Process Biochemistry 43 (2008) 1–7 www.elsevier.com/locate/procbio Development of a continuous process to adapt the textile wastewater treatment by fungi to industrial conditions P. Blánquez, M. Sarrà *, T. Vicent Departament d’Enginyeria Quı́mica, Escola Tècnica Superior d’Enyingeria, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain Received 8 June 2007; received in revised form 27 September 2007; accepted 6 October 2007 Abstract The scale-up of a 10 L air pulsed bioreactor for the continuous treatment of textile wastewater by pellets of the white rot fungus Trametes versicolor has been carried out, based on the geometric similitude with lab-scale bioreactors (0.5 and 1.5 L). Decolourisation experiments of 150 mg L 1 Grey Lanaset G dye solution carried out in the pilot-scale bioreactor showed that in both discontinuous and continuous treatment with an HRT of 48 h, the decolourisation levels were higher than 90%. Some operational changes were carried out in the continuous decolourisation treatment of the dye solution in order to adapt the process to industrial conditions such as, non-sterilization of the dye solution, use of tap water instead of distilled water plus macronutrients and micronutrients and the use of industrial quality co-substrate instead of reagent grade. The pilot system was working continuously during 3 months and over 70 days without sterilization of the dye feeding solution, achieving good decolourisation levels (78% average during the treatment). Continuous treatment of real industrial textile wastewater under non-sterile conditions was carried out during 15 days in the pilot-scale bioreactor, with colour reduction levels between 40 and 60%. These dye concentrations are regarded as environmentally acceptable to be discharged into a municipal wastewater treatment plant if necessary according to the local regulation. # 2007 Published by Elsevier Ltd. Keywords: Textile dye; Pilot-scale; Bioreactor; Trametes versicolor 1. Introduction In recent years many studies have demonstrated the ability of ligninolytic fungi to degrade lignin and a wide range of aromatic compounds as a result of their non-specific extracellular ligninolytic enzyme system. These extracellular enzymes can oxidize persistent organic pollutants including synthetic dyes [1]. Two strategies for the application of such enzymes in the degradation of persistent pollutants have been pursued; the first being the direct transformation of pollutants by active cultures of white rot fungi and the second the use of enzymes produced by the fungi. The advantage of the first strategy with respect to the second is that the enzyme recovery step is avoided. Nevertheless, before an industrial application can be implemented, fungal bioreactors which can be operated under industrial conditions using real wastewaters must be developed. Neither patents nor research works have been found that address this issue. * Corresponding author. Tel.: +34 93 581 27 89; fax: +34 93 581 20 13. E-mail address: Montserrat.Sarra@uab.cat (M. Sarrà). 1359-5113/$ – see front matter # 2007 Published by Elsevier Ltd. doi:10.1016/j.procbio.2007.10.002 A literature search has been carried out to determine the existence of patents related to effluent treatment plants or procedures for decolourisation using white rot fungi. The first patent found is from Chang et al. [2], US patent number 4,554,075. It describes the biodegradation of chloroorganics by Phanerochaete chrysosporium in a rotational reactor, in batch operation under sterile conditions. Similarly, the same authors in the US patent number 4,655,926 [3] describe the procedure for the decolourisation of pulp effluents. They state that the process can also be carried out in continuous mode but they only show results in batch operation. In some other patents such as number US 5,091,089 [4], and number US 6,613,559 B2 [5], the experiments were carried out at Erlenmeyer scale. In the patent request number WO 03/035561 [6], the discontinuous treatment at Erlenmeyer scale under sterile conditions is described but there is no further explanation of how to operate this procedure at industrial scale. Hence, there is no method or procedure ready for immediate industrial