AN INVESTIGATION PROBLEM

ON

“Biodegradation of phenol by native bacteria isolated from

various soil samples and industrial wastes”

For partial fulfillment of the requirement for the degree of

MASTER OF SCIENCE

IN

BIOTECHNOLOGY

Session :( 2009-2011)

SUBMITTED TO: SUBMITTED BY:

Mr. Anil Kumar Roshan Shrestha

Assistant Professor Reg. N0. 09092042

DEPARTMENT OF BIO & NANO TECHNOLOGY

GURU JAMBHESHWAR UNIVERSITY OF

SCIENCE AND TECHNOLOGY

Mr. Anil Kumar, Assistant Professor

No.........…….. Dated…………………

CERTIFICATE I

This is to certify that the investigation problem report entitled, “Biodegradation of

phenol by native bacteria isolated from various oil samples and industrial wastes”

submitted by Roshan Shrestha (Reg.No.-9092042) for the partial fulfilment of the

requirement of the degree of Master of Science in Biotechnology to Department of Bio &

Nano Technology, GJUS&T, Hisar, Haryana-125001 has been carried out under my

supervision and guidance. The assistance and help received during the work has been

fully acknowledged.

Advisor

DEPARTMENT OF BIO AND NANO TECHNOLOGY,

GURU JAMBHESHWAR UNIVERSITY OF SCIENCE AND TECHNOLOGY,

Mr. Anil Kumar, Assistant Professor

No.........…….. Dated…………………

CERTIFICATE II

This is to certify that the investigation problem (MBD 600) entitled “Biodegradation of

phenol by native bacteria isolated from various soil samples and industrial wastes”

submitted by Mr. Roshan Shrestha, Reg. No. 09092042 to the Department of Bio &

Nano Technology, Guru Jambheshwar University of Science & Technology, Hisar for the

partial fulfillment of the requirement of the degree of Master of Science in Biotechnology

has been approved after an oral examination of the same.

External examiner Chairperson

Department of Bio & Nano Technology,

GJU S&T, Hisar.

DEPARTMENT OF BIO AND NANO TECHNOLOGY,

GURU JAMBHESHWAR UNIVERSITY OF SCIENCE AND TECHNOLOGY,

DECLARATION

Certified that the project report entitled “Biodegradation of phenol by native bacteria

isolated from various soil samples and industrial wastes”, submitted by me in partial

fulfilment for the award of degree of M.Sc. in Biotechnology, Guru Jambheshwar

University of Science & Technology, Hisar-125001, Haryana, is a record of the original

work carried out by me. The matter embodied in this report has not been submitted for the

award of any other degree or diploma.

Roshan Shrestha

ACKNOWLEDGEMENT

At the outset, I would like to convey my deepest sense of gratitude and indebtedness to

almighty God and my parents. The happiness of success lies in the sharing it with all who

have helped and inspired me to attain the goal. I would like to express my gratitude to all

those people whose constant guidance and encouragement served as a beacon of light.

I feel privileged to express my heartiest thanks and gratitude to my esteemed advisors Mr.

Anil Kumar, Assistant Professor, Department of Bio and Nano Technology for their

precise and inspiring guidance, valuable and generous suggestions throughout my

project.

It gives me immense pleasure in bringing this acknowledgement to pay my sincere

regards to Prof. Ashok Chaudhury, Chairperson, Department of Bio & Nano Technology,

GJUS&T, Hisar for his valuable support.

I pay my true regards to all the faculty members of the department especially Dr. Vinod

Chhokar, Dr. Namita Singh and Dr. Neeraj Dilbaghi for their kind co-operation.

I recognize with gratitude the invaluable co-operation and immense support provided by

Mr. Vikas Beniwal, Mr. Mahesh Kumar, Mr. Narender Malik, Mr. Rakesh Yadav, Mrs.

Sarika Malik, Ms. Pooja Arora, Mrs. Manju Yadav,Mr. Himanshu Aggarwal, Mr. Rajesh

Yogi and Mr. Ravinder Mehra.

I humbly offer my thanks to the whole staff of the Department of Bio & Nano Technology,

for their help and support during the period.

I truly acknowledge the ready help and co-operation extended to me by all my seniors,

classmates and my friends specially.

Again I iterate a word of thanks to all those hands that helped me in some way or the

other in pursuing this elusive work.

June,2011 Roshan Shrestha

CONTENTS

List of figures……………………………………………………………………….

List of Tables………………………………………………………………………..

CHAPTERS

Chapter 1 INTRODUCTION

Chapter 2 REWIEW OF LITERATURE

Chapter 3 MATERIALS AND METHODS

3.1 Isolating of phenol degrading bacteria

3.2 Characterization of isolated strain by morphological and biochemical tests.

3.3 Immobilization of microbial cells

Chapter 4 RESULTS & DISCUSSIONS

4.1Morhology and biochemical test of isolated strains.

4.2 Residual phenol estimation

4.3 Comparative study of phenol degradation by immobilized and free cells.

Chapter 5 CONCLUSIONS

RERERENCES

List of Tables

Table 1 Microscopic, morphological and

biochemical test of various isolates

Table 2 Standard curve

Table 3 Residual phenol Estimation

Table 4 Calculation of residual phenol

concenteration at end of 6, 12, 18 &

24 hrs incubation by S1G

Table 5 Calculation of residual phenol

concenteration at the end of 6, 12, 18

& 24 hrs incubation by S4A

Table 6 Calculation of residual phenol

concenteration at the end of 6, 12, 18

& 24 hrs incubation by S5C

Table 7 Comparative study of phenol

degradation by immobilized and free

cells.

LISTS OF FIGURES

Fig. No. Content

Fig. 1 Standard Curve

Fig. 2 Residual phenol estimation

Fig. 3 Residual phenol concentration

Fig 4 Phenol degradation by encapsulated cells

and free cells

INTRODUCTION

1. INTRODUCTION

Environmental pollution is an emerging threat and great concern in today’s context

pertaining to its effect on the ecosystem. The worldwide rise in population and the

industrialization during the last few decades have resulted in ecological unbalance and

degradation of the natural resources. One of the most essential natural resources which

have been the worst victim of population explosion and growing 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 persist in the

environment, they are capable of long range transportation, bioaccumulation in human

and animal tissue and biomagnification 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 wastewater.

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, aldrin, dieldrin, heptachlor etc is harm

full for aquatic life and human beings as well. Discharge of cyanide-contained wastewater

to water mass may lead to death of fish and other aquatic life therein. Use of water

containing fluoride can causes mental disorders and stomach ailments and can also

reduces 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 formula which are extremely toxic and found in

different form or together with other elements (EPA, 2004). Phenol is one of the 50

major 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 (CMR 2005). Phenol is considered

to be a toxic compound by the Agency for Toxic Substances and Disease Registry

(Agency for Toxic Substance and Disease Registry 2003) and death among adults has

been reported with ingestion of phenol ranging from 1 to 32 g. Phenol and its derivatives

1

are also generated by various industries, such as petroleum refining, petrochemical, coke

conversion, pharmaceutical, plastic, and resin manufacturing, coal gasification, coke-

oven batteries, and other industries, such as synthetic chemicals, herbicides, pesticides,

antioxidants, pulp-and-paper, photo developing chemicals, etc. (Marrot et al., 2006;

Bodalo et al., 2008; Jayachandran and Kunhi, 2008) in the waste effluents and its

concentration may vary from 1 to 15000 mg l-1. Natural sources of phenol include forest

fire, 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 Control Board of India (CPCB) have prescribed

maximum permissible limits of 3.4 and 5.0 mg l-1, respectively in industrial waste

discharges. Now the associated problem due to phenol is that when it is present in waste

water even in low concentrations can be toxic to some aquatic species and causes taste

and odour problems in drinking water (Rittmann and McCarty, 2001). 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 in environment

and could decrease enzymatic activity as well. Also, it is toxic to fishes and is mortal

between 5 – 25 mg/l for them. Moreover, direct effect of phenol is a blocker for biologic

reaction. 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. 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 an

anaesthetic 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

2

disturbance, vomiting, weakness, weightlessness, muscle pain, hepatic tenderness and

nervous disorder. It is also suspected that exposure to phenol may cause paralysis, cancer

and genetofibre striation. Phenol and its derivatives are toxic and classified as hazardous

materials (Zumriye and Gultac, 1999). These phenolic compounds possess various

degrees of toxicity and their fate in the environment is therefore important (Bollag et al.,

1988).

