INTRODUCTION

1. INTRODUCTION

Life is a delicate phenon~enon on earth, which is supported by

nature. Anything, which impairs nature, is harmful to life too. One of the

most serious problems affecting the modern world is the polluted

environment. Increasing numbe of industries force us to face the

accumulation of waste and toxic materials in the environment. Since this

questions the survival of life itself, degradation studies are of utmost

importance.

The different chemicals discharged by the industries to the

environment can upset the delicate balance of the ecosystem. Minute

quantities of these compounds *entering into the living organisms may

accumulate in the various levels of food chain. Studies about the

degradation and transformation of such compounds and the proper

processing of effluents containing them are essential for a safe and clean

environment. Rachel Carson's "Silent Spring" was a landmark in the public

awareness of potential damage to human and environmental health from

toxic substances. This awareness could arise a main concern about

pollution among the mankind.

The term biodegradatisn has been used to describe

transformations of every type including those that yield products more

complex than the starting material as well as those responsible for the

complete oxidation of organic ccmpounds to CO,, H20, NO3 and other

inorganic compounds (Atlas and Bartha, 1998). Bioremediation refers to

the application of biodegradation reactions to the practical clean up of

compound. The term mineralization has been proposed for describing the

ultimate degradation and recycling of an organic molecule to its mineral

constituents. The underlying prc~cess of bioremediation is the natural

process of biodegradation, which can reduce the concentration of

pollutants and sometimes can completely oxidize the compound.

Microorganisms are especially useful for biodegradation and bioremediation

because of their great metabolic diversity which includes the ability to

metabolise these pollutants. Biodegradation is often considered as safe

and cost effective means of restoriig environmental quality.

Microbiologists have hardl) dipped below the surface of the natural

pool of microbial diversity. When iew organisms have been isolated with

biodegradation efficiency, their bi~lchemical versatility has been found to

be immense. Attempts to det,?rmine microbial diversity in natural

environments are limited by the inability of the microbiologists to culture all

microbes present in a particular er~vironmental sample. Whilst the extent of

this diversity can only be a matter of conjecture at this time, our present

understanding of the biochemical versatility of microbes leads one to

suggest that for any given polltrtant there will be a natural organism

capable of metabolizing it. Howewr, the isolation of that microbe will often

require a targeted intelligent approach to screen the biosphere for its

presence (Wackette and Hershb~rger, 2001). It is from this point that we

have started the present work tcl isolate, screen, select and identify an

organism from soil for the biodegradation and bioremediation of phenol.

1 .l. BIODEGRADATION

Biodegradation and biocatalysis are as old as life and was initiated

3.6 billion years ago. Virtually all theories of early evolution confirm a

prebiotic soup of organic molecules that sewed as precursors for the

molecules that constituted the first life. Self replication requires energy

which is likely to derive from prefcrmed molecules in the prebiotic soup. By

definition, a thermodynamically favourable chemical reaction involves the

transformation of one or more m~~lecules into a thermodynamically stable

molecule.

Introduction 3

Biodegradation of materials involve initial proximity, allowing

adsorption or physical access to the substrate, secretion of extracellular

enzymes to degrade the substrates or uptake via transport systems

followed by intracellular metaboli:;m. The efficiency of biodegradation of

organic compounds is influenced by the type of the organic pollutant, the

nature of the organism, the enzyme involved, the mechanism of

degradation and the nature of the influencing factors.

1.1.1. Organic pollutants

Major contaminants of the ecosystem are the chemicals used in

day today activities and the chemicals produced as essential byproducts of

industries. Xenobiotics are chemical compounds synthesized by humans,

which are not naturally found in living organisms and hence cannot be

metabolized by the organisms. These compounds resist biodegradation or

are incompletely metabolized vtith the result that some xenobiotic

compounds accumulate in the erivironment as recalcitrant which affects

three main portions of the biosph?re viz, air, water and soil. Our present

knowledge of the fate of organic compound in nature is incomplete as it is

extremely difficult to predict the biological impact of chemicals on the

environment. The major xencbiotic compounds include haloalkyl

propellants, halobenzenes, halophenols, halobenzoates, polychlorinated

biphenyls, dioxins, synthetic polymers, alkyl benzene sulfonates,

chlorinated pesticides, chlorinated phenols etc. The chemical structure of

organic pollutants has a protound influence on the abilities of

microorganisms to metabolize them. Some organic compounds are readily

degradable and others are recalcitrant. Low molecular weight

hydrocarbons and alcohols are representative of readily degradable

chemicals. Branched and polynuclear compounds are more difficult to

degrade than simple mono aromatic compounds. Increase in the degree of

halogenation decreases the rate o: biodegradation(At1as and Bartha,1998).

There are many reasons for an organic compound to be recalcitrant.

These are unusual bonds or bond sequences, unusual substitutions like

halogenations, highly condensed ;~romatic ring, excessive molecular size,

failure of a compound to induce the synthesis of degradative enzymes,

failure of a compound to enter into a microbial cell, unavailability of a

compound due to its insolubility and toxicity of a parent compound or its

metabolic products.

A priority list of organic polluyants was constructed by EPA (1979). In

the present study phenol was selected for the biodegradation studies,

which is a member of the priority list and is a common pollutant in the

wastewater of many industries SUCI as resin, oil, pesticide, paper and pulp

industries (Borghei and Hosseini, 2004).

1 .I .2. Microorganisms in biodegradation

Microorganisms reproduce and thus evolve new diversity faster

than macroscopic organisms. Repi.oduction involves constant cell division

and hence eating. Eating in this context is largely biodegradation which

ends when there is nothing more in the local environment to degrade

(Lazcano and Miller. 1996).

Alexander (1 965) formalized the general understanding of the bio-

degradative capacity of microorganism as the principle of microbial

infallibility expressing the empirical observation that no natural organic

compound is totally resistant to biodegradation provided that the

environmental conditions are favou*able.

It is widely acknowledged that more than 99% of the bacteria in a

single soil or sediment sample habe yet to be isolated and characterized.

Enrichment culture is usually adopted as a method to isolate pure culture

for the biodegradation of a particular pollutant. Bacterial flora of the world

can be considered as a supraorganism that metabolises individually and

collectively shares biodegradative genes and evolves collectively to

biodegrade new compounds that enter into environmental niche (Sonea

and Panisset, 1983).

Pseudomonas sp is generally considered as a potent

biodegrading group with brcad capacity to metabolise exotic

compounds. The compounds m~btabolized by Pseudomonas sp include

aliphatic hydrocarbons, aliphatic and aromatic acids, aliphatic and

aromatic compounds with chloro, cyano, nitro and phosphoryl

group substitutions and cyclcl aliphatic compounds. The genus

with the second largest set of substrate metabolized is Arthrobacter

(Parathion, Phenanthrene). The others include Rhodococcus

(Styrene), Flavobacterium (pentac:hlorophenol), Clostridium sp (Phenol),

E. coli (Carbazole), Sphingcmonas sp(Xylene), Alcaligenes sp

(2,4-Dichlorophenoxyaceticacid), Acinetobacter sp (Cyclohexanol),

Thiobacillus (Thiocyanate), Aercmmonas (phenanthrene). Fungi are also

predominant in biodegradation pr.>cesses. They are particularly important

because of their ability to catalyze? novel hydroxylation reactions. Fungi of

the genera Mucor (Hydrocarbon), Fusarium (2- Nitropropane), Rhizopus

(Steroid), Trichoderma (cellulose) Aspergillus ( 2- amino benzoate), were

observed to have substantially higher activity than many of the others

(Wackette and Hershberger, 2001).

