Introduction

Synthetic polymers are considered to be an indispensable gift of modern

science and technology to mankind (Luckachan and Pillai, 2011). Modern world can’t
be imagined without plastics as they have become integral part of contemporary life
due to their vast applications, which extend from small domestic setups to huge
industries plastics are utilized in almost every manufacturing industry in the world
ranging from automobiles to medicine. Unfortunately, this great discovery gets buried
in landfills every day and takes up space because of its non-degradable nature (Shah,
2014). Improper disposal of plastics has threatened natural environment worldwide.

Petrochemical plastics being xenobiotic or recalcitrant to microbial

degradation pose a big threat to ecosystem. Excessive molecular size (50,000 to
1,000,000 Da). Seems to be mainly responsible for the resistance of these chemicals
to biodegradation and their persistence in soil for a long time. Approximately 140
million tons of plastics in consumed every year globally, which necessitate the
processing of about 150 million tons of fossil fuel and directly causes massive
environmental pollution. The waste generated by this pollution can take thousands of
years to naturally deteriorate, if it degrades at all (Hempel et al., 2011).

Huge amount of plastic is also discarded into marine environment every year
killing many marine animals and affecting aquatic flora and fauna. Although plastics
can be disposed of by incineration or recycling, but incineration is very difficult,
dangerous and expensive, and recycling is a long process and not very efficient. If
plastics were made biodegradable then these would no longer accumulate, and
recycling and incineration troubles could be overcome (DeMarco, 2005).

Plastics fit into two categories: thermoplastics and thermosetting plastics these
two have different properties. The difference between these two types of plastics is
that thermoplastics can be heated and shaped over and over again whereas
thermosetting plastics can only be heated and shaped once. As plastics are produced
from non-renewable resources such as petroleum and are not friendly with natural
carbon cycle because of their non-degradable feature, so to overcome this problem,
the production and applications of eco- friendly products such as bioplastics is
mandatory.
Common plastics and uses

 Polypropylene (PP) – Food containers, appliances, car fenders

(bumpers).
 Polystyrene (PS) – Packaging foam, food containers, disposable cups,
plates and cutlery, CD and cassette boxes.
 High Impact Polystyrene (HIPS) – Fridge liners, food packaging,
vending cups.
 Acrylonitrile butadiene styrene (ABS) – Electronic equipment cases
(e.g. computer monitors, printers, keyboards).
 Polyethylene terephthalate (PET) – Carbonated drinks bottles, jars,
plastics film, microwavable packaging.
 Polyester (PES) – Fibers, textiles.
 Polyamides (PA) (Nylons) – Fibers, tooth brush bristles, fishing line,
under the hood car engine moldings.
 Polyvinyl chloride (PVC) – Plumbing pipes and guttering, shower
curtains, window frames, flooring.
 Polyurethanes (PU) – Cushioning foams, thermal insulation foams,
surface coatings, printing rollers. Currently 6th or 7th most commonly
used plastic material, for instance the most commonly used plastic
found in cars.
 Polycarbonate (PC) – Compact discs, eyeglasses, riot shields, security
windows, traffic lights, lenses.
 Polyvinylidene chloride (PVDC) – Food packaging.
 Polyethylene (PE) – Wide range of inexpensive uses including
supermarket bags, plastic bottles.
 Polycarbonate / Acrylonitrile Butadiene Styrene (PC/ABS) – A blend
of PC and ABS that creates a stronger plastic. Car interior and exterior
parts.

Bioplastics have been researched for many years as a substitute to synthetic

plastics. The manifestation of bio-feedstocks and bio-based commodity polymers
production, in tandem with rising oil prices, growing consumer awareness and
recuperating economics, has ushered a new and rousing era of bioplastic
commercialization. Bioplastics are not just one solo substance, they comprise of a
whole family of materials with differing properties and applications. The production
of biodegradable polymers from renewable resources is the need of the hour, in the
face of these ecological facts (Aarthi and Ramana, 2011).

The name biopolymer is presently used for polymers that are either
synthesized by living organisms or formed from substrates obtained from living
organisms. Bioplastics/green plastics may be derived from renewable biomass
sources, such as vegetable oil, corn starch, pea starch or micro biota and
biodegradable. Examples of the first class of biopolymers are naturally occurring
polymers such as cellulose; starch and polyhydroxyalkanoates (PHAs). Among the
second type, there are polylactic acid (PLA), that can be produced from biologically
attained lactic acid, or even polyethylene, produced from ethylene obtained from bio-
ethanol.

Bioplastics are biopolymers with plastic characteristics. Bioplastics

synthesized by living organisms are usually biodegradable; and chemically
synthesized polymers, especially those derived from petroleum, are generally non-
degradable, while those that are “bio-based” (i.e. acquired using a biologically
produced substrate such as bioethanol), have several degrees of biodegradability
polyethylene and polypropylene, whether bio-based or not, are considered non-
biodegradable, even when there have been claims of slow degradation of these
polymers in nature (Corti et al., 2010).

There are expectations to the connection between biological origin and

biodegradability, as not all biopolymers are biodegradable and not all biodegradable
polymers. They are certain plastics obtained from non- biological processes that can
also be biodegraded, such as poly(Caprolactone) and the petroleum derived polymer
poly (butadiene adipate-co-terephthalate) (Queiroz and Collares - Queiroz, 2009).

Some of the naturally occurring essential precursors for bioplastics could be

cellulose, starch, polylactic acid (PLA) and polyhydroxyalkanoates (PHAs). These
materials embrace advance advantages over synthetic plastic precursors as they are
biodegradable and are produced from renewable sources (Bagheriasl, 2012).
Bioplastics are plastic material which are either bio-based, biodegradable, or feature
both properties.
 The potential of bioplastics while shape the future of the plastics industry.
Currently, many diverse biodegradable bioplastics are available, amongst these are
blends having natural polymers, such as starch and cellulose; and polymers
synthesized chemically from different substrates, such as poly (lactic acid), poly
(Caprolactone), and others (Rehm, 2010).

Bioplastic produced by using food grade raw materials which are generally
food products are less attractive because this would severally affect the food chain
and it is highly undesirable by considering scarcity of food resources for constantly
growing world population. PHAs are natural biopolymers produced by numerous
bacteria from different substrates. In sharp contrast to the former bioplastics
mentioned above, these polymers are totally biodegradable, as all microorganisms that
naturally accumulate PHAs can degrade them. Moreover, PHAs can also be degraded
by many other microorganisms, both bacteria and fungi, under either aerobic or
anaerobic conditions. Development and application of bioplastics is still in its infancy
stage but holds great promise for sustainable and eco-friendly future. Considering
dearth of biodegradable plastics, and unfortunately most of the alternatives compete
with food/ feed assets, so production of bioplastic from microbial sources especially
using low cost agro-wastes would be very useful and significant. These biodegradable
plastic materials can retain the desired material properties of conventional synthetic
plastics, and can be completely degraded without leaving and undesirable residue.
The global bioplastics production capacity is set to grow 400% by 2017
(www.Corbion.com/media/.../eubp-facts 400% figures- bioplastics- 2013.pdf).

Polyhydroxyalkanoates (PHA) has gained major attention due to their

structural diversity and close analogy to plastics. Other types of bioplastics are formed
directly or indirectly use food resources for their production but PHAs are produced
directly by microorganisms, preferable bacteria under stringent conditions.
Polyhydroxyalkanoates (PHA) in bacteria had been known since 1926, when Maurice
Lemoigne observed that Bacillus sp. produces an intracellular polymer of
hydroxybutyrate monomers, later called polyhydroxybutyrate. This initial observation
got intensified during 1970’s when the world was facing acute petroleum crisis, and a
scientific movement aimed at discovery of alternative sources of fossil fuel reserves
was undertaken (Prieto, 2007).
 Structure and synthesis of PHA granules: First investigations on purified PHA
granules were done by Williamson and Wilkinson (1958) and also by Griebel et al.,
(1968) who demonstrated that the granules contained proteins and lipids besides the
PHB. Recent studies on the structure of PHA granules and granule-associated proteins
have been especially carried out in Pseudomonas oleovorans by Witholt and co-
workers (De Smet et al., 1983).

Bacterial PHA granules are mostly between 200 and 500 nm in diameter
(Anderson and Dawes, 1990) with thickness of membrane 15-20 nm (Lundgren et al.,
1964). Chemical analyses have shown that inclusion bodies contain approximately
97.5% PHA, 2% protein, and 0.5% lipid (Griebel et al., 1968).

