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Degradation of crystal
violet by bacillus subtilis
a

C. Yatome , T. Ogawa & M. Matsui

Department of chemistry, Faculty of

Engineering , Gifu University , Yanagido 11,
Gifu, 50111, Japan
Published online: 15 Dec 2008.

To cite this article: C. Yatome , T. Ogawa & M. Matsui (1991) Degradation

of crystal violet by bacillus subtilis , Journal of Environmental Science and
Health . Part A: Environmental Science and Engineering and Toxicology: Toxic/
Hazardous Substances and Environmental Engineering, 26:1, 75-87, DOI:
10.1080/10934529109375621
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J. ENVIRON. SCI. HEALTH, A26(l), 75-87 (1991)

DEGRADATION OF CRYSTAL VIOLET BY

Baci11us
subtilis

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Key

words : C r y s t a l v i o l e t , m i c h l e r ' s k e t o n ,
degradation, Bacillus s u b t i 1 i s
C. Yatome, T. Ogawa and M. Matsui
Department of c h e m i s t r y ,
F a c u l t y of E n g i n e e r i n g ,
Gifu U n i v e r s i t y ,
Yanagido 1-1, G i f u 501-11, J a p a n
ABSTRACT

Crystal Violet
dye,

(BV3), a t y p i c a l

triphenylmethane

was d e g r a d e d by growing c e l l s of B a c i l l u s

s u b t i l i s IFO 13719 i n 802 medium, a l t h o u g h

their

growth was i n h i b i t e d a t an i n i t i a l s t a g e of
incubation.

The compound was d e g r a d e d a t a low

c o n c e n t r a t i o n below 7 10-6 mol dm-3 which b r o u g h t

a b o u t t h e i n h i b i t i o n of t h e growth
degradation

product

The major

of BV3 was e x t r a c t e d w i t h b e n z e n e

from t h e r e a c t i o n m i x t u r e and i d e n t i f i e d a s 4,4'bis(dimethylamino)benzophenone (Michler's

by GC-MS.

k e t o n ; MK)

I n a d d i t i o n t o BV3, two o t h e r

triphenylmethane

dyes ( P a r a r o s a n i l l n e and V i c t o r i a

b l u e ) were shown t o be d e g r a d e d by g r o w i n g c e l l s of B.
s u b t i l i s IFO 13719.
75
Copyright 1991 by Marcel Dekker, Ine.

76

YA.TOME, OGAWA, AND MATSUI

INTRODUCTION

Triphenylmethane dyes are used extensively

in the t e x t i l e industry, as the medicine and as the
biological s t a i n .

The degradation

of the dyes has

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received considerable a t t e n t i o n from the viewpoint of

waste water treatment
photoreduction

(1).

The ozonization and

of the dyes have been reported (2-5).

There have r e c e n t l y been the i n v e s t i g a t i o n s into the

d e c o l o r i z a t i o n and N-demethylation r e a c t i o n s of
Crystal Violet (BV3) by microorganisms (6-7).
However,the r a t e of the decolorization of BV3 i s not
so f a s t as expected, and no other d e t a i l e d
i n v e s t i g a t i o n i s reported on the metabolism
(catabolism) of the dye. In the present paper, we
report the microbial

degradation

of BV3 by Baci1lus

s u b t i l i s IFO 13719.

MATERIALS AND METHODS

Chemicals.

The dyes used are shown in TABLE 1.

Commercial C. I. Basic Violet 3, Crystal Violet (BV3),

was purified by preparative
and

thin-layer chromatography

then recrystal1ized from ethanol.

The other dyes

used were of reagent grade, but they were used without

any p u r i f i c a t i o n steps.

Their purity was examined by

thin-layer chromatography and confirmed to be

DEGRADATION OF CRYSTAL VIOLET

77

TABLE 1
Dyes was used

NHHCl

CI

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u jsj/^

C. I . Basic Yellow 2 ; Auramine C. I . Basic Red 9 ; Para

(BY2)
Rosaniline
(BR9)

C. I. Basic Violet 3 ;
Crystal Violet
(BV3)

homogeneous.

