Biodegradation of azo dyes by the yeast Candida zeylanoides in batch aerated cultures

Chemosphere,Vol.

38, No. 11, pp. 2455-2460, 1999 © 1999 Elsevier Science Ltd. All rights reserved 0045-6535/99/$ - see front matter

Pergamon

PII: 80045-6535(98)00448-2

BIODEGRADATION OF AZO DYES BY THE YEAST Candida zeylanoides IN BATCH AERATED CULTURES M.AM. Martins, M.H. Cardoso(a), M.J. Queiroz,M.T. Ramalho* and A.MO.- Campos Instituto de Biotecnologia e Qulmica Fina, Universidade do Minho; (a) Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 471g Braga Codex, Portugal (Received in Germany 15 June 1998; accepted 27 August 1998)

ABSTRACT A number of simple azo dyes was degraded in liquid aerated batch cultures by a strain of the yeast Candida

zeylanoides. The standard decolodzationmedium containedglucose as a carbon and energy source, and its pH was either controlled to 5.0-5.2, or allowed to decrease to 3.2-2.8, in the course of microorganismgrowth. The extent of colour removal in the culture mediumwas assessed throughthe decrease in dye absorbance of the supematants. The extent of colour removal ranged from 44 to 90%, after 7 days, for 5 out of 6 dyes studied in shaked cultures, without pH control, and from 46 to 67%, after 22 hours, for 6 out of 8 dyes in batch experiments,at controlled pH. ©1999 Elsevier Science Ltd. All rights reserved INTRODUCTION

The many studies on biodegradation, environmental impact and health effects of colorant materials, over the two last decades, clearly reveal the complexity of the subject, not only due the structural variety of these compounds, but also as a result of the complex composition of effluents which they contaminate. The experimental research aiming at the bioremediation of their environmental effects has been based in several possible strategies. One of them assesses the effects of dye-containing effluents upon activated sludge, br other adequate biological marker, and examines the efficiency of the treatment processes through the parameters of the treated water. Another approach, which will be used in the present work, aims at the elucidation of the degradation processes of a limited number of dyes by selected microorganisms, under controlled laboratory conditions. It is expected, through a better understanding of the degradation mechanisms, to widen the available range of remediation tools. The biotransformation of azo dyes by environmental microorganisms has been reviewed elsewhere [1]. It is known that many strains of bacteria reductively cleave some dyes, producing aryl amines. Anaerobic conditions are usually refered to as being favourable to the reduction step, but it has been also shown that strains of Bacillus subtifis, Aeromonas hydrophila, Pseudomonas cepacia and of a Flavobacterium sp. can reduce azo dyes in the presence of oxygen [1]. However, the ultimate purpose of complete mineralization of dye molecules can not usually be reached under anaerobic conditions [2,3]. The most promising strategy seems to be a tandem anaerobic-aerobic treatment by an adapted bacterial consortium, as described by Haug et al. [4] who achieved, through this method, the complete mineralization of Mordant Yellow 3. White-rot fungi e.g. Phanerochaete

chrysosporium, are also being investigated as a potential alternative for dye 2455

2456 decolorization. These organisms have been shown to degrade several dyes by oxidative mechanisms, probably involving the ligninolytic enzymatic system [5,6]. Yeasts, on the other hand, are remarkably absent from the literature on dye degradation. Trindade & Angelis [7] refer to azo dye removal by Rhodotorulla yeasts, presumably by an adsorptive interaction with fully grown cells, and Luo & Liu [8] used a yeast strain, isolated from dye-contaminated soil, in the decolorization and degradation of Direct Orange S. The present work provides preliminary evidence on the potential use of ascomycete yeasts for the removal and degradation of azo dyes in aerated glucose-containing culture media. The extent of colour loss brought about by a strain of the yeast Candida zeylanoides, in the presence of glucose as carbon and energy source, is strongly dependent on the dye struture, particularly on the nature and position of substituent groups in the aromatic rings. For some of the dyes tested, the influence of glucose in decolorization extent has also been examined.

