Environmental Pollution 75 (I 992) 273- 278

Degradation of azo dyes by algae

Liu Jinqi & Liu Houtian
('hinese Research Academy 0] Environmental Sciences. Be~jing 100012, People's Republic ~ff (*hina

(Received 19 February 1991; accepted 19 April 1991)

The degradation of azo dyes by algae was evaluated and it was tkmnd that certain
algae can degrade a number ofazo dyes to some extent. The reduction rate appears
to bc related to the molecular structure of the dyes and the species of algae used. The
azo reductase of algae is responsible for degrading azo dyes into aromatic amine by
breaking the azo linkage. The aromatic amine is then subjected to further
metabolism by algae. It is proposed in this paper that in stabilization ponds, algae
can play a direct role in the degradation of azo dyes, rather than only providing
oxygen lbr bacterial growth.

INTRODUCTION The present study was undertaken to investigate the

With the growing use of'a variety of dyes pollution by dye
wastcwater is becoming increasingly serious. There are
about 3000 types of dyes on thc world market. More than
60 000t of dyes are released in wastewater every year all
over the world (ETAI), 1984). Azo compounds are one of
the oldest industrially synthesized organic dyes. Azo
c o m p o u n d s have low biodegradability and greatly
influence natural ecosystems and humans [Chung et al.,
1978). Purification of dye wastewater is a matter of great
concern, and some treatment methods have been
developed (Gu, 1985). Biological methods are the most
widely used as they are simple to use and low in cost.
Bacterial degradation has been the main focus in most
studies on biodegradation o f a z o dyes (Liu & Yang, 1987;
Xian & Yang, 1988). Some investigators have systemically
studied the degradative mechanism of" azo dyes by
bacteria (Brown, 1983a,h, 1987: Pagga & Brown, 1986;
Brown & Hambuger, 1987). They proposed the following
pathway of the degradation of azo dyes: azo compound--
aromatic amine-simple molecules, and then the cleavage
of the azo bond by azo reductase.
There have been few reports regarding degradation of
azo dyes in wastewater by algae. Some investigators
reported that species of Oscillatoria could decolor dye
wastewater (Anon., 1977). Zhu et al. (1979) tried to use
three species of Oscillatoria in a rotating biological
contactor which could satisfactorily remove BOD 5 and
C O D from wastewater. Since the experimental system
was not used under sterile conditions, it was not possible
to distinguish the separate roles of algae or bacteria in the
reduction of azo dyes.
Environ. Pollut. 0269-7491/92/$03"50 ,~ 1991 Elsevier Science
Publishers Lid, England. Printed in Great Britain
273
degradativc mechanism of azo dyes by algae and the
relationship between the biodegradability of azo com-
pounds and their molecular structures.
MATERIALS AND METHODS
Algae
Chlorella p.vrenoidosa, C. vulgaris and Oscillateria tenuis
isolates used in this work were supplied by the Institute
of Hydrobiology, Academia Sinica, Wuhan, People's
Republic of China. They were grown in flasks contain-
ing a sterilized liquid medium containing (g/liter) 0-04
Ca(NO3)2. 0.01 K z H P O 4, 0.025 MgSO,,, 0.02 Na2CO 3,
0.025 Na2SiO 3, 0.003 citric acid and 0.003 ferric citrate.
Simultaneously, 10ppm of a dye was added to the algal
cultures to acclimate the algae. Inoculated flasks were
kept in a culture room at a temperature of 25 _+ I~C (pH
7'5) and with a daily photoperiod of 16h light (6000+
200 Lx) and 8 h darkness.
Decolorizing ability of algae
More than 30 azo compounds were selected in the
decolorization test (Table 1). All the compounds were
chemically pure. Algae (10S/ml) were introduced sepa-
rately into flasks containing an azo compound (20 ppm).
Cultures were maintained under sterile conditions for
96 h in the culture room and then centrifuged at 5000 rpm
for 15min. The supcrnatant was evaluated via a light
absorption method and percentage reduction rates were
calculated. Culture fluid without algae was used as the
control.
274 Liu Jinqi. Liu Houtian

