Indian Journal of Experimental Biology

Vol. 40, January 2002, pp. 101-105

Degradation of predigested distillery effluent by isolated bacterial strains

Neeraj Jain, A

Minocha & C L Verma*

Environmental Science and Technology Division, Central Building Research Institute, Roorkee 247 667, India

Received 17 May 2001; revised 23 August 2001

Batch studies were conducted on degradation of anaerobically digested distillery wastewater by three bacterial strains,
viz.

Xanthomonas !ragariae, Bacillus megateriu/1/

and

Bacillus cereus

in free and immobilized form, isolated from the

activated sludge of a distillery wastewater treatment plant. The removal of COD and colour with all the three strains
increased with time up to 48 hr and only marginal increase in COD and colour removal efficiency was observed beyond this
period up to 72 hI'. After this period removal efficiency remained fairly constant up to 120 hI'. The maximum COD and

colour removal efficiency varied from 66 to 81 % and 65 to 75 %, respectively for both free and immobilized cells of all the
three strains. Thc strain

Bacillus cereus showed the maximum efficiency

of COD (81 %) and colour (75 %) removal out of

the three strains. An interrelationship between the percent COD and colour removal was carried out by correlation and
regression analysis and was justified by high values of coefficient of correlation (r

0.99) for all the cases. The first order

removal rate kinetics was also applied and rate constants were evaluated for COD and colour removal efficiencies.

Water pollution by industrial wastes has become a

major problem in India. Distilleries are one of the
major polluting industries with about 88% of raw
materials ending up as wastes. The large volume of
spent wash (15-20 III of alcohol produced), generated
during the production of alcohol from distillation
section, contributes a major fraction of wastewater
from distilleries. Besides carrying appreciable organic
load, spent wash is dark coloured and highly acidic
with a strong objectionable odour. All of which pose
serious environmental problems that entail treatment
of distillery wastewater prior to dumping into
receiving bodies.
Distillery wastewaters, generally treated with
physico-chemical and biological treatments like
. 1
1
coagl!IatlOn 2
. , adsorptlOn-, reverse osmOSIS, etc. h ave
met with lit Ie success due to high cost and enormous
amount of sludge produced which required further
treatment.
Of the several biological treatment methods
available, anaerobic digestion followed by aerobic
treatment has gained wide acceptability due to
methane recovery in anaerobic step of the treatment.
The anaerobic digestion alternatives included are:
6
conventional digesters4.5, anaerobic filter -8, \,vo
9.lo
phase biomethanation , up-flow anaerobic sludge
11.12
blanket (U ASB) reactors
The ava: lable literature
.
reveals that anaerobic treatment can lead to 60-85 %
.

*Correspondent author

Tel: ++91-1332-82306: Fax: ++91-1332-72272

E-mai!: clverma@cscbri.ren.nic.in

of BOD reduction, but still the substantial amount of

organic pollutants are left behind and require
secondary (aerobic) treatment.
Aerobic treatment of distillery wastewater was also
performed in activated sludge process and bio
kinetics coefficients were evaluatedI3-15. Though
aerobic treatment processes are capable of reducing
about 90 % of the incoming BOD, the effluent still
contains appreciable amount of colour, which poses
serious disposal problems.
In the view of the above problems related to the
treatment of distillery wastewater, there is a need to
find out a techno-economically feasible solution. With
the knowledge of the importance of pure bacterial
cultures and their probable use for the treatment of
industrial wastewater16, the present study deals with
the degradation of anaerobically digested spent wash
by bacterial strains viz. Bacillus cereus, Xanthomonas
Jragariae and Bacillus megaterium in free and
immobilized form.
Materials and Methods

Effluent-Predigested spent wash samples were

collected from a distillery situated in western U.P.
The sample was stored at 4C and sterilized sample
was used through this study. The sample was analysed
for various parameters as de cribed in the Standard
Methodsl7. The main characteristics of spent wash
were: pH = 7.6, COD= 12048 mg/l, BOD=6,880
mg/I, colour = 12,OOO Co-Pt units, total solids =
17,800 mg/I, Kjeldal nitrogen = 98 mgll and
phosphorus = 38 mg/1.