PHA synthase or PHA polymerase (phaC) since the cloning of the PHA
biosynthesis operon from W. eutropha H16 about 27 years ago (Schubert et al., 1988;
Slater et al., 1988; Peoples and Sinskey, 1989). More than 60 different PHA synthase
genes have been sequenced from different bacteria (Rehm and Steinbuchel, 2002).

PHAs are synthesized by many living organisms (Anil-Kumar et al., 2007;

Halami, 2008). The main candidates for the large-scale production of PHAs are plants
and bacteria. Plant cells can only cope with low yields [<10% (w/w) of dry wt.] of
PHSA production. In contrast within bacteria, PHAs are accumulated to levels as high
as 90% (w/w) of the dry cell mass (Verlinden et al., 2007)

These are produced by wide range of bacteria as energy storage compounds

especially during limited nutritional supplies and in the presence of excess carbon
source (Kulkarni et al., 2011; Hungund et al., 2013).

PHAs due to their thermoplastic and elastomeric properties can be transformed

into plastic like materials similar to petrochemical derived plastics, but fully
biodegradable. Ecological awareness has impelled the development of new
biodegradable materials.

In PHA structure R can be hydrogen or hydrocarbon chains of up to around

C13 in length, and X can range from 1 to 3 or more. Varying X and R provides a
broad range of physical and mechanical properties, such as hydrophobicity, glass
transition temperature (Tg), melting point (Tm), and level of crystallinity which can
range from around 70% to very low, giving tremendous stiffness or elasticity as
required (Zhang, 2010).

PHAs are the only waterproof thermoplastic materials available that are fully
biodegraded both in aerobic and anaerobic environments. PHAs are polyesters formed
by poly condensation of carboxylic acids with hydroxyl alcohol.

PHA synthases (phaC) are key enzymes for PHA biosynthesis, which catalyze
the polymerization of 3-hydroxyacyl-CoA (3HACoA) substrates into PHA polyesters
with a concomitant release of CoA. PHA synthases have been cloned and assigned
into four classes based on the primary structures and the substrate specificities (Amara
and Moawad, 2011).

Applications of PHA

Besides being a thermoplastic with properties comparable to that of

polyethylene, PHAs are also completely biodegradable. The ability to produce PHAs
from renewable carbon sources also ensures a sustainable ‘green chemistry’ process.
Much work has been directed to the production of various types of PHAs for
applications as commodity plastics. The ability to further chemically modify the
functional groups in these PHAS broadens their scope of application as biodegradable
polymer as well as bio absorbable materials for biomedical purposes.

Tissue engineering: For application as tissue engineering scaffolds, the

suitable material must possess properties such as biocompatibility, support cell
growth, guide and organize the cells, allow tissue ingrowth and should finally degrade
to non-toxic products (Williams et al., 1999).

Impacts of plastics and Importance of PHA

In the modern society, plastic are the main machineries for the advancement of
the country but they also cause some environmental hazards due to lack of proper
utilization. Side effects: 1. Soil decay, 2.Declining of land fertility, 3. Drainage
problems.

Controlling managements: 1.Recycling of plastics, 2.Appropriate employment,

after using one should through the polymer materials into dustbins, etc. 3.
Amalgamation of biodegradable plastics, i.e., non- biodegradable plastics can be
blended with biodegradable plastics which can be decayed easily by the presence of
microorganisms on or under the ground.

High molecular weight polymers play an important role in nature as structural

or storage components (e.g., cellulose and starch). Other polymers are of immense
importance for technical application (e.g., plastics such as polyethylene or
polystyrene).

Recently, science and industry have focused on the development and

application of biodegradable plastic materials. Biodegradable plastics are defined by
the American Society for Testing and Materials as degradable plastics in which the
degradation results from the action of naturally occurring microorganisms such as
bacteria, fungi, and algae (American Society for Testing and Materials 1992). Our
increased perception of today’s ecological problems as well as legislative pressure
have led to the introduction of these materials. According to a survey, two thirds of
the American population agree with the assertion that plastics pose the greatest threat
to the environment because of their non - biodegradability (Rogers, 1990).
 Review of Literature

Plastic

Plastic, made up of chemicals, is very hazardous for the environment and

additional energy is also required for its extraction and processing. These xenobiotic
plastics are now posing a great threat to humanity and other life forms by
contaminating the land, water, soil and overall environment (Luengo et al., 2003).

Plastic is almost non- biodegradable and has recalcitrant in tons every year due
to all types of anthropogenic activities. Synthetic polymer i.e. plastics are utilized in
every manufacturing industries i.e. printing, coating, food packaging, building
construction products, toys, electric wires and medical devices i.e. gloves, synthetic
catheters etc. The structure of these polymers can be easily modified chemically and
morphologically to different shapes and of varying strength as a fiber or as thin sheets
to suit array needs. This unique property of modification increases the widespread
application of synthetic plastics in different fields. It has high durability and is
resistant to various chemicals (Atlas, 1993).

According to the ‘Oxford Dictionary’ word ‘plastic’ was coined in the mid of
17th century and derived from French ‘Plastique’, Latin ‘Plasticus’ or form Greek
‘Plastikos’/ ‘Plassein’ meaning of all these cognates is ‘able to be molded into
different shapes’ (Joel, 1995).

In 1862, the man made plastic named as “parkesine” was announced by

Alexander parks at International Exhibition in London. This synthetic plastic was
chemically made up of manufacturing based industries affected the lives of people, by
introducing improved packaging material to chemically based textile goods (Flechter,
1990). The man made plastic being a xenobiotic, becomes highly resistant to
microbial degradation (Atlas, 1993)

In 1868 John Wesley Hyatt invented celluloid, derived from cellulose and
alcoholized camphor that could be molded with heat and pressure into a durable
shape. By 1900, movie film was an exploding market for celluloid (Harris, 1981).
 Therefore, much more efforts are required for the development of natural or
biodegradable polymers and to provide a sustainable benefit to both the environment
and living beings (Willett and Shogren, 1995).

Naturally derived renewable resources including green plastics are the topic of
interest for contemporary scientists as it is a substitute of traditional chemical based
plastics. The green plastic must have following properties i.e. it should be derived
from renewable source, biodegradable in nature and must be eco-friendly (Stevens,
2003).

Growth in the human population has let to the accumulation of huge amounts
of non-degradable waste materials across our planet. The accumulation of plastic
wastes has become a major concern in terms of the environment (Guillet, 2002;
Derraik, 2002; Thompson et al., 2004).

Conventional plastics not only take many decades to be decomposed in nature,

but also produced toxins during the process of degradation. For this reason, there is
special interest in producing plastics from materials that can be readily eliminated
from our biosphere in an eco-friendly fashion (Gross and Kalra, 2002).

Currently many industries are highly dependent on fossil fuels as a source of

energy for the production of structural materials like (plastics, foams, coating and
adhesives). Based on recent usage current trends evidence suggested, that an alarming
rate of depletion of these valuable fossil fuels (Zagar, 2000).

Worldwide approximately 140 million tons of plastics per annum is being

consumed. Processing of these plastic used approximately 150 million tons of fossil
fuels, which are difficult to substitute. The challenge to the world is whether we can
substitute the fossil fuel with a sustainable one or not. In this context attention has
focused on research for the production bioplastics (Ajumu et al., 2000).

Bioplastics

Biopolymers are the plastics that are biodegradable and are produced from
bio-based source (i.e.) they are produced from renewable sources. Biodegradability of
the plastic depends on their chemical structure; some plastics are bio-based but are
non-biodegradable. There is a strong carbon-carbon single bond existence in the
structure of certain polymers (i.e.) polyethylene’s are derived from renewable masses
such as sugarcane. The strong bonding of carbon in their structure makes it non-
biodegradable, yet it is recyclable and is produced from bio-based source (Phillips,
2008).

Plastics were reported in literature that the share of biopolymers in the market
is less than 1%. In year 2015 the expected growth rate of the biopolymers is almost
just over 1%. The polymers whose physical and chemical properties undergo
deterioration and are completely degraded by microbial species are termed as
biodegradable polymers. The biodegradable nature of polymers is due to the inherent
oxygen or nitrogen atoms in their backbone chain. It was investigated by American
Society for Testing and materials (ATSM) that biodegradable polymers could be
classified as compostable material only, if it should yield carbon dioxide inorganic
compounds and water at the rate paralleled with the other knoen compostable
materials (Stevens, 2002).

