5 Irene_365.pmd 26-2-2009.pmd


________________________
*Corresponding author, Phone: +62-298-321212,

Fax: +62-298-321433, E-mail: irene_meiti@yahoo.com

ISSN 1978-3477
Volume 2, Number 2, August 2008

   p 73 - 78

Optimum Concentration of Glucose and Orange II for Growth and

Decolorization of Orange II by Enterococcus faecalis ID6017

under Static Culture

V. IRENE MEITINIARTI1*, ENDANG S. SOETARTO2, EKO SUGIHARTO3, AND KRIS HERAWAN TIMOTIUS1

1 Faculty of Biology, Universitas Kristen Satya Wacana, Jalan Diponegoro 52-60, Salatiga 50711, Indonesia;
2 Faculty of Biology, 3 Department of Chemistry, Faculty of Natural Science and Mathematics,

Universitas Gadjah Mada, Jalan Teknika Selatan, Sekip Utara, Kampus Bulaksumur,  Yogyakarta 55281, Indonesia

Growth and decolorization performance of bacterial grown on azodyes-containing-medium is influenced by various

concentrations of carbon sources and azodyes. The optimum level of glucose and Orange II concentration for growth and

Orange II decolorization by Enterococcus faecalis ID6017 are reported in this paper. The experiments were carried out in liquid

static culture as batch experiments. Glucose and Orange II concentrations used in these experiments were 0.45, 0.90, 1.80 g l -1,

and 40, 80, 120 mg l-1, respectively. The specific growth rate and decolorization rate of Orange II by E. faecalis were highest

on the medium which contained at least  0.90 g l-1 glucose. It is necessary to note that glucose above 0.90 g l-1 gave no significant

difference. On the medium containing 0.90 g l-1 glucose and 80 mg l-1 Orange II, E. faecalis grew with the highest specific growth

rate (0.28 h-1) and Orange II decolorization rate (0.47 h-1). The maximum specific growth rate of biomass (µ
max

) and the half-

saturation coefficient (K
S
) under optimal conditions were 0.25 h-1 and 1.5 g.l-1, respectively. The kinetics of decolorization

indicated that the process followed first order kinetics with respect to the initial concentration of Orange II. The inhibition

constant (K
I
) was found to be 750 mg l-1 Orange II, indicating that Orange II concentration at e” 750 mg l-1 would inhibit

bacterial growth to decolorize Orange II.

Key words: Enterococcus faecalis, azodyes, decolorization, glucose, Orange II

_____________________________________________

Orange II is one of synthetic azodyes which has been

widely used for coloring of textiles, food, and cosmetics.

Orange II (Acid-Orange-7 or p-(2-hydroxy-1-naphthylazo)

benzenesulfonic acid sodium salt) has the molec(ular

structure  C
16

H
11

N
2
NaO

4
S. 5H

2
0 (Fig 1: Merck Index 1968).

This azodye has been used as a model substrate for azodye

degradation. Orange II can be degraded by Sphingomonas

sp. 1CX and E. faecalis ID6017 into two main intermediate

products i.e. sulphanilic acid and 1-amino-2-naphthol

(Coughlin et al. 1999; Meitiniarti et al. 2007). Both of them

may be mineralized further.

Azodyes are relatively resistant to microbial degradation

under conditions normally found in waste-water treatment

plants. However, several microorganisms are reported to be

able to transform azodyes into non-colored products or even

to mineralized products (Stolz 2001). Recently, a number of

studies have been focusing on microbial degradation of

azodyes (Kim et al. 1995). This is because physicochemical

methods used for color removal of the effluents show

disadvantages in terms of operational problems, high cost

and sludge production (Kapdan et al. 2000; Kodam et al.

2005). According to Rafii et al. (1990), human intestinal

microbiota generally found in the effluent of waste water

treatment and using of these bacteria for azodyes

degradation has been reported. In industrial waste-water

containing various dyes, azodye degraders should exhibit

decolorizing ability for a wide range of dyes and

environmental conditions (Fang et al. 2005). In regard to

this E. faecalis is a bacterial species which has been reported

to decolorize azodyes (Handayani et al. 2007). Therefore the

decolorization of azodyes is an important topic for study.

Bacterial azodyes degradation is usually initiated by

cleavage of the azo bond under anaerobic conditions

resulting in the formation of aromatic amine colorless

products. Hence the process is called decolorization

(Zimmermann et al. 1982; Tan 2001; van der Zee 2002).

