Decolourization of reactive azo dyes by transformation with Pseudomonas luteola

Bioresource Technology49 (1994) 47-51 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/94/$7.00 ELSEVIER

DECOLOURIZATION OF REACTIVE A Z O DYES BY TRANSFORMATION WITH PSE UDOMONAS L UTE OLA T.L.Hu Dept. of Environmental Science, Feng Chia University, Taichung, Taiwan (Received 22 March 1994; revised version received 24 April 1994; accepted 27 April 1994)

have continued. Ogawa et al. (1978) isolated a bacterium that degraded dye compounds from the sewage system of a dyestuff factory. Kulla (1981) described degradative pathways for sulphonated azo dyes by Pseudomonas strains previously adapted to growth on the corresponding carboxylated azo dyes. Rhodotorula, an oxidative yeast-degrading crystal violet, was isolated by Kwasniewska (1985). Later, Idaka et al. (1987) described the reductive fission of azo bonds by Pseudomonas cepacia. A fungus, Phanerochaete chrysosporium is reported to be able, under aerobic conditions, to decolourize three azo dyes: Orange II, Congo Red and Tropaeolin O (Cripps et al., 1990). The faangal lignin peroxidase was implicated in the decolourization process. Beyond azo dyes, P. chrysosporium ATCC 24725 showed decolourization of direct dyes, reactive dyes, acidic dyes and dispersed dyes (Chen et al., 1991). Paszczynski et al. (1992) described selected Streptomyces spp. that also decolourized azo dyes aerobically, but they were unable to demonstrate the oxidative enzymes involved. Decolourization of azo dyes by a fungal peroxidase system seemed promising, but in all cases the mineralization of azo dyes had to be done in a separate process, as the dye compounds could not be incorporated into the medium; this would be impractical when much dyeing wastewater needs to be treated. According to the literature, most biotransformable or biodegradable azo dyes belonged to the orange or blue colour (Cripps et al., 1991; Pasti-Grigsby et al., 1991), or were those that gave easily and/or more easily decoloured dye compounds; red dyes had seldom, if ever, been investigated. In the present work, the aim was to isolate bacteria that were capable of decolouring azo dyes, and four reactive azo dyes of red colour, commonly used in Taiwan, were chosen to evaluate biotransformation or biodegradation.

Abstract

A bacterium isolated from sludge from dyeing wastewater treatment which removed the colour of reactive azo dyes such as Red G, RBB, RP2B and ~ R P was identified as P. luteola. After shaking incubation for 48 h, P. luteola removed the colour of these dyes during a further 2 days of static incubation, and the fraction of decolourization was 37"4% (Red G), 93"2% (RBB), 92"4% (RP2B) and 88% (V2RP). There was no increased removal of colour when P. luteola was cultured in a low-N medium. No colour was removed under continuous shaking or completely static incubation. According to results from a thin-layer chromatogram, Red G was not degraded and reduction of the azo bond (--N=N--) was the major reason for its decolourization. An extra two spots appeared on the TLC for the other three reactive dyes, which indicated that they were degraded by E luteola; the azoreductase activity was high when RBB was the substrate, but sulphanilic acid was not found in the metabolites. Key words: Azo dyes, azoreductase, decolourization, sulphanilic acid, transformation. INTRODUCTION

Azo dyes are commonly used in the textile, food and cosmetic industries (Michaels & Lewis, 1985, 1986). They are also the most common manufactured synthetic dyes. Commonly used treatments for waste removal inadequately eliminate many azo dyes from effluent waters of textile mills and dyestuff factories (Kimura, 1980). Advanced processes, such as colour adsorption by activated carbon, have been suggested, but not widely applied because of the high cost. Although many aromatic compounds are considered recalcitrant, various microorganisms that are able to degrade azo compounds anaerobically have been isolated. The colourless aromatic amines produced by all these anaerobic microorganisms may be highly toxic and carcinogenic (Meyer, 1981). Although azo dyes were long considered nearly nondegradable or untransformable by bacteria under aerobic conditions (Zimmermann et al., 1982), efforts to isolate microorganisms capable of transforming azo compounds

METHODS Isolation and identification of strains

Bacterial strains were isolated from samples from an activated-sludge system that had been stabilized for six months before the sludge served as the source of isolation (Hu et al., 1991). This system was fed with pH47

48

T. L. Hu

adjusted dyeing wastewater from a dyeing factory near Taichung. An aliquot (10 ml) was vortexed for 5 min, and the standard method of dilution plate count was used to separate single colonies. All colonies appearing coloured and morphologically distinct were isolated and purified. These strains were further tested for their decolouring ability by inoculating them into a dyecontaining broth. Another set of broths without dye served as control. Growth of cells was evaluated by their absorbance at 600 nm and the API 20 NE (Bio Merieux S.A. France) identification system was used.