For number of years, the development of bio-based innovative polymers has

been in progress and a topic of interest by various nano technologists, material
designers and advance material developers. The data analysis shows that the
shipments from Canadian bioplastic industry enhanced by 10.6% from 1995 levels to
9.1 billion in year 1996. Leaver such in 2002 reported that the demand for
biodegradable plastic per year was increased by 30% (Charron, 1999).

Economic concerns for the development of biopolymers must be taken in

concern as the cost competitiveness and the ability of the society to pay for it is the
major factors on which the future of the product depends. Sustainability is also a
major factor connected directly with development of the product. Biodegradable
biopolymers are the source materials that can solve problems, both related to
environmental concerns and can also be significant in numerous other fields. They
can replace plastic either alone or by blending with materials which can enhance their
important physical properties. Polyhydroxyalkanoates is a family of such type of
biodegradable biopolymers (Luengo et al., 2003).

Bioplastics are natural biopolymers that are synthesized and catabolized by

various organisms (Akar et al., 2006; Kragelund et al., 2005; Tokiwa and Calabia,
2004; Kim and Rhee, 2003; Jendrossek and Handrick, 2002). And these materials do
not cause toxic effects in the hits and have certain advantages over petroleum-derived
plastic (Chen and Wu, 2005a, b; Steinbuchel, 2005; Reddy et al., 2003; Williams and
Martin. 2002; Zinn et al., 2001; Hunkeler, 1999; Steinbuchel and Fuchtenbusch,
1998). These biopolymers accumulate as storage materials in microbial cells under
stress conditions (Berlanga et al., 2006).

The most widely produced microbial bioplastics are polyhydroxyalkanoates

(PHAs) and their derivatives (Madison and Huisman, 1999; Witholt and Kessler,
2002; Kim and Lenz, 2001). Beijerinck first observed lucent granules of PHA in
bacterial cells in 1888 (Chowdhury, 1963).

The composition of PHAs was first described by Lemoigne as a

homopolyester of the 3-hydroxybutyric acids, called polyhydroxybutyrate PHB
(Lemoigne, 1926).

Macrae and Wilkinson (1958) reported the rapid biodegradability of PHB

produced by Bacillus megaterium and Bacillus cereus from that time, the interest in
PHB increased dramatically. Later, the research on PHB and other forms of PHAs
began and the potential use of these biopolymers was realized (Braunegg et al., 1998;
Volova, 2004; Scott, 2005; Noda et al., 2005; Pandey et al., 2005; Ren et al., 2005).

Biodegradable plastic is new and very interesting because of its actual

utilization of bacteria to form a biopolymer. Bioplastics are a special type of
biomaterials, derived from plant sources or microbial sources, rather than traditional
petrochemical. Though bioplastic have been made from both plants and microbes, we
focus on microbial bioplastics. Microbial plastics are polyesters that are produced by
a range of microorganisms cultivated under various growth and nutrient conditions.
This polymer accumulates as a storage material such as mobile, amorphous, lipid
granules meant for microbial survival under stressful conditions (Ningthoujam, 2009).

Bacterial plastic is usually defined as an exciting new area of research, where

naturally synthesized bacterial polymer as, lipid storage material PHB is being used as
raw materials for plastic based packaging materials (Madigan et al., 1997).
Historical background

Beijernick first observed PHAs as refractile bodies inside bacterial cells in

1888. However, PHA composition was established by Lemoigne only in 1926.
Though the promises of these biopolymers were recognized in the 1960s, its possible
exploitation was seriously considered only in the late 1970s. In 1976, ICI of England
explored if PHB could be satisfactorily produced by microbial fermentation. In 1993,
Zeneca bioproducts took over ICI’s activities and in 1996 Monsanto bought the
bioplastic production business from Zeneca Monsanto closed down its activities in
1998 but many new players became active in this field since than some prominent
ones are metabolic, Proctor and Gamble, Dupont, General motors, and Toyota etc.
BIOPOL is marketed bioplastic product (Ningthoujam, 2009).

Types of bioplastics

Polyhydroxyalkanoates (PHAs), a type of microbial polyesters, are the best

studied group of biodegradable plastics. Some of the major types of bioplastics are:

Soy-based bioplastic, Starch based bioplastic, Polylactides (mostly from

plants), Poly 3-hydroxy butyrate (PHB), Poly 3-hydroxyalkanoate (PHA), Aliphatic
polyesters, Polylactic acid (PLA).

These have been developed successfully over the few years to meet specific demands
in various field and industries (Byrom, 1991; Chang, 1994; Lee et al., 1994; Lee2,
1996).

These materials offer solution to managing of waste sand in some cases, good
substitute for conventional plastic where mechanical properties are desired (Lee 2,
1996; Ojumu et al., 2004).

Polyhydroxyalkanoates

PHAs are the polymers of hydroxyalkanoates that accumulate as storage

material in many microorganisms such as Bacillus spp, Rhizobium spp, Alcaligenes
spp, Pseudomonas spp, Azotobactor spp, etc. They are attaining considerable
attention as biodegradable polymers and are emerging as a new branch of research
field (Salechizadch and Loosdrecht, 2004).
 A wide variety of microorganisms spread over, more than 90 genera of
bacteria are known to produce PHA (Steinbuchel, 1991; Findlay and White, 1983).
Lemoigne first discovered PHA in 1926 in Bacillus megaterium as hydrophobic
inclusions in the cytoplasm (Eversloh et al., 2001; Anderson and Dawes, 1990). These
inclusion bodies are of high industrial significance, which are optically active, easily
biodegradable with a melting point 180 c and have properties similar to polyethylene
(PE) and polypropylene (PP) (Lee, 1996; Sasikala and Ramana, 1996).

However their high price compared to thermoplastic has limited their use in
many areas. To reduce the cost and make PHAs competitive with the conventional
plastic, continued efforts are being made to improve the strain, growth conditions,
enhancement of PHA production, fermentation parameters, separation, recovery and
also in the genetic engineering aspects (Lee, 1996).

The cost of PHA depends on many factors such as substrate used, productivity
and the recovery process. PHA content of the biomass also effects the cost, as
increase in PHA content from 50% to 88% leads to lowering of recovery cost (Lee
and Choi 1998).

Occurrence of PHA in microorganisms bacteria are capable of accumulating

25-80% PHA of their cellular dry weight; cupriavidus necator formerly called as
(Alcaligenes eutrophus/ Ralstonia eutropha) is one of the high PHA yielding
bacterium, PHA synthesizing genes of which are completely sequenced and cloned.
Various groups of microorganisms such as phototrophs, chemotrophs and a few fungi
are also known to produce PHA. But Enterobacteria are among the organisms, which
do not form PHA, but PHA producing gene from Alcaligens eutrophus has been
cloned and expressed in E.coli (Slater et al., 1988; Schubert et al., 1998).

Many phototrophic organisms like cyanobacteria and chemotrophs are known

to accumulate PHA in considerable amount. Free living and symbiotic nitrogen fixing
bacteria namely Azotobacter spp, Rhizobium spp. also accumulate high concentration
of PHA (65-70%). PHA accumulations by bacteria such as Halophilic and
Archaebacteria have also been reported (Kshama and Shamala, 2003).

PHAs are formed as intracellular inclusions under unbalanced growth

conditions, which is described as poly (β-hydroxyalkanoate) containing different alkyl
groups. Several poly (β-hydroxyalkanoate) and copolymers can be produced under
controlled growth conditions, which have wide applications in medical and packaging
areas (Brandle et al., 1990).

As many as 150 different hydroxyalkanoic acids have been identified and

grouped as follows: 1. 3HA from 3-hydroxy propionic acid (3HP) to 3-hydroxy
hexanoic acid (3HHx). 2. Unsaturated 3-hydroxyalkanoic acids with one double
bond. 3. (3-hydroxy-4 pentanoic acid, 3-hydroxy-7-cis tetra decanoic acid) one or
double bonds (3 hydroxy 5, 8-cis-cis-tetra decanoic acid) in the R pendent group. 4.
Non-3HA such as 4-hydroxybutyric acid (4HB) or (4-hydroxy valeric acid). 5. 3HA
with different functional groups including free carboxyl groups such as malic acid,
esterified alkyl groups (3-hydroxy succinic acid methyl ester acetoxy groups and
other functional groups). 6. Hydroxyalkanoic acid with modification at third carbon
atom, which contribute to the backbone of the polyester are 3-hydroxyalkanoic acids
with a double bond such as 3-hydroxy-2-butenoic acid and various 3HA with methyl
group (Steinbuchel and Valentine, 1995).

