INTRODUCTION:

Natural Rubber (NR) is a biopolymer (poly-1, 4-cis-isoprene) produced by many plant species mostly belonging to Euphorbiaceae or Compositae family and by fungi¹. It has been commercially produced from the latex of tree Hevea brasiliensis (rubber tree) at about several million tons per year². NR contains 90% rubber hydrocarbon along with small amount of proteins, resins, fatty acid, sugars and minerals¹. The chemical name of Natural Rubber is poly-isoprene, since it is a polymer composed of many C₅H₈ isoprene (2-methyl-1, 3- butadiene) units, each containing one double bond in the cis configuration and linked at C1 and C4³. The average composition of the NR latex is 25-30% poly-isoprene, 1-1.8% proteins, 1-2% carbohydrates, 0.5-0.6% polar lipids, 0.4-1.1% neutral lipids, 0.4% amino-acids, 0.4-0.6 inorganic components, etc., and other 50-70% water⁴.

The total rubber production worldwide increased to 26.4 million tons in 2012, a rise of 11.9% from 21.7 million tons in 2009, which comprised of 42.5% NR production and 57.8% production of synthetic rubber⁵. The different products are manufactured by vulcanization of natural rubber. During the manufacturing process, other chemical additives are added along with the vulcanized rubber⁶. The human population is growing exponentially and the products of rubber are widely used in our day to day life which has led to the accumulation of huge amounts of non-degradable residues across our planet over a long period of time affecting the potential survival of many species⁷.

The solution to reducing this issue is to recycle the used products but due to the cross-linking formed during vulcanization the recycling becomes a bit difficult because unlike polythene, it is impossible to simply melt and reshape the NR derived products⁸. The alternative method to overcome the environmental problems is provided by microbial degradation of rubber and its products without causing any deleterious effects on the environment.

MATERIALS AND METHODS:

Collection of Rubber Sample and Microorganism:

The NR sheet (sample) was collected from the Rubber processing unit (Jain Trading Co., Jalandhar) and was carried in the plastic bag to the laboratory of Lovely Profession University to investigate the degradation of NR by the microorganisms.

Vial culture of fungus, Aspergillus niger (NCIM 501) and Phlebia radiate (MTCC 2791) were procured from National Chemical Laboratory and Institute of Microbial Technology, Chandigarh, India respectively.

Freeze-dried culture of bacteria,Pseudomonas aeruginosa (MTCC 4673) and Streptomyces coliecolor (MTCC 1139) in the vacuum sealed glass ampoule were procured from Institute of Microbial Technology, Chandigarh, India.

Revival of Microorganisms:

A fresh fungal culture was prepared by adding a loopful of culture from vial of Aspergillus niger (NCIM 501) into the sterilized potato dextrose broth. The fungus was also grown on potato dextrose agar and was kept in the incubator at 25°C for 4-5 days.

The fungus Phlebia radiata (MTCC 2791), white rot fungi, was revived from the procured vial culture on its specific growth medium malt extract broth and was kept for incubation at 25°C for 10-14 days.

A fresh bacterial culture of Pseudomonas aeruginosa (MTCC 4673) was prepared by streak plate method on cetrimide agar and was placed in an incubator for incubation at 37°C for 2-3 days.

Streptomyces coliecolor (MTCC 1139), was revived from the culture on its specific growth medium CM broth and was kept for incubation at 25°C for 5-7 days. A number of bacterial colonies were isolated using spread plate and a few were picked and pure colonies were isolated by streak plate method by incubating at 25°C for 2-3 days.

Screening of Natural Rubber degradation by using Mineral salt medium (MSM):

Natural Rubber degrading ability of microorganisms were checked in the laboratory conditions by growth experiment in mineral salt medium (MSM), where NR was used as sole source of carbon⁸. NR sheet was sliced into small pieces (20 mm²) to provide more surface area for better degradation.

Five different conical flasks containing MSM were used. In the first flask, only NR piece was added, used as a control. In the second flask, NR pieces were inoculated with Aspergillus niger, In the third flask, NR pieces were inoculated with Pseudomonas aeruginosa and kept for incubation of 90 days on rotary shaker incubator at 27±2°C for fungus and 25°C for bacteria. In the fourth flask, NR pieces were inoculated with Phlebia radiata and in the fifth flask, NR pieces were inoculated with Streptomyces coliecolor and kept for incubation of 45 days on rotary shaker at 27±2°C for fungus and 25°C for bacteria.