1.1.3. Enzymes in biodegradation

Enzymes are extremely versatile catalysts that meet the needs of a

modern world filled with an enornlous diversity of organic structures. Most

of the microbial enzymes of interest in biodegradation fall under the broad

heading of catabolic enzymes. Unlike the usual enzymes catabolic

enzymes generally show broad substrate specificity. Naphthalene

dioxygenase from Pseudomonas sp can catalyze the degradation of at least

72 substrates. They can catalyze substrate dioxygenation, monoxygenation,

and desaturation. These are all oxidation reactions and require the generation

of an activated oxygen species (Resnick et a1 1996). It is typical of a large

class of aromatic hydrocarbon dioxygenases. The other examples are

methane monoxygenase and xylene monoxygenase. Methane

monoxygenase is estimated to react over 100 different substrates

(Lipscomb, 1994). The other major enzymes involved in microbial degradation

are camphor 5-monoxygenase, nitric acid reductase, phenol

hydroxylase, polyphenol oxidase, peroxidase, tyrosinase(oxidoreductases);

dichloromethane dehalogenase(tr,msferases); atrazine chlorohydrolase,

carboxyl esterases(hydrolases); mandelate recemase(isomerase);

aminobenzoate ligase ( ligase) ( Babbit and Gerlt , 1997).

1.1.4. Mechanism of biodegradation and influencing factors

The aliphatic and aromatic: hydrocarbons are biodegradable by a

range of bacteria and fungi af!robically or anaerobically. In aerobic

biodegradation, molecular O2 activate the molecules via oxygenation

reactions. Evidence of anaerobic biodegradation of aromatic hydrocarbons

is also growing. Anaerobic biodegradation rates are slower than aerobic

rates. Most halogenated aliph3tic compounds can be reductively

dehalogenated to less halogenated species. Lightly halogenated aromatics

can be aerobically biodegraded via initial oxygenation reaction.

In aerobic biodegradation of aliphatic compounds usually the initial

attack occurs by enzymes that have a strict requirement for molecular

oxygen i.e monoxygenase or dioxygenase (Atlas and Bartha, 1998).

In the first case one atorr~ as O2 molecule is incorporated to the

alkanes.

In the second case, both atoms of O2 are transferred to the alkane

yielding a labile hydroperoxide intermediate that is subsequently reduced

by NADPH2 to an alcohol and H2Cl.

Once a fatty acid is formed further catabolism occurs by

O-oxidation.

Aromatic compounds are the most common organic compounds

found in nature which are generally very easily 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. On the contrary, most of the alkyl

substituted aromatic compouncs such as isomeric xylenes, cresols,

xylenols etc have been shown to be amenable to microbial degradation.

The critical step in the mt?tabolism of aromatic compounds are the

destruction of the resonance structure by hydroxylation and fission of the

benzoid ring which is achieved t ~ y 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 cat;%bolic pathways converge at catechol.

Catechols are formed as intermejiates from a vast range of substituted

and non-substituted mono and poly aromatic compounds. The initial

reaction of the aromatic compound is catalysed by dioxygenases leading

to the formation of cis-dihydrodiol:; which are then converted to catechols

by the action of a dihydrodiol debydrogenase. This reaction involving the

molecular oxygen occurs mainly in prokaryotes. These dioxygenases are

highly labile enzymes which lack a detailed investigation.

In eukaryotic organisms, t t~e initial reaction is catalysed by a mono

oxygenase forming trans-diol intermediates which derive one hydroxyl

group (OH) from water.

Catechols are cleaved eitt~er by ortho-fission (intradiol i.e., carbon

bond between two hydroxyl groups or by a meta-fission (extra diol i.e.,

between one of the hydroxyl groJps and a non-hydroxylated carbon) as

given in Fig.1. Thus the ring is 3pened and the open ring is degraded

(Cerniglia, 1984).

Fig.1 Ortho and Meta Pathway tor the Degradation of Aromatic Compounds

META-PATHWAY ORTHO-PATHWAY

2 - Hydro*) n~uconic ' COOH coyH Semialdehyde , COOH CHO Cis-Cis C Muconic acid

Oxoprnt - 4 - m ,ae i.

HO,CH, c=o I I H20 COOH Mucunolaclone

HOHC/~~'C=O Oxalo ace tale I I

CH? COOH

1 PYRUVA7E ACETADEHYDE

As a general rule, most of the halo aromatics are degraded through

the formation of the respective ialocatechols, the ring fission of which

takes place via ortho-mode. Cln the other hand, most of the non

halogenated aromatic compounds degraded through meta pathway.

The fission product of or:ho-cleavage would be cis, cis muconic

acid or its derivative depending ~n whether the catehol is substituted or

not. The meta-fission product of catechol would be 2-hydroxy muconic

semialdehyde and the products 3f 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.

The mechanism of degraclation 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, nature of the

organism, type of the enzyme ard also by the external factors affecting

biodegradation. In some cases, through the action of monooxygenase,

aromatic compounds may be corverted into gentisic acid. The fission of

this compound occurs between ,:he hydroxyl and carboxyl groups, i.e.,

meta fission .It has been shovrn in some cases the chloroaromatic

compounds such as 4-chlorobenzoate, 4-chlorophenol and others may get

dechlorinated during the hydroqrlation resulting in the formation of 4-

hydroxy benzoates (4-HBA). This 4 HBA on further hydroxylation will be

converted to protocatechuate acicl(3,4-dihydroxy benzoic acid) which may

be cleaved either through ortho or meta mode.

Several external factors can limit the rate of biodegradation of

organic compounds. These factors may include temperature, pH, oxygen

content and availability, substrate concentration and physical properties of

contaminants. 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 is particularly

important since it inhibits the growth of the organism at higher

concentrations.

1.1.5. Analytical techniques used in biodegradation studies

The monitoring of biodr?gradation and the analysis of the

biodegradation products can be performed by the application of many

analytical techniques. The techniclues include Gas chromatography (GC),

Mass spectroscopy (MS), High Performance Liquid Chromatography

(HPLC) and Fourier Transform Infrared spectroscopy (FT-IR).

1 .I .5.1. Gas Chromatography( GC: )

Gas chromatography is ; 3 versatile tool for the analysis of the

biodegradation products. Separation of the products is effected by the

distribution of components between two phases, which may be either gas

liquid or gas-solid. GC can analyze any component that is volatile or that

can be derivatized to a volatle substance. The retention times of

compounds on particular columns at specified temperature and gas flow

are characteristics of the compounds .The separation of the biomolecules

is influenced by the temperature of the column, which may be constant

during the analysis of the sample

1 .I .5.2. Mass spectroscopy (MS)

Mass spectroscopy is usually used in association with gas

chromatography for biodegradation studies. Mass spectroscopy helps in

the analysis of the each product of biodegradation. It is the most accurate

method for determining the molecular mass of a compound and its

elemental composition. When v3pour of a compound in high vacuum is

bombarded with a moving beam of high energy electrons, ionization of the

molecule takes place where one of the valence electrons of the molecule is

knocked out resulting in the formation of a radical cation which is

designated as M' cation. Each kind of ion has a particular mass to charge

ratio (mlz) value. All positive ions are accelerated in an electric field and

then passed through a magnetic field where they get separated giving rise

12 Chapter I

to a signal for each m/z value. The largest peak is called the base peak

and the intensities of other peaks are expressed relative to it. The particle

of highest mass usually correspontjs to the molecular mass of the original

molecule.

1 . I .5.3. High performance Liquid Chromatography (HPLC)

High performance liquid chromatography is useful for both

analytical and preparative work .Ir contrast to GC, HPLC analysis are not

limited by volatility and thermal stability of the compounds. HPLC is suited

for routine analysis. It can be usec for the rapid separation of labile natural

compounds and high molecular weight compounds. HPLC operates at a

high pressure forcing the mobile phase through the specific column at a

high flow rate to give rapid separation with reduced band broadening due

to small particle size. The speed sensitivity and versatility of HPLC makes

it suitable for the separation of many small molecules of biological interest.

