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Biosorption of simulated dyed effluents by inactivated fungal biomasses

Biosorption of simulated dyed effluents by inactivated fungal biomasses
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  Biosorption of simulated dyed effluents by inactivated fungal biomasses Valeria Prigione, Giovanna Cristina Varese  * , Leonardo Casieri, Valeria Filipello Marchisio Universita`  degli Studi di Torino, Dipartimento di Biologia Vegetale, Viale Mattioli 25, 10125 Torino, Italy Received 15 March 2007; received in revised form 26 June 2007; accepted 27 July 2007Available online 20 September 2007 Abstract Treatment of dyed effluents presents several problems mainly due to the toxicity and recalcitrance of dyestuffs. Innovative technol-ogies, such as biosorption, are needed as alternatives to conventional methods to find inexpensive ways of removing dyes from large vol-umes of effluents. Inactivated biomasses do not require a continuous supply of nutrients and are not sensitive to the toxicity of dyes ortoxic wastes. They can also be regenerated and reused in many cycles and are both safe and environment-friendly. The sorption capacities(SC) of autoclaved biomasses of three Mucorales fungi ( Cunninghamella elegans ,  Rhizomucor pusillus  and  Rhizopus stolonifer ), culturedon two different media, were evaluated against simulated effluents containing concentrations of 1000 and 5000 ppm of a single dye and amix of 10 industrial textile dyes in batch experiments. SC values of up to 532.8 mg of dye g  1 dry weight of biomass were coupled withhigh effluent decolourisation percentages (up to 100%). These biomasses may thus prove to be extremely powerful candidates for dyebiosorption from industrial wastewaters. Even better results were obtained when a column system with the immobilised and inactivatedbiomass of one fungus was employed.   2007 Elsevier Ltd. All rights reserved. Keywords:  Biosorption;  Cunninghamella elegans ;  Rhizomucor pusillus ;  Rhizopus stolonifer ; Synthetic dyes 1. Introduction Synthetic dyes are used extensively in the textile, paperand printing industries and in dyehouses. Dye-containingeffluents are one of the most difficult-to-treat wastewaterson account of their high chemical and biological demands,suspended solids and content in toxic compounds and theaesthetic issues raised by their easily recognized colours(Wesemberg et al., 2003). Dyes released into the environ-ment are toxic for several organisms and a threat to ecosys-tems, mainly because they block out the sunlight and thusreduce photosynthesis and dissolved oxygen concentration(Banat et al., 1996; Aksu, 2005). Prior to their release,therefore, wastewaters are usually treated to bring theirdye concentrations down to nationally permitted levels.Some physical and chemical treatments are effective, buttheir limitations render them unattractive. These includeexcessive usage of chemicals, accumulation of concentratedsludge with obvious disposal problems, expensive plantrequirements, high operational costs and sensitivity to vari-ations in the wastewater input (Aksu, 2005). To cut downthe employment of harmful chemicals, use is often madeof biological methods in which active sludges exploit thedegradative capabilities of bacteria and protozoa, but can-not result in efficient decolourisation.Textile industry wastewaters are the current centre of attention. Their decolourisation is the subject of discussionand regulation in many countries aware that water is avaluable asset which must be protected. The complex aro-matic molecular structures of most dyes make them morestable and hence more difficult to biodegrade. Even if dyesare not the principal component of the wastes discharged inthe processing of textiles, they are not readily removed ordegraded in treatment plants (Easton, 1995; Waters,1995) and most effluents are still coloured when they leavea plant (Loyd et al., 1992). 0960-8524/$ - see front matter    2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2007.07.053 * Corresponding author. Tel.: +39 011 6705964; fax: +39 011 6705962. E-mail address: (G.C. Varese).  