- Email: [email protected]
Biodegradation and detoxification potential of rotating biological contactor (RBC) with Irpex lacteus for remediation of dye-containing wastewater
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/watres
Biodegradation and detoxification potential of rotating biological contactor (RBC) with Irpex lacteus for remediation of dye-containing wastewater Katerina Malachova a,*, Zuzana Rybkova a, Hana Sezimova a, Jiri Cerven a, Cenek Novotny a,b a
Faculty of Science, University of Ostrava, Chittussiho 10, 710 00 Slezska´, Ostrava, Czech Republic ska´ 1083, 142 20 Prague 4, Czech Laboratory of Environmental Biotechnology, Institute of Microbiology ASCR, Vı´den Republic b
article info
abstract
Article history:
Use of fungal organisms in rotating biological contactors (RBC) for bioremediation of liquid
Received 23 March 2013
industrial wastes has so far been limited in spite of their significant biodegradation po-
Received in revised form
tential. The purpose was to investigate the power of RBC using Irpex lacteus for decolor-
26 June 2013
ization and detoxification of industrial dyes and dyeing textile liquors. Recalcitrant dye
Accepted 5 July 2013
Methylene Blue (150 mg L1) was decolorized within 70 days, its mutagenicity removed,
Available online 20 October 2013
and the biological toxicity decreased more than 10-fold. I. lacteus biofilm in the RBC completely decolorized within 26 and 47 days dyeing liquors containing disperse or reac-
Keywords:
tive dyes adjusted to pH4.5 and 5-fold diluted with the growth medium, respectively. Their
Liquid textile wastes
respective biological toxicity values were reduced 10- to 104-fold in dependence of the test
Dye decolorization
used. A battery of toxicity tests comprising Vibrio fisheri, Lemna minor and Sinapis alba was
Genetic toxicity
efficient to monitor the toxicity of textile dyes and wastewaters. Strong decolorization and
Biological toxicity
detoxification power of RBC using I. lacteus biofilms was demonstrated. ª 2013 Elsevier Ltd. All rights reserved.
Rotating biological contactor Irpex lacteus
1.
Introduction
Textile industry produces large volumes of dye-containing effluents that are ineffectively remediated in wastewater treatment plants and are responsible for coloration of streams that negatively affects water life. Biological and genetic toxicity of dyes for bacteria, protozoa, aquatic animals, plants and mammals has been widely documented (Gottlieb et al., 2003; Soni et al., 2006; etc.). Textile wastewaters are extremely variable in composition due to the presence of
various dyes, desizing and scouring agents, detergents, finishing agents and inorganic salts that all can contribute to their toxicity (Dubrow et al., 1996). Consequently, efficient remediation must result in both decolorization and detoxification of the wastewater. Decolorization of dyes with ligninolytic fungi has been proven to be an efficient, cheap and environment-friendly process but their detoxification power has been studied less frequently (e.g. Knapp et al., 2008). A number of chemicallydifferent types of persistent dyes have been shown to be
* Corresponding author. Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 710 00 Slezska´, Ostrava, Czech Republic. Tel.: þ420 2 597092315; fax: þ420 2 597092382. E-mail addresses: [email protected] (K. Malachova), [email protected] (Z. Rybkova), [email protected] (H. Sezimova), [email protected] (J. Cerven), [email protected] (C. Novotny). 0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2013.07.050
7144
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
effectively degraded by various fungal organisms (e.g. Singh, 2006). Methylene Blue (CAS No. 61-73-4, C.I. 52015, MB), a heterocyclic phenothiazine dye, is widely used for dyeing leather and textile materials. Its decolorization strongly depends on the conditions of fungal culture and the efficiency of various ligninolytic fungi is quite variable (Tychanowicz et al., 2004; etc.). Partial to weak decolorizations were reported by various ligninolytic fungi in liquid- and solid-state cultures, by bacteria and the aerobic activated sludge (e.g. Ma et al., 2011). Anaerobic sludge is able to remove the dye but due to reoxidation by air only a low color removal effect was achieved (Ong et al., 2005). Broader adverse effects of MB include eye injury, breathing problems, methemoglobinemia, bacteriostatic and fungicidal activities, and a significant toxicity to aquatic plants, crustaceans and fish (Wainwright et al., 1999 etc.). Because of the persistence and toxicity, MB was chosen in this study as a model industrial dye for testing the efficiency of RBC reactor using Irpex lacteus to decolorize and detoxify dyes and textile effluents. RBC reactors operable in repeated-batch or continuous mode offer advantages in bioremediation of industrial wastewaters due to great surface per unit volume, low power requirement and limited flow clogging (Anderson, 1983). Ligninolytic fungi behave well in RBC but so far few studies have been conducted, comprising only a limited number of fungal strains (e.g. Guimaraes et al., 2005; Axelsson et al., 2006). Biodegradation and detoxification power of I. lacteus in this type of reactor has not been thoroughly investigated. Ecotoxicity of pollutants is usually measured with standard toxicity tests, i.e. bacterial, crustacean, algal and seed germination tests. For instance, Vibrio fischeri bioluminiscence test was used to measure both a decrease and increase of toxicity resulting from degradation of various textile dyes by Trametes versicolor (Ramsay and Nguyen, 2002) or formation of toxic products in the course of anaerobic decolorization of Reactive Black 5 by Enterococcus faecalis and Clostridium butyricum (Gottlieb et al., 2003). Tests with Daphnia spp. were used to monitor decolorization-linked detoxification of phthalocyanine- and azo dyes obtained with Penicillium simplicissimum or of a raw textile effluent treated with horseradish peroxidase (Bergsten-Torralba et al., 2009). A reduction of mutagenicity of Reactive Orange 16 and Disperse Blue 3 dyes in a two-step treatment with activated sludge and a static culture of I. lacteus was monitored by the Ames test (Malachova´ et al., 2006). Our study was undertaken to investigate the dye decolorization and detoxification capacity of I. lacteus under the conditions of RBC reactor using MB and two different textile dyeing liquors containing mixtures of reactive or disperse dyes to test the decolorization and detoxification efficiency. Biological toxicity changes during the treatment were measured with a battery of standard bacterial and plant tests and the change of genetic toxicity with Ames test.
2.
Material and methods
2.1.
Chemicals
The dyeing liquors were obtained from INOTEX a.s., Czech Republic. Wastewater I contained Sumifix Black B 150% (C.I.
Reactive Black 5) (9.82 g L1), Inosin Yellow V-GR 160% (C.I. Reactive Yellow 15) (2.47 g L1), NaCl (75 g L1) and the fixation agent Texalkon MS (7.87 g L1). Wastewater II contained Itosperse Yellow RAP dye mix (5.47 g L1), Itosperse Red RAP dye mix (3.75 g L1), Itosperse Blue RAP dye mix (2.47 g L1), the disperging agent Nicca Sunsolt RF-557 (1 g L1) and acetic acid (0.3 ml L1). Malt extract and agar were purchased from Oxoid, UK, Disperse Blue 3 (DB3, anthraquinone) and Methylene Blue (MB, phenothiazine) dyes from SigmaeAldrich, Czech Republic. Other chemicals were of analytical grade.
2.2.
Microorganism
Irpex lacteus 931 was provided by the Culture Collection of Basidiomycetes, Institute of Microbiology ASCR, Prague and maintained on malt extract-glucose (MEG) medium containing 2% (w/w) agar at 4 C.
2.3.
