Biodegradation of Aromatic Compounds Present in Industrial Wastewater by Bacterial Species

BIODEGRADATION OF
AROMATIC COMPOUNDS
Final Project
AUGUST 28, 2014

AMITY INSTITUTE OF BIOTECHNOLOGY
Amity University Haryana
BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.
Acknowledgement
The joyness, satisfaction and euphoria that comes along with successful completion of any work
would be incomplete unless we mention the people who made it possible. Their constant guidance
and encouragement served as a beam of light and crowed out efforts.
I am indebted to my industry guide Mrs. Neelam Bhola, Helix Biogenesis, Noida U.P. for her
active guidance, valuable advice and constant inspiration and support to complete the project work
effectively.
I would like to express my deep sense of gratitude to Dr. Ravindra Kumar, Helix Biogenesis Noida,
for his co-operation, valuable advice, constant inspiration and support during the course of this
project.
I am also thankful to Mr. A.K. Srivastava for his help and support.
Last but not the least I would like to express my heartfelt gratitude towards Professor Dr. S. M.
Paul Khurana, Director, Amity Institute of Biotechnology, Amity University Haryana. Without his
co-operation and guidance this project would never have been successful.
I am indebted as well, to my parents and family members for their constant encouragement and
moral support.
Sabyasachi Dasgupta
B. Tech Biotechnology (2010-14)
Enroll No: A50204110009
II
Table of Contents
Contents
Abstract ........................................................................................................................................... 1
Introduction ..................................................................................................................................... 2
Physical Properties of Phenol: .................................................................................................... 7
Physical Properties of 8-Hydroxyquinoline: ............................................................................... 9
Properties of Catechol: .............................................................................................................. 10
Outline of the Experiments performed: .................................................................................... 11
Literature Review.......................................................................................................................... 12
Materials and Methods .................................................................................................................. 18
Microbiological Tests ............................................................................................................... 19
Sampling from Industrial Areas: ........................................................................................... 20
Isolation of species by Serial Dilution and Spreading .......................................................... 21
Isolation of species by Streak Plate Method ......................................................................... 22
Biochemical Methods: .............................................................................................................. 23
Indole Test ............................................................................................................................ 25
Methyl Red Test .................................................................................................................... 26
Voges Proskeaur Test ........................................................................................................... 27
Catalase Test ......................................................................................................................... 28
Citrate Test ............................................................................................................................ 29
Gram Staining Test ............................................................................................................... 30
Urea Hydrolysis Test ............................................................................................................ 32
Mannitol Test ........................................................................................................................ 32
Starch Test ............................................................................................................................ 33
Gelatin Test ........................................................................................................................... 34

III
MacConkey's Agar Test ........................................................................................................ 35
Growth at 4C and 25C ....................................................................................................... 36
Growth at 5% and 10% NaCI ............................................................................................... 37
Cetrimide Test ....................................................................................................................... 38
Carbohydrate, Lactose, Sucrose and Dextrose Fermentation ............................................... 39
Bacterial identification based on biochemical tests .................................................................. 40
Biodegradation experiments: .................................................................................................... 41
Phenol Degradation ............................................................................................................... 42
8-Hydroxyquinoline Degradation ......................................................................................... 43
Catechol Degradation............................................................................................................ 45
Protein profile analysis: ............................................................................................................ 47
Cell disruption and protein extraction................................................................................... 48
SDS PAGE for protein analysis ............................................................................................ 49
Result and Discussion ................................................................................................................... 51
Isolation of bacterial species ..................................................................................................... 52
Biochemical tests: ..................................................................................................................... 53
Indoletest ............................................................................................................................... 54
Methyl Red Test .................................................................................................................... 55
Voges-Proskauer test ............................................................................................................ 56
Citrate test result ................................................................................................................... 57
Catalase test result................................................................................................................. 58
Gram staining result .............................................................................................................. 59
Urease test result ................................................................................................................... 61
Mannitol test result ............................................................................................................... 63
Starch test result .................................................................................................................... 64

IV
Gelatin test result .................................................................................................................. 65
MacConkey's Agar Test result .............................................................................................. 66
Growth at 4C and 25C ....................................................................................................... 67
Growth at 5% and 10 % NaCl............................................................................................... 69
Cetrimide agar test result: ..................................................................................................... 71
Carbohydrate fermentation test result ................................................................................... 72
Bacterial Identification based on biochemical test ............................................................... 74
Biodegradation study of different aromatic compounds ........................................................... 75
Microbial Growth Analysis using Spectrophotometric method ........................................... 77
Enzvmes responsible for biodegradation of phenol, catechol and 8-hydroxyquinoline ........... 90
SDS PAGE for Pseudomonas putida grown in phenol at different concentrations ............. 91
SDS PAGE for Pseudomonas aeruginosa grown in phenol at different concentration ........ 92
SDS PAGE RESULT Pseudomonas putida grown in catechol at different concentration .. 93
SDS PAGE RESULT Pseudomonas aeruginosa grown in catechol concentration ............. 94
SDS PAGE RESULT Pseudomonas putida grown in 8-hydroxyquinoline at different
concentration. ........................................................................................................................ 95
SDS PAGE Result for Pseudomonas putida grown in 8-hydroxyquinoline at different
concentration ......................................................................................................................... 96
List of the Enzymes that are responsible for biodegradation of phenol, catechol and 8hydroxyquinoline .................................................................................................................. 97
Bioinformatic analysis of the enzymes produced during aromatic biodegradation in different
bacterial species. ....................................................................................................................... 98
ClustalW2 Alignment of catechol 2,3 dioxygenase found in different bacterial species ..... 99
ClustalW2 alignment of catechol 1,2 dioxygenase enzyme found in different bacterial species
............................................................................................................................................. 102
ClustalW2 alignment of peroxidase enzyme found in different bacterial species .............. 105
ClustalW2 alignment of quinoline -2-oxidoreductase enzyme found in different bacterial

species ................................................................................................................................. 107
Conclusion & Future Prospects .................................................................................................. 109
Conclusion .............................................................................................................................. 110
Future Prospects ...................................................................................................................... 111
Appendix ..................................................................................................................................... 112
References ................................................................................................................................... 122
Abstract
The waste waters from industrial waste contain a number of toxic organic compounds that is
phenol and its derivatives. Their removal requires physic-chemical treatments but here in this
project, the main component that has been used is microorganisms. The microbial strains used for
decontamination of different origin waste water should not only be highly active to one of the
contaminants but they should also be resistant enough to the remainder. Their resistance can be
ensured by the degradation activity of the strains used towards most of the waste products present
in waste water. Many species are known as an effective bio-degradant and can hence be utilized
to remove a number of toxic aromatic compounds from the environment. The present project deals
with processes of degradation, utilization of monohydroxyl derivatives of phenol (catechol and 8hyroxyquinoline), which are the most toxic aromatic pollutants of the environment, their
biochemical studies followed by their analysis.
Introduction
Environmental pollution is an emerging threat and great concern in todays context pertaining to
its effect on the ecosystem. The worldwide rise in population and industrialization during the last
few decades have resulted in ecological imbalance and degradation of natural resources. One of
the most essential natural resources which have the worst victim of population explosion and
industrialization is water. In recent years considerable attention has been paid to industrial wastes
discharged to land and surface water. Industrial effluents often contain various toxic metals
harmful dissolved gases and several organic and inorganic compounds. Organic pollutants
comprise a potential group of chemicals which can be dreadfully hazardous to human health. Many
of these are resistant to degradation. As they are persist in the environment, they are capable of
long range transportation, bioaccumulation in human and animal tissue and bio magnification in
food chain. Huge quantity of waste water generated from human settlement and industrial sectors
accompany the disposal system either as municipal wastewater of industrial waste water. Over 5
million chemical substances produced by industries have been identified and about 12000 of these
are marketed which amount to around half of the total production. Contaminated water by
pesticides, such as DDT, heptachlor etc is harmful for aquatic life and human beings as well.
Discharge of cyanide-contained wastewater to water mass may lead to death of sh and other
aquatic life therein. Use of water containing uoride can causes mental disorders and stomach
ailments and can also reduce agricultural production. Phenol along with other xenobiotic
compounds is one of the most common contaminants present in effluents from process industries.
Phenols are compounds with ArOH which are extremely toxic and found in different form or
together with other elements. Phenol is one of the 50major bulk chemicals produced in the world
and its annual production reached 6.6 billion pounds in 2004 and expected to grow by 6% per year.
Phenol is considered to be a toxic compound by the Agency for toxic Substances and Disease
Registry and death among adults has been reported with ingestion of phenol ranging from 1 to 32
g. Phenol and its derivatives are also generated by various industries such as Petroleum refining,
Petrochemical, coke conversion, pharmaceutical, plastic and resin manufacturing, Coal
gasication, coke-oven batteries, and other industries. Such as synthetic chemicals, herbicides,
pesticides, antioxidants. Pulp and paper, photo developing chemicals, etc. (Marrot et.al, 2008) in
the waste effluents and its concentration may vary from 1 to 15000 mg l-l. Natural sources of
phenol include forest re, natural run off from urban area where asphalt is used as the binding
material and natural decay of lignocellulosic material. United States Environmental Protection
Agency(USEPA) and Central Pollution board of India(CPBl) have prescribed maximum
permissible limits of 3.4 and 5.0 g/l respectively in industrial waste discharges. Now the associated
problem due to phenol is that when it is present in wastewater even in low concentrations can be
toxic to some aquatic species and causes taste and odor problems in drinking water. Inhalation and
dermal contact of phenol causes cardiovascular diseases and severe skin damage while ingestion
can cause serious gastrointestinal damage and oral administration into laboratory animals has also
induced muscle tremors and death. Phenol in solution form, easily passes through the skin, and its
metabolism occurs in the liver, although, it could occur in the lung and kidney too. Phenol is toxic
inenvironmt and could decrease enzymatic activity as well. Also, it is toxic to shes and is mortal
between 5 25 mg/l for them. Phenolic compounds are serious pollutant for rivers (EPA, 2004)
and they have harmful effects such as growth inhibition, decrease of resistance against diseases,
aquatic mortality and increase in growth of weedy plants. Acute exposure of phenol causes central
nervous system disorders. It leads to collapse and coma (Jindrova et.al, 2002).Muscular
convulsions are also noted. A reduction in body temperature is resulted and this is known as
hypothermia. Mucus membrane is highly sensitive to the action of phenol. Acute exposure of
phenol can result in myocardial depression. Whitening and erosion of the skin may also result due
to phenol - exposure. Phenol has anesthetic effect and causes gangrene. Renal damage and
salivation may be induced by continuous exposure to phenol. Exposure to phenol may result in
irritation of the eye, conjunctional swelling, corneal whitening and finally blindness. Other effects
include frothing from nose and mouth followed by headache. Phenol can cause hepatic damage
also. Chronic exposure may result in anorexia, dermal rash, dysphasia, gastrointestinal
disturbance, vomiting, weakness, weightlessness, muscle pain, hepatic tenderness and nervous
disorder. It is also suspected Phenol may cause paralysis, cancer and gene to bre striation. Phenol
and its derivatives are toxic and classied as hazardous materials. These phenolic compounds
possess various degrees of toxicity and their fate in the environment is therefore important. In
recent years, a great deal of research work has been directed toward the development processes in
which enzymes are used to remove phenolic contaminants. Phenol is an antiseptic agent and is
used in surgery, which indicates that they are also toxic to many micro-organisms (Monteiro
A.A.M.G. et.al, 2002). If phenolic pollution goes to underground water, it causes serious
ecological problems. Hence, allowable amount of Phenol in industrial outgoing must not be more
than 0.5 mg/L.
Considering the above issue, the removal of such chemicals from industrial effluents is of great
importance. The conventional method of treatment of phenolics are largely physical and chemical,
like hybrid processes electro catalytic degradation (Yang R.D., et al. 2009) adsorption on to
different matrices, chemical oxidation, solvent extraction or irradiation but these processes led to
secondary effluent problems due to formation of toxic materials such as cyanates, chlorinated
phenols, hydrocarbons, etc. These methods comprise of mainly chlorination, ozonation, solvent
extraction, incineration, chemical oxidation, membrane processes, coagulation, flocculation,