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 (Ghioureliotis and

Nicell, 1999). Phenol is an antiseptic agent and is used in surgery, which indicates that

they are also toxic to many microorganisms (EPA, 1979). 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 methods of treatment of phenolic and nitrate-nitrogen Waste water are

largely physical and chemical processes like hybrid process (Bodalo et al., 2008)

electrocatalytic degradation (Wang et al., 2009) adsorption on to different matrices,

chemical oxidation, solvent extraction or irradiation (Spiker et al., 1992) but these

processes led to secondary effluent problems due to formation of toxic materials such as

cyanates, chlorinated phenols, hydrocarbons, etc. These methods are mainly chlorination,

ozonation, solvent extraction, incineration, chemical oxidation, membrane process,

coagulation, flocculation, adsorption, ion exchange, reverse osmosis, electrolysis, etc.

Biological treatment is attractive due to the potential to almost degrade phenol and other

pollutant while producing innocuous end products, reduced capital and operating costs,

maintains phenol concentrations below the toxic limit. However difficulty arises in such

treatment due to the toxicity of phenol to the microbial population. The biological

degradation of phenol is accomplished through benzene ring cleavage using the enzyme

present in the microorganism. The bacteria express differently when exposed to different

initial phenol concentrations and other conditions. The most efficient Pseudomonas

Putida is capable of using phenol as the sole source of carbon and energy for cell growth

and metabolism degrade phenol via metapathway. That is 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.

3

OBJECTIVES OF WORK DONE:

To isolate the potent phenol degrading Bacteria.

To characterize various bacterial isolates by morphological and biochemical test

To study phenol degradation by cell immobilization method.

To study the growth curve.

4

REVIEW OF LITERTURE

5

2. REVIEW OF LITERATURE

Today Bioremediation is considered as a new tool to eliminate environment pollutions

(EPA, 2004). Bioremediation is defined as the process whereby organic wastes are

biologically degraded under controlled conditions to an innocuous state, or to levels

below concentration limits established by regulatory authorities (Atlas and Bartha, 1998).

For bioremediation to be effective, microorganisms must enzymatically attack the

pollutants and convert them to harmless products. As bioremediation can be effective

only where environmental conditions permit microbial growth and activity, its application

often involves the manipulation of environmental parameters to allow microbial growth

and degradation to proceed at a faster rate. Bioremediation techniques are typically more

economical than traditional methods such as incineration, and some pollutants can be

treated on site, thus reducing exposure risks for clean-up personnel, or potentially wider

exposure as a result of transportation accidents. Most bioremediation systems are run

under aerobic conditions, but running a system under anaerobic conditions may permit

microbial organisms to degrade otherwise recalcitrant molecules also. Microbial growth

and activity are readily affected by pH, temperature, and moisture. Although

microorganisms have been also isolated in extreme conditions, most of them grow

optimally over a narrow range, so that it is important to achieve optimal conditions. If the

soil has too much acid it is possible to rinse the pH by adding lime. Temperature affects

biochemical reactions rates, and the rates of many of them double for each 10 °C rise in

temperature. Above a certain temperature, however, the cells die. Available water is

essential for all the living organisms, and irrigation is needed to achieve the optimal

moisture level.

Hydrocarbons are readily degraded under aerobic conditions, whereas chlorinated

compounds are degraded only in anaerobic ones. To increase the oxygen amount in the

soil it is possible to till or sparge air. In some cases, hydrogen peroxide or magnesium

peroxide can be introduced in the environment. Low soil permeability can impede

movement of water, nutrients, and oxygen; hence, soils with low permeability may not be

appropriate for in situ clean-up techniques.

6

MICROORGANISMS IN PHENOL BIODEGRADATION

Degradation of phenol occurs as a result of the activity of a large number of

microorganisms including bacteria, fungi and actinomycetes. Bacterial species include

Bacillus sp, Pseudomonas sp, Acinetobacter sp, Achromobacter sp etc. Fusarium sp,

Phanerocheate chrysosporium, Corious versicolor, Ralstonia sp, Streptomyces sp etc are

also proved to be efficient fungal groups in phenol biodegradation. (Prieto et al., 2002).

Many studies on biodegradation of phenol come from bacteria. The genus Pseudomonas

is widely applied for the degradation of phenolic compounds. These bacteria are known

for their immense ability to grow on various organic compounds. Phenol biodegradation

studies with the bacterial species have resulted in bringing out the possible mechanism

and also the enzyme involved in the process. The efficiency of the phenol degradation

could be further enhanced by the process of cell immobilization (Annadurai et al., 2000a,

b). Phenol and other phenolic compounds are common constituents of many industrial

effluents. Once a suitable micro organism based process is developed for the effective

degradation of phenol these phenolic effluents can be safely treated and disposed

(Borghei and Hosseini, 2004). Candida tropicalis RETLCrl from the effluent of the

Exxon Mobile Oil Refinery waste water treatment was investigated for phenol

degradation using batch and fed batch fermentation under aerobic condition (Mohd Tuah,

2006). Microbiological degradation of phenol and some of its alkyl derivatives (p-cresol,

4-n-propyl phenol, 4-i –propyl phenol, 4-n-butyl phenol, 4-sec-butyl phenol, 4-t-butyl

phenol and 4-t-octyl phenol) were examined under both

aerobic and anaerobic conditions in seven Japanese paddy soil samples (Atsushi et al.,

2006). The rate of biodegradation of phenol by Klebsiella oxytoca strain was studied. It

was found that K. oxytoca degraded phenol at elevated concentration where 75% of initial

phenol concentration at 100 ppm was degraded within 72 h (Shawabkeh et al., 2007).

Phenol was degraded by Actinobacillus species (Khleifat and Khaled, 2007). They found

that pH 7, the incubation temperature of 35 to 37°C, and the agitation rate of 150 rpm

were the optimal conditions for achieving the higher percentage of phenol degradation.

Succinic acid and glycine as respective carbon and nitrogen source were found to be the

most efficient co-substrates for the removal of phenol. Immobilized Alcaligenes sp d2

was successfully used for the effective treatment of phenolic paper factory effluent

(Nair and Shashidhar, 2007).