INDIAN J EXP BIOL, JANUARY 2002

102

Organisms-The bacterial strains used were

isolated from the activated sludge of a distillery
wastewater treatment plant and were identified as
Bacillus cereus, Xanthomollas fragariae and Bacillus
l8
megaterium . After checking the purity of the strains,
stock cultures of the strains were maintained on
nutrient agar slants at 4C and subcultured
periodically.
Media and culture conditions
I nitially all the
three bacterial strains were grown in basal medium 19
having the following composition (gll):(NH4hS04,
2.6; K2HP04, 1.0; KH2P04, 0.1; MgS04, 0.2; CaCI2,
0.0 I; FeS04, 0.00 i; yeast extract, 0.1 and 2.0 glucose
as a carbon source. The pH of the medium was
adjusted to 7.2 (using HCllNaOH) before autoclaving.
Portions of inoculated medium (150 ml) were
incubated in 500 ml flasks on a rotary shaker at 30C
for 3 days.
Preparation of inocula -Bacterial masses of each
strain were collected by centrifugation in a R23 REMI
research centrifuge at 5000 g for IS min from each
cuilure flask, washed with sterile water and
resuspended in ISO ml of sterile water to give a cell
4
cells/ml.
The above
suspension of 102 10
homogenates obtained as above served as the inocula
for both free and immobilized cells.
Preparation of alginate beads-Immobilization of
each bacterial strain was carried out using calcium
alginate entrapment method. For immobilization, 4 g
bacterial mass was suspended in 100 ml of sterile
water. This homogeneous cell suspension (100 ml)
was added to equal volume of 3% sterilized sodium
alginate solution. After thorough mlxmg, the
homogeneous solution was dropped into a solution of
calcium chloride (0.2 N) by means of hypodermic
needle to get spherical beads of about 3 mm diameter.
These beads were kept in a fresh calcium chloride
solution overnight to achieve complete gelation and
stored at 4C for batch studies after washing with tap
water.
-

Batch process
Batch experiments were carried
out in 500 ml Erlenmeyer flasks containing 200 ml of
sterilized spent wash (1:3 diluted, COD=3000 mg/l
and colour = 3024 Co-Pt units) supplemented with
nutrients at optimum concentration and pH for
Bacillus cereus, Xanthomonas fragariae and Bacillus
2o
megaterium . Glucose as extra carbon source and
NH4Ci as additional nitrogen source were added in the
sample at a concentration of 0.8 % and 0.12%
respectively. KH2P04 was added as a source of
phosphorus at a concentration of 0.24%. The pH of
-

the sample was maintained at 7 in all the cases. For

free cell study, each flask was inoculated with 20 ml
inocula of the strain to study and for immobilized
form, 200 beads were added. Experiments were
carried out in duplicate. All flasks were kept in a
water bath at 30C and the level of dissolved oxygen
was maintained at 1-1.5 mg/l in each flask by
compressed <.iir and checked throughout the
experiment. Contents of each flask were analyzed for
COD and colour at an interval of 2, 4, 6, 8, 10, 12, 24,
48, 72, 96 and 120 hr. Undiluted, without nutrients
supplemented (C, N and P) and non-inoculated
controls were also kept at the same conditions with
the above flasks.
Determination of COD and colo u r
COD was
l7
measured by dichromate method . Colour units were
determined as cobalt-platinum (Co-Pt) units, on
U-3200 spectrophotometer (Hitachi) at 465 nm as
17
described earlier .
-

Results and Discussion

The results are presented in Figs 1-4.

