Process Biochemistry 39 (2004) 1401–1405

Effect of environment factors on dye decolorizationby P. sordida ATCC90872 in a aerated reactor

Yang Ge∗, Liu Yan, Kong Qinge

College of Life Science, Qufu Normal University, Qufu, Shandong 273165, PR China

Received 3 January 2003; received in revised form 20 May 2003; accepted 29 June 2003

Abstract

Biological decolorization of textile dyestuff Basic Blue 22 (C.I. 61512), a phthalocyanine type reactive dyestuff, by the white-rot fungusPhanerochaete sordida ATCC90872 was studied in a rotating biological contactor (RBC). The effects of different operating parametersincluding disc type, rotational speed, glucose and dyestuff concentration on the decolorization performance of white-rot fungi were investigated.The system was operated in repeated-batch mode with 48 h hydraulic retention time. Three different disc types; plastic, metal mesh coveredplastic discs and metal mesh discs were used and the plastic disc was found to be most suitable. The highest decolorization efficiencywas obtained with a rotational speed of 40 rpm. Minimum glucose concentration for 78% decolorization efficiency was 5 g/l. TOC removalefficiency was around 80% for 50–200 mg/l initial dyestuff concentrations and decreased to 52% for 400 mg dyestuff/l.© 2003 Elsevier Ltd. All rights reserved.

Keywords: Decolorization; Dyestuff; Rotating biological contactor;Phanerochaete sordida

1. Introduction

Wood-rotting fungi, characterized by their ability to de-grade lignin and cellulose, are the predominant agents ofwood degradation. Their enzyme capacities provide the po-tential to colonize wood at all stages of decomposition.Some wood-rotting fungi, especially white-rot fungi, appearto have some potential for bioremediation applications dueto their non-specific systems which have been developedfor depolymerization and mineralization of the complex andrecalcitrant polymer of lignin[1–3]. As the lignin degrada-tion systems of these fungi is not substrate specific, they areable to transform and sometimes completely mineralize, avariety of persistent environmental pollutants[4,5]. Amongsuch compounds are many synthetic dyes characterized bytheir high stability in light and during washing, which givesthem recalcitrance to biodegradation[6,7].

The dyestuff, textile, paper, and leather industries, the ma-jor producers and users of dyes, produce effluents that areusually resistant to biological treatment. Between 10 and15% of the total dye consumed in dyeing processes may be

∗ Corresponding author.E-mail address: gyang@home.ipe.ac.cn (Y. Ge).

found in wastewater. Most of these compounds are highlyresistant to microbial attack. Therefore, it is hard to removethem from effluents by means of conventional biologicalwastewater treatments, for example activated sludge treat-ment.

Color can be removed from wastewater by chemicaland physical methods including adsorption, coagulation-flocculation, oxidation and electrochemical methods. Thesemethods are quite expensive and have operational problems.White-rot fungi can degrade a wide variety of recalcitrantcompounds including xenobiotics, lignin and dyestuffs bytheir extracellular lignolytic enzyme system.Phanerochaetesordida, Phanerochaete chrysosporium andCoriolus versi-color are the major white-rot fungi strains mainly used fordelignification and biological decolorization[8,9]. Thereare several advantages of using white-rot fungi for decom-position of recalcitrant compounds. Lignolytic enzymesare substrate non-specific; therefore, they can degrade awide variety of recalcitrant compounds and even complexmixtures of pollutants. Since the enzymes are extracellular,substrate diffusion limitation into the cell, generally en-countered in bacteria, is not observed. White-rot fungi donot require preconditioning to particular pollutants, becauseenzyme secretion depends on nutrient limitation, either ni-trogen or carbon, rather than the presence of pollutants. In

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0032-9592(03)00273-5

1402 Y. Ge et al. / Process Biochemistry 39 (2004) 1401–1405

addition, the extracellular enzyme system enables white-rotfungi, to tolerate high pollutant concentrations. However,the complexity of the biodegradation mechanism of lig-nolytic systems. The requirement for some chemicals thatare unlikely to be present in a wastewater, such as veratrylalcohol and Tween 80, the low pH requirement (4.5) for theoptimum activity of the enzymes are the disadvantages offungal bioremediation.

