Isolation and characterization of bacteria from domestic and industrial waste materials for biopolymer production

Abstract

Dependence on conventional plastics and their boundless usage have resulted in waste accumulation and greenhouse gas emissions. Recent technologies are directed towards the development of bio-green materials that exert negligible side effects on the environment. A biologically-synthesized plastic, polyhydroxybutyrate (PHB) has been attracting major interests due to its similar physical properties to synthetic plastics. Unlike synthetic plastics, PHB is produced from renewable resources and is degraded aerobically by microorganisms to CO2 and H2O upon disposal. PHB is a renowned biodegradable plastic that do not release any toxins or residues in the environment like petroleum based plastics. The present study was undertaken to isolate potential indigenous PHB producing bacteria from different waste materials. The isolated bacteria were tested for desired PHB production along with their biotechnological applications. The aerobic heterotrophic bacterial load of the collected samples ranged in between 5.50×103 and 2.52×107, 3.10×104 and 2.23×107, 7.50×103 and 1.01×107 cfu/g or cfu/ml in NAG (nutrient agar with 1% glucose), PYG and LB media, respectively. The maximum bacterial count (2.52×107cfu/g) was observed in soil of BDR market and minimum (5.5×103cfu/ml) in garments waste water. The highest number (33) of PHB producing bacteria was found in kitchen waste soil and the lowest number (9) was in the Turag River water. Desired indigenous bacteria isolated from different waste materials were screened for maximum PHB production. On the basis of PHB activity, 30 isolates showed better performance among the isolates. Screening for PHB was done by Sudan black B and tested under microscope using sudan black staining. Both Gram positive and Gram negative PHB positive bacteria were found to be associated with studied samples. Among 30 isolates, 21 were Gram positive rods and 9 were Gram negative rods. Gram positive rods were provisionally identified as Bacillus spp. while Gram negative bacteria were identified as Acetobacter, Enterobacter, Klebsiella, Neisseria, Pseudomonas, Rhizobium and Tatumella. Potential 10 isolates were further studied for molecular identification. Among them, 9 genera were matched with their conventional identification except Rhizobium leguminosarum.

Molecularly identified 10 isolates were selected for quantitative PHB activity. The PHB producing capabilities of the isolated indigenous 10 isolates ranged in 3.43 ± 0.24 and 79.75 ± 6.10 μg/ml. All the isolates showed maximum PHB activity after 72 h of incubation except Bacillus megaterium LY6, which showed the highest PHB activity after 96 h of incubation. All the isolates showed decreased PHB production after 72 h of incubation with the increase of the incubation period. Among 10 isolates, Bacillus cereus HB45 showed the highest PHB activity (79.75 ± 6.10 μg/ml) at 72 h of incubation before optimization. Two isolates viz. Bacillus cereus HB45 and Bacillus thuringiensis B43 could produce PHB more than 70 μg/ml among the selected isolates and were optimized for PHB production. Before optimization these isolates could produce PHB 79.75 ± 6.10 and 72.72 ± 6.59 μg/ml, respectively. Through optimization, PHB production increased up to 867.17 ± 7.35 and 955.41 ± 7.35 μg/ml by B. cereus HB45 and B. thuringiensis B43. Before optimization B. cereus HB45 was better than B. thuringiensis B43 but after optimization B. thuringiensis performed better than B. cereus. Characterization of extracted PHB was carried out by FT-IR, which later confirmed the presence of intracellular accumulated polymer and substantiated as PHB. PHB films were produced by Bacillus cereus HB45 and Bacillus thuringiensis B43 using best carbon and nitrogen source. The PHB films produced were very stiff and brittle in appearance and were found to be fragile. Use of bioplastics produced by these bacteria will be helpful to reduce environmental pollution. The result clearly reflected that biotechnologically these two isolates could be useful and have got potential for commercial applications.