1 .I .5.4. Fourier Transform-Infrared Spectroscopy (FT-IR)

Radiation from IR regions is not energetic enough to cause electronic

excitation, but result in the stretching and bending of bonds joining various

atoms. This results in the formaticn of rich array of absorption bands in the

IR spectrum. IR spectra are comn~only described in terms of frequencies of

radiation absorbed rather than their wavelength. The most common unit

used to describe IR spectra is the wave number which represents the

number of vibrations of radiations per centimeter (cm-'). IR spectrum really

helps in the detection of the fundional group in a compound and hence

the nature of the compound. The accuracy of IR spectroscopy is greatly

enhanced by Fourier Transformation (FT), named after the mathematician

J.B. Fourier. Fourier transform is a procedure for interconverting frequency

functions and time or distance functions. In FT-IR information is obtained

from an interferometer which s~~ l i t s the incident beam so that it passes

through both the sample and reference. When the beam is recombined

interference patterns arise because the two path lengths are different. The

interference pattern has the same relationship to normal spectrum and

integral computers use FT to convert the pattern into a spectrum.

In the present study on b~odegradation, gas chromatography (GC)

has been used to separate and analyse the products of biodegradation of

phenol. Mass spectroscopy (MS) has been used along with the GC to

monitor the molecular weight of the products and Fourier Transform

Infrared spectroscopy (FTIR) h3s been used to confirm the chemical

nature of the products.

1.1.6. Biological treatment of wastewater

Liquid wastes are produced everyday by human activities and also

by various agricultural and illdustrial operations. The liquid waste

discharges from sewage drain into surface water, such as rivers lakes and

oceans. Slowly but gradually they percolate into the ground water table.

People use this same water as an alternative source for drinking,

household, industrial irrigation, fish and shellfish production and for

swimming and other recreational activities. Therefore it is crucial to

maintain the quality of these natural waters to the best of our ability.

In the contemporary liqilid waste treatment, the usual criteria of

operations are the reduction in chemical oxygen demand (COD) and

biological oxygen demand (130D) associated with the wastewater.

Chemical oxygen demand is the amount of oxygen consumed for the

chemical oxidation of the wastewater and biological oxygen demand is a

measure of oxygen consumpti3n required for the microbial oxidation of

readily degradable organics and ammonia in sewage. The different stages

in the treatment of ~ndustrial wastewater are referred to as primary,

secondary and tertiary treatmerts.

Primary sewage treatmenl removes only suspended solids. This

removal is achieved in settling tanlts or basins, where the solids are drawn

off from the bottom. In a secondary sewage treatment, a smaller portion of

the dissolved organic matter is mineralised and a larger portion is

converted from a dissolved state ts removable solids. The combination of

primary and secondary treatment:; usually reduces the original COD and

BOD of the sewage by 80-90%. The secondary sewage treatment step

relies on microbial activity. The trsatment may be aerobic and anaerobic

and may be conducted in a large variety of devices.

1 .I .6.1. Trickling Filter System

A simple and relatively inexpensive film-flow-type aerobic sewage

treatment installation is the trickling filter. A boom-type sprinkler distributes

the sewage over a bed of porous naterial. It slowly percolates through the

porous bed and the effluent is c:ollected at the bottom. Dense, slimy

bacterial growth coats the po ro~s material of the filter bed. Zooglea

ramigera and similar bacteria play a principal role in generating the slime

matrix, which accommodates a heterogeneous microbial community

including bacteria, fungi, protozc~a, nematodes and rotifers (Atlas and

Bartha, 1998).

1 .I .6.2. Rotating Biological Contactor

Closely spaced discs, usually manufactured from plastic material

are rotated in a trough containing the sewage effluent. The partially

submerged discs become coated with a microbial slime. Biodisc systems

require less space than trickling filters, are more efficient and stable in

operation and produce no aerosols, but they require a higher initial

investment.

1.1.6.3. Submerged Aerobic Filter!;

In submerged or flooded aerobic filters, effluent flows down through

a bed of small natural or plastic media and air is injected into the base of

the bed. This allows the wastewater to be applied at a much higher rate

than conventional filters. There may be a second filtration stage below the

injection point to remove solids generated in the upper aerobic mixed

zone.

1 .I .6.4. Fluidized bed reactors

In fluidized bed reactors the support materials are fluidized by

pumping wastewater through ths medium or by injecting air or oxygen

suspended cell sewage treatment systems.

1 .I .6.5. Oxidation Lagoons

Oxidation ditches and 1;igoons are low cost treatment systems

within which microorganisms glow as suspended particles in the water

column rather than as biofilms. Oxidation lagoons tend to be inefficient and

require large holding capabilities and long retention times. As oxygenation

is usually achieved by diffusion .dnd by the photosynthetic activity of algae,

these systems need to be shallow and oxygenation is usually incomplete,

with consequent odour problen~s. Performance is strongly influenced by

seasonal temperature fluctuatims and usefulness, therefore is largely

restricted to warmer climates.

1 .I .6.6. Activated Sludge

A popular suspended growth type of liquid waste treatment system

is the activated sludge proc?ss. After primary settling, the sewage,

containing dissolved organic (:ompounds is introduced into an aeration

tank. Air injection andlor mechanical stirring provide aeration. Microbial

activity is maintained at high levels by reintroduction of most of the settled

activated sludge. During the holding period in the aeration tank, vigorous

development of heterotrophic microorganisms takes place.

1 .I .6.7. Anaerobic Digesters

Anaerobic wastewater treatment methods are generally slower but

save energy compared to processes requiring forced aeration. Some

anaerobic treatment systems can also salvage a part of the chemical

energy content of the wastewater by generating biogas, a useful fuel.

Conventional anaerobic digesters are large fermentation tanks designed

for continuous operation under anaerobic conditions. Provisions for

mechanical mixing, heating, gas collection; sludge addition and draw off of

stabilized sludge are also inccarporated. The digester contains high

amounts of suspended organic matter and a considerable part of this

suspended material is bacterial biomass. Fungi and protozoa are present

in low numbers only and are no1 considered to play a significant role in

anaerobic digestion.

1 .I .6.8. Tertiary Treatments

Tertiary liquid waste tre.atments are aimed at the removal of

nonbiodegradable organic pollutants and mineral nutrients. The removal of

nonbiodegradable organic pollutanrs, such as chlorophonols. Polychlorinated

biphenyls and other synthetic pollutants is necessary because of the

potential toxicity of these compo~lnds. Activated carbon filters are used to

remove such materials from seco~idary treated industrial effluents.

1 . I .6.9. Disinfection

The final step in sewage treatment is disinfection, designed to kill

enteropathogenic bacteria or virlrses that were not eliminated during the

previous steps of sewage treatment. Disinfection is commonly

accomplished by chlorination, using either chlorine gas (CI,) or

hypochlorite. Chlorine gas and hypochlorite react with water to give

hypochlorous acid. The hypochlorite (CIO-) ion is the actual disinfectant.

Hypochlorite is a strong oxidant, which is the basis of antibacterial action.

A disadvantage of disinfection by chlorination is that more resistant types

of organic molecules, including some lipids and hydrocarbons are not

oxidised completely but become instead partially chlorinated. Chlorinated

hydrocarbons tend to be toxic and are difficult to mineralise. However,

alternative means of disinfection are more expensive, so chlorination

remains the principal means of sewage disinfection.

1.1.7. Biodegradation of phenol

1 . I .7.1. Phenol as a pollutant

Phenol and its higher horr~ology are aromatic molecules containing

hydroxyl, methyl, amide or sulphmic groups attached to the benzoic ring

culture. The origin of phenol in the environment is both industrial and

natural. Natural sources include 'orest fire, natural run off from urban area

where asphalt is used as the binding material and natural decay of

lignocellulosic material. Phenol rnay also be accumulated from industrial

waste derived from fossil fuel extraction and beneficiation process,

chemical manufacturing process such as phenol manufacturing plants,

pharmaceutical, wood and pestic~de industries.