Medium Two media were used: medium 1, for routine transfer, consisted of glucose (0"5%), peptone (0.3%) and yeast extract (0"3%), with pH adjusted to 7"0+0.2, and medium 2, to monitor loss of colour, had components similar to medium 1, except that the glucose concentration was decreased to 0.125% and 0.01% (w/v) filtersterilized dyes were added. Reactive dyes Four reactive dyes were supplied (Ta Chung Dyeing Company). According to information provided by the company, these four reactive dyes are azo dyes. All dyes had distinctive maximum absorbance at 500 nm (Red G), 530 nm (RP2B) and 551 nm (V2RP). Culture conditions for decolourization Fresh cells ( 18 h, 1 ml, medium 1 ) were inoculated into medium 2 broth ( 100 ml) in a flask (250 ml). A shaking incubation (100 rpm) at 28°C for 48 h was carried out after inoculation, followed by a static incubation for 5-11 days. A sample (5 ml) of culture medium was centrifuged (7000 rpm, 5°C, 10 min) and the supernatant was analysed with a spectrophotometer (Shimadzu, UV-160A). The results are reported as averages of duplicates. Thin-layer chromatography Culture broths were centrifuged and the supernatant (40 ml) concentrated with a vacuum evaporator (Buchi Ratavapor, RE 111, 50°C) to about 2 ml. This sample was applied to an activated-silica thin layer (Silica Gel 60 F254, 20 x 20 cm, thickness 200/zm; E. Merck D-6100, Darmstadt, Germany), and developed with the solution systems n-propanol : water : aqueous ammonia = 90 : 20 : 1 or n-propanol :water:ethyl acetate = 60: 30:1. The results were made visual with UV light at 366 nm. A spot of uninoculated broth was applied each time as the control. Enzyme assay The biomass in culture broth (2 litres) was collected by centrifugation and washed with phosphate buffer (0.1 M) several times. Cells were disrupted in portions (200 ml) by ultrasonic treatment (MSE 20 unistrip) for 15 min in an ice bath. Particle-free cell extracts were made by centrifuging the cell extract for 20 min at 5000 rpm. The protein content of the cell extract and

the azoreductase activity were measured according to Lowry etal. (1951) and Wuhrmann etal. (1980).

Suiphanilic acid detection The supernatant for TLC determination was concentrated (about 10 ml) for detection of sulphanilic acid. A Ct8 column (Baker-10 SPE, 1982) was used to extract and concentrate sulphanilic acid from the culture broth. Sulphanilic acid in the eluted methanol solution was determined with a UV spectrophotometer.

RESULTS AND DISCUSSION

Isolation and identification of dye-decolouring bacteria The initial stage of this research involved the isolation and identification of dye-decolouring bacteria. Cells having yellow and white colours were predominant organisms in the sludge examined; these cells had been adapted to the dyeing wastewater for 6 months in a batch activated-sludge system. These organisms were chosen and tested for their ability to remove colour. The gram-negative rods were identified with the API 20 NE system as Pseudomonas luteola and P. versicularis, respectively (Table 1). P. luteola exhibited a white colony on the agarose medium, and P. versicularis showed a yellowish colony. The nutrient requirements for these two strains were examined according to the growth response (Table 2). When cultured in commercial nutrient broth, the absorbance of these two organisms was less than half of that when cells were cultured in glucose, peptone broth containing 0.3% yeast extract. Therefore, yeast extract was added to the medium for the following experiments (media 1 and 2). Although azo dyes generally contain one or more sulphonic-acid groups on aromatic rings, which might act as detergents and inhibit the growth of microorganisms (Wuhrmann et al., 1980), all four azo dyes that were used showed no effect on the growth of isolates, even when the concentration of dye was as great as 0.2% (w/v) (data not shown). The colour of medium 2 after being treated with P. versicularis remained yellowish, the colour of P. versicularis. It would not be practical for an effluent, after being treated, to retain colour; therefore only an isolate of P. luteola was used for further tests. Removal of dye colour by P. luteola Most loss of colour occurred after incubation for 7 days in total. There was 84.5% colour removal after incubation for 13 days for Red G. The final pH of the culture broth increased from 7"0 to 8"6. To ensure that loss of colour was not simply a function of pH variation, the effect on visible adsorption was assayed between pH 6"0 and 9"0 with a phosphate buffer. The visible adsorption spectra of all dyes tested were unaffected by pH over this range. As the protracted duration of colour removal was considered to be due possibly to lack of oxygen, continuous shaking and

