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TERM PAPERTOPIC:- IMPORTANCE OF

PSEUDOMONAS PUTIDA
D.O.R:-1.11.2010

SUBMITTED BY
SHASHI SHARMA
ROLL NO:- B15
REG. NO:- 11006142
SECTION:- P8003

TABLE OF CONTENTS

 ABSTRACT
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 INTRODUCTION

 ROLE OF MICROBES IN BIOREMEDATION

 OUTLINE MORPHOLOGY OF PESUDOMONAS

PUTIDA
 ROLE OF PESUDOMONAS PUTIDA IN
BIOREMEDATION
 ACCUMULATIO OF POLYHYDROXYLALKONATE
FROM STYRENE AND PHENYLACETIC ACID BY
PSEUDOMONAS PUTIDA
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ABSTRACT

Pseudomonas putida CA-3 is capable of converting the

aromatic hydrocarbon styrene, its metabolite phenylacetic
acid, and glucose into polyhydroxyalkanoate (PHA) when a
limiting concentration of nitrogen (as sodium ammonium
phosphate) is supplied to the growth medium. PHA
accumulation occurs to a low level when the nitrogen
concentration drops below 26.8 mg/liter and increases
rapidly once the nitrogen is no longer detectable in the
growth medium. The depletion of nitrogen and the onset
of PHA accumulation coincided with a decrease in the rate
of substrate utilization and biochemical activity of whole
cells grown on styrene, phenylacetic acid, and glucose.
However, the efficiency of carbon conversion to PHA
dramatically increased once the nitrogen concentration
dropped below 26.8 mg/liter in the growth medium. When
supplied with 67 mg of nitrogen/liter, the carbon-to-
nitrogen (C:N) ratios that result in a maximum yield of
PHA (grams of PHA per gram of carbon) for styrene,
phenylacetic acid, and glucose are 28:1, 21:1, and 18:1,
respectively. In cells grown on styrene and phenylacetic
acid, decreasing the carbon-to-nitrogen ratio below 28:1
and 21:1, respectively, by increasing the nitrogen
concentration and using a fixed carbon concentration
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leads to lower levels of PHA per cell and lower levels of

PHA per batch of cells.

INTRODUCTION

Bioremediation uses naturally occurring microorganisms to degrade

various types of wastes. Like all living creatures, microbes need
nutrients, carbon, and energy to survive and multiply. Such
organisms are capable of breaking down chemicals to obtain food
and energy, typically degrading them into harmless substances such
as carbon dioxide, water, salts, and other innocuous products.

Biological treatment techniques fall into two categories,

biostimulation and bioaugmentation. Biostimulation refers to the
addition of specific nutrients to a waste situation with the hope that
the correct, naturally occurring microbes are present in sufficient
numbers and types to break down the waste effectively. This
assumes that every organism needed to accomplish the desired
treatment results is present. But how can we be certain that those
organisms present are the most suitable to degrade all the materials
present? And, if the naturally occurring organisms present were truly
effective in achieving complete waste breakdown, then why are there
problems at sites?

Let’s look at an example. Grease accumulates in sewers and grease

traps. Grease is biodegradable and sewers contain a large bacteria
population. Nitrogen and phosphorous are in ample supply and most
sewers are aerobic in nature. Yet grease accumulates. If the
principle that all the necessary organisms are always present in
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sufficient number and type, then grease breakdown should occur in

sewers and grease deposits should be rare.

An alternative approach is to use bioaugmentation, which is the

scientific approach to achieving controlled, predictable, and
programmed biodegradation. Bioaugmentation involves the addition
of specifically formulated microorganisms to a waste situation. It is
done in conjunction with the development and monitoring of an ideal
growth environment, in which these selected bacteria can live and
work.

Bioaugmentation allows one to control the nature of the biomass. It

ensures that the proper team of microbes is present in the waste
situation in sufficient type, number, and compatibility to attack the
waste constituents effectively and break them down into their most
basic compounds.

ROLE OF MICROBES IN BIOREMEDATION

The goal in bioremediation is to stimulate microorganisms with

nutrients and other chemicals that will enable them to destroy the
contaminants. The bioremediation systems in operation today reply
on microorganisms native to the contaminated sites, encouraging
them to work by supplying them with the optimum levels of nutrients
and other chemicals essential for their metabolism. Thus, today's
bioremediation systems are limited by the capabilities of the native
microbes. However, researchers are currently investigating ways to
augment contaminated sites with nonnative microbes-including
genetically engineered microorganisms-specially suited to degrading
the contaminants of concern at particular sites. It is possible that this
process, known as bioaugmentation, could expand the range of
possibilities for future bioremediation systems.