All the bacterial strains used show continued
activity until about the end of 120lh hr of the
experiment in free and immobilized forms. The
results clearly indicate that Bacillus cereus exhibits
highest removal efficiency in free and immobilized
forms. However, the difference in the removal
efficiency is not much in both the free and
immobilized forms. It is also e ident that the
performance of free cells is better than that of
immobilized cells. This may be due to easy
availability of the substrate/nutrients and better
oxygen transfer capacity in case of free cells. No
perceptible changes in COD and colour removal were
observed in all the controls. The observed efficiency
of COD and colour removal was almost negligible
(less than 5%).
Interrelationship

between

COD

and

colour

colour removal efficiency increases

with the increase in COD removal efficiency in both
free and immobilized forms (Figs 1-3). As such a
correlation
and
regression
analysis
of
the
experimental data of COD and colour removal
efficiency of free and immobilized cells was carried
out with the presumption that it follows a straight line
relationship of the form:
Ycol=b Xcoo+C
Where, Ycol = percent colour removal
B =slope of the line
Xcoo = percent COD removal
C = intercept on Y-axis
removal-The

JAIN el. al.: PREDIGESTED DISTILLERY EFFLUENT DIGESTED BY BACTERIAL STRAINS

103

The results of the analysis are presented in Figs 4a

and b for free and immobilized forms respectively.
The linear relationship between colour and COD
(Ycol, Xcoo) reduction is justified by high values' of
co-efficient of correlation (r) in both the cases and it
can be attributed that the colour causing materials in
spent wash are organic in nature and are degraded by
the bacterial strains and subsequently results in colour
removal. The plot (Fig. 4a) has a slope of around 1
with COD removal values of 7.8% in free form and

3.6% in immobilized form (Fig. 4b) at colour removal

of 0%. This may be attributed to the degradation of
some organic matter (glucose) which is not colour
causing.

90 .-----

90 .-----,

Kinetics

80

80

70

70

50

50

l( - *

----

l:r---6

1.0

--.-

COD lobservedl

CO Dlcalculotedl

Colourl observedl

80

50

f/

"'

/ ",

//

)It
/./.

...-

COD

-761 1-e -0,06 tl

)(
---- --------

_.. ..-.

/(:,

.- . .!:..

col

_0_._.

COD Icalcul ated I

Jf

-.-.-

Colourlcalculotedl

C olour( obser ved I

u
'
Q)

90

__

__
_L
__
_L
__

r--------------------------- ---------.

(b)

0.80

70

-7211-e-o'OStJ

50

/ I/

50

COD 1 0 bserved!

----

__

.,/---

70

50

/ ./
,

'1J

1.0

)(, i
'(:,i

1.0

)(,/(:'i
I
,I

30

20

l(

*- *

fr--/::.

OL-_L__L-

90 r------'

a.

/"

'-Ki
'/
'I,'i

1.0

Col ourl calculatedl

removal-

/.:u---Y 1-63 11-e-0'0 6tl

co

/"

Av
//
//(:,

50

11 O
(b)

colour

l<---------==-.

20

Q)

and

e -01)7tl
Y
COD-55 11-

30

COD

(a)

(a)

50

of the

Analysis of the data (Figs 1 to 3) exhibits a first order

reaction kinetics for the removal of COD and colour
with time for all the three strains both in free and
immobilized forms. As such, a mathematical formula
21
tion following the expression:

30

if
Kiff

20

1 0f-!i

k
I!<
O
o

__
_L__

__

__
-L

__

20

1.0

60

80

100

120

Time (hOUr)
Fig. 1- Removal of COD and colour as a function of time by
Xalllhomonas fragariae (a) free cells and (b) im mobilized cells

20

1.0

50

Time (houri

80

100

120

Fig. 2-Removal of COD and colour as a function of time by

Bacillus megaterium (a) free cells and (b) immobilized cells

INDIAN J EX!" SIOL. JANUARY

104

Yt = Y max (1- e-kt)

Where, Yt = percent removal of COD or colour at time t
Y max = maximum attainable percent removal of

COD or colour
k = rate constant
t = time in hours
was fitted in the data and reaction rate constant (k)
and Y max were determined by Thomas method22. The
values of Y max and k for COD and colour removal by
Xanthomonas jragariae, Bacillus megaterium and
Bacillus cereus in free and immobilized forms are

2002

shown in Table 1. U sing these parameters, percent

removal of COD and colour was calculated from 2 to
120 hr with the help of the above equation for all the
three strains.
It can be concluded that all three strains
Xanthomonas jragariae, Bacillus megaterium and
Bacillus cereus in free and immobilized forms
showed appreciable amount of COD and colour
removal at optimum culture conditions in a detention
period of 48 hours. The strain Bacillus cereus has
shown the maximum efficiency of COD (81 %) and

90.---- ------,

(0)

....--. !S I ragariae
A--A !!.. m<gaterium

...-......