The performance of microbial decolorization rates

depends on the activity of the microorganism through a

specific enzymatic process and is affected by environmental

conditions, such as substrate concentration, composition

of the medium, pH, carbon and nitrogen sources, and

incubation temperature. Sphingomonas sp. strain 1CX has

an ability to decolorize 20 mg l-1 Orange II, Acid Orange 8,

Acid Orange 10, Acid Red 4, or Acid Red (Coughlin et al.

1997), where as E. faecalis ID6017 has an ability to decolorize

Acid Red 27 and Reactive Red 2 up to concentration of

100 mg l-1 in batch system (Handayani et al. 2007) and

Orange II up to concentration 120 mg l-1 in fedbacth system

(Meitiniarti et al. 2006). Meitiniarti et al. (2005) reported that

E. faecalis produces an Orange II reductase. Several

environmental conditions, such as substrate and co-

substrate concentration, could influence the enzymes activity

(Tan 2001). In this paper, we discuss the optimum level of

glucose as co-substrate and Orange II concentrations on

the growth kinetics of E. faecalis and Orange II decolorization

performance.

Fig 1  Chemical structrure of Orange II.

OH

N=N SO
3
Na.5H

2
O



74   MEITINIARTI  ET AL.                                                                                Microbiol Indones

MATERIALS  AND  METHODS

Microorganism and Culture Condition. E. faecalis

ID6017 was used as a bacterial model for the decolorization

of Orange II and was maintained on a slant media of

Trypticase Soy Agar (TSA) at room temperature (26-28°C)

as a stock culture. E. faecalis was grown on liquid-basal-

medium consisting of (g l-1): 0.25 MgSO
4
.7H

2
O, 1.98

(NH
4
)

2
SO

4
, 5.55 K

2
HPO

4
, 2.13 KH

2
PO

4
, and 0.25 yeast extract.

Glucose was added in concentrations of 0.45, 0.90, and

1.80 g l-1 Orange II (Merck, CI Acid Orange 7, CI 15510,

MW= 440.41) was added in concentrations of 0, 40, 80, and

120 mg l-1.

Inoculum Preparation. 48-h-old TSA slant cultures of

E. faecalis were grown on 500 ml Erlenmeyer flask, containing

basal medium without Orange II, for 24 h on a shaker (150 rpm).

This culture was the pre-culture (inoculum).

Experimental Design and Culture Conditions. The

experiments were carried out in two batchs using liquid

medium for 9 h at room temperature. In the first experiment,

three flasks each containing 80 mg l-1 Orange II medium were

prepared and each flask was added 0.45, 0.90, and 1.80 g l-1

glucose. This experiment was done to determine the glucose

concentration that increased bacterial growth and Orange II

decolorization rate. The glucose concentration which resulted

in increasing growth and decolorization rate, was selected

and used as the limited-substrate-concentration. In the

second group of experiments, the selected glucose

concentration was then supplemented with 40, 80, and

120 mg l-1 Orange II concentrations.  Experiments was car-

ried out in triplicate.

As much as 50 ml aliquots of pre-culture were transfered

to 450 ml medium in a 1 l volume growing vessels (Duran)

with a rubber stopper, and incubated under static condi-

tions at room temperature. At interval of one hour, 10 ml

aliquots of each culture was removed up the late exponential

growth-phase. Parameters examined were concentrations of

glucose, Orange II, cell mass (biomass) and sulphanilic acid

(as a decolorization product of Orange II).

Analytical Methods. Samples were centrifused at 3 326g

for 30 min to separate biomass from the supernatant. The

supernatant was analyzed for glucose, Orange II, and

sulphanilic acid concentrations. After being washed twice,

the biomass was re-suspended into an initial volume (10 ml)

using distilled water and examined spectrophotometrically

(ë
600nm

). The biomass concentration in units of cell dry weight

was calculated using a standard curve of optical density

(OD
600nm

; y-axis) against biomass concentration (cell dry

weight) (mg l-1; x-axis).

Glucose concentrations were determined spectro-

photometrically (ë
540nm

) using the DNS reagent method

(James 1995) and Orange II concentration were determined

spectrophotometrically (ë
482nm

) (Zimmermann et al. 1982).

The sulphanilic acid concentration was analyzed by

injecting five to ten µl sampels of supernatant into a HPLC

Shimadzu model LC-3A chromatograph, equipped with UV-

detector and ODS column (150 mm x 8 mm). The mobile phase

was composed of methanol and acetic acid 0.6% (v/v) = 60:40.