In the light of the aforementioned studies, this study wasdesigned to investigate the effects of several important oper-ating parameters such as, disc type, rotational speed, glucoseand dyestuff concentration on the biological decolorizationof Basic Blue 22 (C.I. 61512) byP. sordida ATCC90872 ina rotating biological contactor (RBC).

2. Materials and methods

2.1. Culture and medium

The white-rot fungus, used for the decolorization stud-ies wasP. sordida ATCC90872 obtained from the AmericaType Culture Collection (ATCC, Rockville, MD). The stockculture was grown on Potato Dextrose Agar at its optimumgrowth temperature (28◦C). The culture was maintained at4◦C and refreshed in every 30–40 days.

The culture used for inoculation of RBC was cultivatedon a shaker in 250 ml Erlenmeyer flasks. Flasks containingmalt extract broth were aseptically inoculated, using 8 mmplugs cut from actively growing culture on an agar plate andincubated at 120 rpm for 4 days at 28◦C. Fungi cultivatedin shake flasks were homogenized for 5 min and 200 mlof homogenized culture was used for inoculation of thereactor.

Synthetic medium was made up of 8 g/l glucose, 0.03 g/lurea, 1.5 g/l KH2PO4, 0.09 g/l CaCl2, 0.6 g/l MgSO4,0.0008 g/l thiamine and 1 ml/l trace elements. The mediumwas carbon-sufficient, but nitrogen deficient. Stock traceelements solution was prepared by dissolving 0.06 gCuSO4·7H2O, 0.04 g Fe2(SO4)3 in 1 l of distilled water[10]. Dyestuff concentration was 200 mg/l which corre-sponds to a loading rate of 0.16 g dyestuff/m3 per day inmost of the experiments, except the experiments with vary-ing dyestuff concentrations. The textile dyestuff used wasa phythalocyanine type reactive dye Basic Blue 22 (C.I.61512) (Fig. 1).

Fig. 1. Chemical structure of the Basic Blue 22.

2.2. Aerated reactor

The biodisc contactor consisted of 15 plastic discs witha diameter of 15 cm each resulting in a total disc surfacearea ofA = 0.362 m2. Small holes (0.5 cm diameter) wereopened on the discs to enhance the immobilization of fungi.The liquid volume in the reactor wasVL = 1.6 l resulting ina surface to liquid ratio ofA/V 200 m2/m3. Rotation speedwas 30 rpm in most of the experiments. The temperature waskept at 28◦C using a heating jacket and pH was adjusted to4.5–5 by adding 3% H2SO4 and 4% NaOH, manually. Thefungi was immobilized on discs, using 8 g/l glucose contain-ing defined medium devoid of dyestuff for biofilm formationon the discs within 3 days. The liquid phase was removedand replaced with fresh medium containing nutrients and thedyestuff after biofilm formation. The reactor was operatedin repeated-batch mode. The reactor content was removedevery 2 days and reloaded with colored fresh media withoutremoving the immobilized fungi from the system. The hy-draulic retention time was 48 h and media was changed sixtimes resulting in 12 days operation period.

2.3. Analytical methods

Samples were withdrawn from the reactor each day andcentrifuged at 12,000 rpm for 5 min to separate mycelia frommedia. TOC, glucose and color measurements were carriedout on the clear supernatant.

A scanning spectrophotometer (Shimadzu UV-2101PC)was used for absorbance measurements. The maximumabsorption wavelength for Basic Blue 22 was 650 nm.Samples were diluted by a factor of 1/4 with distilledwater prior to measurements. Measured absorbance val-ues were used for calculations of decolorization efficien-cies.

Glucose concentration was measured by colorimetricmethods using a Sigma Diagnostic Glucose Kit.

TOC analyses were carried out by using a TOC5000A/5000 Model Shimadzu Total Carbon Analyzer withan ASI5000A/5000 Shimadzu Autosampler.

Triplicate sampling was performed.

3. Results and discussion

3.1. Effect of disc type

Three different types of discs; metal mesh covered plasticdiscs, plastic discs and metal mesh discs were used in or-der to determine the most appropriate disc type that wouldprovide better immobilization of fungi and decolorization.The reactor was operated batch-wise for 24 h with an initialdyestuff concentration of 200 mg/l.

