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International Journal of Environmental Research, Vol. 4, No. 3, July-September, 2010, pp. 519-524 Article Effective utilization and management of coir industrial waste for the production of poly-β - hydroxybutyrate (PHB) using the bacterium Azotobacter Beijerinickii Sathesh Prabu, C. and Murugesan, A . G . * Manonmaniam Sundaranar University, Sri Paramakalyani Centre for Environmental Sciences, Alwarkurichi - 627 412. Tamil Nadu, India Date of Submission: 11-Jul-2009 Code Number: er10058 Abstract Coir pith, a byproduct of coconut fibers and waste material from the coir industry, is stable and not easily degradable due to its high lignin content. Coir pith takes a decade to decompose thereby posing environmental hazard and disposal problem. Pollution by plastics creates an alternative solution to reduce problems. Hence the potential use of coir industrial wastes for production of bioplastics (Poly-(hydroxybutyrate - PHB) as an alternative to plastics was studied. Commercial production of this polymer is limited, however, due to higher cost and longer fermentation process as compared to petrochemical plastics. These concerned make it necessary to use the cheapest and readily available raw materials. Azotobacter beijerinickii used coir industrial waste as a substrate and produced PHB. Production of PHB was maximized at pH 6.5 with 3% coir hydrolysate. The amount of PHB produced by A. beijerinickii was 2.4 ± 0.2 g/L. The yield was 48.19 %. Production of PHB was confirmed by Sudan black B staining under a light microscope, acridine orange staining under a fluorescent microscope and by an infrared spectrometer. This investigation showed that coir industrial waste could be effectively used for the production of PHB.Keywords: Biopolymer, Bioplastic, PHB, Coir waste, Azotobacter beijerinickii Introduction Industrialization, urbanization, improper agricul-tural practices and various anthropogenic activities are responsible for pollution and loss of environmental qual-ity. Problems concerning the global environment have created much attention in developing eco-friendly prod-ucts (Rawte and Mavinkurve, 2002). Biopolymers are one product that can help to overcome problems caused by petrochemical polymers. Biopolymers are generated from renewable natural sources and are often biode-gradable and nontoxic (Poirier, 1999; Flieger et al., 2003). They can be produced by biological systems (microor-ganisms, plants and animals), or chemically synthe-sized from biological materials (sugars, starch, natural fats and oils, etc.) (Flieger et al., 2003). Polyhydroxyalkanoates (PHA) are a good substi-tute for plastics and elastomers. The PHAs have char-acteristics similar to petrochemical plastics but are bio-degradable (Anderson and Dawes, 1990; Doi, 1990a). PHAs are polyesters, which are accumulated as energy and/or carbon storage materials by numerous microor-ganisms, usually when a nutritional component such as nitrogen, phosphorus, sulfur, oxygen, and magne-sium is limited in the presence of an excess carbon source (Anderson and Dawes, 1990; Lee, 1996; Page, 1992; Ribera, et al., 2001; Wang and Lee, 1997). At present, they are produced by microbial fermentation; in the future, production will also be possible by in vitro methods or by agriculture using transgenic plants (Steinbuchel and Fuchtenbusch, 1998). In case of mi-croorganisms, PHA serves as a carbon and energy source during starvation and is rapidly oxidized thereby retarding the degradation of cellular compo-nents (Rawte and Marinkurve, 2001).Poly-(-hydroxybutyrate (PHB), the most commonly found member of the PHA family, is a thermoplastic with many desirable properties. It is biodegradable and the cur-rent market demand for a biodegradable thermoplastic is enormous (Lafferty, et al., 1988 and Khanafari, et al., 2006). The physical properties of the homopoly-mer of PHB are similar to those of polypropylene (melt-ing point, crystallinity, glass transisition and tempera-tures), and represents a stiff and brittle material (Thakor et al., 2006). They can be used in a wide variety of products including containers, bottles, razors and food packaging materials. The latex of PHAs can be used to produce a water-resistant layer for paper, film or cardboard (Hocking and Marchessault, 1994). PHA/ PHB can be very useful in medical applications, such as implants, gauzes, suture filaments, osteosynthetic materials, and a matrix material for slow release drugs and in vitro cell cultures (Zinn et al., 2001; Sudesh, 2004; Chen, 2005; Chen and Wu, 2005a,b; Sudesh and Doi, 2005; Suriyamongkol et al., 2007). Because of the versatile applications as thermoplastic biopolymers, PHA can also be used for natural fiber composites, or as binder in paints, and various applications in medi-cine and pharmacy (Shanks et al., 2004). Though several organisms that accumulate PHA have been identified, commercial production of this polymer is limited. Several factors influence the eco-nomics of biodegradable polymer production. One main factor is cost of the substrate. The ability to pro-duce biodegradable polymers from inexpensive and renewable carbon sources may improve the econom-ics of the process and lower production cost (Hanggi, 1995; Khanfari et al., 2006). One of the cheapest raw materials is coir pith, byproduct of coir processing in-dustry that produces coconut fibers. Large amounts of coir pith accumulate nearby coir processing units (approximately 7.5 million tones annually in India), caus-ing severe disposal problems, fire hazards and ground water contamination due to the release of phenolic compounds (Namasivayam et al., 2001). Coir pith con-tains 87% of organic matter and 13% of ash content (Thampan, 1987). So recycling of industrial wastes is one way of disposal mechanism and another may be resource management. In the present study, to evalu-ate the potential value of coir pith, PHB has been pro-duced from coir industrial waste applying a bacterial species, A. beijerinckii. Materials and Methods A. beijerinickii MTCC 2641 was procured from the Microbial Type Culture Collection and Gene Bank of Institute of Microbial Technology, Chandigarh, In-dia for the production of PHB. A. beijerinckii was grown in the selective medium containing: 0.2g KH 2 PO 4 ; 0.8g K 2 HPO 4 ; 0.2g MgSO 4 ; 0.1g CaSO 4 ; 0.59g Yeast extract; 20g sucrose; FeCl 3 (trace) at 37°C for 24 h. Flasks containing 50 mL of specified medium were inoculated with a loopful of cells. After 48h of growth at 37°C, the medium attained cloudiness and used as inoculum for the production of PHB. Coir industrial waste was collected from the coir pith processing industry located in Tenkasi (Tamil Nadu, India) in a sterile container. The waste material was washed water, sun dried for two days and dried at 80°C for over night in hot air oven. Then dried sample was ground to fine powder so the samples passed through a 35-mesh sieve. Concentration of cellulose, hemicellulose and lignin were estimated (Updegraff, 1969; Goering and Vansoest, 1970). Coir industrial waste was first partially delignified by autoclaving (120°C, 15 lb, 20min). After cooling, it was hydrolyzed by the enzyme (Cellulase, Hi-media). Twelve filter paper ac-tivity units (FPU) of cellulase were added per gram of substrate. The mixture was kept at room temperature (37 ± 2°C) for 24h. The amount of reducing sugar re-leased in the coir waste hydrolysate was estimated spectrophotometrically using dinitrosalicylic acid (DNS) reagent (Miller, 1959). Optimum concentration of the hydrolysate was determined by incubating 5% (V/V) of the culture with 1 to 5% coir waste hydrolysate. Optimum pH for the maximum growth of the organism was studied by incu-bating 5% (V/V) of culture into the specific medium with pH from 4.5 to 8.5. Growth of the organism was measured by determining the dry cell weight (DCW). Culture samples (10 mL) were centrifuged at 2,000 g for 10 min and the cell pellet was washed in deionized water (6,000 g for 5 min) and dried to constant weight (90°C, 24 h), cooled in a desiccator and weighed (Grothe et al., 1999). Culture of A. beijerinckii (5% V/V) was grown for the production of PHB in the medium con-taining the optimized concentration of hydrolysate and pH. The medium was supplemented with meat extract (1%) and NH 4 Cl (0.5%). Flasks were incubated at 37°C for 48 hrs. After incubation, PHB produced was extracted chemically. 