After an incubation period, natural rubber pieces were removed and degradation was identified by weight loss after washing and drying at 50°C in hot air oven for 24 hours.

Confirmation of NR degradation by staining with Schiff’s reagent:

Polyisoprene, a polymer of isoprene, is the primary chemical component of natural rubber in the form of cis-1,4-poly-isoprene. Due to the degradation of cis-1,4-poly-isoprene, the aldehyde and ketone will be released. Schiff’s reagent is utilized for the detection of aldehyde and ketone group. The reaction of Schiff’s reagent with aldehyde and ketone group developed the purple color which determines the degradation of natural rubber polymer.

The confirmation for degradation of cis-1,4-poly-isoprene rubber hydrocarbon chain was obtained by staining treated Natural Rubber pieces with 10 ml of Schiff’s reagent in a tightly stoppered bottle and kept for incubation for 10-30 minutes at room temperature⁸.

Confirmation of NR degradation by Fourier Transform Infrared spectroscopy (FTIR):

The chemical changes occurring on the NR surface as a result of degradation process was confirmed by FTIR analysis⁹.

Confirmation of NR degradation by Scanning Electron Microscopy:

The evidence for degradation of cis-1, 4-poly-isoprene NR hydrocarbon chain was obtained by observing the Natural Rubber pieces under SEM. NR pieces incubated in MSM, which was subjected to degradation, were coated with gold prior to analysis. They were observed under Scanning electron microscope¹⁰. SEM analysis was done at SAI Labs, Thapar University, Patiala, Punjab.

RESULTS AND DISCUSSION:

Weight loss of microbial treated NR pieces:

Weight loss was observed after incubation. NR samples were removed, washed and dried in hot air oven at 50°C for 24 hours. The difference in the final and initial weight was observed and the loss percent was calculated.

Confirmation of rubber degradation by Schiff’s reagent:

The degradation of the Natural Rubber was confirmed by the Schiff’s test. There was no color formation in the natural pieces of control flask after the treatment with Schiff’s reagent. The formation of the Purple color in the NR sample inoculated with fungi is due to the presence of aldehyde and ketone group which is produced by the degradation of poly-isoprene units.

Fig 1(a)

Fig 1(b)

Figure 1: Schiff’s test: (a) Aspergillus niger and (b) Phlebia radiata inoculated NR Pieces

Fig 2(a)

Fig 2(b)

Figure 2: Schiff’s test: (a) Pseudomonas aeruginosaand (b) Streptomyces coliecolor inoculated NR Pieces

Confirmation of NR degradation by FTIR:

The treated NR pieces were subjected to FTIR studies, peaks were observed at the wavelength between 2,858.60 and 1,514.17 cm^-1 having H–C=O:C–H stretch and C=O stretch, which indicates the presence of aldehydes and ketones produced as a result of NR degradation in the treated sample which is absent in NR control, thus confirms the NR degradation.

The comparative analysis of NR control and microbial treated determines the NR degradation. The shifting of FTIR peaks is visible in treated NR samples, in case of Aspergillus niger treated NR sample (fig 4), aldehyde H–C=O:C–H stretch is found at 2733.22 cm^-1 which lie between the 2830-2695 cm^-1 aldehyde IR range and ketone C=O stretch is found at 1699.34 cm^-1 which lie between 1680-1700 cm^-1 ketone IR range. In the case of Phlebia radiata treated NR sample (fig 5), the aldehyde stretch is found at 2731.29 cm^-1 and ketone stretch is found at 1799.65 cm^-1 which lie in the IR range of 1705-1850 cm^-1. The aldehyde and ketone stretch found in the treated NR sample indicates the presence of aldehyde and ketone produced as a result of NR degradation.

In case of Pseudomonas aeruginosa(fig 6), strong peaks at 1680.05cm^-1and medium two peaks at 2733.22cm^-1were obtained showing the presence of carbonyl with (C=O) stretch and an aliphatic aldehyde with (C=O) stretch and aldehyde with (=C-H) stretch respectively.

In case of Streptomyces coliecolor(fig 7), strong peaks were obtained at 1739.85cm^-1and medium two peaks at 2731.29cm^-1 were showing the presence of carbonyl with (C=O) stretch and aldehyde with (C=O) and (=C-H) stretch respectively.

