外文原文-白耙齿菌生物转盘反应器对印染废水的生物降解和解毒能力.pdf

Biodegradation and detoxifi cation potential of rotating biological contactor RBC with Irpex lacteus for remediation of dye containing wastewater Katerina Malachova a Zuzana Rybkovaa Hana Sezimovaa Jiri Cervena Cenek Novotny a b aFaculty of Science University of Ostrava Chittussiho 10 710 00 Slezska Ostrava Czech Republic bLaboratory of Environmental Biotechnology Institute of Microbiology ASCR V de nska 1083 142 20 Prague 4 Czech Republic a r t i c l e i n f o Article history Received 23 March 2013 Received in revised form 26 June 2013 Accepted 5 July 2013 Available online 20 October 2013 Keywords Liquid textile wastes Dye decolorization Genetic toxicity Biological toxicity Rotating biological contactor Irpex lacteus a b s t r a c t Use of fungal organisms in rotating biological contactors RBC for bioremediation of liquid industrial wastes has so far been limited in spite of their signifi cant biodegradation po tential The purpose was to investigate the power of RBC using Irpex lacteus for decolor ization and detoxifi cation of industrial dyes and dyeing textile liquors Recalcitrant dye Methylene Blue 150 mg L 1 was decolorized within 70 days its mutagenicity removed and the biological toxicity decreased more than 10 fold I lacteus biofi lm in the RBC completely decolorized within 26 and 47 days dyeing liquors containing disperse or reac tive dyes adjusted to pH4 5 and 5 fold diluted with the growth medium respectively Their respective biological toxicity values were reduced 10 to 104 fold in dependence of the test used A battery of toxicity tests comprising Vibrio fi sheri Lemna minor and Sinapis alba was effi cient to monitor the toxicity of textile dyes and wastewaters Strong decolorization and detoxifi cation power of RBC using I lacteus biofi lms was demonstrated 2013 Elsevier Ltd All rights reserved 1 Introduction Textile industry produces large volumes of dye containing effl uents 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 Sonietal 2006 etc Textilewastewatersare extremely variable in composition due to the presence of various dyes desizing and scouring agents detergents fi n ishing agents and inorganic salts that all can contribute to their toxicity Dubrow et al 1996 Consequently effi cient remediation must result in both decolorization and detoxifi cation of the wastewater Decolorization of dyes with ligninolytic fungi has been proven to be an effi cient cheap and environment friendly process but their detoxifi cation power has been studied less frequently e g Knapp et al 2008 A number of chemically different 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 420 2 597092315 fax 420 2 597092382 E mail addresses Katerina Malachova osu cz K Malachova Zuzana Rybkova osu cz Z Rybkova Hana Sezimova osu cz H Sezimova jiri cerven osu cz J Cerven novotny biomed cas cz C Novotny Available online at ScienceDirect journal homepage water research 47 2013 7143e7148 0043 1354 e see front matter 2013 Elsevier Ltd All rights reserved http dx doi org 10 1016 j watres 2013 07 050 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 de pends on the conditions of fungal culture and the effi ciency 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 re oxidation by air only a low color removal effect was ach ieved Ong et al 2005 Broader adverse effects of MB include eye injury breathing problems methemoglobinemia bacteriostatic and fungicidal activities and a signifi cant toxicity to aquatic plants crusta ceans and fi sh 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 effi ciency of RBC reactor using Irpex lacteus to decolorize and detoxify dyes and textile effl uents RBC reactors operable in repeated batch or contin uous mode offer advantages in bioremediation of industrial wastewaters due to great surface per unit volume low power requirement and limited fl ow clogging Anderson 1983 Lig ninolytic 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 detoxifi cation power of I lacteus in this type of reactor has not been thoroughly investigated Ecotoxicity of pollutants is usually measured with stan dard toxicity tests i e bacterial crustacean algal and seed germination tests For instance Vibrio fi scheri 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 butyr icum Gottlieb et al 2003 Tests with Daphnia spp were used to monitor decolorization linked detoxifi cation of phthalocy anine and azo dyes obtainedwith Penicilliumsimplicissimum or of a raw textile effl uent 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 lac teus was monitored by the Ames test Malachova et al 2006 Our study was undertaken to investigate the dye decolor ization and detoxifi cation 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 detoxifi cation effi ciency Biologicaltoxicitychangesduringthetreatmentwere 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 Sumifi x Black B 150 C I Reactive Black 5 9 82 g L 1 Inosin Yellow V GR 160 C I Reactive Yellow 15 2 47 g L 1 NaCl 75 g L 1 and the fi xation agent Texalkon MS 7 87 g L 1 Wastewater II contained Ito sperse Yellow RAP dye mix 5 47 g L 1 Itosperse Red RAP dye mix 3 75 g L 1 Itosperse Blue RAP dye mix 2 47 g L 1 the disperging agent Nicca Sunsolt RF 557 1 g L 1 and acetic acid 0 3 ml L 1 Malt extract and agar were purchased from Oxoid UK DisperseBlue3 DB3 anthraquinone andMethyleneBlue 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 contain ing 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 carriedoutasepticallyinMEGmedium perlitre 5gmaltextract 10gglucose pH4 5 at22 Candforcedaerationwithair 50Lh 1 Sterile PUF discs were put horizontally in MEG and inocu lated 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 con tainingonelitre ofMEGmediumwithDB3orMBdyesdissolved at a