C. I. Basic Blue 26 ; Victoria

Blue
(BB26)

A b u f f e r ( S ^ r e n s e n b u f f e r ; pH 7.0)

c o n t a i n i n g 0.03 M p o t a s s i u m p h o s p h a t e a n d 0.03 M
s o d i u m p h o s p h a t e was u s e d t h r o u g h o u t t h i s s t u d y .

Microorganism and culture conditions.

Baci1lus

s u b t i l i s IFO 13719 (ATCC 6051), Nocardia g l o b e r u l a IFO

13510 (ATCC 21292), Pseudomonas c e p a c i a IFO 14595, and
Pseudomonas c r u c i v i a e IFO 12047 were k i n d l y
by I n s t i t u t e f o r Fermentation,

Osaka, Japan.

provided
Nocardia

c o r a l l i n a IAM 12121 (ATCC 4273), E s c h e r i c h i a c o l i IAM

1264 (ATCC 10798) and Pseudomonas s t u t z e r i IAM 12097
were k i n d l y provided by I n s t i t u t e of Applied

78

YATOME, OGAWA, AND MATSUI

Microbiology,

U n i v e r s i t y of Tokyo, J a p a n .

All s t r a i n s

were s t o r e d a t -80*C i n t h e f o l l o w i n g medium ( 2 cm ) :

K2HPO4, 12.6 g ; sodium c i t r a t e , 0.9 g ; MgSO4 7H2O,
0.2 g ; (NH 4 ) 2 SO 4 , 1.8 g; g l y c e r i n , 88 g; d e i o n i z e d
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water, 1 dm , because stock c u l t u r e s maintained on

n u t r i e n t agar s l a n t s a t ca. 5*C r e s u l t e d in t h e
d e c r e a s e of t h e i r a b i l i t y to degrade BV3.
The frozen c u l t u r e s (2 cm ) were p r e c u l t u r e d in a
LB medium (20 cm ) which contained t r y p t o n e ( D i f c o ) , 10
g ; y e a s t e x t r a c t ( D i f c o ) , 5 g ; NaCl, 5 g ; d e i o n i z e d
3

water,1 dm

or a 802 medium (20 cm ) which contained

polypeptone

(Nippon Seiyaku), 10 g ; y e a s t e x t r a c t

( D i f c o ) , 2 g ; MgSO4 7H2O, 1 g ; deionized water, 1

dm . Those p r e c u l t u r e s were incubated under t h e
following c o n d i t i o n s : J3. s u b t i 1 i s ; a t 37'C and pH 7.0
for 16 h. in a 802 medium, _P. cepacia ; a t 37'C and pH
7.0 f o r 18 h. in a 802 medium, _P. s t u t z e r i ; a t 35"C
and pH 7.0 f o r 18 h. in a 802 medium, P. c r u c l v l a e ;
at 30"C and pH 7.0 f o r 18 h.in a 802 medium,
N.coral I l n a and N. g l o b e r u l a ; a t 28*C and pH 7.0 f o r
16 h. in a LB medium, E. col 1 ; a t 37*C and pH 7.0 f o r
18 h. in a LB medium.
C e l l s were h a r v e s t e d from the p r e c u l t u r e s with
c e n t r i f u g a t l o n (3,400 rpm,15 min.), washed twice with
S/Srensen b u f f e r (pH 7.0) and then resuspended

in t h e

DEGRADATION OF CRYSTAL VIOLET

79

same buffer ("washed c e l l s " ) .

The absorbance of

washed c e l l s suspensions was adjusted to approximately

0.8 a t 680 nm and 1 cm path length.
Assay of decolorization.

Decolorization of BV3,

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BY2, BB26 and BR9 by the s t r a i n s was measured

spectrophotometrically.

2
A r e a c t i o n mixture (12 cm )
g

containing precultures (2 cm ), a LB or a 802 medium

3

(5 cm ) and dye (5 cm ) o r a r e a c t i o n mixture (12 cm )

Q

c o n t a i n i n g washed c e l l s s u s p e n s i o n s (2 cm ), S/Srensen
3

o r i g i n a l b u f f e r (5 cm ) and dye (5 cm ) was incubated

with shaking.

