Journal of Scientific & Industrial Research

Vol.58, June 1999, pp 431-435

Biodegradation of Bulk Drug Industrial Effiuents by Microbial

Isolates from Soil+
P Ajay Babu, M Sitaram Kumar, D Ganesh Reddy,
P Mahendra Kumar and A K Sadhukhan'
Biotechnology R&D, Dr Reddy's Research Foundation, Bollaram Road, Miyapur,
Hyderabad 500 050, India
Received: 13 January 1999
Fax No: 040-3045438, e-mail : drf@hdl.vsnl.neLin
Bacteria isolated from soils exposed to bulk drug wastes were used for the aerobic treatment of two bulk drug industrial effluents.
Shake flask experiments followed by scale up studies using individual bacterial isolate reduced COD by 75 per cent. Supplementation of
the native bacteriaillora with a specific culture helped in efficiently reducing COD of bulk drug effluent.

Introduction
Bulk drug industrial effluents are generated through
raw materials used in the process for the manufacture of
various bulk drugs and intermediates. Effluents are typically toxic, colored, inorganic, organic, turbid, with high
suspended solids'. Many organic compounds that are recalcitrant in nature are produced while manufacturing
bulk drugs. Although many of them can be readily treated,
some compounds that are poorly degradable are released
in the effluent. In order to protect the environment from
undesirable toxic materiaF the wastewater must be suitably treated before discharge.
Biological wastewater treatment is the most widely
accepted process due to its ease of handling and economic
feasibility. An attempt was made to establish a biological treatment process for bulk drug industrial effluents
collected from IDA Bollaram, Hyderabad. Cultures were
isolated and selected after intensive screening of soil
samples that was exposed for prolonged periods to bulk
drug wastes.
Biological treatment processes mainly include
fractionation of COD and assessment of significant kinetic and stoichiometric coefficients. COD fractionation
involves identification of inert and biodegradable COD
together with readily biodegradable and slowly biodegradable fractions 3 Emphasis was laid on COD reductions as a means of monitoring the treatment process.
Wastewater treatment depends on the ability of the microorganism to metabolize carbonaceous organic matter
'Corresponding author
'Publication No.82 from DRF

within a specified period of retention in the treatment

process. Most organic compounds are vulnerable to microbial attack. Bacteria and fungi are especially capable
of producing a wide variety of enzymes that can degrade
organic compounds and mineralize the substances4 . Complex mixture of products are produced during the biodegradation of even a single compounds. The break down
products are dependent upon both the micro-organisms
present and the environmental circumstances 5. However,
the particular substance will not be degraded if the relevant microbial strains are absent or fail to grow during
the treatment6 .

Materials and Methods

Twenty-liter samples of two bulk drug effluents were
collected from IDA Bollaram, Hyderabad. Sample A
(COD 11,000 mg/I) was collected from primary sedimentation tank of an effluent treatment plant and Sample B
(COD 14,000 mg/l) from neutralization tank of an effluent treatment plant of another industry. Physico-chemical characters of the samples are mentioned in Table I.

Experiments Conducted
The following experiments were carried out:
I Isolation and screening of bacterial cultures,
2 Chemical treatment of the effluent, and
3 Biological treatment of effluent with individual
microbial isolates.

432

J SCI IND RES VOL.58 JUNE 1999

Table I - Physico-chemical characteristics of sample A and B

SI No.

Sample A(mg/I)

Sample B
(mg/I)

pH

6.8

8.8

Parameter

TS

21260

14930

3
4

IDS

13850

TSS

20350
910

COD

10488

14076

1080

BOD

6164

9894

Chlorides

7320

5320

88

175

Nil

20

210

380

Sulphates

Alkalinity

10

Alkalinity

(Phenolphthalein)
(Methyl orange)

Isolation and Screening of Bacterial Cultures

Cultures were isolated from soil samples collected
from different sites where bulk drug effluents were being treated. One gram of soil was serially diluted to 10. 7

in Bushnell Haas 7 broth (without hydrocarbons) of

the following composition: MgS04 -O.2g; CaCI 20.02g; KH 2P04 1.0g; K 2 HP04- 1.Og; NH 4NO J -1.Og
and FeCI1-0.0Sg; in one litre of water (pH 7.0iO.2).
0.2Sml at"iquot of each dilution (10.5 - 10.7) was transferred onto the surface of Bushnell Haas agar medium containing O.OS ml each of benzene and toluene as carbon sources. Using a sterile L-shaped glass
rod the sample was spread on the entire surface of
the agar. The plates were inverted and a fil ter paper
pad dipped in a mixture of Benzene and Toluene
(I: I) was placed in the lid of the petri plateSto maintain an atmosphere of be nzene and tolue ne. The
plates were incubated in a BOD incubator at 28C
for Sd.
Colonies that appeared after 120 h, were transferred onto Nutrient agar slants and incubated at 28C
for 48 h. The isolates were screened in the effluent
samples A and B to select bacteria capable of reducing COD. A loopful of culture was inoculated into
SOml of Nutrient Broth (Hi -media) in 2S0ml Erlenmeyer conical flask having following composition :
Peptone -S.Og ; Beef extract-I.Sg; Yeast extract -

