Laccase production by selected bacteria species isolated from some aquatic and terrestrial milieu of the Eastern Cape : applications in wastewater treatment

Abstract

Aromatic pollutants are a diverse group of chemicals which are continuously produced from industrialization, urbanization and sophistication in technological advancement. Pristine water source polluted by these chemicls makes the water unsafe for human consumption, and as well disrupts the trophic structure of the aquatic milieu. Physico-chemical treatment techniques employed so far have been accompanied by major drawbacks which have overriden the relative successes recorded, hence, greener, simpler and more efficient methods of pollutant transformation is imperative. The prospect of enzymatic treatment of pollutants has gradually been receiving growing attention in contemporary times due to the their environmental friendliness and production economic feasiblity. Laccase, a multicopper oxidase has heightened its appeal towards environmental and biotechnological applications due to its broad substrate specificity and its requirement of atmospheric molecular oxygen as a cosubstrate and the discharge of water as the byproduct. Hence, this present study was designed to evaluate the biotechnological potentials of laccases produced by some bacteria species from some aquatic biomes of the Eastern Cape Province, South Africa. The laccase-producing bacteria were isolated from selected environmental samples by selective enrichment using selective aromatic compounds as sole carbon source and subsequently, laccase-screening phenolic substrates. The laccase-producing bacteria were identified by molecular techniques as proteobacteria belonging to the following genera: Achromobacter, Bordetella, Citrobacter, Pseudomonas and Stenotrophomonas. Optimisation of laccase production in a submerged fermentation was by traditional and statistical methods, where four isolates (Hb9c; Achromobacter xylosoxidans HWN16, Hb16c; Bordetella bronchisepta HSO16, Berl11b2; Stenotrophomonas maltophilia BIJ16, Ie1c; Citrobacter freundii LLJ16) were evaluated for the fermentative production of laccase from lignocellulosic agroindustrial residues. Predictions from statistical optimisation showed that weakly acidic conditions (pH 5) and low agitation speed (100 rpm) were required for maximum laccase production from mandarin peelings (0.5 g/200 mL) and NaNO3 (0.25 g/200 mL) in Hb9c, maize stover (0.50 g/200 mL) and NaNO3 (0.050 g/200mL) in Berl11b2 while a lower agitation speed (50 rpm) was required for maximum laccase output from 2.0 g/200 mL maize stover and 0.050 g/200 mL KNO3 in Ie1c. However, 2.50 g/200 mL wheat bran, 0.050 g/200 mL yeast extract and 50 rpm agitiation under acidic conditions (pH3) yielded maximum laccase titres in Hb16c. Further characterisation of Hb9c and Ie1c laccase secretions portrayed their polyextremotolerant capacities. They were active at a broad range of tempertaure (0-90 degreesC); with optima at 70°C (Hb9c) and 60°C (Ie1c), pH (3-11); with optima at pH 6 (Ie1c) and pH 8 (Hb9c), respectively, and were equally thermo- and pH-stable. Their activities were either improved or left unabated by high concentrations of cations, detergents, and chloride. In addition, catalytic activities of Hb9c and Ie1c laccase secretions increased when they were preincubated with 2 – 20 percent of fluoride, a potent inhibitor. Consequently, a molecular perspective depicted the isolates to have multiple homologous laccase encoding genes. The enzymes were successfully immobilised on solid supports comprising gelatin and Na-alginate with a recovery of cca. 85 percent residual activity after 8 cycles of oprertional stability experiments. The immobilised laccases were remarkable in the decolourisation of synthetic dyes, albeit, free forms also elicited satisfactory performances. Ultimately, the application of free laccases in denim bleaching, individually or with a blend of a mediator, ABTS, showed that denim colours could be bleached without the need for chemical bleaching agents. The results obtained suggest the bacteria laccases produced from lignocellulosic wastes may serve as potent degraders of phenolic pollutants in water and, may also contribute to the bioeconomy and promote greener techniques for industrial applications