Decolourization of azo dyes by P. luteola

49

Table 1. Identification results of isolated strains with AP120 NE: (a) white colony, (b) yellow colony Physiological characteristics a. Assimilation of CHO Glucose Arabinose Mannose Mannitoi N-acetyl-glucosamine Maltose Glucinate Caprate Adipate Malate Citrate Phenyl-acetate

at

b:~

+ + + + + + + + -

+ + + -

Physiological characteristics

a

b

b. Nitratereductase c. Indol production d. Glucose acidification e. Arginine dihydrolase f. Urease g. fl-Glucosidase h. Protease i. fl-Galactosidase j. Cytochrome oxidase k. Gram stain Name:

+ + -

+ + -

Pseudomonas luteola

P. versicularis

tWhite colony. ~:Yellow colony.

Table 2. Growth of isolated strains in various media Medium

Table 4. Effects of concentration of peptone on colour removal by P. luteola in various dye-containing broths

OD600 n m

Pepton& Nutrient broth Glucose, NH4Ci Glucose, peptone Glucose, peptone, YE"

P. luteola

P. versicularis

1.10 0"03 0.40 2.90

0.32 0"03 0.74 1"60

~Yeast extract. Glucose at 0.5%, peptone and YE at 0"3%.

Table 3. Effect of culture conditions (continuous shaking versus static) on the colour removal by P. luteola pH

ODt00 nm

Dye

48h

48h

% Colour removaP after

120h 48h

Shaking

Static

RG RBB RP2B V2RP RG RBB RP2B VzRP

~Colour removal (%)

2.94 1.47 1.44 -0"40 0"31 0"27 --

8'15 8'21 8.19 -4'17 4'24 4"27 4"25

8.57 0 8"50 63"5 8 " 5 8 10"9 8"21 -4"09 80"5b 4 " 1 5 36"3 4"17 10"8 4"35 --

(A)--Abs of residual broth

Abs of uninoculated broth (A) hFlocculation occurred. --, Not determined.

Dye

% Decolorization oafter

(%) 0.3

0.03

RG RBB RP2B V2RP RG RBB RP2B VzRP

18h

42h

10.8 93.3 74.6 -4.8 93.6 59.3 --

37.4 93.2 92.4 88.0 24.2 94.2 89.0 4.0

"Medium contained yeast extract (based on medium 2). bShaking incubation for 2 days then static incubation for time shown. --, Not determined.

120h 0 56"5 5-0 0 75"5 54-9 23"0 50"6 x 100.

static culturing were c a r d e d out for comparison of colour removal (Table 3). With continuous shaking incubation for 5 days, P. luteola grew well as judged from the absorbance at 600 nm. However, the colour of Red G and V2RP failed to decrease. Static culturing failed to yield good growth of the cells, but 2 3 - 7 6 % of colour was removed, and some dye appeared to be b o u n d to the cells. A continuous shaking condition offered enough dissolved oxygen for the organism to

grow, but the bacteria lacked the ability to remove colour. However, shaking incubation for 2 days and then keeping the cultures under static conditions caused loss of colour for all azo dyes tested. T h e fractions of colour removal in these conditions are shown in Table 4, peptone, 0"3%.

Effect of nitrogen on removal of colour U n d e r nitrogen-limiting, ligninolytic conditions, P. chrysosporium showed a great ability to remove the colour of most azo dyes (Cripps et al., 1990; Spadaro et al., 1992); this mechanism of colour removal is not associated with bacteria such as Pseudomonas sp. (Ogawa et al., 1978; Kulla et al., 1983) or Bacillus sp. (Horitsu et al., 1977), but in all cases the concentrations of azo dyes tested were low. In the present experiments at least 0.01% (100 ppm) o f a z o dyes were used. T h e effect of peptone concentration and comparison of nitrogen sources were used to illustrate the mechanism of colour removal by P. luteola. According to the results of Table 4, when the peptone concentration was decreased to one tenth, the colour removal of V2RP

50

T. L. Hu

was greatly decreased; there was a decrease of 13% for Red G, however, removals of colours of RBB and RP2B were unaffected. There was also no distinction between inorganic or organic nitrogen sources on loss of dye colour (Table 5). These results indicated that decreasing the N concentration did not enhance the colour removal even in the high concentrations of azo dye tested, and the colour removal mechanism of P. luteola was different from that of P. chrysosporium. Since it required a static incubation after 2 days' shaking incubation for colour loss, during completely static incubation anaerobic transformation may occur in microanaerobic zones within aggregates in aerobic systems (Michaels & Lewis, 1986), therefore the colour removed by P. luteola might be due to the structural alteration of the chromophoric azo group. Metabolites of dye compound and azoreductase activity