concentration of 150 mg L 1representing 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 fi scheri test ISO 11348 3 2007 measured bioluminiscence inhibition after a 30 min exposition using a LUMIStox300 luminometer Hach Lange Du sseldorf Ger many 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 deter mined the inhibition of root growth after a 3 d exposure The test was considered to be valid if the germination of the con trol 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 standardwerealsomeasuredtocheckthesensitivityofthe water research 47 2013 7143e71487144 individualtests Inthetests EC50orIC50valueswerecalculated and expressed as a logarithm of the dilution factor determined asavolumepercentage v v ofthedilutedsampleusedinthe 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 Hisereversion 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 asanumberofrevertantcolonies Rt obtained withthetreated samplecomparedtothenumberofrevertantcoloniesobtained with the control sample Rc The index of mutagenicity was calculatedasaratioofRt Rcandatwofoldincreaseoftheindex was considered to be signifi cant Mutation potential repre sentedtheindexofmutagenicityrelatedtotheconcentrationof the tested compound expressed in micrograms Each test was repeatedatleastthree 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 detoxifi cation of model dyes Decolorization of DB3 was used as a forerunner test of the ability of the fresh grown I lacteus biofi lms mounted in the RBC reactor to decolorize recalcitrant dye compounds as the funguswas previously reportedtodecolorizeanddetoxifyDB3 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 con taining MB was added A complete decolorization was ach ieved within 70 days Fig 1 The total amount of fungal biofi lmson 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 d 1g 1dry biomass These data well compare with other fungi namely the decolorization of 5e20 mg MB L 1by Phanerochaete chrys osporium and T versicolor Mazmanci et al 2002 Radha et al 2005 or a partial decolorization of 200 mg MB L 1by Lenti nula 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 decolorizationshownin Fig 1 On Day0 themutageniceffects of the dye were observed in all variants of the Ames test MB induced frameshift and substitution direct and indirect mu tations both with and without metabolic activation However in the tests without metabolic activation MB was concluded to be only potentially mutagenic since a signifi cant twofold in crease of the revertant number was not accomplished due to the toxicity of high sample concentrations for the indicator strains The respective maximalmutagenic 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 confi rmed the genotoxic effects of the intact dye reported by other authors NTP TR 540 2008 The degradation resulted in a complete detoxifi cation the sample removed at Day 70 whentheabsorbancedecreasedtozero exhibitedno 0 0 2 0 4 0 6 0 8 1 1 2 1 4 010203040506070 Days Absorbance 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 0 5 10 15 20 25 30 35 40 45 50 1 2 3 4 1 2 3 4 1 2 3 4 Day 0Day 20Day 70 Mutation potential 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 water research 47 2013 7143e71487145 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 Testsofacutebiologicaltoxicitydemonstrated asignifi cant toxicity of the sample containing intact MB removed on Day 0 The V fi scheri test and the plant tests with L minor and S alba showedthefollowingrespectivetoxicityvalues EC50 18 07 0 31 mg L 1 EC50 12 22 0 55 mg L 1and IC50 100 0 1 mg L 1 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 or ganisms of various trophic levels and confi rmed previous fi ndings 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 dur ing 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 end product concentration was found to be signifi cant F 15 57 p 0 017 The fi tted model y rate of growth 0 0008 conc 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 fi scheri and Daphnia pulex tests Bergsten Torralba et al 2009 Ramsay and Nguyen 2002 3 2 Decolorization and detoxifi cation of textile dyeing liquors Textile wastewaters containing reactive Wastewater I or disperse Wastewater II dyes were completely decolorized in the RBC reactor within47and 26 days respectively Fig 1 The decolorization of Wastewater I containing two reactive dyes a high concentration of NaCl and Texalcon MS fi xation agent was slower than that of Wastewater II containing three disperse dyes and low concentrations of disperging agent and acetic acid The difference ofdecolorization ratesmay refl ecta 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 decoloriza tion of Wastewater II was comparable to the data obtained for decolorization of an untreated textile wastewater by Pleurotus fl abellatus 60e70 decolorization within 10 d Nilsson et al 2006 or a crude effl uent from a dye manufacture by Pleuro tus sanguineus 70 decolorization within 14 d Vanhulle et al 2008 but lower than the decolorization rate of a pigment plant effl uentbyPycnoporuscinnabarinus 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 effl uents when used under the conditions of RBC reactor 0 1 2 3 1 2 3 1 2 3 1 2 3 Day 0Day 20Day 70 Log EC50 or IC50 Fig 3 e Toxicity of Methylene Blue expressed as Log EC50 or IC50 during biodegradation in RBC reactor measured with Vibrio fi scheri 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 4 3 2 1 0 1 2 3 1 2 3 1 2 3 1 2 3 Day 0Day 10Day 26 Log EC50 or IC50 1 0 1 2 3 1 2 3 1 2 3 1 2 3 Day 0Day 40Day 47 Log EC50 or IC50 b a Fig 4 e Toxicity of Wastewater I a and Wastewater II b during biodegradation in RBC reactor measured with Vibrio fi scheri 1 Sinapis alba 2 and Lemna minor 3 The samples withdrawn on Day 40 and Day 47 Wastew