Biodegradation of Pretreated Vulcanized Rubber By Microorganisms

Abstract

Vulcanized rubber products mainly tyres are recalcitrant after their disposal as it persists in the environment due to the presence of different additives in its composition, making it difficult to degrade and presents it as environmental pollutant. Biodegradation of waste tyre rubbers can be a solution for handling the spent tyre rubbers. Main focus of the present study was to check the effects of pretreatments on the biodegradation of vulcanized rubber under different treatment conditions. Screening of vulcanized rubber degrading microbes was performed through enrichment technique and six bacterial strains were isolated from soil and identified through 16S rRNA as Bacillus cereus NK1, Pseudomonas aeruginosa NK2, Xanthomonas sp. NK3, Bacillus badius NK4, Leucobacter komagatae NK5 and Alcaligenes sp. NK6. On the basis of growth of each bacterial strain, it was noticed that Bacillus cereus NK1 showed the best growth in liquid MSM having tyre rubber pieces as the only carbon source in the medium. Bacillus cereus NK1 was selected for optimizing some of the physicochemical parameters further. Different physical (pH, Temperature, Size of inoculum) and biochemical parameters (Effect of different carbon sources and Effect of different nitrogenous sources) on microbial degradation of tyre rubber were optimized for Bacillus cereus NK1. Maximum growth was observed at 35°C for the Bacillus cereus NK1. Remarkable changes were noted in the FTIR spectrum of rubber samples incubated at 35°C with Bacillus cereus NK1. Eroded surface with cracks were easily seen with SEM photographs at 35°C. At pH 7 Bacillus cereus NK1 was showing better growth. Maximum protein concentration was observed for pH 7 on the 28th day of incubation as 332 μg/ml. Maximum changes were observed in the tyre rubber pieces incubated at pH7 as a number of new group were identified in the FTIR spectrum. The intensity of the peaks at 2847.01 cm-1 and 2913.58 cm-1 was decreased more indicating the degradation of the polymeric backbone of the tyre rubber pieces. Similarly, maximum growth was observed at day 28th and the amount of protein was found maximum on 21st day of incubation in case of 10% inoculum. When 10% inoculum was used, there were cracks and erosion on the surface of the tyre rubber pieces followed by 5% inoculum size which was showing less surface roughness. In case of 10% inoculum almost disappearance of peak at 1427.97 cm-1 was observed with the formation of peak at 1462.09 cm-1 and 1551.98 cm-1. The presence of organic carbon source in the medium was helpful in increasing the biodegradation of tyre rubber pieces more as compare to the inorganic source of extra carbon in the medium. Among all the carbon sources checked, sucrose showed the maximum growth density. An increase in the protein concentration was observed in case of sucrose on 28th day of incubation with the values of 1165 μg/ml. Scanning electron micrographs of test samples when compared with the control, it revealed that extra carbon source either organic or inorganic both gave more space for biodegradation of tyre rubber pieces to be degraded by Bacillus cereus NK1. Significant changes were observed in the spectral lines at 900-1700 cm-1 range. Maximum growth was observed in case of urea and protein concentration was 1080 μg/ml for the nitrogenous source urea on 21st day of incubation. By SEM analysis it was shown that presence of both either organic or inorganic nitrogenous sources in the medium increased the biodeterioration of rubber pieces by Bacillus cereus NK1. Spectacular changes in the region of 1500-1750 cm-1 were revealed in the FTIR spectra of tyre rubber treated in the presence of urea as an additional nitrogen source. Peaks at 1551.45, 1588.53 cm-1, 1688.38 cm-1 & 1725.67 cm-1 were observed showing the biodeterioration of the tyre rubber polymer by the action of Bacillus cereus NK1 in the presence of urea as an extra nitrogen source. Another bacterial strain Bacillus sp. S10 isolated previously from sewage sledge through enrichment technique in liquid mineral salt medium supplemented with polyisoprene rubber. The capability of isolate Bacillus sp. S10 to degrade tyre rubber was checked at different temperatures and pHs and it was observed that Bacillus sp. S10 showed better growth at pH7 and 35°C temperature in the presence of tyre rubber pieces as the only carbon source in liquid medium. Also Sturm test was conducted to check out the biodegradation of tyre rubber pieces by Bacillus cereus NK1 and Bacillus sp. S10 separately and in combination, in term of evolution of carbon dioxide. Degradation of tyre rubber pieces was tested after soil burial for a certain time period under ambient environmental condition. In the present study soil burial technique was used to determine the biodegradation of tyre rubber pieces. Pieces of tyre rubber were taken from the soil containers after 10 months of soil burial and were analyzed. SE micrographs of the tyre rubber pieces after treatment with the optimized physicochemical parameters of Bacillus cereus NK1 showed changes on the surface of tyre pieces. There was a notable change on the surface of rubber pieces treated with Bacillus cereus NK1, FTIR analysis of the controlled tyre rubber pieces and optimized conditioned Bacillus cereus NK1 treated tyre rubber pieces. Different pretreatments (thermal, UV, surfactant, chemical and biological) were given to the tyre rubber pieces and these pieces were then subjected to the shake flask experiments. Maximum growth was observed at day 21st for 100°C, followed by 80°C at 28th day of shake flask incubation with Bacillus cereus NK1. Very little changes were observed in case of thermal pretreatment at 40°C and 60°C. The results of thermal pretreatment of rubber at 100°C, revealed the appearance of new small peaks at 1736.06 cm-1, 1640.44 cm-1 and 1580.10 cm-1. Similarly, maximum growth was observed at 21st day after 15 hrs of UV pretreatment for vulcanized rubber. Dense colonization of the Bacillus cereus NK1 was clearly observed on the tyre rubber pieces irradiated for 15 hour for UV pretreatment, showing severe deterioration of the rubber pieces as the microbe grows to embed in the matrix of tyre rubber pieces. Maximum surface deterioration was shown by the T-80 pretreated tyre rubber pieces and incubated with the Bacillus cereus NK1. Three consecutive peaks at 1008.47 cm-1, 1033.12 cm-1 and 1076.90 cm-1 were also identified. Maximum surface degradation was observed in the case of DMSO. Surface rupturing and presence of holes were observed in the rubber samples pretreated with chloroform, methylated spirit and THF. In case of DMSO pretreated tyre rubber pieces, transmittance at 1428.25 cm-1, 2848.50 cm-1 and 2916.33 cm-1 decreased, showing the degradation of carbon and methyl group stretching. On comparison of tyre rubber pieces treated with DMSO with Bacillus cereus NK1 incubated DMSO pretreated tyre rubber pieces, peak shifting 2916.33 cm-1 to 2918.58 cm-1 was revealed. Peak intensity was decreased at 1015.40 cm-1, also disappearance of some peaks at 800-1000 cm-1 was observed. With the fungal pretreatment, followed by Bacillus cereus NK1 treatment, it was examined through FTIR spectra that the shifting of peak from 2915.29 cm-1 and 2847.51 cm-1 to 2918.06 cm-1 and 2850.06 cm-1; also the intensity of peaks were decreased due to the microbial treatment. When the pretreatment of tyre rubber pieces was done by fungal strain followed by Thiobacillus sp. and the shake flask incubation was performed with Bacillus cereus NK1. A wide peak appeared at 3275.12 cm-1. Shifting of peaks from 1535.72 cm-1 and 1642.85 cm-1 to 1542.34 cm-1 and 1626.39 cm-1 and formation of a new peak at 1723.59 cm-1 also occurred.