degradation of textile effluent: High operational stability of the continuous process

degradation of textile effluent: High operational stability of the continuous process

Biochemical Engineering Journal 93 (2015) 17–24 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.else...

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Biochemical Engineering Journal 93 (2015) 17–24

Contents lists available at ScienceDirect

Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Regular Article

Combination of chemical and enzymatic treatment for efficient decolorization/degradation of textile effluent: High operational stability of the continuous process Meenu Chhabra a , Saroj Mishra b,∗ , Trichur Ramaswamy Sreekrishnan b a b

Centre for Biologically Inspired System Science, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan 342011, India Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi 110016, India

a r t i c l e

i n f o

Article history: Received 7 June 2014 Received in revised form 28 August 2014 Accepted 13 September 2014 Available online 20 September 2014 Keywords: Textile effluent Aerobic processes Bioremediation Cyathus bulleri laccase Membrane bioreactors Waste-water treatment

a b s t r a c t Textile effluent is characterized by high colour, chemical oxygen demand and conductivity due to presence of a large number of recalcitrant compounds. The effluent is often discharged without an effective treatment leading to contamination of water bodies. In the present study, a combination of mediator assisted laccase and chemical treatment was used for decolorization of effluent from a local textile mill in a continuous enzyme membrane reactor (EMR). Treatment of the effluent first with laccase and ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) lead to about 60% decolorization but was accompanied by membrane fouling. Addition of alum further coagulated 90% of the residual colour but the process was associated with dye sludge formation and ABTS could not be recovered from the treated effluent. Reversal of the treatment sequence was effective in that 85% decolorization was achieved in the EMR and the process could be operated for over a period of 15 days. No sludge formation was noticed and membrane fouling was negligible. Most importantly, about 60% ABTS could be recovered from the treated effluent. Analysis of the treated effluent by mass spectrometery indicated extensive breakdown of the dye molecules by laccase and ABTS and the breakdown products were neither toxic nor mutagenic as assessed by measurement of the oxygen consumption rate and the standard Ames test. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The textile sector is of great economic importance in Asia and Europe. It represents close to a fifth of India’s exports revenue and employs 35 million people. However, an important issue related to this sector is the generation of huge volumes of coloured water emanating from the dyeing and finishing sections. The dye waters if discharged untreated in natural water bodies pose serious environmental hazard. The textile effluent is highly variable in its composition and contains unused dyes (8–20%), dyeing auxiliaries, inorganic salts and other chemicals that improve adherence to the fibres [1]. Around 100,000 synthetic dyes are commercially available and these are of complex chemical nature making them recalcitrant and persistent in natural water bodies [2]. Presence of colour in effluent streams is also an aesthetic nuisance. In addition to the toxicity of the dye molecules, dyes absorb light and reduce its availability for aquatic photosynthesizing systems. This results in

∗ Corresponding author. Tel.: +91 11 2659 1007; fax: +91 11 2658 2282. E-mail addresses: [email protected], [email protected] (S. Mishra). http://dx.doi.org/10.1016/j.bej.2014.09.007 1369-703X/© 2014 Elsevier B.V. All rights reserved.

oxygen deprivation and disruption of aquatic food chain. Thus, dye removal is of major concern during treatment of textile effluent. Different types of physico-chemical treatments or a combination of these have been tried to effectively treat the textile wastewaters. These include adsorption by carbon, chemical or electrocoagulation, electrochemical oxidation, Fenton treatment, ozonation, ultrafiltration etc. [3]. The major drawback of the physical methods is that the pollutants are not degraded but transferred from one phase to the other. The chemical methods such as electrolysis and ozonation lead to formation of concentrated sludge or toxic end products. Oxidants, such as the Fenton reagent oxidize the pollutants by generating reactive hydroxyl radicals and higher oxidized iron species but are associated with sludge formation at high pH values of the effluent. Most of these methods are also associated with high operational costs [3]. Biological treatment involves use of either intact cells or enzymes and the former depends on the availability of nutrients as well as optimum growth conditions [4]. Textile effluent is generally nutrient deficient and does not support microbial growth. There are a number of reports on the use of cells for decolorization and biosorption of dyes from textile effluent [5–9]. But, this treatment is of limited use as the