Fig. 2shows the variation of decolorization efficiency withtime for different disc types. When plastic discs were used,absorbance decreased fromAo = 1.455 to Ae = 0.0028

Y. Ge et al. / Process Biochemistry 39 (2004) 1401–1405 1403

0

20

40

60

80

100

120

0 5 10 15 20 25 30

Time(hours)

Dec

olor

izat

ion

Effi

cien

cy(%

)

Fig. 2. Effect of disc type on decolorization of Basic Blue 22 RBC. (�)Plastic discs, (�) metal mesh covered plastic discs, (�) metal mesh discs.

within 24 h, a decolorization efficiency of 98%. The plasticdiscs were covered with metal meshes to provide a roughsurface for better fungal immobilization. A thick biofilm for-mation was observed and a 98% decolorization efficiencywas obtained within 24 h. When metal mesh discs were usedin the reactor, the biofilm was very thin and absorbance de-creased fromAo = 1.443 toAe = 0.814 with only 43% de-colorization efficiency in 24 h. Plastic and metal mesh cov-ered plastic discs resulted in almost the same decolorizationefficiency and biofilm formation. For the sake of simplicity,plastic discs without metal mesh covers were used for theother parts of this study.

3.2. Performance of repeated-batch operation

Fig. 3 shows the variation of decolorization and glucoseremoval efficiencies with time in repeated-batch operationof the RBC. The decolorization efficiency decreased withrepeated media loading. For the first two media loadings,the decolorization efficiency was 72 and 80%, respectively.However, 50% efficiency was obtained for the next fourloadings. A similar situation was observed for glucose re-moval efficiency. The efficiency was 80% at the beginningof the operation and then decreased to 55%, after the thirdmedia change. Diffusion limitations with high biofilm thick-nesses may be one possible explanation for this result. Thebiofilm thickness increased as the system was repeatedlyloaded with glucose containing media resulting in diffusionlimitations for transport of carbon sources and lignolytic en-zymes causing low decolorization efficiencies. Another pos-sible explanation may be the strong relationship betweennutrient depletion and decolorization in fungal bioremedia-tion. Decolorization takes place during the stationary growthphase, when nutrients are consumed to very low concentra-tions. Since the glucose concentration was high because ofinefficient glucose utilization by the fungi during the lastloads, enzyme secretion and therefore, decolorization effi-ciency dropped. This problem may be eliminated by control-ling the biofilm thickness and carbon source concentration.

0

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Time,(days)

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4050

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7080

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Glu

cose

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Effi

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Fig. 3. Dyestuff and glucose removal performance of the RBC operatedin repeated-batch mode.

3.3. Effect of glucose concentration

Glucose concentrations used in decolorization studieswith fungi are generally around 15–20 g/l[11]. It is un-likely to find such a high glucose concentration in a textilewastewater and also, it is not feasible to add large concen-trations of glucose into the wastewater. In order to minimizethe glucose concentration, decolorization performance ofP. sordida ATCC90872 at 5 and 2 g/l glucose concentra-tions were investigated. Four different runs were arrangedto evaluate the effects of growth and decolorization phaseglucose concentrations. TOC and color removal efficienciesare presented inTable 1.

In the first experiment, fungi was grown at 5 g/l glucoseconcentration and then the glucose concentration was then

Table 1Effect of initial glucose concentration on the dyestuff and TOC removalefficiencies

Runnumber

Glucose concentration (mg/l) ECOLOUR

(%)ETOC

(%)

Growth phase Decolorization phase

1 5 2 54 612 5 5 55 593 8 5 78 744 8 2 66 61

1404 Y. Ge et al. / Process Biochemistry 39 (2004) 1401–1405

decreased to 2 g/l in the decolorization phase in order tokeep the fungi in stationary growth phase. Decolorizationand TOC removal efficiencies were 54 and 61%, respec-tively. The glucose concentration in both growth and decol-orization phase was 5 g/l in the second experiment whichresulted in a decolorization efficiency of 55% and a TOCremoval efficiency of 59%. Decreasing glucose concentra-tion from 5 to 2 g/l, in the decolorization phase did nothave significant effect on decolorization and TOC removalefficiencies.

In order to investigate the effect of growth phase glucoseconcentration on decolorization efficiency, fungi was grownat 8 g/l glucose concentration in dyestuff-free media. Theglucose concentration was then decreased to 5 g/l, in the de-colorization phase The efficiencies obtained for color andTOC removal were 77 and 73%, respectively. When the glu-cose concentration was decreased to 2 g/l in the decoloriza-tion phase, the efficiencies were 66% for decolorization and61% for TOC removal, indicating that 2 g/l glucose con-centration was not sufficient to sustain the activity of fungiduring decolorization.