Table I. Degradation of azo compounds by Chlorella vulgaris

m _ .
m _ _

Number Azo compound Characteristics of structure Observed Decolorization

wavelength (%)
(rim)

I Benzene azo-l-naphthyl amine 5-NH 2 445 63

2 Alkaline orange 2,4-(N H 2)2 447 50
3 4-Aminoazo benzene 4-N H, 374 48
4 p-Hydroxy azo benzene 4-OH 347 55
5 4-[(p-Nitrophenyl)-azo]-l-naphthol 4-OH 477 67
6 Sudan Ill 6"-O1! 348 41
7 Sudan II 4,6qCH 0z 485 5
8 Para-methyl red 4'-N-(CIt 3)z 466 0
9 Methyl red A.R. 4'-N-(('H3) 2 430 0
10 Methyl yellow 4-N-(CH3) 2 446 18
11 4-Methoxy azo benzene 4'-O-(' H .~ 350 20
12 :~-Naphthylamine azo-benzene
p-sulfonic acid 4"-SO3Na. 5'-NH 2 470 40
13 Eriochrome blackT 5-SO~Na, 7-OH, W-OH 527 76
14 Eriochrome blueSE 3',6'-(SO3Na1,, 6-OH, 1',8'-(OH), 530 95
15 Orange I I'-SO.~Na, 5-OH 475 62
16 C.I. Direct blue71 4,7-(SO.~Na),, 2".6"-(SO3Na),, 8"'-OH, 3'"-NH 2 587 I(X)
17 C.I. Direct red23 7'-S(-)~Na, 2"-SO.~Na. 6'-OH, 4"-OH 507 88
18 C.I. Direct orange26 6'-SO.~Na, 3"-SO.~Na, 8'-OH, I"-OH 494 80
19 C.I. Direct blackl9 4.6-1N112)2, I"-OH, 3".6"-(SO~Na),, 8"-NH,,
1"",5'"'-(N H,), 514 79
20 C.I. Direct brown2 6"-SO.~Na, 2"-NH 2. 4-OH, 8"-O}t 391 65
21 Solochrome black 4-SO.~Na, I-OH, 4',8'-(OH), 533 60
22 FD&C Red No. 2 5-SO~Na. 2",7"-(SO3 N a)2 522 20
23 Benzyl orange 4-SO~K 441 7
24 Alizarin yellow GG ind. 3-NO, 484 7
25 Acid yellow GG * 354 17
26 Acid red G 402 19
27 Acid blue GBA 531 25
28 Mordant yellowGG 358 24
29 Mordant blueBFT 428 17
30 Mordant redB 510 35
31 Polar brilliant redB 526 64

* - - indicate that structure of the dye is not known, to the knowledge of the authors.

Utilization of dyes Preparation and activity of azo reductase

Culture fluid without c a r b o n and nitrogen, or with only
one-half or one-fifth o f the normal content o f c a r b o n and
nitrogen, was used to evaluate utilization o f two dyes by
algae. Then C. vulgaris, C. pyrenoidosa and O. temds were
tested individually for their ability to degrade Eriochrome
blueSE and blackT. Decolorizing activity was used to
explain algal ability to degrade azo dyes (rag dye/dry wt
per day).
Examination of the reductive cleavage of azo bond
After incubation with algae for 96 h the culture fluid was
centrifuged. Some o f the suspension was assayed with the
UV-visible spectrum m e t h o d (Shimadzu UV-240). Sub-
sequently the suspension was run through a column
G.D.X.-104, dehydrated, and analyzed with F T . 1 R
(Nicolet, Varian China Ltd, Sunnyvale, California).
Paraffin oil, free o f water, was used as solvent.
The azo rcductase preparation was made according to the
procedure described by ldaka et al. (1987). The reductase
activity was then studied.
Degradation of aniline by algae
Since aniline was considered likely to be formed by the
degradation o f azo dyestuffs, it was the present authors'
object to investigate its algal degradability. Algal cultures,
treated with aniline, were centrifuged, with the culture
fluid without algae used as a control. The suspension was
analyzed with an H P L C system (Varian 5500) equipped
with a UV-200 detector and M i c r o p a k spc-13 column
(4.6 m m x 150 mm). The mesh size o f the packing was 5 m
and the mobile phase was c o m p o s e d o f H 2 0 and C H 3 O H
with various ratios changing from 35:65 ( H 2 0 : C H 3 O H )
to 0:100 ( H 2 0 : C H 3 O H ) . The solvent gradient was
2%,rain.
 Degradation of azo dyes by algae 275