Available online at Bioresource Technology 99 (2008) 3559–3567  Many studies have focused on microorganisms that bio-degrade dyes and bioremediation is becoming an environ-ment-friendly and cost-competitive alternative for dyewastewater treatment. A wide variety of microorganisms,especially fungi, degrade different classes of dyes. Theapplication of biodegradation to industrial effluents, how-ever, is limited due to the long time required and the needfor microorganisms to function in strictly controlledenvironments.Alternatives to conventional methods must thus beexplored to find inexpensive ways of removing dyes fromlarge volumes of effluents (Aksu, 2005). Biosorption isviewed as one such alternative. Biosorption of a broadrange of pollutants by sea weeds, bacteria, yeasts andhyphal fungi has been investigated and fungi seem the mostpromising (Fu and Viraraghavan, 2001; Knapp et al.,2001). According to Van Driessel and Christov (2002)and Aksu (2005), biosorption encompasses a number of metabolism-independent processes (physical and chemicaladsorption, electrostatic interaction, ion exchange, com-plexation, chelation and micro precipitation) that mainlytake place in the cell wall. The main attractions of biosorp-tion are its high selectivity and efficiency, cost effectivenessand good removal from large volumes (Aksu, 2005).Both living and dead biomass can be used to removehazardous organics. Dead cells are obviously preferablefor wastewater treatment since they are not affected bytoxic wastes and chemicals and do not pollute the environ-ment by releasing toxins and/or propagules (Aksu, 2005).Dead and dried biomass can be stored for long periods atroom temperature with little risk of putrefaction. Thismakes it easier to use and transport. Dead biomass is alsogenerated as a waste product from established industrialprocesses (Kapoor and Viraraghavan, 1995).This paper illustrates the sorption capacities (SC) of thedead biomasses of three Mucorales fungi as displayed inbatch experiments with simulated effluents containing1000 and 5000 ppm of a single dye and a mix of 10 direct,reactive and acid dyes widely used in the textile industry,namely anthraquinonic, azoic and phthalocyanin chro-mophores. Biomasses were cultured on one medium withglucose as the carbon source and another with cheaperstarch, since the production of biomass for industrial-scalebioremediation is expensive and the use of media withalternative carbon sources is a desirable objective. Indus-trial application was mimicked by immobilising the bio-mass of one fungus ( Cunninghamella elegans ) in calciumalginate to run an experiment on biosorption in a columnsystem. 2. Methods  2.1. Test organismsC. elegans  Lendner (MUT 2861),  Rhizomucor pusillus (Lindt) Schipper (MUT 2229) and  Rhizopus stolonifer (Ehrenberg) Vuillemin (MUT 1515) were obtained fromthe  Mycotheca Universitatis Taurinensis  Collection(MUT, University of Turin, Department of Plant Biology).Starting cultures were lyophilised and cryopreserved untiluse. They were revitalized on Malt Extract Agar andmature conidia for the inocula and biomass productionwere obtained from cultures grown on the same mediumin the dark at 24   C for 1 week.  2.2. Dyes and preparation of simulated effluents Nine out of the 10 dyes used in this study were kindlyprovided by Clariant Italia S.p.a., the model dye RBBRwas purchased from Sigma–Aldrich (St. Louis, MO). Theirstructures (where available), chemical groups and maxi-mum absorbance peak wavelengths are listed in Table 1.Stock solutions 20000 ppm concentrated prepared bydissolving each dye powder in distilled water were sterilisedby 0.2  l m pore filters and stored at 4   C until use. Sincereactive dyes are released into effluents after hydrolysationof reactive groups occurring in dyeing bath conditions,industrial dyes B41, B49, B214 and R243 were hydrolysedby heating at 80   C for 2 h in a 0.1 M Na 2 CO 3  solution,and then neutralized.Stock solutions of the direct azo dye R80, the reactiveazo dye B214 and the anthraquinonic reactive model dyeRBBR were added to a 9 g l  1 NaCl saline solution toobtain final concentrations of 1000 and 5000 ppm. Com-plex effluent was prepared by adding all 10 dyes to one sal-ine solution to obtain a final concentration of 5000 ppm.R80 and B214 were chosen to produce simulated efflu-ents because they are generally unaffected by conventionalwastewater treatment procedures (personal communica-tion), and are direct and reactive dyes widely employed inthe textile industry, RBBR was chosen because it is anon-industrial dye that serves as a model of the anthraqui-nonic class. The complex effluent was employed sinceindustrial effluents usually contain a mixture of dyes deriv-ing from the baths of different machines. Moreover all thedyes were dissolved in saline solution since textile effluentsare usually characterised by high salt concentrations.  