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Decolorization of synthetic and real textile wastewater by the use of white-rot fungi
Enzyme and Microbial Technology 38 (2006) 94–100
Decolorization of synthetic and real textile wastewater by the use of white-rot fungi I. Nilsson a , A. M¨oller a , B. Mattiasson a , M.S.T. Rubindamayugi b , U. Welander a,∗ a
Department of Biotechnology, Center of Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden b Department of Botany, Applied Microbiology Unit, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania Received 31 August 2004; received in revised form 13 April 2005; accepted 28 April 2005
Abstract Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h. The absorbance at 584 nm, the wavelength at which the wastewater absorbed the most, decreased from 0.3 in the inlet to approximately 0.1 in the effluent from the reactor. © 2005 Published by Elsevier Inc. Keywords: Biological rotating contactor reactor; Textile dyes; Pleurotus flabellatus; Trametes versicolor; Reactive Blue 4; Reactive Red 2
1. Introduction Wastewater from textile industries constitute a threat to the environment in large parts of the world. The degradation products of textile dyes are often carcinogenic [1,2]. Furthermore, the absorption of light due to textile dyes creates problems to photosynthetic aquatic plants and algae. Earlier studies have shown that many reactive dyes are not degraded in ordinary aerobic sewage treatment processes and that they can be discharged from the treatment plant unaffected [3,4]. The reactive dyes are highly water-soluble polyaromatic molecules, which means that their adsorption to solids is relatively poor [5]. Some dyes are decolorized under anaerobic conditions. The effluent might, however, be ∗ Corresponding author. Present address: School of Technology and Design, Department of Chemistry, V¨axj¨o University, Vejdes Plats 6, SE-351 95 V¨axj¨o, Sweden. Tel.: +46 470 70 88 21; fax: +46 470 70 87 56. E-mail address: [email protected] (U. Welander).
0141-0229/$ – see front matter © 2005 Published by Elsevier Inc. doi:10.1016/j.enzmictec.2005.04.020
toxic [6]. Furthermore, there might be a risk for reverse colorization when anaerobic degradation products are exposed to oxygen [7]. When studying degradation of organic molecules, textile dyes may be regarded as a poorly chosen example, since they are once being selected for their color but also for being resistant to external factors. Dyes on cloths are selected to be resistant to, e.g. UV light, and therefore they constitute a challenging group of chemicals when designing degradation processes. A key feature in the degradation processes, at least the aerobic ones, involves generation of activated oxygen forms that can carry out the initial attack on the stable structure. After the first modification, there are often easier steps where more organisms can be beneficial. Wood rotting fungi have interesting properties in the sense that they are capable to degrade lignin which is a polymeric structure with a lot of aromatic rings. Lignin is regarded as stable. The fungi have been shown to excrete certain enzymes that catalyze the formation
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Fig. 1. The chemical structures of Reactive Red 2 (the drawing to the left) and Reactive Blue 4 (the drawing to the right).
of activated oxygen, and then the process is initiated. The lack of selectivity among these enzymes with regard to the aromatic compounds that are attacked is the background to the interest to use wood rotting fungi also for treating textile dyes. Measurements of the enzyme activity in the medium is, however, not always a reliable parameter for determination of decolorization capacity, since most of the enzymes might be retained in the fungal slime layer [8]. The fungi do need to be supplied with an external carbon source in order to be able to degrade polycyclic aromatic hydrocarbons [9]. White-rot fungi have been used in studies concerning decolorization of different textile dyes [10–15]. Relatively few experiments have, however, been performed using continuous processes with white-rot fungi for treatment of wastewater. Rotating biological contactors have been studied regarding treatment of kraft pulp bleach plant effluent [16,17] and munition waste [18]. A fluidized bed reactor with Trametes versicolor immobilized in calcium alginate gel has furthermore been used for treatment of Kraft bleach effluents [19]. The aim of the study was to develop an efficient and relatively simple continuous process based on white-rot fungi for decolorization of textile dyes. Two dyes, Reactive Blue 4 and Reactive Red 2, from two of the most commonly used groups of reactive dyes were used as model substances in the experiments (Fig. 1). Four different white-rot fungi strains Phanerochaete chrysosporium, T. versicolor, Pleurotus ostreatus and Pleurotus sajor-caju were screened on colored agar plates for their capability to degrade the dyes and the most efficient strains in regard to decolorization were then chosen for experiments in liquid phase. Real textile wastewater from a textile industry in Tanzania was furthermore treated by Pleurotus flabellatus, a fungus which was isolated from the environment in Tanzania.
2. Material and methods 2.1. Microorganisms The fungi used in this study were T. versicolor PRL 572, P. ostreatus MUCL 29527, P. sajor-caju MUCL 29757, P. chrysosporium DSM 1556 and P. flabellatus (Prairie Regional Laboratory (PRL), Mycoth´eque de l’Univerist´e Cathaolique de Louvain (MUCL), Deutsche Sammalung von Mikroorganismen (DSM)). T. versicolor, P. ostreatus,
P. sajor-caju and P. chrysosporium were kindly provided by Dr. P.-O. Nyman, Department of Biochemistry, Lund University. A pure culture of P. flabellatus was obtained from the fungal collection of the Department of Botany, Applied Microbiology Unit, University of Dar es Salaam, Tanzania. 2.2. Analyses The samples were filtered through GF/A glass-fiber filters before the absorbance was measured on Ultrospec 1000 or Ultrospec 3000 spectrophotometers (Phamacia Biotech, Uppsala, Sweden) during the first part of the study with synthetic wastewater. The peak absorbance was measured at 595 nm for Reactive Blue 4 and 538 nm for Reactive Red 2. Scanning was performed between 200 and 800 nm. Tap water was used as reference. The absorbance was measured at the peak wavelength 584 nm after centrifugation of the samples for 5 min at 12,000 × g when real wastewater was studied. Distilled water was used as reference. Scanning could unfortunately not be performed due to practical reasons. 2.3. Storage of strains of fungi The fungi were stored on maltagar plates made on a medium consisting of 10 g/l malt extract and 10 g/l agar dissolved in distilled water. New plates were inoculated once a month to ensure viability of the isolates. 2.4. Evaluation of dye decolorization potential of different fungal strains Four different white-rot fungi strains T. versicolor, P. ostreatus, P. sajor-caju and P. chrysosporium were screened for their capability to degrade poly-R 478, Reactive Blue 4 (an anthraquinone dye) and Reactive Red 2 (an azo dye) (Fig. 1). Poly-R 478 was used as a control dye to check that the fungi had not lost enzyme activity during storage on malt agar plates [20,21]. The experiment was performed on solid media plates made on media which contained 0.2 g/l dye, 10 g/l agar and for half the number of plates 10 g/l of malt extract. The media were autoclaved and poured into petridishes. Each plate was then inoculated with a piece of malt agar overgrown with fungal mycelia. The decolorization of
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the plates was studied as a function of time. The radius of the decolorized area around the inoculum was measured with a ruler. The colored plates were used for a continuous control of the enzyme productivity of the fungi.