TLC was used to assess metabolite formation after the dye-containing broths (medium 2) were cultured with P. luteola (Table 6). Uninoculated broth alone and with

0"01% dye were also tested as negative controls. There was no coloured metabolite observed for all dyes tested. According to the TLC, all dyes except Red G produced two extra spots compared to the control. This result showed that Red G was still an intact molecule after treatment with P. luteola; the reason for Red G becoming colourless must have been reduction of the azo linkage to a single bond (--N--N--). The initial step in biodegradation of azo compounds is a reduction cleavage of the azo group. Under anaerobic conditions this reaction is catalysed by various biological systems (Walker, 1970; Meyer, 1981), and the enzyme azoreductase is considered to be sensitive to oxygen (Wuhrmann et al., 1980). However, azo dyes could be reduced under aerobic or microaerophilic conditions; Horitsu et al. (1977) demonstrated that PAAB was reduced by Bacillus subtilis under aerobic conditions. Idaka et al. (1978) reported the apparent aerobic reduction of simple azo compounds by Aeromonas hydrophilia. A strictly aerobic bacterium (Flavobacterium) reduced 4,4'-dicarboxy-azobenzene to two molecules of amino-benzoic acid under aerated conditions that excluded any anoxic situation in the external medium around the cell (Kulla et al., 1984). In

Table 5. Effect of nitrogen source on the decolourization by P.. luteola

N source" (0.3%)

Dye

(NH4)2504 RG NH4CI Peptone

RBB RP2B V2RP RG RBB RP2B V2RP

% Decolourizationh after

Table 7. Total activity and specific activity of crude azoreductase a

2 days

2 days

7 days

Dyeh

1.52 1.57 1.52 -1"75 1"75 1"78 --

24.0 26.0 7.2 71"0 0 43'5 14"5 78'0

33"5 95.7 85.1

Total activity (Amg dye/ (liter/min))'

Specific activity (Amg dye/ (mg protein/min)) d

RG RBB RP2B V2RP

0 0.94 0"38 0"41

0 2.60 × 10 -4 1.06 × 10 -4 1"12 × 10 -4

OD6o0 nm

47-0 94-0 93"3

aMedium contained yeast extract (based on medium 2). bShaking then static incubation process. -- Not determined.

aActivity was measured after shaking incubation for 24 h. bCultures contained dye at 0.01% concentration. 'Total activity is defined as the changes of dye (mg/liter) during assay period. dprotein content of cell extract as described in Methods.

Table 6. TLC Rf values of metabolites ofRG, RBB, RP2B and V2RP after incubation with P. luteola a

Dyes (Rf)

Rf values Uninoculated

Culture of P. luteola

Broth

Broth + dye

Broth

Broth + dye

RG(--) h

0, 0.28, 0.31

0

RBB(0-51)h

0, 0.28, 0"31

0

RP2B(0-43, 0"63)h

0, 0.28, 0.31

0

V2RP(0"48)'

0, 0.67

0, 0.06, 0.20, 0.25, 0"28, 0.55, 0.69 0, 0.06, 0"20, 0.25, 0.28, 0.55, 0"69 0, 0-06, 0.20, 0.25, 0-25, 0"55, 0"69 0, 0-67

0, 0.06, 0.20, 0.25, 0.28, 0-55, 0.69 0, 0-06, 0-20, 0.25, 0.28, 0.39, 0.45, 0-69 0, 0.06, 0-20, 0.15, 0.28, 0-39, 0.45, 0-55, 0.69 0, 0"62, 0"67, 0-73

0, 0.48, 0"67

"Shaken incubation 2 days, static incubation 11 days in medium 2. hSolvent system; n-propanol: H20: ethyl acetate -- 60: 30:10 (v/v/v). 'Solvent system; n-propanol:H20 :ethyl = 60:30:10 (v/v/v). --, No movement of dye. Underlined spots indicate the new ones.

Decolourization of azo dyes by R luteola order to prove that the colour removal of the azo dyes tested had occurred via a similar pathway, the azoreductase activity was also determined (Table 7). P. luteola showed no azoreductase activity towards Red G. This result coincided with the result of TLC, which also indicated that the Red G was not broken down by this organism. The two extra spots on the TLC for each azo dye (Table 6) except RPB corresponded to the azoreductase activity. Sulphanilic acid, a metabolite of azo compounds, was not detected by the method used; other extraction methods for sulphanilic acid will be further tested.

ACKNOWLEDGEMENT I thank the National Science Council of the Republic of China for support (NSC80-0421-B-035-01Z).

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