Regardless of whether the microbes are native or newly introduced to

the site, an understanding of how they destroy contaminants is
critical to understanding bioremediation. The types of microbial
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processes that will be employed in the cleanup dictate what

nutritional supplements the bioremediation system must supply.
Furthermore, the byproducts of microbial processes can provide
indicators that the bioremediation is successful.

Microorganisms gain energy by catalyzing energy-producing

chemical reactions that involve breaking chemical reactions that
involve breaking chemical bonds and transferring electrons away
from the contaminant. The type of chemical reaction is called an
oxidation-reduction reaction: the organic contaminant is oxidized, the
technical term for losing electrons; correspondingly, the chemical
that gains the electrons is reduced. The contaminant is called the
electron donor, while the electron recipient is called the electron
acceptor. The energy gained from these electron transfers is then
"invested", along with some electrons and carbon from the
contaminant, to produce more cells. These two materials — the
electron donor and acceptor — are essential for cell growth and are
comply called the primary substrates.

.
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IMPORTANCE OF Pseudomonas putida IN

BIOREMEDATION:-

Description and significance

Pseudomonas putida is a rod-shaped, flagellated,

gram-negative bacterium that is found in most soil
and water habitats where there is oxygen. It grows
optimally at 25-30 C and can be easily isolated.
Pseudomonas putida has several strains including
the KT2440, a strain that colonizes the plant roots
in which there is a mutual relationship between
the plant and bacteria. The surface of the root,
rhizosphere, allows the bacteria to thrive from the
root nutrients. In turn, the Pseudomonas putida
induces plant growth and protects the plants from
pathogens. Because Pseudomonas putida assist in
promoting plant development, researchers use it
in bioengineering research to develop
biopesticides and to the improve plant health. [17]
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Pseudomonas putida has a very diverse aerobic

metabolism that is able to degrade organic
solvents such as toluene and also to convert
styrene oil to biodegradable plastic
Polyhydroxyalkanoates (PHA). This helps degrade
the polystyrene foam which was thought to be
non-biodegradable. Due to the bacteria’s strong
appetite for organic pollutants, researchers are
attracted to using Pseudomonas putida as the
“laboratory ‘workhorse’ for research on bacteria-
remediated soil processes”. [4] This bacteria is
unique because it has the most genes involved in
breaking down aromatic or aliphatic hydrocarbons
which are hazardous chemicals caused by burning
fuel, coal, tobacco, and other organic matter.
There is great interest in sequencing the genome
of Pseudomonas putida due to its strong effect in
bioremediation.

Aside from aiding in bioremediation, Pseudomonas

putida is very helpful in the research of different
species in the genus Pseudomonas, especially
Pseudomonas aeruginosa, a pathogenic bacterium
that is one of the leading fatal diseases in humans.
Researchers find that Pseudomonas putida,
although saprophytic, can aid in the research on
cystic fibrosis which is caused by Pseudomonas
aeruginosa. The two bacteria are very closely
related and share similar sequenced genomes
(approximately 85% are shared), except
Pseudomonas putida lack the genes that
determine virulence. Because of its nonpathogenic
nature, many researchers find Pseudomonas
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putida very beneficial to research due to its

versatility and ease of handling.

Genome structure

In 1995, the scientists at The Institute for Genomic

Research in Germany decoded the first complete
genome sequence of Pseudomonas putida. Thirty
microbial strains have been completed and fully
sequenced, while another seventy-five are in the
process of being sequenced. Through the genome
analysis, Pseudomonas putida is found to have
approximately 6.2 million DNA base pairs. Among
the Pseudomonas putida, the strain F1 is
5,959,964 nucleotides long and contains 61%
guanine and cytosine content and 39% adenine
and thymine content. While another important
strain, KT2440 is 6,181,863 nucleotides long. [2]
Pseudomonas putida has a circular genome where
at least eighty genes in oxidative reductases, a
family of enzymes, are involved in decomposing
substances in the environment. Moreover, the
majority of the genes are for detecting chemical
signals in the surroundings so it can quickly
respond to toxins. This bacterium also has many
important plasmids, such as the sequenced TOL
and OCT plasmid, which play an important role in
the degradation of pollutants. [5, 6] Nonetheless,
not all plasmids are helpful in bioremediation.
Some create a disadvantage for Pseudomonas
putida because it reduces the growth rate and is
useless in function such as the plasmid R68-45.
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Cell structure and metabolism