implementation for the biodegradation of pollutant compounds or decolourisation of large volumes of industrial 2 P. Blánquez et al. / Process Biochemistry 43 (2008) 1–7 wastewaters at full-scale under non-sterile conditions and continuous mode. Most of research works on dyes biodegradation are conducted at Erlenmeyer scale [7,8]. Screening of different fungi has been carried out demonstrating the great biodegradation potential for different types of dyes [9–11]. Nevertheless, some works have been published about dyes biodegradation by white rot fungi in bioreactor under continuous mode and sterile conditions using culture mediums similar to those used at laboratory scale using Erlenmeyer flasks, without optimization for further application at full-scale [12–15]. Although good decolourisation yields are obtained, there are few works of decolourisation processes in bioreactor under non-sterile conditions. Yang and Yu [16] obtained decolourisation yields for the dye Red 533 with P. chrysosporium between 87 and 100%. Hai et al. [17] developed a submerged membrane fungi reactor for synthetic textile wastewater treatment with Coriolus versicolor under non-sterile conditions. However, no papers related to the continuous treatment of real industrial textile wastewaters in bioreactor by white rot fungi were found. Thus, the main purpose of the present research was to investigate the possibility to develop the process at industrial conditions. To do so, the following objectives must be fulfilled: (1) scale-up a 10 L air pulsed bioreactor, based on the knowledge and experience obtained in our previous research working with lab-scale air pulsed bioreactors, in continuous mode and with periodic biomass renovations [18,19]; (2) undertake continuous treatment of the textile dye Grey Lanaset G under non-sterile conditions in the pilot-scale bioreactor with pellets of Trametes versicolor; (3) continuous treatment of real industrial textile wastewater under non-sterile conditions in the pilot-scale bioreactor. 2.4. Equipment and operating conditions Lab-scale bioreactors. Two glass bioreactors with a useful volume of 1500 and 500 mL, were used to carry out the decolourisation experiments [18]. Biomass fluidization and biomass-liquid phase homogenization conditions were maintained by air pulses generated by an electrovalve [22]. The bioreactor was equipped with a pH controller in order to maintain pH at 4.5 and the temperature was maintained at 25 8C. Operating conditions. Two types of experiments were carried out, (1) batch and (2) continuous. For both types of experiments the start-up of the bioreactor was carried out loading the bioreactor with the batch medium. Then the culture medium was inoculated with an amount of pellets equivalent to 3.2 g dry weight L 1. In the continuous experiments after a batch stage and once the glucose concentration in the reactor was 2 g L 1 approximately the continuous stage was switched on. The hydraulic retention time (HRT) was variable depending on the experiment, and it is detailed in each case. The biomass, in pellet form and maintained under fluidizing conditions by air pulses, was retained in the bioreactor throughout the experiment with no loss in the effluent. Mixture time determination. For the determination of the mixture time the bioreactor is filled with water and the aeration system is set-up by air pulses. A dye pulse of the dye Grey Lanaset G (50 mL of solution containing 0.25 mg of dye) is introduced by the bottom of the bioreactor. From this moment samples are taken from the top of the bioreactor and absorbance is measured. Mixture time is determined as the one corresponding to the stability in absorbance measurements. 2.5. Industrial textile wastewater A spent dying bath was kindly supplied by Artèxtil S.A. (Sabadell, Spain). The composition of the spent bath is unknown, only the initial dying bath composition is defined: esterol 126 1.0%, citric acid 1.5 g L 1, amplex DG 1.0 g L 1, antifoam SAE 0.3%, Grey Lanaset G 0.7491%, Yellow Lanaset 2R 0.2677%, Red Lanaset G 0.1257%. Weight percentages are related to fiber weight. A volume of 600 L of dying bath was used to dye 60 kg of wool. 2.6. Colour determination 2. Materials and methods Spectrophotometric measurements were carried out at the visible maximum absorbance, 590 nm on a PV 8620 Philips spectrophotometer. 