All experiments were performed in duplicate at room temperature.
2.5. Carrier materials
Two cylindrical reactors with conical bottoms were used for continuous experiments with T. versicolor (Fig. 2). One of the reactors was used for studies of decolorization of Reactive Red 2 (reactor 1) while the other was used for studies of decolorization of Reactive Blue 4 (reactor 2). The reactors had a liquid volume of 1 l. They were autoclaved and then filled with natural luffa sponge overgrown with fungus mycelia. A medium with the same composition as described above in the paragraph about the luffa sponge batch experiments was prepared, autoclaved and cooled to room temperature. The medium was then pumped into the reactors with a flow rate of 12 ml/h during the first 13 days. The flow rate was then decreased to 5.2 ml/h in order to examine if the absorbance at the peak wavelengths could be further decreased. The dye concentration in the medium into reactor 1 was 0.5 g/l during the whole study (26 days) while it was 0.5 g/l into reactor 2 day 1–26 and 0.2 g/l day 27–34. The air-phase of the reactors were renewed by the aid of aquarium pumps connected with tubes at the bottom of the reactors. The air was led into and out from the reactors through sterile filters (0.2 m) in order to avoid contamination of the reactors. The medium was pumped from e-flasks sealed with cotton stoppers and the outlet was collected in the same kind of vessels. The ends of the tubes were washed with 70% ethanol when the medium or outlet flasks were exchanged. All flasks and tubes were autoclaved before use. The oxygen concentration of the reactor contents was approximately 7 mg/l while the pH
Two different carrier materials were used during the experiments, natural luffa sponge and birch wood. The sponge was boiled and sliced into 3 cm thick slices which were then autoclaved together with 250 ml of a malt extract medium (10 g/l) in jars with a volume of 2 l. The sponge was inoculated with malt agar pieces overgrown with fungus. The jars were stored closed in dark until a considerable amount of biomass was established. The jars were rotated once a day so that the sponges were soaked in the malt extract. The birch wood carrier had the shape of circular discs with a diameter of 2.5 cm for the batch experiments and 5 cm for the continuous experiments. The thickness of the discs was 0.5 cm in all experiments. The discs were placed in beakers, which were filled with water and covered with aluminum foil, and autoclaved during 20 min at 121 ◦ C. The discs were then placed on malt agar plates overgrown with fungus. The carriers were overgrown with fungal mycelia after a period of 1–3 weeks. 2.6. Batch experiments The decolorization of Reactive Blue 4 and Reactive Red 2 were studied in batch experiments using T. versicolor. The experiments were performed in 250 ml e-flasks. The flasks were filled with 50 or 100 ml of medium, sealed with cotton stoppers and autoclaved. The smaller volume was used for the experiments with wooden carrier. A medium containing 0.5 or 0.2 g/l of dye, 3 g/l glucose, 0.24 g/l KNO3 , 0.1 g/l K2 HPO4 , 0.1 g/l KCl, 0.11 g/l MgSO4 ·H2 O, 2.4 mg/l Fe(EDTA), 5 ml/l of a trace metal solution, 1 mg/l thiamine and 10 ml/l of a phosphate/citric acid buffer was used in the experiments with natural luffa sponge. The buffer contained 78.5 g/l Na2 HPO4 ·2H2 O and 214.6 g/l citric acid while the trace metal solution contained 200 mg/l FeSO4 ·7H2 O, 10 mg/l ZnSO4 ·7H2 O, 3 mg/l MnCl2 ·4H2 O, 20 mg/l CoCl2 ·6H2 O, 1 mg/l CuCl2 ·2H2 O, 2 mg/l NiCl2 ·6H2 O, 500 mg/l Na2 MoO4 ·2H2 O and 30 mg/l H3 BO3 . Distilled water was used for all solutions. Two different media were used in the experiments with birch wood discs. One of the media had the same composition as the medium described above and the other medium contained 0.2 or 0.5 g/l of dye and 10 ml/l of the phosphate citric acid buffer. Tap water was used for the latter medium in order to ensure the presence of enough amounts of trace metals. Carriers overgrown with fungus were then added. The flasks were placed on a shaking table having a rate of 120 rpm. The pH was approximately 3 in all experiments. Autoclaved controls were set up with autoclaved carriers in all experiments in order to study abiotic dye removal.
2.7. Continuous experiments
Fig. 2. The design of the reactors filled with natural luffa sponge. The height of the reactor is 35 cm, while the diameter is 12 cm.
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of wastewater from the bleaching process. Nutrients and an extra carbon source were added to the water according to the following: 0.75 g/l glucose, 0.25 ml/l acetic acid, 0.23 g/l NH4 Cl, 0.06 g/l NaH2 PO4 ·2H2 O, 0.28 g/l NaCl, 0.37 g/l KCl, 0.04 g/l MgCl2 ·6H2 O, 0.02 g/l CaCl2 ·2H2 O and 0.20 g/l yeast extract. The pH of the textile wastewater was set to between 3 and 4, the optimal pH range for the fungus. The air-phase of the reactor was renewed with an air-pump. The experiments was continued for 10 days. All reactor experiments were performed at room temperature. Fig. 3. The design of the biological rotating contactor reactor. The diameter of the reactor cylinder is 7 cm while its length is 29 cm.