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dye only passes from the liquid phase to the solid cell material. A number of microbial systems can also employ aerobic, anaerobic, or sequential aerobic–anaerobic pathways to attack the azo bonds or to convert it to amines through low specificity azo reductases [10]. The resulting molecules have been shown to be more toxic and in some cases mutagenic [11] thus limiting the use of such treatments. Combination of physico-chemical and biological methods has been suggested as an important alternative for development of sustainable treatment processes [12]. Enzymes of lignin degradation have been reported [5,13] to be very effective in decolorization of textile dyes and simulated effluent as these can act under varying conditions. Laccases, in particular, find extensive use in decolorization of a variety of dyes [14]. The range of its activity can be further increased by inclusion of small molecules called as mediators [15]. However, application of laccases is restricted on large scale as many of the substances present in the effluent inhibit enzyme activity [16]. The mediators used are often expensive and add to the toxicity of the treated effluent. Thus, an effective strategy needs to be employed which can address stability of laccase and mediator recovery during the treatment process. In the present study, a combination of coagulation and laccase treatment was used to address these issues. Coagulation has been widely used in the literature for removal of organic substrates [17,18] and two mechanisms have been put forward for its action. The most plausible mechanism is the binding of the metal to anionic sites in the organic molecules to neutralize the charge resulting in precipitation and the other possible mechanism is the adsorption of organic substrates on metal hydroxide precipitates. Recovery of mediator can be accomplished by addition of salts, as has been demonstrated in an earlier study [19]. In this context, the objective of the present work was to evaluate two different combination treatments for decolorization and decomposition of real effluent in a continuous enzyme membrane reactor (EMR). Results were interpreted in terms of retention of laccase activity, operational stability of the system, decolorization efficiency, mediator recovery and membrane fouling.

2. Materials and methods 2.1. Characterization of effluent The effluent originating from the dyeing bath was collected from a local textile mill located in Ghaziabad, UP, India. The effluent was characterized in terms of pH, chemical oxygen demand (COD), total dissolved solids (TDS), total suspended solids (TSS), colour units (in Pt–Co units), total Kjeldahl nitrogen (TKN) and conductivity as per standard methods [20].

2.2. Microbial culture and laccase production Laccase was produced using the basidiomycete fungus Cyathus bulleri, procured from Canadian type culture collection. The culture was maintained on malt extract agar plates and stored at 4 ◦ C. Enzyme production was carried out in basal liquid medium as described previously [21] using 2,6-dimethyl aniline as an inducer. The basal liquid medium contained glucose (10 g/l), ammonium tartarate (1.81 g/l) as a nitrogen source, 2,2-dimethyl succinic acid (14.6 g/l), KH2 PO4 (20 g/l), MgSO4 (5 g/l), CaCl2 (1 g/l), MnSO4 ·4H2 O (0.5 g/l) and NaCl (1 g/l). Other trace elements and vitamins were added as described previously [21]. The medium was inoculated with 1 cm disc of the fungal culture scraped from the growing end of the mycelium cultivated on an agar plate. The flasks were incubated at 26 ◦ C under static conditions. The cultures were harvested on day 5 and clear culture filtrate obtained after removal of the