I.Sg, NaCI -S.Og and distilled water one litre (pH

7.4 0.2). The flasks were incubated at 28C for 48
h, on a rotary shaker at 200 rpm (stroke I.S in). Microbial characteristics viz. growth, shape and motility were observed under a phase contrast microscope
(Olympus CH-2).
For primary screening process, 20 ml of the above
seed was inoculated into 80m I of effluent A and B
and incubated at 28C on a rotary shaker at 200 rpm.
After 48 h incubation, the flasks were checked microscopically for their characteristics and compared
with that observed in the initial inoculum. Cultures
with relatively good growth / motility in the effluent
sample were selected and incubated for 240 hand
checked for their ability to reduce COD.
The cultures selected from primary screen were
subjected to secondary screening process for confirmation of COD reductions. Five cultures showing
good COD reductions were shortlisted and allotted
DRCC numbers, as follows:
I
2
3
4
5

DRCC - 165, gram +ve coccobacilli,

DRCC - 166, gram - ve cocci,
DRCC - 167, gram -ve cocci,
DRCC - 168, gram +ve motile curved bacteria, and
DRCC - 169 , gram +ve coccobacil li (*DRCC
Dr Reddy 's Culture Collection).

COD AnaLysis

Closed reflux colorimetric method9 (E.Merck) was

adopted for analysis. Sampling was carried out at 0,
48, 120, 192 and 240 h, respectively. 5 ml of the
sample was centrifuged at 10000 rpm for 10 min and
the supernatant was filtered through 0.45 ~ filter lO
for the estimation of soluble COD. Merck's COD
solution A and B were added in the manner prescribed, followed by the addition of suitably diluted
sample making the total volume up to 5 m\. The
Merck 's digestion tubes were placed in Merc k's
thermoreactor at 148C and refluxed for 2 h. After
cooling, optical density of the sample was read at
S8S nm using Spectrophotometer (Spectronic 20D)
with Icm diam cuvette. The resulting aD multiplied
by a factor of 4600 gave the COD in mg I\,

BABU el al. : BIODEGRADATION OF BULK DRUG INDUSTRIAL EFFLUENTS

Chemical Treatment

-~

100 ml of sample A and B were treated with 200

mg each of alum at pH 7.0, ferrous sulphate at pH
6.0, and lime for 30 min under constant stirring". At
the end of each process, the samples were centrifuged at 10000 rpm for 15 min to remove the floc
formed and the supernatant was taken up for subsequent treatments. The resultant samples were analysed for COD, TS, TDS, and TSS'2.

Biological Treatment Using Soil Isolates

..

Biological treatment was initiated using five individual DRCC cultures. Twenty per cent inoculum
of each culture grown in nutrient broth was added to
the effluent taken in 500 ml Erlenmeyer flasks. These
were then incubated at 28 0.5C on a rotary shaker
for 240 h. Control flask without DRCC cultures was
also run for the same period. pH was maintained
between 7.2 - 7.5 and evaporation losses were made
up every 24 h with demineralised water. COD analysis was carried out at 0 h and after 48, 120, 192 and
240 h of treatment, respectively.

Scale Up Studies
10 I scale-up of the process was attempted in a
12 I plastic carbouy. Air was sparged through a ring
sparger and agitation( I 00 rpm) carried out with an
overhead stirrer having two, four bladed impellers
Isolate that gave high COD reduction in shake flask
was selected for the scale up. Seed inoculum was
developed in two stages: 100ml of NB in 500 ml
Erlenmeyer conical flask was inoculated with a
loopful of culture and incubated for 24 h (stage I)
which was then transferred to 2 I NB in a 5 I Aspira-

tor bottle (stage II). pH of the effluent was adjusted

to 7.4; aeration and agitation was started 30 min
before inoculation. 100ml samples were withdrawn
into a conical flask after inoculation and incubated
on shaker. 10 I control effluent without any added
culture was also run in parallel. pH was maintained
between 7.2 - 7.5 and evaporation losses were made
up every 24 h with demineralised water. COD analysis of the shake flask and scale up samples was carried out at 0, 120, and 240 h, respecti vely.