PHAs are polymers with (R)-3-hydroxy monomer unit. The monomers of

short chain length PHA (SCL– PHA) consist of 3-5 carbon atoms and monomers of
medium length (MCL – PHA) consist of 6-14 carbon atoms. The molecular weight of
these polymers ranges between 2×105 and 3× 106 and number of monomers (n)
varies from 1,000 to 30,000. D (-) -3-hydroxy butyric acid (3HB) remained only
known constituent of PHA until constituents other than 3HB were reported, based on
chemical analysis 3HV and 3 HHX were detected later as constituents of PHA
produced Bacillus species (Findlay and White, 1983).

3-hydroxy octanoic acid (3HO) was detected as constituent of PHA in

Pseudomonas oleovorans (De Smet et al., 1983). Terminology of PHA with the
discovery of new hydroxyl alkanoic acids, terminologies used by different researchers
are as follows: 1. Poly (hydroxyalkanoates) [P(HA)] general. 2. Poly
(hydroxybutyrate-co-hydroxyvalerate) [P (HB-CO-HV)] is for copolymer. 3. Poly
(hydroxyl-butyrate-co-hydroxypropionate-co-hydroxyvalerate), [P(HB-COHP-CO-
HV)] for terpolyesters. 4. Short chain length PHA (SCL-PHA) 3-5 carbons. 5.
Medium chain length PHA (MCL-PHA) 6-16 carbons. 6. Long chain length PHA
(LCL-PHA) > 16 carbons (Sasikala and Ramana, 1996).
 Non-PHA with unsaturated or substitute side chains have not yet been
detected and also LCL-PHA is yet to be detected (Steinbuchel and Valentine, 1995).

Copolymer incorporation of different hydroxyalkanoates units into the P (HB)

results in PHA copolymer which has improved physical properties than the
homopolymer. Many bacteria are known to synthesize copolymer with other
hydroxyalkanoates of medium chain length depending on their intrinsic PHA
biosynthesis pathway and the carbon source. A copolymer of P (HB-CO-HV) has
been investigated most extensively among the PHA copolymers and applied to
commercial products. The incorporation of hydroxyl valerate (HV) into P (HB) results
in P (HB-CO-HV) (Marchessault, 1996).

The ring opening polymerization reactions of 4 membered lactones in the

presence of a catalyst, can lead to chemical synthesis of PHA. High molecular weight
P(HB) and P(HV) were synthesized from butyrolactone and valerolactone,
respectively. Propionaldehyde and malonic acid were reacted first to form pentanoic
acid via knoevenager reaction followed by bromination and lactonization yielding
valerolactone, which was polymerized by a suitable catalyst to get P(HV) (Sasikala
and Ramana, 1996).

Metabolism PHA is an intracellular material synthesized due to unbalanced

nutrient condition during the stationary phase of growth when the cells become
limited of essential nutrients but have an excess of carbon source (Dawes and Senior,
1973).

During nutrient limiting condition NADH accumulates in the cell and exerts
feed back repression on enzymes of the TCA cycle. Acetyl coenzyme A (Co A)
accumulates and acetyl Co A acetyl transferase is induced to initiate P (HB) synthesis
(Page, 1989).

Polymerization of acetyl Co A consumers NADH and P (HB) synthesis

acquires the role of an electron sink (Tsuge, 2002). PHA biosynthesis pathway
mainly there are three naturally occurring PHA biosynthesis pathways in bacteria
synthesis of PHA monomers from acetyl Co A is the common metabolic pathway
found in many bacteria.
 PHAs are biodegradable biopolymers that are produced by various bacteria
due to nutrient depletion conditions and they are stored intracellular as energy reserve
(Abe et al., 1990).

Polyhydroxybutyrate (PHB) which is one of the PHA homopolymer, is

commonly found in various bacterial genera and it was first discovered by Lemoigne
in 1926 in Bacillus sp (Lemoigne, 1926).

PHAs produced by bacteria are broadly classified into two groups: 1) Short-
chain-length PHAs (SCL-PHA) that mainly consists of monomers containing 4 to 5
carbon atoms, 2) Medium-chain-length PHAs (MCL-PHA) which contain 6 to 14
carbon atoms (Dekonig, 1993).

Commercially PHA is available as SCL-PHA which is a copolymer of

hydroxybutyrate and hydroxyvalerate – P (HB-CO-HV). P (HB), which is commonly
produced by bacteria, is crystalline, brittle and has poor film forming and mechanical
properties (Hocking and Marchessault, 1994).

PHA are accumulated in the cells as discrete granules, the size and number per
cell vary depending on the different species. The granules appear as highly refractive
inclusions under electron microscopic observation. In Alcaligenes eutrophus, 8 to 13
granules were observed per cell, with diameter ranging from 0.2 to 0.5µm (Byrom,
1994).

As PHAs are insoluble in water, the polymers are accumulated in intracellular

granules inside the cells and the polymerization of these soluble intermediates into
insoluble molecules prevents the leakage of valuable compound out of bacterial cell
(Peters and Rehm, 2005).

Phospholipids and proteins form a layer over the surface of a PHA granule and
in the interface of granules the most dominant compound seen is phas in a class of
proteins known to influence the number and size of PHA granules (Potter et al., 2002;
Potter and Steinbuchel, 2005).

Biodegradability of PHA

The property that distinguishes PHA from petroleum based plastics is their
biodegradability. PHA are degraded upon exposure to soil, compost, or marine
sediment. Biodegradation is dependent on a number of factors such as microbial
activity of the environment, and the exposed surface area, moisture, temperature, pH,
molecular weight (Boopathy, 2000). For PHA, polymer composition and crystallinity
also assume importance (Lee, 1996b).

The nature of the monomer units also has been found to affect degradation.
Copolymers containing PHB monomer units have been found to be degraded more
rapidly than either PHB or 3HB-co-3HV copolymers. Microorganisms secrete
enzymes that break down the polymer into its molecular building blocks, called
hydroxyacids, which are utilized as a carbon source for growth. The principal enzyme
for degradation of PHB and oligomers derived from the polymer is PHB
depolymerase. Studies on the extracellular PHB depolymerase of Alcaligenes faecalis
have indicated it to be an endo type hydrolase. Other prominent organisms in which
PHB depolymerase has been identified and worked upon are Rhodospirillum rubrum,
B. megaterium, A. beijerinckii, and Pseudomonas lemoigne. Biodegradation of PHA
under aerobic conditions results in carbon dioxide and water, whereas in anaerobic
conditions the degradation products are carbon dioxide and methane.

Properties of PHA

PHAs differ in their physical and chemical characteristics due to their

monomer content. The chain length of the monomer influences hydrophobicity,
melting point, glass transition temperature and degree of crystallinity (Crank et al.,
2004).

The PHB structure has been found to show similar degree of crystallinity to
polypropylene (Holmes et al., 1985). And was the major reason for the gaining so
much of interest as alternatives to petrochemical plastics. MCL-PHAs on the other
hand are elastomers with low crystallinity and higher elasticity but their tensile
strength is low while their elongation to breaking point is high (Doi et al., 1995).

Copolymers like PHBv, PHBHx etc. are less stiff then PHB and retain the
most of the mechanical properties of PHB (Padarmshoke et al., 2004). Depending on
the percentage of different monomers incorporated into these copolymers they are
hard crystalline to elastic.
 a) Physical properties

The physical property of P (3HB) has been well studied and compared with common
commodity plastics. The mechanical properties of P (3HB), i.e., the young’s modulus
(5GPa) and the tensile strength (43MPa) are comparable to those of polypropylene
(PP). However, the elongation to break for P (3HB) (5%) is poor compared to that of
PP (500%). Therefore, P (3HB) is stiffer and more brittle in comparison to PP.
Although natural P (3HB) is stiff and brittle, the P (3HB) film prepared using cold-
drawing procedures have better mechanical properties (Iwata et al., 2003).

b) Mechanical properties

The mechanical properties of P (3HB-CO-3HV) are dependent on the molar

ratio of 3HV as described earlier. The value P (3HB-CO-3HV) becomes more
flexible. At the same time, these copolymers are tougher due to the increase of
impact strength. In general, the tensile strength decreases gradually as the 3HV molar
ratio increases. The incorporation of 4HB monomers results in copolymers having
various physical properties that range from highly crystalline to elastomeric (Saito et
al., 1994).