Biodegradation in RBC reactor
The rotating biological contactor (RBC) reactor consisted of a glass vessel and a horizontal driving axis with six 1-cm thick polyurethane foam (PUF) discs (diameter 8 cm, rotation speed 2 rpm, 40% of disc volume immersed). The experiments were carried out aseptically in MEG medium (per litre: 5 g malt extract, 10 g glucose, pH 4.5) at 22 C and forced aeration with air (50 L h1). Sterile PUF discs were put horizontally in MEG and inoculated with a homogenate (Ultra-Turrax T25 mixer, IKA Werk, Germany, 20 s) of a 7-d-old, static MEG culture grown at 28 C (10% v/v inoculum). The discs were colonized with the fungus (7 d, 28 C) and then mounted aseptically in the reactor containing one litre of MEG medium with DB3 or MB dyes dissolved at a concentration of 150 mg L1 representing the respective dye concentrations of 0.56 and 0.47 mM. Their decolorization was measured spectrophotometrically at 645 nm and 505/ 580 nm, respectively. Wastewaters I and II were adjusted to pH 4.5 and used 5-fold diluted with MEG. Their decolorization was measured at respective maxima of 575 and 425 nm. The fungal biomass on the discs was estimated gravimetrically at the end of the experiment as dry biomass.
2.4.
Biological toxicity tests
The acute biological toxicity was estimated using bacterial luminiscence, aquatic plant growth and seed germination as the endpoints. V. fischeri test (ISO 11348-3, 2007) measured bioluminiscence inhibition after a 30-min exposition using a LUMIStox300 luminometer (Hach-Lange, Du¨sseldorf, Germany). Lemna minor test (ISO CD, 20079, 2005) determined growth inhibition of fronds, the exposition time was 7 days. The Phytotoxkit Sinapis alba test (ISO 11269-1, 1993) determined the inhibition of root growth after a 3-d exposure. The test was considered to be valid if the germination of the control was 90%. The stimulation effect of endproducts was evaluated by using a linear model. A positive toxic effect was evaluated in the tests against negative controls containing only the culture medium. Positive controls using toxicants recommended in the corresponding ISO standard were also measured to check the sensitivity of the
7145
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
Absorbance
1,2 1 0,8 0,6 0,4 0,2 0 0
10
20
30
40
50
60
70
Days
Fig. 1 e Time course of decolorization of Methylene Blue (oeo) and textile dyeing liquors containing reactive dyes (CeC, Wastewater I) and disperse dyes (:e:, Wastewater II) in RBC reactor with Irpex lacteus. Absorbance was measured at the corresponding absorption maxima: Methylene Blue 580 nm, Wastewater I 575 nm, Wastewater II 475 nm. The absorbance values represent the means of three samples.
individual tests. In the tests, EC50 or IC50 values were calculated and expressed as a logarithm of the dilution factor determined as a volume percentage (v/v %) of the diluted sample used in the test relative to the original undiluted sample removed from the reactor. This enabled us to compare the changes of EC50 and IC50 measured before and after degradation.
2.5.
Mutagenicity (Ames) test
Mutagenicity was detected using a plate-incorporation version of the Salmonella typhimurium Hise reversion assay used with or without the in vitro metabolic activation with the rsat liver S9 microsomal fraction and cofactor mixture (OECD Test No. 471, 1997). The auxotrophic strains TA100 and TA98 were used for detection of the base substitution mutations and frameshift mutations, respectively. The mutagenic activity was expressed as a number of revertant colonies (Rt) obtained with the treated sample compared to the number of revertant colonies obtained with the control sample (Rc). The index of mutagenicity was calculated as a ratio of Rt/Rc and a twofold increase of the index was considered to be significant. Mutation potential represented the index of mutagenicity related to the concentration of the tested compound expressed in micrograms. Each test was repeated at least three times using two replicate plates for each sample and the results were calculated using SALM software (Broekhoven and Nestmann, 1991).
3.
Results and discussion
3.1.
Decolorization and detoxification of model dyes
Decolorization of DB3 was used as a forerunner test of the ability of the fresh-grown I. lacteus biofilms mounted in the
RBC reactor to decolorize recalcitrant dye compounds as the fungus was previously reported to decolorize and detoxify DB3 (Malachova´ et al., 2006). The dye was completely decolorized within 25 days (data not shown). Then the reactor was washed with MEG medium and, subsequently, a batch of MEG containing MB was added. A complete decolorization was achieved within 70 days (Fig. 1). The total amount of fungal biofilms on the surface of PUF discs in RBC after decolorization of MB was 7.53 g dry biomass and the average decolorization rate was calculated to be 0.34 mg MB d1 g1 dry biomass. These data well compare with other fungi, namely, the decolorization of 5e20 mg MB L1 by Phanerochaete chrysosporium and T. versicolor (Mazmanci et al., 2002; Radha et al., 2005) or a partial decolorization of 200 mg MB L1 by Lentinula edodes (Boer et al., 2004). Sampling at Day 0, 20 and 70 (cf. Figs. 2 and 3) intended to measure the toxicity of intact MB, degradation intermediates and the endproduct in keeping with the time course of MB decolorization shown in Fig. 1. On Day 0, the mutagenic effects of the dye were observed in all variants of the Ames test. MB induced frameshift and substitution, direct and indirect mutations both with and without metabolic activation. However in the tests without metabolic activation MB was concluded to be only potentially mutagenic since a significant, twofold increase of the revertant number was not accomplished due to the toxicity of high sample concentrations for the indicator strains. The respective maximal mutagenic activities obtained with TA100 and TA98 strains expressed as the index of mutagenicity were 1.54 (25 mg MB per plate) and 1.85 (2.5 mg MB per plate). In the tests with metabolic activation, the values of the index of mutagenicity were even higher: TA100 1.78 (25 mg MB per plate), TA98 3.85 (2.5 mg MB per plate). The results thus confirmed the genotoxic effects of the intact dye reported by other authors (NTP TR 540, 2008). The degradation resulted in a complete detoxification: the sample removed at Day 70, when the absorbance decreased to zero, exhibited no
50 45 40 Mutation potential
1,4
35 30 25 20 15 10 5 0
1
2 3 Day 0
4
1
2 3 Day 20
4
1
2 3 Day 70
4
Fig. 2 e Mutagenicity of Methylene Blue during biodegradation in RBC reactor measured with Salmonella typhimurium His- test with and without S9 activation. Samples were withdrawn on Day 0, Day 20 (intermediates) and Day 70 (endproducts): TA100-S9 (1); TA98-S9 (2); TA100 D S9 (3); TA98 D S9 (4).
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
3
a3
2
2
Log EC50 or IC50
Log EC50 or IC50
7146
1
1
0
0 2 Day 0
3
1
2 3 Day 20
1
2 Day 70
3
Fig. 3 e Toxicity of Methylene Blue expressed as Log EC50 or IC50 during biodegradation in RBC reactor measured with Vibrio fischeri (1), Sinapis alba (2), and Lemna minor (3). Samples were withdrawn on Day 0, Day 20 and Day 70 to measure the toxicity of intact dye, intermediates and endproducts of biodegradation, respectively.