1 . I .7.2. Chemistry of phenol

Synonyms: Carbolic acid, Hydroxybenzene, phenic

monohydtoxybenzene, phenic acid, phenylic acid,

phenyl hydroxide, oxybenzene, benzenol,

monophe,iol, phenyl hydrate, phenylic alcohol,

Baker's P and S, phenol alcohol.

Chemical formula: C6 H6O

Phenols contain an OH group attached directly to an

aromatic ring.

Properties: They may be colorless solids or thick liquids, often

contains a pink tint owing to the presence of

oxidation ~roducts. Phenol is a hygroscopic,

crystalline sdid with distinctive odor and is acidic.

Molecular weight of phenol is 94.11, the density is

1.072 and tbe boiling point is 181 .g°C.

1 .I .7.3. Toxicity of phenol

Acute exposure of phenol causes central nervous system

disorders. It leads to collapse an3 coma. Muscular convulsions are also

noted. A reduction in body temp~rature is resulted and this is known as

hypothermia. Mucus membrane is highly sensitive to the action of phenol.

Muscle weakness and tremors are observed. Acute exposure of phenol

can result in myocardial depression also. Phenol causes a burning effect

on skin. Whitening and erosion c4 the skin may also be resulted due to

phenol exposure. Phenol has an anaesthetic effect and causes gangrene

also. Renal damage and saliv;%tion may be induced by continuous

exposure to phenol.

Exposure to phenol may rt?sult in irritation of the eye, conjunctional

swelling, corneal whitening and finally blindness. Other effects include frothing

from nose and mouth followed b l headache. Phenol can cause hepatic

damage also. Chronic exposure may result in anorexia, dermal rash,

dysphasia, gastrointestinal disturbalice, 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 is an antiseptic agent and is used in surgery, which indicate

that they are also toxic to many microorganisms. (EPA, 1979)

1.1.7.4. Microorganisms in the bicdegradation of phenol

Degradation of phenol oc3urs as a result of the activity of a large

number of microorganisms inclllding bacteria, fungi and actinomycetes.

These include Bacillus sp, Pseudomonas sp, Acinetobacter sp,

Achromobacter sp etc. Fusarivm sp, Phanerocheate chrysosporium,

Corious versicolor, Ralstonia sp Streptomyces sp etc are also proved to

be efficient fungal groups in phenol biodegradation. However, these

microorganisms suffer from substrate inhibition at higher concentration of

phenol, whereby the growth is inhibited (Prieto et a/., 2002). Measures

have been taken to improve such limitations resulting in the advancement

of novel biodegradation process

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 resultetl 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 a1 , 2000 a and b). Phenol and other phenolic

compounds are common constituents of many industrial effluents. Once a

suitable microorganism based process is developed for the effective

degradation of phenol these phenolic effluents can be safely treated and

disposed. (Borghei and Hosseini, 2004)

1 .I .7.5. Enzymes in the biodegradation of phenol

Microbial degradation of phenol takes place through different

pathways which involves various enzymes. The important enzymes in

phenol biodegradation include oxygenases, peroxidases and phenol

hydroxylases.

Oxygenases include monoxygenases and dioxygenases. Polyphenol

oxidase is a (EC 1.14.18.1) monoxygt?nase which catalyses the 0-hydroxylation

of phenols and the oxidation of 0-dihydric phenols to 0-quinones using

molecular oxygen. Laccases are phenol oxidases which utilize molecular

oxygen. They are known to have the ability to oxidize polyphenols,

methoxy substituted phenols, dian~ines and a variety of other components.

(Kadhim,1999). The mechanism >y which polyphenol oxidase catalyses

the conversion of monophenols to 0-quinones involves the hydroxylation

of monophenols followed by dehydrogenation to form 0-quinones. These

quinones undergo spontaneous nonenzymatic polymerization in water,

eventually forming water insoluble polymers which can be separated from

water by filtration (Edwards eta/., 1999).

Peroxidases can act on >hen01 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 polymer:.

Phenol hydroxylase (E. C 14. 1.3.7) catalyses the degradation of

phenol via two different pathways initiated either by ortho or meta

cleavage. Aerobically, phenol is first converted to catechol, a reaction

which is catalyzed by a monoxygenase. Subsequently, the catechol is

degraded via an 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 Bhydro cis muconic semi aldehyde for the latter

(Gurujeyalakshmi and Oriel, 1988).

1.1.8. Biological treatment of phenolic wastewater

Phenol and other phen01'~: compounds are common constituents of

many industrial effluents from cht:mical operations such as polymeric resin

production, oil refining, pulp ant1 paper manufacturing etc (Borghei and

Hasseini, 2004). Phenolic compourKfs are hazardous pdlutants that are toxic at

relatively low cmcentration. Accunlulation of such chemicals, which could not

be assimilated creates toxicity botfi for Nora and fauna. Even at extremely low

concentrations, phenol imparts objectionable taste and odor to drinking water.

So wastewater containing phenols and other toxic compwnds need careful

treatment before discharging intc the receiving wateWies (Santos and

Linardi, 2003). The techniques used for treatment can be physico chemical or

biological.

1.1.8.1. Physico-chemical methcds

Physical treatment is malnly done through adsorption with charcoal,

chitosan etc, while the chemical processes include oxidation reactions

using ozone, ozoneluv, ozoncJH20,, photolysis, heterogeneous photo

catalysis using HeOd uv, wet z,ir oxidation, super critical water oxidation

and electro chemical oxidation (Annadurai eta/., 2000 a and b).

1.1.8.2. Biological methods

Objective of any biological treatment is to remove soluble or

colloidal or suspended biologically transformable organics in the

wastewater. This is achieved by bringing active microorganisms in contact

with the wastewater. Biological treatment may be aerobic or anaerobic.

It may also be suspended or attached process.

A variety of techniques have been used for the clean up of phenol

contaminated waters and soils. Biodegradation is given the most attention

due to its environmental friendly approach and its ability to mineralize toxic

organic compounds (Prpich and Dauglis, 2005). The phenolic wastewater

may be treated with many biological methods. Free cells as well as

immobilized cells were reported to be very effective in phenolic waste

removal (Torres et al., 1998). Enzymes immobilized in capillary bioreactors

can also be used for the treatment of phenolic wastewater. Many reports,

such as application of fluidized bed reactor (Gonzalez et a/., 2001), moving

bed biofilm reactor (Hosseini anc Borghei, 2005) and rotating perforated

tube biofilm reactor (Kargi and Eker, 2005) suggested the use of microbial

cells in the treatment of phenolic effluents.

1.2. REVIEW OF LITERATURE

Microbial transformations are reactions of organic compounds

catalyzed by microorganisms. The use of microbial catalysts in the

biodegradation of organic compounds has advanced significantly during the

past three decades. It has been found that large numbers of microbes

co-exist in almost all natural environments, particularly in soils, waters and

sewage. Many natural and synthetic organic chemicals are readily

biodegradable in natural environment. The intensity of biodegradation is

influenced by several factors such as nutrients, oxygen, pH, composition,

concentration and bioavailability of the contaminants, chemical and physical

characteristics and the pollution history of the contaminated environment

(Schinner and Margesin, 2001).

1.2.1 Microbial degradation of organic compounds

The presence of organic c:ompounds in the environment creates

serious ecological and health problems through the pollution of ground and

surface water. Such compounds present problems in wastewater treatment

plants also. In order to overcome these adverse effects scientists have

developed many biodegradation processes mediated by microorganisms

and their enzymes. During the n~etabolism of aromatic compounds the

microorganisms use molecular oxygen to hydroxylate aromatic compounds

and to perform oxidative cleavage of the aromatic ring. During the

hydroxylation and cleavage of aromatic compounds some microorganisms

produce enzymes called monoxygenases or dioxygenases that transform

aromatic compounds into variety of products (Cinar, 2004). A better

understanding of the degradative pathways and yields of these organic

compounds is needed especially with respect to waste disposal problems.