2.1. Microorganism 2.7. Glucose determination T. versicolor was obtained from the American Type Culture Collection (ATCC # 42530). The fungus was maintained on 2% malt agar slants at 25 8C until use. Subcultures were routinely prepared as required from the mother culture. Pellets of T. versicolor were obtained as described previously [20] with a 3 mm diameter approximately. Glucose concentrations were measured with an YSI 2000 enzymatic analyzer from Yellow Springs Instruments and Co. 2.2. Media cultures Batch medium. The batch medium contained per liter: 8 g glucose, 1.9 g NH4Cl, 11 mL of a supplemented medium [21] and 0.15 g Grey Lanaset G dye. The pH was adjusted to 4.5 with 0.5 M NaOH and the solution was sterilized at 120 8C for 30 min. Continuous medium. The continuous simulated feeding wastewater consisted of dye (0.15 g L 1), glucose (0.31 g glucose (g dry weight) 1) and supplemented medium (11 mL L 1). 2.3. Chemicals Grey Lanaset G, which is a commercial mixture of several metal complex dyes (Cr and Co), was complimentarily supplied by Ciba (Ref. 080173.5). All other chemicals were reagent grade. 2.8. Laccase activity Laccase activity was measured using a modified version [23] of the method for the determination of manganese peroxidase [24], where 2,6-dimethoxyphenol (DMP) is oxidized by laccase, even in the absence of a cofactor. Conversely, oxidation by manganese peroxidase (MnP) requires the presence of H2O2 as a cofactor and catallytically active Mn2+. One activity unit (AU) was defined as the number of micromoles of DMP oxidized per minute. The DMP extinction coefficient was 10,000 M 1 cm 1. 2.9. Intracellular laccase activity Biomass was washed with water, filtrated, and resuspended in a 250 mM sodium malonate buffer at pH 4.5. Samples of 3 mL were taken and disrupted in a Constant Cell Disruption Systems (Constant Systems Ltd.) by one shot at 2.86 atm. Finally the mixture was centrifuged for 30 min at 12000 rpm and 4 8C. Laccase assay was applied to the clear liquid. P. Blánquez et al. / Process Biochemistry 43 (2008) 1–7 3. Results and discussion 3.1. Pilot plant bioreactor design The good results obtained at lab-scale in the continuous decolourisation process of the dye Grey Lanaset G by T. versicolor with air-pulsed bioreactors [18,19] encouraged us to carry out the design of a pilot plant air-pulsed bed bioreactor with a useful volume of 10 L in order to proceed with the decolourisation process of bigger volumes of wastewater. KLa was not used as scaling criteria because one of the main characteristics of the lab-scale bioreactors used for this purpose is the presence of excess oxygen in the liquid medium, since both, mix and pellets of T. versicolor fluidization are obtained by means of air flow. Moreover, oxygen consumption is low, given that during the continuous decolourisation process there is no growth of the biomass due to the lack of nitrogen in the feeding solution. As lab-scale bioreactors, the pilot-scale bioreactor comprises three parts: The lower part, where air is introduced into the system and is distributed by means of a holed plate, which should have a pore size under 1 mm, so as to retain the fungal pellets in the bioreactor; a central cylindrical part; and the head of the bioreactor, with a diameter larger than that of the central part, to facilitate the gas–liquid phase separation and to minimize foam formation. This part has different orifices in the top for instrumentation, an air outlet, a glass tube for biomass renovation and an acid and alkali inlet in order to maintain pH. 3 The head of the bioreactor must also have a lateral orifice for the effluent outlet, maintaining constant volume. Typical ratios between the bioreactor head and body diameters have not been found in the literature, neither height ratios between both parts of the bioreactor have been found, but in fact these ratios may vary for the industrial fluidized bioreactors. In our case, for the pilot-scale bioreactor design total height was taken into account, as it should be placed in a temperaturecontrolled room. The pilot plant bioreactor was designed with these restrictions and with a progressive expansion angle from the central part to the head of the reactor of 608. Fig. 1 shows the scheme of the pilot experimental system and the dimensions of the pilot bioreactor. 