mutagenic effect (Fig. 2). A similar removal of mutagenicity by I. lacteus growing in static liquid cultures was demonstrated for Reactive Orange 16 azo dye (Malachova´ et al., 2006). Tests of acute biological toxicity demonstrated a significant toxicity of the sample containing intact MB removed on Day 0. The V. fischeri test and the plant tests with L. minor and S. alba showed the following respective toxicity values: EC50 ¼ 18.07 0.31 mg L1, EC50 ¼ 12.22 0.55 mg L1 and IC50 ¼ 100 0.1 mg L1 (Fig. 3). The germination of seeds of S. alba was 5- and 8-fold less sensitive to the toxic effect of the dye than the bacterial luminiscence and growth of L. minor, respectively. These results showed that MB was toxic for organisms of various trophic levels and confirmed previous findings of the adverse side-effects (e.g. NTP TR 540, 2008; Wainwright et al., 1999). The EC50 values demonstrated a good sensitivity of the test battery and were comparable to those obtained for various azo dyes with crustaceans Daphnia magna and Desmocaris trispinosa (Ogugbue and Oranusi, 2006; Verma, 2008). The decrease of biological toxicity of MB during biodegradation, expressed as EC50 or IC50, exceeded one order of magnitude. In the tests with L. minor up to a 10% stimulation of growth of fronds, compared to the control, was observed with the sample withdrawn at Day 70; probably, the degradation endproducts were usable as nutrients by the plants. The stimulation effect along the gradient of endproduct concentration was found to be significant (F ¼ 15.57, p ¼ 0.017). The fitted model: y (rate of growth) ¼ 0.0008 conc þ 0.137. Similar studies reported both decrease and increase of biological toxicity after the treatment of various dyes with T. versicolor and Penicillium simplicissimus, when monitored with V. fischeri and Daphnia pulex tests (Bergsten-Torralba et al., 2009; Ramsay and Nguyen, 2002).
3.2. Decolorization and detoxification of textile dyeing liquors Textile wastewaters containing reactive (Wastewater I) or disperse (Wastewater II) dyes were completely decolorized in
-1
b
1
2 3 Day 0
1
2 3 Day 40
1
2 3 Day 47
3 2
Log EC50 or IC50
1
1 0 -1 -2 -3 -4
1
2 3 Day 0
1
2 3 Day 10
1
2 3 Day 26
Fig. 4 e Toxicity of Wastewater I (a) and Wastewater II (b) during biodegradation in RBC reactor measured with Vibrio fischeri (1), Sinapis alba (2) and Lemna minor (3). The samples withdrawn on Day 40 and Day 47 (Wastewater I) and Day 10 and Day 26 (Wastewater II) represented 50 and 100% decolorization to measure the toxicity of biodegradation intermediates and endproducts, respectively.
the RBC reactor within 47 and 26 days, respectively (Fig. 1). The decolorization of Wastewater I containing two reactive dyes, a high concentration of NaCl and Texalcon MS fixation agent was slower than that of Wastewater II containing three disperse dyes and low concentrations of disperging agent and acetic acid. The difference of decolorization rates may reflect a high NaCl concentration in Wastewater I and the presence of dispersant in Wastewater II (cf. Novotny´ et al., 2003). It is not easy to compare the decolorization of wastewaters with other studies due to different compositions of wastewaters and various fungi and reactor types used. The rate of decolorization of Wastewater II was comparable to the data obtained for decolorization of an untreated textile wastewater by Pleurotus flabellatus (60e70% decolorization within 10 d; Nilsson et al., 2006) or a crude effluent from a dye manufacture by Pleurotus sanguineus (70% decolorization within 14 d; Vanhulle et al., 2008) but lower than the decolorization rate of a pigment plant effluent by Pycnoporus cinnabarinus (100% decolorization within 3 d; Schliephake et al., 1993). I. lacteus was able to completely remove the color of both Wastewater I and II and demonstrated a strong potential for decolorization of true industrial effluents when used under the conditions of RBC reactor.
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
Neither of the wastewaters tested exhibited a genetic toxicity when measured with the Ames test with or without S9 activation before the treatment. In order to check the possibility of formation of genotoxic degradation products, samples were removed when the absorbance decreased below 50% of the initial value (intermediates) and at the end of the experiment (endproducts), i.e. on Days 40 and 47 for Wastewater I and on Days 10 and 26 for Wastewater II, respectively (cf. Fig. 1). The results indicated that no genotoxic intermediates or end-products were formed (data not shown). The biological detoxification of Wastewater I and II was again measured with V. fischeri, L. minor and S. alba. A significant toxicity of Wastewater I was detected with all three tests, S. alba being the least sensitive of the tests used (Fig. 4A). The degradation led to a 10-fold decrease of toxicity measured with V. fischeri and L. minor. The severe inhibition of growth of L. minor by Wastewater I was probably caused by a high concentration of NaCl in the dyeing liquor as salinities exceeding 1.66% had been reported toxic for the plant (Haller et al., 1974). Interestingly, the high toxicity was removed by I. lacteus suggesting an important desalting capacity of fungal biofilms (Fig. 4A). The respective initial toxicity values of Wastewater II were 5e10-fold inferior to those of Wastewater I when measured with V. fischeri and L. minor. On the other hand, the toxicity of Wastewater II measured by germination of S. alba seeds exceeded that of Wastewater I more than 103-fold (Fig. 4A, B). The degradation decreased the toxicity of Wastewater II measured with Vibrio fisheri and S. alba 10- and 104fold, respectively, but the toxicity for L. minor remained the same, suggesting that the latter toxic effect was probably not caused by the dyes but by other compounds that were not degraded by the fungus (Fig. 4B). The results demonstrated a strong detoxification power of I. lacteus comparable, for instance, to T. versicolor capable to decrease the toxicity of Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Brilliant Red 3G-P dyes (Ramsay and Nguyen, 2002) or of a textile effluent from dyeing with cochineal extracts (Arroyo-Figueroa et al., 2011). The battery of the toxicity tests was applied to monitor the decrease of genetic and biological toxicity during biodegradation of dyes. The sensitivity of the individual tests to various toxicants differed: V. fischeri test was the most sensitive to MB, L. minor test to Wastewater I, and S. alba test to Wastewater II. These differences evidently reflected the involvement of various biological processes targeted by the toxicants in the individual tests: bioluminiscence, growth, and seed germination. This fact stressed the importance of using batteries of tests that include various endpoints and organisms of different trophic levels for the evaluation of environmental toxicity.
4.
Conclusions
I. lacteus biofilms used in the RBC-type reactor with PUF discs demonstrated a high efficiency of decolorization and detoxification of recalcitrant, medium-toxic dyes and textile dye mixtures in industrial dyeing liquors and confirmed a potential of this fungal technology for remediation of textile wastewaters. No production of genetically or biologically
7147
toxic compounds was observed during the dye removal. The endproducts of MB degradation were usable as nutrients for growth of L. minor and S. alba. A battery of acute toxicity tests comprising the bioluminiscence test and plant growth- and seed germination tests showed good sensitivity for monitoring the toxicity of textile dyes, comparable to often used water crustacean tests. Different sensitivities of the individual tests towards particular dyes and dye mixtures stressed the importance of using various biological targets for the assessment of danger represented by textile waste effluents.