mycelium was used as a source of laccase. The specific activity of laccase was 2.35 U/mg extracellular protein. 2.3. Screening of mediators for decolorization of dyeing bath effluent Effluent from the dyeing bath was diluted to one-fifth of the original strength with sodium phosphate buffer, pH 5.6. Decolorization was performed in a 25 ml conical flask containing 5 ml of the diluted effluent. Laccase was added to a final activity of 100 U/l while the synthetic and the natural mediators (Supplementary Fig. 1) were added at their minimum effective concentrations as optimized previously [13,19] for simulated dye mixture. The mediators and their concentrations were 2 -azinobis-(3-ethylbenzothiazoline-6sulfonic acid or ABTS (100 ␮M), 1-hydroxybenzotriazole or 1-HOBT (500 ␮M), violuric acid (100 ␮M), acetovanillone (500 ␮M), acetosyringone (100 ␮M) or syringaldehyde (100 ␮M). Incubations were carried out at 28◦ C, 100 rpm for 72 h. Samples were removed at regular intervals and percent decolorization determined by monitoring absorbance at 515 nm. This was based on maximum absorption displayed by the effluent at this wavelength. After identifying the most suitable mediator (ABTS), laccase was varied (10–500 U/l) to arrive at a concentration leading to maximum decolorization of the diluted effluent. Similarly, ABTS concentration was varied from 0–200 ␮M at the optimized level of laccase. Decolorization was monitored by measuring absorption at 515 nm. 2.4. Evaluation of toxicity and mutagenicity of the treated effluent The respiratory toxicity of the untreated and laccase–ABTS treated effluent was evaluated by measuring the decline in oxygen consumption rate (OCR) of Pseudomonas putida [13]. Briefly, a 16 h old P. putida culture was incubated with the test sample for a period of 2 h. Next, the incubated culture was mixed with sterile nutrient broth (previously purged with air for 30 min) in an air tight assembly fitted with a dissolved oxygen metre (Oxi 315, WTW Weinheim, Germany). The decline in oxygen concentration was monitored and plotted as a function of time. The slope of the linear region provided OCR. The toxicity of the sample was correlated with its ability to cause decline in OCR. Distilled water and 4-chlorophenol served as negative and positive controls respectively. The mutagenicity was assessed by standard Ames test using Salmonella typhimurium TA 98 obtained from IMTECH Chandigarh, India. The procedure involved the standard plate incorporation assay [22] and mutagenicity was determined by estimating the fold increase in his+ revertants in the presence of test chemical as opposed to the spontaneous revertants. Distilled water and 4nitro-o-phenylenediamine served as negative and positive controls respectively. 2.5. Decolorization of effluent using alum Decolorization of the enzyme treated effluent or the raw effluent was performed using alum. The optimum concentration of alum and pH were determined first. Enzyme treated or the raw effluent (5 ml) was dispensed in 25 ml conical flasks and alum was added in the concentration range of 0–1 g/l. Flasks were incubated in an incubator shaker at 100 rpm for 2 h. After visible precipitation, settling time of 1 h was given. Clear supernatant was taken and absorbance was measured at 515 nm. The effect of pH was monitored by adjusting the pH of the effluent at different values ranging from pH 4–12 using 1 N HCl or 1 N NaOH. Alum was then added to each flask at a fixed concentration of 0.15 g/l.

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2.6. Analytical methods

2.7. Batch studies

Laccase activity was determined using ABTS as a substrate [23] and measuring oxidation of ABTS to its cation radical (ε420 , 36,000 M−1 cm−1 ). One unit of enzyme activity is defined as the amount of enzyme that released 1 ␮mol of product per min under standard assay conditions. The mass spectrometry of laccase + ABTS treated effluent was carried out using QStar electrospray ionization high resolution mass spectrometer (Applied Biosystems). The untreated and the treated effluent were filtered using 0.45 ␮m filter and injected into the system. Mass spectrometer was operated in positive ion mode and the settings were as follows: ion spray voltage: 5500 V, nebulizer gas: 35 lb/in2 , curtain gas: 30 lb/in2 , Declustering potential: 60 V, focusing potential: 265 V.

