The ability of gellan gum-immobilised cells of the heavy metal-tolerant bacterium Alcaligenes sp. AQ05-001 to utilise both heavy metal-free and heavy metal-polluted feathers (HMPFs) as substrates to produce keratinase enzyme was studied. Optimisation of the media pH, incubation temperature and immobilisation parameters (bead size, bead number, gellan gum concentration) was determined for the best possible production of keratinase using the one-factor-at-a-time technique. The results showed that the immobilised cells could tolerate a broader range of heavy metal concentrations and produced higher keratinase activity at a gellan gum concentration of 0.8% (w/v), a bead size of 3 mm, bead number of 250, pH of 8 and temperature of 30 °C. The entrapped bacterium was used repeatedly for ten cycles to produce keratinase using feathers polluted with 25 ppm of Co, Cu and Ag as substrates without the need for desorption. However, its inability to tolerate/utilise feathers polluted with Hg, Pb, and Zn above 5 ppm, and Ag and Cd above 10 ppm resulted in a considerable decrease in keratinase production. Furthermore, the immobilised cells could retain approximately 95% of their keratinase production capacity when 5 ppm of Co, Cu, and Ag, and 10 ppm of As and Cd were used to pollute feathers. When the feathers containing a mixture of Ag, Co, and Cu at 25 ppm each and Hg, Ni, Pb, and Zn at 5 ppm each were used as substrates, the immobilised cells maintained their operational stability and biological activity (keratinase production) at the end of 3rd and 4th cycles, respectively. The study indicates that HMPF can be effectively utilised as a substrate by the immobilised-cell system of Alcaligenes sp. AQ05-001 for the semi-continuous production of keratinase enzyme.
Biodegradation of agricultural wastes, generated annually from poultry farms and slaughterhouses, can solve the pollution problem and at the same time yield valuable degradation products. But these wastes also constitute environmental nuisance, especially in Malaysia where their illegal disposal on heavy metal contaminated soils poses a serious biodegradation issue as feather tends to accumulate heavy metals from the surrounding environment. Further, continuous use of feather wastes as cheap biosorbent material for the removal of heavy metals from effluents has contributed to the rising amount of polluted feathers, which has necessitated the search for heavy metal-tolerant feather degrading strains. Isolation, characterization and application of a novel heavy metal-tolerant feather-degrading bacterium, identified by 16S RNA sequencing as Alcaligenes sp. AQ05-001 in degradation of heavy metal polluted recalcitrant agricultural wastes, have been reported. Physico-cultural conditions influencing its activities were studied using one-factor-at-a-time and a statistical optimisation approach. Complete degradation of 5 g/L feather was achieved with pH 8, 2% inoculum at 27 °C and incubation period of 36 h. The medium optimisation after the response surface methodology (RSM) resulted in a 10-fold increase in keratinase production (88.4 U/mL) over the initial 8.85 U/mL when supplemented with 0.5% (w/v) sucrose, 0.15% (w/v) ammonium bicarbonate, 0.3% (w/v) skim milk, and 0.01% (w/v) urea. Under optimum conditions, the bacterium was able to degrade heavy metal polluted feathers completely and produced valuable keratinase and protein-rich hydrolysates. About 83% of the feathers polluted with a mixture of highly toxic metals were degraded with high keratinase activities. The heavy metal tolerance ability of this bacterium can be harnessed not only in keratinase production but also in the bioremediation of heavy metal-polluted feather wastes.