Acknowledgments The provision of textile wastewaters by INOTEX s.r.o., Czech Republic is gratefully acknowledged. We thank R. Por´ızka, M. Holesova´, L. Vasutova´ and I. Falgentra¨gerova´ for help in realization of the experiments. We acknowledge the financial support from the following projects: IAAX00200901 (Grant Agency of the ASCR), Institutional Research Concept No. AV0Z50200510, SGS 19/PrF/2012, Inst. Environ. Technol. project CZ.1.05/2.1.00/03.0100 realized within Research and Development for Innovations Operational Programme cofinanced by Structural Funds of EU and the Czech Republic.
references
Anderson, J.G., 1983. Immobilized cell and film reactor systems for filamentous fungi. In: Smith, J.E., Berry, D.R., Kristiansen, B. (Eds.), The Filamentous Fungi, vol. 4. Arnold Ltd, London, pp. 145e167. Arroyo-Figueroa, G., Ruiz-Aguilar, G.M.L., Lo´pez-Martı´nez, L., Gonza´les-Sa´nchez, G., Cuevas-Rodrı´guez, G., Rodrı´guezVa´zquez, R., 2011. Treatment of a textile effluent from dyeing with cochineal extracts using Trametes versicolor fungus. Sci. World J. 11, 1005e1016. Axelsson, J., Nilsson, U., Terrazas, E., Alvarez, T.A., Welander, U., 2006. Decolorization of the textile dyes Reactive Red 2 and Reactive Blue 4 using Bjerkandera sp. strain BOL13 in a continuous rotating biological contactor reactor. Enzyme Microb. Technol. 39, 32e37. Bergsten-Torralba, L.R., Nishikawa, M.M., Baptista, D.F., Magalhaes, D.P., da Silva, M., 2009. Decolorization of different textile dyes by Penicillium simplicissimus and toxicity evaluation after fungal treatment. Braz. J. Microbiol. 40, 808e817. Boer, C.G., Obici, L., de Souza, C.G.M., Peralta, R.M., 2004. Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme. Bioresour. Technol. 94, 107e112. Broekhoven, L.H., Nestmann, E.R., 1991. Statistical Analysis of the Salmonella mutagenicity assay. In: Krewski, D., Franklin, C. (Eds.), Statistics in Toxicology. Gordon and Breach Science Publishers, Amsterdam, pp. 28e34. Dubrow, S.F., Boardman, G.D., Michelsen, D.L., 1996. Chemical pretreatment and aerobic-anaerobic degradation of textile dye wastewater. In: Reife, A., Freeman, H.S. (Eds.), Environmental Chemistry of Dyes and Pigments. John Wiley & Sons Inc, New York, pp. 75e104.
7148
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
Gottlieb, A., Shaw, C., Smith, A., Wheatley, A., Forsythe, S., 2003. The toxicity of textile reactive azo dyes after hydrolysis and decolourisation. J. Biotechnol. 101, 49e56. Guimaraes, C., Porto, P., Oliveira, R., Mota, M., 2005. Continuous decolorization of a sugar refinery wastewater in a modified rotating biological contactor with Phanerochaete chrysosporium immobilized on polyurethane foam disks. Process. Biochem. 40, 535e540. Haller, W.T., Sutton, D.L., Barlowe, W.C., 1974. Effects of salinity on growth of several aquatic macrophytes. Ecology 55, 891e894. ISO 11269-1, 1993. Determination of the Effects of Pollutants on Soil Flora-method for the Measurement of Inhibition of Root Growth. ISO Standards Catalogue, TC 190 Soil quality. ISO CD 20079, 2005. Water Quality e Determination of the Toxic Effect of Water Constituents and Waste Water to Duckweed (Lemna Minor) e Duckweed Growth Inhibition Test. ISO Standards Catalogue, TC 147 Water quality. ISO 11348-3, 2007. Water Quality e Determination of the Inhibitory Effect of Water Samples on the Light Emission of Vibrio fischeri (Luminescent Bacteria Test). ISO Standards Catalogue, TC 147 Water quality. Knapp, J.S., Vantoch-Wood, E.J., Zhang, F., 2008. Use of woodrotting fungi for the decolorization of dyes and industrial effluents. In: Gadd, G.M. (Ed.), Fungi in Bioremediation. Cambridge University Press, Cambridge, pp. 242e304. Ma, D.Y., Wang, X.H., Song, C., Wang, S.G., Fan, M.H., Li, X.M., 2011. Aerobic granulation for methylene blue biodegradation in a sequencing batch reactor. Desalination 276, 233e238. Svobodova´, K., kova´, Z., Novotny´, C., Malachova´, K., Pavlı´c Lednicka´, S., Musı´lkova´, E., 2006. Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus Irpex lacteus. Environ. Mol. Mutagen 47, 533e540. Mazmanci, M.A., Unyayar, A., Ekiz, H.I., 2002. Decolorization of methylene blue by white rot fungus Coriolus versicolor. Fresenius Environ. Bull. 11, 254e258. Nilsson, I., Mooler, A., Mattiasson, B., Rubindamayugi, M.S.T., Welander, U., 2006. Decolorization of synthetic and real textile wastewater by the use of white rot fungi. Enzyme Microb. Technol. 38, 94e100. Rawal, B., Bhatt, M., Patel, M., Sa sek, V., Novotny´, C., Molitoris, H.P., 2003. Screening of fungal strains for remediation of water and soil contaminated with synthetic dyes. In: NATO Advanced Research Workshop “The Utilization of Bioremediation to Reduce Soil Contamination: Problems and Solutions. Kluwer, pp. 143e148.
NTP TR 540, 2008. Toxicology and Carcinogenesis Studies of Methylene Blue Trihydrate. NIH Publication, pp. 149e165. No. 08-4429. OECD Test No. 471, 1997. Bacterial Reverse MutationTest. In: OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, pp. 1e11. Ogugbue, C.J., Oranusi, N.A., 2006. Toxicity of azo dyes to the freshwater shrimp (Desmocaris trispinosa). Int. J. Nat. Appl. Sci. (IJNAS) 1, 37e44. Ong, S.A., Toorisaka, E., Hirata, M., Hano, T., 2005. Biodegradation of redox dye methylene blue by up-flow anaerobic sludge blanket reactor. J. Hazard. Mater. 124, 88e94. Radha, K.V., Regupathi, I., Arunagiri, A., Murugeshan, T., 2005. Decolorization studies of synthetic dyes using Phanerochaete chrysosporium and their kinetics. Process. Biochem. 40, 3337e3345. Ramsay, J.A., Nguyen, T., 2002. Decoloration of textile dyes by Trametes versicolor and its effect on dye toxicity. Biotechnol. Lett. 24, 1757e1761. Schliephake, K., Lonergan, G.T., Jones, C.L., Mainwaring, D.E., 1993. Decolourisation of a pigment plant effluent by Pycnoporus cinnabarinus in a packed-bed bioreactor. Biotechnol. Lett. 15, 1185e1188. Singh, H., 2006. Mycoremediation-fungal Bioremediation. Wiley Interscience, Hoboken, pp. 357e483. Soni, P., Sharma, S., Sharma, S., Kumar, S., Sharma, K.P., 2006. A comparative study on the toxic effects of textile dye wastewaters (untreated and treated) on mortality and RBC of a freshwater fish Gambusia affinis (Baird and Gerard). J. Environ. Biol. 27, 623e628. Tychanowicz, G.K., Zilly, A., de Souza, C.G.M., Peralta, R.M., 2004. Decolourisation of industrial dyes by solid-state cultures of Pleurotus pulmonarius. Process. Biochem. 39, 855e859. Vanhulle, S., Trovaslet, M., Enaud, E., Lucas, M., Taghavi, S., van der Lelie, D., van Aken, B., Foret, M., Onderwater, R., Wesenberg, D., Agathos, S.N., Schneider, Y.-J., Corbisier, A.-M., 2008. Decolorization, cytotoxicity, and genotoxicity reduction during a combined ozonation/fungal treatment of dyecontaminated wastewater. Environ. Sci. Technol. 42, 584e589. Verma, V., 2008. Acute toxicity assessment of textile dyes and textile and dye industrial effluents using Daphnia magna bioassay. Toxicol. Ind. Health 24, 491e500. Wainwright, M., Phoenix, D.A., Gaskell, M., Marshall, B., 1999. Photobactericidal activity of methylene blue derivatives against vancomycin-resistant Enterococcus spp. J. Antimicrob. Chemother. 44, 823e825.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/watres
Biodegradation and detoxification potential of rotating biological contactor (RBC) with Irpex lacteus for remediation of dye-containing wastewater Katerina Malachova a,*, Zuzana Rybkova a, Hana Sezimova a, Jiri Cerven a, Cenek Novotny a,b a
Faculty of Science, University of Ostrava, Chittussiho 10, 710 00 Slezska´, Ostrava, Czech Republic ska´ 1083, 142 20 Prague 4, Czech Laboratory of Environmental Biotechnology, Institute of Microbiology ASCR, Vı´den Republic b
article info
abstract
Article history:
Use of fungal organisms in rotating biological contactors (RBC) for bioremediation of liquid
Received 23 March 2013
industrial wastes has so far been limited in spite of their significant biodegradation po-
Received in revised form
tential. The purpose was to investigate the power of RBC using Irpex lacteus for decolor-
26 June 2013
ization and detoxification of industrial dyes and dyeing textile liquors. Recalcitrant dye
Accepted 5 July 2013
Methylene Blue (150 mg L1) was decolorized within 70 days, its mutagenicity removed,
Available online 20 October 2013
and the biological toxicity decreased more than 10-fold. I. lacteus biofilm in the RBC completely decolorized within 26 and 47 days dyeing liquors containing disperse or reac-
Keywords:
tive dyes adjusted to pH4.5 and 5-fold diluted with the growth medium, respectively. Their
Liquid textile wastes
respective biological toxicity values were reduced 10- to 104-fold in dependence of the test
Dye decolorization
used. A battery of toxicity tests comprising Vibrio fisheri, Lemna minor and Sinapis alba was
Genetic toxicity
efficient to monitor the toxicity of textile dyes and wastewaters. Strong decolorization and
Biological toxicity
detoxification power of RBC using I. lacteus biofilms was demonstrated. ª 2013 Elsevier Ltd. All rights reserved.