2.7.1. Enzyme treatment followed by alum coagulation of effluent Effluent was diluted to one-fifth of the original strength using sodium phosphate buffer, pH 5.6. The initial O.D.515 was 0.9. Enzymatic treatment was performed as described in Section 2.3. Residual colour in the enzyme treated effluent was removed using alum (0.15 g/l), pH 9. Incubation was performed at 100 rpm for 2 h followed by settling time of 1 h. Decolorization was monitored by measuring absorbance at 515 nm. 2.7.2. Decolorization of the effluent using alum followed by enzyme treatment For this study, the pH of the undiluted effluent was set at 9.0. Coagulation was performed as described in Section 2.5 with 0.15 g/l of alum. The coagulated dye was collected and reconstituted in

Fig. 1. Schematic diagram of the sequential treatment processes for the decolorization of coloured textile effluent: (A) laccase + ABTS treatment in an EMR followed by colour coagulation using alum. (B) Alum mediated coagulation, dye sludge reconstitution in phosphate buffer followed by laccase + ABTS treatment in an EMR.

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phosphate buffer (5× the original effluent volume) and pH set at 5.6. This was next treated with laccase (10 U/l) and ABTS (100 ␮M). Absorbance was read at 515 nm to determine the extent of decolorization.

2.8. Continuous studies For continuous studies, effluent was treated using a combination of methods. Two different sequences were followed; one involved enzymatic treatment in an EMR followed by alum coagulation, and the other, alum coagulation followed by laccase and mediator treatment in an EMR. The schematic outline of the same is given in Fig. 1A and B respectively and the details of the reactor operation are given below.

Fig. 2. Screening mediators for the treatment of textile effluent.

3. Results 2.8.1. Enzymatic treatment followed by alum coagulation The effluent was diluted to one-fifth the original strength using sodium phosphate buffer, pH 5.6. The initial O.D.515 was 0.9. Enzymatic treatment was performed with laccase and ABTS in a continuous mode in an EMR (1 l capacity and 250 ml working volume) comprising of a polyacrylonitrile membrane of 20 kDa molecular weight cut-off and a cross flow module (Fig. 1A). Feed solution consisted of the diluted effluent supplemented with ABTS (100 ␮M) and laccase (10 U/l). The reactor was operated at a hydraulic retention time of 10 h (based on earlier optimization studies, data not shown). The system was operated for a period of 7 days. During the course of the reactor operation, samples were withdrawn at specific time intervals and assayed for % decolorization and residual laccase activity. The effluent emerging from the outlet stream of the reactor was next treated with alum (0.15 g/l, pH 9). ABTS was recovered from the clear upper phase by addition of 20% (w/v) ammonium sulfate. The concentration of the recovered ABTS was determined by converting it to radical cation using excess laccase as described previously [19]. In order to rule out the possibility of dye adsorbance by the membrane, reactor was operated with the feed solution consisting only of the effluent. At the end of the operation, fouling was determined by monitoring percent decrease in permeability of water through the membrane.

2.8.2. Alum coagulation followed by laccase and ABTS treatment In this case, the original effluent was first subjected to alum treatment (0.15 g/l) as described in Section 2.7.2. The coagulated dye was next reconstituted in phosphate buffer (pH 5.6). ABTS (100 ␮M) and laccase (10 U/l) were added and the effluent was treated in a membrane reactor (Fig. 1B) for 15 days. The scale of operation was the same as that in Section 2.8.1. The hydraulic retention time (HRT) was kept at 10 h. Samples were collected at regular intervals and percent decolorization and residual laccase activity determined. The permeate collected over a period of 7 days was processed for ABTS recovery by addition of 20% (w/v) ammonium sulfate. ABTS recovery and membrane fouling were determined as described in Section 2.8.1.