2.3. Fungal biomass preparation and batch biosorptionexperiments The two media used for biomass production were EQ(20 g l  1 glucose, 2 g l  1 ammonium tartrate, 2 g l  1 KH 2 PO 4 , 0.5 g l  1 MgSO 4  Æ  7H 2 O, 0.1 g l  1 CaCl 2  Æ  2H 2 O,10 ml mineral stock) and ST, in which 18 g l  1 potatostarch is used instead of glucose (Fluka, St. Louis, MO).Since the literature (Ellis et al., 1974) and preliminarytests had shown that  R. stolonifer  cannot use starch as acarbon source, it was cultured on EQ only.Each isolate was inoculated as a conidial suspension(final concentration of 1  ·  10 5 conidia ml  1 ) in several500 ml Erlenmeyer flasks containing 300 ml of medium,and incubated at 30   C for 7 days. To avoid produc-tion of aerial mycelium, rich in hydrophobins and poorly 3560  V. Prigione et al. / Bioresource Technology 99 (2008) 3559–3567   Table 1Dyes used in the study, their commercial and C.I. name, chromophore, chemical group, wavelength of maximum visible absorbance ( k max ) and chemical structure (where available)CommonnameCommercial name C.I. name Chromophore Chemicalgroup k max  (nm) Chemical structureB113 Nylosan bleu marineN-RBL P 187Acid blue113Azoic Acid 541 NN N NNaSO 3 NaSO 3 HN B214 Drimaren bleu marineX-GN CDGReactiveblue 214Azoic Reactive 607B225 Nylosan bleu F-2RFLP 160Acid blue225Anthraquinonic Acid 590–626B41 Drimaren turquoiseX-B CDGReactiveblue 41Phtalocyanin Reactive 616–666B49 Drimaren bleu P-3RLNGRReactiveblue 49Anthraquinonic Reactive 586–625 NaSO 3 NNN ClNaSO 3 OONaSO 3 NNHNHHHN B81 Solar bleu G P 280 Direct blue81Azoic Direct 577  OHNHNNaSO 3 N NN NNaSO 3 OH NaSO 3 NaSO 3 N R111 Scarlet nylosanF-3GL 130Acid red111Azoic Acid 499 NaSO 3  NaSO 3 OHN NH 3 C OOOS N NCH 3  CH 3 R243 Drimaren redX-6BN CDGReactivered 243Azoic Reactive 517R80 Solar red BA P 150 Direct red80Azoic Direct 540 NaSO 3 NN NNaSO 3 NNaSO 3 OHNCHNaSO 3  N NNNaSO 3 NNaSO 3 OHNHO RBBR Remazol brilliantblue RReactiveblue 19Anthraquinonic Reactive 593  OONHSOHOONH 2 SO 3 HN SO 2 CH 2 CH 2 OSO 3--  V .P r  i     g i    o n e  e  t   a l    . /   B  i    or  e  s  o ur  c  e T  e  c  h   n o l    o  g  y 9   9    (  2   0   0   8    )   3   5   5   9  – 3   5   6   7    3    5    6   1     adsorbent, biomasses were cultured in agitated condition at150 rpm with a Minitron Infors orbital shaker (Bottmin-gen, CH). After incubation biomasses were collected witha sieve (150  l m pore), rinsed several times with distilledsterilised water to remove residual medium and then inac-tivated in a 9 g l  1 NaCl solution by autoclaving at 121   Cfor 30 min. They were then collected in sterile conditionsand rinsed as already described.Each biomass was weighed and 3 g fresh weight wasplaced in 50 ml Erlenmeyer flasks containing 30 ml of sim-ulated effluent. The flasks were incubated at 30   C in agi-tated conditions (150 rpm). Each trial was performed intriplicate.After 24 h of incubation, the biomass of each samplewas filtered on paper filters (Wattman type 1), dried at65   C until its weight reached the equilibrium, and thesorption capacity (SC), a parameter that takes into consid-eration the maximum sorbent capability, was calculated asfollows:SC  ¼  mg of dye removed = g of biomass  ð dw Þ The milligrams of dye removed were calculated subtract-ing the residual amount of dye at the end of the experiment(obtained from the decolourisation percentage, describedbelow) from the initial amount of dye.After 2, 6 and 24 h, 300  l l of effluent was taken fromeach sample, centrifuged at 14000 rpm for 5 min, to removedisturbing mycelial fragments, and examined with a spec-trophotometer (Amersham Bioscences Ultrospec 3300Pro, Fairfield, CT) to acquire the complete absorbancespectra