RESULTS
A

Characteristic chemical structures and the degree of

decolorization for 31 azo compounds by C. vulgaris are
given in Table !. Fourteen of the samples were decolorized
by 50°/,, or greater. C.I. Direct blue71 was completely
decolorized.
These results suggest that the degree of azo dye
decolorization by algae seems to be related to the
molecular structures of the dyes. Compounds 1-6 with a
hydroxy or amine group were readily decolorized.
Compounds 12-21 with an amino or hydroxy group
might counteract the inhibition of the SO3H group on azo
reduction. From these results it is considered that the
hydroxy or amino group might make an important
'7'
contribution to determine the ability of" algae to
decolorize the above compounds. On the other hand, the
compounds 7- 11 and 23-24 have a lower reduction rate.
From this it is suggested that the methyl, methoxy, nitro
or sulfo derivatives are hardly decolorizcd.
Figure 1 shows the decolorizing activity of algae in all
the culture fluids containing different contents of
inorganic carbon and nitrogen. The highest activity is
observed in the culture fluid which is free of inorganic
carbon and nitrogen. This suggests that the algae can
utilize Erichrome blueSE and blackT as its sole source of
carbon and nitrogen, and that the process of azo
compound degradation is related to the physiological
metabolism of the algae.
An obvious change of UV-visible absorption spectrum 200 300 400 500 600
of azo dyes occurred after the treatment by O. tenuis and Fig. 2. UV spectra of azo dye before and after algal treatment.
C. wdgaris (Fig. 2), indicating a change of the molecular (A) Eriochrome blackT; (B) Eriochrome blueSE.---- , Control;
.... , after C rul~aris action: .... , after ('. pyrenoidosa action;
structure of the azo compound. Infrared analysis was
• after O. tenuis action.
introduced to identify the structural variation of Erio-
chrome blueSE after algal action. The results are shown in
Fig. 3. Compared with Fig. 2, a new wide peak appears in within thc range 1630-1575cm -1 (shaded parts of
the range 3500 3100cm -~ on the spectra of Figs 3 and 4. figures). It is suggested that algal action results in the
It represents the stretching vibration of aromatic amine. cleavage ot" azo linkage of Eriochrome blueSE and the
The stretching vibration of the azo bond diminishes formation of aromatic amine.
C vulgaris and Eriochrome blueSE were selected to
~.6
study the activity of azo reductase (Table 2). The enzyme
preparation appeared to have azo reduction activity. In
1.4
the case of adding N A D H or N A D P H the reductase
~ 1.2 activity results in a marked increase. It is evident that
N A D H or N A D P H can act as an electron donor for the
azo reductase in breaking down the azo bond, and
N A D P H is more effective than N A D H . Moreover, the
T. 0.6 unacclimated algae display a lower azo reductase activity
compared with the acclimated algae (Fig. 4). These
:~ 0.4
findings suggest that the azo reductase of C. vulgaris is an
o 0-2 induced enzyme and the substrate itself can act as a kind
of inducer. The induction causes a marked enhancement
A B, C A B C
of the enzyme activity.
Fig. I. Utilization of azo dye by algae. (A) O. tenuis; (B) C. After being treated by C. t,ulgaris or O. tenuis under
vulgaris; (C) C. pyrenoidosa. ~ , No inorganic carbon and
nitrogen: l , ~ carbon and nitrogen of normal content; I~, sterile conditions, the aniline was quantified by the H P L C
carbon and nitrogen of normal content. (a) Eriochrome blueSE; system (Fig. 5). Algal action caused 100% loss of aniline
(b) Eriochrome blackT. and no other organic compounds were detected• It is
276 Liu Jinqi. Liu Houtian

003 L
£ 4 ¸

0.20'
L_ I
0.43' Jl
E 3
I /t Jl
0 /
0-66
J /
J/ J /
0-69 E
2 /
I3/ J /
J /

o: L.
0
J

//
J
Jj

0.61 O
U jl
/ J
J I
a J J
0-92 C < /

G. G. T I
D D Q. I~L T I
0 0
1"23
u Z Z
t-
z z
+ + •4- +
~ O.ll a. fit. 0.. n
D
O

0-43 Fig. 4. The change of azo reductase activity before and after
acclimation. I~, Enzyme activity ofacclimated algae; Fq, enzyme
0-741
activity of unacclimated algae.