7

Many studies on biodegradation of phenol using pure and mixed cultures have been

reported (Collins et al., 2005; Dursun and Tepe, 2005; Marrot, 2006; Shen et al., 2009;

Laowansiri et al., 2008; Celik et al., 2008; Santos et al., 2009). In India also a

considerable amount of study on phenol biodegradation has been undertaken like the

studies of Dhagat et al. (2002); Chandra and Rathore (2002); Ambujom and Manilal

(2004); Shetty et al. (2007); Jayachandran and Kunhi (2008). Biological processes are

generally preferred due to their lower operational costs and the possibility of complete

mineralization (Agarry et al. 2008; Brar et al. 2006). A number of microorganisms have

been shown to utilize phenol and its derivatives as sole sources of carbon and energy at

varying concentrations, under aerobic conditions. They include several bacterial cultures

(Babu et al. 1995b; Chung et al. 2003; Karigar et al. 2006), a few yeast strains (Agarry et

al. 2008; and mycelial fungi (; Santos et al. 2003; Yordanova et al. 2009). Removal of

phenol also has been carried out using activated sludge systems for the last several years

(Amor et al. 2005; Marrot et al. 2008). But, high phenol loading rates and fluctuations in

phenol loads have been reported to cause the breakdown of these systems (Watanabe et

al. 2000).

The major difficulty encountered in the microbial treatment of phenol-containing

wastewater is the substrate inhibition of microbial growth and concomitant hindrance in

the biodegradation. To overcome these problems immobilization of the degrading

organism(s) has been proposed as an effective strategy (Keweloh et al. 1989). The main

advantages of using immobilized cells, rather than free suspended ones, include the

retention in the reactor of higher concentrations of microorganisms, protection of cells

against toxicity and prevention of suspended microbial biomass in the effluent. Moreover,

immobilization of microbial cells provides, in general, high degradation efficiency and

good operational stability. Aerobic granulation of the degrading organism(s) as a new

form of cell immobilization for exploitation in phenol wastewater treatment in sequencing

batch reactors also has been gaining importance, recently (Tay et al. 2004). The

aggregation of microbial cells into compact self-immobilized granules can serve as an

effective protection against phenol toxicity. However, the traditional immobilization

technique of entrapment of microorganisms in gel polymers has been shown to be more

efficient in resisting the phenol toxicity and improving the degradation rates. Several

different polymeric materials have been tried as matrices for immobilization. Ca-alginate

has been used as the gel matrix in several studies for encapsulating strains of bacteria,

8

yeasts, and mycelial fungi (Abd-El-Haleem et al. 2003; Santos et al. 2003). Other

polymers which were occasionally used for immobilizing phenol-degrading

microorganisms were agar, polyacrylamide (PAA), polyacrylamide hydrazide (PAAH),

polyvinyl alcohol (PVA), polysulfone hollow fiber membrane, and modified

polyacrylonitrile membrane (Chen et al. 2002; Yordanova et al. 2009). All the phenol-

degrading strains exhibited improved ability, to varying degrees, in the immobilized

systems than as freely suspended cells. However, studies on comparison of the suitability

and efficiency of different matrices for immobilization of phenol degrading organisms are

very rarely found in literature (Bettmann and Rehm 1984). Particularly, a comparative

appraisal of the calcium alginate and agar immobilized microorganisms has been very

scarcely found (Karigar et al. 2006). Pseudomonas sp. strain CP4, a laboratory isolate

was found to be a potent degrader of phenol, isomers of cresol, and other aromatics under

shake flask conditions (Ahamad and Kunhi 1999; Ahamad et al. 2001; Babu et al. 1995b)

and was an efficient partner of a mixed culture with Pseudomonas aeruginosa strain 3mT

in the degradation of mixtures of 3-chlorobenzoate (3-CBA) and phenol/cresols (Babu et

al. 1995a).

MECHANISM OF PHENOL BIODEGRADATION

Generally aromatic compounds are broken down by natural bacteria. However, polycyclic

aromatic compounds are more recalcitrant. Derivatisation of aromatic nuclei with various

substituents particularly with halogens makes them more recalcitrant. There are reports on

many microorganisms capable of degrading phenol through the action of variety of

enzymes. These enzymes may include oxygenases hydroxylases, peroxidases, tyrosinases

and oxidases.

The critical step in the metabolism of aromatic compounds is the destruction of the

resonance structure by hydroxylation and fission of the benzoid ring which is achieved by

dioxygenase-catalysed reactions in the aerobic systems. Based on the substrate that is

attacked by the ring cleaving enzyme dioxygenase, the aromatic metabolism can be

grouped as catechol pathway, gentisate pathway, and proto catechaute pathway. In all

these pathways, the ring activation by the introduction of hydroxyl groups is followed by

the enzymatic ring cleavage. The ring fission products, then undergoes transformations

leading to the general metabolic pathways of the organisms. Most of the aromatic

9

catabolic pathways converge at catechol. Catechols are formed as intermediates from a

vast range of substituted and nonsubstituted mono and poly aromatic compounds.

Aerobically, phenol also is first converted to catechol, and subsequently, the catechol is

degraded via ortho or meta fission to intermediates of central metabolism. The initial ring

fission is catalysed by an ortho cleaving enzyme, catechol 1, 2 dioxygenase or by a meta

cleaving enzyme catechol 2,3 dioxygenase, where the product of ring fission is a cis-

muconic acid for the former and 2-hydro Cis- muconic semi aldehyde for the latter

(Gurujeyalakshmi and Oriel, 1988). Streptomyces setonii (ATCC 39116) degraded

aromatic compounds such as phenol or benzoate via an ortho cleavage pathway using

catechol 1,2 dioxygenase (An et al., 2001). These dioxygenases are highly labile

enzymes and there requires a detailed investigation into its structural properties. A

bacterial strain, Serratia plymuthica was able to tolerate phenol up to a concentration of

1050 mg/L. Phenol was degraded through ortho pathway and the crude extract showed

the presence of ring cleaving enzyme catechol 1, 2-dioxygenase (Nilotpala and Ingle,

2007). Catechols are cleaved either by ortho-fission (intradiol, that is, carbon bond

between two hydroxyl groups or by a meta-fission (extra diol, that is, between one of the

hydroxyl groups and a non-hydroxylated carbon) as given in Figures 2 and 3. Thus the

ring is opened and the open ring is degraded (Cerniglia, 1984). As a general rule, most of

the halo aromatics are degraded through the formation of the respective halocatechols, the

ring fission of which takes place via ortho-mode. On the other hand, most of the non

halogenated aromatic compounds are degraded through meta pathway.

10

Figure 3. Meta pathway of phenol degradation. Figure 2. Meta pathway of phenol degradation.

The fission product of ortho-cleavage would be cis, cis muconic acid or its derivative

depending on whether the catechol is substituted or not. The meta-fission product of

catechol would be 2-hydroxy muconic semialdehyde and the products of both ortho and

meta pathways are further metabolized as intermediates of TCA cycle. Ortho pathway is

the most productive pathway for the organism as it involves less expenditure of energy.