of the effluents and calculate the percentage of removed dye (DP, decolourisation percentage) as follows:DP  ¼  100   ð Abs 0    Abs t  Þ = Abs 0 where Abs 0  is the absorbance at time 0 and Abs t  is theabsorbance at time  t , at the maximum visible wavelength( k max ) of each dye (Table 1). The absorbance of the com-plex effluent was measured at 588 nm, namely the wave-length that corresponds to the maximum absorbance invisible light.Simulated effluents without biomass were used as theabiotic control and to assess decolourisation other thanthat due to biosorption (e.g., photobleaching or complexa-tion). The influence of the incubation time on the biosorp-tion yield was assessed from the increase in decolourisationfrom the 2nd to the 24th h, calculated as (DP at T24    DPat T2)  Æ  100/DP at T24.The significance of differences ( P  6 0.05) between theSC values and between the DP values at 2, 6 and 24 hwas calculated with the Mann–Whitney test (SYSTAT 10for windows SPSS Inc., 2000).  2.4. Biomass immobilisation and biosorption experiment in acolumn system This experiment was conducted with  C. elegans  only.A 2% solution of alginic acid sodium salt (from brownalgae – Fluka, St. Louis, MO), inoculated with a conidialsuspension (final concentration 2.5  ·  10 4 conidia ml  1 )and continuously shaken with a magnetic agitator wasdripped with a peristaltic pump (model SP311 VELP Scien-tifica, Milan) into a continuously shaken 0.25 M calciumchloride solution. The calcium alginate beads (2–3 mmdiameter) thus produced were completely hardened byshaking for about 1 h in calcium chloride. They were thenrinsed with a saline solution to eliminate the free propa-gules and excess calcium chloride.Thirty grams of beads in 250 ml of ST medium placed in500 ml Erlenmayer flasks, were incubated for 7 days at30   C in agitated conditions (130 rpm) with a MinitronInfors orbital shaker (Bottmingen, CH), collected with ametal sieve (150  l m pore), rinsed with saline solution andinactivated by autoclaving at 121   C for 30 min.About 300 g of the beads thus obtained, correspondingto 50 g of biomass (wet weight) were packed in a 5 cmdiameter, 54 cm tall glass column connected to a flask con-taining 500 ml complex effluent kept circulating at a con-stant 20 ml min  1 by a peristaltic pump (model SP311VELP Scientifica, Milan). One ml of effluent was with-drawn from the system after 30 min, 1–6 and 24 h and itsDP was calculated as in the batch experiments. 3. Results 3.1. Batch biosorption experiments The SC values obtained by biomasses of the three spe-cies at the end of the experiment ranged from 167.9 to532 mg of dye g  1 of biomass dry weight (Table 2). TheSCs of   R. pusillus  and  R. stolonifer  cultivated on EQ incontact with B214 and  R. stolonifer  cultivated on EQ incontact with the complex effluent were more than 500 mgof dye g  1 of biomass dry weight.Table 2 shows that the species displayed different SCvalues in the same conditions, that the SC of a biomasswas not the same on the two media, and that the sorptionefficiency of a biomass cultured on the same medium variedfrom one dye to another. The complex effluent wasadsorbed more efficiently than the single-dye effluents: of these, B214 was removed most efficiently.The DP values obtained for the 1000 and 5000 ppmeffluents are set out in Tables 3 and 4. Substantial decolo-urisation of the 1000 ppm effluents was nearly alwaysachieved after 24 h. R80 was completely removed by allthree species and DPs from 98.2% to 100% were observedfor B214. RBBR was the most difficult to remove, exceptby  R. stolonifer ; the lowest yields were obtained with  C. ele- gans : 57.8% when cultured on EQ and 63.2% when culturedon ST. The DP values were much lower (from 35.3% to81%) for the 5000 ppm effluents, though those for the com-plex effluent treated with the  C. elegans  and  R. pusillus  bio-masses (88.6% to 92.1%) were close to the maxima for the1000 ppm effluents. 3562  V. Prigione et al. / Bioresource Technology 99 (2008) 3559–3567   More than 50% of the total decolourisation was usuallyachieved after 2 h (increase from 2 h to 24 h <50%; Tables3 and 4). Examples of absorbance spectra of 5000 ppmeffluents at the beginning of the experiment and after 2 h,6 h and 24 h are shown in Fig. 1. No differences in theshape of those of R80, B214 and RBBR effluents wereobserved, whereas the spectrum of the complex effluent dis-played some differences between 540 nm and 580 nm due todissimilar dye removal (Fig. 1). 