Global Research Vol II Iss II 8

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Journal of Global Research Computer Science & Technology

(JGRCST)
Vol.-II, Issue-II, October 2014
ISSN: 2349 - 5170
Separation and Characterization of Biological Degradation of selected

Pharmaceuticals in Water
Rajesh Kumar
OPJS University, Rajasthan, India
1 Introduction
The organic pollutants like pharmaceutical drugs and pesticides are group of persistent
contaminants of environmental and toxicology with great social concern. The difference
between pharmaceuticals and pesticides with respect to environmental release is that
pharmaceuticals have the potential for ubiquitous direct release into the environment due
to different humans activities. Due to worldwide use of pharmaceutical products in large
quantities, they have been identified in a wide variety of environmental media and biota
(1-8). The persistent and bio-accumulative nature of pharmaceutical products have been
recognized particularly in aquatic ecosystems, where the stepwise accumulation in soils,
sediment, fish and humans and degraded and concentrated through food-chain is rather
common (9-11).
The distribution of pharmaceuticals is a large function of their production volumes,

which can rival those for many pesticides. The pharmaceutical drugs with their
respective metabolites and transformation products will collectively referred to as
pharmaceuticals. The pharmaceuticals are continually enter into the environment
through sewage water treatment (23, 24) which cause to contaminate the ground
water and surface water(25, 26) . The presence of numerous drugs in aquatic
environment sharing the specific mode of action could lead to significant effects on
humans (26, 27) and marine organisms. There is a little literature on the occurrence
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and effects of degraded drugs in the environment more data exist for antibiotics than
for any other therapeutic class. This is a result of their extensive use in both human
(23, 24) therapy and animal husbandry, their morrow easily detected effects and
points and their grater chances of introducing into the environment, not just by
sewage treatment plants.
Pharmaceuticals are designed to target specific metabolic pathways in humans and
domestic animals, they can have numerous often unknown effects on metabolic
systems of non-target organisms especially invertebrates. Although many non-target
organisms share certain receptors with humans, effects on non-target organisms are
usually unknown. It is important to recognize that many drugs, their specific modes
of action even in the target species are also unknown without knowing to mode of
action of the degraded product, it is impossible to assess the toxicity tests.
Pharmaceuticals will refer to non-biologic drug. The number of biologics approved
by USFDA is growing and their fate in the environment is unknown. Pharmaceutical
drugs are chemicals used for diagnosis treatment, alteration or prevention of disease
health condition of the human body. The drugs are usually designed with specific
mode of action in mind, they can also have numerous side effects on non-target
organisms. The world combines literature has addressed only a very small
percentage of degraded pharmaceuticals compounds.
Pharmaceuticals are continually released into the environment in enormous
quantities as a result of their manufacture, use (via excretion, mainly in urine and
feces) and disposal of unused / unwanted drugs those that have disposed both
directly into the domestic sewage system and via burial in landfill. Although largely
unknown there is evidence that large quantities of prescription and nonprescription
over-the-counter drugs are never consumed (28) and many of these are undoubtedly
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eventually disposed down toilets or via domestic refuse. The possibility that
pharmaceuticals can enter the environment from a number of different routes and
possibly cause untoward effects in biota has been in the literature for several decades
(1,2,3,4,6) . The evidence support the case that drugs refractory to degradation and
transformation (1) do indeed have the potential to reach the environment. But study
on the degradation product pharmaceuticals in the environment is limited.
Pharmaceutical enter into the environment are degraded and forum a new compound
due to environmental condition. The degraded product- metabolites and conjugates
from eukaryotic and prokaryotic metabolism and from physicochemical alterationadd to the already complex picture of thousands of highly bioactive chemicals. The
concentrations of degraded products are increased through food chain (10). The
degraded products can give more side effects on marine organisms and humans.
Compounds surviving the various phases of metabolism and other degradative or
sequestering actions (environmental persistence) can then pose an exposure risk for
organisms in the environment. Even the less/ nontoxic conjugates can later be
converted back to the original bioactive compounds via enzymatic or chemical
hydrolysis. Some degradation products can even be more bioactive than the parent
compound. Therefore conjugates can essentially act as storage reservoirs from which
the free drugs can alter be released into the environment (6, 12, and 29).
Several authors reported the distribution of pharmaceuticals in ecosystem in