Phenol hydroxylase (E. C 14. 1.3.7) catalyses the degradation of phenol via two different

pathways initiated either by ortho or meta cleavage. There are many reports on phenol

hydroxylase and catechol 2, 3 dioxygenase involved in the biodegradation of phenol

(Leonard and Lindley, 1999). Hublik and Schinner (2000) reported the characterization of

11

laccase from Penurious ostreatus. The enzyme was purified to homogeneity and was

characterized. It was a monomeric protein with a molecular weight of 67 KD and with an

isoelectric point of 3.6. They observed that the laccase retained most of its activity in high

ionic buffer, pH.10, 20°C temperature in the presence of 10 mM benzoic acid and with

35% ethylene glycol. The degradation of phenolic compounds by immobilised laccase

from Streptomyces psammoticus was evaluated and confirmed by thin layer

chromatography and nuclear magnetic resonance spectroscopy (Niladevi and Prema,

2007). Polyphenol oxidase is a (EC 1.14.18.1) monoxygenase which catalyses the O-

hydroxylation of phenols and the oxidation of O-dihydric phenols to O-quinones using

molecular oxygen. Laccase are phenol oxidases which utilize molecular oxygen. They are

known to have the ability to oxidize polyphenols; meta substituted phenols, diamines and

a variety of other components (Kadhim, 1999). The mechanism by which polyphenol

oxidase catalyses the conversion of monophenols to O-quinones involves the

hydroxylation of monophenols followed by dehydrogenation to form O-quinones. These

quinones undergo spontaneous nonenzymatic polymerization in water, eventually

forming water insoluble polymers which can be separated from water by filtration

(Edwards et al., 1999). There were various reports on the exploitation of polyphenol

oxidase in the detoxification of the phenols. The interest in polyphenol oxidase had been

fueled by their potential uses in detoxification of environmental pollutants (Bollag et al.,

1988). Production of useful chemicals from lignin (Burton et al., 1993) by polyphenol

oxidase was also reported. Garzillo et al. (1998) reported a polyphenol oxidase from the

white rot fungus Trametes trogii. It was an enzyme with molecular weight 70 KD. The

purified enzyme oxidised a number of phenolic compounds. This multicopper oxidases

had a wide range of substrate specificity. Coprinus macrorhizus and Arthromyces

ramosus were proved to be effective in removing phenol and phenolic compounds from

water (Wu et al., 1998). Of the various enzymes acting on phenol, polyphenol oxidase

was the most important one probably because of its increasing demand in lignin

degradation (Garzillo et al., 1998). The non specific nature of the polyphenol oxidase was

also discussed by Schneider et al. (1999). Immobilised polyphenol oxidase on chitosan

coated polysulphone capillary membranes were used for improved phenolic effluent

bioremediation (Edwards et al., 1999).

They also highlighted the removal of quinones and other polymerized products using

chitosan. Polyphenol oxidases were widely distributed in many plants and fungal species

(Robles et al., 2000). They suggested the possibility of using a polyphenol oxidase

12

producing strain of the hyphomycete Chalara paradoxa in the detoxification of olive mill

wastewater. Sakurai et al. (2001) showed that the peroxidase from Coprinus cinereus

could be used for the removal of Bisphenol. Polymerization of the bisphenol by the

enzyme was utilized here. Manophenols in aqueous solution could also be removed by

peroxidase catalysed oxidation (Xia et al., 2003). Certain actinomyces and Streptomyces

strains could produce tyrosinase enzyme, which oxidized halogen substituted phenols.

Peroxidases could catalyse the transformation of phenol and halogenated phenols.

Peroxidases such as those from Arthrobacter and Streptomyces strains were being

reported as the phenol degrading enzymes (Fetzner and Lingens, 1994). The peroxidase

catalysed polymerization process was proved to be very effective in eliminating phenol

and a variety of other aromatic pollutants from waste waters (Ghioureliotis and Nicell,

1999). Peroxidases can act on phenol and other aromatic compounds through oxidative

coupling. In presence of hydrogen peroxide two equivalents of phenol are converted by

each equivalent of enzyme into highly reactive radical species. Once they are formed,

they react with one another to yield phenolic polymers. Tyrosinase catalyzes the

oxidation of phenols involving the formation of orthoquinones. The mechanism of the

enzymatic action of tyrosinase on various phenols was discussed in detail by Siegbahn

(2003). The mechanism of degradation of an organic compound may be unusual (Jenisch-

Anton, 1999). The mechanism of degradation is generally decided by the nature of the

organic compound, its solubility, and nature of the organism, type of the enzyme and also

by the external factors affecting biodegradation. In some cases, through

the action of monooxygenase, aromatic com-pounds may be converted into gentisic acid.

The fission of this compound occurs between the hydroxyl and carboxyl groups, that is,

meta fission. It has been shown in some cases that chloroaromatic compounds such as 4-

chlorobenzoate, 4-chlorophenol and others may get dechlorinated during the

hydroxylation resulting in the formation of 4–hydroxy benzoates (4-HBA). This 4 HBA

on further hydroxylation will be converted to protocatechuate

acid (3,4-dihydroxy benzoic acid), which may be cleaved either through ortho or meta

mode.

Each of these factors should be optimized for the selected organism for the maximum

degradation of the organic compound of choice. The optimization of the substrate

concentration in phenol biodegradation is particularly important since it inhibits the

growth of the organism at higher concentrations. Biotechnology for hazardous waste

management involves the development of biological systems that catalyse the

detoxification, degradation or decontamination of environmental pollutants.

13

MATERIALS AND METHODS

14

3. MATERIALS AND METHODOLOGY

3.1 GLASSWARES:

Autoclaved beakers, Flasks, Petri plates, Glass rods, Pestle and mortar, Centrifuge tubes

and Eppendorf tubes

3.2 INSTRUMENTS AND EQUIPMENT REQUIRED:

BOD Incubator SONAR

Autoclave NSW, New Delhi

Laminar Air Flow Atlantis, New Delhi

Weighing Machine A&D Company Ltd

Centrifuge REMI, Mumbai

Vortex LABNET

Water Bath POPULAR TRADERS, Ambala Cantt

Microwave oven L.G.

Refrigerator L.G.

µpH system- 361 Systronics

15

3.3 METHODS

3.3.1 METHODS OF ISOLATING PHENOL DEGRADING BACTERIA

Soil samples were collected from various places of India, the most potent phenol degrader

was isolated by following procedure.

1 gm of soil sample was added to 9ml of autoclaved distilled water and the resulting

mixture was centrifuged. Then 5 ml of this media was inoculated to 250 ml of new BHM

broth supplemented with phenol at the rate of 600mg/L at 37°C for another one week.

Again, 10 ml of the second passage was inoculated into the new BHM broth media and

incubated in the above mentioned situation. These passages were repeated until turbidity

was obtained from bacteria growth, which was not due to mixed sedimentation with the

first media. After the last passage, it was cultured on phenol agar media as an isolate and

the bacterium was isolated as a colony alone. Regarding water samples, same method was

applied, except that 10 ml water was mixed with 100 ml phenol broth media and all

previously mentioned stages were repeated (Koutny et al., 2003).

The compositions of Bushnell’s Hass Medium in g l-1 were

K2HPO4 - 0.1

NaCl - 0.1

CaCl2.2H2O - (0.015)

MgSO4 - 0.2

FeCl3 - 0.02

NH4Cl - 2.00

Phenol - 0.66

3.4 EXPERIMENTAL PROCEDURE:

The isolated bacteria were suspended in Bushnell’s Hass broth medium supplemented

with 600mg/l phenol at pH 7. The suspension cultures of the isolated bacteria were

inoculated in BHM containing 600 mg l-1 initial concentration of phenol to compare their

phenol degrading efficiency. The line of experiment was designed according to a previous

16

study of Wael et al. (2003), where he had isolated six pure bacterial strains from a coke

processing unit waste water. The major modification was the initial phenol concentration

of 600 mg l-1, which he had earlier taken as 100 mg l-1. The residual phenol concentration

was monitored at different time intervals spectrophotometrically according to the method

described by Yang and Humphrey (1975). The strain degrading phenol to a greater extent

within a relatively short time was selected as efficient phenol degrader among the isolates

for the optimization studies of the physical environment.

3.5 ISOLATION AND IDENTIFICATION OF THE BACTERIA:

Eight bacterial strains (S1G, S2B, S1H, D1, D5, S4A, S4C, and S5C) were found to be

growing in Bushnell’s Hash Medium containing 600 mg l-1 of phenol during the early

screening for the investigation.

S1G and S1H are isolated from the soil sample of Soya Industry, Indore.