PHA production in microorganisms

Growth conditions Accumulation of PHA in bacteria proceeds under nitrogen

or phosphorus limiting condition and in the presence of excess of carbon. It is difficult
to compare PHA produced by different organisms due to the wide range of PHA,
diversity in production, variety of substrates and different growth conditions.
Optimization of growth conditions of microorganisms is very important to get
maximum PHA yields. Grothe and coworkers (1999) investigated the effect of
temperature, pH and ionic strength of the medium, type of nitrogen source, carbon
and nitrogen ratio on PHA production. Optimum temperature for the growth of
Alcaligenes latus and for the PHA synthesis appeared to be 33ºC. They also found
that initial pH of 6.5 increases the growth rate of A. latus and it was 0.075gh-1,
maximum sucrose utilization was 0.38gh-1, where 63% of P (HB) was accumulated
(Grothe et al., 1999).

Optimization of nutrient limiting condition and PHA production by Rhizobium

meliloti and its mutant strain has been reported using urea as nitrogen source to obtain
60% of PHA (Kshama et al., 2004). A. eutropus, has been used for optimal
production of P (HB), a homopolymer which is accumulated under nitrogen limitation
(Morinaga et al., 1978). Azotobacter beijerinckii produces PHA under oxygen
limitation compared to nitrogen or phosphorus limitation (Ward et al., 1977).

A wide variety of PHA copolymers are synthesized in Bacillus spp. from

fermentation of different carbon sources (Valappil et al., 2007; Anil Kumar et al.,
2007; Labuzek and Radecka 2001).

Besides the importance of nutrients and carbon and other physical growth
parameters (pH, temperature), other survival mechanism of microorganism seems to
play a significant role in the production of PHA. PHA supports the survival of
microorganisms under stress conditions such as osmotic pressure or UV radiation
(Brandl et al., 1990).

In general, cells containing PHA have a better survival rate than others and
PHA also plays physiological function during the sporulation in Bacillus (Williamson
and Wilkinson, 1958) and encystment in Azotobacter (Dawes, 1974).

Substrate and Precursor for PHA synthesis PHA production is based on the
substrate used and it has received attention as they grow on renewable resources
where bacteria can utilize waste generated from food, agricultural and industrial
process and fatty acids as carbon source. High carbon content substrates are used for
the growth of the organism. Sugars such as glucose and sucrose are main carbon
sources used in PHA production.

Carbon sources Beet molasses, maltose, corn syrup, cane molasses, malt
extract, and palm oil mill effluents have been used as substrates for polymer
production. Type of PHA produced depends on the organism and the substrate used.
PHA production from glucose and sucrose has been optimized. The development of
technology by using cheaper carbon sources would be a key factor for further
reducing the PHA cost (Tsuge, 2002).

The cost of the carbon source contributes significantly to the overall

production of PHA (Yamane, 1992). Use of cheaper carbon source can lower the
production cost. When glucose was used at a concentration of 157gl-1, PHA content
was 77%, with productivity of 3.2 gl-1h-1 and the cost of carbon source was 38% of
the total production cost (Choi and Lee, 1999).

When cornstarch was used as a substrate under the same conditions,

production obtained was 1.19gl-1, and the cost of carbon source was 15% lower
compared to glucose. Crude carbon substrates such as cane molasses, beet molasses,
whey, plant oils; starch (corn and tapioca), cellulose and hemicelluloses can be used
as excellent substrates for the growth and polymer production by bacteria. The
copolymer production also depends on the substrate and the organism used. The co
substrate acts as a precursor for the polymer production.

High Cell Density cultivation High-density cultivation has been carried out to
enhance PHA productivity with reduced culture time and increased cell concentration
and the type of copolymer. This not only increases the cell concentration but also
reduces cost of downstream processing (Yamane et al., 1996).

Fed batch culture has been the most popular culture system to reach high cell
density and P (HB) content (Kim et al., 1994; Suzuki et al., 1986b).

In high cell density culture, optimal concentration of nutrients, pH, dissolved

oxygen, antifoam, agitation, and aeration are maintained during fermentation (Kim et
al., 1992).

Yamane et al., 1996 carried out fed batch culture of A. latus to achieve the
high cell density to reduce the culture time, where in the sucrose solution and
inorganic medium with the NH3 solution was fed periodically during fermentation.
This resulted in 142 gl-1 of cell and 68.4 gl-1 of P (HB) in 18 h with initial cell
concentration of 13.7gl-1. Methanol has also been used as a sole carbon and energy
source for the production of P (HB) by Methylobacterium extorquens. Methanol was
added to the medium as a carbon source (0.01gl-1) and P (HB) obtained was 46% of
the total biomass weight (Bourque et al., 1995).

Pseudomonas oleovorans was also grown to obtain mcl-PHA using n-octane

as carbon source leading to maximum biomass concentration of 37gl-1 by feeding
nitrogen in combination with various metal ions. Final cell density achieved was 112
gl-1 but PHA content was low (Kellerhals et al., 1999).
 Efficient production of intracellular fermentation products requires both fast
growth of the organism and accumulation of cells to high cell concentration and high
yield. Recombinant E. coli containing A. eutrophus PHA biosynthesis gene has been
employed for the production of P (HB) by fed-batch to get 77 gl-1 with the
productivity of 2 gl-1 h-1 (Wang and Lee, 1998).

Application

The ability to produce PHAs from renewable carbon sources and the use of
specialized carbon sources for tailor made polymer biosynthesis ensures a sustainable
green chemistry process which can result in polymer with distinctive properties.
PHAs are hydrophobic, oxygen impermeable and can be used for biodegradable
packaging materials such as compost bags and food packaging (Lee, 1996a).

The films can be used as laminates for the manufacture of compostable

personal hygiene articles. PHAs can be woven into fibers as well as to construct
materials such as nonwoven fabrics. PHB and its copolymers such as P (3HB-CO-
3HV) can also be used as hot melt adhesives (Madison and Huisman, 1999).

Addition of tackifiers and its cross-linking using UV radiation was found to

improve the strength of the PHAs resulting in pressure sensitive adhesives.
Unsaturated PHAs can be used for the manufacture of biodegradable rubber using
cross-linking chemicals such as sulfur or peroxides and electron beam source (Lenz
and Marchessault, 2005).

The use of tall oil, linseed oil, rape seed oil as substrates are used for
manufacture of low molecular weight MCL-PHAs finds application as low viscosity
paints as well as solvent free paints (Chen and Wu, 2005).

Materials with better tear resistance, tensile strength and flexibility can be
obtained by varying the cross-linking conditions (Henne et al., 1999).

PHAs can be used to replace petro-chemical polymers in toner and developer

compositions or as ion conducting polymers. They can also be used as latex for paper
coating applications as well as to produce dairy cream substitutes or flavor delivery
agents in foods. Cheese coatings is technically demanding process which involves
number of functions such as mechanical and hygienic protection, semi-permeability
for water, co2 and other flavoring components, easy applicability, long stability etc.,
PHAs can be used to replace synthetic plastic based latex coatings for cheese which
enables functional protection like ripening control, mechanical and bacterial
protection (Vander Walle et al., 2001; Sudesh and Doi, 2005).

Polymer blending is considered advantageous for commercialization of PHB

and helps to modify the required properties using conventional technology at low
cost. The blending of polymers capitalizes the maximum possible performance of the
blend (Yu et al., 2006).

The blends of PHB with other oligomers or with flexible polymers with
plasticizers can improve the material properties such as impact strength, amphilicity
and biodegradability. PHB has been found to be miscible with poly(ethylene oxide),
poly(vinyledene fluoride), poly(vinyl acetate), poly(epichlorohydrin) as well as
partially miscible with poly(vinyl alcohol), poly(L- lactide) and poly(caprolactone)
(Ikejima et al., 1999); (Zhao et al., 2004); Xu et al., 2006).

These films were reported to show improved ‘elongation to break’ property

and smooth surface useful for tissue engineering applications. PHAs is also being
explored for biomedical applications such as wound management drug delivery,
urological stents, barrier material for guided tissue regeneration in periodontitis and
computer assisted tomography and ultra sound imaging (Zinn et al., 2001).

PHB is optically active and solutions containing them can rotate the plane
polarized light. The unique chiral geometry has not been exploited for commercial
use. The hydrolyzed PHB polymer was reported to be active only in its chiral form
and the material could be used as building block in the organic synthesis of many
drugs (Xu et al., 2006).
 Materials and Methods

Collection of samples and isolation of bacteria

Different types of soil samples were collected from College campus in Theni
(District). 1g of soil Sample was suspended in 10 ml sterile distilled water and it was
diluted serially from 10-2 to 10-6. Aliquots 0.1 ml from each dilution was spread over
the surface of nutrient agar plates. The plates were incubated at 37ºC for 24 hours.