adsorption, ion exchange, reverse osmosis, electrolysis and a plethora of other similar processes.
Biological treatment is attractive owing to the fact that the potential to almost degrade phenol and
other pollutants while producing innocuous end products can be utilized to maintain phenol
concentrations below the toxic limit at a reduced capital and overall maintenance cost. However,
difficulty arises in treatment due to the toxicity of aromatic compounds to the microbial population.
Their biological degradation is achieved through benzene ring cleavage using the enzyme present
in the microorganisms. The bacteria expresses differently when exposed to differential aromatic
concentrations and other conditions. The microorganisms are capable of using phenol and its
derivatives as the sole source of carbon and energy for cell growth and metabolism degrade phenol
via meta-pathway. In this process the benzene ring of phenol is dehydroxylated to form catechol
derivative and the ring is then opened through meta-oxidation. The final products are molecules
that can enter the tricarboxylic acid cycle.
Physical Properties of Phenol:
1) Molecular Structure:
2) Chemical Formula: C6H5OH

3) Other Names:
Carbolic Acid, Benzenol, Phenylic Acid, Hydroxybenzene, Phenic

Acid.
4) Solubility in Water: Phenol has a limited solubility (8.3g/100 ml at 20C) in water.

5) Acidity/Basicity:
Phenol is slightly Acidic in Nature
6) Appearance:
White Crystal
7) Molecular Weight: 94.11
8) Toxicity of Phenol: Acute exposure of Phenol is known to cause the following effects:
Disorders of Central Nervous System Leading to collapse and coma.
Hypothermia Reduction in body temperature.
Acute exposure of phenol can result in myocardial depression.
Muscle weakness and tremors have also been observed.
Phenol causes a burning effect on skin. As a result whitening and erosion of the
skin may also result.
Exposure to phenol may result in irritation of the eye, corneal whitening finally
leading to permanent blindness.
Phenol can cause gastrointestinal disturbance, vomiting, weakness, loss of weight

and muscle pain.
It is also suspected that exposure to phenol may cause paralysis and even cancer.
All such effects have led to classification of Phenol and its derivatives as highly toxic and
hazardous.
Physical Properties of 8-Hydroxyquinoline:
2) Product Name:
8-Hydroxyquinoline
3) Other Names:
8-Quinolinol; Quinolin-8-ol
4) Molecular Formula:
C9H7NO
5) Molecular Weight:
145.16
6) Medicine Class Appearance:
Off-White Needle Crystals
7) Purity (HPLC, GC):
99.8% minimum
8) Melting Point:
73 75 C
9) Water Solubility:
Slightly Soluble
10) Toxicity: Exposure to 8-Hydroxyquinoline is known to cause the following effects:
Potential Acute Health Effects: Hazardous in case of ingestion or inhalation.

Slightly hazardous in case of skin contact and eye contact (irritant, permeator).
Developmental Toxicity: The substance may be toxic to CNS and cause organ
damage. LD50 value of 1200 mg/kg was reported for oral administration to rodents.
Potential Chronic Health Effects:

a) Carcinogenic Effect: Deemed Not Classifiable for human by IARC.
b) Mutagenic Effect: Mutagenic for mammalian somatic cells. Mutagenic for
bacteria and/or yeast.
10
Properties of Catechol:
2) General Characteristics: Catechol is a colorless crystal with a phenolic odor (HSDB,

1993). It easily sublimes and can react with oxidizing materials. Catechol is soluble in
water, alcohol, carbon tetrachloride, hot benzene, chloroform, and ether. It is slightly
soluble in cold benzene and very soluble in pyridine and aqueous alkalies.
3) Molecular Weight:
110.11
4) Boiling Point:
245.5 C
5) Melting Point:
105 C
6) Flash Point:
261 F
7) Vapor Density:
3.79 (air=1)
8) Vapor Pressure:
0.03 mm Hg at 20 C
9) Conversion Factor:
1 ppm = 4.5 mg/m3
10) Toxicity of Catechol:
Catechol
is
Non-Carcinogenic.
Catechol
causes
methemoglobinemia. Systemic toxicity is similar to that of phenol; however, catechol may

be more likely to cause convulsions and hypertension. Direct contact is highly irritating to
the eyes and skin. Acute exposures can cause skin burns, headaches, nausea, muscle
twitching and convulsions. Catechol is a Central Nervous System depressant and increases
blood pressure as observed in animal studies.
11
Outline of the Experiments performed:
Samples were collected from various locations.
Species were isolated using microbiological tools and techniques.
Strains and species are grown in different aromatic compounds.
The following series of biochemical analyses are performed:

1. Protein profile analysis of different enzymes produced during normal conditions
and when aromatic compounds are present, and
2. Their bioinformatics analysis
Literature Review
12
13
Over the last two decades significant advances have been made in our understanding of the
anaerobic biodegradability of monoaromatic hydrocarbons. It is now known that compounds such
as benzene, toluene, and phenol can be biodegraded in the absence of Oxygen by a broad diversity
of organisms. These compounds have been shown to serve as carbon and energy sources for
bacteria growing phototrophically or respiratorily with nitrate, manganese and ferric iron sulfate
or carbon dioxide as the sole electron acceptor. In addition, it has also been recently shown by
studies that complete degradation of monoaromatic hydrocarbons can also be coupled to the
respiration of oxyanions of chlorine such as perchlorate or chlorate, or to the reduction of the
quinone moieties of humic substances. Many pure cultures of hydrocarbon degrading anaerobes
now exist and some novel biochemical and genetic pathways have been identified. In general, a
fumarate addition reaction is used as the initial activation step of the catabolic processes of the
corresponding monoaromatic hydrocarbon compounds. However, other reactions may
alternatively be involved depending on the electron acceptor utilized or the compound being
degraded. In case of toluene, fumarate addition to the methyl group mediated by benzylsuccinate
synthase appears to be the universal mechanism of activation and is now known to be utilized by
anoxygenic phototrophs, nitrate-reducing, Fe(III) reducing, sulfate-reducing, methanogenic
cultures. Many of these biochemical pathways produce unique extracellular intermediates that can
be utilized as biomarkers for the monitoring of hydrocarbon degradation in natural environments.
The group of scientists involved in the study did not perform any test on phenol derivative
degradation and their effect on cell growth. (Chakraborty R, et al. 2004).
The effect of adaptation of Psuedomonas putida F1 ATCC 700007 (Pp F1) to the biodegradation
of benzene (B), toluene (T) and phenol (P) was studied by another team of researchers. The
adaptation of microorganism to BTP decreased the biodegradation time from 24 hours to 6 hours
14
for benzene (90 mg/l) and toluene (90 mg/l), and from 90 to 18 hours for phenol (50 mg/l).
Andrews kinetic model for single substrate was solved by them to obtain specific growth rates,
half saturation and substrate inhibition constant. Cell growth using toluene (max, T = 0.61) and
benzene (max,
= 0.62) as carbon sources were better and faster than the growth in phenol
(max, P = 0.051). For the substrate mixtures, a sum kinetics model was used and the interaction
parameters were determined. These models provided an excellent prediction of growth kinetics
and the interaction between these substrates. Toluene inhibited the utilization of benzene
(IT, B = 5.16) much more than benzene inhibits the utilization of toluene (IB, T = 0.14) enhances the
biodegradation of phenol, and phenol inhibits the biodegradation of benzene (IP, B = 1.08) and
toluene. Scientists have given the effect of aromatic compounds on each others degradation by P.
putida but they have not studied the genes that affect this process (IP, T = 1.03). Besides this they
have studied the effect of physiological changes on biodegradation of these compounds (Tarik
Abuhamed, et al. 2004).
These group of researchers started with an acclimatized mixed microbial culture, predominantly
Pseudomonas sp. enriched from a sewage treatment plant. Its potential to simultaneously degrade
mixtures of phenol and m-cresol was investigated during its growth in batch shake flasks. A 22 full
factorial design with the two substrates at two different levels and different initial concentration
ranges (low and high), was employed by them to carry out the biodegradation experiments. The
substrates phenol and m-cresol were completely utilized within 21 hours when present at low
concentrations of 100 mg/L for each, and at a high concentration of 600 mg/L for each, a maximum
time of 187 hours was observed for their removal. The biodegradation results also showed that the
presence of phenol in low concentration range (300 100 mg/L) did not inhibit m-cresol
biodegradation. Whereas, the presence of m-cresol inhibited phenol biodegradation by the culture.
15
Moreover, irrespective of the concentrations used, phenol was degraded preferentially and earlier
than m-cresol. A sum kinetics model was used to describe the variation in the substrate specific
degradation rates, which gave a high co-efficient of determination value (R2 > 0.98) at the low
concentration range of the substrates. From the estimated interaction parameter values obtained
from this model, the inhibitory effect of phenol on m-cresol degradation by the culture was found
to be more pronounced compared to that of m-cresol on phenol. This study showed a good potential
of the indigenous mixed culture in degrading mixed substrate of phenolics but lacked the study of
effect of pH and temperature on both substrate consumption (Pichiah Saravanan, et al. 2008).
The kinetics of biodegradation of single phenol and sodium salicylate (SA) and their binary
mixtures in water by suspended Pseudomonas putida CCRC 14365 was studied at 30 C and pH
7.0. Experiments were performed at different total substrate concentrations (0 4.25 mM) and /or
mole fractions of phenol. The initial cell concentration was fixed at 0.025 g/L. Based on the
parameters of the Haldane model for specific growth rate of the cells on single phenol and SA
(correlation coefficient R2 > 0.9737), phenol had larger degradation rate than SA, whereas the
inhibition of P. putida by phenol was less significant than by SA. That is, the cells were more
favored to degrade phenol than SA under comparable conditions. On the other hand, the specific
growth rate of the cells on binary substrates could be described by an extended Haldane equation
(R2 = 0.9256). The substrate interactions were thus discussed according to the modeled parameters.
The dynamics in the biodegradation of single and binary substrate systems was finally analyzed.
The scientist group did not analyzed proteins associated with phenol metabolism (Ruey-Shin
Juang, et al. 2006).
Biological degradation of phenol and catechol by a bacterial strain of Pseudomonas putida (MTCC
1194) in basal salt medium (BSM) was investi gated in shake-flask experiments at 29.9 0.3 C
16
and pH of approximately 7.1. The lyophilized cultures of P. putida (MTCC 1194) were revived
and exposed to increasing concentrations of phenol, and catechol in shake-flasks. This bacterial
strain could be acclimatized to the concentrations of 1000 and 500 mg/l for phenol and catechol,
respectively, over a period of three months. The higher the concentration of phenol or catechol,
the longer was the lag period. The well-acclimatized culture of P. putida (MTCC 1194) degraded
the initial phenol concentration of 1000 mg/l and initial catechol concentration of 500 mg/l
completely in 162 and 94 hours respectively. Both the phenol and catechol were observed to be
inhibitory compounds. Monods and linearized Haldanes model could not represent the growth
kinetics over the studied concentration range. However, Haldanes growth kinetics model could
be fitted to the growth kinetics data well for the entire concentration range. Further, the decay
coefficients have been found to be 0.0056 and 0.0067 h1 for the growth on phenol and catechol,
respectively. Besides, the yield coefficient for the growth on phenol and catechol were found to be
0.65 and 0.50 mg/mg, respectively. It is in view that the above information would be useful for
modeling and designing the units treating phenol and catechol containing wastewaters (Arinjay
Kumar et al. 2005).
Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methyl substituted phenols.
These compounds are degraded by the set of enzymes encoded by the plasmid located dmp-operon.
The sequences of all the fifteen structural genes required to encode the nine enzymes of the
catabolic pathway have been determined and the corresponding proteins have been purified. In
this review the interplay between the genetic analysis and biochemical characterization of the
catabolic pathway is emphasized. The first step in the pathway, the conversion of phenol to
catechol, is catalyzed by a novel multicomponent oxygenases, particularly methane
monooxygenase (EC 1.14.13.25) in their paper. The other enzymes encoded by the operon are
17
those of the well-known meta-cleavage pathway for catechol, and include the recently discovered
meta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties
of these meta-pathway enzymes, and isofuctional enzymes from other aromatic degraders, are
summarized. Analysis of the sequences of the pathway proteins, many of which are unique to the
meta-pathway, suggests new approaches to the study of these generally little-characterized
enzymes. Furthermore, biochemical studies of some of these enzymes suggests that physical
associations between meta-pathway enzymes play an important role. In addition to the pathway
enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to XylR regulator
of toluene and xylene catabolism is discussed. But they have not (Powlowski J. and Shingler V.
1994).
The present thesis will analyze and carry out necessary experiment to know the microorganism
involved in aromatic degradation and their ability to grow in higher concentration of aromatic
compounds and the proteins involved in phenol and its derivative degradation.
Materials and Methods
18