Germany (26, 30) and in other countries (19). But there is not much literature on the
degradation and metabolite products of pharmaceuticals in the environment in India
and in other countries.
Therefore it is important to understand that fate of biological degradation
and its
metabolites products of pharmaceutical in water from sewage treatment plant(STP) and
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drinking water and soils Therefore identifying the metabolic and biological degradation
products of pharmaceuticals is essential to understand the impact on humans and marine
organisms by using instruments like LC-MS and Gas Chromatograph equipped with Mass
spectrometer(GC-MS). And also this study may be helpful to remove the pharmaceuticals
in water from sewage treatment plants and the effluents from pharmaceutical industries.
2 Literature Survey
a)
Rationale of the study supported by cited literature.
b) Hypothesis the organic pollutants like pharmaceutical drugs and pesticides are
group of persistent contaminants of environmental and toxicology with great
social concern. The difference between pharmaceuticals and pesticides with
respect to environmental release is that pharmaceuticals have the potential for
ubiquitous direct release into the environment due to different humans activities.
Due to worldwide use of pharmaceutical products in large quantities, they have
been identified in a wide variety of environmental media and biota (1-8). The
persistent and bioaccumulative nature of pharmaceutical products have been
recognized particularly in aquatic ecosystems, where the stepwise accumulation
in soils, sediment, fish and humans and degraded and concentrated through foodchain is rather common (9-11).
The distribution of pharmaceuticals is a large function of their production volumes,

which can rival those for many pesticides. The pharmaceutical drugs with their
respective metabolites and transformation products will collectively referred to as
pharmaceuticals. The pharmaceuticals are continually enter into the environment
through sewage water treatment (23,24) which cause to contaminate the ground
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water and surface water (25,26) . The presence of numerous drugs in aquatic
environment sharing the specific mode of action could lead to significant effects on
humans (26,27) and marine organisms.
There is a little literature on the occurrence and biological degradation and

metabolites of pharmaceuticals in the environment in India and other countries. This
is the result of attempt to carry out this research problem.
b) Current status of research and development in the subject (both international and
national status)Several authors reported the distribution of pharmaceuticals in
ecosystem in Germany (1-5) and in other countries (6,7). But there is not much
literature on the degradation products, Metabolites of pharmaceuticals in the
environment in India and in other countries. Therefore it is important to understand
that fate of biological degradation, Metabolites of pharmaceutical products in water
from sewage treatment plants (STP), water and soil and further need to study the
characterization.
3 Methodology
3.1 Materials
The selected pharmaceuticals were procured from Sigma Aldrich chemicals.
1) Sodium diatrizoate dehydrate, acetamidophenol sigmaultra (paracetamol),
cetrizine, ciprofloxacin, Meclofenomic acid, Bezafibrate, sulfamethoxazole.
2) Methanol, hexane, benzene, ethyl acetate and chloroform are glass distilled
and use for extraction of degraded pharmaceutical product from samples by solid
phase extraction method (SPE).
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3) Solid phase extraction column C-18 (SPE).
3.2
Source
1) Sewage water was collected in sterile jar and used for isolation of different type
of drug tolerant bacteria and fungus.
2) Water effluent from juggat pharma was collected in sterile jar and used for
isolation of different type drug tolerant bacteria and fungus.
3.3
Isolation
3.3.1 Isolation of bacteria: effluent from pharmaceutical industries and from pesit
drainage were taken and inoculated on agar plate containing known concentration of
standard drug.
Nutrient Agar preparation

For 1 L of nutrient agar
Component
Amount(g)
Beef extract
Peptone
Nacl
Agar for bacterial culture
15
Distilled water
1000(ml)
Bacterial plate preparation
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Agar was autoclaved and cooled down to 600c then Standard (0.2 gm/100ml
media) was mixed well.
It was poured in sterilized petriplate in LAF and left for solidifying
1ml of 10-6 diluted sample water was poured and using spread plate technique
inoculation was performed.
It was kept for 48 hour in incubator at 37 0c.
Bacterial culture were obtained
Pure culture of bacteria