3.1. Characterization of the effluent The effluent from the dyeing bath was characterized and the results are shown in Supplementary Table 1. As observed, COD, colour, conductivity and total nitrogen content were much higher than the permissible levels and thus the effluent was not considered suitable for discharge. However, as per the OCR data and the Ames test, it was neither toxic nor mutagenic. 3.2. Screening of mediators for decolorization of effluent Amongst the different mediators tested, combination of laccase and ABTS led to 60% decolorization of the diluted effluent in 10 h. Other synthetic mediators such as 1-HOBT and violuric acid performed better than the natural mediators syringaldehyde and acetovanillone and led to 28–35% decolorization in the first 24 h (Fig. 2). Acetosyringone, a natural phenolic compound, gave 35% decolorization. Further incubation for another 24 h led to increase in decolorization to 50% with 1-HOBT. For other synthetic and natural mediators, no significant increase was observed. Prolonged incubations did not lead to any further increase in the extent of decolorization. It was also observed that if laccase alone was used (no mediator), no decolorization took place. Thus, laccase and ABTS combination was selected for further study. At a fixed concentration of ABTS (100 ␮M), when laccase was varied from 10 to 500 U/l, effective decolorization of 60% was achieved even at 10 U/l (data not shown). No significant increase in decolorization was observed at enhanced enzyme levels. At 10 U/l of laccase, a maximum decolorization of 62% was achieved even when ABTS was increased to 200 ␮M. Thus laccase units and ABTS were optimized at 10 U/l and 100 ␮M respectively for treatment of effluent. The untreated and the treated effluent were analyzed by mass spectrometry and the results are shown in Fig. 3A and B. As seen, the intensity of the high molecular weight compounds (m/z 535–903) was reduced after treatment (Fig. 3B) with more bands appearing in the 100–400 m/z range. The relative intensities were also reduced indicating effective degradation of many complex effluent molecules by laccase and ABTS. 3.3. Toxicity and mutagenicity assessment

2.9. Statistical analysis All batch experiments were done in triplicates and the reported results are an average of three runs with a standard deviation between 5 and 7%. The continuous decolorization experiments in an EMR were done in duplicates and the results reported are an average of two experiments with standard deviation between 8 and 10%.

The toxicity and mutagenicity of the laccase + ABTS treated effluent was determined. No decrease in OCR of P. putida was observed in presence of either the untreated or the treated effluent relative to control experiment. Similarly, no increase in His+ S. typhimurium revertants was observed over the spontaneous revertants by standard plate incorporation assay with either the untreated or the treated effluent. This led us to conclude that the

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Fig. 3. Mass spectra profile of the effluent (A) before enzymatic and (B) after enzymatic treatment.

treatment with laccase + ABTS did not impart any mutagenic or toxic status to the effluent and thus the treatment was safe. 3.4. Decolorization using alum

and the mediator treatment were next given to the coagulated dye reconstituted in phosphate buffer, pH 5.6. This laccase + ABTS treatment this time resulted in 95% decolorization. An overall 85% decolorization was achieved with this combination.

From the optimization studies, we found that alum could mediate good colour coagulation at a concentration of 0.15 g/l. At an alum concentration <0.15 g/l, the colour removal was low (20–23%) while above this concentration the bulkiness of the settled layer/sludge increased. With an increase in alum concentration from 0.05 to 1 g/l, percent decolorization increased from 22% to 52% (Fig. 4A). We chose to use the concentration of 0.15 g/l for subsequent experiments. The alum mediated coagulation was pH dependent (Fig. 4B) and maximum precipitation occurred at pH 9.0. At this pH value and concentration of 0.15 g/l alum, nearly 70% residual colour removal could be achieved from a previous enzyme + mediator treated solution (Fig. 4A and B). On the other hand, when raw effluent was treated with alum (0.15 g/l, pH 9), nearly 90% colour coagulation was achieved. 3.5. Batch studies of combination treatment Two sequences were followed here. In the first sequence, batch studies were carried out at small scale (5 ml diluted effluent in a 25 ml flask) using laccase treatment first followed by alum coagulation. The enzyme treated effluent was next subjected to coagulation by alum. With this combination, an overall 80% decolorization was achieved. In the second sequence, alum coagulation was followed by laccase treatment. About 90% colour coagulation was achieved by addition of alum to the undiluted effluent (pH 9.0). The enzyme

Fig. 4. Effect of alum dose (A) and pH (B) on colour removal from laccase and ABTS treated effluent.