Rotating biological contactor Irpex lacteus
1.
Introduction
Textile industry produces large volumes of dye-containing effluents that are ineffectively remediated in wastewater treatment plants and are responsible for coloration of streams that negatively affects water life. Biological and genetic toxicity of dyes for bacteria, protozoa, aquatic animals, plants and mammals has been widely documented (Gottlieb et al., 2003; Soni et al., 2006; etc.). Textile wastewaters are extremely variable in composition due to the presence of
various dyes, desizing and scouring agents, detergents, finishing agents and inorganic salts that all can contribute to their toxicity (Dubrow et al., 1996). Consequently, efficient remediation must result in both decolorization and detoxification of the wastewater. Decolorization of dyes with ligninolytic fungi has been proven to be an efficient, cheap and environment-friendly process but their detoxification power has been studied less frequently (e.g. Knapp et al., 2008). A number of chemicallydifferent types of persistent dyes have been shown to be
* Corresponding author. Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 710 00 Slezska´, Ostrava, Czech Republic. Tel.: þ420 2 597092315; fax: þ420 2 597092382. E-mail addresses: [email protected] (K. Malachova), [email protected] (Z. Rybkova), [email protected] (H. Sezimova), [email protected] (J. Cerven), [email protected] (C. Novotny). 0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2013.07.050
7144
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
effectively degraded by various fungal organisms (e.g. Singh, 2006). Methylene Blue (CAS No. 61-73-4, C.I. 52015, MB), a heterocyclic phenothiazine dye, is widely used for dyeing leather and textile materials. Its decolorization strongly depends on the conditions of fungal culture and the efficiency of various ligninolytic fungi is quite variable (Tychanowicz et al., 2004; etc.). Partial to weak decolorizations were reported by various ligninolytic fungi in liquid- and solid-state cultures, by bacteria and the aerobic activated sludge (e.g. Ma et al., 2011). Anaerobic sludge is able to remove the dye but due to reoxidation by air only a low color removal effect was achieved (Ong et al., 2005). Broader adverse effects of MB include eye injury, breathing problems, methemoglobinemia, bacteriostatic and fungicidal activities, and a significant toxicity to aquatic plants, crustaceans and fish (Wainwright et al., 1999 etc.). Because of the persistence and toxicity, MB was chosen in this study as a model industrial dye for testing the efficiency of RBC reactor using Irpex lacteus to decolorize and detoxify dyes and textile effluents. RBC reactors operable in repeated-batch or continuous mode offer advantages in bioremediation of industrial wastewaters due to great surface per unit volume, low power requirement and limited flow clogging (Anderson, 1983). Ligninolytic fungi behave well in RBC but so far few studies have been conducted, comprising only a limited number of fungal strains (e.g. Guimaraes et al., 2005; Axelsson et al., 2006). Biodegradation and detoxification power of I. lacteus in this type of reactor has not been thoroughly investigated. Ecotoxicity of pollutants is usually measured with standard toxicity tests, i.e. bacterial, crustacean, algal and seed germination tests. For instance, Vibrio fischeri bioluminiscence test was used to measure both a decrease and increase of toxicity resulting from degradation of various textile dyes by Trametes versicolor (Ramsay and Nguyen, 2002) or formation of toxic products in the course of anaerobic decolorization of Reactive Black 5 by Enterococcus faecalis and Clostridium butyricum (Gottlieb et al., 2003). Tests with Daphnia spp. were used to monitor decolorization-linked detoxification of phthalocyanine- and azo dyes obtained with Penicillium simplicissimum or of a raw textile effluent treated with horseradish peroxidase (Bergsten-Torralba et al., 2009). A reduction of mutagenicity of Reactive Orange 16 and Disperse Blue 3 dyes in a two-step treatment with activated sludge and a static culture of I. lacteus was monitored by the Ames test (Malachova´ et al., 2006). Our study was undertaken to investigate the dye decolorization and detoxification capacity of I. lacteus under the conditions of RBC reactor using MB and two different textile dyeing liquors containing mixtures of reactive or disperse dyes to test the decolorization and detoxification efficiency. Biological toxicity changes during the treatment were measured with a battery of standard bacterial and plant tests and the change of genetic toxicity with Ames test.
2.
Material and methods
2.1.
Chemicals
The dyeing liquors were obtained from INOTEX a.s., Czech Republic. Wastewater I contained Sumifix Black B 150% (C.I.
Reactive Black 5) (9.82 g L1), Inosin Yellow V-GR 160% (C.I. Reactive Yellow 15) (2.47 g L1), NaCl (75 g L1) and the fixation agent Texalkon MS (7.87 g L1). Wastewater II contained Itosperse Yellow RAP dye mix (5.47 g L1), Itosperse Red RAP dye mix (3.75 g L1), Itosperse Blue RAP dye mix (2.47 g L1), the disperging agent Nicca Sunsolt RF-557 (1 g L1) and acetic acid (0.3 ml L1). Malt extract and agar were purchased from Oxoid, UK, Disperse Blue 3 (DB3, anthraquinone) and Methylene Blue (MB, phenothiazine) dyes from SigmaeAldrich, Czech Republic. Other chemicals were of analytical grade.
2.2.
Microorganism
Irpex lacteus 931 was provided by the Culture Collection of Basidiomycetes, Institute of Microbiology ASCR, Prague and maintained on malt extract-glucose (MEG) medium containing 2% (w/w) agar at 4 C.
2.3.