Microbiological Tests
19
20
Sampling from Industrial Areas:

Waste waters were collected from different industrial effluent near Indraprastha area, Delhi. Here
different types of industries like chemical, pharmaceutical, petrochemical, food industries are
present. So effluents contain various types of contaminants that mostly include aromatic
compounds and polycyclic aromatic hydrocarbons.
Figure 1 : Yamuna River, Indraprastha Industrial Area
21
Isolation of species by Serial Dilution and Spreading

Serial dilutions are an accurate method of making solutions of low molar concentrations. A stock
solution with a molarity greater than that which is required is accurately diluted using a suitable
solvent. Since measuring small volumes of solution is prone to error, an F series of dilutions are
performed in order to gradually reduce the concentration of the solution from that of the stock
solution. Then 50 l of the lowest two molar concentrations of this is spread on LB agar plate and
kept overnight.
Procedure
Five test tubes were obtained and 9 ml of sterilized water were pipetted into each tube.
From the stock solution (waste water), 1 ml of waste water was pipetted into test tube
number 1 with 9 ml of sterilized water and mixed properly.
Then, 1ml from test tube number 1 was pipetted into test tube number 2 with 9 ml of
sterilized water and mixed properly.
Then, 1 ml from test tube number 2 was pipetted into test tube number 3 with 9 ml of
sterilized water and mixed properly
Then, 1 ml from test tube number 3 was pipetted into test tube number 4 with 9 ml of
Then, 1ml from test tube number 4 was pipetted into test tube number 5 with 9 ml of
Then picked up test tube number 3 and 4 and took out 50 l from each and spread it on LB
agar plate and marked it properly. The plates were stored overnight in an incubator at
37 C.
22
Isolation of species by Streak Plate Method

A small amount of sample is transferred onto the surface of a suitable solid agar medium either by
loop or transfer needle. This is often streaked in a way to provide successive dilutions and
ultimately to have well isolated colonies. Streaking may be done in any of the ways. In each case
the sample becomes progressively diluted and at the end of streaking, one would expect the well
isolated colonies.
Procedure
In previous step, spreading plates containing colonies were prepared for two dilutions 10
3
and 104. These plates were used for streaking.
Prepared Lurea Bertini Broth.

Inside Laminar Airflow Bench, sterilize inoculating loop and picked up single isolated
colony from 103 dilution and inoculated it into LB broth, and kept overnight in an
incubator.
Similarly performed the same procedure with 104 dilution.
Labelled the LB agar containing petridish on the flipside.
Sterilized the transfer loop before obtaining a specimen from LB broth containing 103
and 104 dilutions.
Collected a sample of specimen using the sterile loop.
Inserted the loop into the culture tube and removed a loopful of broth.
Streaked both the plates, then kept them overnight in the incubator at 37 C.

Biochemical Methods:
23
24
These are groups of tests used to identify unknown species in a collected sample. The methods
tested on the present sample are enlisted below:
Indole Test
Methyl Red Test
Voges Proskauer Test
Citrate Test
Catalase Test
Gram Staining Test
Urea Hydrolysis Test
Mannitol Fermentation Test
Starch Hydrolysis Test
Gelatinase Test
MacConkeys Agar Test
Growth at 4 C and at 25 C
Growth at 5% NaCl and 10% NaCl
Cetrimide Test
Carbohydrate fermentation Tests:

1) Lactose Fermentation
2) Dextrose Fermentation
3) Sucrose Fermentation
25
Indole Test
Principle:
The amino acid Tryptophan can be broken down by enzyme Trytophanase to form indole, pyruvic
acid and ammonia as end products. Tryptophanase differentiates indole-positive enterics, such as
E. coli and P. vulgaris from indole-negative enterics, such as S. marcescens (MacFaddin and Jean
F. 1980).
Media and Reagents:
Media with Tryptophan or in Peptone water medium and Klovacs Reagent.
Method:
1) Innoculate medium and incubate at 37 C for 24 48 hours.
2) Post incubation add 5 drops of Kovacs Reagent to the surface. Do not shake or stir the tube.
Expected Results:
Positive Test: Kovacs reagent combines with indole and turns the surface red.
Negative Test: No Red color development is observed.
26
Methyl Red Test

Principle:
Methyl red test is used to identify enteric bacteria based on their pattern of glucose metabolism. If
they use mixed acid pathways and produce acidic products, then they are said to test methyl-redpositive. If they use butylene glycol pathway and produce neutral end products, then they are said
to test methyl-red-negative (MacFaddin K. 2001).
Media and Reagents:
Glucose phosphate broth and methyl red indicator.
Method:
1) Inoculate broth and incubate at 37 C for 2 5 days.
2) Transfer 2.5 ml of inoculation to another tube and add 5 drops of methyl red.
3) A small sample is rolled between the palms of the hands to disperse methyl red.
Expected results:
Positive Test: acids + methyl red red solution
Negative Test: neutral end products + methyl red yellow color
27
Voges Proskeaur Test

Principle:
It is used to identify enteric bacteria based on their pattern of glucose metabolism. The enterics
that produce neutral end-products, such as acetoin are detected by the VP test (Manual of Clinical
Microbiology, 5th Edition).
Media and Reagent:
Glucose phosphate broth and Reagent A (contains alpha-naphthol) Reagent B (contains KOH).
Method:
1) Inoculated medium and incubate at 37 C for 48 hours.
2) After incubation, transferred 2.5 ml of inoculate to another tube.
3) Added 6 drops of Reagent A and 2 drops of Reagent B and mixed gently.
4) The mixture is allowed to sit still for 10 15 minutes to allow time color development.
Expected Results:
Positive Test: acetoin + alpha-napthol + KOH Red Color
Negative Test: alpha-napthol + KOH Copper color
28
Catalase Test
Principle:
Catalase is an enzyme that decomposes Hydrogen Peroxide (H2O2) into oxygen and water.
Excluding the Streptococci sp. most aerobic and facilitative anaerobic bacteria possesses catalytic
activity (MacFaddin J.F, 1980).
Hydrogen peroxide forms as one of the oxidative end products of aerobic carbohydrate
metabolism. Catalase converts hydrogen peroxide into water and oxygen. The catalase test is
commonly used to differentiate streptococci (negative) or staphylococci (positive).
Reagents and Equipment:
3% Hydrogen peroxide, clean glass slide. Dropper, Bacteriological Loop
Method:
1. With loop or applicator stick, transferred cells from the center of a well-isolated colony to
glass slide.
2. Added 1-2 drops of the 3% Hydrogen peroxide to the bacterial
Expected Result:
Positive Test: Rapid appearance of sustained gas bubbles
Negative Test: No gas bubble production.
29
Citrate Test
Principle: Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme
citrase. Citrase is produced by some bacteria such as E. aerogenes but not by others like E. coli
(MacFaddin, J. F., 1980).
Media and Reagent: Simmon's Citrate Agar. It has citrase as the only carbon source and pH
indicator bromothymol blue.
Method:
1) Pour the agar in boiling tubes and kept it at an angle of 45.
2) After solidification, inoculate the slant and incubate at 37C for 24-48 hours.
Expected Results:
Positive Test: Growth and color changes to blue.
Negative Test: No growth and color remains green.
30
Gram Staining Test

Principle: Gram staining, the most widely used staining procedure in bacteriology, is a complex
and differential staining procedure. Through a series of staining and decolorization steps,
organisms in the Bacteria are differentiated according to cell wall composition. Gram-positive
bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall). These stain
purple. Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of wall), and
high lipid content. These stain pink.( Saviola, B., and Bishai, W. December 1, 2000).
Media and reagent:
Primary Stain: Crystal Violet Staining Reagent, Gram's Iodine, Decolorizing Agent (ethanol),
safranin
Method:
1) Picked up a colony from two plates 10"3 and 10"4 dilution and placed it on two glass slides.
2) "Heat-fix" the slide with the specimen by passing it over a heat source, such as a flame,
several times using a forceps. The slide should be passed very quickly through the flame
and not be heated excessively. Place slide on the staining tray.
3) Flood the fixed smear with crystal violet solution and allow to remain for 1 minute.
4) Rinsed off the crystal violet with distilled or tap water.
5) Rinsed off the crystal violet with distilled or tap water.
6) Flood the slide with iodine solution. Allow to remain for one minute.
7) Rinsed off the iodine solution with distilled or tap water.
8) Flood the slide with decolorizcr for one to five seconds.
9) Rinse off the decolorizer with distilled or tap water.
10) Flood the slide with safranin. Allow to remain for 30 seconds.
11) Rinsed off the safranin with distilled or tap w ater.