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maintained at 50% up to 14 days. Membrane fouling was avoided and nearly 60% of ABTS was recovered from permeate. Sludge formation was totally avoided. The overall decolorization was 85%. A comparison of the two treatment sequences is given in Table 1.

4. Discussion

Fig. 5. Continuous treatment of the effluent in an EMR showing decolorization and laccase activity profile. (A) Raw effluent subjected to laccase and ABTS treatment in an EMR. (B) Reconstituted effluent after alum treatment subjected to laccase and ABTS treatment in an EMR. Table 1 Comparison of the sequence of treatments for colour removal from textile effluent. Parameter

Continuous laccase + mediator treatment followed by chemical treatment

Chemical treatment followed by continuous laccase + mediator treatment

Final decolorization (%) ABTS recovery (%) Dye sludge formation Membrane fouling after 7 days (%) Laccase activity after 7 days (%) Operation time

80 0 Yes 70 5 Short term operation due to high membrane fouling Does not represent a sustainable approach

85 60 No 0 65 >15 days with no membrane fouling

Conclusion

Presents a sustainable approach

3.6. Continuous treatment of the effluent 3.6.1. Laccase and ABTS treatment followed by alum treatment Treatment of the diluted effluent with laccase and ABTS in an EMR resulted in 60% decolorization for a period of 3 days after which a drop in decolorization was observed. The reactor efficiency dropped at the end of day 7 and only 18% decolorization could be achieved. Concomitant with this was a loss of laccase activity in the outlet stream (Fig. 5A). Attempt to treat the emerging outlet stream (after 3 days) with alum resulted in sludge formation. The process could not be operated for a long period due to excess membrane fouling. Under these conditions, ABTS could not be recovered from the supernatant. The major features are summarized in Table 1. 3.6.2. Alum coagulation followed by laccase and ABTS treatment The sequence involving coagulation by alum followed by laccase and ABTS treatment presented a successful approach as the reconstituted dye was decolorized to 95%. Nearly 90% decolorization could be maintained for a period of 14 days (Fig. 5B). After an initial drop in laccase activity (by 40%), the activity was stably

Treatment of textile effluent by biological agents (cells or enzymes) would be highly desirable but remains a challenging task as variability in the effluent characteristics does not allow developing a generalized treatment approach [1]. Also, biological agents (cells or enzymes) are more sensitive to the complex and toxic ingredients of the effluent. Although microbial consortia have been developed for treatment of textile wastewaters [24], the process does not continue for long due to acid formation and generation of toxic compounds. The dyes are not completely degraded under these conditions. Thus, a combination of processes has to be applied to address the problems of colour, toxicity and sludge formation. Combination of laccase and mediators for treatment of individual dyes and dye mixture has been reported [13,25–27]. Benzina et al. [28] reported decolorization of two textile industry effluents using laccase and 1-HOBT. However, there is no report on the use of laccase and mediator for continuous treatment of real effluent. In an attempt to develop a continuous system for treatment of real effluent, a number of synthetic and natural mediators were first evaluated for effective decolorization. ABTS showed a rapid and higher percent decolorization (up to 60%) compared to other mediators. Low concentrations of laccase were employed and micromolar concentrations of ABTS were used for the process. This was consistent with our previous findings wherein C. bulleri laccase + ABTS were effective on both individual dyes and simulated dye mixture [13]. The salt tolerant property of this laccase [29] could be responsible for its effectiveness on the real effluent. It is a well-accepted observation that waste waters from textile industry contain high concentrations of Na2 SO4 or NaCl [30] and majority of laccases are chloride sensitive. Analysis of the treated effluent by mass spectrometry indicated degradation of a number of high molecular weight compounds (m/z 535–903). The relative intensity of many of the other peaks with m/z between 50 and 300 m/z increased suggesting extensive breakdown of the dyes. The products of degradation were analyzed for mutagenicity by Ames test and found to be non-mutagenic. The removal of residual colour (40%) was attempted using alum. Alum was found to coagulate the colour optimally at pH 9.0 and this could be explained by the concentration-pH aluminium-species diagram [31]. Around acidic pH, monomerichydroxoaluminium cations are the primary species while towards higher pH, these co-exist with aluminium hydroxide precipitates and polymeric hydroxoaluminium species. As a result, positively charged precipitates are formed. In alkaline pH range, monomeric hydroxoaluminium anions are the main species [32] and can act to retrieve the dyes leaving behind chloride/sulfate ions present in the waste waters [30]. Coagulation using alum and other inorganic coagulants is a common practice [33–35]. However, to the best of our knowledge this is the first report where it has been successfully combined with laccase treatment. Since the enzymatic treatment followed by alum precipitation lead to sludge formation, the treatment sequence was reversed. The main advantage of this reversed scheme was that 90% of the dyes were precipitated initially by alum while laccase inhibitory ions (sulfate, chlorides) were left behind in the supernatant. Laccase could act effectively on the reconstituted dyes in the second part of the treatment. EMR was used for the continuous process of effluent decolorization. The utility of EMR for continuous decolorization of simulated effluent was shown in previous studies as well [19]. EMR’s, in