EXPERIMENTAL Azo dyes. Azo dyes derived from 2-naphtholaminoazobenzene and p-N,N-dimethylaminoazobenzene (figure 1) were synthesized and characterized by conventional methods. Microorganism and growth conditions. The strain of C. zeylanoides (strain UM 2, collection of the Biology Department) used in the present work, was isolated from dye polluted soil near the waste water treatment facilities of a textile finishing plant located in the neighbouring town of Vila Nova de Famalic~o. Yeasts with azo dye decolorizing potential were screened through a protocol involving inoculation of YEPD/agar plates, containing solubilized dye, with samples of the supernatant of a soil suspension. After 3-4 days incubation at 25°C, colonies which decolorized the agar were selected for confirmation experiments and.identification. The plating medium contained, per litre, yeast extract (5 g), bactopeptone (10 g), glucose (20 g) and agar (20 g). The final pH was 5.2, and chloramphenicol (0.05% w/v) was added as bacterial growth inhibitor. Glucose was from Merck and other medium components were purchased from Difco. The C. zeylanoides strain was identified through the usual morphological and physiological tests [9]. Medium for dye decolorization. The liquid medium for decolorization experiments contained, per litre, yeast extract (1 g), bactopeptone (10 g), glucose (20 g) and 10-50 ppm dye, according to its water solubility. The dye stock solutions were made up 25-fold concentrated either in water or ethanol, if water-insoluble, and were filter-sterilized prior to addition to the sterile culture medium. Cell growth and dye monitoring. Aliquots collected aseptically from the cultures were used for cell growth (OD640) and dye monitoring. For the latter procedure cell-free samples were diluted, as required, in 0.1M acetate buffer, pH 5 , and their absorbance, at the observed Xmax, read against a blank containing the same buffer concentration in distilled water. At the dyes Xmax there was no interference of other medium components. Decolorization in shaked cultures without pH control. were carried out in 250 ml conical flasks, containing 100 ml sterile Ioopful of freshly grown cells taken from an YEPD/agar slant. The at 25 oc, in an orbital incubator shaker, for several days. Aliquots for biomass and dye monitoring as described above.

Preliminary batch experiments liquid medium inoculated with a flasks were shaken at 150 rpm, collected at intervals were used

2457

Batch decolorizaUon with pH control. For batch decolorization experiments with pH control 900 ml volumes of medium were dispensed to the 1 L vessel of a Biolab bioreactor (B. Braun Biotech International). The sterile medium was directly inoculated with a suspension of cells from a fresh slant, and the pH was kept at 5.2-5.0 by a pH controller connected to concentrated (5M) hydrochloric acid and sodium hydroxide reservoirs. The culture, at room temperature, was aerated (200 ml air/min.~ and shaken (150 rpm/min) by a motor-driven stirrer. Cell growth and decolorization were monitored in aliquots collected over 22 hours.

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Figure 1. Structuresof the testeddyes RESULTS AND DISCUSSION The dye disappearence in shaken batch cultures, along with cell growth and pH, were monitored over seven days, in glucose-containing medium. In order to detect the relative effects of cell wall adsorption and biotransformation on colour removal, the supematant absorbance, at dye Xmax, was recorded in the late exponencial phase and after seven days incubation. The growth curves obtained in the presence of dyes 1.a, l.b, 2.a, 2.b, 2.e and 2.£ and the data on colour removal are displayed in figures 2 and 3. 10 ,_

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0,01 0

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40

60

80

1O0

120

140

160

180

Time (h)

Figure 2. Growthcurvesin shakencultures,withoutpH control, in the absence (~a)and in the presenceof dyes 1.a (ll), 1.b, (r-I),2.a (A), 2.b (Z~),2.e (0) and 2.f (0)

2458

100 8O 60

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40 20 ~ , 0

-~0 h Time 168 h 1 .a

1.b

2.a

2.b

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DYE Figure 3. Percentagesof dye in culture supernatants in the late exponentialgrowth phase (c.a, 20 h) and after 7 days For most of the dyes in this set of experiments, extensive decolorization was observed. However, the small decrease in the concentration of the dye 2.f in the culture medium occured during the exponential growth phase only, remaining virtually unchanged thereafter. It seems, therefore, that this dye, which is the less polar and has an electron-donating methyl substituent group in ring A (Fig. 1) is quite recalcitrant under the conditions of the assay. In contrast, 90% of the dye l : a was removed, with little cell wall adsorption, in the exponential phase of growth, suggesting a very fast (less than 20 hours) biodegradation process. The pH of the culture medium decreased, over the incubation period, from an initial value of 5,0-5.2 to 3.2-2.8 (data not shown). The bioreactor experiments were restricted to a 22h incubation period, in which about two thirds of dye l . b was removed from the culture medium. Samples collected at the end of the adaptative phase, in the late exponential phase and after 22 h, were selected for analysis of results. The growth curves are shown in figure 4 and the percentages of colour removal after the selected incubation periods are depicted in figure 5. From figure 4 it is apparent that the specific growth rates of the microorganism in the exponential phase are higher in the presence of dyes with a p-sulphonic substituent (l.a, 2.a) than for the m-substituted analogs (l.b, 2.b). This parameter is unrelated, however, to final OD640. Growth inhibition was particularly severe for the carboxylic dyes (2.c, 2.d) and, to a lesser extent, for the methyl-substituted dye 2.f. The percentages of colour removal were close to, or higher than 50%, except for dyes 2.b and 2.f. The latter was, as in shaken cultures, the most recalcitrant (only 27% colour loss). Considering the diferences in the incubation periods, the results from the batch bioreactor experiments are in good agreement with those from the shaken cultures. The most obvious exception is observed with the dye 2.b. The colour loss reached 70% in shaken cultures and only 20% in the controlled pH experiment, despite a reasonable cell growth; in addition, the decrease in dye absorbance occured exclusively during the exponential growth phase (data not shown). This

