A. niger after 30 days=28.0% comparable with the control (Table 1, Figure 3).

*Values
Significant at PC 0.05 level.
In this respect, Ismail et al. [13], showed high degradation rate of poly (cis-1, 4-isoprene) rubber ( Figure 4) by Aspergillus terreus 31.04% and A. flavus 28.73% after 30 days and comparable with the control. The ability was also determined by measuring the increase in protein content of each fungus, reduction in molecular weight and inherent viscosity and observing the growth using scanning electron microscopy (SEM).    Roy et al. [17] stated also that significant decrease in the molecular weight of the natural rubber was treated with Aspergillus sp. after 20 weeks (MW 2.70 × 10 5 in the control to 1.90 × 10 4 ). Indicating that Aspergillus sp. was able to degrade the natural rubber.

Scanning Electron Microscopy (SEM)
The 15 and 30 days old inoculated natural rubber samples with P. chrysogenum and A. niger were photographed using scanning electron microscopy (SEM). These SEM microphotographs clearly indicate the colonization of the fungus on the natural rubber surface (Figure 3).
In this respect, the colonization and/or growth on the rubber surface using by SEM micrographs have been observed for Aspergillus terreus, Aspergillus flavus and Myceliophthora thermophila [13], Aspergillus sp. and Pseudomonas sp. [17].

Conclusion
The degradation process of natural rubber of C. procera by P. chrysogenum and A. niger were proven by the increase in protein content, decrease in both molecular weight and inherent viscosity, and by observing the fungal growth on the surface of natural rubber using scanning electron microscopy (SEM). The current results demonstrate that the degrading fungal isolates could be used as promising tools for the removal of disposal of rubber products.

Analysis parameters
Growth diameter (cm) Protein content mg/g dry wt.
Viscosity measurements dl/g Molecular weight g/ mol × 10 4 X ± SD X ± SD