Pure culture of bacteria was done using streaking method in slant culture.
3.3.2 Isolation of fungus: effluent from pharmaceutical industries and from pesit
standard drug.
MRBA preparation
For 1 L of MRBA
Component
Amount(g)
Peptone
KH2pPO4
Dextrose
10
Agar
15
Streptomycin
0.03
Rose Bengal
0.013
Distilled water
1000(ml)
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Fungal plate preparation

o
MRBA was autoclave and cooled down to 600c then Standard (0.2 gm/100ml
media) was mixed well.
It was poured in sterilized petriplate in LAF and left for solidifying
1ml of 10-6 diluted sample water was poured and using spread plate technique
inoculation was performed.
It was kept for 48 hour in incubator at 37 0c.
Fungal culture were obtained
Pure culture of fungus

Pure culture of bacteria was done using streaking method in test tube on MRBA.
3.4 Drug sensitivity method

o
Eight Nutrient agar plate were prepared with each having 8 wells,
Drug tolerance bacteria were taken and inoculated on these plates.
7 micro liter of each standard were dropped in these wells and kept for
incubation for 48 hour.
Clearance zone was observed and drug sensitivity test was performed for
checking bacterial tolerance.
3.5 Bacterial physical characterization

Color: From basic observation.
Gram stain:
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Smear preparations: Smear was prepared using aseptic technique.
Gram Staining Procedure:

o
Cover with Crystal Violet for 20 seconds (Primary Stain).
Gently rinse off the stain with water and shake off the excess.
Cover with Gram's Iodine for one minute (Mordant).
Pour off the Gram's iodine.
Run 95% Ethyl Alcohol down the slide until the solvent runs clear (about 10-20)
(Decolorizing Agent).
Rinse with water to stop the action of the alcohol. Cover With Safranin for 20
second (Counter Stain).
Gently rinse off the stain with water and clean off the bottom of the slide with
95% alcohol.
Slides were viewed under microscope and bacteria were characterized.
3.6 Bacterial Biochemical characterization

I) Indole test
It detects ability of bacteria to breakdown tryptophan to indole.
II)Methyl red test
It detects ability of bacteria to produce and maintain stable acid end product from glucose
fermentation.
III)Vogues proskauer test
Use to detect production of acetylmethylcarbinol (acetoin) from pyruvic acid during
glucose fermentation.
IV)Simmonss citrate test
It detects capability to utilize citrate as carbon source.
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3.7 TLC
Optical density bacterial( 0.06) culture were taken and centrifused to form pelet and
supernatant.palet was taken and mixed with standard drug . Then degradation was studied
with help of TLC method.
3.8 Bioagumentation
Known concentration of pharmaceutical standard sodium ditrizoate dihydrate and
acetaminophin sigmaultra (paracetamol), citrizine, ciprofloxacin were inoculated with 0.6
OD of bacterial prime culture of known volume. The degradation was analyzed by with
cod and degradation product was extracted through SPE column for analysis by HPLC.
Prime culture
For bacterial growth studies
o
Prime culture media was prepared
For 1 L of Prime culture

Component
Amount(g)
Beef extract
Peptone
Nacl
Distilled water
1000(ml)
Tolerant bacteria were inoculated and kept for 48 hour of incubation.
OD was found by spectrometer.
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Analysis
4.1 Analysis by COD method
o
Reagents
o
Standard potassium dichromate solution, 0.04167M: Dissolve 12.259 g

K2Cr2O7, primary standard grade, previously dried at 150C for 2 h, in
distilled water and dilute to 1000 mL. This reagent undergoes a sixelectron
reduction
reaction;
the
equivalent
concentration
is
6 X 0.04167M or 0.2500N.
o
Sulfuric acid reagent: Add Ag2SO4, reagent or technical grade, crystals

or powder, to conc H2SO4 at the rate of 5.5 g Ag2SO4 /kg H2 SO4. Let
stand 1 to 2 d to dissolve. Mix.
Ferroin indicator solution: Dissolve 1.485 g 1,10-phenanthroline

monohydrate and 695 mg FeSO47H2O in distilled water and dilute to
100 mL. This indicator solution may be purchased already prepared.*
Standard ferrous ammonium sulfate (FAS) titrant, approximately