Pseudomonas putida is a rod-shaped,

nonsporeforming, gram-negative bacteria that
utilizes aerobic metabolism. This bacterium also
has multiple polar flagella for motility. The flagella
have a waveform that is usually 2 to 3 wavelengths
long. Pseudomonas putida is sensitive to the
environment and suppresses the changes in the
direction of flagella rotation upon sensing
chemoattractants. This is very helpful in guiding
the Pseudomonas putida to propel towards the
seeds of the plants which provides nutrients to the
bacterial cells.

Pseudomonas putida is able to tolerate

environmental stresses due to its diverse control
of proteins including protein and peptide secretion
and trafficking, protein modification and repair,
protein folding and stabilization, and degradation
of proteins, peptides, and glycopeptidesSome
important proteins include the global regulatory
proteins which link the pathway genes to the cell
status. Pseudomonas putida exercises a very
complex metabolism, the proteins control a
particular pathway that not only depends on the
signal received, but also the specific promoters
and regulators in the pathway. And in turn, once
the signals are received, it informs the cell of the
oxygen and nutrient availability. Another
important protein is the Crc protein which is part
of the signal transduction pathway moderating the
carbon metabolism. It also functions in biofilm
production.
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Pseudomonas putida has metabolism functions in

biodegradable plastics. Styrene degradation in
Pseudomonas putida CA-3 degrades styrene in two
pathways 1) vinyl side chain oxidation and 2)
attack on the aromatic nucleus of the molecule.
Pseudomonas putida also has sideospores, an iron
chelating compound that allows the bacteria to
enhance levels of iron and promote the active
transport chain. Strains of Pseudomonas putida
have outer membrane receptor proteins that help
transport the iron complex to the sideospores,
specifically known as pyoverdines, which are found
in the bacterial cell. From there the iron is used in
metabolic processes where oxygen is the electron
acceptor. Oxygen serves as a good electron
acceptor. The oxygen byproducts, however, are
toxic to the bacteria including superoxide and
hydrogen peroxide. In response, Pseudomonas
putida produces catalase to protect the cell from
the reactive properties of the byproducts.

In addition, Pseudomonas putida has important

lipids that are developed as an adaptation
mechanism to respond to physical and chemical
stresses. The bacteria is able to change its degree
of fatty acid saturation, the cyclopropane fatty
acids formation, and the cis-trans isomerization. In
different phases, the cell changes its
characteristics to better respond to the
environment. During the transition from growth to
stationary phase, there is a higher degree of
saturation of fatty acid and a higher membrane
fluidity which improves substrate uptake, thus
regulating the cell. [9] All these characteristics
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allow Pseudomonas putida to survive deadly toxins

in the soil and allow it to thrive in contaminated
areas. Its metabolism allows these bacteria to
convert harmful organic solvents to nontoxic
composites which are so essential to
bioremediation.

In addition to the ability for P. putida to degrade

synthetic compounds, it can also use an
alternative metabolic pathway such as the Entner-
Doudoroff pathway. In this pathway, P. putida
degrades common hexoses, such as glucose and
gluconate, to yield one net ATP for every glucose
molecule degraded. This is in contrast to the two
net ATP produced for every glucose molecule
degraded in the classic glycolysis pathway.

The Entner-Doudoroff pathway begins by

converting glucose to gluconate-6-phosphate
through two intermediates. The first intermediate
is gluconate which is then converted to 2-
ketogluconate. 2-ketogluconate is then converted
to gluconate-6-phosphate. It should be noted that
in some cases, gluconate-6-phosphate can be
produced directly via phosphorylation of
gluconate. The gluconate-6-phosphate is converted
to 2-Keto-3-deoxy-gluconate-6-phosphate (KDGP).
Finally, KDGP is converted to triosephosphate and
pyruvate. Interestingly, P. putida has many
alternative pathways that it can utilize to produce
energy, yet it does not use them and mainly relies
on the Entner-Doudoroff pathway outlined above.
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Ecology