3. Results was between 3 and 4. Two reactors with the same design as described above were used as sterile controls. Another continuous reactor with a design of a rotating biological contactor was operated with a medium consisting of 0.2 g/l of dye and 10 ml/l of the phosphate citric buffer described under the batch experiments (Fig. 3). It had a total volume of 1 l and a liquid volume of 400 ml. Nine wood discs overgrown with T. versicolor were placed on an axis, which rotated with a speed of 20 rpm. The air volume was renewed with the aid of an aquarium pump giving a flow rate of 900 cm3 /min. The air was led into and out from the reactor through sterile filters (0.2 m) in order to avoid contamination. A second reactor of the same design as the reactor described above was used as a sterile control. The retention time in the reactors were 3 days during days 1–10 after which it was halved. However, due to an increase of absorbance in the outlet from the reactor inoculated with fungus, the flow rate was reset to its original value on day 12. The pH was around 3. The process was running for 18 days with Reactive Blue 4 and 10 days with Reactive Red 2. The reactor parts were autoclaved before assemblage and the same routine as described above was used for collection of the outlet and exchange of medium and outlet flasks. A seventh reactor with a volume of 1.5 l was used in order to study decolorization of dyes in real textile wastewater from a textile industry in Tanzania. The reactor was autoclaved and packed with natural luffa sponge inoculated with P. flabellatus. Autoclaved textile wastewater was then pumped into it with a flow rate of 59 ml/h giving a hydraulic retention time of 26 h. The water was a mixture of 750 ml/l of wastewater from the dyeing process and 250 ml/l
3.1. Evaluation of dye decolorization potential of different fungi The experiment with poly-R 478 showed that all strains had some extracellular enzyme activity. P. chrysosporium and T. versicolor were shown to be the most efficient strains in regard to decolorization of Reactive Blue 4 and Reactive Red 2 (Table 1). These strains were therefore used in the batch and continuous experiments in order to study decolorization of the dyes in water phase. P. chrysosporium did, however, not decolorize the dyes in water phase so the paper is therefore focused on T. versicolor. 3.2. Batch experiments T. versicolor was shown to be able to decolorize Reactive Red 2 as well as Reactive Blue 4 when natural luffa sponge was used as carrier material (Figs. 4 and 5). The experiments in which birch wood was used as carrier showed that the efficiency of T. versicolor regarding decolorization of Reactive Blue 4 was not depending on the medium used. No measurable improvement in decolorization of the dye was found when the glucose based medium was used in comparison to the medium based on that the fungus is using the wood as carbon source (Fig. 6). The poor medium containing no extra carbon source was used in the experiment with Reactive Red 2. No color change was obtained in that experiment. The carriers overgrown with fungus became strongly colored in the initial phase of the experiments but they were
Table 1 The results from the plate experiments with T. versicolor (T.v.), P. chrysosporium (P.c.), P. ostreatus (P.o.) and P. sajor-caju (P.s.) Day
Reactive Blue 4
Reactive Red 2
Agar
3 4 6 7 8
Agar + malt
Agar
Agar + malt
T.v.
P.c.
P.o.
P.s.
T.v.
P.c.
P.o.
P.s.
T.v.
P.c.
P.o.
P.s.
T.v.
P.c.
P.o.
P.s.
0.4 0.6 1.5 1.5 2.0
0.1 0.4 2.5 4.0 4.0
0.1 0.2 1.0 1.0 1.0
0.1 0.2 0.2 1.0 1.0
0.4 1.0 2.5 2.5 3.5
0.1 0.1 3.5 4.0 4.0
0.05 0.2 1.0 1.0 1.5
0.1 0.1 0.3 1.0 1.5
0.2 0.4 1.5 1.5 2.0
0.05 0.05 0.4 0.5 4.0
0 0 0 0 0
0 0 0 0 0
0.3 0.6 2.0 3.0 3.5
0 0 2.0 2.0 2.0
0 0 0 0.05 0.05
0 0 0 0 1.5
The radii of the decolorized areas of the plates is given in centimeter. The results are mean values from two plates. The radius of the plates is 4.0 cm.
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The decrease in absorbance of the controls is due to adsorption of the dyes to the carrier surface. 3.3. Continuous experiments
Fig. 4. The results from the batch experiment using T. versicolor on natural luffa sponge for decolorization of Reactive Blue 4. The legends are constructed according to the following examples Blue 0.2 I stands for the first flask containing medium with a dye concentration of 0.2 g/l, Blue 0.2 II stands the second flask containing medium with a dye concentration of 0.2 g/l.
Fig. 5. The results from the batch experiment using T. versicolor on natural sponge for decolorization of Reactive Red 2. The legends are constructed according to the following examples Red 0.2 I stands for the first flask containing medium with a dye concentration of 0.2 g/l, Red 0.2 II stands for the second flask containing medium with a dye concentration of 0.2 g/l.
decolorized during the experiments while the carriers used in the controls stayed strongly colored. This difference most probably depends on decolorization of the dyes taking place due to the action of fungal enzymes during the experiments.
Fig. 6. The results from batch experiment using T. versicolor on birch wood discs for decolorization of Reactive Blue 4. The medium contained only dye, tap water and phosphate/citric acid buffer. The legends are constructed according to the following examples 0.2 g/l I stands for the first flask containing medium with a dye concentration of 0.2 g/l, 0.2 g/l II stands for the second flask containing medium with a dye concentration of 0.2 g/l.
The absorbance at 595 nm decreased with around 85% during treatment under the first 15 days of the experiment with decolorization of Reactive Blue 4 in the reactor containing natural luffa sponge. The scanning of the absorbance between 200 and 800 nm showed that a peak at 257 nm obtained when the influent was scanned was flattened to a hump in the effluent while a plateau in the area 349–380 nm disappeared during treatment. The absorbance increased after 15 days probably depending on that the reactor became infected. Growth of black biomass was detected on day 34. A piece of the sponge overgrown with fungus taken from the reactor was then placed on a colored malt agar plate to further check the decolorization capacity of the fungus. No decolorization was detected which indicates that the fungal enzyme production was disturbed or that the enzyme was destroyed by the bacteria. The absorbance at 538 nm decreased with around 80% during treatment under the first 15 days of the experiment with decolorization of Reactive Red 2 in the reactor containing natural luffa sponge. Scanning of the absorbance between 200 and 800 nm showed that a peak at 281 nm obtained when the influent was scanned disappeared during treatment. A plateau in the area 312–319 nm was furthermore diminished to a hump. The absorbance increased after 15 days depending on the same kind of problems as mentioned above. The absorbance at 595 nm decreased with approximately 70% at a hydraulic retention time of 3 days in the experiment in which T. versicolor was used for decolorization of
Fig. 7. The absorbance at 595 nm of the inlet to and outlet from the biological contactor reactor and the absorbance of the outlet from the control reactor during the experiment with Reactive Blue 4. The flow rate was doubled on day 10 and reset to the original value on day 12.
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Fig. 8. The results from scanning of the inlet to and outlet from the biological rotating biological contactor reactor.
Fig. 9. The bottle to the left contains a sample of untreated textile wastewater while the bottle to the right contains a sample of the outlet from the reactor inoculated with P. flabellatus.
Reactive Blue 4 in the biological contactor reactor (Fig. 7). Scanning of the absorbance did furthermore show that a peak at 257 nm and a plateau in the area 344–385 nm obtained when the inlet was scanned disappeared during the treatment. A plateau was, however, formed around 250 nm (Fig. 8). No measurable difference in absorbance at 538 nm was found between the effluents from the control reactor and the reactor inoculated with fungus in the experiment with Reactive Red 2. The absorbance at 584 nm decreased with between 60 and 70% during the treatment of real wastewater (Fig. 9). The oxygen concentration of the reactor content was 8 mg/l. A microscopic study of the effluent from the reactor during the latter phase of the experiment showed that some bacteria and protozoa were present.