A f t e r an incubati'on, the r e s i d u a l dye

in a mixture was e x t r a c t e d with 1-butanol (5 cm ). The

absorbance of the e x t r a c t was measured a t A max of a
dye solved In 1-butanol s a t u r a t e d with water.
D e c o l o r i z a t i o n r a t e was c a l c u l a t e d a s follows :
D e c o l o r i z a t i o n (JO = ( i n i t i a l absorbance) - (observed
absorbance) / ( i n i t i a l absorbance) x 100.

Reaction of B. subtl1 Is with BV3. A reaction

mixture (120 cm3) containing B_. s u b t i l i s precultures

(20 cm3),802 medium (50 cm3) and BV3 (SxlO~ 6 mol dm'3,
50 cm3) was incubated with shaking at 37*C for 24 h.
The reaction mixture was centrifuged at 3,000rpm for
30 min., and the supernatants collected were adsorbed
onto a column (2.7 cm x 15 cm) of Extrelut 20 (E.
Merck AG, Darmstadt, Federal Republich of Germany) in

80

YATOME, OGAWA, AND MATSUI

the dark.

Holding t h e column a t room t e m p e r a t u r e f o r

20 min., benzene (60 cm ) was passed through t h e

column.

The r e a c t i o n product in t h e benzene e f f l u e n t

was i d e n t i f e d by gas chromatograph-mass s p e c t r o m e t r y .

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Chromatography.

An e f f l u e n t was c o n c e n t r a t e d and

s u b j e c t e d t o t h i n l a y e r chromatography (TLC ; S i l i c a
gel 60 F 2 54'

2 c m x 5 c m > E- M e r c k A G )

The

chromatogram was developed with e t h y l a c e t a t e - h e x a n e (6

: 4 v/v).

Spots were d e t e c t e d with UV l i g h t (254 nm).

Gas chromatograph-mass spectrometry (GC-MS).

P r e l i m i n a r y gas chromatographic a n a l y s i s of t h e
e f f l u e n t was c a r r i e d out with a Shimazu gas
chromatograph 4CPTF, equipped with a flame i o n i z a t i o n
d e t e c t o r and 1 m by 3 mm s t a i n l e s s s t e l l column packed
with S i l i c o n e OV-17 (2 %) on chromosorb WAW DMCS. The
i n j e c t i o n p o r t , column, and d e t e c t o r t e m p e r a t u r e s were
s e t a t 300 , 270 ,and 300*C, r e s p e c t i v e l y . The
3
-1
n i t r o g e n gas flow r a t e was 30 cm min . Gas
chromatograph-mass s p e c t r o m e t r y was performed on a
Shimazu mass s p e c t r o m e t e r model QP-1000 o p e r a t e d under
computer c o n t r o l .

E l e c t r o n impact mass s p e c t r a were

obtained a t 20 eV i o n i z i n g energy.

The GC c o n d i t i o n s

d e s c r i b e d above were a l s o used f o r g l a s s column GC-MS.

3
1
Helium a t a flow r a t e of 30 cm min was used as
c a r r i e r gas.

DEGRADATION OF CRYSTAL VIOLET

81

RESULTS AND DISCUSSION

D e c o l o r i z a t i o n o f BV3 by B. s u b t i l i s and o t h e r

strains.

D e c o l o r i z a t i o n of BV3 by B. s u b t i 1 i s , E.

c o l i , and_P. s t u t z e r i i s shown in FIG.1-3.

BV3 was

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remarkably d e c o l o r i z e d by B. s u b t i 1 i s a f t e r 8 h.,
although t h e growth of c e l l s was low beyond d e t e c t i o n
(B. s u b t i 1 i s e x h i b i t e d poor growth to a l e v e l below
detection).

But t h e compound was completely

d e c o l o r i z e d in 24 h., when t h e b a c t e r i a l growth was

abundant

(FIG. 1 . ) . The i n i t i a l c o n c e n t r a t i o n of j ^ .

s u b t i I i s in t h e medium was 0.4 mg (dry weight) cm

The d e c o l o r i z a t i o n of BV3 was observed only a t a low
c o n c e n t r a t i o n below 7.0 x 10" mol dm" . The growth
of t h e bacterium was completely i n h i b i t e d a t 1.5 - 2.0
x 10

mol d m .

The d e c o l o r i z a t i o n of BV3 was

s i m i l a r l y observed with N. c o r a l 1ina and_N. g l o b e r u l a ,

although t h e i r growth was poor.