10 mL of culture was centrifuged at 2,000 g for 10 min. The collected pellet was treated with 10 mL of sodium hypochlorite and the mixture was incubated at 30°C for 2 hrs. After incubation, the mixture was centrifuged at 2,000 g for 15 min and washed with deion-ized water, acetone, methanol respectively for wash-ing and extraction. After washing, the pellet was dis-solved in 5 ml of boiling chloroform; chloroform was evaporated by pouring the solution on sterile glass tray kept at 4°C. After evaporation the powder was collected for further analysis (Rawate and Mavinkurve, 2002; Kumar and Prabakaran, 2005). The dried PHB was weighed and the yield was defined as a percent-age of the dried PHB to DCW. PHB granules in the cells were determined by Sudan black B staining under a light microscope (Norris and Swain, 1971). PHB granules were determined by fluo-rescence staining method using acridine orange (Kumar and Prabakaran, 2005). For infrared analysis of PHB, 1 mg of sample was ground well with 10 mg of spectral pure anhydrous potassium bromide crystals and the powder was made into a pellet. The relative intensity of transmitted light energy was measured against the wave-length of absorption 4000 - 400 cm -1 using JOEL-FT IR-4000 plus double beam spectrometer. Data were subjected to a one-way analysis of vari-ance (ANOVA) and Tukey test using SPSS -11 pack-age to determine the level of significance of variation in bacterial growth and PHB production caused by the variables, coir hydrolysate concentration and pH. All experimental data were expressed as mean ± SD. Prob-ability (P) values of < 0.01was considered significant. Results and Discussion PHB, a biodegradable thermoplastic are accumu-lated by a wide range of bacteria as carbon/energy or reducing-power storage material (Salehizadeh and Loosdrecht, 2004). Their synthesis is seen as an at-tractive system for the sustained production of large amounts of biodegradable polymers at low cost (Poirier, 2002). Coir pith is a byproduct of the coir processing industry posing disposal problems (Christopher, et al., 2007). In the present study, we estimated the chemical composition of the coir pith. Coir contained 42.14 ± 3.2 % of cellulose, 43.26 ± 4.1 % of lignin and 0.86 ± 0.07% of hemicellulose. Lignocellulose is the most abundant constituent of coir pith and provides simple sugars on hydrolysis. The reducing sugar content in the coir in-dustrial waste hydrolysate was 30 ± 2.4 %.The compo-sition and properties of coir industrial waste vary de-pending on the maturity of the coconut, method of extraction and disposal, period between extraction and disposal. Coir pith obtained from fully mature nuts has higher amounts of polysaccharides and fewer water soluble salts compared with younger nuts (Savithri and Khan, 1994). A study of Murugesan and Ruby (2004) revealed that cassava starch industrial waste could be used for the production PHB employing mi-crobes such as R. eutropha and Arthrobacter viscosus. Kocer, et al. (2003) obtained PHB from corn oil acids and a mixture of glucose (15 g/L) and acetic acid (2.5 g/ L) employing Pseudomonas sp. and Alcaligenes eutrophus. Poirier (2002) summarized that the applica-tion of autoretransgenic plants for the PHB synthesis. In the present study, among the different concentra-tions of coir hydrolysate tested, the maximum yield was noted in medium containing 3% of the coir hydrolysate [Figure - 1]. The differences in DCW of A. beijerinckii due to hydrolysate concentrations were significant at the 1 % level (F=114.24; d.f=4; P< 0.01). A. beijerinckii showed maximum growth at pH 6.5 in which the highest DCW (g/100mL), 0.498 ± 0.04 was observed, where as it was 0.280 ± 0.04, 0.31 ± 0.06, 0.296 ± 0.02 and 0.198 ± 0.04 at the pH of 4.5, 5.5, 7.5 and 8.5 respectively. Optimiza-tion of fermentation conditions has been used to sub-stantially enhance yield and productivity of many bioprocesses (Chisti and Young, 1999). The pH values may have affected the bioavailability of some trace ele-ments; pH values that differed even slightly from the optimum value reduced culture performance (Grothe, et al., 1999). When R. eutropha MTCC 1285 was grown at pH 8.0 with sago effluents and molasses as raw materi-als, more PHB was produced as compared to produc-tion at pH 9.0 (Kumar and Prabakaran, 2005). In this study, the amount of DCW was optimized at pH 6.5. Yield of PHB (%) was calculated only from the DCW of the bacterial cells determined. The differences in DCW of A. beijerinckii due to pH were significant at 1 % level (F=21.02; d.f=4; P< 0.01). In the present study, production of PHB was quali-tatively confirmed by the formation of a thin layer on extraction with chloroform which may be due to the presence of interlinked PHB granules. In the absence of PHB production, such a film was not formed. It was weighed (dry) and the amount of PHB produced was calculated. Chloroform extraction has been widely used to recover PHB with a high degree of purity without polymer degradation (Ramasamy et al., 1994). PHB can be extracted with hypochlorite solution, which is a highly degradable procedure and decreases