4. Discussion T. versicolor has earlier been shown to decolorize a number of dyes either completely or partially in shake flasks [14]. The decolorization was measured as the decrease of absorbance at the peak wavelength of each dye. Microtox assays showed that the toxicity of the solutions was either unchanged or decreased after degradation for most of the dyes. Two of the dyes, Cibacron Brilliant Yellow 3B-A and Congo Red, did, however, become very toxic after treatment
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[14]. Eighteen fungal strains were studied regarding their potential to decolorize three azo dyes and two phthalocyanine dyes [12]. Only three of the strains, Bjerkandera adusta, T. versicolor and P. chrysosporium were found to be able to decolorize all the dyes studied. The screening of the fungi was performed on colored agar plates while the degradation tests were performed in 500 ml conical flasks either statically or under agitation [12]. The sensitivity of T. versicolor towards infections has earlier been shown by Borchert and Libra [11]. They found that the metabolic capability of the fungus decreased under non-sterile conditions as compared to when growing under sterile conditions. They used batch and sequencing batch reactors with homogenized mycelia for the degradation of some textile dyes. A larger amount of biomass was, however, found to decrease the negative effect of an infection probably depending on that higher concentrations of enzymes were produced which could compensate for the enzyme molecules being destroyed by bacteria [8]. Experiments were also carried out in order to study the possibility to use T. versicolor growing on different solid lignocellulosic support materials for decolorization of dyes. The purposes were to produce cheap inocula for use in textile wastewater treatment processes and to give the fungus an advantage over bacteria [8]. The experiments showed a fast growth of the fungus on sterilized substrates containing starch, e.g. rye or wheat while little or no growth was found on sterilized substrates containing no starch, e.g. saw dust even after 4 weeks. These results differ from the ones obtained by Sclosser et al. [22]. They got growth on wheat straw and beech wood chips within 14 days when studying a submersed culture of a T. versicolor strain. The results from the latter study are in agreement with those obtained in the present study since the fungus was shown to able to grow on birch wood discs even if only the blue dye was decolorized. The metabolic potential obviously differ from one strain of T. versicolor to another. It is of an interest that decolorization of some dyes might take place without addition of an easily degradable carbon source. This may help suppressing the growth of disturbing bacteria in the process. The wastewater treatment process will also be cheaper if an external carbon source does not have to be added. Further research is needed concerning the potential of other white-rot fungi species than T. versicolor in regard to decolorization of different textile dyes without addition of, e.g. glucose. It would also be of importance to evaluate the possibility to decolorize real textile wastewater containing different kinds of dyes with the aid of white-rot fungus in a process without addition of an easily degradable carbon source, e.g. glucose. A process in which lignocellulosic material is used as growth support and carbon source might has a potential for practical use. Further research is, however, needed in order to find fungal strains with an optimal metabolic potential as well as to develop new reactor designs which can be utilized with, e.g. cotton waste as support material.
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Reactor systems with immobilized mycelia were used in the continuous experiments in the present study since earlier results indicate that enzyme secretion is better in this kind of systems than in systems built on suspended cultures [23]. T. versicolor has furthermore been shown to efficiently decolorize Everzol Turquoise Blue G, a phtalocyanine dye, when a rotating biological contactor was used in batch mode [13]. The concentrations of the dyes used during our experiments were in the same order of magnitude as the concentrations used in earlier studies [11,25]. The results from the scanning of the absorbance between 200 and 800 nm of the influents to and effluents from the reactors indicate that the aromatic structure of both dyes was changed due to microbial reactions. No earlier publications concerning decolorization of Reactive Blue 2 and Reactive Red 4 using T. versicolor has been found by the authors. The efficient decolorization of the real wastewater using P. flabellatus is interesting. It is to our knowledge the first time this species has been used for studies of decolorization of textile dyes. The fungus was isolated from the environment in Tanzania, which means that it is adapted to the climate prevailing in many countries, which have a large textile industry. The present study indicates that decolorization of textile dyes in real wastewater by the aid of white-rot fungi is a method with potential for practical use. The indication that P. flabellatus was not disturbed by the presence of bacteria and protozoa is of importance. Unfortunately there was no time to evaluate if the bacteria contributed to the decolorization. Further studies are needed with P. flabellatus as well as other fungi species isolated from relevant environments. Studies of a combined treatment with fungi and bacteria would also be interesting. Many fungal strains are known to degrade complex structures difficult for bacteria to handle. Intermediates are, however, often formed which might be degraded by bacteria. Liquid chromatography–mass spectrometry analyses would be helpful during this kind of studies in order to make an evaluation of the fate of the dye molecules possible.
Acknowledgement SIDA/SAREC is gratefully acknowledged for its financial support, Erik Andersson is gratefully acknowledged for valuable discussions.