BV3 was not

d e c o l o r i z e d by E. c o l i , although t h e bacterium grew

remarkbly (FIG.2.).

The same phenomenon obserbed in

c u l t u r e s of P. c e p a c i a and P. c r u c i v i a e wi th BV3. P.
s t u t z e r i n e i t h e r grew, nor d e c o l o r i z e d in a BV3 medium
(FIG. 3.).

In t h e course of t h e experiments d e s c r i b e d

above, t h e a c t i v i t y t o d e c o l o r i z e d BV3 appeared to be

gram-specific.

The a c t i v i t y was observed in

B . s u b t i l i s c u l t u r e s were incubated in 802 medium,but

82

YATOME, OGAWA, AND MATSUI

I n i t i a l BV3 cone. = 2.1 x 10"&mol m-3

pH 7.0, a t 37'C

1.0

100

E
c ^
O

* 3

c
o
0.5

Q,

a
N
o

o
o

0.0

CU
Q

Incubation
FIG. 1.

6
time,

24

h.

Decolorization of BV3 by Baci1lus subti1 is

(IFO 13719)

Initial BV3 cone = 2.1 x 10-6mol dra-3 Q

pH 7.0, at 37'C

1.0

100

Onm

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>

CO

>
3

0.5

"a
\_
o

Ol

k.

o
in
2

Incubation
FIG. 2 .

v/^0.0
24

t i m e , h.

D e c o l o r i z a t i o n of BV3 by E s c h e r i c h i a c o l i
(IAM 1 2 6 4 )

DEGRADATION OF CRYSTAL VIOLET

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100

c
o
"a

83

1.0

I n i t i a l BV3 cone. = 2.1 x 10"6mol dm"3

pH 7.0, a t 37'C

E a.
c >
o
J0.5

50

S 3

8 8 8SS22
2

Incubation
FIG. 3.

24

0.0 o

time, h.

D e c o l o r l z a t i o n of BV3 by Pseudomonas
s t u U e r i CIAM 12097)

that was not observed In the c u l t u r e s were incubated

in minimum medium (Spezizen medium: (NH^gSO., 2 g ;
K2HPO4, 14 g ; KH2PO4, 6 g ; sodium c i t r a t e , 1 g ;
MgSO4 7H2O, 0.2 g ; glucose, 6 g ; ion exchanged
water, 1 dm ). The a c t i v i t y to d e c o l o r i z e BV3 was not
a l s o observed in washed c e l l s c u l t u r e s were incubated
in S^irensen b u f f e r .

On the other hand, the enzyme

a c t i v i t y b r i n g s about t h e d e c o l o r i z a t i o n of BV3 was

not considered

to be e x t r a c e l l u l a r , because t h e

a c t i v i t y was not observed in t h e f i l t e r e d f l u i d

( e x t r a c e l l u l a r f l u i d ) of B . s u b t i l i s c u l t u r e s in 802
medium was concentrated 5 fold with c o l l o g i o n bag
(pore s i z e

84

YATOME, OGAWA, AND MATSUI

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c a.

24

0.0

Incubation time, h.
FIG. 4.

Decolorization of BR9 by Baci1lus subtil is

(IFO 13719)

I n i t i a l BB26 cone. = 2.1 x 10-6mol

pH 7.0, a t 37*C

n-3 O

1.0

I
10

0.5
c

2
4
6
Incubation time,
FIG. 5.

'0* 0.0
8
h.

D e c o l o r i z a t i o n of BB26 by B a c i l l u s s u b t i l i s
(IFO 13719)

DEGRADATION OF CRYSTAL VIOLET

85

TABLE 2
Specific a c t i v i t y (SA) of B. s u b t i I i s for the
decolorization
SA
_Q

_1

nmol dm
min
mg
(mg : d r y weight of c e l l s )
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BR9

BV3

BB26

0.01

0.03

0.05

_1

D e c o l o r i z a t i o n of BY2, a t y p i c a l diphenylmethane
dye, was not observed in any s t r a i n s , in a s i m i l a r
manner as i s shown in FIG. 2.
Decolorization

o f BR9 a n d BB26 b y B. s u b t i l i s .

D e c o l o r i z a t i o n of BR9 and BB26 by B_. s u b t i 1 i s i s shown

in FIG. 4 - 5 .