3.1.1. Air supply system The air supplied to the bioreactor is used for fungal activity maintenance and for the biomass-liquid phase homogenization, but the needs for homogenization are greater than those for the maintenance, so the air is supplied by pulses, achieving enough oxygen for fungal maintenance, homogenization in the bioreactor and thereby avoiding any hooking of the fungus to the walls or instrumentation in the bioreactor. The pulsed airflow was generated as previously described [20] and the pulse frequency (0.16 s 1) was the same as for the lab-scale bioreactors. In order to establish the airflow in the pilot bioreactor, the air rates for the lab-scale bioreactors were taken into account. For the 0.5 L volume bioreactor, the air rate Fig. 1. Scheme of the pilot experimental system. The dimensions are (cm): a, 7; b, 9; c, 3.5; d, 9.5; e, 33; f, 3; g, 9; h, 6; Øi (int), 18; Øj (int), 10; Øk (int), 140. 4 P. Blánquez et al. / Process Biochemistry 43 (2008) 1–7 Table 1 Air rates and mixing times in the lab-scale (0.5 and 1.5 L) and pilot (10 L) bioreactors Volume (L) Air flow (L h 1) Air rate (cm s 1) Mixing time (min) 0.5 1.5 10 12 30 40 0.17 0.19 0.14 4.5 2.5 6 was 0.17 cm s 1 and for the 1.5 L volume bioreactor, it was 0.19 cm s 1, so for the pilot plant bioreactor it was fixed at 0.18 cm s 1, corresponding to an air flow rate of 50 L h 1. But after system start up, the flow rate should be decreased to 40 L h 1 due to foam formation. The air rate corresponding to this flow rate was 0.14 cm s 1, as shown in Table 1. The airflow rate is not constant, in 1 s 66.6 cm3 of air is introduced into the bioreactor, which leads to an increase of the level of liquid in the bioreactor. This increase is minimized by widening the head of the bioreactor, therefore the level of liquid after the air pulse only increases 2.6 mm. 3.2. Batch operation Fig. 2a shows the results obtained in a batch fungal decolourisation process in the pilot plant bioreactor with an initial dye concentration of 150 mg L 1. It is observed that after 24 h of treatment the decolourisation percentage was over 90%, and at the end of the run the decolourisation level was 94%. Extracellular laccase activity increased from the beginning of the process up to 1838 AU L 1 at the end of the run, and glucose was almost totally consumed during the first 3 days of treatment. If these results are compared with those obtained in the 0.5 L working volume bioreactor (Fig. 2b), it is observed that the behavior of the system was quite similar. The main difference is the glucose consumption rate, which was higher for the pilot-scale bioreactor. In the lab-scale bioreactor total depletion of glucose was achieved after 5 days of treatment. This fact may be due to the better oxygen transfer in the pilot plant bioreactor. It was also observed that decolourisation percentages over 90% were achieved more quickly than in labscale bioreactors, although laccase activity levels were similar in both cases. At the end of the batch process in the pilot bioreactor, a low fluidization of the biomass was observed due to an increase in biomass resulting from the growth of the fungus. No significant effect was observed in the fluidization of the biomass in lab-scale bioreactors due to fungal growth. To avoid fluidization operational problems in the pilot-scale bioreactor the initial inoculum was reduced for the following experiments from 3.2 to 2.5 g dry weight L 1, reducing the biomass requirement by 20%, and checking that the biomass reduction had no influence on the decolourisation yield. 