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[3] Carliell CM, Barclay SJ, Buckley CA. Treatment of exhausted reactive dyebath effluent using anaerobic digestion: laboratory and fullscale trials. Water SA 1996;22:225–33. [4] Panswad T, Luangdilok W. Decolorization of reactive dyes with different molecular structures under different environmental conditions. Water Res 2000;34:4177–84. [5] Ganesh R, Boardman GD, Tincher WC. Fate of azo dyes in sludges. Water Res 1994;28:1367–76. [6] Brown MA, DeVito SC. Predicting azo dye toxicity. Crit Rev Environ Sci Technol 1993;23:249–324. [7] Knapp JS, Newby PS. The microbiological decolorization of an industrial effluent containing a diazo-linked chromophore. Water Res 1995;29:1807–9. [8] Libra JA, Borchert M, Banit S. Competition strategies for the decolorization of a teile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions. Biotech Bioeng 2003;82:736–44. [9] Cerniglia CE. Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation. J Ind Microbiol Biotechnol 1997;19:324–33. ¨ [10] Mazmanci MA, Unyayar A. Decolorisation of Reactive Black 5 by Funalia trogii immobilised on Luffa cylindrica sponge. Process Biochem 2005;40:337–42. [11] Borchert M, Libra JA. Decolourization of reactive dyes by the white-rot fungus Trametes versicolor in sequencing batch reactors. Biotechnol Bioeng 2001;75:313–21. [12] Heinfling A, Bergbauer M, Szewzyk U. Biodegradation of azo and phtalocyanine dyes by Trametes versicolor and Bjerkandera adusta. Appl Microbiol Biotechnol 1997;48:261–6. [13] Kapdan IK, Kargi F. Biological decolourization of textile dyestuff containing wastewater by Coriolus versicolor in a rotating biological contactor. Enzyme Microb Technol 2002;30:195–9. [14] Ramsay JA, Nguyen T. Decolorisation of textile dyes by Trametes versicolor and its effect on dye toxicity. Biotechnol Lett 2002;24:1757–61. [15] Wesenberg D, Buchon F, Agathos SN. Degradation of dye-containing textile effluent by the agaric white-rot fungus Clitocybula dusenii. Biotechnol Lett 2002;24:989–93. [16] Eaton DC, Chang H, Joyce TW, Jeffries TW, Kirk TK. Method obtains fungal reduction of the color of extractionstage kraft bleach effluents. Tappi 1982;65:89–92. [17] Yin C, Joyce TW, Chang HM. Kinetics of bleach plant effluent decolorization by Phanerochaete chrysosporium. J Biotechnol 1989;10:67–76. [18] Sublette KL, Ganapathy EV, Schwartz S. Degradation of munitions wastes by Phanerochaete chrysosporium. Appl Biochem Biotechnol 1992;31:302–7. [19] Pallerla S, Chambers RP. Characaterization of a Ca-alginate immobilized Trametes versicolor bioreactor for decolorization and AOX reduction of paper mill effluents. Bioresour Technol 1997;60: 1–8. [20] Field JA, De Long E, Feijo Costa G, De Bont JAM. Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white-rot fungi. Appl Environ Microbiol 1992;58:2219–26. [21] Field JA, De Long E, Feijo Costa G, De Bont JAM. Screening for lignolytic fungi applicable to the biodegradation of xenobiotics. Trends Biotechnol 1993;11:44–9. [22] Schlosser D, Grey R, Fritsche W. Patterns of lignolytic enzymes in Trametes versicolor: distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood. Appl Microbiol Biotechnol 1997;47:412–8. [23] Venkatadri R, Irvin RL. Effect of agitation on ligninase activity and ligninase production by Phanerochete chrysosporium. Appl Environ Microbiol 1990;9:2684–91. [25] Yang G, Liu Y, Qinge K. Effect of environment factors on dye decolorization by P.sordia ATCC90872 in an aerated reactor. Process Biochem 2004;39:1401–5.
Decolorization of synthetic and real textile wastewater by the use of white-rot fungi I. Nilsson a , A. M¨oller a , B. Mattiasson a , M.S.T. Rubindamayugi b , U. Welander a,∗ a
Department of Biotechnology, Center of Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden b Department of Botany, Applied Microbiology Unit, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania Received 31 August 2004; received in revised form 13 April 2005; accepted 28 April 2005
Abstract Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h. The absorbance at 584 nm, the wavelength at which the wastewater absorbed the most, decreased from 0.3 in the inlet to approximately 0.1 in the effluent from the reactor. © 2005 Published by Elsevier Inc. Keywords: Biological rotating contactor reactor; Textile dyes; Pleurotus flabellatus; Trametes versicolor; Reactive Blue 4; Reactive Red 2
1. Introduction Wastewater from textile industries constitute a threat to the environment in large parts of the world. The degradation products of textile dyes are often carcinogenic [1,2]. Furthermore, the absorption of light due to textile dyes creates problems to photosynthetic aquatic plants and algae. Earlier studies have shown that many reactive dyes are not degraded in ordinary aerobic sewage treatment processes and that they can be discharged from the treatment plant unaffected [3,4]. The reactive dyes are highly water-soluble polyaromatic molecules, which means that their adsorption to solids is relatively poor [5]. Some dyes are decolorized under anaerobic conditions. The effluent might, however, be ∗ Corresponding author. Present address: School of Technology and Design, Department of Chemistry, V¨axj¨o University, Vejdes Plats 6, SE-351 95 V¨axj¨o, Sweden. Tel.: +46 470 70 88 21; fax: +46 470 70 87 56. E-mail address: [email protected] (U. Welander).
0141-0229/$ – see front matter © 2005 Published by Elsevier Inc. doi:10.1016/j.enzmictec.2005.04.020
toxic [6]. Furthermore, there might be a risk for reverse colorization when anaerobic degradation products are exposed to oxygen [7]. When studying degradation of organic molecules, textile dyes may be regarded as a poorly chosen example, since they are once being selected for their color but also for being resistant to external factors. Dyes on cloths are selected to be resistant to, e.g. UV light, and therefore they constitute a challenging group of chemicals when designing degradation processes. A key feature in the degradation processes, at least the aerobic ones, involves generation of activated oxygen forms that can carry out the initial attack on the stable structure. After the first modification, there are often easier steps where more organisms can be beneficial. Wood rotting fungi have interesting properties in the sense that they are capable to degrade lignin which is a polymeric structure with a lot of aromatic rings. Lignin is regarded as stable. The fungi have been shown to excrete certain enzymes that catalyze the formation
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Fig. 1. The chemical structures of Reactive Red 2 (the drawing to the left) and Reactive Blue 4 (the drawing to the right).
of activated oxygen, and then the process is initiated. The lack of selectivity among these enzymes with regard to the aromatic compounds that are attacked is the background to the interest to use wood rotting fungi also for treating textile dyes. Measurements of the enzyme activity in the medium is, however, not always a reliable parameter for determination of decolorization capacity, since most of the enzymes might be retained in the fungal slime layer [8]. The fungi do need to be supplied with an external carbon source in order to be able to degrade polycyclic aromatic hydrocarbons [9]. White-rot fungi have been used in studies concerning decolorization of different textile dyes [10–15]. Relatively few experiments have, however, been performed using continuous processes with white-rot fungi for treatment of wastewater. Rotating biological contactors have been studied regarding treatment of kraft pulp bleach plant effluent [16,17] and munition waste [18]. A fluidized bed reactor with Trametes versicolor immobilized in calcium alginate gel has furthermore been used for treatment of Kraft bleach effluents [19]. The aim of the study was to develop an efficient and relatively simple continuous process based on white-rot fungi for decolorization of textile dyes. Two dyes, Reactive Blue 4 and Reactive Red 2, from two of the most commonly used groups of reactive dyes were used as model substances in the experiments (Fig. 1). Four different white-rot fungi strains Phanerochaete chrysosporium, T. versicolor, Pleurotus ostreatus and Pleurotus sajor-caju were screened on colored agar plates for their capability to degrade the dyes and the most efficient strains in regard to decolorization were then chosen for experiments in liquid phase. Real textile wastewater from a textile industry in Tanzania was furthermore treated by Pleurotus flabellatus, a fungus which was isolated from the environment in Tanzania.
