Isolation and characterization of biomass modifying enzymes for bioremediation and production of “green specialty products”

Abstract

Environmental pollution has been a major problem and poses many challenges to sustainable development to the chemical-based industrial development in both developed and developing countries. Leather industry which extensively uses lime and sodium sulfide in dehairing hides and skins is currently facing serious environmental pollution problems. Lime and sodium sulfide heavily contribute to increased odour, effluent toxicity, health hazards to the tannery workers, production of poisonous sludge with disposal challenges and blockage to sewerage pipes. Similarly, use of organic dyes in industries pose pollution problems in the form of colored wastewater discharge into water bodies resulting into high turbidity, increased chemical oxygen demand (COD) and reduced light penetration. Furthermore, dyes are not only recalcitrant and refractory pollutants but also toxic, mutagenic and carcinogenic. To address these concerns, the present research affords the opportunity to isolate crude alkaline protease enzyme from extremophile bacteria from Lake Bogoria, Kenya and apply the isolated enzyme in processing leather and bioremediation of industrial wastewater containing organic dyes as part of the process of developing biotechnology for solving environmental problems. Four (4) protease producing bacterial strains were isolated from Soil samples collected from different parts of Lake Bogoria in the Kenyan Rift Valley. Biochemical test and phylogenetic characterization indicated that the four isolates were all associated mainly with members of the Bacillus Cereus. The submerged fermentation process parameters such as pH, temperature, incubation time, different substrates and concentrations that influence production of protease enzyme were analyzed and optimized for large scale enzyme production. Decolorization and biodegradation of Malachite Green (MG) dye and Reactive Black 5 (RB5) dye was studied using crude alkaline protease enzyme from isolate 1-p. From the dynamic batch vii experiments carried out for dye decolorization, over 98% decolorization efficiency was achieved within 24 hours for MG using an initial dye concentration of 1.0 x 10-5M. Similarly, over 98% decolorization was achieved within 120 hours for RB5 using an initial dye concentration of 1.0 x 10-4M. Experimental results revealed that the decolorization process was highly dependent on contact time, initial dye concentration, aqueous solution temperature and pH. Biodegradation of dyes was monitored by UV-VIS spectrophotometer and the resultant metabolites confirmed by Thin Layer Chromatography (TLC), Liquid Chromatography–Hybrid Quadrupole Time of Flight Mass Spectrometry (LC–QToF-MS) and Gas Chromatography/Mass Spectrometry (GC - MS). The results obtained revealed that the enzymatic degradation of MG and RB5 by crude alkaline protease enzyme from strain 1-p resulted in complete mineralization and benzene ring-removal; the latter known to be responsible for the organic dye toxicity. Kinetic study results revealed that first-order kinetic equation was the appropriate equation to describe decolorization of both MG and RB5 dye. Michaelis-Menten kinetics, Lineweaver–Burk plot and Eadie-Hofstee plot models were used to establish the kinetic parameters for the dye decolorization process. Lineweaver–Burk plot provided the best theoretical correlation of the experimental data for the decolorization of both dyes. The maximum rate (Vmax) and Michaelis-Menten constant (Km) were found to be 17.70 mg l-1 h-1 and 124 mg l-1, respectively for MG dye and 3.975 mg l-1 h-1 and 548.06 mg l-1 for RB5, respectively using Lineweaver–Burk plot. The results provide evidence that the crude enzyme from Bacillus cereus strain 1-p is an effective and potential candidate for industrial wastewater treatment The efficacy of the isolated crude alkaline protease enzyme from Bacillus cereus strain 1-p to dehair cow hide and descale Nile perch (Lates niloticus) skin was investigated. Factors affecting enzyme dehairing process such as temperature, pH and incubation time were determined and viii optimized. Industrial demonstrations of large scale beam house dehairing process (trial) was successfully accomplished whereby a 21 kg full bull hide was 100% chemical free dehaired and degreased using the isolated crude alkaline protease enzyme within 8 hours compared to the conventional method which take between 2- 6 days. Furthermore, 10 pieces (9.5 kg) of Nile perch (Lates niloticus) skin were successfully descaled and degreased within 2 hours. 1.1 kg of quality hair and 0.98 kg of valuable scales removed during dehairing/descaling process were readily recovered while still in good condition suitable for further use. The envisaged recoveries of hair and scales should greatly contribute to the reduction of BOD, COD and TDS loads in effluents. Physical-mechanical characteristics of the resultant leather after full processing was analyzed and the results compared with the set Kenya Bureau of Standards (KEBS) standards. The results revealed that the quality of the final leather from bull hide was within the set KEBS standards. In view of the results obtained and considerations of environment protection, application of crude alkaline protease enzyme from Bacillus cereus strain 1-p is promising to yield a clean technology which completely eliminates the traditional use of sodium sulfide and lime in dehairing. These will greatly reducing the pungent smell which is characteristic of tanneries across the world without compromising the quality of leather. This can be seen as a means to producing ―green‖ leather through a ―green leather‖ process. Furthermore, the same enzyme can be utilized in bioremediation of organic dye in wastewater effluent.