3.3. Continuous operation Fig. 3a shows the results obtained in a continuous decolourisation process with the pilot-scale bioreactor. After 3 days of batch operation, the continuous process was switched Fig. 2. Time course of glucose concentration (~), laccase activity (*) and percentage of colour reduction (&) during the discontinuous biodegradation process of the dye in (a) pilot-scale bioreactor (10 L) and (b) lab-scale bioreactor (0.5 L). on, and it was maintained for 10 more days with a hydraulic retention time of 48 h. In this experiment intra- and extracellular laccase activity were measured. Maximum extracellular enzyme activity (4192 AU L 1) was reached after 9 days of treatment and was much higher than the obtained in the batch process (1838 AU L 1). The decolourisation percentage was maintained approximately constant at 90%. Intracellular enzyme activity presented the same profile as extracellular enzyme activity. If this experiment is compared with the one carried out in a lab-scale bioreactor (Fig. 3b) it can be observed that the profiles are very similar, but when working with the pilot plant bioreactor the extracellular enzymatic activity was twice as high as the activity in the lab-scale bioreactors. The greatest colour reduction was obtained during start-up, where pellets turned completely dark due to the initial dye adsorption on the biomass. After 24 h the pellets became discoloured, even during the continuous process when the dye was continuously fed. This indicates that decolourisation was not due to an adsorption process as previously evidenced [20]. The results obtained in continuous mode show that the behavior of the pilot-scale bioreactor is similar to that of lab scale ones, achieving good decolourisation percentages. P. Blánquez et al. / Process Biochemistry 43 (2008) 1–7 Fig. 3. Time course of glucose concentration (~), laccase extracellular activity (*), laccase intracellular activity (^) and percentage of colour reduction (&) during the continuous biodegradation process of the dye in (a) pilot-scale bioreactor (10 L) and (b) lab-scale bioreactor (0.5 L). HRT: 48 h. 3.4. Pilot-scale treatment adaptation to industrial conditions In order to check the robustness of the pilot-scale treatment and to adapt the developed process at lab-scale to industrial conditions, some operational changes were carried out in a continuous experimental run, such as non-sterilization of the dye wastewater, use of tap water instead of distilled water plus micronutrients and macronutrients and the use of industrial 5 quality co-substrate instead of reagent grade. Fig. 4 shows the results obtained in this experiment. After a 3-day batch period, once glucose concentration in the reactor was 2 g L 1 approximately, the continuous stage was switched on. Until day 31 the HRT was maintained at 48 h, and the decolourisation percentage was over 85%. From day 21 one third of the biomass was removed once per week [19], establishing therefore a cellular retention time (CRT) of 21 days. The arrows in Fig. 4 indicate the moment of partial biomass renovations. On day 28 the second biomass renovation was carried out, but inactive biomass was added to the bioreactor instead of fresh biomass, in order to analyze the response of the system to withstand any possible problems related to biomass state. A direct consequence of this perturbation was a decrease of the decolourisation percentage from 85 to 55%, corresponding to one third reduction in the decolourisation percentage, showing the effect of the inactive biomass. From this moment, to evaluate the recovery of the process, on day 31 the HRT was increased, and the following biomass renovations were carried out with active biomass, allowing an increase in the decolourisation percentage. An increase in enzymatic activity was also observed in each biomass renovation. As previously described [20], no direct relationship between extracellular enzyme activity and decolourisation rate exist, but a minimum laccase concentration (150 AU L 1) is necessary to carry out the decolourisation process. On day 41 of treatment, when the decolourisation was 77%, the system was considered to have recovered sufficiently and it was ready to be used to analyze a new perturbation. The feeding of the dye solution was carried out under non-sterile conditions. Despite this, the system continued to improve and achieved decolourisation percentages over 80% on day 51 of treatment. From that moment macronutrients and micronutrients were removed from the feeding solution, the distilled water was replaced by tap water and HRT was