Treatment of phenolic effluents with immobilised enzymes

Phenolic effluents are produced by many industries including pulp and paper processing, coal conversion and dyeing and textiles. Such effluents pollute receiving waters due to toxicity, BOD and high colour. Because present treatment methods are expensive and often inefficient, the use of immobilised microbial cells and enzymes as alternative treatment methods were investigated. The effect of white-rot fungi and phenol oxidase enzymes on 3 industrial effluents were compared. The effluents were two from a cotton cleaning mill, hydroxide (OH) and sulphide (S) ; the fraction >1000 D from the Ei stage of a kraft pulp mill. An artificial coal conversion effluent was also studied. Effluents added to batch cultures of white-rot fungi with glucose as a carbon source, were decolorised by 75-85% in 6 d (Coriolus versicolor K) and by 70-75% (Stereum hirsutum, Coriolus versico1 or B). Successive additions of OH (1% v/v) to batch cultures of C.versicolor(K) at days 0, 8 and 15 were decolorised by 80-85% but at gradually reduced rates. OH diluted to 2% (v/v) and 10% was decolorised by C.versicoIor(K) but 20% OH was toxic. Laccase production was induced by both high and low molecular weight phenolic compounds. The enzyme was routinely produced in 1 litre shake flasks (with a 2 cm glass bead) and was induced by the addition of2,5-xylidine after 7 d growth of C.versicolor(K). Soluble laccase and horseradish peroxidase (HRP) removed colour from OH, E and S effluents. Colour removed by HRP was 61% (OH), 36% (E) and 51% (S), colour removed by laccase was 36% (OH), 40% (E) and 30% (S), in 2-4 d. Soluble laccase,222 U/ml, could also precipitate 51 mg/l/h phenol from artificial coal conversion effluent at pH 6.0, although the optimum for activity was pH 4-5. However in all cases rapid and irreversible enzyme inactivation occurred. Entrapment of laccase in alginate beads improved decolorisation by factors of 2.1 (OH) and 1.5 (E) , entrapment of HRP improved decolorisation by 1.1 (OH), 1.5 (E) and 0 (S). Copolymerisationof laccase or HRP with tyrosine produced insoluble polymers with enzyme activity. Entrapment of these copolymers in gel beads further increased the efficiency of decolorisation of E effluent by laccase and HRP. Decolorisation by entrapped enzymes increased with increasing bead: effluent ratios (v/v) and the maximum of 86% decolorisation of OH effluent was achieved with an HRP bead:eff1uent ratio of 1:2.5. However, all enzyme preparations were released from the alginate beads at 3uch a rate that beads could not be used to decolorise more than one batch of effluent. Laccase could be immobilised on activated carbon by covalent coupling using a water soluble diimide. Up to 50 mg of laccase protein per g support was bound, but at the highest protein levels the expressed enzyme activity decreased. The maximum bound activity was obtained at11.5 mg laccase/g support. Bound laccase (CIL) was not eluted from carbon by washing with 10 mM buffer (pH 4-9) and was stable to salt concentrations up to 1M. The pH profile was unchanged but the temperature range of activity was broadened. The activation energy of CIL (17.61 kJ/M C>2) was decreased compared with soluble laccase (22.28 kJ/M 02) indicating the possibility of conformational changes during binding of laccase to carbon. Laccase bound to carbon retained 20% of its initial activity after oxidation of 7 batches of 2,6-dimethoxyphenol (DMP) and 55% of activity was retained after continuous oxidation of 9 1 of DMP in a fluidised bed reactor. In batch incubations with E effluent, CIL removed 57% of colour at an average rate of 94 CU/h/U enzyme. In the continuous fluidised bed system the removal was higher, 115 CU/h/U enzyme, however, continuous recirculation of effluent through the column for 5-8 h was necessary to achieve high colour removal by the immobilised laccase.