The flow rate was 0.7 ml min-1. The eluent were monitored

using a UV detector at 276 nm (Chang et al. 2001; Supaka et

al. 2004).

Determination of Kinetic Parameters (k, µµµµµ, µµµµµ
max

, K
S
, and

K
I
). The ability of bacterial strains to decolorize Orange II

was determined by calculating the decolorization rate used

equation (1) (Wuhrmann et al. 1980):

 Note: k is decolorization rate (decrease of color intensity);
C

0
 and C are color concentrations (mg l-1) at t

0 
and t; t

0 
and t

are time (h).

The substrate/co-substrate consumption and decreasing

of decolorization product (sulphanilic acid) was calculated

using equation (2):

Note: S is substrate/co-substrate consumption (mg); S
0
 is

glucose/Orange II concentration (mg l-1) at t
0
; S

t
 is the lowest

glucose/Orange II concentration or the highest sulphanilic

acid concentration (mg l-1); S
e
 is the lowest sulphanilic acid

concentration (mg l-1); t
0
,
 
t, and t

e
 are time (h).

The specific growth rate (µ ) is determined from the cell

mass as a first order with the following equation:

Integration of equation 3 yields:

where X  and X
0
 are biomass concentrations at time t  and

t=0.

Generally, the growth rate is restricted by the concentration

of the growth-limiting substrate that can be described by the

Monod Equation:

where µ
max

 is the maximum specific growth rate when  S>>K
S
.

The constant  K
S
 is the saturation constant or half-velocity

constant and is equal to the concentration of the rate-

limiting substrate when the specific rate of growth is equal

to 50% of the maximum.

In this paper, the Monod equation modified by the Hanes

model (called the Hanes-Monod Model) was used to analyze

the values of µ
max

 and K
S
 (Brandt 2001):

A plot of against is linear with a slope of  1/u and an

intercept on the y-axis of 

max

1

µ

.

When a substrate is inhibited by its own biodegradation,

like Orange II, the Monod model was derivatived by

incorporating the inhibition constant K
I
. Among the substrate

inhibition models, the Andrew’s equation (7) is most widely

used (Okpokwasili and Nweke 2005):

S = S
0
-S

t
     or    S = S

t
-S

e

SK

S

S
+

=
.

max
µ

µ

maxmax
µµµ

S
KSS

+=

 ln (C
0
-C)

(t-t
0
)

k =  ———————

,X
dt

dX
µ=    

0
XX =    at t=0

t
eXX

µ

0
=,ln

0

t
X

X
µ=    or



Volume 2, 2008                                          Microbiol Indones   75

where K
I
 is the inhibition constant (calculated by Microsoft

Excel Software in this paper).

RESULTS

Optimal Level of Glucose. The addition of various

concentration of glucose (0.45 to 1.80 g l-1) did not give

any significant difference in the specific growth rate, biomass

production of  E. faecalis or the concentration of sulphanilic

acid (Table 1 and Fig 2). However, as shown in Table 1, the

decolorization of Orange II was significantly influenced by

the addition of glucose. By calculating glucose consumption

in three initial glucose concentrations, we showed that by

increasing the glucose concentration, from 0.90 to 1.80 g l-1, this

did not efficiently increased the specific growth rate, biomass

production, and Orange II reduction by  E. faecalis. Thus, in

the second experiment, we used optimal glucose concentration

i.e. 900 mg l-1.

Optimal Level of Orange II. The specific growth rate of

E. faecalis in Orange II concentration from 40 to 120 mg l-1

the optimal glucose concentration was not significantly

different to that of the control (Fig 3). Based on the specific

growth rate of E. faecalis, our results shown that by

increasing the Orange II concentration did not increase the

specific growth rate. By calculating the kinetic parameter of

growth our results show that the best specific growth rate of

E. faecalis was on the 80 mg l-1 Orange II-containing-

medium (Table 2). In the Orange II 120 mg l-1 containing

medium, the decolorization rate and capacity were higher

than the lower Orange II concentration (40-80 mg l-1) (Table 2).