Pseudomonas putida are significant to the

environment due to its complex metabolism and
ability to control pollution. There is a high
versatility of bacterial communities towards
contaminations which is further increased by
certain catabolic sequences on the TOL plasmids in
the cell. [7] Even the plasmids are important in
sensing the environmental stress. Some of the
environmental stresses are caused by benzene,
xylene, and toluene, the main components of
gasoline and are major sources of water
contamination. Pseudomonas putida can degrade
the hydrocarbons of these organic solvents
through oxidative reactions therefore placing
Pseudomonas putida as one of the most important
microbes in bioremediation.

Pseudomonas putida also interacts with other

organisms in the soil. One such interaction with
Saccharomyces cerevisiae in the rhizosphere led to
beneficial effects on the state of the Pseudomonas
putida. Fungi Saccharomyces cerevisiae produced
the necessary glucose and also maintained the pH
which was both favorable to the bacteria
Pseudomonas putida. [16] The complex interaction
of Pseudomonas putida and Saccaromyces
cerevisiae together regulate plant health.
Moreover, the bacteria itself is a great maintainer
of abundant plant life. The production of the
siderophores, such as pyoverdine and pyochelin,
protect the plants from fungal pathogens. The
mutual relationship benefits both partners. While
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Pseudomonas putida is able to reside in the plant

seed and rhizosphere, the plant is, in turn,
protected from plant pathogens and able to obtain
vital nutrients from the bacteria.

Application to Biotechnology

Pseudomonas putida has the ability to produce

Poly-3-hydroxyalkanoates (PHA) from the aromatic
hydrocarbon styrene. Styrene, a major
environment toxic pollutant, is released in millions
of kilograms a year from industrial sites and is
known to cause spinal tract irritation, muscle
weakness, and narcosis in humans and other
mammals. The conversion to PHA allows the cure
of styrene pollution.

Because PHA is beneficial to society, it serves in

medical applications such as tissue engineering
and also as antibiotics and vitamins. PHA is also
very environmental friendly, oil and grease
resistant, and has a long shelf life therefore it is
also used in everyday items such as plastic
utensils and other disposable items. Unlike
styrene, PHA can readily break down in soil or
water. Commonly used styrofoam, aka polystyrene
foam, is transformed into biodegradable plastic
through the Pseudomonas putida’s complex
metabolism.

Styrofoam is at first converted to styrene oil where

it is introduced to Pseudomonas putida to convert
to PHA. [20] Within Pseudomonas putida, PHA
accumulates under unbalanced growth conditions
as a means of intracellular storage, storing excess
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carbon and energy. These PHA polymers are

synthesized by enzyme PHA synthase which is
bound to the surface of the PHA granules and uses
coenzyme A thioesters of hydroxyalkanoic acids as
substrates.

Current Research

In a recent study, Pseudomonas putida strains

were compared to each other to determine the
phylogenetic relationships. Because the
Pseudomonas putida strain KT2440 genome is fully
sequenced it serves as a standard reference to
compare with other Pseudomonas putida strains.
They carried out a study that assessed the utility
of Pseudomonas putida strain KT2440-based high-
density DNA microrarrays for transcriptomics
studies of DSM 6125, DSM 3931, DSM 291, and
S12, along with other non-sequenced strains.
Transciptomics allow researchers to gain careful
insight into the complex metabolic and cellular
systems of the bacteria. In conclusion, they were
able to draw similarities between the different
strains, for instance, proving that strains DSM
6125 was completely identical to that of DSM 3931

In another research study, Pseudomonas putida

strain RB1500 and strain RB1501 (which were
created from Pseudomonas putida strain KT2440)
were tested in South Carolina to determine
whether light will be produced in the strains in
response to trinitrotoluene (TNT), and whether
production of light means detection of TNT. The
technology will then be used for land mine
detection in soils. A concern during the experiment
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was if Pseudomonas putida will present an

ecological hazard; due to the nonpathogenic
characteristics of the soil bacteria, however,
researchers were assured that it will not cause any
hazards to the environment or to the society.

