Biodegradation of Aromatic Compounds Present in Industrial Wastewater by Bacterial Species

8/10/2019 Biodegradation of Aromatic Compounds Present in Industrial Wastewater by Bacterial Species

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BIODEGRADATION OF

AROMATIC COMPOUNDS

Final Project

AUGUST 28, 2014AMITY INSTITUTE OF BIOTECHNOLOGY

Amity University Haryana


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BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP. I

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


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BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP. 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


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BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.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


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BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP.IV

Gelatin test result .................................................................................................................. 65

MacConkey's Agar Test result .............................................................................................. 66

Growth at 4 C 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 8-

hydroxyquinoline .................................................................................................................. 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


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BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP. V

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


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BIODEGRADATION OF AROMATIC COMPOUNDS BY BACTERIAL SP. 1

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 8-

hyroxyquinoline), which are the most toxic aromatic pollutants of the environment, their

biochemical studies followed by their analysis.


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Introduction


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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 oflong 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


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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


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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


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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.


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Physical Properties of Phenol:

1) Molecular Structure:

2) Chemical Formula : C 6H5OH

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 20 C) 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.


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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.


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Physical Properties of 8-Hydroxyquinoline:


2) Product Name: 8-Hydroxyquinoline

3) Other Names: 8-Quinolinol; Quinolin-8-ol

4) Molecular Formula: C9H7 NO

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.


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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/m 3

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.


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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


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Literature Review


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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 anaerobesnow 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


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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 gr owth rates,

half saturation and substrate inhibition constant. Cell growth using toluene ( max, T = 0.61) and

benzene ( max, B = 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 (I B, T = 0.14) enhances the

biodegradation of phenol, and phenol inhibits the biodegradation of benzene (I P, B = 1.08) and

toluene. Scientists have given the effect of aromatic compounds on each others degra dation by P.

putida but they have not studied the genes that affect this process (I P, 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 2 2 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.


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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


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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 h 1 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


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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.


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Materials and Methods


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Microbiological Tests


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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


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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 ofsterilized water and mixed properly.

Then, 1ml from test tube number 4 was pipetted into test tube number 5 with 9 ml of

sterilized water and mixed properly.

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.


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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 10 4. These plates were used for streaking.

Prepared Lurea Bertini Broth.

Inside Laminar Airflow Bench, sterilize inoculating loop and picked up single isolated

colony from 10 3 dilution and inoculated it into LB broth, and kept overnight in an

incubator.

Similarly performed the same procedure with 10 4 dilution.

Labelled the LB agar containing petridish on the flipside.

Sterilized the transfer loop before obtaining a specimen from LB broth containing 10 3

and 10 4 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.


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Biochemical Methods:


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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


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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.


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Methyl Red Test

Principle:

Methyl red test is used to identify enteric bacteria based on their pattern of glucose metabolism. Ifthey use mixed acid pathways and produce acidic products, then they are said to test methyl-red-

positive. 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


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Voges Proskeaur Test

Principle:

It is used to identify enteric bacteria based on their pattern of glucose metabolism. The entericsthat produce neutral end-products, such as acetoin are detected by the VP test (Manual of Clinical

Microbiology, 5 th 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


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Catalase Test

Principle:

Catalase is an enzyme that decomposes Hydrogen Peroxide (H 2O2) 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.


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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.


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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.


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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.


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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.


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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.


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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 (atleast 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.


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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.


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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 Minimum( C) Optimum( C) Maximum( C) Comments

Psychrophile Below 0 10 15 Below 20Grows best at relatively

low Temperature

Psychrotroph H 15 30 Above 25

Ability to grow at low

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.


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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.


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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.


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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 37 C 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 iffermentation 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.


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Bacterial identification based on biochemical tests

Bergey's Manual Of Determinative Bacteriology (Seventh Edition) by

Robert S. Breed 1, E. G. D. Murray 2, Nathan R. Smith 3

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".