reduced. The system continued to work correctly indicating that the changes did not affect the operation. From day 59 of treatment, when the decolourisation was 78%, the co-substrate reagent grade glucose was replaced by industrial quality glucose and no change was observed either in the system response. On day 66 HRT was increased again and the glucose concentration feeding was reduced from 3 to 2 g L 1, which Fig. 4. Time course of glucose concentration (~), laccase activity (*) and percentage of colour reduction (&) during the continuous biodegradation process of the dye under non-sterile conditions in the pilot-scale bioreactor (10 L). 6 P. Blánquez et al. / Process Biochemistry 43 (2008) 1–7 means a 33% of reduction with respect to the glucose concentration corresponding to the experimentally determined consumption rate [18]. This reduction in the glucose feeding concentration resulted in a decrease in the decolourisation yield from 70 to 60%. These results are in agreement with Dosoretz et al. [25] who stated that in glucose-limited cultures the fungus produces proteases that reduce enzyme concentration, which could affect decolourisation. From day 90 the glucose concentration was reestablished to the corresponding consumption rate and the system recovered, achieving decolourisation percentages over 85%, and finally, during the last period, after day 96 HRT was decreased to 48 h. Thus, the process had been carried out over 70 days without sterilization of the dye feeding solution, achieving good decolourisation levels (average during the treatment 78%) and overcoming various problems that decreased the decolourisation percentage to 60%. It also shows that the feeding glucose concentration must be the corresponding to the consumption rate. Some other authors have also worked under non-sterile conditions in continuous or semi-continuous mode achieving similar decolourisation percentages, but the operation times are lower than the one of the present work. Yang and Yu [16] maintained the decolourisation process of Red 533 dispersed dye in a biofilm bioreactor with P. chrysosporium immobilized on foam material for 11 days under non-sterile conditions. The decolourisation level was 100% during the first 8 days and dropped to 87% remaining stable for the rest of the experiment period (3 days). The experiments were not continued further but the authors stated that the acidification of the medium (pH 3.1) by the fungal activity minimized any contamination risk. Leidig et al. [26] described the continuous biotransformation of the dye Poly R-478 with encapsulated T. versicolor under nonsterile conditions. The polyvinyl alcohol capsule protected the fungus and the peroxidases from bacterial attack for about 6 weeks. Borchert and Libra [27] described the decolourisation of black five reactive dye in a sequencing batch process with T. versicolor under non-sterile conditions. The experiment was maintained for 55 days (five cycles). The first four cycles achieved high degrees of decolourisation (91–98%), but in the fifth cycle the decolourisation dropped to 72%, followed by an almost total loss of decolourisation ability in the subsequent cycle due to the presence of bacteria in the culture. Libra et al. [28] continued their research on different strategies for synthetic textile wastewater treatment at Erlenmeyer scale to maintain the decolourisation efficiency at high levels under non-sterile conditions. High decolourisation percentages were achieved growing T. versicolor on sterilized grains as sole substrate and support material during two cycles, but in the third one the decolourisation percentage dropped to 55%. 