S2B was isolated from Auto market Hisar.

S4A was isolated from Pesticide factory Hisar.

S5C was isolated from Alcohol industry Hisar,

D1 was isolated from soil sample of Palwal and

D5 was isolated from underground water of Hisar.

Microscopic observation and growth characteristics as well as biochemical tests of the

isolated bacteria were studied.

3.5.1 GRAM’S STAINING:

Gram’s stain developed by Christian Gram in 1884 is the most widely employed staining

method in bacteriology. It is an example of differential staining. This is a useful method

of identifying and classifying bacteria in two major groups:

-Gram Positive

- Gram Negative

The method relies on the cell structure differences between the two groups. The Gram

positive cell wall consist of 20-80nm thick homogenous layer of peptidoglycan (murein)

lying outside the plasma membrane. The peptidoglycan itself is not stained instead it

seems to act as a permeability barrier preventing loss of Crystal Violet. In contrast the

Gram negative cell wall is quite complex. It has a 2-7nm peptidoglycan layer covered by

a 7-8 nm thick outer membrane.

17

During the procedure the bacteria are first stained with crystal violet and next treated with

iodine to promote dye retention. When Gram positive are then treated with ethanol, the

alcohol is thought to shrink the pores of thick peptidoglycan. Thus the dye iodine

complex is retained during this short decolorization step and the bacteria remain purple.

In contrast the Gram negative peptidoglycan is very thin, not as highly cross linked and

has larger pores. Alcohol treatment may also extract enough lipids from the Gram

negative outer membrane to increase its porosity further. For these reasons, alcohol more

readily removes the purple Crystal Violet Iodine complex from Gram negative bacteria.

Thus Gram negative bacteria are then easily stained red or pink by the counterstain

saffranin.

3.5.1.1 PROCEDURE:

1. Initially a smear of bacterial culture was prepared on a slide, air dried and then

heat fixed in order to immobilize the bacteria.

2. Smear was then placed in a small jar containing Crystal Violet solution and was

allowed to stand for 1min.

3. After 1min slide was washed with distilled water and then dipped in iodine

solution for 1min.

4. After 1min decolorizer was applied drop wise until no more color flowed from

smear.

5. Slide was then washed with distilled water and then stained with saffranin and

kept for 1min.

6. Slide was then washed with distilled water and made dry.

7. Finally, the slide was examined under oil immersion objective microscope for

staining.

3.5.1.2 INTERPRETATION:

After staining if the bacterial strain was found to be purple it is Gram positive otherwise

Gram Negative

3.5.2 CITRATE TEST (SIMMON’S CITRATE TEST)

The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow

utilizing citrate as its sole carbon and energy source. Simmon's media contains

bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at

18

acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6).

Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color.

Growth of bacteria in the media leads to development of a Prussian blue color (positive

citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative.

3.5.2.1 PROCEDURE:

1. A loopful of bacteria was streaked onto a citrate agar slant.

2. It was incubated 24 to 48 hours with a loose cap.

3.5.2.2 INTERPRETATION:

After incubation a positive reaction is indicated by a slant with a Prussian blue color. A

negative slant will have no growth of bacteria and will remain green.

3.5.3 UREASE TEST

Urease broth is a differential medium that tests the ability of an organism to produce an

exoenzyme, called urease that hydrolyzes urea to ammonia and carbon dioxide.  The

broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and

the pH indicator phenol red.  Phenol red turns yellow in an acidic environment and

fuchsia in an alkaline environment.  If the urea in the broth is degraded and ammonia is

produced, an alkaline environment is created, and the media turns pink.

Many enterics can hydrolyze urea; however, only a few can degrade urea rapidly.  These

are known as “rapid urease-positive” organisms.  Members of the genus Proteus are

included among these organisms.

Urea broth is formulated to test for rapid urease-positive organisms.  The restrictive

amount of nutrients coupled with the use of pH buffers prevent all but rapid urease-

positive organisms from producing enough ammonia to turn the phenol red pink.

3.5.3.1 PROCEDURE:

1.    Inoculate a urea tube with 3 loopful of slant culture.

2.    Incubate 24 hours, observe for reaction.

3.    A pink color formation indicates the breakdown of urea to ammonia and CO2

3.5.3.2 INTERPRETATION

19

Phenol red indicator is added to the broth so that when the pH reaches 8.4 (due to

ammonia production) the tube will turn from an orange color to a pink color, indicating

the choice of organism is Urease positive.

3.5.4 EOSINE METHYLENE BLUE (EMB) AGAR

DESCRIPTION:

A selective medium for gram-negative bacteria Levine’s EMB agar contains methylene

blue, which inhibits gram-positive bacteria. Differential for enterics: will differentiate

lactose fermenters from nonfermenters.

3.5.4.1 PROCEDURE:

One loopful of culture was inoculated in EMB agar slant.

3.5.4.2 INTERPRETATION:

After incubation: Lactose nonfermenters will have cream colored colonies. Lactose

fermenters will have pinkish colonies, sometimes with dark centres. E. coli often has a

greenish metallic sheen.

3.6 Immobilization of Microbial Cells

20

Cells of mixed culture collected from soils containing pollutants or specific culture (pure)

isolated from the pollutant containing soil are immobilized in/on solid matrix.

Acclimization of microorganisms is done by increasing the pollutant concentration (say

of phenol) gradually during culture preparation. The acclimized culture is used for the

immobilization in/on the solid matrix. Immobilization of cells means that the cells have

been confined or localized so that it can be reused continuously. These exhibit totally

different hydrodynamic characteristics than surrounding environment. Living cells

produce enzymes (biological catalysts) to catalyze cellular reactions vital to the organism.

The microorganisms are normally immobilized on natural and synthetic supports. Various

types of solid matrices like polyacrylamide gel, Ca alginate, porous glass, plastic beads,

activated carbon, sand, charcoal, diatomaceous earth, cement balls made of coal ash,

cellulose, polymeric materials, polymeric ions, chitosan, lignins, chitins, coal, collagens

etc. have been used for immobilization of whole cells. In the recent years, the

immobilization of biocatalysts with polyvalent salts of alginic acids has received much

attention because of low cost of alginate and the mild conditions of immobilization.

Techniques of immobilization are broadly classified into four categories namely: covalent

bonding, cross-linking (chemical methods), entrapment and adsorption (physical

methods). Covalent binding most extensively used technique, where cells or enzymes are

covalently linked to the support through the groups in them or through the functional

groups in the support material. In the cross-linking technique, the cells are immobilized

through chemical cross-linking using homo as well as hetero-bifunctional cross-linking

agents. Adsorption is the simplest of all techniques and does not alter the activity of the

bound cells. Adsorption involves adhesion or condensation of the cells to the surface of a

carrier. The diving force causing immobilization is the combined hydrophobic

interactions, hydrogen bonding and salt bridge formation between the adsorbent and cells.

Entrapment within gels or fiber is a convenient method for reactions involving low

molecular weight substrates and mainly used for immobilization of whole cells. This

method is nothing but the polymerization of the unsaturated monomers in the presence of

cells results in the entrapment of the cells within the interstitial spaces of the gel

3.6.1 PROCEDURE OF CELL IMMOBILIZATION:

21

a. Alginate was dissolved in boiling water and autoclaved at 121°C for 15 min.

b. Cells were harvested during the mid-logarithmic growth phase by centrifugation

(5000 g, 10 min)

c. The Cells mass was resuspended in 2 ml of saline and added to 100 ml of

sterilized alginate solution. This alginate/cell mixture (with stirring) was extruded

drop by drop into a cold, sterile 0.2 M CaCl2 solution through a sterile 5 ml

pipette.

d. Gel beads of approximately 2 mm diameter were obtained.

e. The beads were hardened by resuspending into a fresh CaCl2 solution for 24 h at

4°C with gentle agitation.

f. Finally these beads were washed with distilled water to remove excess calcium

ions and unentrapped cells.

g. Then the beads were transferred to 50 ml production medium and cultivated for

the required time.

