Environment Friendly Leather Making and Recovery of Value Added Products from Solid Tannery Waste

Leather industries contribute substantially to the environmental pollution due to huge quantum of solid and liquid waste generated. It is a chemical processing industry facing major environmental challenges, with most crucial one of waste minimization. Chemicals used in leather processing steps i.e., tanning are mostly health hazardous posing serious threats to ecosystem. Research is under way to develop alternative low cost, low waste, cleaner process technologies by reducing the pollution and chemical load on the environment. Intensive efforts are being directed towards use of enzymes as plausible alternative technology for soaking, dehairing and bating processes. Use of cheaper, less toxic and biodegradable proteases for dehairing may reduce or completely eliminate the use of sodium sulfide producing equally fine quality leather. Therefore, the aim of this current research work was to produce bacterial proteases that can be used for dehairing goat skin and hides as an alternative environment friendly process that may replace conventional method of using sodium sulfide in larger amounts. In the present study, thirteen indigenous bacterial strains were isolated from soil samples of Pakistan and screened for their proteolytic potential by measuring zone sizes of protein hydrolysis on nutrient agar plates with 1% casein as substrate. Bacterial strains RM1and ASM1 produced largest zone sizes of hydrolysis of 29mm and 22mm respectively on 1% casein agar plates after flooding with 10% TCA in qualitative protease assay and were selected for further studies. Based on morphological, biochemical and 16S rRNA gene analysis these two strains ASM1 and RM1 were taxonomically characterized as Bacillus cereus and Bacillus subtilis with the nucleotide sequences submitted in NCBI database under accession number KJ600795 and KT004404 respectively. Both strains were cultured in shake flasks and different nutritional and cultural parameters were optimized by one factor at a time strategy in the batch experiments for maximum protease activity. Five different media with different protease substrates were assayed for higher enzyme production. Bacillus cereus ASM1 grows best in M4 medium with beef extract as protein substrate with 8.7 U/mg specific activity. The alkaline pH value of 9 was suitable for maximum protease activity along with 1% glucose as C-Source, inoculum size 2% (v/v ratio) at mesophilic temperature range (30-37⁰C). Maximum enzyme production with a specific activity of 9.58 U/mg was observed after 48 hours of incubation at optimized conditions for ASM1. For Bacillus subtilis RM1, maximum proteases production with a specific activity of 9.82 U/mg was achieved in M3 medium with malt extract as main carbon and nitrogen source. Other optimized conditions include 1% dextrose as best C-Source in 1% inoculum size at pH 6-7 after 48h incubation. Crude enzyme from both of the strains produced at optimized conditions of enzyme production was used in dehairing application of goat skin. Results for dehairing suggested that RM1 was having better efficacy than ASM1, showing complete removal of hairs after time of 24 h incubation. ASM1 can be used in degradation of trimmings and shavings from untanned hides but collagenolytic nature makes it unsuitable for dehairing. RM1 shows ability to produce thermo-tolerant proteases in bulk quantity by growing at high temperature up to 55oC as it was isolated from the soil bank of a hot water spring of Kashmir, Pakistan. Enzyme from Bacillus subtilis RM1 was purified by two step purification process after ammonium sulfate precipitation and gel filtration column chromatography. Overall yield of the enzyme was increased up to 7.53 folds with a specific activity of 55.125 U/mg (25%) after purification. Purified enzyme retained its catalytic activity over a broad range of temperature (5°C to 65°C) and pH (5-8). EDTA inhibited the efficiency of enzyme confirming it as a metallo-protease while SDS-PAGE analysis confirmed the size of protease as 28.24 kDa. Purified protease exhibited excellent stability and activity in the presence of polar solvents and detergents. Moreover, it retained significant enzymatic activity for higher concentrations of commercially available detergents suggesting that it could be used both in leather and textile industries with ecological benefits. Taking all together, it can be concluded that protease enzyme of Bacillus subtilis RM1 could be an attractive candidate for dehairing. Only about 20-25% of wet salted hides/skins are converted into commercial leather, while large percentage is lost in wasteland as leather shavings, buffing dust, trimmings and splits, fat, soluble proteins and solid suspended pollutants. The harmful effects of the traditional disposal methods for tannery wastes have created a dire need and demand for more cost effective and environmentally acceptable alternatives. The most ideal way to minimize the waste generated is to convert them in to reusable items. This has motivated the research community to search for more innovative uses and applications of tannery wastes. As the wastes are highly proteinaceous, a wide range of value added products can be retrieved making the leather industry as an integrated business with value creation opportunities. Present work also deals with the aim of developing an efficient and value-added process for the management of untanned trimmings, produced in large quantities during the pre-tanning operations. The objective is to extract collagen proteins from tannery waste, treat and to convert the isolated collagen into useful products. For this grounded limed, delimed-bated and alkali treated trimmings were dissolved to make a collagen solution using pepsin. The physico-chemical properties of the resultant sponges and effects of the processing steps on the sponges prepared from un-tanned hides were studied. Results showed that bating and alkaline treatments given to proteinaceous fiber networks after liming have considerable effects on the resultant properties of sponges. SDS-PAGE analysis showed the molecular weights of all three were maintained in the range as of a commercial Type 1 collagen i.e., 100-200kDa. Bating appears to be the best treatment among different treatments as it yields the sponges with best characteristics of water absorption and visco-elasticity. Also, it has positive effect on reducing the apparent density of sponges, within desirable density ranges. Thus, the preliminary results of this study suggest feasibility of using these biobased collagen sponges prepared from un-tanned hides as absorbants that can be commercially viable.