0.25M: Dissolve 98 g Fe(NH4)2(SO4)26H2O in distilled water. Add 20
mL conc H2SO4, cool, and dilute to 1000 mL.
Standardize this solution daily against standard K2Cr2O7 solution.
Add all reagents to the refluxing flask open to the atmosphere without
the condenser attached for 2 hour. Find dichromate utilized by titration
with FAS.
4.2 Extraction of degraded compound by SPE column

o
Column conditioning: One column of acetone, one column of methnol,

one column of distilled water was passed through SPE column.
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o 20 ml of each solution was passed through SPE column after

conditioning and then it was extracted with 10ml of methanol.
4.3 Analysis by HPLC method

Samples were analyzed.
5. Result and Discussion

5.1 Isolation of Fungus
Isolation of fungus: effluent from pharmaceutical industries and from pesit drainage were
taken and inoculated on MRBA plate containing known concentration of standard drug
Plate prepared by spread plate technique
Fungi on Diclofinac fungi on glaciphage
fungi on Nimuslide fungi on Ranitidine
These four fungi were isolated on MRBA media which contains different drugs.
5.2 Isolation of bacteria
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Isolation of bacteria and fungus: effluent from pharmaceutical industries and from pesit
standard drug.
Isolation from Pesit Drainage
DICLOFINAC
RANITIDINE
GLACIPHASE
MIXTURE OF
NIMUSULIDE
MEDIA WITHOUT DRUG
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DICLOFIONAC
GLACIPHAGE,
NIMUSULIDE RANITIDINE
These bacteria were isolated from PESIT drainage and pharmaceutical industrial effluent
Isolation of Bacteria from Industrial Effluent
Ciprofloxacin
acetaminophin
ditrizoate dehydrate Cetrizine sigmaultra

(paracetamol)
5.3 Bacterial physical and biochemical characterization

Bacterial physical and biochemical characterization were performed .bacterial tolerance
were checked by drug sensitivity method.
CHARACTERIZATION OF BACTERIA ISOLATED FROM PESIT DRAINAGE

Tolerance
bacteria
symbol
Color
Gram
test
Indole
test
Methyl red
test
Voges
proskauer test
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Test
DICLOFIONAC
Creamy
GLACIPHAGE
White
NIMUSULIDE
+ve
cocci
+ve
cocci
+ve
cocci
+ve
cocci
+ve
-ve
+ve
-ve
+ve
-ve
+ve
-ve
+ve
+ve
+ve
+ve
+ve
-ve
+ve
-ve
Creamy
+ cocci
+ ve
-ve
+ ve
Brown
+ cocci
+ve
-ve
+ve
Brown
N
RANITIDINE
Brown
R
MEDIA
-ve
MW
WITHOUT
DRUG
MIXTURE OF
MA
ALL ABOVE
DRUG
MW
MA
MW
MA
Indol test
D
Methyl Red Test

N
MW
MA
MW
MA
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Voges Test
Simmons Citrate Test
Bacterial Biochemical characterization result

I) Indole test : all bacteria were breaking down tryptophan to indole.
II)Methyl red test: nimuslide bacteria produce and maintain stable acid end product
from glucose fermentation , other do not.
III)Voges proskauer test : all bacteria are producing acetylmethylcarbinol (acetoin)
from pyruvic acid during glucose fermentation.
IV)Simmonss citrate test : bacteriag rown on nimesulide media have the capability to
utilize citrate as carbon source, other are not.
CHARACTERIZATION OF BACTERIA ISOLATED FROM PESIT DRAINAGE