the mo-lecular weight of the isolated granules presumably by the removal of their outer layer (Winfered and Robards, 1973). In this study, A. beijerinckii produced 2.4 ± 0.2 g/L of PHB with the yield of 48.19 %. In the present study the coir waste hydrolysate was supplemented with meat extract for the maximum production of PHB because Khanafari, et al. (2006) ob-tained maximum production of PHB in cultures of A. chrococcum 1735 grown in meat extract supplemented whey broth medium under suitable conditions. In this study, for PHB production, cultures were not shaken because O 2 values increased and decreased yield (Katie, et al., 2003; Khanafari, et al., 2006). The increasing O 2 values by shaking at 122 rpm decreased PHB yield from 4.43 to 0.04 g/L by A. chroococcum (Khanafari et al., 2006). In the present study, Sudan Black B staining also clearly showed the presence of PHB granules. Light microscope investigations of the cells, stained with Sudan Black B provide easy detections of PHA in the cells. In Sudan black B staining, lipid inclusion gran-ules are stained blue - black or blue-grey, whilst the bacterial cytoplasm is stained light pink (Collee et al., 1989). Discrete granules of PHA generally occurred in the cytoplasm as inclusion bodies of irregular morphol-ogy with a diameter of about 0.2 - 0.5 nm (Vincenzini et al., 1990). Proteins and lipids associated with PHA gran-ules apparently originate from a 2 nm thick coat sur-rounded the granules.The proteins associated with PHA possess PHA synthease and depolymerase ac-tivities (Doi, 1990b). Production of PHB was identi-fied with fluorescence; cells became enlarged and the granules were observed. Normal cells were not en-larged. PHB granules exhibited a strong orange fluo-rescence which was stained by the Nile Blue A (Oste and Holt, 1982). FTIR spectrum of PHB was recorded in the range of 4000-400 cm -1 and spectroscopic analy-sis showed the presence of broad bands responding to the groups OH, C-O and C=O indicating the struc-ture similar to PHB [Figure - 2]. At low frequency, the functional groups can be identified by their characteristics vibration transmit-tance makes the FTIR spectrum the simplest, most rapid and often most reliable means for assigning a compound to a class. The FTIR absorption peak val-ues of PHB from A. beijerinckii ranged from 4000 - 3122.19/cm (Intra molecular H bond), 2238/cm - 2924/ cm (O - H stretching), 1710/cm (C = O stretching), 1401.03/ cm (O=H tertiary alcohol), 1076/cm (C-O stretching). PHB production from the organisms was confirmed under the FTIR spectroscopy (Mazarevica et al., 1984). The native PHB granules possess defi-nite structural features and their crystal structure could be revealed by FTIR absorption and under an electron microscope (Merrick et al., 1965). The FTIR spectrum analysis of the PHB product clearly reveals its purity. The absorbance peak values obtained were compared with the available literature values and confirmed the product as PHB. The peak values obtained in this study coincides with previous results (Ramsay, et al., 1994; Pal and Paul, 2002; Kumar and Prabhakaran, 2005; Oliveira et al., 2007). Conclusion The two factors increasing production cost of PHB are the substrate used for the polymer production and the down stream process. There are a number of coir industries in India. The waste material generated from their processing causes serious environmental haz-ards. Use of cheap carbon sources would bring down the polymer cost. Agricultural wastes like beet and cane molasses, malt extract, corn syrup, wheat bran and dairy wastes like cheese whey could be used for culti-vating bacteria accumulating PHA. The cost of the substrate can be lowered by using the lignocellulosic wastes as a substrate for PHB production. However, in the production of PHB, the cost of the enzyme to be used for hydrolysis (highly preferred process) must be reduced and their efficiency increased in order to make the process economically feasible. This study provided valuable information about the coir indus-trial waste utilization and as an inexpensive potential substrate for the production of eco-friendly plastic. Acknowledgements The authors gratefully acknowledge the authori-ties of Department of Chemistry, Manonmaniam Sundaranar University, Tirunelveli for the FTIR analy-sis. C.S.P. is thankful to the Jawaharlal Nehru Memo-rial Fund, New Delhi for providing financial support to conclude this work.[50] References
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