3.4. Effect of rotation speed on decolorization

Agitation was reported to have an adverse effect on thestability of the lignolytic enzymes. A high mixing rate de-creases fungal growth and lignolytic enzyme activity[12].In the present study, mixing and aeration were provided bythe rotation of the discs. Therefore, rotational speed wasconsidered as an important parameter affecting the systemperformance.

Fig. 4depicts the effect of rotational speed (rpm) on decol-orization and TOC removal efficiencies. The decolorizationefficiency increased with increasing rotational speed. At lowrotational speeds such as 10 rpm (revolutions per minute)and 20 rpm, color removal efficiencies were around 34 and

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66

68

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decolorizationTOC

Fig. 4. Effect of rotational speed on the decolorization and TOC removalefficiency.

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0 100 200 300 400 500

Dyestuff Concentration (mg/L)

TO

C R

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ffici

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Fig. 5. Effect of dyestuff concentration on the TOC removal efficiency.

36%, respectively. TOC removal efficiencies were 67% for10 rpm and 71% for 20 rpm. These results indicated that aer-ation was not sufficient for decolorization and TOC removal,at low rotational speeds such as 10 and 20 rpm. At higherrotational speeds, such as 40 and 50 rpm, decolorization andTOC removal efficiencies were approximately 77 and 78%,respectively. Higher rotational speeds provided better aera-tion, resulting in increased decolorization efficiency. Sincethere was no significant increase in color and TOC removalefficiencies at higher rotational speeds, the optimum rota-tional speed was determined as 40 rpm.

3.5. Effect of dyestuff concentration

Extracellular, substrate non-specific enzyme system helpsfungi to tolerate high concentrations of toxic compounds.Considering this characteristic of fungi, the maximaldyestuff concentration that can be tolerated byP. sordidaATCC90872 was determined. The RBC was operated at 50,100, 200 and 400 mg/l initial dyestuff concentrations. Therotational speed and glucose concentrations were 30 rpmand 5 g/l, respectively.

The variation of TOC removal efficiencies with initialdyestuff concentration is depicted inFig. 5. TOC re-moval efficiencies were almost the same, around 80% for50–200 mg/l initial dyestuff concentrations. However, with400 mg/l initial dyestuff concentration, the TOC removalefficiency dropped to 52%. Therefore, for efficient decol-orization of Basic Blue 22, byP. sordida, the dyestuffconcentration should be lower than 200 mg/l.

4. Conclusions

Decolorization efficiency of a textile dyestuff byP. sor-dida, a white-rot fungi strain in a rotating biodisc contac-tor varies depending on biofilm thickness, rotational speedand carbon source concentration. Diffusion of glucose intothe biofilm and also release of enzymes would be limitedas biofilm thickness increased. Control of biofilm thick-

Y. Ge et al. / Process Biochemistry 39 (2004) 1401–1405 1405

ness may be essential for effective decolorization. Glucoseconcentration had a strong effect on decolorization of thedyestuff the by fungi. Cultivation of the fungi at high glu-cose concentration (8 g/l) resulted in better fungal growthand higher decolorization efficiency when compared to thatobtained with a low glucose concentration (5 g/l). It was ob-served that, 5 g/l glucose concentration in the decolorizationphase was sufficient to obtain 78% decolorization efficiency.Lower glucose concentrations, either at growth or decol-orization phase, caused insufficient growth or loss of fungalactivity, leading to a decrease in color removal efficiency.

The rotational speed was found to be another importantoperational parameter. The system performance increasedwith increasing rotational speed. Decolorization efficiencywas 77% at 40–50 rpm, while it was only 35% at 10–20 rpm.Higher rotational speeds provided better aeration resultingin increased decolorization efficiency. Similar effects wereobserved in TOC removal efficiency, which increased from72 to 80% with an increase in rotational speed from 20 to50 rpm.

The maximum tolerable dyestuff concentration byP. sor-dida ATCC90872 was 200 mg/l. TOC removal efficiencywas around 80% for dyestuff concentrations between 50 and200 mg/l and decreased to 52% at an initial dyestuff con-centration of 400 mg/l. Higher dyestuff concentrations couldhave toxic effects on fungi.

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