Figure 3: FTIR of NR control

Figure 4: FTIR of NR treated with Aspergillus niger

Figure 5: FTIR of NR treated with Phlebia radiata

Figure 6: FTIR of NR treated with Pseudomonas aeruginosa

Figure 7: FTIR of NR treated with Streptomyces coliecolor

Confirmation of NR degradation by Scanning Electron Microscopy (SEM):

NR pieces were observed under SEM (JEOL, JSM-6510LV) to analyze biodegradation. The surface morphology was smooth in control NR piece when observed under SEM. While as variation in surface morphology was observed in treated NR samples. The formation of cavities and biofilm formation were observed at 1500X and 1000x. which indicates that NR pieces were degraded by the microbial treatment.

Figure 8: SEM image of NR control (1500x)

Figure 9: SEM images of NR treated with Aspergillus niger showing degradation (1000X)

Figure 10: SEM images of NR treated with Phlebia radiata showing degradation (1500X)

Figure 11: SEM images of NR treated with Pseudomonas aeruginosa(1000X)

Figure 12: SEM images of NR treated with Streptomyces coliecolor(1000X)

DISCUSSION:

In the present study, degradation of NR pieces is carried out by the rubber degrading micro-organisms. The results unveiled that fungal; Aspergillus niger and Phlebia radiataand bacterial species Pseudomonas aeruginosaand Streptomyces coliecolor are capable of degrading the NR. The degradation of Natural Rubber was studied by conducting the growth experiment in MSM and the degradation was confirmed by staining method, weight loss, SEM and FTIR analysis. In the period of 90 days, both the fungal and bacterial species have shown partial degradation of natural rubber (table 1). Comparatively maximum degradation was shown by fungi, Aspergillus niger, which were observed 27.27% on the scale of natural rubber weight loss. Minimum degradation was done by bacteria, Pseudomonas aeruginosa, which were observed 15.38% on the scale of natural rubber weight loss.

This result explained that fungus has been shown better degradation capability over bacterial strain. The staining with the Schiff’s reagent revealed that sample NR pieces turned purple and there was no color formation in the control. The formation of the color in the NR sample is due to the presence of aldehyde and ketone group which is produced by the degradation of poly (cis-1,4-isoprene) units. G Nayanashree study⁸on biodegradation of NR showed the similar result. In their experiment. Aspergillus niger was shown 28.3% in weight loss of natural rubber after the three months of incubation. They got similar results of Schiff’s test and SEM analysis which confirmed the degradation of rubber.

The SEM results, fig 8,9,10,11 and 12, showed that there were morphological changes on the surface which were confirmed by the formation of cavities and bio-film formation which indicated NR samples were degraded. The degradation of NR was also confirmed by the FTIR study which detects the chemical changes in the degraded NR sample. Similar attempt has been done¹¹to study microbial degradation of poly-isoprene rubber. They screened various microbes to check their ability to degrade poly-isoprene rubber (NR latex gloves). The analysis of degradation was done through SEM and FTIR spectroscopy showed changes in surface morphology, like the appearance of pits and cracks, and marked difference in transmittance spectra of sample and control due to changes in the functional groups.

CONCLUSION:

NR products are used very commonly in our daily life. During manufacturing of rubber products, some chemical additives are added along with the vulcanized rubber to make them temperature resistant. After using these products, their disposal is world-wide solid waste problem which gets accumulated in the environment and show its adverse effect. An alternative way to overcome this problem is to subject these products to microbial degradation. From this study, it is concluded that Aspergillus niger, Phlebia radiata, Pseudomonas aeruginosa and Streptomyces coliecolor are rubber degrading microorganisms. They can be explored to degrade NR effectively without causing any deleterious effects on the environment and Aspergillus niger as the potent degrader of Natural Rubber.

ACKNOWLEDGEMENT:

The work was supported by Lovely Professional University, Phagwara, Punjab, India. I would like to thank SAI Labs, Thapar University, Patiala, Punjab, India for supporting us to complete SEM work.

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Received on 21.06.2017 Modified on 24.07.2017

Research J. Pharm. and Tech 2017; 10(11): 3939-3944.

DOI: 10.5958/0974-360X.2017.00715.6

S. No	Fungal Strain	No of Days	Initial weight (gm)	Final weight (gm)	Weight Loss (gm)	Weight Loss in %
1	Aspergillus niger	90	0.77	0.56	0.21	27.27
2	Phlebia radiata	90	0.70	0.54	0.16	22.85
3	Pseudomonas aeruginosa	90	0.52	0.44	0.08	15.38
4	Streptomyces coliecolor	90	0.66	0.52	0.14	21.21