1 Ixite Professor Emerilus, Cnrnell University, Geneva, New York2 Research Professor, University of Western Ontario.London, Ontario, Canada3 Senior Bacteriologist, Retired, Plant Industry Station,(U. S. Department of Agriculture, Beltsville, Maryland


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Biodegradation experiments:


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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):

Molarity ( ) Molecular Weight Volume(ml)Weight (g)=

1000

M

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


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8-Hydroxyquinoline Degradation

The biodegradation potential of the selected strains were evaluated in medium containing 8-

Hydroxyquinoline 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):

Molarity ( ) MolecularWeight Volume(ml)Weight (g)=

1000

M

Serial No Concentration8-Hydroxyquinoline

amount( l)

1. 2 mM 14.52

2. 4 mM 29.04

3. 6 mM 43.56

4. 8 mM 58.085. 10 mM 72.60

Table 3. Calculation of amount of 8-hydroxyquinoline for different Concentrations

Biomass Analysis


intervals of time (Gibson D.T., et al, 1968).


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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:

(Initial 8-Hydroxyquinoline)-(Residual 8-Hydroxyquinoline)Degradation (%) 100(Initial 8-Hydroxyquinoline)


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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):

Molarity( ) Molecular Weight Volume (ml)Weight (g)=

1000

M

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).


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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:

Initial catechol Residual CatecholDegradation (%)= 100

Initial catechol


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Protein profile analysis:


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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.


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SDS PAGE for protein analysis

Principle

SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its moleculeshave 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


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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.

___________


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Result and Discussion


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Isolation of bacterial species

Species are isolated using microbiological techniques spreading and streaking.

Figure 3. Spread plates for 10 -3 isolation Figure 4. Spread Plates for 10 -4 isolation

Figure 5. Streaked Plates for 10 -3 isolation Figure 6. Streaked Plates for 10 -4 isolation


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Biochemical tests:


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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)


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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.


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Voges-Proskauer test

Copper color of the medium indicates negative VP test result. This means that the organism doesnot 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 Metabolism Pyruvic Acid

Pyruvic acid No Acetoin

No acetoin + added alpha-naphthol + added KOH copper color

Figure 10. Methyl test result for 10 -3 and 10 -4 isolates (-

Figure 11. V-P test for 10 -3 and 10 -4 isolate (-ve)


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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

color changed with growth)

Figure 13. Citrate test result for 10 -4 isolate

(Positive color changed with growth)


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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)


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Gram staining result

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 15. Gram Negative Cell wall structure


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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)


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Urease test result

Result : Organisms that produce urease will turn phenol red (pH 6.8) 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:

(NH 2)2CO + 2 H 2O Urease CO 2 + H 2O + 2 NH 3

Figure 18. Urease test result for 10 -3 dilution. (negative result). No color

change indicates that the organism does not produce urease.


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Figure 19. Urease test result for 10 -4 dilution. Color change from

yellow to pink ndicates that the organism produces urease (+ve

result.)


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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.


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Starch test result

This test is used to detect the enzyme amylase, which breaks down. starch. Ae f 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 e

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.


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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

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.


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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 growand 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.


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Growth at 4 C 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 4 C for 10 -4 dilution (-ve result). No

growth is seen

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

growth is seen


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Figure 30. Growth of bacteria at 25 C for 10 -3 dilution (+ve result).

Growth is seen.

Figure 31. Growth of bacteria at 25 C for 10 -4 dilution (+ve result).

Growth is seen


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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


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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

indicates bacteria cannot ferment Lactose. (-ve result)

Figure 39. Lactose test for 10 -4 dilution. Color change

from pink to yellow indicates a positive result.


79/132


Figure 40. Dextrose test for both 10 -3 and 10 -4 dilution.

Color change from pink to yellow indicates positive

result.

Figure 41. Sucrose test for both 10 -3 and 10 -4 dilu

Color change from pink-red to yellow indicates positiv

result. Bacteria can ferment sucrose.


80/132


Bacterial Identification based on biochemical test

After studying BERGEY'S MANUAL of DETERMINATIVE BA

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Biodegradation of Aromatic Compounds Present in Industrial Wastewater by Bacterial Species

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