2. Material and methods 2.1. Microorganisms The fungi used in this study were T. versicolor PRL 572, P. ostreatus MUCL 29527, P. sajor-caju MUCL 29757, P. chrysosporium DSM 1556 and P. flabellatus (Prairie Regional Laboratory (PRL), Mycoth´eque de l’Univerist´e Cathaolique de Louvain (MUCL), Deutsche Sammalung von Mikroorganismen (DSM)). T. versicolor, P. ostreatus,
P. sajor-caju and P. chrysosporium were kindly provided by Dr. P.-O. Nyman, Department of Biochemistry, Lund University. A pure culture of P. flabellatus was obtained from the fungal collection of the Department of Botany, Applied Microbiology Unit, University of Dar es Salaam, Tanzania. 2.2. Analyses The samples were filtered through GF/A glass-fiber filters before the absorbance was measured on Ultrospec 1000 or Ultrospec 3000 spectrophotometers (Phamacia Biotech, Uppsala, Sweden) during the first part of the study with synthetic wastewater. The peak absorbance was measured at 595 nm for Reactive Blue 4 and 538 nm for Reactive Red 2. Scanning was performed between 200 and 800 nm. Tap water was used as reference. The absorbance was measured at the peak wavelength 584 nm after centrifugation of the samples for 5 min at 12,000 × g when real wastewater was studied. Distilled water was used as reference. Scanning could unfortunately not be performed due to practical reasons. 2.3. Storage of strains of fungi The fungi were stored on maltagar plates made on a medium consisting of 10 g/l malt extract and 10 g/l agar dissolved in distilled water. New plates were inoculated once a month to ensure viability of the isolates. 2.4. Evaluation of dye decolorization potential of different fungal strains Four different white-rot fungi strains T. versicolor, P. ostreatus, P. sajor-caju and P. chrysosporium were screened for their capability to degrade poly-R 478, Reactive Blue 4 (an anthraquinone dye) and Reactive Red 2 (an azo dye) (Fig. 1). Poly-R 478 was used as a control dye to check that the fungi had not lost enzyme activity during storage on malt agar plates [20,21]. The experiment was performed on solid media plates made on media which contained 0.2 g/l dye, 10 g/l agar and for half the number of plates 10 g/l of malt extract. The media were autoclaved and poured into petridishes. Each plate was then inoculated with a piece of malt agar overgrown with fungal mycelia. The decolorization of
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the plates was studied as a function of time. The radius of the decolorized area around the inoculum was measured with a ruler. The colored plates were used for a continuous control of the enzyme productivity of the fungi.
All experiments were performed in duplicate at room temperature.
2.5. Carrier materials
Two cylindrical reactors with conical bottoms were used for continuous experiments with T. versicolor (Fig. 2). One of the reactors was used for studies of decolorization of Reactive Red 2 (reactor 1) while the other was used for studies of decolorization of Reactive Blue 4 (reactor 2). The reactors had a liquid volume of 1 l. They were autoclaved and then filled with natural luffa sponge overgrown with fungus mycelia. A medium with the same composition as described above in the paragraph about the luffa sponge batch experiments was prepared, autoclaved and cooled to room temperature. The medium was then pumped into the reactors with a flow rate of 12 ml/h during the first 13 days. The flow rate was then decreased to 5.2 ml/h in order to examine if the absorbance at the peak wavelengths could be further decreased. The dye concentration in the medium into reactor 1 was 0.5 g/l during the whole study (26 days) while it was 0.5 g/l into reactor 2 day 1–26 and 0.2 g/l day 27–34. The air-phase of the reactors were renewed by the aid of aquarium pumps connected with tubes at the bottom of the reactors. The air was led into and out from the reactors through sterile filters (0.2 m) in order to avoid contamination of the reactors. The medium was pumped from e-flasks sealed with cotton stoppers and the outlet was collected in the same kind of vessels. The ends of the tubes were washed with 70% ethanol when the medium or outlet flasks were exchanged. All flasks and tubes were autoclaved before use. The oxygen concentration of the reactor contents was approximately 7 mg/l while the pH
Two different carrier materials were used during the experiments, natural luffa sponge and birch wood. The sponge was boiled and sliced into 3 cm thick slices which were then autoclaved together with 250 ml of a malt extract medium (10 g/l) in jars with a volume of 2 l. The sponge was inoculated with malt agar pieces overgrown with fungus. The jars were stored closed in dark until a considerable amount of biomass was established. The jars were rotated once a day so that the sponges were soaked in the malt extract. The birch wood carrier had the shape of circular discs with a diameter of 2.5 cm for the batch experiments and 5 cm for the continuous experiments. The thickness of the discs was 0.5 cm in all experiments. The discs were placed in beakers, which were filled with water and covered with aluminum foil, and autoclaved during 20 min at 121 ◦ C. The discs were then placed on malt agar plates overgrown with fungus. The carriers were overgrown with fungal mycelia after a period of 1–3 weeks. 2.6. Batch experiments The decolorization of Reactive Blue 4 and Reactive Red 2 were studied in batch experiments using T. versicolor. The experiments were performed in 250 ml e-flasks. The flasks were filled with 50 or 100 ml of medium, sealed with cotton stoppers and autoclaved. The smaller volume was used for the experiments with wooden carrier. A medium containing 0.5 or 0.2 g/l of dye, 3 g/l glucose, 0.24 g/l KNO3 , 0.1 g/l K2 HPO4 , 0.1 g/l KCl, 0.11 g/l MgSO4 ·H2 O, 2.4 mg/l Fe(EDTA), 5 ml/l of a trace metal solution, 1 mg/l thiamine and 10 ml/l of a phosphate/citric acid buffer was used in the experiments with natural luffa sponge. The buffer contained 78.5 g/l Na2 HPO4 ·2H2 O and 214.6 g/l citric acid while the trace metal solution contained 200 mg/l FeSO4 ·7H2 O, 10 mg/l ZnSO4 ·7H2 O, 3 mg/l MnCl2 ·4H2 O, 20 mg/l CoCl2 ·6H2 O, 1 mg/l CuCl2 ·2H2 O, 2 mg/l NiCl2 ·6H2 O, 500 mg/l Na2 MoO4 ·2H2 O and 30 mg/l H3 BO3 . Distilled water was used for all solutions. Two different media were used in the experiments with birch wood discs. One of the media had the same composition as the medium described above and the other medium contained 0.2 or 0.5 g/l of dye and 10 ml/l of the phosphate citric acid buffer. Tap water was used for the latter medium in order to ensure the presence of enough amounts of trace metals. Carriers overgrown with fungus were then added. The flasks were placed on a shaking table having a rate of 120 rpm. The pH was approximately 3 in all experiments. Autoclaved controls were set up with autoclaved carriers in all experiments in order to study abiotic dye removal.
2.7. Continuous experiments
Fig. 2. The design of the reactors filled with natural luffa sponge. The height of the reactor is 35 cm, while the diameter is 12 cm.