A research proved that Pseudomonas putida N-acyl

homoserine lactone quorum sensing regulation
involves Lon protease. Screening of a transposon
mutant bank of Pseudomonas putida was done to
identify regulators that were concerned in
negative regulation of acyl homoserine lactones
quorum sensing. After the experiment, three Tn5
mutants were identified. One mutant had the
transposon located in the Lon protease gene,
which plays an important role in degradation of
misfolded proteins. In brief, the Lon protease is a
negative regulator of acyl homoserine lactone
production.

A 2005 research article stated that Pseudomonas

putida is associated with clinical infections in pre-
mature babies. Babies that were admitted into
Special Care Nursery (SCN) were fed through
catheters. These catheters went through a heparin
flush procedure in order to keep the pathway clear
for fluids to enter the body. During this procedure,
the environment conditions within the tube
allowed P. putida to grow. This growth of bacteria
caused bloodstream infections within the baby.
Although heparin has antibiotic activity, P. putida
is resistant and is able to grow.
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Accumulation of Polyhydroxyalkanoate from Styrene and

Phenylacetic Acid by Pseudomonas putida CA-3
Patrick G. Ward,1 Guy de Roo,2 and Kevin E. O'Connor1*
MATERIALS AND METHODS

Media and growth conditions.

P. putida CA-3 cultures were grown in 250-ml conical

flasks containing 50 ml of E2 medium (32) at 30°C, with
shaking at 200 rpm. For PHA accumulation studies, the
inorganic nitrogen source (NaNH4HPO4· ·4H2O) was
supplied at between 0.125 g/liter (8.4 mg of nitrogen/liter)
and 2.0 g/liter (134 mg of nitrogen/liter) (E2 N-lim). For
PHA accumulation studies, P. putida CA-3 was grown in
batch culture with 50 ml of E2 N-lim medium for 48 h
unless otherwise stated. Phenylacetic acid and glucose
were added directly to the growth medium after
autoclaving. For inhibition experiments, the required
concentration of 2-bromooctanoic acid was added to the
medium before inoculation.

Styrene is a volatile liquid that is poorly soluble in water

(maximum solubility, 0.3 g/liter at 30°C). Therefore,
styrene (20 to 200 mg, equivalent to 0.4 to 4.0 g per liter
of medium) was placed in a glass tube (10 mm in diameter
by 60 mm in length) fused to the central base of the flask
and transferred to the culture through the vapor phase.
The concentration of styrene in the air and liquid was
measured by gas chromatography analysis.
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Analysis of styrene concentration in the headspace and

growth medium.

Styrene concentration in the headspace was analyzed by

taking 100-μl samples from the headspace and manually
injecting them into a Fisons GC-8000 series gas
chromatograph (GC) equipped with a 30-m by 0.25-mm
HP-1-0.25 μm column (Hewlett-Packard) operating in split
mode (split ratio, 8:1) with temperature programming
(50°C for 1 min, increments of 10°C/min up to 140°C, and 1
min at 140°C). The retention time obtained for styrene
with this method was 5.2 min. The styrene concentration
in the liquid medium was analyzed by removing 1-ml
samples and extracting the styrene into a 1-ml mixture of
hexane and ethanol in the ratio of 95:5. The organic layer
was then removed, and a sample was run on a GC as
above. The limit of detection of styrene with gas
chromatography is 0.001 g/liter.

PHA monomer determination.

PHA monomer composition was determined by using a

previously described method (9). Samples were analyzed
on a GC. For peak identification, PHA standards from
Pseudomonas oleovorans were used. PHA monomer
composition was confirmed by GC-mass spectrometry as
described previously The composition of PHA was also
confirmed by 13C and 1H nuclear magnetic resonance
(NMR) analysis of the polymer.
HPLC.
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The concentration of phenylacetic acid in the growth

medium at various points in the growth curve of P. putida
CA-3 was analyzed by high-performance liquid
chromatography (HPLC). A 1-ml sample was taken from
the flask and centrifuged at 20,000 × g for 10 min at 4°C.
The supernatant was filtered prior to HPLC analysis by
using a 0.45-μm-pore-size nylon filter. Samples were
analyzed by HPLC using a C8 Lichrospher 125- by 4.0-mm
column and a Hewlett-Packard HP1100 instrument
equipped with an Agilent 1100 Series diode array
detector. The samples were eluted with a 90% 0.2 M
K2HPO4-10% isopropanol mix at a flow rate of 1.0 ml/min.
The retention time for phenylacetic acid was 7.1 min.