3.5. Continuous treatment of industrial textile wastewater Finally, to evaluate the ability of the system to treat real industrial textile wastewater continuously under non-sterile conditions, a preliminary experiment was carried out. No papers related to the continuous treatment of real industrial Fig. 5. Time course of glucose concentration (~), laccase activity (*) and percentage of colour reduction (&) during the continuous biodegradation process of a real textile wastewater under non-sterile conditions in the pilotscale bioreactor (10 L). TRH: 48 h. textile wastewater in bioreactor under non-sterile conditions by white rot fungi were found. Libra et al. [28] and Hai et al. [17] obtained good decolourisation yields under non-sterile conditions of synthetic textile wastewater, but they did not try with real textile wastewater, and Nilsson et al. [29] showed the decolourisation of a real textile wastewater by Pleurotus flabellatus grown on luffa sponge packed in a continuous reactor but under sterile conditions. In the preliminary experiment the start-up medium consisted of wastewater supplemented with the nutrients corresponding to the batch medium defined in materials and methods. The culture medium was inoculated with pellets, 2.5 dry weight L 1, and after 3-day start-up period, the continuous operation, feeding real textile industrial wastewater under non-sterile conditions, was switched on with a HRT of 48 h. The glucose was fed separately under sterile conditions. Results obtained are shown in Fig. 5. Industrial wastewater is not as coloured as synthetic wastewater. Industrial wastewater shows lower initial absorbance (0.370 units at 590 nm) than synthetic wastewater with 150 mg L 1 of the dye Grey Lanaset G (equivalent to 1.8 units at 590 nm). The maximum colour reduction obtained during the continuous treatment of industrial wastewater was 60%, between days 4 and 6 of treatment, but it decreased to 40% and was maintained until the end of the run (15 days). The decrease in the colour reduction percentage may be due to a huge bacteria contamination of the feeding tank detected from day 10. The feeding tank was maintained at 25 8C during several days and very probably the bacteria contamination was present in the system before the detection. Even the low colour reduction percentage obtained in our experiment, the treated textile wastewater is regarded as environmentally acceptable to be discharged into a municipal wastewater treatment plant if necessary according to the local regulation (DOG 3894-29.5.2003). Similar results were obtained by Nilsson et al. [29], the HRT was 25 h and the experiment was run for 10 days. P. Blánquez et al. / Process Biochemistry 43 (2008) 1–7 4. Conclusions Decolourisation experiments of 150 mg L 1 Grey Lanaset G dye solution by pellets of T. versicolor were carried out in the pilot-scale bioreactor (10 L) showing that in both discontinuous and continuous treatment with an HRT of 48 h, the decolourisation levels were higher than 90%. The pilot system for the continuous treatment of Grey Lanaset G solution was adapted to industrial conditions making the following improvements: non-sterilization of the synthetic dye solution, macronutrients and micronutrients elimination in the dye solution and industrial quality glucose used as a cosubstrate. The system was operating during 3 months, facing up to provoked disturbances, obtaining colour removal percentages upper than 70%. Real industrial textile wastewater was successfully treated continuously during 15 days under nonsterile conditions, with colour reduction levels between 40 and 60%. Acknowledgements The present work was funded by the Spanish Commission of Science and Technology (Project PPQ2000-0645-C02-01) and the Spanish Commission of Education and Science (Project CTQ2004-01459). The authors wish to thank the financial support of DURSI 2005SGR 00220 Generalitat de Catalunya. The Department of Chemical Engineering of the Universitat Autònoma de Barcelona is the Unit of Biochemical Engineering of the Xarxa de Referència en Biotecnologia (XRB) de la Generalitat de Catalunya. References [1] Reddy CA. The potential for white rot fungi in the treatment of pollutants. Curr Opin Biotech 1995;6. pp. 328–328. [2] Chang H, Joyce TW, Kirk TK, Huynh V. Process of degrading chloroorganics by white rot fungi. Patent No. 4,554,075, 1985. [3] Chang H, Joyce TW, Kirk TK, Huynh V. Process of treating effluent from a pulp or papermaking operation. Patent No. 4,655,926, 1987. [4] Shen HP, Mou DG, Lim KK, Feng P, Chen CH. Microbial decolorization of wastewater. Patent No. 091,089, 1992. [5] Raghukumar C, Shailaja MS. Simultaneous decolourisation and detoxification of molasses spent wash using novel white rot-lignin-modifying fungus Flavodon