3.2. Biosorption experiment in the column After 30 min, 3500 ppm (70%) of the complex effluenthad been removed. Complete decolorisation was reachedafter 6 h (Fig. 2). 4. Discussion These results show that  C. elegans ,  R. pusillus  and  R.stolonifer  biomasses are endowed with a high ability toabsorb industrial dyes R80 and B214 (SC from 221.4 to519.2 mg of dye g  1 dry weight). The added value of thisresult stems from the widespread employment of these dyesand the difficulties posed by their physical and chemicalremoval.The literature data (Table 5) make it clear that deter-mined SC values are well above those obtained by otherworkers using both live or inactivated biomasses and otherdyes (Fu and Viraraghavan, 2000, 2002a; O’Mahony et al.,2002; Zhang et al., 2003; Aksu, 2005), whereas they arecomparable with the theoretical maxima postulated for Rhizopus oryzae  (Aksu and Tezer, 2000; Aksu and Caga-tay, 2006). It must also be borne in mind that 1000 and5000 ppm concentrations are much higher than the800 ppm maximum used by the researchers quoted in Table5. They are also close to those of real industrial effluents,which were more congruently simulated in our study bythe inclusion of a complex effluent composed of 10 dyesand the use of concentrated saline solutions so as to intro-duce a real parameter that bars the attainment of good bio-sorption yields (Aksu, 2005). The high fungus SC valueswere accompanied by DPs that often resulted in changes Table 3Decolourisation percentage of R80, B214 and RBBR 1000 ppm effluents after 2 h (T2), 6 h (T6) and 24 h (T24) incubation by the biomasses of the speciescultured on EQ ( Cunninghamella elegans ,  Rhizomucor pusillus  and  Rhizopus stolonifer ) and ST medium ( Cunninghamella elegans  and  Rhizomucor pusillus )and increase of decolourisation from T2 to T24Species Dye Medium Decolourisation percentage (Mean ± SD) Increase of decolourisation from T2 to T24 (%)T2 T6 T24 Cunninghamella elegans  R80 EQ 83.2 ± 4.9 a 99.6 ± 0.3 b 100.0 ± 0.0 b 16.8ST 73.6 ± 1.9 a 98.2 ± 0.4 b 100.0 ± 0.0 c 27.0B214 EQ 88.2 ± 1.3 a 95.3 ± 0.2 b 99.0 ± 0.1 c 12.2ST 91.7 ± 1.4 a 95.5 ± 0.4 b 98.8 ± 0.3 c 7.7RBBR EQ 22.7 ± 3.6 a 38.0 ± 2.2 b 57.8 ± 0.9 c 60.7ST 28.8 ± 2.7 a 46.1 ± 4.1 b 63.2 ± 3.6 c 54.4 Rhizomucor pusillus  R80 EQ 99.5 ± 0.2 a 99.9 ± 0.0 b 100.0 ± 0.0 c 0.5ST 72.0 ± 4.0 a 99.7 ± 0.2 b 99.7 ± 0.1 b 27.8B214 EQ 98.4 ± 0.1 a 98.6 ± 0.1 a 100.0 ± 0.0 b 1.6ST 98.3 ± 0.3 a 99.1 ± 0.1 b,c 98.7 ± 0.3 a,c  – RBBR EQ 60.2 ± 8.6 a 84.6 ± 6.2 b 97.9 ± 1.4 c 38.5ST 48.6 ± 14.5 a 67.1 ± 12.8 ab 84.2 ± 4.6 b 42.3 Rhizopus stolonifer  R80 EQ 87.2 ± 5.5 a 99.5 ± 0.2 b 100.0 ± 0.0 c 12.8B214 EQ 97.5 ± 0.1  a 97.9 ± 0.2 a 98.2 ± 0.1 b 0.7RBBR EQ 97.8 ± 0.5 a 98.5 ± 0.3 ab 98.8 ± 0.3 b 1.0 a–c Significant differences between decolourisation percentages at T2, T6 and T24.Table 2Sorption capacity (mg of dye g  1 of biomass dry weight) of 5000 ppm R80, B214, RBBR and complex effluents by inactivated biomasses cultured on EQ( Cunninghamella elegans ,  Rhizomucor pusillus  and  Rhizopus stolonifer ) and ST medium ( Cunninghamella elegans  and  Rhizomucor pusillus )Dyes  Cunninghamella elegans Rhizomucor pusillus Rhizopus stolonifer EQ ST EQ ST EQR80 278.7 ± 8.7 aA 432.5 ±18.5 *aA 365.4 ± 20.0 bA 386.8 ± 59.8 aA 221.4 ± 24.4 cA B214 327.6 ± 7.7 aB 427.8 ± 25.2 *aA 532.8 ± 14.5 bB 403.9 ± 37.8 *bA 506.7 ± 33.7 bB RBBR 176.1 ± 27.4 aC 273.3 ± 16.6 *aB 167.9 ± 35.8 aC 300.9 ± 21.6 *aB 232.0 ± 23.1 bA Complex effluent 393.1 ± 7.2 aD 498.8 ± 2.8 *aC 410.5 ± 35.4 aA 453.8 ± 12.4 *bA 519.2 ± 37.1 bB ( * ) Significant differences between the SC of a species cultured on different media.  a,b, . . . Significant differences between the SC for the same effluent of different species cultured on the same medium.  A,B, . . . Significant differences between the SC for different effluents of the same species cultured on the samemedium. V. Prigione et al. / Bioresource Technology 99 (2008) 3559–3567   3563
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