The Maximum Specific Growth Rate (µµµµµ
max

), the Half-

Maximum Saturation Coefficient (K
s
), and the Inhibition

Constant (K
I
). By calculating µ

max
 and K

s
 values using the

Hanes-Monod model for E. faecalis growing on medium

containing various glucose consentrations, we obtained

values of µ
max

 and K
s
  of 0.22 h-1 and 0.05 g l-1, respectively

(Fig 4). By similar methods, the values of µ
max

 and K
s
 for

E. faecalis growth on medium containing various Orange II

concentrations could be determined. The values of µ
max

 and

K
s
 were 0.25 h-1 and 1.5 mg l-1, respectively (Fig 5). Based on

the kinetic parameters obtained: m (0.26, 0.28, and 0.25 h-1 for

40, 80, and 120 mg l-1 Orange II, respectively), µ
max

 (0.25),

and K
s
 (1.5) with various initial S (Orange II) concentrations,

the K
I
 value could be calculated using microsoft excel

programme by inserting the assumed K
I
 value as a variable.

After 15 iterations, the best fit (i.e between the m values

calculated by Hanes-Monod method and the m Haldane,

correlation coefficient R2 ≅ 1) gave an Orange II K
I 
value of

750 mg l-1.

DISCUSSION

As shown by the data in Table 1 and Fig 2, increasing the

glucose concentration from 0.45 to 0.90 g l-1, increased

specific growth rate and biomass production. However,

increasing the glucose concentration from 0.90 to 1.80 g l-1

did not significantly increase the specific growth rate and

I

S
K

S
SK

S
2max

++

⋅= µµ

Table 1  The influence of glucose on Enterococcus faecalis ID6017 growth and its performance for Orange II decolorization in the medium

containing 80 mg l-1 Orange II and several concentrations of glucose

Parameter of growth and decolorization
Glucose concentration (g l-1)

0.45 0.90 1.80

Specific growth rate  (h-1)

Biomass produced (mg)

Glucose consumption (g)

Orange II reduction (mg)

Decolorization rate (h-1)

Capacity of decolorization (mg substrate/mg cell d.w.)

Concentration of sulphanilic acid (mg l-1)

  0.18 + 0.01

        29.3 + 2.518.04*

   0.39 + 0.005

65.9 + 4.4

0.51 + 0,1

3.65 + 0.3

32.5 + 0.3

 0.20 + 0.02

        30.1 + 7.422.86*

   0.67 + 0.007

71.1 + 3.2

0.78 + 0.2

3.11 + 0.1

  35.5 + 0.12

 0.21 + 0.02

        31.3 + 3.716.52*

   0.79 + 0.009

72.5 + 8.6

0.77 + 0.2

4.39 + 0.5

 36.7 + 0.71

*biomass produced during decolorization of Orange II.

B
io

m
a
ss

 c
o

n
c
n

 (
m

g
 l

-1
)

5 0

4 5

4 0

3 5

3 0

2 5

2 0

1 5

1 0

5

0
1 2 3 4 5 6 7 80

Incubation time (h)

y1= 13.091e0.1797x

R2= 0.991

y2= 12.628e0.2042x

R2= 0.9513

y3= 8.8155e0.2086x

R2= 0.9845

a

 ( ) µ1= 0.1797 h-1; ( ) µ2= 0.2042 h-1; ( ) µ3= 0.2086 h-1

1 2 3 4 5 6

L
n
 O

ra
n
g
e
 I

I 
c
o
n
c
n
 (

m
g
 l

-1
)

5

4 . 5

4

3 . 5

3

2 . 5

2

1 . 5

1

0 . 5

0

0

y3= -0.7782x + 6.1293

R2= 0.9666

y2= -0.7832x + 6.1398

R2= 0.9616

y1= -0.5039x + 5.3186

R2= 0.9479

Incubation time (h)

b

 ( ) k1= 0.509 h-1; ( ) k2= 0.7832 h-1; ( ) k3= 0.7782 h-1

Fig 2  Specific growth rate (a) and Orange II decolorization rate, (b) during Enterococcus faecalis growing on the medium containing

Orange II and 0.45 g l-1 glucose ( ), 0.90 g l-1 glucose ( ), and 1.80 g l-1 glucos ( ).



76   MEITINIARTI  ET AL.                                                                                Microbiol Indones

biomass produced by the bacteria. This might be because

the glucose concentration of 0.90 g l-1 is adequate to support

the growth of E. faecalis. It was also shown from glucose

consumption and biomass production that these were low

during bacterial growth. For their growth, bacteria require C

and N sources in certain proportions (Thingstad and Lignell

1997). In the 0.90 g l-1 glucose containing medium, the

proportions of C and N sources were suitable for bacterial

growth. Thus, the bacteria grew fast and produced more

biomass in this medium.

amount of glucose was related to the rate of decolorization

and the amount of reduced Orange II.