Tolerance
symbol
Color
bacteria
Gram
Indole
Methyl
Voges
Citrate
test
test
red test
proskauer
Test
test
Cetrizine
CT
Creamy
+ve
-ve
-ve
-ve
-ve
-ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
cocci
Cifrofloxacin
CF
White
+ve
cocci
acetamidophenol
sigmaultra
Creamy
+ve
cocci
(paracetamol)
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Sodium
White
ditrizoate
+ve
-ve
+ve
-ve
-ve
cocci
dehydrate
Bacterial Biochemical characterization result
I) Indole test : no bacteria were breaking down tryptophan to indole.
II) Methyl red test: ciprofloxacin and ditrizoate bacteria produce and maintain stable
acid end product from glucose fermentation, other do not.
III) Voges proskauer test: all bacteria are producing acetylmethylcarbinol (acetoin)
from pyruvic acid during glucose fermentation.
IV)Simmonss citrate test : none bacteria have the capability to utilize citrate as carbon
source.
CT
CF
CF
Vouges test
Indol test
CF
CT
CT
CF
CT
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Methyl Red Test
Simmons Citrate Test.
5.3 Drug sensitivity method
Fig. known concentration of all eight standards
1.
2.
Cetrizine
3. Ciprofloxacin
5. Methyl propionic acid 7. Paracetamol
Bezafibrate 4. Sulfamethoxazole 6. Meclofenomic acid 8. Sodium ditrizoate
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Observation:
Clear zone large :4
Clear zone small :3
Clear zone very
small:2
Observation:
Clear zone large:5
Clear zone small :4,3
Clear zone very
small:1,7,6

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Observation:
Clear zone large:4
Clear zone small :3
Clear zone very
small:2
Observation:
Clear zone large:4
Clear zone small :3
Clear zone very small:2
Observation:
Clear zone large:no
Clear zone small :5
Clear zone very
small:8,1
Observation:
Clear zone large:4
Clear zone very small:no
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Observation:
Clear zone large:4
Clear zone small :3,2,1
Clear zone very small: 6
Observation:
Clear zone large:4
Clear zone very small:2,8
Inferences : all the bacteria were recheked for all eight standards.
5.4 Bioagumentation
Known concentration of pharmaceutical standard sodium ditrizoate
dehydrate and
acetaminophin sigmaultra (paracetamol), citrizine, ciprofloxacin were inoculated with 0.6

OD of bacterial prime culture of known volume. The degradation was analyzed by with
cod and degradation product was extracted through SPE column for analysis by HPLC.
OPTICAL DENSITY OF PRIME CULTURE (at 540nm)

TIME
24 hour
48 hour
72 hour
96 hour
120 hour
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Cetrizine
0.34
0.54
0.68
0.75
0.78
Cifrofloxacin
0.25
0.60
0.66
0.72
0.80
acetamidophenol
0.32
0.64
0.70
0.74
0.76
0.28
0.50
0.60
0.66
0.67
sigmaultra
(paracetamol)
sodium ditrizoate
dehydrate
The
bacteria
grown
in
presences
of
selected
pharma
0.9
0.8
0.7
0.6
cetrizine
0.5
ciprofloxacin
0.4
paracetamol
0.3
ditrizoate
0.2
0.1
0
24 hour
48 hour
72 hour
96 hour
120 hour
Inferences : Maximum growth was shown at around 48 hour so bacteria were in log phase
so 0.60 OD bacterial culture were taken for bioaugumentation.
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5.5 Chemical Oxygen Demand

Standard procedure for determining COD was followed.
COD of effluent with cetrizine tolerance bacteria

TIME
0hour
24hour
48hour
72hour
96hour
SAMPLE
1400
600
400
200
100
CONTROL
1400
800
600
400
300
1400
1200
1000
800
SAMPLE
600
CONTROL
400
200
0
0Hr
24
48
72
96
COD of effluent with ciprofloxacin tolerance bacteria
TIME
0hour
24hour
48hour
72hour
96hour
SAMPLE
2000
1200
1000
800
400
CONTROL
2000
1800
1600
1400
1300
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2000
1500
SAMPLE
1000
CONTROL
500
0
0hour
24hour
48hour
72hour
96hour
COD of Acetaminophin standard solution with Acetaminophin tolerance bacteria
DAY
14
18
22
32
SAMPLE
3420
792
460
300
200
180
CONTROL
3420
1528
1320
840
480
290
3500
3000
2500
2000
SAMPLE
1500
CONTROL
1000
500
0
0day
14
18
22
32
COD of Sodium Ditrizoate Dihydrate standard solution with Sodium Ditrizoate