Glucose determination assays.

Glucose concentrations in the growth medium were

measured by using a 3,5-dinitrosalicylic acid method as
described previously (2).

Nitrogen determination assays.

The nitrogen concentration (as ammonium ion) in the

medium was measured by the method described by
Scheiner Biochemical assays.

The oxygen consumption of P. putida CA-3 cells was

monitored in a biological oxygen monitor (Rank Brothers
Ltd., Cambridge, England) as reported previously. The cell
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dry weight (CDW) in oxygen consumption assays varied

between 0.15 and 0.3 g/liter. The activity of the styrene
monooxygenase enzyme was monitored by measuring
indigo formation from indole by P. putida CA-3 whole cells
as previously described Unwashed cells were added to a
0.25 mM solution of indole at a CDW concentration of
approximately 0.1 g/liter. The formation of indigo was
measured at 5-min intervals over 30 min.

Isolation and analysis of PHA polymers.

PHA polymers were isolated by using a previously

13
described method (9). C NMR spectrum analysis was
performed in a Bruker advance 400-MHz NMR
13
spectrometer operating at 111 MHz for C measurements
at room temperature. NMR analysis was obtained from
25% (wt/vol) chloroform solutions, and a delay time
between pulses of 2.0 s was applied. Differential scanning
calorimetry experiments were carried out in a Perkin-
Elmer Pyris Diamond system. Two scans were performed
by using a 20°C/min heating rate and a 200°C/min cooling
rate (quenching) between runs. Thermograms were
obtained in the range of −60 to +75°C under helium
purge. The glass transition temperature (Tg) values were
taken at the inflection point on the transition of second
scans. Thermogravimetric analysis investigation was
carried out with a Perkin-Elmer Pyris 1 TGA over a
temperature range of 30 to 600°C and a rate of 10°C/min.
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The average molecular weights, molecular weight

distribution, and polydispersity index were determined by
using a Waters gel permeation chromatograph equipped
with a refractive index detector (series 410). Polymer
Laboratories gel columns (500 Å) conditioned at 30°C
were used to elute samples (1 g/liter) at a 1-ml/min HPLC
grade tetrahydrofuran flow rate. Ten samples of
polystyrene standards having molecular masses ranging
from 950,000 to 1,340 Da were used for calibration.

RESULTS AND DISCUSSION

P. putida CA-3 is capable of transforming styrene into PHA
when supplied with E2 mineral medium with a low
nitrogen concentration of 67 mg of nitrogen/liter. Under
these conditions, P. putida CA-3 was found to accumulate
PHA intracellularly in discrete granules (Fig. (Fig.1).1).
The PHA accumulated from styrene, as measured by GC-
mass spectrometry, is composed of (R)-3-hydroxyhexanoic
acid, (R)-3-hydroxyoctanoic acid, and (R)-3-
hydroxydecanoic acid monomers in a ratio of 3:27:70. The
monomer composition was further confirmed by 1H and 13
C
NMR analysis of the polymer (data not shown). P. putida
CA-3 is also capable of transforming phenylacetic acid and
glucose to PHA. The monomer composition of the PHA
accumulated from these substrates is virtually identical to
that of PHA accumulated from styrene.
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Monitoring microbial cellular activity and PHA

accumulation over time.

PHA accumulation by P. putida CA-3 was monitored over a

48-h period when either styrene (1.85 g of carbon/liter),
phenylacetic acid (1.4 g of carbon/liter), or glucose (0.8 g
of carbon/liter) was supplied at 2.0 g/liter to medium
containing nitrogen at a concentration of 67 mg/liter (Fig.
(Fig.2).2). The depletion of the nitrogen concentration
below approximately 26.8 mg/liter coincided with the
onset of PHA accumulation from all three carbon sources
tested. This observation is in agreement with other
studies where a limiting concentration of nitrogen
resulted in the onset of PHA accumulation (9, 21, 35).
However, it was not until the nitrogen concentration in
the growth medium fell below the detectable limit (0.1 mg
of nitrogen/liter) that a dramatic increase in the level of
PHA accumulation occurred (Fig. (Fig.2).2). Interestingly,
the rate of nitrogen depletion was lower in incubations
with cells grown on styrene than in incubations with cells
grown on phenylacetic acid and glucose (Fig. (Fig.22).