12) Dried the slide on bibulous paper or absorbent paper and place in an upright position.
13) Microscopically examine the slide for bacterial organisms under a I0X objective.
14) Observed several fields on the slide for bacterial organisms.
Expected result:
Gram-positive bacteria stain deep violet to blue
Gram-negative bacteria stain pink to red.
31
32
Urea Hydrolysis Test

Principle: Some bacteria produce urease, an enzyme capable of breaking down urea and produce
alkaline end products. This distinguishes Proteus from other bacteria (Benini, Stefano et.al., 1999).
Media and Reagent:
Urea Broth with phenol red
Method:
Inoculate the media with a loop and incubate at 37C for 24 hours.
Expected Results
Positive Test: Production of alkaline end products = pinkish red color
Negative Test: No color change.
Mannitol Test
Principle: Mannitol Salt Agar contains 7.5% NaCl, which is inhibitory to most bacteria. Bacteria
that can grow on this agar can be differentiated based on mannitol fermentation. Fermentation of
mannitol results in acidic products which turn phenol red pH indicator from red to yellow.( John
A. Washington, et.al ; 1970 April).
Media and reagent:
MSA and phenol red indicator
Method:
Streaked MSA plate and incubated at 37C for 2 days.
Expected Results:
Positive Test: Mannitol fermentation occurred = growth and color changed to yellow
Negative Test: No mannitol fermentation = may or may not grow and no color change.
33
Starch Test
Principle: Starch molecules are too large to enter the bacterial cell, so some bacteria secrete
exoenzymes to degrade starch into subunits that can then be utilized by the organism
(Alfred.E.Brown. 2007).
Material and Method:
Starch agar is a simple nutritive medium with starch added. Since no color change occurs in the
medium when organisms hydrolyze starch, added iodine to the plate after incubation. Iodine turns
blue, purple, or black (depending on the concentration of iodine) in the presence of starch.
Expected Result:
A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.
34
Gelatin Test
Principle: Some bacteria produce Gelatinase enzyme that hydrolyzes gelatin (M J Pickett et.al.
1991 October; 29).
Method:
The gelatin stab method employs nutrient gelatin deep tubes that contain 12% gelatin. A heavy
inoculum from a pure culture of the test organism is stabbed into the media. The gelatin media is
incubated for at least 48 hours, and then placed into the refrigerator for approximately 30 minutes.
Expected Result:
Positive Test: If the organism has produced sufficient Gelatinase, the tube will remain liquid (at
least partially) and not solidify in the refrigerator. A Positive Test result is recorded.
Negative Test: If the gelatin is still intact (the bacteria did not produce Gelatinase), the media will
solidify in the refrigerator and a Negative Test result is recorded.
Some organisms may produce Gelatinase in rather small quantities. Thus, a tube with a negative
Gelatinase result should be reincubated for 30dys. Whenever desired, the tube may be refrigerated
and results observed. If the tube is still negative after 30days of incubation (completely solidifies
when refrigerated), it can be reasonably concluded that this organism does not produce Gelatinase.
35
MacConkey's Agar Test

Principle: MacConkey's agar is a selective and differential medium selective medium for gram ve bacteria (bile sail & crystal violet inhibit the growth of gram +ve bacteria).( Leininger, H.V;
(1976).
Materials: Test sugar: lactose. pH indicator neutral red (yellow in alkaline, pink in acidic pH).
Method:
1. Inoculated MacConkey's agar plate with the test organism by streaking.
2. Incubated the plate at 35oC for 24 hrs.
Expected Result:
Pink colonies: lactose fermenter
Pale colonies: lactose non-fermenter.
36
Growth at 4C and 25C

Principle: The cold-loving organisms are psychrophilics defined by their ability to grow at 0
degrees. A variant of a psychrophile (which usually has an optimum T of 10-15 degrees) is a
psychrotroph which grows at 0 degrees but displays an optimum T in the mesophile range, nearer
room temperature. Psychrotrophs are the scourge of food storage in refrigerators since they are
invariably brought in from their mesophilic habitats and continue to grow in the refrigerated
environment where they spoil the food. Of course, they grow slower at 2 degrees than at 25 degrees
(Bachoon, Dave S. et.al, 2008).
Group
Comments
Minimum(C) Optimum(C) Maximum(C)
Psychrophile
Below 0
1015
Below 20
Grows best at relatively

low Temperature
Ability to grow at low
Psychrotroph
1530
Above 25
Temperatures but prefer

moderate ones.
Table 1:
Temperature require for growth (C)
Method:
1. Prepared LB Agar plates and marked the two plates as 10"' for 4C and 10'* for 25C.
2. Streaked the plates with pure isolated cultures.
3. Incubated one plate at 4C and another plate at 25C for overnight in incubator.
Expected Result:
If it grows at 4C then it is a psychrophile microorganism and if it grows at 25C then it is a
psychrotroph.
37
Growth at 5% and 10% NaCI

Principle: A halophile is an organism that can grow in higher salt concentrations than the norm.
Based on optimal saline environments halophilic organisms can be grouped into three categories:
(i) extreme halophiles, (ii) moderate halophiles, and (iii) lightly halophilic or halotolerant
organisms. They can tolerate from 3% salinity to 35% saline environment. (Yiang et.al, 2008)
Materials: LB agar and NaCI.
Method:
1. Prepared 4 LB agar plates with 5% and 10% NaCI for two dilutions and marked them as
growth at 5% and 10% NaCI"
2. Streaked the culture on the plates and incubated them for 24Hr at 37C in an incubator.
Expected Result:
If growth occurs on the plates means species is halophiles and if not result is negative.
38
Cetrimide Test
Principle: Enzymatic Digest of Gelatin provides the nitrogen, vitamins, and carbon in Cetrimide
Agar. Magnesium Chloride and Potassium Chloride enhance the production of pyocyanin and
fluorescein.Cetrimide (cetyltrimethylammonium bromide) is the selective agent. Cetrimide acts as
a quaternary ammonium cationic detergent causing nitrogen and phosphorous to be released from
bacterial cells other than Pseudomonas aeruginosa. Agar is the solidifying agent. Glycerol is
supplemented as a source of carbon (Zilligan, P. H. 1995).
Materials: Enzymatic Digest of Gelatin, Cetrimide (Cetyltrimemylammonium Bromide, Glycerol
Method:
Inoculated species colonies directly on Cetrimide Agar by the streak method from nonselective
medium or the clinical specimen.
When plating directly from the specimen, the inoculum level will vary.
Expected Results:
Examine plates or tubes for the presence of characteristic blue, blue-green, or yellow-green
pigment. If both pyocyanin and fluorescein, then it is positive result. If not it is deemed to be a
negative result.
39
Carbohydrate, Lactose, Sucrose and Dextrose Fermentation

Principle:
In this set of tests, you will be able to determine if the bacterium can ferment dextrose, can
hydrolyze lactose (into glucose and galactose and then ferment either of the monomers released,
usually only the glucose), and can hydrolyze sucrose (into glucose and fructose and then ferment
either of the monomers released). Fermentation simply uses an organic molecule as an electron
acceptor, with the result being the production of organic acids (and a pH change in the medium).
One will also be able to determine if the bacterium can produce a gas (usually C02) during the
fermentation process.(Zugh R. et.al. ,1953).
Materials:
Peptone, NaCI, and different carbohydrate with phenol red.
Method:
1. Using aseptic technique, transfer a small inoculum of each of your assigned bacteria into
each of the three broths (glucose, sucrose, lactose).
2. Incubate the inoculated broths at 37C for 24-48 hours.
3. Observe each broth and note the result.
Expected Result:
Growth with red color (i.e., no change in color compared to the uninoculated control) - indicates
the bacterium cannot ferment the sugar in the tube (either cannot ferment either of the monomers
or cannot hydrolyze the dimer to release monomers) or does not produce any organic acids if
fermentation does take place.
Growth with yellow color - acid produced (lower pH changes the phenol red pH indicator in the
broth to yellow); this indicates the bacterium CAN ferment the sugar and, if the sugar is sucrose
or lactose, can also hydrolyze that sugar to release "fermentable" monomers.
40
Bacterial identification based on biochemical tests

Bergey's Manual Of Determinative Bacteriology (Seventh Edition) by
Robert S. Breed1, E. G. D. Murray2, Nathan R. Smith3
was referred for bacterial identification based on the test results of biochemical tests performed.
The identified microorganism for two different dilutions 10"3 and 10*4 has been discussed in
"RESULT AND DISCUSSION Section".
Ixite Professor Emerilus, Cnrnell University, Geneva, New York

Research Professor, University of Western Ontario.London, Ontario, Canada
3
Senior Bacteriologist, Retired, Plant Industry Station,(U. S. Department of Agriculture, Beltsville, Maryland
2

Biodegradation experiments:
41
42
Phenol Degradation
The biodegradation potential of the selected strains were evaluated in medium containing phenol
at different concentrations (from 2mM to lOmM concentration) by incubating it at 37C in an
incubator shaker(200rpm) and then by monitoring growth of biomass and analyzing residual
phenol.
Formula to calculate phenol amount in LB Broth (50ml):
Weight (g)=
Molarity (M ) Molecular Weight Volume(ml)

1000
Serial No
Concentration
Phenol amount(l)
1.
2 mM
9.41
2.
4 mM
18.82
3.
6 mM
28.23
4.
8 mM
37.64
5.
10 mM
47.05
Table 2.
Calculation of amount of Phenol for different concentrations
Biomass Analysis
In case of bacteria, biomass (turbidity) was monitored by measuring OD at 600 nm at regular
intervals of time (Harayama, S. and K. N. Timmis. 1989).
Phenol analysis:
Residual Phenol was monitored by measuring OD at 269nm at an interval of 3 Hrs ,and Phenol
degradation % was calculated by: Initial phenol - Residual phenol xlOO Initial phenol
43
8-Hydroxyquinoline Degradation
The biodegradation potential of the selected strains were evaluated in medium containing 8Hydroxyquinoline at different concentration from 2mM to lOmM concentration by incubating it
at 37C in an incubator shaker(200rpm) and then by monitoring growth of biomass and analyzing
residual 8- Hydroxyquinoline .
Formula to calculate 8- Hydroxyquinoline amount in LB Broth(50ml):
Weight (g)=
Molarity (M ) Molecular Weight Volume(ml)

1000
8-Hydroxyquinoline
Serial No
Concentration
1.
2 mM
14.52
2.
4 mM
29.04
3.
6 mM
43.56
4.
8 mM
58.08
5.
10 mM
72.60
Table 3.
amount(l)
Calculation of amount of 8-hydroxyquinoline for different Concentrations
Biomass Analysis
intervals of time (Gibson D.T., et al, 1968).
44
8-Hydroxyquinoline analysis
Residual 8-Hydroquinoltne was monitored by:
1. Take aliquot containing catechol and mixed 1 ml of Sodium nitrite solution in a volumetric
flask.
2. Add 1 ml of 4.8M sulphuric acid solution.then added 2 ml of neutral red solution and
maintain volume by adding distilled water upto 10 ml.
3. Incubated for 30 sec and then take O.D at 540nm.
8-Hydroquinoline degradation % was calculated by:

Degradation (%)
(Initial 8-Hydroxyquinoline)-(Residual 8-Hydroxyquinoline)

100
(Initial 8-Hydroxyquinoline)
45
Catechol Degradation
The biodegradation potential of the selected strains were evaluated in medium containing Catechol
at different concentration from 2mM to l0mM concentration by incubating it at 37C in an
incubator shaker(200rpm) and then by monitoring growth of biomass and analyzing residual
catechol .
Formula to calculate Catechol amount in LB Broth (50ml):
Weight (g)=
Molarity(M ) Molecular Weight Volume (ml)