The increase in Orange II concentration from 40 to

80 mg l-1 raised the specific growth rate of E. faecalis.

However, when Orange II was increased up to 120 mg l-1,

it decreased the specific growth rate of E. faecalis. These

results show that E. faecalis  did not use Orange II as a

growth substrate and might be toxic for bacterial growth.

The highest specific growth rate of E. faecalis was on the

medium containing 80 mg l-1 Orange II. This might be due to

the fact that in 80 mg l-1 Orange II concentration, the bacteria

B
io

m
a
s 

c
o

n
c
n

 (
m

g
 l

-1
)

8 0

6 0

4 0

2 0

0

2 4 8 1 060

Incubation time (h)

Fig 3  The growth of Enterococcus faecalis on the medium with-
out Orange II ( ), with 40 mg l-1; ( ), 80 mg l-1; ( ), and 120 mg

l-1 ( ), Orange II. Bars indicated standard error where P=0.05.

  0.25 + 0.03

33.72 + 7.50

       23.90*

756.2 + 22.9

74.45 + 6.38

      62.00

  0.32 + 0.05

  2.77 + 0.57

 37.02 + 0,71

In contrast to the influence of glucose on bacterial

growth, the decolorization of Orange II was significantly

influenced by the addition of glucose. The consumption of

glucose was related with the rate of decolorization and the

amount of reduced Orange II. This means that additional

glucose as an energy source is needed by the E. faecalis to

decompose Orange II i.e. decolorization of Orange II is an

energy consuming activity.

The addition of glucose did not give any significant

difference on sulphanilic acid formation. Based on the

concentration of sulphanilic acid at the end of cultivation, it

showed that the concentration of sulphanilic acid was quite

high. This is because this bacteria can not metabolise

sulphanilic acid (Meitiniarti et al. 2007), although the added

Table 2  The influence of Orange II on Enterococcus faecalis ID6017 growth and its performance for Orange II decolourization in the

medium contained 0.90 g l-1 glucose and several concentrations of Orange II

Parameter of growth and decolorization
Orange II concentration (mg l-1)

0 4 0 8 0 120

Specific growth rate (h-1)

Biomass produced (mg)

Glucose consumption (g)

Orange II reduction (mg)

Orange II reduction (%)

Decolorization rate (h-1)

Dye removal capacity (mg substrate/mg cell d.w)

Concentration of sulphanilic acid (mg l-1)

        0.28 + 0.03

      51.77 + 8.10

      695.6 + 12.9

n.d

n.d

n.d

n.d

  0.26 + 0.02

45.04 + 11.90

      25.10*

718.2 + 38.6

33.63 + 4.70

      84.00

  0.95 + 0.14

  1.55 + 0.19

  24.2 + 2.9

 0.28 + 0.02

34.1 + 7.1

       22.9*

732.7 + 25.0

64.22 + 6.38

      80.27

  0.47 + 0.07

  2.64 + 0.32

34.02 + 3.09

*biomass produced during removal of Orange II, n.d: not determined.

1 0

8

6

4

2

0
0 0 . 5 1 . 0 1 . 5 2 . 0

G
lu

c
o

se
 c

o
n

c
n

/s
p

e
c
if

ic
 g

ro
w

th
 r

a
te

 (
S

/µ
)

Glucose concentration (S) (g l-1)

y= 4.6111x + 0.2503

R2= 0.9961

Fig 4  A plot of glucose concentration/specific growth rate (S/µ)

vs glucose concentration (S) resulting in a straight line with slope

equal to 1/µ
max

= 4.61 and an intercept of K
S
/µ

max
= 0.25 for various co-

substrate concentrations on growing medium of Enterococcus faecalis.

O
r
a

n
g

e
 I

I 
c
o

n
c
n

/s
p

e
c
if

ic
 g

ro
w

th
 r

a
te

(S
/µ

)

6 0 0

5 0 0

400

3 0 0

2 0 0

100

0
0 5 0 1 0 0 150

Fig 5  A plot Orange II concentration/specific growth rate (S/µ)

vs Orange II concentration (S) resulting in a straight line with a slope

equal to 1/µ
max

= 3.93 and an intercept of K
S
/µ

max
= 5.89 for various

substrate concentrations on growing medium of Enterococcus faecalis.