Screening for PHA- production bacteria

Rapid screening of isolates for PHA production by plate assay method

The bacterial colonies were examined for PHA accumulation by staining with
Sudan Black B (0.3 g in 70% ethanol) by using rapid screening method. All the
isolates were qualitatively tested for PHA production using Sudan Black B dye.
Ethanolic solution of (0.05%) Sudan Black B was spread over the colonies and the
plates kept undisturbed for 30 minutes. They are washed with ethanol (96%) to
remove the excess stain from the colonies. The dark blue colored colonies were taken
as positive for PHA production.

Identification of Bacteria

The identification of bacteria was performed on the basis microscopic

examination and biochemical test according to Bergey’s Manual of Determinative
Bacteriology.

Microscopic Observation

Gram’s staining

The bacterial isolates were identified morphologically based on their shape

using Gram’s staining and the isolation were categorized as either gram positive or
gram negative organisms. The glass slide was cleaned. A thin smear of the given
culture was made. The smear was air dried and heat fixed.
Motility test

Motility of the organism was observed by the hanging drop method. A cavity
slide and the cover slip were cleaned and a small amount of Vaseline was placed in
each corner of the cover slip. In the center of the cover slip a drop of bacterial culture
was placed and observed under the microscope. If the cell movement is there it
indicates motile otherwise non-motile.

Spore staining

Smear was prepared on a glass slide and subjected to malachite green and kept
in water bath for 20 minutes. Then the slide was washed and subjected with safranin
for 30 seconds and the stain was washed immediately and allowed the slide to air dry
and observed under the microscope.

Biochemical Characterization

The isolated organisms were subjected to various biochemical tests such as:

Indole Production Test

The indole production was performed to bacterial isolates to determine the

ability of the organisms to split indole from the amino acid tryptophan. A loop full of
isolated bacterial culture was inoculated aseptically into the tryptone broth and
incubated for 24 hours at 37℃. After incubation 1ml of Kovac’s reagent was added
and the results were observed. If it developed red and pink color ring on the top, it
indicates indole positive. Whereas, if there is no pink color it indicated indole
negative result.

Methyl Red Test

MR-VP medium was prepared and 5 ml of medium was transformed in test

tube and sterilized. A loopful of the culture was inoculated in MR-VP medium and
inoculated at 37℃ for 24 hours. After incubation methyl red reagent was added and
the results were observed. If it is shows red color formation, indicates positive result
or if there was no red color it indicates negative.
Voges-Proskauer Test

Voges-Proskauer test is used to detect acetone in a bacterial broth culture. The

test is performed by alpha naphthol and potassium hydroxide to the Voges-Proskauer
broth which has been inoculated with bacterial culture. The isolates were inoculated
in MR-VP broth and the tubes were incubated for 24 hours at 37℃. After incubation,
1ml of 4% potassium hydroxide and 3ml of 5% of naphthol solution in absolute
ethanol was added and the result were observed. If it shows pink in color it indicates
positive result where absence of pink color. It indicates VP negative result.

Citrate utilization Test

Citrate Utilization test is used to detect the ability of bacterial to utilize citrate
as a carbon source. The Simmons citrate agar was prepared and inoculated with the
isolated culture and incubated at 37℃ for 24 hours. After incubation the result were
observed. If it changes from green color to blue. It indicates citrate positive. Whereas,
it remains green in color. It indicates citrate negative.

Catalase Test

Catalase test is used to detect the availability of catalase production by the

organisms. Transfer small quantity of culture to glass slide. Add one drop of 3%
Hydrogen Peroxide (H2O2) on the culture with the help of Pasteur pipette and
observed for bubble formation. The bubble is denoted as catalase positive. Absence of
bubbles is denoted as catalase negative.

Oxidase Test

A loopful of culture was placed on an oxidase disc (B-Dimethyl aniline) and

observed the result. If it shows violet in color, it indicates positive. Whereas, if there
was no violet color formation, it indicates oxidase negative.

Starch Hydrolysis Test

Starch agar plates were prepared and the test organisms were
inoculated with a single line streak in the Centre of the plate. The plates were
inoculated at 37℃ for 24 hours, after incubation iodine solution was added and the
result was observed. If it shows zone formation around the bacterial culture, it
indicates positive result. Whereas, if there was no zone formation, it indicates
negative result.

Gelatin hydrolysis test

Gelatin agar medium was prepared. The medium was poured into the sterile
petriplates. The isolates were inoculated separately into the plates and incubated at
30±2ºC for 4 days. After the incubation, HgC12 solution was flooded over the
medium. The formation of clear zone around the culture is indicates the positive
results.

Triple Sugar Iron Test

Triple sugar iron agar medium was prepared. The medium was poured into the
sterile test tubes and allowed to solidify. The cultures were inoculated into the tubes
and incubated at 30±2ºC for 24 hours, the result were noted.

Hydrogen sulfide production test

SIM agar medium was prepared. The medium was poured into the sterile test
tubes. The isolates were inoculated separately into the test tubes and incubated at
30±2ºC for 4 days. The hydrogen sulfide production was observed.

Optimization of PHA producing bacteria

Optimization for PHA production at various Incubation time by Bacillus sp. and
Rhizobium sp.

Effect of incubation time on PHA production was determined by using

bacterial isolates in minimal medium. The culture was inoculated and incubated at
37ºC for different interval of time such as 12, 24, 36, 48 and 60 hours for PHA
production. After incubation, 10 ml culture was drawn and estimated for the PHA
production by using the formula

PHA accumulation (%) = Dry weight of PHA (g/l) / Cell dry weight g/l) *
100.
Optimization for PHA production at pH by Bacilus sp. and Rhizobium sp.

Effect of pH on PHA production was determined by using bacterial isolates in

minimal medium with variable pH (adjusted 5, 6, 7, 8 and 9). The culture was
inoculated and incubated at 37ºC for 48 hours for PHA production. After incubation,
10 ml culture was drawn and estimated for the PHA production

Optimization for PHA production at various temperature by Bacillus sp. and

Rhizobium sp.

Effect of temperature on PHA production was determined by using bacterial isolates

in minimal medium. The culture was inoculated and incubation at different
temperature such as 30ºC, 35ºC, 40ºC, 45ºC and 50ºC for PHA production. After
incubation, 10 ml culture was drawn and estimated for the PHA production.

Optimization for PHA production at Carbon sources by Bacillus sp. and

Rhizobium sp.

Carbon sources such as Glucose, Sucrose, Maltose and Dextrose were used as
a carbon sources to induce the PHA production at a various concentration like 0.1%,
0.2%, 0.3%, 0.4% and 0.5% in minimal medium. After media preparation, the culture
were inoculated and incubated at 37ºC for 48 hours for PHA production. After
incubation, 10ml culture was drawn and estimated for the PHA production.

Optimization for PHA production at Nitrogen sources by Bacillus sp. and

Rhizobium sp.

Nitrogen sources such as Malt extract, Ammonium sulphate, Ammonium

nitrate and Urea were as nitrogen sources to induce the PHA production at a various
concentration like 0.1%, 0.2%, 0.3%, 0.4% and 0.5% in minimal medium. After
media preparation, the culture were inoculated and incubated at 37ºC for 48 hours for
PHA production. After incubation, 10 ml culture was drawn and estimated for the
PHA production.
Mass cultivation

Minimal medium was prepared and incubation time, optimum pH, temperature
and carbon source and nitrogen source the culture was inoculated and incubated at
37ºC in a rotatory shaker at 150 rpm for 48 hours.

Cell dry weight:

After incubation, bacterial culture centrifuged (10,000 rpm) for 15 mints. cell
pellets, dried to determine dry cell weight (DCW) as a g/l unit.

PHA Extraction:

Bacterial cells containing polymer were collected after centrifugation at 4000

rpm for 10 min. Then pellet was resuspended in equal volume of 4% sodium
hypochlorite and incubated at 37ºC for 24 hours. Pellet was washed with acetone,
ethanol and water to remove the unwanted materials. The whole mixture was
centrifuged again and the supernatant was discarded. Finally polymer granules were
dissolving in chloroform.