flavus. Patent No. 6,613,559 B2, 2003. [6] Vanhulle S, Lucas M, Mertens V, Gobeaux B, Corbisier AM, Bols CM, Buchon F, Wesenberg D, Agathos S. Sustainable process for the treatment and detoxification of liquid waste. Patent request No. WO 03/035561 A2, 2003. [7] Máximo C, Costa-Ferreira M. Decolourisation of reactive textile dyes by Irpex Lacteus and lignin modifying enzymes. Process Biochem 2004;39(11):1475–9. [8] Yesislada O, Asma D, Cing S. Decolorization of textile dyes by fungal pellets. Process Biochem 2003;38(6):933–8. [9] Swamy J, Ramsay A. The evaluation of white rot fungi in the decoloration of textile dyes. Enzyme Microb Tech 1999;24:130–7. 7 [10] Moreira MT, Mielgo I, Feijoo G, Lema JM. Evaluation of different fungi strains in the decolourisation of synthetic dyes. Biotechnol Lett 2000;22:1499–503. [11] Chagas EP, Durrant TR. Decolourisation of azo dyes by Phanerochaete chrysosporium and Pleurotus sajorcaju. Enzyme Microb Tech 2001;29: 575–9. [12] Zhang FM, Knapp JS, Kelvin NT. Development of bioreactor systems for decolorization of Orange II using white rot fungus. Enzyme Microb Technol 1999;24:48–53. [13] Mielgo I, Moreira MT, Feijoo G, Lema JM. Biodegradation of a polymeric dye in a pulsed bed bioreactor by immobilised Phanerochaete chrysosporium. Water Res 2002;36:1896–901. [14] Rodrı́guez Couto S, Sanromán MA, Hofer D, Gübitz GM. Stainless steel sponge: a novel carrier for the immobilisation of white-rot fungus Trametes hirsuta for decolourization of textile dyes. Bioresour Technol 2004;95:67–72. [15] Romero S, Blánquez P, Caminal G, Font X, Sarrà M, Gabarrell X, Vicent T. Different approaches to improving the textile dye degradation capacity of Trametes versicolor. Biochem Eng J 2006;31:42–7. [16] Yang FC, Yu JT. Development of a bioreactor system using immobilized white rot fungus for decolorization. Part II: continuous decolorization tests. Bioprocess Eng 1996;16:6–11. [17] Hai FI, Yamamoto K, Fukushi K. Development of a submerged membrane fungi reactor for textile wastewater treatment. Desalination 2005;192: 315–22. [18] Blánquez P, Caminal G, Sarrà M, Vicent T. The effect of the HRT on the decolourisation of the Grey Lanaset G textile dye by Trametes versicolor. Chem Eng J 2007;126:163–9. [19] Blánquez P, Sarrà M, Vicent T. Study of the cellular retention time and the partial biomass renovation in a fungal decolourisation continuous process. Water Res 2006;40:1650–6. [20] Blánquez P, Casas N, Font X, Gabarrell X, Sarrà M, Caminal G, Vicent T. Mechanism of textile metal dye biotransformation by Trametes versicolor. Water Res 2004;38:2166–72. [21] Kirk TK, Schulz E, Connors WJ, Lorenz LF, Zeikus JG. Influence of cultural parameters on lignin metabolism by Phanerochaete chrysosporium. Arch Microbiol 1978;117:277–84. [22] Feijoo G, Dosoretz C, Lema JM. Production of lignin peroxidase from Phanerochaete chrysosporium in packed bed bioreactors with recycling. Biotechnol Tech 1994;8(55):365–8. [23] Kaal EEJ, de Jong E, Field JA. Stimulation of ligninolytic peroxidase activity by nitrogen nutrients in the white rot fungus Bjerkandera sp. strain BOS55. Appl Environ Microb 1993;59:4031–6. [24] Paszczynski A, Crawford RL, Huynh VB. Manganese peroxidase of Phanerochaete chrysosporium: purification. Method Enzymol 1988;161: 264–70. [25] Dosoretz CG, Dass SB, Reddy CA, Grethlein HE. Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium. Appl Environ Microb 1990;56(11):3429–34. [26] Leidig E, Prusse U, Vorlop K, Winter J. Biotransformation of Poly R-478 by continuous cultures of PVAL-encapsulated Trametes versicolor under non-sterile conditions. Bioprocess Eng 1999;21(1):5–12. [27] Borchert M, Libra JA. Decolorization of reactive dyes by the white rot fungus Trametes versicolor in sequencing batch reactors. Biotechnol Bioeng 2001;75:313–21. [28] Libra JA, Borchert M, Banit S. Competition strategies for the decolourisation of a textile-reactive dye with the white rot fungi Trametes versicolor under non-sterile conditions. Biotechnol Bioeng 2003;82(6): 736–44. [29] Nilsson I, Möller A, Mattiasson B, Rubindamayugi MTS, Welander U. Decolorization of synthetic and real textile wastewater by the use of whiterot fungi. Enzyme Microb Technol 2005;38(1/2):94–100.