Orange II concentration (S) (mg l-1)



Volume 2, 2008                                          Microbiol Indones   77

nutrient requirement was fullfilled and they could tolerate

Orange II concentration. So, they could grow favorably.

Based on the decolorization rate (Table 2), our results

show that the increase in Orange II concentration also

increased the decolorization rate. This is because the

reductase enzyme activity of E. faecalis to remove or reduce

Orange II, was related to substrate concentration (Orange

II).  The kinetic analysis of decolorization indicated that the

decolorization process followed first order kinetics with

respect to the initial concentration of Orange II. E. faecalis

had the highest Orange II decolorization rate on the medium

containing 80 mg l-1 Orange II because E. faecalis could

not use Orange II as a growing substrate. At a higher

concentration, Orange II did inhibit E. faecalis growth.

As shown on Table 2, the increase in the decolorization

rate at higher Orange II concentrations would not decrease

sulphanilic acid concentration. It seems that although the

sulphanilic acid concentration increased due to increasing

of Orange II decolorization,  E. faecalis could not metabolise

sulphanilic acid. This would result in the accumulation of

sulphanilic acid in the medium. Mendez-Paz et al. (2005)

reported that sulphanilic acid produced by Orange II

decolorization was not degrade futher and it would then

be accumulated in the medium.

The µ
max

 value obtained from E. faecalis, grown on

medium containing various glucose concentrations, was

similar to the specific growth rate of E. faecalis which was

grown on the medium contained of 0.9-1.8 g l-1 glucose

concentration. This result showed that by increasing the

glucose concentration up to 1.86 g l-1, it would not efficiently

increase the growth of E. faecalis. A low value of K
s
 obtained

here indicated that bacteria had a very high affinity for

glucose as a carbon substrate. Hence, the bacterial growth

would not be affected until the glucose concentration

declined to a very low level. This result also showed that

using glucose at an optimal concentration is quite economic

when applied in waste-water-treatment plants.

The µ
max

 values of E. faecalis growth with various

Orange II concentrations was similar with the specific growth

rate of E. faecalis grown on the medium containing lower

than 40 mg l-1 Orange II concentration. This result indicated

that Orange II was not a growth substrate for E. faecalis.

Futhermore, when the K
s
 value of Orange II was compared

with that of glucose, it showed that bacterial affinity to

Orange II was lower than to glucose. This result indicated

that E. faecalis would preferentially use glucose on the

medium for rapid growth (Thingstad and Lignell 1997).

Although a decrease in Orange II concentration occurred in

these experiments, it is not be due to Orange II consumption

as a substrate. Orange II might be used together with direct

growing supporting substrate via cometabolism.

Based on the value (750 mg l-1) of inhibition coefficient

K
I 
obtained in this experiment, it is shown that Orange II at a

concentration of ≥ 750 mg l-1 would inhibit growth of E.

faecalis. The K
I 
value of Orange II also indicated that although

µ of E. faecalis on the medium contained of 120 mg l-1 Orange II

decreased, Orange II concentration used in this experiment

(40, 80, and 120 mg l-1) was too low to inhibit the growth of E.

faecalis.

Based on our results, it can be concluded that by

increasing the glucose (as a co-substrate) concentration up

to 0.90 g l-1 and Orange II up to 80 mg l-1, we could increase

the specific growth rate and Orange II decolorizatin rate of

E. faecalis. An  increase in Orange II concentration above

80 mg l-1 would decrease the specific growth rate of E.

faecalis, and when the concentration was increased to more

than 750 mg l-1, the growth of bacteria will be inhibited.

Although the actual dye concentration in effluent is not

more than 750 mg l-1, we can predict that the specific growth

rate of bacteria will decrease when the Orange II concentration

was more than 80 mg l-1. Moreover, our results show that E.

faecalis could not consume sulfanilic acid as one of the

intermediate products of azodye degradation. Accordingly,

it is suggested that this bacteria should not be used alone in

dye-contaminated waste-water treatment, but should be

mixed with other bacterial species which could consume

intermediate products of azodye degradation and had the

ability to degrade other kinds of azodyes.

REFERENCES

Brandt BW. 2001. Realistic characterization of biodegradation.

[doctoral thesis]. Amsterdam: Vrije Universiteit.

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78   MEITINIARTI  ET AL.                                                                                Microbiol Indones

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