Biodegradation in RBC reactor
The rotating biological contactor (RBC) reactor consisted of a glass vessel and a horizontal driving axis with six 1-cm thick polyurethane foam (PUF) discs (diameter 8 cm, rotation speed 2 rpm, 40% of disc volume immersed). The experiments were carried out aseptically in MEG medium (per litre: 5 g malt extract, 10 g glucose, pH 4.5) at 22 C and forced aeration with air (50 L h1). Sterile PUF discs were put horizontally in MEG and inoculated with a homogenate (Ultra-Turrax T25 mixer, IKA Werk, Germany, 20 s) of a 7-d-old, static MEG culture grown at 28 C (10% v/v inoculum). The discs were colonized with the fungus (7 d, 28 C) and then mounted aseptically in the reactor containing one litre of MEG medium with DB3 or MB dyes dissolved at a concentration of 150 mg L1 representing the respective dye concentrations of 0.56 and 0.47 mM. Their decolorization was measured spectrophotometrically at 645 nm and 505/ 580 nm, respectively. Wastewaters I and II were adjusted to pH 4.5 and used 5-fold diluted with MEG. Their decolorization was measured at respective maxima of 575 and 425 nm. The fungal biomass on the discs was estimated gravimetrically at the end of the experiment as dry biomass.
2.4.
Biological toxicity tests
The acute biological toxicity was estimated using bacterial luminiscence, aquatic plant growth and seed germination as the endpoints. V. fischeri test (ISO 11348-3, 2007) measured bioluminiscence inhibition after a 30-min exposition using a LUMIStox300 luminometer (Hach-Lange, Du¨sseldorf, Germany). Lemna minor test (ISO CD, 20079, 2005) determined growth inhibition of fronds, the exposition time was 7 days. The Phytotoxkit Sinapis alba test (ISO 11269-1, 1993) determined the inhibition of root growth after a 3-d exposure. The test was considered to be valid if the germination of the control was 90%. The stimulation effect of endproducts was evaluated by using a linear model. A positive toxic effect was evaluated in the tests against negative controls containing only the culture medium. Positive controls using toxicants recommended in the corresponding ISO standard were also measured to check the sensitivity of the
7145
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
Absorbance
1,2 1 0,8 0,6 0,4 0,2 0 0
10
20
30
40
50
60
70
Days
Fig. 1 e Time course of decolorization of Methylene Blue (oeo) and textile dyeing liquors containing reactive dyes (CeC, Wastewater I) and disperse dyes (:e:, Wastewater II) in RBC reactor with Irpex lacteus. Absorbance was measured at the corresponding absorption maxima: Methylene Blue 580 nm, Wastewater I 575 nm, Wastewater II 475 nm. The absorbance values represent the means of three samples.
individual tests. In the tests, EC50 or IC50 values were calculated and expressed as a logarithm of the dilution factor determined as a volume percentage (v/v %) of the diluted sample used in the test relative to the original undiluted sample removed from the reactor. This enabled us to compare the changes of EC50 and IC50 measured before and after degradation.
2.5.
Mutagenicity (Ames) test
Mutagenicity was detected using a plate-incorporation version of the Salmonella typhimurium Hise reversion assay used with or without the in vitro metabolic activation with the rsat liver S9 microsomal fraction and cofactor mixture (OECD Test No. 471, 1997). The auxotrophic strains TA100 and TA98 were used for detection of the base substitution mutations and frameshift mutations, respectively. The mutagenic activity was expressed as a number of revertant colonies (Rt) obtained with the treated sample compared to the number of revertant colonies obtained with the control sample (Rc). The index of mutagenicity was calculated as a ratio of Rt/Rc and a twofold increase of the index was considered to be significant. Mutation potential represented the index of mutagenicity related to the concentration of the tested compound expressed in micrograms. Each test was repeated at least three times using two replicate plates for each sample and the results were calculated using SALM software (Broekhoven and Nestmann, 1991).
3.
Results and discussion
3.1.
Decolorization and detoxification of model dyes
Decolorization of DB3 was used as a forerunner test of the ability of the fresh-grown I. lacteus biofilms mounted in the
RBC reactor to decolorize recalcitrant dye compounds as the fungus was previously reported to decolorize and detoxify DB3 (Malachova´ et al., 2006). The dye was completely decolorized within 25 days (data not shown). Then the reactor was washed with MEG medium and, subsequently, a batch of MEG containing MB was added. A complete decolorization was achieved within 70 days (Fig. 1). The total amount of fungal biofilms on the surface of PUF discs in RBC after decolorization of MB was 7.53 g dry biomass and the average decolorization rate was calculated to be 0.34 mg MB d1 g1 dry biomass. These data well compare with other fungi, namely, the decolorization of 5e20 mg MB L1 by Phanerochaete chrysosporium and T. versicolor (Mazmanci et al., 2002; Radha et al., 2005) or a partial decolorization of 200 mg MB L1 by Lentinula edodes (Boer et al., 2004). Sampling at Day 0, 20 and 70 (cf. Figs. 2 and 3) intended to measure the toxicity of intact MB, degradation intermediates and the endproduct in keeping with the time course of MB decolorization shown in Fig. 1. On Day 0, the mutagenic effects of the dye were observed in all variants of the Ames test. MB induced frameshift and substitution, direct and indirect mutations both with and without metabolic activation. However in the tests without metabolic activation MB was concluded to be only potentially mutagenic since a significant, twofold increase of the revertant number was not accomplished due to the toxicity of high sample concentrations for the indicator strains. The respective maximal mutagenic activities obtained with TA100 and TA98 strains expressed as the index of mutagenicity were 1.54 (25 mg MB per plate) and 1.85 (2.5 mg MB per plate). In the tests with metabolic activation, the values of the index of mutagenicity were even higher: TA100 1.78 (25 mg MB per plate), TA98 3.85 (2.5 mg MB per plate). The results thus confirmed the genotoxic effects of the intact dye reported by other authors (NTP TR 540, 2008). The degradation resulted in a complete detoxification: the sample removed at Day 70, when the absorbance decreased to zero, exhibited no
50 45 40 Mutation potential
1,4
35 30 25 20 15 10 5 0
1
2 3 Day 0
4
1
2 3 Day 20
4
1
2 3 Day 70
4
Fig. 2 e Mutagenicity of Methylene Blue during biodegradation in RBC reactor measured with Salmonella typhimurium His- test with and without S9 activation. Samples were withdrawn on Day 0, Day 20 (intermediates) and Day 70 (endproducts): TA100-S9 (1); TA98-S9 (2); TA100 D S9 (3); TA98 D S9 (4).
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
3
a3
2
2
Log EC50 or IC50
Log EC50 or IC50
7146
1
1
0
0 2 Day 0
3
1
2 3 Day 20
1
2 Day 70
3
Fig. 3 e Toxicity of Methylene Blue expressed as Log EC50 or IC50 during biodegradation in RBC reactor measured with Vibrio fischeri (1), Sinapis alba (2), and Lemna minor (3). Samples were withdrawn on Day 0, Day 20 and Day 70 to measure the toxicity of intact dye, intermediates and endproducts of biodegradation, respectively.