70

Regression line
95"1.Confidence limi1

60

50

Ycol .101 XCOD-n

* - * COD

1.0

r"099

1.0

I 0 bserved)

---- COD Icatcutated)

Colourlobserved)

20

-.-- Colourlcalculated)

>
0

-0

'"
L.

go

5 o
0
-0 00

r----

(b)

CO

1-"

70

60

//'

//
/
/1

./

"".

[j'11-e o-o 3t)

'*/-_---1<-----t.

-.",,.- 6.

. -'
/.....-----Y

_.

_ ._._ ._._

,/,/
/11

30

II
'I. I/j

It!
V

20 -

'
o

LL--LI
L--LI__
-LI__
I __
L--L
LI __

__

20

70

1.0

,t!

30

L.

50

, .

1.0

'"
CL

(b)

60

"S211- e-O.021l
Col

'1.1

50

c
:'.!

1.0

60

lime I hour)

80

1 00

120

Fig. 3--Removal of COD and colour as a function of time by

Bacilllls cere1ls (a) free cells and (b) immobilized cells

20

30

1.0

50

60

Percent COD removal

Fig. 4--1nterrelationship between

70

percent COD

ft'movui by (a) free cells and (b) immobilized cells

80

90

and colour

JAIN

et. at.:

PREDIGESTED DISTILLERY EFFLUENT DIGESTED BY BACTERlAL STRAINS

105

Table 1- Values of kinetic constants

Bacterial strains

COD removal

Xanthomonas fragariae
Bacillus megaterium
Bacillus cereus

Colour removal

Y max

Y max

(%)

(h")

(%)

k
'
(h- )

F-cells

76

0.07

73

0.06

I-cells

72

0.05

68

0.05

F-cells

76

0.06

72

0.05

I-cells

65

0.07

63

0.06

F-cells

82

0.04

79

0.03

I-cells

83

0.03

82

0.02

F-cells: Free cells; I-cells: Immobilized cells

colour (75 %) removal out of the three strains. The

efficiency of free cells was found to be better than the
immobilized
cells
In
all
the
cases.
The
interrelationship between percent COD and colour
removal follows a straight-line equation, which is
justified by high values of coefficient of correlation.
The values of first order reaction rate constant (k) and
Y max are calculated and can be utilized to design
treatment plants for the desired COD and colour
removal
efficiency
for
predigested
distillery
wastewater, after continuous reactor studies.

Vaidyanathan R T, Meenambal T & Gokuldas K, Bio-kinetic

co-efficients for design of two stage anaerobic digester to
treat distillery waste,

Indian J Environ Hit", 37 (1995) 237.

10 Cho Y K, Performance of two stage methane digester for

alcohol stillage derived from sugarcane molasses,

II

Biotechnol

Letl, 5 (1983) 555.

Shin H S, Bae B U, Lee J J & Paik B C, Anaerobic digestion
of distillery wastewater in a two phase UASB system,

Water

Sci Technol, 25 (1992) 361.

12 Jayanth K S & Ramanujan T K, Applicability of upflow
anaerobic sludge blanket reactor for treatment of distillery
spent wash,

Indian J Env Protec, 15 (1994) 106.

13 Shah V B, Joshi J B & Kulkarni P R, Aerobic biological

treatment of distillery waste: kinetics and microbiological

Acknowledgement

analysis,

Indian Chemical Engineer, 31 (1989) 61.

The authors are thankful to the Director, Central

Building Research Institute for his permjssion to
publish the work.

14

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