Quantification of bacterial growth and dry weight:

Cell growth was monitered by measuring the optical density (O.D) at 600 nm
using spectrophotometer ten milliliter culture medium was centrifuged at 10,000 rpm
4ºC for 15 min and cell pellet was washed with 10 ml distilled water. Cell pellet was
harvested by centrifugation and dried at 105ºC for 48 h, or till constant weight was
obtained cell mass concentration was determined by the standard calibration curve
between OD 600 nm and cell dry weight.

PHA accumulation (%) = Dry weight of extracted PHA (g/l) / Dry cell weight
(g/l)* 100%

Quantification for standard PHA:

Standard PHA sample (0.02-0.1g) was digested by heating in concentrated H2

SO4 at 100ºC for 10 min estimated at 235 nm in UV visible spectrophotometer to
determine slope and easily calculate factor = 1/ slope by referring to the standard
curve, the quantity of PHA produced was determined.
Characterization of polymer by FTIR:

FTIR spectrum was taken using Fourier transform IR spectrophotometer

Schimadzu – (8400 s). The spectra were recorded in range of 4000-6000 cm-1.

Result and Discussion

Isolation of bacteria

The serial dilution technique was employed to isolate bacteria from soil
samples. In totality, 10 different bacteria species were isolated.

Screening of PHA – Production bacteria

The bacterial isolates were stained by Sudan black staining method and
observed microscopically. A total of 10 strains were found to accumulate PHA
granules after observing microscopically by Sudan black staining method. All 10
isolates were also tested for PHA production following the viable colony screening
method based on the intensity of staining. The black stained isolates were ranked in
terms of + symbol. The poorly stained colonies were indicated with + symbol,
medium stained colonies as ++ symbol, strongly stained colonies as +++ symbol
while excellently stained as ++++ symbol. Out of the 10 isolates screened for PHA
production. 2 showed excellent staining by Sudan black in staining method.

Juan et al (1998) employed viable colony screening method for the rapid
detection and isolation of PHA producing bacterial strains by using 0.02% alcoholic
solution of Sudan Black B. Colonies unable to incorporate the Sudan Black B
appeared white, while PHA procedures appeared bluish black. Hartman (1940) was
the first to suggest the use of Sudan Black B, as a bacterial fat stain. Subsequently,
Burdon et al (1942a) confirmed the greater value of this dye and modified the
procedure for demonstrating intracellular fatty material in bacteria by preparing
microscopic slides of bacteria stained with alcoholic Sudan Black B solution and
counterstained with safranin.
Identification of Bacteria

The bacteria were isolated from soil. It was enriched in nutrient agar and yeast
mannitol salt agar plate. The bacterial strain was named as strain 1 strain 2.

The bacterial strain A4 was gram positive, rod shaped bacteria, motile, sporulating
bacteria. The biochemical analysis showed that the strain was positive for Methyl red,
oxidase, catalase, Indole, starch hydrolysation, Vogesproskaur, citrate utilization,
Gelatin hydrolysis, Triple sugar iron test, Hydrogen sulfide production test. After
conformation the selected strain was screened for PHA production.

The bacterial strain A5 was gram negative, rod shaped bacteria, motile, sporulating
bacteria. The biochemical analysis showed that the strain was positive for Methyl red,
oxidase, catalase, indole, citrate utilization, Triple sugar iron test. The biochemical
analysis showed that the strain was negative for Vogesproskaur, starch hydrolysation,
Gelatin hydrolysis, Hydrogen sulfide production test.

Optimization of PHA producing bacteria

Optimization for PHA production at various Incubation time by Bacillus sp. and
Rhizobium sp.

Optimization of incubation time for better growth was analysed. The

experimental result showed that the highest PHA production was obtained 48 hours
by Bacillus sp. and Rhizobium sp.

Optimization for PHA production at pH by Bacilus sp. and Rhizobium sp.

Effect of pH for better growth was analyzed. The experimental result showed
that highest PHA production was obtained at pH 7 by Bacillus sp. and Rhizobium sp.

Optimization for PHA production at various temperature by Bacillus sp. and

Rhizobium sp.

Effect of temperature for better growth was analyzed. The experimental result
showed that highest PHA production was obtained at 40ºC by Bacillus sp. and
Rhizobium sp.
Optimization for PHA production at Carbon sources by Bacillus sp. and
Rhizobium sp.

Effect of carbon source for better growth was analyzed. It was observed
Bacillus sp. showed maximum activity by Glucose, and Rhizobium sp. showed
maximum activity in sucrose.

Optimization for PHA production at Nitrogen sources by Bacillus sp. and

Rhizobium sp.

Effect of nitrogen source for the better growth was analyzed. It was observed
Bacillus sp. showed maximum activity in ammonium nitrate and Rhizobium sp.
showed maximum activity in malt extract.
 Tables

Table 5.1. Screening of PHA producing bacteria

S. No Sample Source PHA Accumulation

(Sudan staining)
1. A1 +

2. A2 ++

3. A3 ++

4. A4 ++++

5. A5 +++

6. A6 +

7. A7 ++

8. A8 ++

9. A9 +

10. A10 ++

Table 5.2. Identification of isolated bacteria

 Test A4 A5
Morphological characters
Gram staining + -
Shape Rod Rod
Spore staining + +
Motility test + +
Biochemical test
Methyl red + +
Vogesproskaur + -
Indole test + +
Oxidase test + +
Catalase test + +
Citrate utilization + +
Starch hydrolysis test + -
Gelatin hydrolysis test + -
Triple sugar iron test + +
Hydrogen sulfide production test + -

Table 5.3.1 Optimization of PHA production at various incubation time by

Bacillus sp.

Incubation Time Cell dry weight of Cell dry weight PHA

 PHA (g/l) accumulation (%)
(hrs) (g/l)
12 0.3 0.4 75
24 0.6 0.8 75
36 0.8 1 80
48 1.2 1.5 80
60 1 1.3 76.92

Table 5.3.2 Optimization of PHA production at pH by Bacillus sp.

pH Cell dry weight of Cell dry weight PHA accumulation

PHA (g/l) (g/l) (%)
5 0.2 0.3 66.66
6 0.4 0.5 80
7 0.8 1 80
8 0.7 0.8 87.5
9 0.5 0.6 83.33

Table 5.3.3 Optimization of PHA-production at various temperature by Bacillus

sp.

Temperature Cell dry weight of Cell dry weight PHA accumulation

(ºC) PHA (g/l) (g/l) (%)
30 0.1 0.2 50
35 0.2 0.3 66.66
40 0.6 0.8 75
45 0.4 0.7 57.14
50 0.2 0.4 50

Table 5.3.4.1a Optimization of glucose for PHA production by Bacillus sp.

Glucose Cell dry weight Cell dry weight PHA accumulation

 Concentration (%) of PHA (g/l) (g/l) (%)
0.1 1 1.2 83.33
0.2 1 1.3 76.92
0.3 0.3 0.6 50
0.4 0.2 0.6 33.33
0.5 0.1 0.5 20

Table 5.3.4.1b Optimization of sucrose for PHA production by Bacillus sp.

Sucrose Cell dry weight Cell dry weight PHA accumulation

Concentration (%) of PHA (g/l) (g/l) (%)
0.1 0.2 0.3 66.66
0.2 0.7 0.9 77.77
0.3 0.7 1 70
0.4 0.4 0.6 66.66
0.5 0.3 0.5 60

Table 5.3.4.1c Optimization of Maltose for PHA production by Bacillus sp.

Maltose Cell dry weight Cell dry weight PHA accumulation

Concentration (%) of PHA (g/l) (g/l) (%)
0.1 0.2 0.5 40
0.2 0.4 0.6 66.66
0.3 0.6 0.8 75
0.4 0.4 0.6 66.66
0.5 0.2 0.4 50

Table 5.3.4.1d Optimization of Dextrose for PHA production by Bacillus sp.

 Dextrose Cell dry weight of Cell dry weight PHA
Concentration (%) PHA (gl) (g/l) accumulation (%)
0.1 0.2 0.4 50
0.2 0.3 0.5 60
0.3 0.7 0.9 77.77
0.4 0.4 0.8 50
0.5 0.2 0.6 33.33

Table 5.3.5.1a Optimization of Malt extract for PHA production by Bacillus sp.

Malt extract Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)
0.1 0.1 0.2 50
0.2 0.2 0.3 66.66
0.3 0.4 0.6 66.66
0.4 0.2 0.4 50
0.5 0.1 0.3 33.33

Table 5.3.5.1b Optimization of Ammonium sulphate for PHA production by

Bacillus sp.

