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 typi-cally toxic, colored, inorganic, organic, turbid, with high suspended solids'. Many organic compounds that are re-calcitrant 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 suit-ably 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 biologi-cal 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 ki-netic and stoichiometric coefficients. COD fractionation involves identification of inert and biodegradable COD together with readily biodegradable and slowly biode-gradable fractions3 Emphasis was laid on COD reduc-tions as a means of monitoring the treatment process.

Wastewater treatment depends on the ability of the mi-croorganism 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 mi-crobial attack. Bacteria and fungi are especially capable of producing a wide variety of enzymes that can degrade organic compounds and mineralize the substances4 . Com-plex mixture of products are produced during the bio-degradation of even a single compounds. The break down products are dependent upon both the micro-organisms present and the environmental circumstances5. However, the particular substance will not be degraded if the rel-evant 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 sedimen-tation tank of an effluent treatment plant and Sample B (COD 14,000 mg/l) from neutralization tank of an efflu-ent treatment plant of another industry. Physico-chemi-cal 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. Parameter Sample A(mg/I) Sample B (mg/I)

pH 6.8 8.8

2 TS 21260 14930

3 IDS 20350 13850

4 TSS 910 1080

5 COD 10488 14076

6 BOD 6164 9894

7 Chlorides 7320 5320

8 Sulphates 88 175

9 Alkalinity

(Phenolphthalein) Nil 20

10 Alkalinity

(Methyl orange) 210 380

Isolation and Screening of Bacterial Cultures

Cultures were isolated from soil samples collected from different sites where bulk drug effluents were be-ing treated. One gram of soil was serially diluted to 10.7

in Bushnell Haas7 broth (without hydrocarbons) of the following composition: MgS04 -O.2g; CaCI2-0.02g; KH2P04 1.0g; K2 HP04 - 1.Og; NH4NOJ -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 trans-ferred onto the surface of Bushnell Haas agar me-dium containing O.OS ml each of benzene and tolu-ene 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 plateS to main-tain an atmosphere of benzene and toluene. The plates were incubated in a BOD incubator at 28C for Sd.

Colonies that appeared after 120 h, were trans-ferred 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 reduc-ing COD. A loopful of culture was inoculated into SOml of Nutrient Broth (Hi -media) in 2S0ml Erlen-meyer 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). Mi-crobial characteristics viz. growth, shape and motil-ity 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 mi-croscopically 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 confir-mation of COD reductions. Five cultures showing good COD reductions were shortlisted and allotted DRCC numbers, as follows:

I DRCC - 165, gram +ve coccobacilli , 2 DRCC - 166, gram - ve cocci, 3 DRCC - 167, gram -ve cocci, 4 DRCC - 168, gram +ve motile curved bacteria, and 5 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 pre-scribed, followed by the addition of suitably diluted sample making the total volume up to 5 m\. The Merck 's digestion tubes were placed in Merck'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\,

I -~

..

BABU el al. : BIODEGRADATION OF BULK DRUG INDUSTRIAL EFFLUENTS 433

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 centri-fuged at 10000 rpm for 15 min to remove the floc formed and the supernatant was taken up for subse-quent treatments. The resultant samples were ana-lysed for COD, TS, TDS, and TSS'2.

Biological Treatment Using Soil Isolates

Biological treatment was initiated using five in-dividual 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 analy-sis 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 analy-sis of the shake flask and scale up samples was car-ried out at 0, 120, and 240 h, respecti vely.

Results and Discussion

Different biochemical mechanisms are responsi-ble for the microbial degradation of compounds present in the effluent. The concentration and types of constituents present in the effluent play an impor-tant role in the response of microorganisms to the mixture of organic compounds. The results of chemi-cal treatment compared to untreated effluent are pre-sented in Table 2.

The TDS of the sample A and B increased by about 15 - 19 per cent after chemical treatment with con-comitant decrease in TSS, however, no significant decrease in soluble COD was observed. Results of biological treatment of sample A and B with indi-vidual 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 re-corded a decrease in COD of 20 - 25 per cent. The

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

Parameter Saml2le- A Sample - B Before treatment After treatment Before treatment After treatmen

( mg /I ) ( mg / I ) ( mg / i ) (mg / I)

TS 2 1260 22902 14930 21610 TDS 20350 22430 13850 216 10 TSS 9 10 472 1080 COD 10488 10304 14076 13064

434 J SCI IND RES VOL.58 JUNE 1999

o 165 166 167 168 169 CONTROL

DRCC CULTURES

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

80

70

.Z 0 60 1= u :J 0

50 !JJ IX 0 0 U 40 I-Z !JJ U IX 30 !JJ 0..

20

10

0 165 166 167 168 169 CONTROL

DRCC CULTURES

Figure 2-COD reduction vs DRCC cu ltures in Sample 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 scale-up study. COD reduction of 72 per cent was achieved in sample A, as compared to 74 per cent in shake flasks, run in parallel, while in sample B it was 64.8

] .2000

Cl :::J , .... (. _ . ~ \ JU .' o bJJ U E

- W\'JO

D DRCC 165 + Sample A ~ CONTROL : Sample A Age (hours)

Figure 3-Kinetics of COD reductions of sample A in presence of DRCC 165

14000

l Z1}3C<

IOC)()

BODO

Cl :::J 6C'30 O~ U g 4C:)1)

,OOC!

D DRCC 165 + Sample 13 ~ CONTROL : Sample 13

Age (hours) 192

Figure 4-Kinetics of COD reductions of Sample B in presence of DRCC 165

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-chemical character-istics, concentration of the chemicals and the spe-cific bacteria capable of withstanding the adverse conditions such as high TDS , COD, presence of sol-vents, antibiotics, etc. Non-uniformity of the efflu-ents 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.

...

BAB U et al. : BIODEGRADATION OF BULK DRUG INDUSTRIAL EFFLUENTS 435

, '1000

12000

10000 '

~ 8000 O...J o ~ 6000 U.s

4000

20'JO

o

o DRCC 165 + Sample A ~ CONTROL : Sample A

24C Age (hours)

Fi gure 5-Scale up study :COD reductions 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 organ-isms present in the effluents may not be sufficient to effectively degrade it. Results of the biological treat-ment indicate that the effluents need to be supple-mented with DRCC 165 in order to establish an effi-cient treatment process.

Further, COD reduction of the effluents, to meet permissible limits may be possible by usi ng a differ-ent group of bacteria. Work is in progress for identi-fication and characterization of DRCC 165.

Acknowledgements We are thankful to Mr Hemanth Sarnaik and Ms

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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' t'.lU

o DRCC 165 + Sample B Age (hours ) :;/,(: I:SI CONTROL ; Sample B

Figure 6-Scale up sludy: COD reducti ons of sample B in presence of DRCC 165

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