22

RESULTS AND DISCUSSION

4. RESULT & DISCUSSION

23

4.1 The morphology and biochemical test of the isolated bacteria were studied and

following results were obtained.

S1G was found to be Gram +ve with cocci shaped which could utilize lactose and urea

but was found –ve to citrate.

S2B was found to be gram +ve and oval shaped which could utilize lactose but was found

–ve to both urea and citrate.

S1H was found to be Gram –ve and rod shaped which could utilize lactose but was –ve to

both urea and citrate.

D1 was found to Gram –ve and rod shaped which can neither utilize lactose, citrate nor

urea.

D5 was found to Gram –ve cocci bacilli which could utilize lactose and citrate but was

found –ve to urea.

S4A was found to Gram –ve and rod shaped which can utilize lactose but was found –ve

to both urea and citrate.

S4C was found to Gram +ve and cocci shaped which is positive to citrate but was found –

ve to lactose and urea.

S5 was found to Gram +ve and rod shaped which could utilize lactose but was found –ve

to both urea and citrate.

The details of the above description are summarized in the Table no 1.

4.2 RESIDUAL PHENOL ESTIMATION

The standard curve was plotted (O.D Vs concenteration of phenol). O.D was obtained by

spectrophotometric measurement at 630nm. This standard curve (Figure no.1) was used

to calculate the residual phenol i.e. the remaining phenol after degradation by isolated

strains by using the formula Y=0.110x + 0.041, where Y=O.D. Now the value of X was

obtained which was again multiplied by the difference in concentration, here the

difference is 200mg/l

The 3 most potent bacteria capable of degrading phenol were found to be S1G, S4A and

S5C having the degradation rate of 97.27%, 89.39% and 84.54% respectively.

The residual phenol concenteration of all 8 isolates is shown in Table 3.

24

4.4 RESIDUAL PHENOL CONCENTERATION mgl-1 after 6, 12, 18 and 24 hr

incubation (wavelength 630nm)

The Strain S1G was found to degrade 21.67% after incubation of 6 hr further incubation

degrades 97.27% at the end of 24 hr.

The rate of degradation is shown in table no.4

The strain S4A was found to degrade 18.49% phenol after incubation of 6hr which further

increases to 89.39% at the end of 24 hr incubation.

The rate of degradation is shown in Table no.5.

The strain S5C was found to degrade 12.42% phenol after incubation of 6 hr which

further increases to 85.54% at the end of 24 hr incubation.

The rate of degradation is shown in table no.6.

4.5 COMPARATIVE STUDY OF PHENOL DEGRADATION BY IMMOBILIZED

CELLS ANF FREE CELLS.

To study the enhanced biodegradation of phenol the cells after centrifuged at 5000rpm for

10min. Were immobilized and incubated at 37oC and the broth containing live cells which

equals to the wet weight of centrifuged cells were incubated at 37oC and the degradation

studies were compared. The data is shown in table no.7 and graph is shown in Figure no.3

Table - 1: MICROSCOPIC, GRAM STAIN AND BIOCHEMICAL CHARACTERS

OF THE ISOLATED STRAINS:

Microscopic

Examination

Gram stain Simmonn

Citrate

Chrisren’s

urease test

Lactose test

25

S1G Cocci Gram +ve -ve Weakly +ve +ve

S2B Oval shaped Gram +ve -ve -ve +ve

S1H Rod shaped Gram –ve -ve -ve +ve

D1 Rod shaped Gram –ve -ve -ve -ve

D5 Cocci bacilli Gram –ve +ve -ve +ve

S4A Rod shaped Gram –ve -ve -ve +ve

S4C Cocci Gram +ve +ve -ve -ve

S5 Rod shaped Gram +ve -ve -ve +ve

Table 2: STANDARD CURVE AT 630nm

Concentration (mg/l) O.D

200 01.14

400 0.272

26

600 0.375

800 0.479

1000 0.589

Figure 1 : Standard Curve (O.D Vs Concenteration)

Table 3: RESIDUAL PHENOL ESTIMATION

The spectrophotometric measurement was done at wavelength 630nm

Strain O.D Residual Phenol (mg/l)

S1G 0.05 16.37

27

S2B 0.170 234.54

S1H 0.158 212.72

S4A 0.076 63.63

S4C 0.186 263.63

S5C 0.092 92.72

D1 0.180 252.72

D5 0.194 278.18

Figure 2: Residual Phenol Estimation

The 3 most potent bacteria capable of degrading phenol were found to be S1G, S4A and S5C having the degradation rate of 97.27%, 89.39% and 84.54% respectively.

28

Table - 4: RESIDUAL PHENOL CONCENTERATION mgl-1 after 6, 12, 18 and 24 hr incubation (Wavelength 630nm)

Strain Time(hrs) 6 12 18 24

O.D(630nm) 0.30 0.21 0.12 0.05

S1G Residual Phenol(mg/l) 470.90 307.27 143.63 16.37

% Removal of phenol. 21.67 48.17 76.06 97.27

Table - 5: RESIDUAL PHENOL CONCENTERATION mgl-1 AFTER 6, 12, 18 and 24 hr incubation (Wavelength 630nm)

Strain Time(hrs) 6 12 18 24

S4A O.D (630nm) 0.31 0.22 0.15 0.076

Residual phenol (gm/l) 489.09 325.45 198.18 63.63

% Removal of phenol 18049 45.375 66.97 89.39

29

Table - 6: RESIDUAL PHENOL CONCENTERATION mgl-1 AFTER 6, 12, 18 and 24 hr incubation (Wavelength 630nm)

Strain Time(hrs) 6 12 18 24

O.D (630nm) 0.33 0.24 0.17 0.092

S5C Residual phenol (gm/l) 525.45 361.81 234.54 92.72

% Removal of Phenol 12.425 39.69 60.91 85.54

Figure 3: Phenol degradation by three most potent isolated at different time

The above graph shows that S1G was the most potent phenol degrader with degradation

of 97.27% after the end of 24hrs while the strains S4A and S5C were found to degrade

89.39% and 84.54% respectively.

30

Table 7: COMPARATIVE STUDY OF PHENOL DEGRADATON BY IMMOBILIZED S1G AND FREE S1G CELLS (Wavelength 630nm).

Phenol degradation study by immobilized cells

Time(Hrs) 6 12 18

O.D 0.29 0.185 0.05

Residual

Phenol(mg/l)

452.72 261.81 16.63

Phenol Degradation Study by live cells

Time (Hrs) 6 12 18

O.D 0.30 0.21 0.12

Residual

Phenol(mg/l)

470.90 307.27 143.63

Figure 4: Comparative study of the phenol degradation by immobilized and free cells

31

CONCLUSION

32

CONCLUSION

Phenol and its components are extremely toxic and can easily be isolated from different

industrial sewage such as oil refinery, petrochemical industry and mines, especially

collier and chemical factories. Hence the presence of these compounds in the

environment could cause environmental pollution, especially in water resources. In the

past, physicochemical method was used for the elimination of phenol and its compounds,

but today, bioremediation is preferable. The aim of this study is to isolate and identify

phenol degrading bacteria from industrial effluents and study the phenol degradation rate.