Ammonium Cell dry weight of Cell dry weight PHA

sulphate PHA (gl) (g/l) accumulation (%)
Concentration (%)
0.1 0.2 0.3 66.66
0.2 0.3 0.4 75
0.3 0.4 0.6 66.66
0.4 0.2 0.4 50
0.5 0.2 0.4 50

Table 5.3.5.1c Optimization of Ammonium Nitrate for PHA production by

Bacillus sp.

Ammonium Cell dry weight of Cell dry weight PHA

 nitrate PHA (gl) (g/l) accumulation (%)
Concentration (%)
0.1 0.2 0.4 50
0.2 0.3 0.5 60
0.3 0.8 0.9 88.88
0.4 0.5 0.8 62.5
0.5 0.1 0.6 16.66

Table 5.3.5.1d Optimization of Urea for PHA production by Bacillus sp.

Urea Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)
0.1 0.1 0.2 50
0.2 0.2 0.3 66.66
0.3 0.8 0.9 88.88
0.4 0.4 0.6 66.66
0.5 0.3 0.5 60

Table 5.3.1 Optimization of PHA production at various incubation time by

Rhizobium sp.

Incubation Time Cell dry weight of Cell dry weight PHA

(hrs) PHA (gl) (g/l) accumulation (%)
12 0.4 0.6 66.66
24 0.5 0.8 62.5
36 0.8 1 80
48 1 1.2 83.33
60 1.3 1.5 86.66
Table 5.3.2 Optimization of PHA production at pH by Rhizobium sp.

pH Cell dry weight of Cell dry weight PHA accumulation

PHA (gl) (g/l) (%)
5 0.3 0.4 75
6 0.4 0.5 80
 7 0.5 0.7 71.42
8 0.4 0.6 66.66
9 0.2 0.4 50

Table 5.3.3 Optimization of PHA-production at various temperature by

Rhizobium sp.

Temperature Cell dry weight of Cell dry weight PHA

(ºC) PHA (gl) (g/l) accumulation (%)

30 0.1 0.2 50
35 0.3 0.5 60
40 0.5 0.6 83.33
45 0.2 0.4 50
50 0.1 0.2 50

Table 5.3.4.2a Optimization of glucose for PHA production by Rhizobium sp.

Glucose Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)

0.1 0.3 0.4 75

0.2 0.7 0.9 77.77
0.3 0.5 0.7 71.42
0.4 0.4 0.6 66.66
0.5 0.2 0.4 50
Table 5.3.4.2b Optimization of sucrose for PHA production by Rhizobium sp.

Sucrose Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)

0.1 0.1 0.4 25

0.2 0.2 0.5 40
0.3 0.6 0.8 75
 0.4 0.2 0.5 40
0.5 0.1 0.3 33.33

Table 5.3.4.2c Optimization of maltose for PHA production by Rhizobium sp.

Maltose Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)

0.1 0.2 0.5 40

0.2 0.4 0.9 44.44
0.3 0.5 0.7 71.42
0.4 0.3 0.5 60
0.5 0.1 0.3 33.33

Table 5.3.4.2d Optimization of dextrose for PHA production by Rhizobium sp.

Dextrose Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)

0.1 0.2 0.3 66.66

0.2 0.3 0.5 60
0.3 0.4 0.8 50
0.4 0.3 0.7 42.85
0.5 0.2 0.6 33.33
Table 5.3.5.2a Optimization of malt extract for PHA production by Rhizobium
sp.

Malt extract Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)

0.1 0.2 0.4 50

0.2 0.4 0.5 80
0.3 0.7 0.8 87.5
0.4 0.4 0.6 66.66
 0.5 0.3 0.6 50

Table 5.3.5.2b Optimization of ammonium sulphate for PHA production by

Rhizobium sp.

Ammonium Cell dry weight of Cell dry weight PHA

sulphate PHA (gl) (g/l) accumulation (%)
Concentration (%)

0.1 0.3 0.4 75

0.2 0.7 0.8 87.5
0.3 1.1 1.3 84.61
0.4 0.8 1 80
0.5 0.5 0.7 71.42

Ammonium Cell dry weight of Cell dry weight PHA

nitrate PHA (gl) (g/l) accumulation (%)
Concentration (%)

0.1 0.2 0.5 40

0.2 0.4 0.9 44.44
0.3 0.5 0.6 83.33
0.4 0.3 0.5 60
0.5 0.1 0.3 33.33
Table 5.3.5.2c Optimization of ammonium nitrate for PHA production by
Rhizobium sp.

Table 5.3.5.2d Optimization of urea for PHA production by Rhizobium sp.

Urea Cell dry weight of Cell dry weight PHA

Concentration (%) PHA (gl) (g/l) accumulation (%)

0.1 0.3 0.5 60

0.2 0.5 0.7 71.42
0.3 0.3 0.6 50
0.4 0.2 0.5 40
0.5 0.1 0.3 33.33

Bar Chats
Table 5.3.1 Optimization of PHA production at various incubation time by
Bacillus sp. and Rhizobium sp.
 1.4

1.2

1
PHA Weight (g/l)

0.8

0.6 Bacillus sp.

Rhizobium sp.
0.4

0.2

0
12 24 36 48 60

Incubation Time (hrs)

Table 5.3.2 Optimization of PHA production at pH by Bacillus sp. and

Rhizobium sp.

0.8

0.7

0.6

0.5
PHA Weight (g/l)

0.4
Bacillus sp.
0.3 Rhizobium sp.

0.2

0.1

0
5 6 7 8 9

pH

Table 5.3.3 Optimization of PHA-production at various temperature by Bacillus

sp. and Rhizobium sp.
 0.6

0.5

0.4
PHA Weight (g/l)

0.3
Bacillus sp.
Rhizobium sp.
0.2

0.1

0
30 35 40 45 50

Temperature (ºC)

Table 5.3.4 Optimization of glucose for PHA production by Bacillus sp. and
Rhizobium sp.

1
0.9
0.8
0.7
PHA Weight (g/l)

0.6
0.5
Bacillus sp.
0.4
Rhizobium sp.
0.3
0.2
0.1
0
0.1 0.2 0.3 0.4 0.5

Concentration of Glucose (%)

Table 5.3.4 Optimization of sucrose for PHA production by Bacillus sp. and
Rhizobium sp.
 0.7

0.6

0.5
PHA Weight (g/l)

0.4

0.3 Bacillus sp.

Rhizobium sp.
0.2

0.1

0
0.1 0.2 0.3 0.4 0.5

Concentration of Sucrose (%)

Table 5.3.4 Optimization of maltose for PHA production by Bacillus sp. and
Rhizobium sp.

0.6

0.5

0.4
PHA Weight (g/l)

0.3
Bacillus sp.
Rhizobium sp.
0.2

0.1

0
0.1 0.2 0.3 0.4 0.5

Concentration of Maltose (%)

Table 5.3.4 Optimization of dextrose for PHA production by Bacillus sp. and
Rhizobium sp.
 0.7

0.6

0.5

0.4
PHA Weight (g/l)

0.3 Bacillus sp.

Rhizobium sp.
0.2

0.1

0
0.1 0.2 0.3 0.4 0.5

Concentration of Dextrose (%)

Table 5.3.5 Optimization of malt extract for PHA production by Bacillus sp. and
Rhizobium sp.

0.7

0.6

0.5
PHA Weight (g/l)

0.4

0.3 Bacillus sp.

Rhizobium sp.
0.2

0.1

0
0.1 0.2 0.3 0.4 0.5

Concentration of malt extract (%)

Table 5.3.5 Optimization of ammonium sulphate for PHA production by Bacillus

sp. and Rhizobium sp.
 1.2

0.8
PHA Weight (g/l)

0.6
Bacillus sp.
Rhizobium sp.
0.4

0.2

0
0.1 0.2 0.3 0.4 0.5

Concentration of Ammonium sulphate (%)

Table 5.3.5 Optimization of ammonium nitrate for PHA production by Bacillus

sp. and Rhizobium sp.

0.8

0.7

0.6
PHA Weight (g/l)

0.5

0.4
Bacillus sp.
0.3 Rhizobium sp.

0.2

0.1

0
0.1 0.2 0.3 0.4 0.5

Concentration of Ammonium nitrate (%)

Table 5.3.5 Optimization of urea for PHA production by Bacillus sp. and
Rhizobium sp.
 0.8

0.7

0.6

0.5
PHA Weight (g/l)

0.4
Bacillus sp.
0.3 Rhizobium sp.

0.2

0.1

0
0.1 0.2 0.3 0.4 0.5

Concentration of Urea (%)