1.o41 (2) ability of algae to utilize dyes--this is determined by

[

algal physiological characteristics; and 13) effects of

1.311 environmental conditions. We report our results from
rt
investigations on factors 1 and 2 here.
Some authors have studied the effect o f d y c structure on
bacterial degradability. The distinct degradability of azo
dyes with different structures by bacteria was assessed by
0"29' Urushigawa and Yonczawa (1977). They discovered that
azo compounds with an hydroxy or amino group are
0'42' more likely to be reliably degraded than those with a
i methyl, methoxy, sulfo or nitro group. In the present
0.57 ' study more than 30 azo compounds were chosen to test
their decolorizing rate. Most of the results support
0'71 Urushigawa and Yonezawa's 11977) findings. A new
4000 3480 2960 2440 1920 1 4 0 0 880 360
phcnomcnon, however, was found in the present authors'
Wave n u m b e r (cm -1) experiment. Amino or hydroxy groups in some azo
Fig. 3. Infrared spectra ofazo dye before and after algal action. compounds can act somewhat against the inhibition of
(1) Infrared spectrum of paraffin oil; (II) infrared spectrum of sulfo groups on azo reduction.
Eriochrome blueSE before algal action: (iI1) infrared spectrum
of Eriochrome blueSE after C. vulgaris action: [IV) infrared
spectrum of Eriochrome blueSE after O. tenui.~ action. Table 2. Decolorization activity of alga azo reductase

Azo reductase Decolorization Activity

proposed that all the selected algae can degrade aniline (%) (mg dye/mg dry
effectively and turn it into some simple inorganic material. wt per hour)

I'~nzyme solution 50 2'60

Enzyme solution
DISCUSSION + NADPH (0.01 nmol) 94 3.80
Imzyme solution
There are three limiting factors found in the degradative + NADH (0.01 nmoll 85 3.40
NADPH 0 0
pathway of azo dyes by algae: (!) degradability of d y e s - - NADtl 8 0.25
this is mainly dependent on the structural features of dyes;
 Degradation of azo dyes by algae 277

A B C D

0 5 10 =5 10 .~ 10 5 10
R e t e n t i o n t i m e (rain)
Fig. 5. HPLC of aniline before and after algal treatment. (A) Before inoculating algae: (B) after C. p.vrenoidosa action: (C) after ('.
culgaris action: (D) after O. tenuis action.