Till date several works are in progress to isolate new and efficient microbial strain that

have ability to degrade phenol. We report here a new, isolate as a potential selected strain

to utilize phenol as sole source of carbon and energy. The degradation ability of isolate

was checked up to 600mg/l concentration. The determination of intermediates of phenol

degradation was also determined. This work has provided a useful guideline in evaluating

potential phenol biodegrades isolated from environment. Different methods have been

used for the elimination of phenol, but the use of bacteria can be one of the cheap and

secure methods. Bacteria with rapid reproduction in the presence of phenol and its

compound have shown extraordinary ability in phenol elimination. So with isolation,

purifying and growing of species which has high ability of phenol elimination, they can

be used in areas with phenol pollution. Different bacteria of different genus have been

isolated as phenol degrading. Most of them, which chiefly belong to family

Pseudomonaceae, are gram negative. Kounty et al. (2003) isolated phenol degrading

bacteria from Siberia soils. They found out that the permanent genus of phenol degrading

in these soils is Pseudomonas and especially, Pseudomonas putida. Although, this agrees

with the finding, their vast distribution in soils and the ability of phenol or phenol

compounds to be eliminated was done by Williams and Sayers (1994) and Torres et al.

(1999).

33

REFERENCES

i

REFERENCE

Abd-El-Haleem D, Beshay U, Abdelhamid AO, Moawad H, Zaki S (2003) Effects of

mixed nitrogen sources on biodegradation of phenol by immobilized Acinetobacter sp.

strain W-17. Afr J Biotechnol 2:8–12

Agarry SE, Durojaiye AO, Solomon BO (2008) Microbial degradation of phenols: a

review. Int J Environ Pollut 32:12–28

Ahamad PYA, Kunhi AAM (1996) Degradation of phenol through ortho-cleavage

pathway by Pseudomonas stutzeri strain SPC-2. Lett Appl Microbiol 22:26–29.

Ahamad PYA, Kunhi AAM (1999) Degradation of high concentrations of cresols by

Pseudomonas sp. CP4. World J Microbiol Biotechnol 15:28–283.

Ahamad PYA, Kunhi AAM, Divakar S (2001) New metabolic pathway for o-cresol

degradation by Pseudomonas sp. CP4 as evidenced by H NMR spectroscopic studies.

World J Microbiol Biotechnol 17:371–377.

Ahamad PYA, Varadaraj MC, Kunhi AAM (1996) Isolation and characterisation of

phenol and cresol degrading pseudomonads. In: Kahlon RS (ed) Perspectives in

microbiology. National Agricultural Technology Information Centre, Ludhiana, pp 35–41

Ali S, Lafuente RF and Cowan DA (1998) Meta-pathway degradation of phenolics by

Thermophilic Bacilli. Enzyme Microb. Technol. 23:462-468.

Ambujom S. and Manilal V.B.: Phenol degradation by a stable aerobic consortium and

its bacterial isolates. Biotech. Letters, 17, 443-448 (2004).

Anselmo AM, Cabral JMS, Novais JM (1989) The adsorption of Fusarium flocciferum

spores on celite particles and their use in the degradation of phenol. Appl Microbiol

Biotechnol 31:200–203.

ii

Ariana F, Elke B and Thomas B (2004). Rapid monitoring of the biodegradation of

phenol-like compounds by the Candida maltose using BOD measurements. Int.

Biodeterior. Biodegrad. 54: 69- 76.

Babu KS, Ajith-Kumar PV, Kunhi AAM (1995a) Simultaneous degradation of 3-

chlorobenzoate and phenolic compounds by a defined mixed culture of Pseudomonas spp.

World J Microbiol Biotechnol 11:148–152.

Babu KS, Ajith-Kumar PV, Kunhi AAM (1995b) Mineralisation of phenol and its

derivatives by Pseudomnas sp. strain CP4. World J Microbiol Biotechnol. 11:661–664.

Bajaj M, Gallert C, Winter J (2008) Biodegradation of high phenol containing synthetic

wastewater by an aerobic fixed bed reactor. Bioresour Technol 99:8376–8381

Bandhyopadhyay K, Das D, Maiti BR (1999) Solid matrix characterization of

immobilized Pseudomonas putida MTCC 1194 used for phenol degradation. Appl

Microbiol Biotechnol

Bettmann H, Rehm HJ (1984) Degradation of phenol by polymer entrapped

microorganisms. Appl Microbiol Biotechnol 20:285–290

Bettmann H, Rehm HJ (1985) Continuous degradation of phenol(s) by Pseudomonas

putida P8 entrapped in polyacrylamide- hydrazide. Appl Microbiol Biotechnol 22: 389–

493

Biodegradation 54:69–76

Bodalo, A., Gomez J.L., Gomez M., Leon G., Hidalgo A.M. and Ruiz M.A.: Phenol

removal from water by hybrid processes: Study of the membrane process step.

Desalination, 223, 323-329 (2008).

Brar SK, Verma M, Surampalli RY, Misra S, Tyagi RD, Meunier N, et Blais J-F (2006)

Bioremediation of hazardouswastes—a review. Pract Period Hazard Toxicol Radioact

Waste Manag 10:59–72

iii

C Karigar, A Mahesh, M Nagenahalli, Yun DJ (2006) Phenol degradation by

immobilized cells of Arthrobacter citreus. Biodegradation 17:47–55

Celik, G.Y., Aslim B. and Beyatli Y.: Enhanced crude oil biodegradation and rhamnolipid

production by Pseudomonas stutzeri strain G11 in the presence of Tween-80 and Triton

X-100. J. Environ. Biol., 29, 867-870 (2008).

Chandra, R. and Rathore B.: Phenol degradation by bacterial isolates and consortium

isolated from anaerobically treated distillery effluent sludge. J. Ecophysiol. Occupl. Hlth.,

2, 143-151 (2002).

Chen KC, Lin YH, Chen WH, Liu YC (2002) Degradation of phenol by PAA-

immobilized Candida tropicalis. Enzyme Microb Technol. 31:490–497 Chung T-P, Tseng

H-Y, Juang R-S (2003) Mass transfer effect and intermediate detection for phenol

degradation in immobilized Pseudomonas putida systems. Process Biochem 38:1497–

1507

CMR (2005) Phenol: chemical profile. Chemical Market Reporter, pp 34–35 Dainty AL,

Goulding KH, Robinson PK, Simpkins I, Trevan MD (1985) Stability of alginate-

immobilized algal cells. Biotechnol Bioeng 28:210–216.

Collins, G., Clare F. and Sharon M., Therese M. and Flaherty V.O.: Anaerobic biological

treatment of phenolics wastewater at 15-18 oC. Water Res., 39, 1614 -1620 (2005).

cutaneum R57. Process Biochem. 38: 915-920.

Dhagat, N., Nene L., Kapley A., Purohit H.J. and Bal A.S.: Role of nitrogen level on

phenol degrading microorganisms in fixed film bioreactor. Pollut. Res., 21, 447-450

(2002).

Dursun, Y. and Tepe O.: Internal mass transfer effect on biodegradation of phenol by Ca-

alginate immobilized Ralstonia eutropha. J. Hazardous Material, 126, 105-111 (2005).

Eduardo A, Bastos R, Moon D (2000). Salt tolerant phenol-degrading microorganisms

isolated from Amazonian soil sample. Appl. Environ Microbiol. 174: 346-352.

iv

Fialova A, Boschke E, Bley T (2004) Rapid monitoring of the biodegradation of phenol-

like compounds by the yeast Candida maltosa using BOD measurements. Int Biodeterior

Fitzhugh TW, & Richter BD (2004). Quenching urban thirst: growing cities and their

impacts on freshwater ecosystems. BioScience, 54(8): 741-754.