1000
Serial No
Concentration
Catechol amount(l)
1.
2 mM
11.01
2.
4 mM
22.02
3.
6 mM
33.03
4.
8 mM
44.04
5.
10 mM
55.05
Biomass Analysis
intervals of time (Dagley S, 1985).
46
Catechol analysis
Residual catechol was monitored by the following procedure:
1. Take aliquote containing eateehol and mixed 1 ml of nitrite solution in a volumetric flask.
2. Add 1 ml og 4.8M sulphuric acid solution
3. Added 2 ml of neutral red solution and maintained volume by adding distilled water upto
10 ml.
4. Incubated for 30 sec and then take O.D at 540nm.
Catechol degradation % was calculated by:
Degradation (%)=
Initial catechol Residual Catechol

100
Initial catechol

Protein profile analysis:
47
Cell disruption and protein extraction

1. .Harvested bacterial cells by centrifugation (5000 rpm/5 min), aspirate supernatant.
2. Suspend the pellet of bacterial cell culture in 0.75 ml of cold extraction buffer (200 l).
3. Add 10 l of triton -X-100 and 20 l of lysozyme.
4. Mix the content by inversion and incubate for 20 min at room temperature.
5. Ccntrifuge 12,000 rpm for 20min at 4C.
6. Take supernatant as a crude protein extract. If protein solution is too dilute then:
Procedure
1. Precipitate protein with 10% TCA.
2. Incubate on ice for 30 min .
3. Centrifugeat 10000*g for 5 min.
4. Wash precipitate with ethanol-cther (1:1).
5. Dissolve precipitate In distilled water.
6. Mix with SDS disruption mix buffer .Boil for 5 min and cool it.
48
49
SDS PAGE for protein analysis

Principle
SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its molecules
have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in
proportion to its relative molecuar mass. The negative charges on SDS destroy most of the complex
structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in
an electric field. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller
molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured
polypeptides, the final separation of proteins is dependent almost entirely on the differences in
relative molecular mass of polypeptides. In a gel of uniform density the relative migration distance
of a protein is negatively proportional to the log of its mass. If proteins of known mass are run
simultaneously with the unknowns, the relationship among the masses of unknown and known
proteins can be estimated. Protein separation by SDS-PAGE can be used to estimate relative
abundance of major proteins in a sample, and to determine the distribution of proteins among
fraction.
Method
1. 1 .Cleaned glass plates with soap and water, then with ethanol. Assemble the glass plates and
spacers.
2. 2.Sealed all the three sides using agarose<0.8%) and allowed to stand for 10-15 min.
3. Prepared resolving gel solution and quickly poured it into the glass plates about SA of the
whole length.Allowed it to solidify for 30 min.
4. Again prepared slacking gel solution and poured it on the top of polymerized resolving
gel.Placed the comb over the top and then allowed it to solidify for 30 min.
50
Loading the gel:

1. Attach the large gel plates containing the polymerized gel to the apparatus via the clips
provided. Pour Tris-glycine SDS buffer into the upper and lower chambers.
2. Remove bubbles trapped at the bottom of the glass plates in the large gel with a syringe.
3. Mixcd in equal ratio Sample disruption mix and sample and heated it for 10-15 min at 65
degree C and then loaded it into the wells..
4. Run the gel at a constant current of 20 mA. After the dye front enters the resolving gel,turned
the current up to 30 mA.
5. Removed the gel from the cassette and then the plates are separated and the gel is dropped into
a staining dish containing staining solution and kept it for 24 hr at room temperature.
6. After 24 hr put the Gel into the destaining solution for 1-2 hr and then visualized it.
___________
Result and Discussion
51

Isolation of bacterial species
Species are isolated using microbiological techniques spreading and streaking.
Figure 3. Spread plates for 10-3 isolation
Figure 5. Streaked Plates for 10-3 isolation
Figure 4. Spread Plates for 10-4 isolation
Figure 6. Streaked Plates for 10-4 isolation
52

Biochemical tests:
53
54
Indoletest
No color change is observed. Negative result for both the isolates means that the bacterial species
under investigation do not have the ability to split indole from the amino acid tryptophan. This
division is performed by a chain of a number of different intracellular enzymes, a system generally
referred to as "tryptophanase(C MacFaddin, Jean F; 1980).
Figure 7.
Indole and Tryptophanase reaction
Figure 8. Indole test for 10-4 isolate (-ve)
Figure 9. Indole test for 10-3 isolate (-ve)
55
Methyl Red Test

All enteric bacteria initially produce pyruvic acid from glucose metabolism.
Glucose Glucose Metabolism Pyruvic acid
Positive result: Some enterics subsequently use the mixed acid pathway to metabolize Pyruvic
acid to other acids (lactic, acetic, and formic acids):
Pyruvic acid Mixed Acid Pathways lactic, acetic, and formic acids
Many acids (pH 4.2) + added methyl red = red color
Hence if red color appears the Bacteria are said to test methyl-red-positive.( Harden, A. 1906)
Negative result: Other enterics subsequently use butylene glycol pathway to metabolize Pyruvic
acid to neutral end-products.
Pyruvic acid Butylene Glycol Pathway neutral end-products.
Neutral end-products (pH 6.0) + added methyl red yellow color
These Bacteria are called methyl-red-negative.
Result Obtained: No color change is observed and hence the

result is negative. It indicates that the organism does not uses
the mixed acid fermentation pathway to convert glucose into
stable acidic end-product. Thus when methyl red is added, in
the absence of acidic end products, the methyl red changes to
yellow color.
56
Figure 10. Methyl test result for 10-3 and 10-4 isolates (-ve)
Voges-Proskauer test
Copper color of the medium indicates negative VP test result. This means that the organism does
not produce acetoin by utilizing the butylene glycol pathway (Voges 0., and B. Proskauer. 1898).
Positive Test Reaction:
Glucose Glucose Metabolism Pyruvic Acid
Pyruvic acid Acetoin
Acetoin + added alpha-naphthol + added KOH red color
Negative Test Reaction:
Glucose Glucose MetabolismPyruvic Acid
Pyruvic acid No Acetoin
No acetoin + added alpha-naphthol + added KOH copper color
Figure 11. V-P test for 10-3 and 10-4 isolate (-ve)
57
Citrate test result

Positive Result: Growth on the medium even without color change is considered as positive. A
color change in the medium would be observed if the test organism produces acid or alkali during
its growth. The usual color change observed is from green (neutral) to blue (alkaline).
Negative Result: No growth observed.
Test results: The color changes from green to blue. This means positive result and indicates that
the organism contains the enzyme citratase which breaks down citrate releasing pyruvate, sodium
bicarbonate (NaHCO3) as well as ammonia (NH3). This results in an alkaline pH. Thus there is a
change of the mediums color from green to blue (Koser, S. A. 1924).
Figure 12. Citrate test result for 10-3 isolate (Positive
Figure 13. Citrate test result for 10-4 isolate
color changed with growth)
(Positive color changed with growth)
58
Catalase test result

Showing positive result for both the isolates, bubb1e formation is indicative of presence of catalase
enzyme in organism. (Woese, C. R. (1987).
Figure 14. Catalase test result for 10-3 and 10-4 isolate (Positive result)
59
Gram staining result
Figure 15. Gram Negative Cell wall structure
Result: Gram-negative bacteria takes pink stain because they do not retain the crystal violet dye
in the Gram stain protocol. Gram-negative bacteria will thus appear red or pink following a Gram
stain procedure due to the effects of the counterstain safranin.( Woese, C. R. (1987b ).
Figure 16. Gram staining result for 10-3 dilution (Gram ve rod shaped)
Figure 17. Gram staining result for 10-4 dilution (Gram ve rod shaped)
60
61
Urease test result

Result: Organisms that produce urease will turn phenol red (pH6.8) to hot pink colour due to
the ammonia produced upon hydrolysis of urea. Those that do not produce urease result in no color
change as no ammonia is produced (Woese, C. R. (1987)).
Reaction:
(NH2)2CO + 2 H2O
Urease
CO2 + H2O + 2 NH3
Figure 18. Urease test result for 10-3 dilution. (negative result). No color
change indicates that the organism does not produce urease.
Figure 19. Urease test result for 10-4 dilution. Color change from
yellow to pink ndicates that the organism produces urease (+ve
result.)
62
63
Mannitol test result

Result: No growth and no color change indicates that the organism cant ferment mannitol .But if
grown on mannitol agar and color changed from red to yellow indicates that the organism in it can
Ferment mannitol that results in acidic products which turn phenol red pH indicator from red to
yellow.( Priest, F. G. 1977.).
Figure 20. Mannitol test result for 10-3 dilution.
Figure 21. Mannitol test result for 10-4 dilution.
64
Starch test result

This test is used to detect the enzyme amylase, which breaks down. starch. Aef incubalion the
plate is treated with Grams iodine. If starch has been hydrolyzed (broken down) then them is 3
clear zone around the bacterial growth produce the exoenzyme amylase which cleaves the starch
into di- and monosaccharides( Bird, R., and R. H. Hopkins. 1954. ).; if it has not been hydrolyzed
then there is a black/blue area indicating the presence of starch that means the organism has not
utilized starch.
Figure 22. Starch test result for 10-3 dilution (-ve test indicates that even
after adding Iodine there is no clearing around the bacterial growth.
This implies that the organism has not hydrolysed starch.
Figure 23. Starch test result for 10-4 dilution (-ve test indicates
that even after adding Iodine there is no clearing around the
bacterial growth. This implies that the organism has not
hydrolysed starch.
65
Gelatin test result

The gelatin is the substrate for the determination of the ability of an organism to produce
gelatinases, which are proteolytic-like enzymes active in the liquefaction of gelatin. (Lautrop, H.
1956).
Figure 24. Gelatin test result for 10-4 dilution (+ve test indicates that
the organism has produced sufficient Gelatinase. The tube remained
liquid and did not solidify under refrigeration.
Figure 25. Gelatin test result for 10-3 dilution (-ve test indicates that
the organism has not produced Gelatinase. The tube solidified under
refrigeration.
66
MacConkey's Agar Test result

Whenever bacterial colonies are growing on MacConkeys AgarBacteria, known as lactose
fermenters, eat the medias lactose, and, in the process, create an acidic end product that causes
the pH indicator, neutral red, to turn pink. With MacConkeys, it is not the media that changes
color, but rather the actual colonies of lactose fermenting bacteria that appear pink. Non-lactose
fermenting bacteria will be colorless (or, if they have any color, will be their natural color rather
than pink). (Schauer Cynthia (2007).
Figure 26. Mac Conckeys agar test result for 10-3 dilution. No growth
and no color change indicates negative results.
Figure 27. Mac Conckeys agar test result for 10-4 dilution. No
growth and no color change indicates negative results.
67
Growth at 4C and 25C