mutagenic effect (Fig. 2). A similar removal of mutagenicity by I. lacteus growing in static liquid cultures was demonstrated for Reactive Orange 16 azo dye (Malachova´ et al., 2006). Tests of acute biological toxicity demonstrated a significant toxicity of the sample containing intact MB removed on Day 0. The V. fischeri test and the plant tests with L. minor and S. alba showed the following respective toxicity values: EC50 ¼ 18.07 0.31 mg L1, EC50 ¼ 12.22 0.55 mg L1 and IC50 ¼ 100 0.1 mg L1 (Fig. 3). The germination of seeds of S. alba was 5- and 8-fold less sensitive to the toxic effect of the dye than the bacterial luminiscence and growth of L. minor, respectively. These results showed that MB was toxic for organisms of various trophic levels and confirmed previous findings of the adverse side-effects (e.g. NTP TR 540, 2008; Wainwright et al., 1999). The EC50 values demonstrated a good sensitivity of the test battery and were comparable to those obtained for various azo dyes with crustaceans Daphnia magna and Desmocaris trispinosa (Ogugbue and Oranusi, 2006; Verma, 2008). The decrease of biological toxicity of MB during biodegradation, expressed as EC50 or IC50, exceeded one order of magnitude. In the tests with L. minor up to a 10% stimulation of growth of fronds, compared to the control, was observed with the sample withdrawn at Day 70; probably, the degradation endproducts were usable as nutrients by the plants. The stimulation effect along the gradient of endproduct concentration was found to be significant (F ¼ 15.57, p ¼ 0.017). The fitted model: y (rate of growth) ¼ 0.0008 conc þ 0.137. Similar studies reported both decrease and increase of biological toxicity after the treatment of various dyes with T. versicolor and Penicillium simplicissimus, when monitored with V. fischeri and Daphnia pulex tests (Bergsten-Torralba et al., 2009; Ramsay and Nguyen, 2002).
3.2. Decolorization and detoxification of textile dyeing liquors Textile wastewaters containing reactive (Wastewater I) or disperse (Wastewater II) dyes were completely decolorized in
-1
b
1
2 3 Day 0
1
2 3 Day 40
1
2 3 Day 47
3 2
Log EC50 or IC50
1
1 0 -1 -2 -3 -4
1
2 3 Day 0
1
2 3 Day 10
1
2 3 Day 26
Fig. 4 e Toxicity of Wastewater I (a) and Wastewater II (b) during biodegradation in RBC reactor measured with Vibrio fischeri (1), Sinapis alba (2) and Lemna minor (3). The samples withdrawn on Day 40 and Day 47 (Wastewater I) and Day 10 and Day 26 (Wastewater II) represented 50 and 100% decolorization to measure the toxicity of biodegradation intermediates and endproducts, respectively.
the RBC reactor within 47 and 26 days, respectively (Fig. 1). The decolorization of Wastewater I containing two reactive dyes, a high concentration of NaCl and Texalcon MS fixation agent was slower than that of Wastewater II containing three disperse dyes and low concentrations of disperging agent and acetic acid. The difference of decolorization rates may reflect a high NaCl concentration in Wastewater I and the presence of dispersant in Wastewater II (cf. Novotny´ et al., 2003). It is not easy to compare the decolorization of wastewaters with other studies due to different compositions of wastewaters and various fungi and reactor types used. The rate of decolorization of Wastewater II was comparable to the data obtained for decolorization of an untreated textile wastewater by Pleurotus flabellatus (60e70% decolorization within 10 d; Nilsson et al., 2006) or a crude effluent from a dye manufacture by Pleurotus sanguineus (70% decolorization within 14 d; Vanhulle et al., 2008) but lower than the decolorization rate of a pigment plant effluent by Pycnoporus cinnabarinus (100% decolorization within 3 d; Schliephake et al., 1993). I. lacteus was able to completely remove the color of both Wastewater I and II and demonstrated a strong potential for decolorization of true industrial effluents when used under the conditions of RBC reactor.
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
Neither of the wastewaters tested exhibited a genetic toxicity when measured with the Ames test with or without S9 activation before the treatment. In order to check the possibility of formation of genotoxic degradation products, samples were removed when the absorbance decreased below 50% of the initial value (intermediates) and at the end of the experiment (endproducts), i.e. on Days 40 and 47 for Wastewater I and on Days 10 and 26 for Wastewater II, respectively (cf. Fig. 1). The results indicated that no genotoxic intermediates or end-products were formed (data not shown). The biological detoxification of Wastewater I and II was again measured with V. fischeri, L. minor and S. alba. A significant toxicity of Wastewater I was detected with all three tests, S. alba being the least sensitive of the tests used (Fig. 4A). The degradation led to a 10-fold decrease of toxicity measured with V. fischeri and L. minor. The severe inhibition of growth of L. minor by Wastewater I was probably caused by a high concentration of NaCl in the dyeing liquor as salinities exceeding 1.66% had been reported toxic for the plant (Haller et al., 1974). Interestingly, the high toxicity was removed by I. lacteus suggesting an important desalting capacity of fungal biofilms (Fig. 4A). The respective initial toxicity values of Wastewater II were 5e10-fold inferior to those of Wastewater I when measured with V. fischeri and L. minor. On the other hand, the toxicity of Wastewater II measured by germination of S. alba seeds exceeded that of Wastewater I more than 103-fold (Fig. 4A, B). The degradation decreased the toxicity of Wastewater II measured with Vibrio fisheri and S. alba 10- and 104fold, respectively, but the toxicity for L. minor remained the same, suggesting that the latter toxic effect was probably not caused by the dyes but by other compounds that were not degraded by the fungus (Fig. 4B). The results demonstrated a strong detoxification power of I. lacteus comparable, for instance, to T. versicolor capable to decrease the toxicity of Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Brilliant Red 3G-P dyes (Ramsay and Nguyen, 2002) or of a textile effluent from dyeing with cochineal extracts (Arroyo-Figueroa et al., 2011). The battery of the toxicity tests was applied to monitor the decrease of genetic and biological toxicity during biodegradation of dyes. The sensitivity of the individual tests to various toxicants differed: V. fischeri test was the most sensitive to MB, L. minor test to Wastewater I, and S. alba test to Wastewater II. These differences evidently reflected the involvement of various biological processes targeted by the toxicants in the individual tests: bioluminiscence, growth, and seed germination. This fact stressed the importance of using batteries of tests that include various endpoints and organisms of different trophic levels for the evaluation of environmental toxicity.
4.
Conclusions
I. lacteus biofilms used in the RBC-type reactor with PUF discs demonstrated a high efficiency of decolorization and detoxification of recalcitrant, medium-toxic dyes and textile dye mixtures in industrial dyeing liquors and confirmed a potential of this fungal technology for remediation of textile wastewaters. No production of genetically or biologically
7147
toxic compounds was observed during the dye removal. The endproducts of MB degradation were usable as nutrients for growth of L. minor and S. alba. A battery of acute toxicity tests comprising the bioluminiscence test and plant growth- and seed germination tests showed good sensitivity for monitoring the toxicity of textile dyes, comparable to often used water crustacean tests. Different sensitivities of the individual tests towards particular dyes and dye mixtures stressed the importance of using various biological targets for the assessment of danger represented by textile waste effluents.