Godjevargova T, Ivanova D, Alexieva Z, and Dimova N (2003). Biodegradation of toxic

organic components from industrial phenol production wastewater by free and

immobilized Trichosporon

Gurusamy, A, Lai Y.L. and Jiunn F.L.: Biodegradation of phenol by Pseudomonas

pictorum on immobilized with chitin. Afr. J. Biotechnol.,6, 296-303 (2007).

Hannaford AM, Kuek C (1999) Aerobic batch degradation of phenol using immobilized

Pseudomonas putida. J Ind Microbiol Biotechnol. 22:121–126.

Jayachandran, V.P. and A Kunhi .A.M.: Degradation of 3-chlorobenzoate and phenol

singly and in mixture by a mixed culture of two orthopathway- following Pseudomonas

strains. J. Industrial Micro. Biotech, (2008).

John DM, White GF (2003). Mechanism for biotransformation of nonyl phenol

polyethoxylates to xenoestrogens in Pseudomonase putida. Appl. Environ. Microbiol.

180: 4332-4338.

Jua´rez-Ramı´rez C, Ruiz-Ordaz N, Cristiani-Urbina E, Galı´ndez-Mayer J (2001)

Degradation kinetics of phenol by immobilized cells of Candida tropicalis in a fluidized

bedreactor. World J Microbiol Biotechnol 17:697–705.

Kapoor A, Kumar R, Kumar A, Sharma A, Prasad S (1998)Application of immobilized

mixed bacterial culture for the degradation of phenol present in oil refinery effluent. J

Environ Sci Health A 33:1009–1021.

Kar, S., Swaminathan T. and Baradarajan A. : Studies on biodegradation of a mixture of

toxic and non toxic pollutant using Arthrobacter species. Bioproc. Biosys. Engg., 15, 195-

199 (1996).

v

Karigar, C., Mahesh A., Nagenahalli M. and Yun D.J.: Phenol degradation by

immobilized cells of Arthrobacter citreus. Biodegradation, 17, 47-55 (2006).

Keweloh H, Heipieper HJ, Rehm HJ (1989) Protection of bacteria against toxicity of

phenol by immobilization in calcium alginate. Appl Microbiol Biotechnol 31:383–389

Khaled, Khleifat M.: Biodegradation of phenol by Ewingella americana: Effect of carbon

starvation and some growth conditions. Process Biochemistry, 41, 2010-2016 (2006).

Koutny M, Ruzicka J, Chlachula J (2003). Screening for phenol degrading bacteria in the

pristine solids of south Siberia. Appl. Soil Ecol. 23: 79-83.

L Amor, M Eiroa, C Kennes, MC Veiga (2005) Phenol biodegradation and its effect on

the nitrification process. Water Res 39:2915–2920

Laowansiri, S., Vinitnantharat S., Chaiprasert P. and Ha S.R.: Anaerobic degradation

kinetics of reactive dye with different carbon sources. J.Environ. Biol., 29, 309-314

(2008).

Ma r ro t , B. , Ba r r i os -Mar t ine z A., Mou in an d l P. N. Roch e: Biodegradation of

high phenol concentration by activated sludge in an immersed membrane bioreactor.

Biochem. Eng. J., 30,

Morrison RM, Boyd RN (1992). Organic chemistry, 6th ed. 1069-1098. Powlowski J and

Shingler V (1994). Genetic and biochemistry of phenol degradation by Pseudomonase sp.

CF600. Biodegradation, 5: 219- 236.

Polymenakou, P.N. and Stephanou E.G.: Effect of temperature and additional carbon

sources on phenol degradation by an indigenous soil Pseudomonad. Biodegradation, 16,

403-413 (2005).

Quintana MG, Didion C, Dalton H (1997). Colorimetric method for a rapid detection of

oxygenated aromatic biotransformation product. Biotechnol. Tech. 11: 585-587.

Rittmann, B.E. and McCarty P.L.: Environmental Biotechnology: Principles and

Applications New York: McGraw-Hill (2001).

vi

Rosa, M., Peter S., Cathrin S. and Anne M.G.: Arthrobacter psychrophenolicus sp.

isolated from an alpine ice cave. Int. J. Syst. Evol. Microbiol., 54, 2067-2072 (2004).

Santos, V.L., Andrea de S.M., Braga D.T. and Santoro M.M.: Phenol degradation by

Aureobasidium pullulans FE13 isolated from industrial effluents. J. Hazardous Material,

16, 1413-1420 (2009).

Santos, V.L., Heilbuth N.M., Braga D.T., Monteiro A.S. and Linardi V.R.J.: Phenol

degradation by a Graphium sp. FIB4 isolated from industrial effluents. Basic Microbiol.,

43, 238-248 (2003).

Shen, J., Rui He, Yu H., Wang L., Zhang J., Sun X., Li J., Han W. and Lu Xu:

Biodegradation of 2,4,6-trinitrophenol (picric acid) in a biological aerated filter (BAF).

Bioresource Technol., 11, 1922-1930 (2009).

Shetty, Vidya K., Ramanjaneyulu R. and Srinikethan G.: Biological phenol removal using

immobilized cells in a pulsed plate bioreactor: Effect of dilution rate and influent phenol

concentration. J. Hazardous Material, 149, 452-459 (2007).

Spiker, K., Crawford L. and Thiel C.: Oxidation of phenolic and non-phenolic substrates

by the lignin peroxidase of Streptomyces viridosporous T7A. Appl. Microb. Biotechnol.,

37, 518-523 (1992).

Wael, S.E.S., Mohamed K.I., Mohamed A.S., Fawkia E.B., Naoya O., Hiroshi S., and

Akikazu A.: Isolation and characterization of phenolcatabolizing bacteria from a coking

plant. Biosci. Biotechnol. Biochem., 67, 2026-2029 (2003).

Wang, Ya-qiong, Bin Gu and Wen-lin Xu: Electro-catalytic degradation of phenol on

several metal-oxide anodes. J. Hazardous Material, 162, 1159-1164 (2009).

Wantanabe K, Hino S, Takahashi N (1996). Effect of exogenous phenol-degrading

bacteria on performance and ecosystem of activated sludge. J. Ferment. Bioeng. 82: 291-

298.

Wantanabe KS, Yamamoto S, Hino S (1998). Population dynamics of phenol-degrading

bacteria in activated sludge determined by gyrBtargeted quantitative PCR. Appl. Environ.

Microbiol. 64: 1203-1209.

vii

Whiteley A, Mark J (2000). Bacterial Community structure and physiological state within

an industrial phenol bioremediation system. Appl. Environ. Microbiol. 66: 2401-2407.

Whiteley AS, Wiles S, Lilley K, Philip J, Babailey MJ (2001). Ecological and

physiological analyses of Pseudomonad species within a phenol remediation system, J.

Microbiol. Methods, 44: 79-88.

Williams P.A and Sayers J.R (1994). The evolution of pathways for aromatic

hydrocarbon oxidation in Pseudomonas. Biodegradation, 5: 195-217.

Xiangchun Q, Hanchang S, Yongming Z (2004). Biodegradation of 2, 4-dichlorophenol

and phenol in airlift inner-loop bioreactor immobilized with Achromobacter sp. Sep.

Purif. Technol. 34: 97-103

Yang, R.D. and Humphrey A.E.: Dynamic and steady state studies of phenol

biodegradation in pure and mixed cultures. Biotechnol. Bioeng., 17, 1211-1235 (1975).

viii