Jenisch-Anton et a/., (1999) worked on the biodegradation of high

molecular weight aliphatic ether using a culture of Rhodococcus ruber.

A detailed investigation of this biodegradation revealed that the degradative

pathway was unusual and it involves mid chain oxidation mechanism

resulting in the formation of alkenes. Heinfling et a/., (1997) reported that

Trametes versicolor and Bjerkandera adusta could degrade azo and

phthalocyanine dyes. During the degradation of these dyes by the above

organism, the toxicity of the dye was significantly reduced. Specific activities

of the exoenzyme preparation with the dyes were determined and compared

to oxidation rates by commercial horse radish peroxidase.

Biodegradation of polychlorinated biphenyls by Arthrobacter sp was

reported by Gilbert and Crowley (1998). Similarly biodegradation of

nitrobenzene to aniline by Commamonas acidovorans (Peres et a/., 1998)

and biodegradation of toluene t ~ y Pseudomonas putida in trickling filter

(Peixoto and Mota, 1998) were also being attempted.

An efficient microbial consortium was developed and was found to

be promising in the biodegradation of atrazine (Gouz et a/., 2000). In

another study using a microbial c~3nsortium Manson eta/., (2000) observed

that they could effectively degrade a mixture of benzene, toluene, ethyl

benzene and the mixed xylenes (BTEX). The study was carried out in a

continuously fed, completely mixed bioreactor in the presence of activated

carbon. The bacteria on the activated carbon surface were constantly in a

flux between adsorbent and free phase.

Perei et a/., (2001) reported that the sulfanilic acid could be

biodegraded by Pseudomonas ~aucimobilis. The isolate could grow on

sulfanilic acid as its sole carbon and nitrogen source and metabolized the

target compound to biomass. Cinar (2004) reported the anaerobic

degradation of catechol with Dt?sulfatomaculum. Table 1 gives a list of

various organic compounds and the micro organisms capable of

degrading them.

Table 1. Microorganisms in the biodegradation of organic compounds

1.2.2 Microorganisms in the biodegradation of phenol

Reports on the biodegradation of aromatic compounds have been

documented from the beginning of 20% century. Microorganisms that

degrade phenol were isolated ;is early as 1908 (Evans, 1947). The

microbiological treatment of industrial effluents depends on the oxidative

activities of the microorganisms. 4s the industrial effluents usually contain

mixtures of other organic compour~ds, problems arise due to incompatibility

and toxic effects. Effluents containing higher concentration of phenol could

be growth inhibitory even to organisms capable of using them as a substrate

for growth (Evans, 1963). However, scientists could isolate many

microorganisms degrading organic: compounds. A number of bacteria and

fungi are capable of degrading phenol and other compounds (Table 2).

Phenol and benzoic acids were converted to non-recalcitrant catechol

by some soil bacteria (Evans, 1947). Phenol degrading Themophilic Bacillus

sp converted catechol to hydroxyrnuconic semi aldehyde by metacleavage

routes (Cemiglia, 1984). Phenol degrading activity was studied in 41 fungal

species comprising a total of 77 strains isolated from seawater in South Asiatic

region and fresh water of the lake, Skader.

Bacillus stearothermophilusBR219 degraded phenol at levels of

15 mM (Gurujayalakshmi and Oriel 1988) .It was a river sediment isolate and

showed growth inhibition at higher concentrations of phenol. Bollag et a/.,

reported the detoxification of phenol by the fungus Rhizoctonia praticola

(1 988). Fresh extracts of Agaricus bisporous was used to hydroxylate and

oxidize a range of phenolic subslrates (Burton et a/., 1993). Kennes and

Lema(1994) suggested the use of Phanerocheate chrysosporium for

bioremediation and the fungus c:ould remove phenol and other related

compounds from samples. Psetrdomonas putida (ATCC 17484) could

degrade phenol at lower concentrations, but higher concentrations of phenol

inhibited the growth of the bacterium (Allsop et a/., 1993). 500 ppm of

phenol could be utilized by Dseuodomonas putida MTCC 1194

(Bandopadhyay et al., 1993).

An aerobic mixed culture degrading phenol was developed and

maintained in a fed batch reactor feeding phenol 500 mg/l/day (Ambujam

and Manilal, 1995). Phenol decomposition by immobilized membranes was

reported by Bodzek et a/., (1996) Immobilized Bacillus stearothemophilus

cells could oxidise phenol which coulcl be used as a biosensor operating at high

temperature (Rella et al., 1996). Pseudomonas putida DSM 549 and

Trichosporon beigelii were able to utilize phenol as the sole energy and

carbon source. The dynamics of microbial growth was studied here

(Gotz and Reuss, 1997). The alga Ochromonas danica showed the ability

to metabolize mixtures of phenols ltnown to pose an environmental hazard

(Semple and Cain, 1997). Thermol~hilic Bacilli capable of degrading phenol

as the sole carbon source were isolated from sewage effluent.

(Ali et a/., 1998).

Coprinus macrorhizus and Arthromyces ramosus were proved to be

effective in removing phenol and pher~olic compounds from water in the presence

of hydrogen peroxide. The method was successfully applied for the treatment of

real wastewaters (Wu etal., 1998). F henol degradation by Ralstonia eufropha

was modeled in different culture modes to assess phenol feeding in

biotechnological depollution processes. A controlled fed batch approach

was used for phenol decomposition (Leonard and Lindley, 1999). Agaricus

bisporous enzymatic preparations were immobilized to treat phenols in

industrial effluents. This was an efficient bioreactor which could completely

bring about the removal of the coloured quinones and associated polymers

from the permeate (Edwards et a/., 1999). Culture filtrate extracted from

Coriolus versicolor grown on whe.3 bran supplemented with yeast extract

was used to remove a range of phenolic compounds (Kadhim et ab, 1999).

Phenol degradation by Pseudomoqas putida ATCC 49451 in batch cultures

were investigated over a wide range of phenol concentrations (25-800 mg/l).

(Mordocco, et a/., 1999). Under optimum conditions Pseudomonas pictorium

degraded phenol at a concentration of 2911 in 33 h. (Chitra et al., 1995).

Batch and continuous culture sti~dies on Pseudomonas cepacia revealed

the phenol utilization capacity (Ghadhi and Sangodkar, 1995). The

maximum concentration of phenol to be degraded by Alcaligenes strain P5

was 0.29 mM. (Baek etal., 2001).

Phenol biodegradation by immobilized Pseudomonas sp was

reported by Banerjee et al., (2001). Biodegradation of phenol in presence of

heavy metals by Acinetobacter calcoaceticus AH strain was observed

(Nakamura and Sawda, 2000). Removal of Olive mill waste water using

Phanerocheate chrysosporium, Aspergillus niger, Aspergellus terreus and

Geotrichum candidum was founa to be effective in waste water treatment.

The individual removal of phenol compounds from olive mill wastewater by

microorganisms was carried out (Garcia et al., 2000). Hublic and Schinner

(2000) reported the use of Pleurotus ostreatus for the continuous elimination

of phenolic pollutants from aqueous solutions. The treatment in a packed

bed reactor was followed by filtration of the formed precipitate.

Pseudomonas putida Al'CC 11172 could remove phenol in an

external loop inversed fluidized bed airlift bioreactor (Loh and Leu, 2001).

Phenol utilization by Acinetobacter sp was studied by Hao et a/., (2002).