Acknowledgments The provision of textile wastewaters by INOTEX s.r.o., Czech Republic is gratefully acknowledged. We thank R. Por´ızka, M. Holesova´, L. Vasutova´ and I. Falgentra¨gerova´ for help in realization of the experiments. We acknowledge the financial support from the following projects: IAAX00200901 (Grant Agency of the ASCR), Institutional Research Concept No. AV0Z50200510, SGS 19/PrF/2012, Inst. Environ. Technol. project CZ.1.05/2.1.00/03.0100 realized within Research and Development for Innovations Operational Programme cofinanced by Structural Funds of EU and the Czech Republic.
references
Anderson, J.G., 1983. Immobilized cell and film reactor systems for filamentous fungi. In: Smith, J.E., Berry, D.R., Kristiansen, B. (Eds.), The Filamentous Fungi, vol. 4. Arnold Ltd, London, pp. 145e167. Arroyo-Figueroa, G., Ruiz-Aguilar, G.M.L., Lo´pez-Martı´nez, L., Gonza´les-Sa´nchez, G., Cuevas-Rodrı´guez, G., Rodrı´guezVa´zquez, R., 2011. Treatment of a textile effluent from dyeing with cochineal extracts using Trametes versicolor fungus. Sci. World J. 11, 1005e1016. Axelsson, J., Nilsson, U., Terrazas, E., Alvarez, T.A., Welander, U., 2006. Decolorization of the textile dyes Reactive Red 2 and Reactive Blue 4 using Bjerkandera sp. strain BOL13 in a continuous rotating biological contactor reactor. Enzyme Microb. Technol. 39, 32e37. Bergsten-Torralba, L.R., Nishikawa, M.M., Baptista, D.F., Magalhaes, D.P., da Silva, M., 2009. Decolorization of different textile dyes by Penicillium simplicissimus and toxicity evaluation after fungal treatment. Braz. J. Microbiol. 40, 808e817. Boer, C.G., Obici, L., de Souza, C.G.M., Peralta, R.M., 2004. Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme. Bioresour. Technol. 94, 107e112. Broekhoven, L.H., Nestmann, E.R., 1991. Statistical Analysis of the Salmonella mutagenicity assay. In: Krewski, D., Franklin, C. (Eds.), Statistics in Toxicology. Gordon and Breach Science Publishers, Amsterdam, pp. 28e34. Dubrow, S.F., Boardman, G.D., Michelsen, D.L., 1996. Chemical pretreatment and aerobic-anaerobic degradation of textile dye wastewater. In: Reife, A., Freeman, H.S. (Eds.), Environmental Chemistry of Dyes and Pigments. John Wiley & Sons Inc, New York, pp. 75e104.
7148
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3 e7 1 4 8
Gottlieb, A., Shaw, C., Smith, A., Wheatley, A., Forsythe, S., 2003. The toxicity of textile reactive azo dyes after hydrolysis and decolourisation. J. Biotechnol. 101, 49e56. Guimaraes, C., Porto, P., Oliveira, R., Mota, M., 2005. Continuous decolorization of a sugar refinery wastewater in a modified rotating biological contactor with Phanerochaete chrysosporium immobilized on polyurethane foam disks. Process. Biochem. 40, 535e540. Haller, W.T., Sutton, D.L., Barlowe, W.C., 1974. Effects of salinity on growth of several aquatic macrophytes. Ecology 55, 891e894. ISO 11269-1, 1993. Determination of the Effects of Pollutants on Soil Flora-method for the Measurement of Inhibition of Root Growth. ISO Standards Catalogue, TC 190 Soil quality. ISO CD 20079, 2005. Water Quality e Determination of the Toxic Effect of Water Constituents and Waste Water to Duckweed (Lemna Minor) e Duckweed Growth Inhibition Test. ISO Standards Catalogue, TC 147 Water quality. ISO 11348-3, 2007. Water Quality e Determination of the Inhibitory Effect of Water Samples on the Light Emission of Vibrio fischeri (Luminescent Bacteria Test). ISO Standards Catalogue, TC 147 Water quality. Knapp, J.S., Vantoch-Wood, E.J., Zhang, F., 2008. Use of woodrotting fungi for the decolorization of dyes and industrial effluents. In: Gadd, G.M. (Ed.), Fungi in Bioremediation. Cambridge University Press, Cambridge, pp. 242e304. Ma, D.Y., Wang, X.H., Song, C., Wang, S.G., Fan, M.H., Li, X.M., 2011. Aerobic granulation for methylene blue biodegradation in a sequencing batch reactor. Desalination 276, 233e238. Svobodova´, K., kova´, Z., Novotny´, C., Malachova´, K., Pavlı´c Lednicka´, S., Musı´lkova´, E., 2006. Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus Irpex lacteus. Environ. Mol. Mutagen 47, 533e540. Mazmanci, M.A., Unyayar, A., Ekiz, H.I., 2002. Decolorization of methylene blue by white rot fungus Coriolus versicolor. Fresenius Environ. Bull. 11, 254e258. Nilsson, I., Mooler, A., Mattiasson, B., Rubindamayugi, M.S.T., Welander, U., 2006. Decolorization of synthetic and real textile wastewater by the use of white rot fungi. Enzyme Microb. Technol. 38, 94e100. Rawal, B., Bhatt, M., Patel, M., Sa sek, V., Novotny´, C., Molitoris, H.P., 2003. Screening of fungal strains for remediation of water and soil contaminated with synthetic dyes. In: NATO Advanced Research Workshop “The Utilization of Bioremediation to Reduce Soil Contamination: Problems and Solutions. Kluwer, pp. 143e148.
NTP TR 540, 2008. Toxicology and Carcinogenesis Studies of Methylene Blue Trihydrate. NIH Publication, pp. 149e165. No. 08-4429. OECD Test No. 471, 1997. Bacterial Reverse MutationTest. In: OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, pp. 1e11. Ogugbue, C.J., Oranusi, N.A., 2006. Toxicity of azo dyes to the freshwater shrimp (Desmocaris trispinosa). Int. J. Nat. Appl. Sci. (IJNAS) 1, 37e44. Ong, S.A., Toorisaka, E., Hirata, M., Hano, T., 2005. Biodegradation of redox dye methylene blue by up-flow anaerobic sludge blanket reactor. J. Hazard. Mater. 124, 88e94. Radha, K.V., Regupathi, I., Arunagiri, A., Murugeshan, T., 2005. Decolorization studies of synthetic dyes using Phanerochaete chrysosporium and their kinetics. Process. Biochem. 40, 3337e3345. Ramsay, J.A., Nguyen, T., 2002. Decoloration of textile dyes by Trametes versicolor and its effect on dye toxicity. Biotechnol. Lett. 24, 1757e1761. Schliephake, K., Lonergan, G.T., Jones, C.L., Mainwaring, D.E., 1993. Decolourisation of a pigment plant effluent by Pycnoporus cinnabarinus in a packed-bed bioreactor. Biotechnol. Lett. 15, 1185e1188. Singh, H., 2006. Mycoremediation-fungal Bioremediation. Wiley Interscience, Hoboken, pp. 357e483. Soni, P., Sharma, S., Sharma, S., Kumar, S., Sharma, K.P., 2006. A comparative study on the toxic effects of textile dye wastewaters (untreated and treated) on mortality and RBC of a freshwater fish Gambusia affinis (Baird and Gerard). J. Environ. Biol. 27, 623e628. Tychanowicz, G.K., Zilly, A., de Souza, C.G.M., Peralta, R.M., 2004. Decolourisation of industrial dyes by solid-state cultures of Pleurotus pulmonarius. Process. Biochem. 39, 855e859. Vanhulle, S., Trovaslet, M., Enaud, E., Lucas, M., Taghavi, S., van der Lelie, D., van Aken, B., Foret, M., Onderwater, R., Wesenberg, D., Agathos, S.N., Schneider, Y.-J., Corbisier, A.-M., 2008. Decolorization, cytotoxicity, and genotoxicity reduction during a combined ozonation/fungal treatment of dyecontaminated wastewater. Environ. Sci. Technol. 42, 584e589. Verma, V., 2008. Acute toxicity assessment of textile dyes and textile and dye industrial effluents using Daphnia magna bioassay. Toxicol. Ind. Health 24, 491e500. Wainwright, M., Phoenix, D.A., Gaskell, M., Marshall, B., 1999. Photobactericidal activity of methylene blue derivatives against vancomycin-resistant Enterococcus spp. J. Antimicrob. Chemother. 44, 823e825.