It has been identified that the variability in reduction compounds can be utilized as sole sources of carbon and
rate o f a z o dyes by bacteria is partly due to differences of nitrogen by the algae. In the reduction of azo dyes by
the dye transportation rate into bacterial cells. Two steps algae the azo bridge is broken down by azo reductase and
might be considered. (11 Transport of dye from external aromatic amine arises as a cleavage product. This
medium through the cell wall to the plasma membrane. dcgradative pathway is mostly similar to the reduction
This step may be governed by an adsorption-dcsorption mechanism of bacteria (Urushigawa & Yonezawa, 1977;
equilibrium of the dye at the cell wall. (2) Membrane Kulla, 1983). Many researchers have probed for the end-
transportation. This is related to the molecular structures product of aromatic amines after bacterial treatment.
of dyes. Kulla 11983) utilized Ct4-1abeled substrates and Pseudo-
The inhibitory effect of sulfonic acid substitution on monas strain KF46 or K22 for the test. He concluded that
bacterial reduction rate of dye may be due to the impeded some aromatic amines can be completely degraded to
transport (Wuhrmann, 1980). It was found in the present carbon dioxide. Most of them, however, are turned into
study that algal degradation of sulfated dyes is poor. This other intermediates. In the case of the authors' experi-
may also be due to difficulty in permeation of the dyes to ments it has been found that algae can also utilize
the algal membrane. aromatic amine (aniline). The following scheme is
The authors' experiment revealed that some azo assumed to illustrate the whole degradative process o f a z o
dyes by algae, but some of the details need further study to
ascertain the end-product.
Azo compound R-~--. N : N ~ R ' Progress has been made in the treatment of dye
wastewater in terms of the stabilization pond method. It is
Azo reductase commonly accepted that bacteria are the main consumers
of organic compounds in the algae-bacteria degradation
system. Algae, however, primarily contribute to photo-
synthesis and serve as an oxygen source for the aerobic
H H action of bacteria (Govidan, 1979). The results presented
in this study, however, suggest that algae also have a direct
effect on the degradation of azo dyes, From these
investigations it follows that the role of algal action also
needs to be considered in the treatment of dye wastewater
R ~ N H 2 H2N--~R' in stabilization ponds.
Aromatic
amine /,,', ,'l',
t i \
I¢/ I~ \ q
NH 2 ,~ R NH 2 ,~ R' REFERENCES
3HC CH3 Anon. (1977). Decoloration of dye waste water by Oscillatoria.
Environ. Sci., 2, 39--45.
CH 3 Brown, D. (1983a). The aerobic biodegradability of primary
3H C >
/ aromatic amines. ('hemosphere, 12, 404- 14.
Brown, D. (1983b). The degradation of dyestuff. Part I: Primary
biodegradation under anaerobic conditions. Chemosphere,
Simple compound or CO 2 12, 397-404.
278 Liu Jinqi. Liu Houtian
Brown, D. & Hambuger, B. (1987). The degradation of dyestuff:
Part 3 Investigation of their ultimate dcgradability. Chemo-
sphere, 16, 1539-53.
(?hung, K.-T.. Fulk, G. E. & Egan, M. (1978). Rcduction o f a z o
dycs by intestinal anaerobes. Appl. & Fnviron. Mi('rohiol., 35,
558--62.
ETA D ( Ecological and Toxological Association of the I)ycst ulls
Manufacturing Industry) (1984). Agricultural use of sludge
contaminated with colorants. Ecological Sub-Committee
Project E3016.
Govidan, V. S. (1979). Studies on the treatment of textile mill
wastewaters by stabilization pond method. Indian .L l-reiron.
Health, 21, 321-3 I.
Gu, D. Y. (1985). Treatment ~?/ Dye 14"aste Water. Publishing
House of Chinese Architectural Industry, Beijing.
Idaka, E., Horitsu, H. & Ogawa, T. (1987). Some properties of
azoreductase produced by Psez,bmzonas ~eparia. Bull.
Ent'iron. ('ontam. Toxicol., 39, 982-9.
Kulla, H. G. (1983). Interference of aromatic sulfo groups in the
microbiol dcgradation of the azo dyes orange I and orange II.
Arch. Microbiol., 135, 1-7.
I.iu. Z. P. & Yang, H. F. 11987). Reduction of azo dyes by
microorganisms. Environ. Pollut. & Control 9, 2-5.
Pagga, U. & Brown, D. [ 1986~. The degradation of dyestuff. Part
11: Behavior of dyestuff in aerobic biodcgradation tests.
Uhemosphere, 15, 479 91.
Urushigawa, Y. & Yonezawa, Y. (1977). Chemo-biological
interactions in biological purification system II -Biodcgra-
dation of azo compound by activated sludge. Bull. Eneiron.
Contain. Toxicol., 17, 214 18.
Wuhrmann, K. [ 19801. Investigation on rate-determining factors
in the microbial reduction of azo dyes. Eur. J. Microbiol.
Biotcctmol., 9, 325-38.
Xian, It. J. & Yang, H. F. (1988). Decoloratization of dyes by
microorganisms. Acta Scientiae Circumstantiae, 8, 266-74.
Zhu, Y. K., Xie, S. Q. & Dong, J. G. (19791. Primary test of
treating dye waste water by rotating algal disc. Era'iron. Sci., 6,
37 41.