Results and Discussion

Different biochemical mechanisms are responsible for the microbial degradation of compounds
present in the effluent. The concentration and types
of constituents present in the effluent play an important role in the response of microorganisms to the
mixture of organic compounds. The results of chemical treatment compared to untreated effluent are presented in Table 2.
The TDS of the sample A and B increased by about
15 - 19 per cent after chemical treatment with concomitant decrease in TSS, however, no significant
decrease in soluble COD was observed. Results of
biological treatment of sample A and B with individual bacterial isolates are illustrated in Figure I
and Figure 2 (average of three experiments).
DRCC 165, a gram positive coccobacilli lowered
COD by 75 per cent in both sample A and B. While
COD reductions of 63 - 70 per cent in sample A and
B were obtained with other cultures . Control
(un inoculated), containing native bacterial flora recorded a decrease in COD of 20 - 25 per cent. The

Table 2-Characteristics of sample A and B before and after chemical treatment

Parameter

TS
TDS
TSS
COD

Saml2le- A
After treatment
Before treatment
( mg /I )
( mg / I )
2 1260
20350
9 10
10488

22902
22430
472
10304

433

Before treatment
( mg / i )
14930
13850
1080
14076

Sample - B
After treatmen
(mg / I)
21610
216 10
13064

J SCI IND RES VOL.58 JUNE 1999

434

] .2000

:::J , .... (.

Cl

. ~ \ JU

.'

bJJ

U E
-

W\'JO

DRCC 165 + Sample A

CONTROL : Sample A

Age (hours)

Figure 3-Kinetics of COD reductions of sample A in presence of

DRCC 165

o
165

169

168

167

166

CONTROL

DRCC CULTURES
14 000

Figure I-COD reduction vs DRCC cu ltures in sample A

l Z1}3C<

IOC) ()
BODO
Cl

:::J

6C'30

4C:)1)

O~

80
70
.Z
0

1=
u

:J
0
!JJ

IX

,OOC!

60

50

0
0

I-

40

!JJ

Age (hours)

192

Figure 4-Kinetics of COD reductions of Sample B in presence of

DRCC 165

!JJ

U
IX

DRCC 165 + Sample 13

CONTROL : Sample 13

30

0..

20

10
0
165

166

167

168

169

CONTROL

DRCC CULTURES

Figure 2-COD reduction vs DRCC cu ltures in Samp le B

experiment was termi nated at 240 h as no further

significant COD reductions were noticed . Kinetics
of COD reduction with reference to control is shown
in Figure 3 and Figure 4.
Figure 5 and 6 represent the results of 10 I scaleup study. COD reduction of 72 per cent was ac hieved
in sample A, as compared to 74 per cent in shake
flask s, run in parallel, whil e in sample B it was 64.8

per cent as compared to 66 per cent in shake flask.

Thus the results of shake flask were reproduced in
scale-up for both sample A and sample B.
The efficiency of biodegradation of the effluents
depends mainly on its physico-chem ical characteristics, concentration of the chemicals and the specific bacteria capable of withstanding the adverse
conditi ons such as high TDS , COD, presence of solvents, antibiotics, etc. Non-uniformity of the effluents generated is, a major problem faced by the bulk
of drug industry. The nature and the characteristics
of the effluent changes depending on the type and
quantity of product being manufactured and also the
process adopted. This is further complicated by the
use of different intermediates in the process.

...

435

BAB U et al. : BIODEGRADATIO N OF B UL K DRUG IND USTRIAL EFFLUENTS

, '1000

' t'.lU

12000
10000
~

'

8000

O...J

~ 6000

U.s

4000
20'JO

DRCC 165 + Sample A

CONTROL : Sample A

24C
Age ( hours)

o DRCC 165 + Sample B

Age ( hours )

:;/,(:

I:SI CONTROL ; Sample B

Fi gure 5-Scale up study :COD redu ction s of sample A in presence of

DRCC 165

Our experiments indicate that DRCC 165 was the

best soil isolate capable of reducing COD up to 75
per cent of sample A and B. Thus the native organisms present in the effluents may not be sufficient to
effectively degrade it. Results of the biological treatment indicate that the effluents need to be supplemented with DRCC 165 in order to establish an efficient treatment process.
Further, COD reduction of the effluents, to meet
permissible limits may be possible by usi ng a different group of bacteria. Work is in progress for identification and characterization of DRCC 165.

Acknowledgements
We are thankful to Mr Hemanth Sarnaik and M s
P V Prashanthi for their help. We are also grateful to
Dr A Venkateswarlu , President, Dr Reddy's Research
Foundation, for the encouragement.

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