Phenol biodegradation and kinetic models were reported by Abuhamed et a/.,

(2003). Biodegradation of phenol by an Alcaligenes sp d2 was observed up

to 120 mM concentration (Nair and Shashidhar, 2004). Graphium A & 2 and

Fusarium FEI 1 presented 75% phenol degradation when lOmM phenol was

used (Santos and Linardi, 2004). A moving bed biofilm reactor, in the report of

Borghei and Hosseini (2004) could efficiently remove phenol. Pseudomonas

putida ATCC 17484 was used for the biodegradation of phenolic industrial

wastewater in a fluidized bed bioreactor. The experiments were carried out in

batch and continuous modes (Gonzalez etal., 2001).

Biodegradation of phenol by the yeast strain, Rhodotorula gustines

was reported to utilize phenol as the sole carbon source (Katayama etal.,

1994). The psychrotrophic bacteril~m Pseudomonas putida ATCC 49451

could degrade phenol in continuo~~s and batch cultures (Wang and Loh,

1999). Phenol consumption of Acinetobacter johnsonii was reported by

Hoyle et a/., (1995). The eukaryotic alga Ochromonas danica grew on

phenol as the sole carbon source ir axenic culture and removed the phenol

carbon from the growth medium (Sl?mple and Cain, 1996). Some strains of

bacteria from the river sediments were capable of degrading phenol(Paula

and Young, 1998). Rhodococcus rvythropolis UPV-I was able to degrade

phenol in synthetic and industrial wastewater in immobilized conditions

(Prieto et al., 2002). Trichospofix, cutaneum R 57 was used in the

biodegradation of phenol in indusvial waste waters (Godjevargova, 2003).

Kargi and Eker (2005) reported the removal of 2,4 dichlorophenol from

synthetic wastewater in a rotcting perforated tube biofilm reactor.

A microbial consortium was capable of enhanced biodegradation of phenol in a

bioreactor. (Prpich and Dauglis, 20C15). Phenol degradation in a batch reactor

was studied using the pure culture of Bacillus brevis (Arutchelvan et al., 2006).

Table 2: Microorganisms in the biodegradation of phenolic compounds

Microorganisms

Bacillus stearothermophilus Gurujeyalakshmi and O!iel(1988)

Pseudomonas putida Allsop el a/.. (1993)

Agaricus bislorus Bulton el al., (1993)

Okeke el al., (1993)

Ambujam and Manilal (1995)

Phenol Acinetoback r iohnsonii Hovle et al.. H995)

7 2- chdrophenol Pseudomanits putida Overmeyar and Rehm (1995)

1 10 1 Penta, chlorophenol I Lentinula ecbdes I Okeke et a/., (1997) I

8 Phenol

Ochromona.; danica Semple and Cain(1997)

Phomvdium valderianum Shashirekha et al., (1997)

13 Phenol Bacillus so Ali etal.. 119981

1 14 1 Phenol I Rhimtonia ~raticola I Bollaa et al.. (1988) I

9 1 Phenol I ~ s e ~ a s so I Gotz and Reuss119971

Pseudomon as sp

Trametes tngii Gaaillo et aL, (1998)

Pseudomonas putida Loh and Wang (1998)

Torres eta/, (1998)

Phenol Pseudomon?~ putida Mordocco et a/., (1999)

Coriolus versicolor Kadhim etal., (1999)

Bodzek et al., (1996)

Pseudomonas putida

Pseudwnonas putida

Phenol Pseudomas pprctorium

Phenol. Nilrophenol Nocardoides

Phenol Phanerocheate chrysosporium

Phenol Pleumtus a:treatus

Phenol Pseudwnonas putiida

21

Wana and Loh 11999) I Zumrive and Gullac (1999) 1

Phenol

Annadurai et al., ( 2 W ) 1

Ralstonia el rfropha I Leonard et al, (1999)

Garc a el aL. 120001

22

HuMik and Schinner 120001 1

Phenol Coprinus ci! lereus

Loh and Tar (2000) I

Schneider st al., (1999)

30

32

33

34

35

36

37

38

39

40

41

31 1 Pheno I Chalara oar 3doxa I Robles eta!. 120001

Phenol

Phenol

Phenol

Phenol

Phenol

Phenol

BisphendA

Phenol, chlorophenol

Phenol

Phenol

Phenol

Ac~neloDacr ?r CalCOaCehCus Nakamura and Sawada (20001

Streptomycf!~ setonii

Alcaligenes sp

Pseudomonss sp

Pseudomonas putida

Pseudomonas putiia

Copnus chereus

Acinetobact ?r sp

Rhc4xoccus erythmpolis

Trichospom? cutaneum

Termitomyc~?~ albuminosus

An et al., (2001)

Baek etal.. (21331)

Gmzalez el al., (2001)

Loh and Jun (2001)

Petruschka et al., (2001)

Sakurai et al., (2001)

Hao et al., ( 2 W )

Prieto el al., (2002)

Godievargova et a/., (2003)

Johjima etal., (2003)

42 1 2,4 MI^ phend I ~ i e d culture

Mixed Fungi Atagana et al., (2004)

Phenol Pseudomas wfida Hamed et al., (2004)

Quan et d., (20%)

43 1 CMom phenol Psedwmas %Ma I Farigh~an (2003)

Santos and Linardi (2006)

Pseudomonas putida Kargi and Eker(2M)S)

Bacillus brevis Arutchekan etab, (2006)

4. Nonvl ohenol Clavanmis ar~uat~ca Moeder et al.. 12006b

44 1 Chloro ~henol I Achrwnobacter so 1 Xianadun et U.. 12003\

47

1.2.3. Enzymes in the biodegradation of phenol

Phenol ( Alcaligenes sp I Nair and Shashidhar (2W)

Phenol and its derivatives are toxic and classified as hazardous

materials (Zumriye and Gultac, 19!39). These phenolic compounds posses

various degrees of toxicity and their fate in the environment is therefore

important. (Bollag et a/. 1988). Irt recent years, a great deal of research

work has been directed toward the development processes in which

enzymes are used to remove pkenolic contaminants (Ghioureliotis and

Nicell, 1999).

There are reports on many microorganisms capable of degrading

phenol through the action of var~ety of enzymes. These enzymes may

include hydroxylases, peroxidases, tyrosinases and oxidases. 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 en~rironrnental pollutants (Bollag et. al., 1988)

and the team attempted polyphend oxidase mediated detoxification of phenolic

compounds. Production of useful chemicals from lignin (Burton et. a/., 1993) by

polyphenol oxidase was also reportelj.

Certain actinomyces ancl 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

(Fetzner and Lingens, 1994) reported the presence of a phenol degrading

polyphenol oxidase in bacteria.

Garzillo eta/., (1998) reported a polyphenot oxidase from the white

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

The purified enzyme oxidised a number of phenolic compounds.This

multicopper oxidases had a wide range of substrate specificity. Coprinus

macrorhizus and Arthromyces r.mosus were proved to be effective in

removing phenol and phenolic compounds from water (Wu et a/., 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 a/., 1998) The peroxidase catalysed polymerization

process was proved to be very effective in eliminating phenol and a variety

of other aromatic pollutants lrom waste waters (Ghioureliotis and

Nice11,1999). The non specific nature of the polyphenol oxidase was also

discussed by Schneider eta/., (1939).

The mechanism by which polyphenol oxidase catalysed the conversion

of monophenols to o-quinones was explained in detail by Edwards et ab,

(1999). Upon characterizing polyphenol oxidase from Coprinus cinereus

they established that it was of ti8 KD molecular mass and was with an

isoelectric pH 4.

Conversion of monophendr; to o-quinones involves the hydroxylation of

moncjphenols with O2 to yield catechots (Cresolase activity) and the

subsequent dehydrogenation of the catechols with O2 to form o-quinones

(Catechdase activity). The o-quinones undergo spontaneous non enzymatic

polymerization in water eventually +orrning water insoluble polymers which

couM be separated from solution by filtration. lmmobi l i i pdyphend oxidase

on chitosan coated polysulphone capillary membranes were used for improved

phenolic effluent bioremediation. Theq also highlighted the removal of quinones

and other polymerized products using chitosan.