Microorganisms growing on LB Agar at temperature 25C and not at 4C indicates that the
organism is a psychrotroph and not a psychrophiles because the optimum temperature for the
psychrotroph is 25C and minimum growth temperature is 4C.In this no growth is seen at 4C
but there is maximum growth at 25C.So the organism is a Psychrotroph. (Gutierrez, M.C. et.al.
2002)
Figure 28. Growth of bacteria at 4C for 10-4 dilution (-ve result). No

growth is seen
Figure 29. Growth of bacteria at 4C for 10-4 dilution (-ve result). No

growth is seen
Figure 30. Growth of bacteria at 25C for 10-3 dilution (+ve result).
Growth is seen.
Figure 31. Growth of bacteria at 25C for 10-4 dilution (+ve result).
Growth is seen
68
69
Growth at 5% and 10 % NaCl

Halobacterium is a group of Archaea that have a high tolerance for elevated levels of salinity.
Some species of halobacteria have acidic proteins that resist the denaturing effects of
salts.(Gabwy, . L. & Hadleyc, . J. (1957).
Figure 32. Growth in 5% NaCl of 10-3 dilution. Growth was seen,

which indicated that the microbe is salt tolerant.
Figure 33. Growth in 10% NaCl of 10-3 dilution. Growth was seen

70
71
Cetrimide agar test result:

Some organisms produces a number of water-soluble pigments, including the yellow-green or
yellow-brown uorescent pigment pyoverdin (uorescein). When pyoverdin combines with the
blue water-soluble pigment pyocyanin, the bright green color characteristic is created. Agar
containing Cetrimide has been used successfully to isolate these types of organisms .( King, E. 0.,
M. K. Ward, and E. E. Raney. 1954).
Figure 36. Cetrimide test for 10-3 dilution. Change in color and growth
shows positive result.
Figure 37. Cetrimide test for 10-4 dilution. Change in color and growth
shows positive result.
72
Carbohydrate fermentation test result

Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate
utilization tests are designed to detect the change in pH which would occur if fermentation of the
given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH indicator
(phenol red) to turn yellow. If the bacteria do not ferment the carbohydrate then the media remains
red. If gas is produced as a by product of fermentation, then the Durham tube will have a bubble
in it.( Muhammad ferhan et.al.2002).
Figure 38. Lactose test for 10-3 dilution. No color change
Figure 39. Lactose test for 10-4 dilution. Color change
indicates bacteria cannot ferment Lactose. (-ve result)
from pink to yellow indicates a positive result.
73
Figure 40. Dextrose test for both 10-3 and 10-4 dilution.
Figure 41. Sucrose test for both 10-3 and 10-4 dilution.
Color change from pink to yellow indicates positive
Color change from pink-red to yellow indicates positive
result.
result. Bacteria can ferment sucrose.
74
Bacterial Identification based on biochemical test

After studying BERGEY'S MANUAL of DETERMINATIVE BACTERIOLOGY BY ROBERT
S. BREED and analysed various research papers on the related topic (N .M.E. Eissa et.al.20l0) ,
the species that is identied for both the dilutions are:
1. l0-3 dilution is identied as Pseudomonas putida.
2. 10-4 dilution is identied as Pseudomonas aerugjnosa.
Biodegradation study of different aromatic compounds
75
76
The minimal medium has been used in the degradation studies and the pH of the medium was
adjusted to 7.0 with 2M NaOH. Phenol and other two aromatic compounds catechol and 8hydroquinoline was added to the minimal medium aer sterilization. Minimal media together with
phenol was used for biodegradation studies. The isolates (lml )were added into the medium(50ml)
containing different concentration of these aromatic compounds and and incubated at 37C for
24h with agitation at 120 rpm.Then taken O.D at
different time intervals and calculated
degradation percentage of these aromatic compounds ,following results were obtained.
77
Microbial Growth Analysis using Spectrophotometric

method
O.D. (600 nm)
Growth Curve for 10-3 dilution (600 nm) in phenol

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
O.D. after
256 hrs.
0.144
0.367
1.046
1.4
1.409
1.302
4mM
0.07
0.357
0.873
1.232
1.336
1.2
6mM
0.136
0.27
0.423
0.491
0.645
0.5
8mM
0.099
0.19
0.42
0.443
0.527
0.49
10mM
0.086
0.237
0.238
0.226
0.216
0.2
2mM
Time in hours
2mM
4mM
6mM
8mM
10mM
O.D. (600 nm)
Growth curve for 10-4 dilution (600 nm) in phenol

2.5
2
1.5
1
0.5
0
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
O.D. after
256 hrs
2 mM
0.779
0.965
1.816
1.92
1.93
1.7
4 mM
0.739
0.91
1.715
1.809
1.9
1.5
6 mM
0.563
0.638
1.688
1.653
1.724
1.424
8 mM
0.53
0.703
1.306
1.448
1.585
1.255
10 mM
0.465
0.549
1.226
1.386
1.545
1.165
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
78
O.D. for 10-3 dilution for phenol degradation

3.5
O.D. (269 mm)
3
2.5
2
1.5
1
0.5
0
Initial O.D.
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
O.D. for 10-4 dilution (269 nm) for phenol degradation

3.5
O.D. (269 mm)
3
2.5
2
1.5
1
0.5
0
Initial O.D.
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
O.D. after
64 hrs
O.D. after
128 hrs
79
Phenol degradation % of 10-3 dilution

90
80
%degradation
70
60
50
40
30
20
10
0
8hrs
16 hrs
32 hrs
64 hrs
128 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
Phenol degradation % of 10-4 dilution

90
80
%degradation
70
60
50
40
30
20
10
0
8hrs
16 hrs
32 hrs
64 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
128 hrs
80
The growth curve for Pseudomonas putida shows that the organism is using phenol as a carbon
source and O.D is increasing till 128 hours then there is a sudden decrease in O.D that means after
certain period accumulated phenol is acting as a toxic compound thus inhibiting the growth of this
species at different phenol concentration.
When analysed the phenol concentration in the growth medium at the same time (8 hrs, 16 hrs) at
269 nm ,it was found that the phenol concentration is decreasing with increase time period which
indicates that phenol is being consumed by the organism .At different phenol concentration O.D
increases with increase in phenol cone but with increase in time O.D decreases with corresponding
concentration .
Using Phenol Degradtion% Formula based on O.D at 269 nm it was observed that degradation%
is highest in 2mM cone than at l0mM concentration. This may be because of high conc of phenol
is inhibiting the growth and thus degradation metabolism pathway.
Same is seen when the experiment was done with Pseudomonas aeruginosa. The growth curve for
Pseudomonas aeruginosa shows that the organism is using phenol as a carbon source and O.D is
increasing till 128 hours then there is a sudden decrease in O.D that means after certain period
accumulated phenol is acting as a toxic compound thus inhibiting the growth of this species at
different phenol concentration.But O.D values are more than previous sp. means Pseudomonas
aeruginosa can use phenol more potently.
When analysed the phenol concentration in the growth medium at the same time (8 hrs, 16 hrs) at
269 nm, it was found that the phenol concentration is decreasing with increase time period which
indicates that phenol is being consumed by the organism .At different phenol concentration O.D
increases with increase in phenol cone but with increase in time O.D decreases with corresponding
concentration.
Using Phenol Degradtion% Formula based on O.D at 269 nm it was observed that degradation %
is highest in 2mM concentration than at 10 mM concentration , this may be because of high cone
of phenol is inhibiting the growth and thus degradation metabolism pathway .But % degradation
is less than in Pseudomonas putida
81
Growth Curve for 10-3 dilution (600 nm) in catechol

1.2
1
O.D. (600 nm)
0.8
0.6
0.4
0.2
0
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
2mM
0.317
0.556
0.739
4mM
0.292
0.526
0.704
6mM
0.233
0.454
0.581
8mM
0.2
0.366
0.499
10mM
0.115
0.338
0.298
O.D. after
64 hrs
O.D. after
128 hrs
O.D. after
256 hrs.
0.95
1.06
0.76
0.921
1.034
0.96
0.746
0.857
0.56
0.633
0.751
0.5
0.371
0.43
0.31
Time in hours
2mM
4mM
6mM
8mM
10mM
Growth Curve for 10-4 dilution (600 nm) in catechol

1.4
1.2
1
O.D. (600 nm)
0.8
0.6
0.4
0.2
0
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
O.D. after
256 hrs.
2mM
0.4
0.8
0.994
1.2
1.306
1.1
4mM
0.313
0.542
0.744
0.978
1.089
0.97
6mM
0.208
0.468
0.629
0.788
0.899
0.689
8mM
0.214
0.366
0.508
0.665
0.76
0.65
10mM
0.154
0.251
0.359
0.465
0.528
0.45
Time in hours
2mM
4mM
6mM
8mM
10mM
82
O.D. for 10-3 dilution (540 nm) for catechol degradation

4.5
4
O.D. (540nm)
3.5
3
2.5
2
1.5
1
0.5
0
Initial O.D.
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
O.D. for 10-4 dilution (540 nm) for catechol degradation

4.5
4
O.D. (540 nm)
3.5
3
2.5
2
1.5
1
0.5
0
Initial O.D.
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
O.D. after
64 hrs
O.D. after
128 hrs
83
Catechol degradation % of 10-3 dilution

80
70
%degradation
60
50
40
30
20
10
0
8hrs
16 hrs
32 hrs
64 hrs
128 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
Catechol degradation % of 10-4 dilution

80
70
%degradation
60
50
40
30
20
10
0
8hrs
16 hrs
32 hrs
64 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
128 hrs
84
The growth curve for Pseudomonas putida shows that the organism is using catechol as a carbon
source and O.D is increasing till 128 hours then there is a sudden decrease in O.D that means after
certain period accumulated catechol is acting as a toxic compound thus inhibiting the growth of
this species at different catechol concentration but O.D value is less than that in phenol
concentration.
When analysed the catechol concentration in the growth medium at the same time (8 hrs, 16 hrs)
at 269 nm, it was found that the catechol concentration is decreasing with increasing time period
which indicates that catechol is being consumed by the organism. Using catechol Degradtion%
Formula based on O.D at 540 nm it was observed that degradation % is highest in 2mM cone than
at 10 mM concentration, this may be because of substrate concentration increases and it accelerates
degradation metabolism pathway. Degradation % is less here.
Same is seen when the experiment was done with Pseudomonas aeruginosa.
The growth curve for Pseudomonas aeruginosa shows that the organism is using phenol as a
carbon source and O.D is increasing till 128 hours then there is a sudden decrease in O.D that
means after certain period accumulated catechol is acting as a toxic compound thus inhibiting the
growth of this species at different catechol concentration. But O.D values are more than previous
sp. means Pseudomonas aeruginosa can use catechol less potently.
When analysed the catechol concentration in the growth medium at the same time (8 hrs, 16 hrs)
at 540 nm, it was found that the catechol concentration is decreasing with increase time period
which indicates that catechol is being consumed by the organism .At different catechol
concentration O.D increases with increase in catechol cone but with increase in time O.D decreases
with corresponding concentration.
Using catechol Degradation% Formula based on O.D at 540 nm it was observed that degradation
% is highest in 2mM cone than at 10mM concentration ,this may be because of high concentration
of catechol inhibits the growth of bacteria. But % degradation is less than in Pseudomonas putida
and is also less than in bacteria grown in phenol containing medium.
85
O.D. (600 nm)
Growth Curve for 10-3 dilution (600 nm) in 8-hydroxyquinoline