BR9 and BB26 were d e c o l o r i z e d in

c u l t u r e s of J3. s u b t i 1 i s , while growth of c e l l s was

remarkably observed.

The dyes were

completely

d e c o l o r i z e d by 24 h.. Despite good growth of t h e

c e l l s , the d e c o l o r i z a t i o n r a t e of BR9 was a l i t t l e
slower than t h a t of BB26 and BV3.

The s p e c i f i c

a c t i v i t y (SA) of J . s u b t i 1 i s f o r t h e d e c o l o r i z a t i o n of
BR9, BV3, and BB26 i s shown in TABLE 2.

SA increased

in t h e o r d e r , BB26 > BV3 > BR9.

Detection of a degradation product (MK) of BV3 by
13. subti I Is.

The benzene e f f l u e n t was subjected to

TLC ( S i l i c a gel 60 F 2 5 4 ) .
The

Four fraction was taken.

Rf value (0.68) of the f i r s t f r a c t i o n was

86

YATOME, OGAWA, AND MATSUI

Column, Silicone OV-17

Col. Temp., 270'C
N 2 , 30 ml min-l

Product MK

Authentic HK

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c
0*

>

a>

Retention

FIG. 6.

time, min.

Gas chromatograph of the degradation product

from BV3

identical to that of authentic MK.

The u l t r a v i o l e t

spectrum corresponding to the MK (\max 345 nm) was

obtained from the f r a c t i o n .

The analysis of the

second, third, and fourth fraction showed the media

components.

Next', the benzen effluent was applied on

GC-MS. The major product (about 95 a yield) in the

effluent showed an.identical retention time (3* 12")
on gas chromatograph to those of authentic MK, as
shown in FIG. 6 and the peaks of the corresponding
fraction appeared at m/z ( r e l . i n t e n s i t y ) ; 269

DEGRADATION OF CRYSTAL VIOLET

(M+l,

1 9 ) , 2 6 8 <M+, 1 0 0 ) , 2 6 7 ( M - l , 2 3 ) , 2 5 1 ( 1 8 ) , 2 2 4

(29) and 148 ( 7 2 ) .

coincided with
hand,

87

The mass numbers of peaks

t h e s e o f a u t h e n t i c MK.

leuco c r y s t a l

violet

were

On t h e o t h e r

was n o t d e t e c t e d on s i l i c a

gel TLC. We a r e p r e s e n t l y a t t e m p t i n g to i d e n t i f y
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a d d i t i o n a l p r o d u c t s and to c l a r i f y f u r t h e r d e g r a d a t i o n
of MK.

REFERENCES
1. C. R. Nelson and R. A. H i t e s , Aromatic Amines in
and near t h e B u f f a l o River. Environ. S c i . Technol.,
14, 1147-1149, 1980
2. J . J . P o r t e r and S. B. Spears JR, The
Photodecomposition of C. I . Basic Green 4. T e x t i l e
Chemist and C o l o r i s t , 2, 191-195, 1970
3. R. Nakamura and M. Hida, P h o t o r e a c t i o n of C r y s t a l
V i o l e t i n S o l u t i o n . J. Soc. F i b . Technol. Japan,
38, 183-190, 1982
4. N. Kuramoto and T. K i t o , The C o n t r i b u t i o n of
S i n g l e t Oxygen t o t h e Photofading of
Triphenylmethane and Related Dyes. Dyes and
Pigments, 3 49-58, 1982
5. M. Matsui, H. Nakabayashi, K. S h i b a t a , and Y.
Takase, Ozonization of Triphenylmethane Dyes.
B u l l . Chem. Soc. Jpn, 57, 3312-3316, 1984
6. K. Kwasniewska, Biodegradation of C r y s t a l V i o l e t
(hexamethyl- - r o s a n i l i n e c h l o r i d e ) by O x i d a t i v e Red
Yeasts. B u l l . Environ. Contam. Toxicol. 34, 323330, 1985
7. J. A. Bumpus and B. J. Brock, Biodegradation of
C r y s t a l V i o l e t by t h e White Rot Fungus. Appl.
Environ M i c r o b i o l . , 54, 1143-1150, 1988
Date Received:
Date Accepted:

08/13/90
09/17/90