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A novel concept of membrane bioreactor based on polymeric ionic liquids laccase membrane has been implemented in batch process for decolorization of the anthraquinonic dye Remazol Brillant Blue R (RBBR). New laccase immobilization strategy has been optimized by casting the enzyme into a polymeric inclusion membrane (PIM) using ionic liquids (ILs) and polyvinyl chloride (PVC) leading to laccase polymeric IL membrane (PILM). Four different ILs (1-octyl-3-metylimidazolium bis(trifluoromethylsulfonyl)imide, [OMIM][NTF2]; cholinium bis(trifluoromethylsulfonyl)imide, [Ch ol][NTF2]; cholinium dihydrogenphosphate, [Chol][H2PO4] and hydroxyethylammonium formate, [HEA][Fo]) have been screened and mixed to constitute the active phase of the support of PIM. This strategy has been fully succeeded since high laccase immobilization rates were recorded (about 98%) when using the optimal mixture containing three ILs (45% [OMIM][NTf2]/5% [Chol][NTf2]/2.5% [HEA][Fo]) and supplemented by 0.5% glutaraldehyde. It was found that such mixture contributes to increase the stability and reusability of laccase-PILM during eight successive assays in a batch discontinued stirred reactor. Decolorization rate of 75% has been reached in the batch decolorization process of RBBR with high reusability yield.

BACKGROUND The bacterial Stenotrophomonas maltophilia AAP56 laccase (SmLac) has been purified from cell extracts and its spectral and kinetic properties studied. It shows a high decolorization rate (99%) of the diazoic dye Reactive Black 5 (RB5) in the presence of redox mediators such as ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), ASGN (Acetosyringone), SGA (Syringaldehyde) and HOBT (1-Hydroxy-Benzotriazole). The optimal pH has been determined as neutral or basic compatible with textile wastewater pH for further treatment application. RESULTS This paper reports the characterization of the catalytic center of purified SmLac as not a classic blue laccase but a yellow one. Its redox potential has been determined at 638 mV, which indicates that the enzyme is a versatile laccase able to oxidase a wide range of substrates. Performance of the enzyme as catalyst for RB5 decolorization in association with a redox mediator has been demonstrated to be a promising bioremediation process since it presents high degradation ability (almost 99% in 1 h reaction time). The study also demonstrated, by electrochemical studies of redox potential of both mediators/protein and following the driving electron force, the importance of the choice of redox mediator and the limiting step in the laccase mediated reaction in azo dye decolorization. CONCLUSION It was demonstrated that the bacterial laccase SmLac has great potential for biotechnological applications (such as azo dye decolorization) compared with Trametes versicolor laccase. © 2013 Society of Chemical Industry

The fungus Sclerotinia sclerotiorum produces invertase activity during cultivation on many agroindustrial residues. The molasses induced invertase was purified by DEAE-cellulose chromatography. The molecular mass of the purified enzyme was estimated at 48 kDa. Optimal temperature was determined at 60 °C and thermal stability up to 65 °C. The enzyme was stable between pH 2.0 and 8.0; optimum pH was about 5.5. Apparent K(m) and V(max) for sucrose were estimated to be respectively 5.8 mM and 0.11 μmol/min. The invertase was activated by β-mercaptoethanol. Free enzyme exhibited 80 % of its original activity after two month's storage at 4 °C and 50 % after 1 week at 25 °C. In order to investigate an industrial application, the enzyme was immobilized on alginate and examined for invert sugar production by molasses hydrolysis in a continuous bioreactor. The yield of immobilized invertase was about 78 % and the activity yield was 59 %. Interestingly the immobilized enzyme hydrolyzed beet molasses consuming nearly all sucrose. It retained all of its initial activity after being used for 4 cycles and about 65 % at the sixth cycle. Regarding productivity; 20 g/l of molasses by-product gave the best invert sugar production 46.21 g/day/100 g substrate related to optimal sucrose conversion of 41.6 %.

A bacterial strain AAP56, isolated from a polluted soil (from Kelibia city) and identified as Stentrophomonas maltophilia, was particularly interesting for its ability to decolorize recalcitrant dyes of an industrial effluent: SITEX Black. The final percentage decolorization 60% was shown by bacterial culture after incubation in LB medium at 30°C under shaking conditions. The decolorization was closely correlated with the metabolic bacterial growth. The replacement of yeast extract in LB medium composition by soya flour was clearly efficient to enhance the percentage decolorization by 20% and also to reduce the growth medium cost 60-fold. The bacteria were able to reduce 23% from the initial COD and 28% from the initial BOD5 of the effluent. The immobilization of bacterial cells in calcium alginate beads improved by 25% the effectiveness of the biotransformation within 24 h in batch conditions. The potential of a downflow fixed column reactor (DFCR) to decolorize SITEX Black was evaluated under dilution rate. The best decolorization percentage (82%) was recorded at 0.3 h−1. This bioprocess seems to be a potentially useful method to remediate the colored textile wastewater.

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO4 in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K m = 700 μM), and pyrocatechol (K m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.