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
O.D. after
256 hrs.
2mM
0.272
0.28
0.289
0.32
0.338
0.255
4mM
0.261
0.27
0.279
0.29
0.32
0.25
6mM
0.256
0.266
0.27
0.273
0.289
0.25
8mM
0.21
0.247
0.256
0.266
0.28
0.185
10mM
0.194
0.23
0.251
0.261
0.272
0.17
Time in hours
2mM
4mM
6mM
8mM
10mM
Growth Curve for 10-4 dilution (600 nm) in 8-hydroxyquinoline

0.45
0.4
0.35
O.D. (600 nm)
0.3
0.25
0.2
0.15
0.1
0.05
0
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
O.D. after
256 hrs.
2mM
0.29
0.298
0.32
0.336
0.389
0.319
4mM
0.275
0.283
0.296
0.31
0.359
0.312
6mM
0.267
0.273
0.288
0.295
0.338
0.298
8mM
0.25
0.264
0.28
0.281
0.304
0.246
10mM
0.243
0.256
0.275
0.285
0.288
0.229
Time in hours
2mM
4mM
6mM
8mM
10mM
86
O.D. for 10-3 dilution (540 nm) for 8-hydroxyquinoline degradation

4.5
4
O.D. (540nm)
3.5
3
2.5
2
1.5
1
0.5
0
Initial O.D.
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
O.D. after
64 hrs
O.D. after
128 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
O.D. for 10-4 dilution (540 nm) for 8-hydroxyquinoline

4.5
4
O.D. (540 nm)
3.5
3
2.5
2
1.5
1
0.5
0
Initial O.D.
O.D. after
8 hrs
O.D. after
16 hrs
O.D. after
32 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
O.D. after
64 hrs
O.D. after
128 hrs
87
8-hydroxyquinoline degradation % of 10-3 dilution

80
70
%degradation
60
50
40
30
20
10
0
8hrs
16 hrs
32 hrs
64 hrs
128 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
8-hydroxy quinoline degradation % of 10-4 dilution

80
70
%degradation
60
50
40
30
20
10
0
8hrs
16 hrs
32 hrs
64 hrs
Time in hours
2 mM
4 mM
6 mM
8 mM
10 mM
128 hrs
88
The growth curve for Pseudomonas putida shows that the organism is using 8- hydroxyquinoline
as a carbon source and O.D is increasing till 128 hours then there is a sudden decrease in O.D that
means after certain period accumulated 8-hydroxyquinoline is acting as a toxic compound thus
inhibiting the growth of this species at different 8-hydroxyquinoline concentrations but O.D value
is less than that in phenol and catechol cone.
When analysed the 8-hydroquinoline concentration in the growth medium at the same time (8 hrs,
16 hrs) at 540 nm, it was found that the 8-hydroxyquinoline concentration is decreasing with
increasing time period which indicates that 8-hydroxyquinoline is being consumed by the
organism.
Using 8-hydroxyquinoline Degradtion% Formula based on O.D at 540 nm it was observed that
degradation % is highest in 2mM cone than at 10mM concentration, this may be because of
substrate cone increases and it accelerates degradation metabolism pathway. Degradation % is
more here than in catechol but less than in phenol.
Same is seen when the experiment was done with Pseudomonas aeruginosa.
The growth curve for Pseudomonas aeruginosa shows that the organism is using 8hydroxyquinoline as a carbon source and O.D is increasing till 128 hours then there is a sudden
decrease in O.D that means after certain period accumulated 8-hydroxyquinoline is acting as a
toxic compound thus inhibiting the growth of this species at different 8-hydroxyquinoline
concentration. But O.D values are more than previous sp. means Pseudomonas aeruginosa can use
8-hydroquinoline more potently.
When analysed the 8-hydroxyquinoline concentration in the growth medium at the same time (8
hrs, 16 hrs) at 540 nm ,it was found that the 8-hydroxyquinoline concentration is decreasing with
increase time period which indicates that 8-hydroxyquinoline is being consumed by the organism
.At different 8-hydroxyquinoline concentration O.D increases with increase in 8hydroxyquinoline concentration but with increase in time O.D decreases with corresponding
concentration.
89
Using 8-hydroquinoline Degradtion% Formula based on O.D at 540 nm it was observed that
degradation % is highest in 2mM cone than at 10mM concentration, this may be because of
substrate concentration increasing and it accelerates degradation metabolism pathway. But %
degradation is more than in Pseudomonas putida
Result: As catechol is 1.5 times more toxic than phenol so degradation in case of catechol and 8hydroquinoline is less than that in phenol in growth medium.

Enzvmes responsible for biodegradation of phenol, catechol and 8-hydroxyquinoline
90
91
To determine which enzymes are produced in the two species SDS PAGE is performed. The
following sections illustrate the results obtained. A final list of the enzymes are recorded in Table
5.
SDS PAGE for Pseudomonas putida grown in phenol at

different concentrations
120 KDa
98 KDa
88 KDa
68 KDa
33 KDa
19 KDa
Markers
2 mM
4 mM
6 mM
8 mM
10 mM
Result: In this SDS PAGE result protein bands of 33KDa and 40KDa are visible which means
three enzymes Catechol2,3 dioxygenase , catechol 1,2 dioxygenase , peroxidase are produced by
Pseudomonas putida during its growth in Phenol containing medium .But the 33KDa band is not
so thick so may be both Catechol2,3 dioxygenase , catechol 1,2 dioxygenase are produced or the
organism is producing only one enzyme.
92
SDS PAGE for Pseudomonas aeruginosa grown in

phenol at different concentration
97 KDa
85 KDa
76 KDa
68 KDa
42 KDa
33 KDa
20 KDa
Markers
2 mM
4 mM
6 mM
8 mM
10 mM
Result: In this SDS PAGE result protein bands of 33KDa are visible which means two enzymes
Catechol 2, 3 dioxygenase , catechol 1, 2 dioxygenase are produced by Pseudomonas aeruginosa
during its growth in Phenol containing medium because 33KDa band is very thick here, but th
organism is not producing peroxidase enzyme. This means Pseudomonas aeruginosa does
notutilize phenol using peroxidase enzyme.
93
SDS PAGE RESULT Pseudomonas putida grown in

catechol at different concentration
97 KDa
88 KDa
75 KDa
67 KDa
55 KDa
33 KDa
25 KDa
14 KDa
6 KDa
Markers
2 mM
4 mM
Waste well
6 mM
8 mM
10 mM
Catechol 2, 3 dioxygenase , catechol 1, 2 dioxygenase are produced by Pseudomonas putida during
its growth in catechol containing medium .But it is not clear which enzyme is being produced by
the organism either Catechol 2,3 dioxygenase or catechol 1,2 dioxygenase or both.
94
SDS PAGE RESULT Pseudomonas aeruginosa grown

in catechol concentration
125 KDa
97 KDa
72 KDa
65 KDa
33 KDa
25 KDa
19 KDa
Markers
2 mM
4 mM
6 mM
8 mM
10 mM
Catechol 2,3 dioxygenase , catechol 1,2 dioxygenase are produced by Pseudomonas aeruginosa
during its growth in catechol containing medium .But it is not clear which enzyme is being
produced by the organism either Catechol 2,3 dioxygenase or catechol 1,2 dioxygenase or both.
95
SDS PAGE RESULT Pseudomonas putida grown in 8hydroxyquinoline at different concentration.
85 KDa
55 KDa
30 KDa
20 KDa
15 KDa
Markers:
2 mM
4 mM
6 mM
8 mM
10
M
Result: In this SDS PAGE result protein bands of 85 KDa, 30 KDa, 20 KDa are visible which
means one enzyme quinoline-2-oxidoreductase is produced by Pseudomonas putida during its
growth in 8-hydroxyquinoline containing medium
96
SDS PAGE Result for Pseudomonas putida grown in 8hydroxyquinoline at different concentration
125 KDa
97 KDa
85 KDa
55 KDa
30 KDa
20 KDa
6 KDa
3 KDa
Markers:
2 mM
4 mM
6 mM
8 mM
10 mM
Result: In this SDS PAGE result protein bands of 85 KDa, 30 KDa, 20 KDa are visible which
means one enzyme quinoline-2-oxidoreductase is produced by Pseudomonas aeruginosa during
its growth in 8-hydroxyquinoline containing medium
97
List of the Enzymes that are responsible for

biodegradation of phenol, catechol and 8hydroxyquinoline
Sl No.
Compound
Enzyme
Catechol 2,3 dioxygenase
Enzyme
Information
M.W. 33KDa
References
Ali et al. (1998)
(4 subunits)
M.W. 33
1
Phenol
KDa (2
An et al. (2001)
subunits)
Catechol 1,2 peroxidase
M.W. 40
Sakurai et al.
KDa (1 unit)
(2001)
M.W. 33
2
KDa (4
Ali et al. (1998)
Subunits)
Catechol
M.W. 33
KDa (2
An et al. (2001)
Subunits)
M.W. 420
KDa (3
3
8-
Quinoline 2-
Subunits)
hydroxyquinoline
oxidoreductase
L85 KDa
Loh KC (2000)
M30 KDa
S20KDa
Table 5: Enzymes produced during aromatic degradation (Wang S.J. and Loh K.C., 1990), (Wu
Y., 1998) & (Xia Z., 2003)
98
Bioinformatic analysis of the enzymes produced during aromatic biodegradation in different

bacterial species.
99
ClustalW2 Alignment of catechol 2,3 dioxygenase

found in different bacterial species
100
101
Result: Protein FASTA sequence of enzyme catehol 2,3 dioxygenase in 10 bacterial species:
Novosphinsobium aromaticivorans,
Thermaerobacter marianensis,
Ralstonia picketti,
Meiothermus ruber DSM 1279,
Leptothrix cholodnii SP-6,
Geobacillus sp. Y4.1MC1,
Kvrpidia tusciae DSM 2912,
Thauera sp. MZlT,
Pseudomonas putida, and
Pseudomonas aeruginosa
were taken from NCBI and then performed CLUSTALW2 multiple sequence alignment. There are
30 conserved amino acids in all ,50 strongly similar amino acids and 22 weakly similar amino
acids that indicates that is somewhat conserved and have also evolved in these particular organisms
and they perform the same activity i.e degradation of phenol. The phylogenetic tree of the above
aligned genes in these organism is given below:
102
ClustalW2 alignment of catechol 1,2 dioxygenase

enzyme found in different bacterial species
103
104
Result: Protein FASTA sequence of enzyme catehol 2,3 dioxygenase in 10 bacterial species:
Paracoccus sp. FLY-8
Rhodocnccus iostii RHA1,
Burkholderia sp. CCGE1002,
Ralstonia pickettii 12D,
Marinomonas mediterranea MMB-1,
Methylobacterium sp.,
Alicvcliphilus denitrificans BC,
Enterobacter asburiae LF7a,
Pseudomonas putida, and
Pseudomonas aeruginosa
were taken from NCBI and then performed CLUSTALW2 multiple sequence alignment.There are 37
conserved amino acids in all ,44 strongly similar amino acids and 20 weakly similar amino acids that
indicates that is somewhat conserved and have also evolved in these particular organisms and they
perform the same activity i.e degradation of phenol. The phylogenetic tree of the above aligned genes
in these organism is given below:
105
ClustalW2 alignment of peroxidase enzyme found in