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

laccase from Pleurotus ostreatus. l'he enzyme was purified to homogeneity

and was characterized. It was a monomeric protein with a molecular weight

of 67KD and 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.

Polyphenol oxidases were widely distributed in many plants and fungal

species (Robles et a/., 2000). They suggested the possibility of using a

polyphenol oxidase producing stra.n of the hyphomycete Chalara paradoxa

in the detoxification of olive mill wastewater.

Streptomyces setonii (ATCC 391 16) degraded aromatic compounds

such as phenol or benzoate via an ortho cleavage pathway using catechol

1,2 dioxygenase ( An et al., 2001). Sakurai et a/., (2001) showed that the

peroxidase from Coprinus cinen?us could be used for the removal of

Bisphenol. Polymerization of thc! bisphenol by the enzyme was utilized

here. Manophenols in aqueous solution could also be removed by

peroxidase catalysed oxidation (Xis et a/., 2003).Most of the polyphenol

oxidases reported were isolated f r ~ m fungi. Reports on bacterial polyphenol

oxidases are rare.

Tyrosinase catalyzes the oxidation of phenols involving the formation

of orthoquinones. The mechanisn~ of the enzymatic action of tyrosinase on

various phenols was discussed in detail by Siegbahn (2003).

Enzyme browning was a natural biochemical process in which

specific enzymes catalysed the transformation of phenol into brown or black

polymers. Primary step was thc: enzymatic formation of benzoquinone,

usually a coloured compound ant1 rapidly these quinones get oxidised and

condensed themselves to undergo polymerization. This polymerization is

catalyzed by polyphenol oxidase. (Leonardes eta/., 2005). Montero et a/.,

(2001) characterized the polyl~henol oxidase of prawn. The enzyme

precipitation was carried out by ammonium sulphate fractionation at a

concentration of 40%. Isoelectric focussing of the purified enzyme

showed the bands at pH 5. The enzyme was most active at pH-5 and 8.

Partial purification and <characterization of polyphenol oxidase

from peppermint showed that the optimum pH was 7 and the temperature

was 30°C. Km of the enzyme was 6.25 x 1 0 ' ~ m ~ with catechol and the

enzyme was found to be inhibited by sodium metabisulfite. In a similar

study conducted in medlar fruits (Dineer et a/., 2002) the optimum pH

was 6.5 and the optimum temperature was 35 '~ . Purification of

polyphenol oxidase from coffee beans was carried out by 30% (NH&

SO4 fractionation followed by dialysis and ion exchange chromatogaphy

with DEAE columns. The molec~llar weight of the enzyme was found to

be 64 KD (Goulart et ab, 2003).

Zavarzina eta/., (2004) used polyphenol oxidase of Panus figrinus

for the biotransformaticn of $oil humic acids. It was shown that

purified polyphenol oxidase was capable of both polymerizing and

depolymerizing humic acids in vitro. The direction of transformation

depends on the nature and properties of humic acids. Polyphenol

oxidase was used for the fabrication of sensitive amperometric

biosensors for phenolic compounds.

Leonardes et a/., (2005) sludied the polyphenol oxidase in the

sunflower seed which degraded chlorogenic acid. Chlorogenic acid was a

natural phenol formed by one molctcule of coffee acid and one molecule

of quinic acid. Chlorogenic acicl was a natural biologically active

compound which was widely used as an antioxidant. It was also directly

involved in enzyme browning. Enzymatic degradation of chlorogenic and

coffee acids encouraged studies about potential biotechnological

application of polyphenol oxidase enzyme in the treatment of effluents

containing natural phenols. In an attempt to characterize the polyphenol

oxidase from Victoria grape, (Rapcianu eta/., 2006) the optimum pH was

5.5 and the optimum temperature was 25OC. The polyphenol oxidase

extract showed high pressure stability.

Table 3. Enzymes in the biodegradat ion o f phenolic compounds

SI. No.

Polypheriol Oxidase Burton eta/., (1 993)

Polyphw~ol Oxidase Cano et al(1997)

Phenol chlorophenol Phenol Clxidase Okeke et a/, (1997)

1

5 1 Phenol I Polypheriol oxidase I Shashirekha eta/., (1 997) I

Type of Phenol

6 1 Phenol I Catechol2,3 dioxygenase I Ali eta/., (1998) I

Phenol

Enzyme Reference

Phenol hydroxylase

7

Gunrjeyalakshmi and Oriel (1 988)

; 1 1 Poiypher!ol oxidase Ga~i l l0 et ab, (1 998)

12 1 Phenol I Poiypheriol oxidase I Edwards eta/,, (1999)

Phenol

Pemxidase

10

13 1 Phenol I ~accase I Kadhirn eta/., (1 998) I

Ghioureliitis and Niiell(1998)

1 Laccase 1 Schneider eta/., (1999) 1 1 Phenol

Methoxyphenol Laccase Setti eta[, (1999)

Phenol Laccase Hublik and Schinner (2000)

Laccase

Phenol

Bollag et a/., (1 998)

11

Horse ra(lish peroxkiase

Phenol I Horse ra(lsh peroxkiase I Zahida eta/., (1998)

17

Wu et a/., (1 998)

19

Phenol

20

24 1 Lignophenols I Peroxidase I Xia eta/., (2003) I

18

Phenol

22

Laccase

Phenol I Catechol1,Z dixygenase I An et a/., (2001)

21 1 Phenol I P~lypher~ol oxidase I Steffens (2002)

Bis phenol

Robles eta/., (2000)

Polyphenol oxidase

Phenol

Luke and Burton (2001)

Peroxidase

23 1 Phenol

Sakurai eta/., (2001)

Phenol oxkiase

Tyrosinae I Xiangchun (2003)

Johjima eta/., (2003)

1.2.4 Biological treatment of phenolic wastewater

Phenol pollution is associated with pulp mills, coal mines,

refineries, wood preservation p l a ~ t and various chemical industries as

well as their waste waters (Paula .and Young, 1998). There are a series

of examples in which phenols appear as contaminants in process stream

and their selective removal is required for waste minimization (Sun and

Payne, 1996). The different methods involved in phenolic effluent

treatment can be divided into: Physcio chemical methods and Biological

methods.

1.2.4.1. Physico chemical methods

Physical treatment of phenolic wastewater was mainly done

through adsorption. Activated carbon and chitosan had been reported to

adsorb phenolics from various effluents (Annadurai et ab, 2000 a and b)

The adsorption by chemicals depended on the mode of contact with a

sorbate in the reactor. With the high cost of activated carbon, charcoal

was found to be a better alternative. Charcoal treatment was suitable for

small scale industries.

1.2.4.2. Chemical methods

Chemical processes included oxidation reactions using ozone;

ozoneIUV, ozonelH202, The method was disadvantageous with initial

high cost of ozone. Air was an inexpensive oxidising agent, but the

reaction was very slow. Kadhim t?t a/., (1999) suggested that the same

cannot be adopted because of high cost, incomplete purification and

formation of toxic intermediates.

1.2.4.3. Biological treatment methods

Objective of any biological treatment is to remove soluble or

colloidal or suspended biologically transformable organics in the

wastewater. Of the various waste treatment processes available,

biological treatment methods are attractive because they are able to

degrade wastewater resulting in lower concentrations of organics

(Nakamura and Sawada, 2000). In future technologies microbial systems

might be potent tools to deal with environmental pollutants.

Biotechnology for hazardous waste management involves the

development of systems that use biological catalysts to detoxify, degrade

or accumulate environmental pollutants. Biotechnology offered a number

of strategies for waste treatment.

i) Improvement of existing processes by application of adapted or

engineered microbial strains.

ii) Use of adapted or genetically engineered microorganisms to treat

contaminated soil, groundwater or aquifers.

iii) Construction of bioreactors containing biofilms of suitable

organism or one of immob~lized biocatalysts in the detoxification

of environmental chemicals.

iv) Development of biosenso1,s to detect trace amounts of toxic

organics or heavy metals.

v) Recovery of products from wastes (Fetzner and Lingens, 1994).