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of wastewater from the bleaching process. Nutrients and an extra carbon source were added to the water according to the following: 0.75 g/l glucose, 0.25 ml/l acetic acid, 0.23 g/l NH4 Cl, 0.06 g/l NaH2 PO4 ·2H2 O, 0.28 g/l NaCl, 0.37 g/l KCl, 0.04 g/l MgCl2 ·6H2 O, 0.02 g/l CaCl2 ·2H2 O and 0.20 g/l yeast extract. The pH of the textile wastewater was set to between 3 and 4, the optimal pH range for the fungus. The air-phase of the reactor was renewed with an air-pump. The experiments was continued for 10 days. All reactor experiments were performed at room temperature. Fig. 3. The design of the biological rotating contactor reactor. The diameter of the reactor cylinder is 7 cm while its length is 29 cm.
3. Results was between 3 and 4. Two reactors with the same design as described above were used as sterile controls. Another continuous reactor with a design of a rotating biological contactor was operated with a medium consisting of 0.2 g/l of dye and 10 ml/l of the phosphate citric buffer described under the batch experiments (Fig. 3). It had a total volume of 1 l and a liquid volume of 400 ml. Nine wood discs overgrown with T. versicolor were placed on an axis, which rotated with a speed of 20 rpm. The air volume was renewed with the aid of an aquarium pump giving a flow rate of 900 cm3 /min. The air was led into and out from the reactor through sterile filters (0.2 m) in order to avoid contamination. A second reactor of the same design as the reactor described above was used as a sterile control. The retention time in the reactors were 3 days during days 1–10 after which it was halved. However, due to an increase of absorbance in the outlet from the reactor inoculated with fungus, the flow rate was reset to its original value on day 12. The pH was around 3. The process was running for 18 days with Reactive Blue 4 and 10 days with Reactive Red 2. The reactor parts were autoclaved before assemblage and the same routine as described above was used for collection of the outlet and exchange of medium and outlet flasks. A seventh reactor with a volume of 1.5 l was used in order to study decolorization of dyes in real textile wastewater from a textile industry in Tanzania. The reactor was autoclaved and packed with natural luffa sponge inoculated with P. flabellatus. Autoclaved textile wastewater was then pumped into it with a flow rate of 59 ml/h giving a hydraulic retention time of 26 h. The water was a mixture of 750 ml/l of wastewater from the dyeing process and 250 ml/l
3.1. Evaluation of dye decolorization potential of different fungi The experiment with poly-R 478 showed that all strains had some extracellular enzyme activity. P. chrysosporium and T. versicolor were shown to be the most efficient strains in regard to decolorization of Reactive Blue 4 and Reactive Red 2 (Table 1). These strains were therefore used in the batch and continuous experiments in order to study decolorization of the dyes in water phase. P. chrysosporium did, however, not decolorize the dyes in water phase so the paper is therefore focused on T. versicolor. 3.2. Batch experiments T. versicolor was shown to be able to decolorize Reactive Red 2 as well as Reactive Blue 4 when natural luffa sponge was used as carrier material (Figs. 4 and 5). The experiments in which birch wood was used as carrier showed that the efficiency of T. versicolor regarding decolorization of Reactive Blue 4 was not depending on the medium used. No measurable improvement in decolorization of the dye was found when the glucose based medium was used in comparison to the medium based on that the fungus is using the wood as carbon source (Fig. 6). The poor medium containing no extra carbon source was used in the experiment with Reactive Red 2. No color change was obtained in that experiment. The carriers overgrown with fungus became strongly colored in the initial phase of the experiments but they were
Table 1 The results from the plate experiments with T. versicolor (T.v.), P. chrysosporium (P.c.), P. ostreatus (P.o.) and P. sajor-caju (P.s.) Day
Reactive Blue 4
Reactive Red 2
Agar
3 4 6 7 8
Agar + malt
Agar
Agar + malt
T.v.
P.c.
P.o.
P.s.
T.v.
P.c.
P.o.
P.s.
T.v.
P.c.
P.o.
P.s.
T.v.
P.c.
P.o.
P.s.
0.4 0.6 1.5 1.5 2.0
0.1 0.4 2.5 4.0 4.0
0.1 0.2 1.0 1.0 1.0
0.1 0.2 0.2 1.0 1.0
0.4 1.0 2.5 2.5 3.5
0.1 0.1 3.5 4.0 4.0
0.05 0.2 1.0 1.0 1.5
0.1 0.1 0.3 1.0 1.5
0.2 0.4 1.5 1.5 2.0
0.05 0.05 0.4 0.5 4.0
0 0 0 0 0
0 0 0 0 0
0.3 0.6 2.0 3.0 3.5
0 0 2.0 2.0 2.0
0 0 0 0.05 0.05
0 0 0 0 1.5
The radii of the decolorized areas of the plates is given in centimeter. The results are mean values from two plates. The radius of the plates is 4.0 cm.
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The decrease in absorbance of the controls is due to adsorption of the dyes to the carrier surface. 3.3. Continuous experiments
Fig. 4. The results from the batch experiment using T. versicolor on natural luffa sponge for decolorization of Reactive Blue 4. The legends are constructed according to the following examples Blue 0.2 I stands for the first flask containing medium with a dye concentration of 0.2 g/l, Blue 0.2 II stands the second flask containing medium with a dye concentration of 0.2 g/l.
Fig. 5. The results from the batch experiment using T. versicolor on natural sponge for decolorization of Reactive Red 2. The legends are constructed according to the following examples Red 0.2 I stands for the first flask containing medium with a dye concentration of 0.2 g/l, Red 0.2 II stands for the second flask containing medium with a dye concentration of 0.2 g/l.
decolorized during the experiments while the carriers used in the controls stayed strongly colored. This difference most probably depends on decolorization of the dyes taking place due to the action of fungal enzymes during the experiments.
Fig. 6. The results from batch experiment using T. versicolor on birch wood discs for decolorization of Reactive Blue 4. The medium contained only dye, tap water and phosphate/citric acid buffer. The legends are constructed according to the following examples 0.2 g/l I stands for the first flask containing medium with a dye concentration of 0.2 g/l, 0.2 g/l II stands for the second flask containing medium with a dye concentration of 0.2 g/l.