different bacterial species
106
Result: The ClustalW2 alignment in the protein sequence of this enzyme did'nt give significant
result.So phylogenetic tree is shown among these species:
Methvlobacterium extorquens DSM 13060,
Marinithermus hvdrothermalis DSM 14884,
Planctomvces brasiliensis DSM 5305,
Cyclobacterium marinum DSM 745,
Rhodopirellula baltica SH 1,
Intrasporaneium calvum DSM 43043,
Bacillus amvloliquefaciens TA208,
Streptomvces zinciresistens K42,
Pseudomonas aeruginosa, and
Pseudomonas putida SI6
and only one amino acid is strongly and one amino acid is weakly similar among these species.
107
ClustalW2 alignment of quinoline -2-oxidoreductase

enzyme found in different bacterial species
This enzyme is found in only two species Pseudomonas aeruginosa and Pseudomonas putida. The
ClustalW2 alignment is shown below:
108
The phylogenetic tree is as shown below:
Result: There is 90% conserved amino acids in the protein sequence that code for quinoline -2oxidoreductase in these two bacterial species.
The similarity and conserved amino acids among the species for different enzymes suggests that
these organism are also capable of aromatic degradation. These different organisms can also
grow in waste waters produced by chemical and pharmaceutical industries.
Conclusion & Future Prospects
109
110
Conclusion
After the gloom of impending disaster and environmental calamity which was present in the 1960s,
the threat from recalcitrant organic chemicals such as catechol .phenol etc. has diminished to some
extent. Extensive microbiological and biochemical research in this project suggest that the
aromatic degrading isolate Pseudomonas putida and Pseudomonas aeruginosa appears to have
greater potential for enhanced phenol and phenolic compounds removal through utilization of them
as sole source of carbon and energy.
Resistance against a high concentration of phenol, catechol and 8-hydroxyquinoline facilitates its
use for biological treatment system of wastewater, with high % degradation of such a high
concentration of phenol, catechol and 8- hydroxyquinoline. Here it has been reported that these
isolates are capable of growth at relatively high aromatic concentrations together with the analysis
of functional properties relevant to the application of this organism to the biodegradation of
aromatic wastes (phenolics).
The enzymes that are responsible for degradation of these aromatic compounds are produced
during their growth in phenolic compound containing medium have greatest importance in waste
water treatment plant. The other species that contain the enzymes responsible for aromatic
degradation can also be used in waste water treatment plant. The genes that code these enzymes
can be isolated and transformed in easily growing bacteria and can be used for biodegradation and
thus removal of these compounds in waste water produced by chemical and pharmaceutical
industries.
111
Future Prospects
The genes that encode for biodegradation of many recalcitrant organic chemicals are often encoded
on plasmids, Sometimes only one or two enzymes are encoded on a plasmid with the rest on the
bacterial chromosome while in others on entire pathway is plasmid encoded. Plasmids can often
be transferred easily between organisms, evm ones that are not closely related and this transfer
offers a mains by which genetic recombination can occur. This happens in nature but it can also
be made to happen in the laboratory giving scientists the potential to develop new bacterial strains
with altered and enhanced degradative ability Genetic engineering may have a role in creating
bacterial strains with entirely new metabolic activities by combining genes from different
organisms to produce an entirely new metabolic pathway. The potential application of these strains
in clearing up waste water containing aromatic compounds or other pollution incidents has been
patented and much hyped' like superbugs r In practice it seems that most of these genetically
engineered organisms will really offer much advantage over mixed cultures developed by
conventional enrichment culture. or indeed over the native microflora. Such strains are more likely
to be of use in the more carefully controlled conditions of special treatment plants.
Appendix
112
113
1. L.B. Broth: composition in g/l

Sl No.
Components
Quantity (g/l)
Tryptone
10
Yeast Extract
Sodium Chloride
10
Final pH
7.0 0.2 at 25C
2. L.B. Agar: Composition in g/l

Sl No.
Components
Quantity (g/l)
10
Yeast Extract
Sodium Chloride
10
Agar
15
C5
Final pH
7.0 0.2 at 25C
3. Peptone Water: Composition in g/l

Sl No.
Components
Quantity (g/l)
Peptone
10
Sodium Chloride
Distilled Water
1 Litre
pH
7.0 at 25C
114
4. Glucose phosphate broth: Composition in g/l

Sl No.
Components
Quantity (g/l)
Peptone
10
Dextrose
Dipotassium Hydrogen Phosphate
Distilled Water
1 Litre
pH
6.9 0.2 at 25C
5. Simons Citrate Agar: Composition in g/l

Sl No.
Components
Quantity (g/l)
Distiled Water
0.2 Litre
MgSO4.7H2o
1.0
NH4H2O
1.0
K2HPO4
0.2
Sodium Citrate
2.0
NaCl
0.05
Agar
15.0
Bromothymol Blue
0.8
pH
7.0
115
6. Urea Broth: Composition in g/l

Sl No.
Components
Quantity (g/l)
Urea
20
Yeast Extract
Dextrose
Na2HPO4
9.5
KH2PO4
9.1
Phenol Red
1.0
Distilled Water
1 Litre
7. Mannitol Agar: Composition in g/l

Sl No.
Components
Quantity (g/l)
Beef Extract
1.0
D-Mannitol
10
Sodium Chloride
75
Phenol Red
Few drops
Agar
15
pH
7.4 0.2 at 25C
116
8. Starch Broth: Composition in g/l

Sl No.
Components
Quantity (g/l)
Tryptone
10
Beef Extract
10
K2HPO4
Starch
30
Agar
20
Distilled Water
1 Litre
9. Gelatin Agar: Composition in g/l

Sl No.
Components
Quantity (g/l)
Beef Extract
Tryptone
Gelatin
40
Distilled Water
1 Litre
pH
7.0
117
10. Mac Conkeys agar: Composition in g/l

Sl No.
Components
Quantity (g/l)
Peptone
20
Sodium Chloride
Bile Salt
1.5
Lactose
10
Neutral red solution
10
Crystal Violet
0.001
Agar
13.5
Distilled Water
1 Litre
11. Carbohydrate broth: Composition in g/l

Sl No.
Components
Quantity (g/l)
Peptone
10
Sodium Chloride
Phenol Red
0.018
Carbohydrates (Glucose, Sucrose or Lactose)
Distilled Water
1 Litre
pH
7.0

12. Cell Lysis Buffer: Composition in g/l
Sl No.
Components
Quantity
Tris pH
50 mM at pH 8.0
Glycerol
10%
Triton X-100
0.1%
Lysozyme 1mM PMSF
100 g/ml
MgCl final concentration
2 mM
13. 5 Sample Buffer

Sl No.
Components
Quantity
SDS
10% w/v
Dithiotheritol, or beta-mercapto-ethanol
10 mM
Glycerol
20% v/v
Tris-HCl, pH 6.8
0.2 M
Bromophenol blue
0.05% w/v
14. 1 Running Buffer (1 Litre)

Sl No.
Components
Quantity (g/l)
Tris HCl
25 mM
Glycine
200 mM
SDS (w/v)
0.1%
118
119
15. Stacking and Resolving gel mix

Sl No.
Components
7%
10%
12%
15%
H2 O
15.3 ml
12.3 ml
10.2 ml
7.2 ml
7.5 ml
7.5 ml
7.5 ml
7.5 ml
0.15 ml
0.15 ml
0.15 ml
0.15 ml
6.9 ml
9.9 ml
12.0 ml
15.0 ml
0.15 ml
0.15 ml
0.15 ml
0.15
0.02 ml
0.02 ml
0.02 ml
0.02 ml
1.5 M Tris HCl, resolving gel

2
(pH 8.8), stacking gel (pH

6.8)
10% (w/v) SDS

Acrylamide/Bis-acrylamide
4
(30% / 0.8% w/v)
10% (w/v) ammonium
5
persulfate (APS)
6
TEMED
16. Sample disruption SDS mix

Sl No.
Components
Quantity
SDS
2%
Glycerol
20%
Tris-HCl, pH 6.8
20 mM
Ethylene Diamine Tetraacetic Acid (EDTA)
2 mM
Dithiothreitol (DTT)
160 mM
Bromophenol blue dye
0.1 mg/ml

17. Staining Solution (100 ml)
Sl No.
Components
Quantity
Coomassie Blue Dye
0.25 g
Methanol
40 ml
Glacial Acetic Acid
7 ml
18. Destaining Solution (100 ml)

Sl No.
Components
Quantity (g/l)
Methanol
40 ml
Glacial Acetic Acid
7.5 ml
19. Protein Extraction Buffer (100 ml)

Sl No.
Components
Quantity (g/l)
Phosphate Buffer
50 mM
Sodium Chloride
300 mM
Beta MarcaptoEthanol
10 mM
Imidazole
10 mM
120

20. The minimal medium (g/l)
Sl No.
Components
Quantity (g/l)
K2HPO4
4.36
NaH2PO4
3.45
NH4Cl
1.0
MgSO4.6H2O
0.912
Trace Salts Solution
1ml/L
21. Trace Salts Solution (g/100 ml)

Sl No.
Components
Quantity (g/l)
CaCl2.H2O
4.77
FeSO4.7H2O
0.37
CoCl2.6H2O
0.37
MnCl2.4H2O
0.10
Na2MoO4.2H2O
0.02
Prepared and stored in dark bottles for 68 weeks
121
References
122
123
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Biodegradation of Aromatic Compounds Present in Industrial Wastewater by Bacterial Species

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BIODEGRADATION OF

AUGUST 28, 2014

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

ClustalW2 alignment of quinoline -2-oxidoreductase enzyme found in different bacterial

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

extraction, incineration, chemical oxidation, membrane processes, coagulation, flocculation,

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Physical Properties of Phenol:

2) Chemical Formula: C6H5OH

Carbolic Acid, Benzenol, Phenylic Acid, Hydroxybenzene, Phenic

4) Solubility in Water: Phenol has a limited solubility (8.3g/100 ml at 20C) in water.

Phenol is slightly Acidic in Nature

7) Molecular Weight: 94.11

Disorders of Central Nervous System Leading to collapse and coma.

Hypothermia Reduction in body temperature.

Acute exposure of phenol can result in myocardial depression.

Muscle weakness and tremors have also been observed.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Phenol can cause gastrointestinal disturbance, vomiting, weakness, loss of weight

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Physical Properties of 8-Hydroxyquinoline:

6) Medicine Class Appearance:

Off-White Needle Crystals

7) Purity (HPLC, GC):

10) Toxicity: Exposure to 8-Hydroxyquinoline is known to cause the following effects:

Potential Acute Health Effects: Hazardous in case of ingestion or inhalation.

Potential Chronic Health Effects:

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

2) General Characteristics: Catechol is a colorless crystal with a phenolic odor (HSDB,

1 ppm = 4.5 mg/m3

10) Toxicity of Catechol:

methemoglobinemia. Systemic toxicity is similar to that of phenol; however, catechol may

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Outline of the Experiments performed:

Samples were collected from various locations.

Species were isolated using microbiological tools and techniques.

Strains and species are grown in different aromatic compounds.

The following series of biochemical analyses are performed:

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Materials and Methods

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Sampling from Industrial Areas:

Figure 1 : Yamuna River, Indraprastha Industrial Area

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Isolation of species by Serial Dilution and Spreading

BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.

Isolation of species by Streak Plate Method