2459 result

is consistent

with

a pH effect

on the degradation

process,

which

is currently

under

investigation

0.01 cl

5

10

15

20

25

Time (h) Figure 4.Growth curves in batch cultures, at pH 5.0-5.2, in the absence (60) and in the presence of dyes 1 .a (s), 1 .b @), 2.a (A), 2.b

(A),2.c (+), 2.d (0 ), 2.e (0) and 2.f (0).

80 60 40 20

0

a

u

Phase

DYE Figure 5. Percentage of dye in the supematants of glucose-containing

culture medium in the late lag phase (a), the

late exponential phase (b) and atier 22 hours (c) ; idem, in basal medium without glucose, after 22 hours (d)

The influence monitoring

of glucose,

dye removal

usually

in bioreactor

a prefered experiments,

carbon and energy

source, was assessed

using basal medium,

without

glucose.

by The

results were similar for dye 2.c (47% and 48% dye removal with and without glucose, respectively)

2460 but, in the case of dyes 1.a and 2.e the presence of glucose was clearly beneficial: colour loss reached 57% for both dyes in glucose-containing medium and only 25% or 38%, respectively, in basal medium (figure 5). CONCLUSION Though further research is necessary to improve the conditions for decolorization of azo dyes, and to elucidate the underlying mechanism, our results show that the ascomycete yeast C. zeylanoides displays a considerable capacity in colour removal of several sulphonic and nonsulphonic dyes, in synthetic nutrient medium, at acidic pH. REFERENCES 1. K.-T. Chung and S.E. Stevens, Jr.. Degradation of azo dyes by environmental microorganisms and helminths, Environ. Toxicol. Chemistry, 12, 2121-2132 (1993) and referencestherein. 2. S.F. Dubrow, G.D. Boardman and D.L. Michelsen. In Environmental Chemistry of Dyes and Pigments, (Edited by A. Reife and H.S. Freeman), chapter 5. John Wiley & Sons, New York (1996). 3. F. Malpei. I coloranti organici: biodegradazione ed implicazione nel trattamento delle acque-Parte I, /A /ngeneria Ambientale, XXV (4) 190-199 (1996). 4. W. Haug, A. Schmidt, B. Nortemann, C.D. Hempel, A. Stoitz & H.-J. Knackmuss. Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium, Appl. Environ. Microbiology, 57, 3144-3149 (1991). 5. A. Paszczynski, M.B. Pasty-Gdsby, S. Goszczynski, R.L. Crawford and D.L. Crawford. Mineralization of sulfonaterd azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Strepomyces chromofuscus,Appl. Environ. Microbiology, 58, 3598-3604 (1992). 6. M.B. Pasti-Grisby, A. Paszczynski, S. Goszczynski, D.L Crawford and R.L. Crawford. Influence of aromatic substitution patterns on azo dye degradability by Strepomyces spp. and Phanerochaete chrysosporium. Appl. Environ. Microbiology, 68, 3605-3613 (1992). 7. R.C. Tdndade and D.F. Angelis, Removal of azo dyes for Rhodotorula: relationships with pH and substantivity index. 7th. International Symposium on Microbial Ecology, Santos, S. Paulo, Brazil, 27 August-01 September, Abstract P324.86 (1995). 8. Z. Luo and M. Liu. Degradation and decoloration of Direct Orange S waste water by yeast S-36, Zhongguo Jishui Paishui, 12(4), 12-13 (1996). 9. J.A. Barnett, R.W. Payne and D. Yarrow. Yeasts: Characteristicsand Identification, 2nd edition. Cambridge University Press, Cambridge (1990). Acknowledgements: The authors are grateful to N.O.B. Martins for the preliminarywork on the identification of several yeast strains, to Dr. C. Le~o, of the Biology Department, for her expert advice in the course of this work and critical reading of the manuscript, and acknowledge financial support from Praxis XXII212.11QUI/44194,FEDER and University of Minho.