The effluent containing phenolic compounds was treated in a

continuous stirred tank reactor. Low, moderate and high level phenol

concentrations were examined in this (Allsop, 1993). The phenol

degrading mixed culture was developed in a fed-batch activated sludge

inoculated with sewage obtained from a sewage pumping station (Manila1

and Ambujam, 1995). Immobilised Phanerocheate chrysosporium was

used in a packed bed reactor for the degradation of chlorinated

phenols(Pal et 81.. 1995). The biodegradation of phenols and cyanides

using polyacrylonitrate membranes with immobilized enzymes was

carried out by Bodzek et aL, (1996). Phenol degradation in continuous

cultures utilized a stirred tank reactor. Pseudomonas putida was used

here (Gotz and Reuss, 1997). Ccb-disposal was a cost effective option

and was reliant on the solid-state refuse fermentation to attenuate the

industrial waste waterlsludge (Daneel and Senior, 1998). A capillary

membrane bioreactor had been dttveloped and tested for the removal of

phenolic compounds from synthetic and industrial effluents. Polyphenol

oxidase was immobilized on singlt? capillary membranes in a small-scale

bioreactor using two morphologically different polymeric membranes.

One had a novel structure with rlo supporting external skin layer. This

membrane allowed greater flux and was shown to facilitate high

efficiency in removal of reaction products from the reactor (Edwards

et a/., 1999).

Phenol degradation of inclustrial effluents was done by fed-batch

fermentations. Two fed-batch cultures in self-cycling fermentation

systems was employed for this (Leonard et a/., 1999). A bubble column

reactor was designed for the rernoval of phenolic contaminants on lab

scale (Mordocco et ab, 1999). Internally skinned polysulphone capillary

membranes were coated with l'iscous chitosan gel and used as an

immobilization matrix for polyi~henol oxidase. Bench-scale, single

capillary membrane bioreactors were used to determine the influence of

chitosan (Edwards eta/., 1999).

The continuous biodegradation of phenol was carried out with a

fluidized bed bioreactor (FBB) wing immobilized Pseudomonas putida

(Gonzalez et ab, 2001). An external loop inverse fluidized bed airlift

bioreactor was constructed, characterized and tested for treating high

strength phenolic wastewater. E!xpanded polystyrene beads were used

as supporting materials for immobilizing Pseudomonas putida ATCC

11 172. A unique feature of this ibioreactor was the installation of a globe

valve between the rise and downcomer sections. The advantage of

EIFBAB was demonstrated fcmr batch biodegradation of phenol at

concentrations of upto 3000mgl' (Loh and Jun, 2001). Sequencing batch

reactor was used for the treatrnent of wastewater containing phenolic

derivatives (Quan et a/., 2003).

Sonobiodegradation (of phenolic polymers was reported by

Entezari and Petrier (2003). Moving bed biofilm bioreactors were used to

treat phenolic wastewater. The results showed that MBBR has good

resistance to shock loads and returned to steady state condition within

two or three cycles of retention time. Microscopic examinations showed

that the main bacterial culture attached to carrier elements and biofilms

were of filamentous type (Hosseini and Borghei, 2005). A rotating

perforated tube biofilm reactor (RTBR) was used in continuous mode for

removal of 2,4 dichlorophenol and toxicity from synthetic waste water

(Kargi and Eker, 2005).

Table 4: Biological treatment of phenolic wastewater

Type of beatmeni

Livingston et ab, (1993)

Chakravarti and Maiti (1 997)

Phenol and chbrophenol 1mmot)ilized cells of Pseudomonas Torres et al., (1998)

2

Garcia et a/, (2000)

Phenolic wastewater from Treatment with purified laccase Campos et a/., (2001)

Phenolic wastewater 1 Immobilized Pseudomonas pktorium I Chtra et a/., (1995)

oil mill Raktonia sp and Pseudomanas sp

Phenolic wastewater from Fluidized bed reactor with Gonzalez et al.,

3 1 Phenolic coke wastewater I irnmobilied enzyme membranes I Bodzek etal., (1996)

11 1 Phenolic waste water- High 1 External loop inversed fluidized bed I Loh and Jun (2001) 1

12

13

14 cells of Trichosporon cutaneum

batch reactor with immcbilized mixed culture

tube biofilm reactor

. strength

Phenol containing wastewater from plastic industy

Phenolic wastewater from resin manufacturing industries

Phenolic wastewater

Quan et al., (2003)

Xiangchun et al., (2003)

Hosseini and Borghei (2005)

Kargi andEker (2005)

air lii ~ioreactor

Polyrnerizatimn and precipitation with pemxidase

Treatment with immobilized cells of Flhotiococcus erythmpollis

Trea~ment with free and immobilized

Sakurai etal(2001)

Prieto et al., (2002)

Godjevargova (2003)

1.2.5. Significance of the study

Biodegradation generally llads to detoxification of a chemical. The

susceptibility to biodegradation of a new compound is of great interest when

judging its potential for environmctntal pollution. Detoxification mechanisms

vary widely depending upon the c:ompound, the microbial species involved

and the environmental conditions present. Thus comprehensive knowledge

of the range of contaminants present and their fate mechanisms in the

system being considered is essential. The detoxification reactions catalyzed

by the microorganisms can be enhanced through engineered systems.

Phenol and other phenolic compounds are common constituents of

many industrial effluents from chemical operations such as polymeric resin

production, oil refining pesticides, pulp and paper manufacturing etc. Phenol

is a priority pollutant (EPA, 197E1) and has many adverse effects on the

ecosystem. It imparts objectionable taste to drinking water. Aqueous

phenolic wastes have been treated for many years by biological treatment

processes such as aerobic and anaerobic systems. Aerobic treatment of

industrial wastewater is a viable technology due to rapid development of

high rate reactors such as biofilm reactors (Borghei and Hosseini, 2004).

Though several of the above mentioned methods have been tried for

the removal of phenol and phenolic derivatives from the environment, they

were found to be expensive artd difficult in practising because of the

incomplete removal of phenol and production of toxic intermediates.

Hence in the present study an attempt was made to isolate and

identify an efficient phenol degrading organism. On selecting the strain the

mechanism involved in the degradation of phenol and the optimum

conditions for the effective rem~val of phenol can be evaluated. The

selected strain can be effectively used for the safe disposal of the phenolic

effluent from the paper factory.

Objectives

The following objectives were considered for the present study:

Isolation and screening 01 an efficient bacterial strain capable of

phenol degradation.

Purification and identification of selected strain.

Study of the growth kinetics of the selected organism in the Mineral

Salt Phenol Medium( MSPM)

Optimisation of the conditions for the maximum removal of phenol

by the organism

Process study on biodegrad,3tion by extraction of products by solvent

extraction and analysis by GCMS and nTR spectroscopy.

Isolation of enzyme from tho selected strain

Purification of enzyme by ammonium sulfate fractionation and ion

exchange chromatography.

Evaluation of the effect of partially purified enzyme in the removal

of phenol from Mineral salt rnedium.

Immobilization of the organism and the effect of immobilized

organism in the removal of phenol from mineral salt phenol medium.

Characterization of the ph!/sico-chemical and biological parameters

of the phenol containing effluent.

Application of the free cells of the selected strain in the treatment

of phenol containing effluent.

12. Application of the immobilized cells of the selected strain in

treating the phenol containing effluent by both batch and

continuous methods.

13. Designing a bioreactor :;ystem for the effective treatment of the

phenol containing effluent.

14. Treatment of the phenol containing effluent with the designed

bioreactor.