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different configurations, have been found useful in batch decolorization [36] and in laccase/mediator recovery coupled cycles [37]. The results of the two treatment regimes (chemical and enzymatic) were evaluated in a continuous process using an EMR. In the first sequence, the enzymatic treatment for colour removal could be carried out for 3 days after which loss in laccase activity lead to drop in decolorization and this was attributed to presence of inhibitory substances in the effluent. Moreover, the permeate collected after 3 days, when subjected to alum treatment, led to sludge formation. There was no recovery of ABTS and membrane fouling stopped the reactor operation. However, on performing the alum coagulation of the dyes in the raw effluent, possible laccase inhibitors and other molecules remained in the supernatant which was discarded. Reconstituted dye could be successfully treated in an EMR for 15 days at nearly 95% decolorization. The problem of membrane fouling was completely avoided and a final decolorization efficiency of 85% was noted. A comparison of the two processes, shown in Table 1, indicated the second combination treatment to be highly efficient when compared to the conventional activated sludge system (decolorization rate of 30–50% with 40–60% COD removal) or the biofilm system (decolorization rate 50–60% with 40–60% COD removal) [38]. The overall decolorization efficiency with the combination treatment was also higher than reported for Pleurotus flabellatus on the real effluent (60–70% decolorization under 25 h HRT) [7] making the process extremely attractive for large scale treatment. 5. Conclusions A combination treatment involving alum coagulation followed by ABTS assisted laccase treatment is suitable for treatment of textile effluent because: • it ensures selective removal of dyes from the effluent which can be successfully treated with low concentrations of laccase and ABTS • it is not accompanied by sludge formation and the process can be carried out continuously on large volumes in a membrane reactor with minimal membrane fouling • it also provides for recovery and reuse of ABTS from the permeate. Acknowledgements SM and TRS acknowledge financial assistance from Department of Biotechnology, Government of India. Council of Scientific and Industrial Research is acknowledged for providing senior research fellowship to MC. Prof. M.L. Gulrajani is gratefully acknowledged for providing the industrial effluent for the present study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2014.09.007. References [1] A.B. dos Santos, F.J. Cervantes, J.B. van Lier, Review paper on current technologies for decolourisation of textile wastewaters: perspectives for anaerobic biotechnology, Bioresour. Technol. 98 (2007) 2369–2385. [2] H. Zollinger, Colour Chemistry – Synthesis, Properties and Applications of Organic Dyes and Pigments, VCH, New York, 1987, pp. 92–100. [3] T. Robinson, G. McMullan, R. Marchant, P. Nigam, Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative, Bioresour. Technol. 77 (2001) 247–255. [4] S.L. Moreira-Neto, S.I. Mussatto, K.M.G. Machado, A.M.F. Milagres, Decolorization of salt-alkaline effluent with industrial reactive dyes by laccase-producing basidiomycetes strains, Lett. Appl. Microbiol. 56 (2013) 283–290.

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