Dihydrate tolerance bacteria
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DAY
12
16
20
26
32
SAMPLE
980
464
320
210
200
190
CONTROL
980
598
498
300
240
210
1000
800
600
SAMPLE
400
CONTROL
200
0
0
12
16
20
26
32
Fig. SPE setup
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Extraction: SPE C18 cartridges were used for the extraction of standard drug from water
after conditioned the column with ethyl acetate, methanol and water and residue eluted
with methanol.
6. Conclusions
We have isolated the bacteria (9 types) and fungi (4 types) from the domestic waste and
also from the pharmaceutical industrial effluents. We have used two bacterial from the
isolated and used for the degradation of the pharmaceuticals. We have studied
biochemical charectistics of the isolated Bactria. The rate of the degradation of the
pharmaceutical compounds was observed by measuring the chemical oxygen demand and
bacterial growth in the selected medium was recorded. We have established the SPE
extraction method for the extraction of the pharmaceutical compounds. The samples are
used for the analysis of pharmaceutical compounds by HPLC(the work is in progress).
(Based on the outcome of this work, I have submitted detailed major research project to
the DST for the financial support).
7. Acknowledgements
We thank to the PESIT Management, Principal, R& D Director for the financial support
and also we thank to Head of the department of Biotechnology for the encouragement and
support to carry out the project work in the department.
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Global Research

Journal of Global Research Computer Science & Technology

Separation and Characterization of Biological Degradation of selected

The distribution of pharmaceuticals is a large function of their production volumes,

Journal of Global Research Computer Science & Technology

Journal of Global Research Computer Science & Technology

Several authors reported the distribution of pharmaceuticals in ecosystem in

metabolites products of pharmaceutical in water from sewage treatment plant(STP) and

Journal of Global Research Computer Science & Technology

Rationale of the study supported by cited literature.

The distribution of pharmaceuticals is a large function of their production volumes,

Journal of Global Research Computer Science & Technology

There is a little literature on the occurrence and biological degradation and

Journal of Global Research Computer Science & Technology

3) Solid phase extraction column C-18 (SPE).

Nutrient Agar preparation

Agar for bacterial culture

Bacterial plate preparation

Journal of Global Research Computer Science & Technology

It was poured in sterilized petriplate in LAF and left for solidifying

It was kept for 48 hour in incubator at 37 0c.

Bacterial culture were obtained

Pure culture of bacteria

Journal of Global Research Computer Science & Technology

Fungal plate preparation

It was poured in sterilized petriplate in LAF and left for solidifying

It was kept for 48 hour in incubator at 37 0c.

Fungal culture were obtained

Pure culture of fungus

3.4 Drug sensitivity method

Drug tolerance bacteria were taken and inoculated on these plates.

3.5 Bacterial physical characterization

Journal of Global Research Computer Science & Technology

Smear preparations: Smear was prepared using aseptic technique.

Gram Staining Procedure:

Cover with Crystal Violet for 20 seconds (Primary Stain).

Cover with Gram's Iodine for one minute (Mordant).

Pour off the Gram's iodine.

Slides were viewed under microscope and bacteria were characterized.

3.6 Bacterial Biochemical characterization

Journal of Global Research Computer Science & Technology

Prime culture media was prepared

For 1 L of Prime culture

Tolerant bacteria were inoculated and kept for 48 hour of incubation.

OD was found by spectrometer.

Journal of Global Research Computer Science & Technology

Standard potassium dichromate solution, 0.04167M: Dissolve 12.259 g

Sulfuric acid reagent: Add Ag2SO4, reagent or technical grade, crystals

Ferroin indicator solution: Dissolve 1.485 g 1,10-phenanthroline

Standard ferrous ammonium sulfate (FAS) titrant, approximately

Standardize this solution daily against standard K2Cr2O7 solution.

4.2 Extraction of degraded compound by SPE column

Column conditioning: One column of acetone, one column of methnol,

Journal of Global Research Computer Science & Technology

o 20 ml of each solution was passed through SPE column after

4.3 Analysis by HPLC method

5. Result and Discussion

Fungi on Diclofinac fungi on glaciphage

fungi on Nimuslide fungi on Ranitidine

5.2 Isolation of bacteria

Journal of Global Research Computer Science & Technology

Isolation from Pesit Drainage

MEDIA WITHOUT DRUG

Journal of Global Research Computer Science & Technology