The absorbance at 595 nm decreased with around 85% during treatment under the first 15 days of the experiment with decolorization of Reactive Blue 4 in the reactor containing natural luffa sponge. The scanning of the absorbance between 200 and 800 nm showed that a peak at 257 nm obtained when the influent was scanned was flattened to a hump in the effluent while a plateau in the area 349–380 nm disappeared during treatment. The absorbance increased after 15 days probably depending on that the reactor became infected. Growth of black biomass was detected on day 34. A piece of the sponge overgrown with fungus taken from the reactor was then placed on a colored malt agar plate to further check the decolorization capacity of the fungus. No decolorization was detected which indicates that the fungal enzyme production was disturbed or that the enzyme was destroyed by the bacteria. The absorbance at 538 nm decreased with around 80% during treatment under the first 15 days of the experiment with decolorization of Reactive Red 2 in the reactor containing natural luffa sponge. Scanning of the absorbance between 200 and 800 nm showed that a peak at 281 nm obtained when the influent was scanned disappeared during treatment. A plateau in the area 312–319 nm was furthermore diminished to a hump. The absorbance increased after 15 days depending on the same kind of problems as mentioned above. The absorbance at 595 nm decreased with approximately 70% at a hydraulic retention time of 3 days in the experiment in which T. versicolor was used for decolorization of
Fig. 7. The absorbance at 595 nm of the inlet to and outlet from the biological contactor reactor and the absorbance of the outlet from the control reactor during the experiment with Reactive Blue 4. The flow rate was doubled on day 10 and reset to the original value on day 12.
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Fig. 8. The results from scanning of the inlet to and outlet from the biological rotating biological contactor reactor.
Fig. 9. The bottle to the left contains a sample of untreated textile wastewater while the bottle to the right contains a sample of the outlet from the reactor inoculated with P. flabellatus.
Reactive Blue 4 in the biological contactor reactor (Fig. 7). Scanning of the absorbance did furthermore show that a peak at 257 nm and a plateau in the area 344–385 nm obtained when the inlet was scanned disappeared during the treatment. A plateau was, however, formed around 250 nm (Fig. 8). No measurable difference in absorbance at 538 nm was found between the effluents from the control reactor and the reactor inoculated with fungus in the experiment with Reactive Red 2. The absorbance at 584 nm decreased with between 60 and 70% during the treatment of real wastewater (Fig. 9). The oxygen concentration of the reactor content was 8 mg/l. A microscopic study of the effluent from the reactor during the latter phase of the experiment showed that some bacteria and protozoa were present.
4. Discussion T. versicolor has earlier been shown to decolorize a number of dyes either completely or partially in shake flasks [14]. The decolorization was measured as the decrease of absorbance at the peak wavelength of each dye. Microtox assays showed that the toxicity of the solutions was either unchanged or decreased after degradation for most of the dyes. Two of the dyes, Cibacron Brilliant Yellow 3B-A and Congo Red, did, however, become very toxic after treatment
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[14]. Eighteen fungal strains were studied regarding their potential to decolorize three azo dyes and two phthalocyanine dyes [12]. Only three of the strains, Bjerkandera adusta, T. versicolor and P. chrysosporium were found to be able to decolorize all the dyes studied. The screening of the fungi was performed on colored agar plates while the degradation tests were performed in 500 ml conical flasks either statically or under agitation [12]. The sensitivity of T. versicolor towards infections has earlier been shown by Borchert and Libra [11]. They found that the metabolic capability of the fungus decreased under non-sterile conditions as compared to when growing under sterile conditions. They used batch and sequencing batch reactors with homogenized mycelia for the degradation of some textile dyes. A larger amount of biomass was, however, found to decrease the negative effect of an infection probably depending on that higher concentrations of enzymes were produced which could compensate for the enzyme molecules being destroyed by bacteria [8]. Experiments were also carried out in order to study the possibility to use T. versicolor growing on different solid lignocellulosic support materials for decolorization of dyes. The purposes were to produce cheap inocula for use in textile wastewater treatment processes and to give the fungus an advantage over bacteria [8]. The experiments showed a fast growth of the fungus on sterilized substrates containing starch, e.g. rye or wheat while little or no growth was found on sterilized substrates containing no starch, e.g. saw dust even after 4 weeks. These results differ from the ones obtained by Sclosser et al. [22]. They got growth on wheat straw and beech wood chips within 14 days when studying a submersed culture of a T. versicolor strain. The results from the latter study are in agreement with those obtained in the present study since the fungus was shown to able to grow on birch wood discs even if only the blue dye was decolorized. The metabolic potential obviously differ from one strain of T. versicolor to another. It is of an interest that decolorization of some dyes might take place without addition of an easily degradable carbon source. This may help suppressing the growth of disturbing bacteria in the process. The wastewater treatment process will also be cheaper if an external carbon source does not have to be added. Further research is needed concerning the potential of other white-rot fungi species than T. versicolor in regard to decolorization of different textile dyes without addition of, e.g. glucose. It would also be of importance to evaluate the possibility to decolorize real textile wastewater containing different kinds of dyes with the aid of white-rot fungus in a process without addition of an easily degradable carbon source, e.g. glucose. A process in which lignocellulosic material is used as growth support and carbon source might has a potential for practical use. Further research is, however, needed in order to find fungal strains with an optimal metabolic potential as well as to develop new reactor designs which can be utilized with, e.g. cotton waste as support material.
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Reactor systems with immobilized mycelia were used in the continuous experiments in the present study since earlier results indicate that enzyme secretion is better in this kind of systems than in systems built on suspended cultures [23]. T. versicolor has furthermore been shown to efficiently decolorize Everzol Turquoise Blue G, a phtalocyanine dye, when a rotating biological contactor was used in batch mode [13]. The concentrations of the dyes used during our experiments were in the same order of magnitude as the concentrations used in earlier studies [11,25]. The results from the scanning of the absorbance between 200 and 800 nm of the influents to and effluents from the reactors indicate that the aromatic structure of both dyes was changed due to microbial reactions. No earlier publications concerning decolorization of Reactive Blue 2 and Reactive Red 4 using T. versicolor has been found by the authors. The efficient decolorization of the real wastewater using P. flabellatus is interesting. It is to our knowledge the first time this species has been used for studies of decolorization of textile dyes. The fungus was isolated from the environment in Tanzania, which means that it is adapted to the climate prevailing in many countries, which have a large textile industry. The present study indicates that decolorization of textile dyes in real wastewater by the aid of white-rot fungi is a method with potential for practical use. The indication that P. flabellatus was not disturbed by the presence of bacteria and protozoa is of importance. Unfortunately there was no time to evaluate if the bacteria contributed to the decolorization. Further studies are needed with P. flabellatus as well as other fungi species isolated from relevant environments. Studies of a combined treatment with fungi and bacteria would also be interesting. Many fungal strains are known to degrade complex structures difficult for bacteria to handle. Intermediates are, however, often formed which might be degraded by bacteria. Liquid chromatography–mass spectrometry analyses would be helpful during this kind of studies in order to make an evaluation of the fate of the dye molecules possible.
Acknowledgement SIDA/SAREC is gratefully acknowledged for its financial support, Erik Andersson is gratefully acknowledged for valuable discussions.
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