PHA accumulation by P. putida CA-3 when 2.0 g of styrene

(A), phenylacetic acid (B), or glucose (C)/liter is supplied
to 50 ml of growth medium containing 67 mg of
nitrogen/liter. Biomass (in grams per liter) (♦), PHA
accumulation (□), concentration of carbon source in the
medium (in grams per liter) (▵), and nitrogen (supplied as
sodium ammonium phosphate) concentration (in
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milligrams per liter) ( ) were all monitored over a 48-h

period. All data are averages of results from at least three
independent determinations. The concentrations of
styrene in the growth medium and in the air above the
growth medium were experimentally determined to be
0.02 and 0.004 g/liter, respectively, at time zero. While
the concentration of styrene in the air above the medium
remained at 0.004 g/liter throughout the growth curve,
styrene was no longer detectable in the growth medium
after time zero.

Nitrogen accounts for approximately 12% of the dry

weight of Pseudomonas cells not containing PHA After 48
h of growth, the total CDWs achieved by cells grown on
styrene, phenylacetic acid, and glucose were 0.768, 0.824,
and 0.608 g/liter, respectively. However, 0.195, 0.247, and
0.091 g of these CDWs are accounted for by PHA.Thus, the
total CDWs minus the weight of PHA correspond to CDWs
between 0.52 and 0.58 g/liter, which are in good
agreement with the CDW value of 0.56 g/liter expected for
Pseudomonas cells grown with 67 mg of nitrogen/liter but
not containing PHA. The rate of carbon utilization was
monitored over the 48-h growth period. Due to the
insoluble nature of styrene, it was supplied as a gas. The
concentration of styrene in the growth medium at time
zero was 0.02 g/liter. At each time point in the growth
curve after time zero, styrene was not detectable in the
growth medium. The styrene concentration in the
headspace remained constant throughout the growth
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curve at 0.004 g/liter. Thus, it was not possible to monitor

styrene depletion in the growth medium over time. The
rates of phenylacetic acid and glucose depletion were
0.11 and 0.12 g/liter/h, respectively, during the initial 6 h
of growth. The rates of substrate depletion fell to 0.05
and 0.056 g/liter/h for cells grown on phenylacetic acid
and glucose, respectively, during the first 16 h of growth

In an attempt to establish the biochemical activity of P.

putida CA-3 cells before and during PHA accumulation,
biochemical assays measuring rates of oxygen
consumption and styrene monooxygenase activity by
whole cells were performed at various time points in the
growth curve (Tables 1 and 2). On all of the carbon
sources tested, the rate of oxygen consumption fell
between 3.09- and 3.78-fold when the nitrogen
concentration was no longer detectable and PHA
accumulation began to increase dramatically. The rate of
oxygen consumption continued to decrease for all carbon
sources during PHA accumulation. A similar pattern was
observed for styrene monooxygenase activity of cells
grown on styrene, where a 2.1-fold decrease in styrene
monooxygenase coincided with the depletion of nitrogen
in the growth medium

The rate of oxygen consumption of whole cells

of P. putida CA-3 harvested at various time
points after inoculationa
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Styrene monooxygenase activity of whole cells

of P. putida CA-3 harvested at various time
points after inoculationa
Interestingly, while the biochemical activity of the cells
with respect to carbon utilization, oxygen utilization, and
styrene monooxygenase activity decreased over time, the
rate of carbon conversion to PHA increased over time (,
the rate of phenylacetic acid consumption by growing
cells decreases 2.2-fold in the first 16 h of growth, while
the rate of carbon-to-PHA conversion increases
approximately 100-fold in the same time period). This
result implies that the efficiency of carbon source-to-PHA
conversion increases over time under inorganic nitrogen
limitation. The change in biochemical activity is in keeping
with previous reports for changes in enzyme activity in
the late log or early stationary phases of growth or under
nutrient limitation. However, this is to our knowledge the
first time that the catabolic activity of PHA-accumulating
cells has been shown to dramatically decrease during PHA
accumulation.

When the efficiencies of the conversion of carbon source

to PHA were compared over the entire growth curve
across the three growth substrates, 0.099, 0.118, and
0.046 g of PHA per g of styrene, phenylacetic acid, and
glucose, respectively, was converted to PHA by P. putida
CA-3. This is equivalent to 0.11, 0.17, and 0.115 g of PHA
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per g of carbon supplied. The differences in the

efficiencies of carbon source-to-PHA conversion across the
three substrates may be explained by the influence of
carbon concentration or carbon-to-nitrogen ratio. Thus,
the effect of carbon supply to the growth medium was
examined.

