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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 INTRODUCTION

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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 leads to lower levels of PHA per cell and lower levels of PHA per batch of cells.

INTRODUCTION

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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 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.

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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 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

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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:-

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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]

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

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Pseudomonas 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.

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

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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.

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 allow Pseudomonas putida to survive deadly toxins in the soil and allow it to thrive in contaminated areas. Its metabolism

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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.

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

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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 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 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.

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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 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

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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.

Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3Patrick 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

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through the vapor phase. The concentration of styrene in the air and liquid was

measured by gas chromatography analysis.

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.

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

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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 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.

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PHA polymers were isolated by using a previously described method (9). 13C

NMR spectrum analysis was performed in a Bruker advance 400-MHz NMR

spectrometer operating at 111 MHz for 13C 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. 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 DISCUSSIONP. 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 13C NMR analysis of the polymer

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(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.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 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

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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 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

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

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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 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 of styrene (♦), phenylacetic acid (•), or

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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 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

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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). REFRENCESES1)

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