The effect of carbon supply on PHA accumulation by P.

putida CA-3.

PHA accumulation was measured in cultures containing 67

mg of nitrogen/liter while the concentration of the carbon
sources supplied was varied. Little PHA was observed on
any of the carbon sources until the C:N ratios exceeded
approximately 9:1 for cells grown on glucose, 10:1 for
cells grown on phenylacetic acid, and 14:1 for cells grown
on styrene (Fig. (Fig.3).3). Thereafter, PHA accumulation
increased to maximal levels of 0.11 g/g of carbon for
styrene, 0.17 g/g of carbon for phenylacetic acid, and 0.22
g/g of carbon for glucose.. These results correspond to
optimum C:N ratios for PHA accumulation of 28:1, 21:1,
and 18:1 for cells grown on styrene, phenylacetic acid,
and glucose, respectively, when supplied with 67 mg of
nitrogen/liter.

PHA accumulation by P. putida CA-3 as a function of the

amount of carbon (in grams per liter) supplied in the form
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of styrene (♦), phenylacetic acid (•), or glucose ( ) to 50

ml of growth medium containing 67 mg of nitrogen/liter.
(more ...)
The PHA levels in cells grown on styrene are always lower
than those in cells grown on phenylacetic acid (Fig.
(Fig.3).3). This result is surprising, as phenylacetic acid is
a known intermediate in the styrene catabolic pathway.
However, styrene is present in the growth medium at a
much lower concentration than phenylacetic acid due to
its supply as a gas and its low solubility in aqueous
solution. The concentration of substrates in the growth
medium is known to affect the concentration of key PHA
intermediates, such as (R)-3-hydroxyalkanoic acids in the
cytoplasm, and ultimately the PHA content of the cell (28).

Effect of sodium ammonium phosphate concentration on

PHA accumulation in P. putida CA-3.

In an attempt to determine the maximum yield of PHA

from aromatic carbon substrates, the initial nitrogen
concentration in the growth medium was altered while the
initial concentration of the carbon source was kept
constant (2.0 or 4.0 g/liter).

When supplied with 2 g of the carbon source/liter, the

highest PHA content of cells (percent CDW) was observed
at low nitrogen concentrations However, PHA yields from
styrene and phenylacetic acid were low due to poor
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growth of the cells. Total PHA yields increased

dramatically to a maximum at a nitrogen concentration of
67 mg/liter, establishing an optimal C:N ratio of 28:1 when
2 g of styrene/liter was supplied and 21:1 when 2 g of
phenylacetic acid/liter was supplied.

FUTURE SCOPE
PESUDOMNAS PUTIDA CAN BE USED AS
DEGRDATION COMPOUND FOR VARIOUS OTHER
HAZARIDIOUS POLLUTENTS IN FUTURE. ITS VAST
VARIETY OF DECOMPOSITION POWER HAS PROVED
THAT IT CAN BE HELPFULL DEGRADING COMPLEX
COMPOUND RATHER THAN POLYSTYRENE. LARGE
OIL SPILLS CAN BE ALSO CONTROLLED IF ITS
DEGRADATION IS MORE PONOUNCED IN SEAS .
PSEUDOMONAS PUTIDA CAN ALSO APPLIED IN
SAVING HUMAN LIFES BY ITS DETERMINATION IN
DETECTING POWERFULL EXPLOSIVES LIKE ITS
BEING USED IN DETECTING TNT(trinitro toluene).

REFRENCESES

1) Kowalski, H. “U.S. – German Research Consortium Sequences

Genome of Versatile Soil Microbe”. J.Craig Venter Archive.CH
December 2002. http://www.tigr.org/news/pr_12_02_02.shtml

2) Muller, R. “Bacterial Degradation of Xenobiotics”. Microbial

Control of Pollution. March 1992. Volume 48.
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3) Härtig, C., Loffhagen, N., Harms, H. “Formation of trans Fatty

Acids Is Not Involved in Growth-Linked Membrane Adaptation of
Pseudomonas putida”. Applied and Enbironmental

Microbiohttp://aem.asm.org/cgi/content/abs.