CHAPTER I

INTRODUCTION

1.1 History and Background

1.1.1 Plastics
Plastics are man- made long chain polymeric molecules similar in many ways to natural
resins found in trees and other plants (Scott,1999). Society is fundamentally ambivalent to the
use of plastics. On the other hand, plastics are uniquely exible materials that have seen them
occupy a huge range of functions, from simple packing materials to complex engineering
components(Jim and Alexandre et al ,2013) .The history of plastic begins from 1862 by
Alexander Parkes. The main raw material in plastic production is petroleum. The properties of
plastic which is high molecular weight and tightly bonded together make the plastic not
degradable, their disposal become difficult and give negative impact on the environment
(Sharmila et al., 2011). During the 1980s, the solid waste problem emerged as a potential crisis
in many areas of the US because of increasing amounts of municipal solid waste(MSW),
shrinking landfill capacity, rising costs and strong public opposition to new solid waste facility
sittings (Regan,Creighton et al 1990).In 1960 plastics made u less than half a percent of US
MSW generation.By 2010 they made up to 12.4% and only 8.2% is recovered.(US EPA,2011)






1.1.2 Biogradable plastics
Biogradable plastics were introduced in the 1980s to find ways to produce non-
petroleum based plastics as well as to reduce the environmental effects because of the increased
landfill( Gironi and Piemonte2010).According to European Bioplastics, a plastic material is
defined as a bioplastic if it is either biobased, biodegradable, or features both properties. The
term biobased means that the part of material or product is derived from biomass. Meanwhile,
biodegradation is a chemical process which could be degraded by the microorganism in the
environment when proper conditions such as the sunlight, moisture, oxygen etc are available
convert materials into natural substances such as water, carbon dioxide, and compost (Abe and
Doi, 2002).

1.1.3 Poly-(3-hydroxybutyrate), PHB
Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a polymer belonging to
the polyesters class that was first isolated and characterized in 1925 by French microbiologist
Maurice Lemoigne. PHB is produced by microorganisms (like Ralstonia eutrophus or Bacillus
megaterium) apparently in response to conditions of physiological stress. The polymer is
primarily a product of carbon assimilation (from glucose or starch) and is employed by
microorganisms as a form of energy storage molecule to be metabolized when other common
energy sources are not available. Microbial biosynthesis of PHB starts with the condensation of
two molecules of acetyl-CoA to give acetoacetyl-CoA which is subsequently reduced to
hydroxybutyryl-CoA. This latter compound is then used as a monomer to polymerize PHB.
(Lemoigne,2009)
The poly-3-hydroxybutyrate (P3HB) form of PHB is probably the most common type of
polyhydroxyalkanoate, but many other polymers of this class are produced by a variety of
organisms: these include poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV),
polyhydroxyhexanoate (PHH),polyhydroxyoctanoate (PHO) and their copolymers.
(Lemoigne,2009)
Poly-(3-hydroxybutyrate), PHB is one of the most important members of PHAs.
According to Li et al. (1999), PHB is an intracellular carbon and energy storage material
prodeuced by many microorganisms under unfavorable growth condition such as limitation of
(NH
4
)
2
SO
4
, PO
3
2-
, Mg
2+
and oxygen. PHB is synthesized from acetyl-CoA using three enzymatic
steps (Paramjit and Nitika, 2011). It is abiodegradable thermoplastic polyester which can be used
in various ways like the conventional nondegradable plastics (Li et al., 1999). The chemical
structure of PHB is:



The properties of PHB are elastomeric, insoluble in water, nontoxic, biocompatible, and
piezoelectric, with high degree of polymerization (Samantary, Nayak and Mallick, 2011).
Besides, PHB is also resistant to water and ultraviolet radiation and impermeable to oxygen In
addition, PHB is a partially crystalline material with high melting temperature and high degree of
crystallinity. PHB is stiff and brittle. PHB does not contain any residues of catalyst. PHB is
perfectly isotactic and does not include any chain branching. It is not water soluble but is 100%
biodegradable. PHB has low permeable for O
2
, H
2
O and CO
2
(Samantary, Nayak and Mallick,
2011).
1.1.4 Biodegradability of PHB

Biodegradation of PHB is dependent upon a number of factors such as the
microbial activity of the environment and the exposed surface area. In addition, temperature, pH,
molecular weight and crystallinity are important factors. Biodegradation starts when
microorganisms begin growing on the surface of the plastic and secrete enzymes that break down
the polymer into its molecular building blocks, called hydroxyacids. The hydroxyacids are then
taken up by the microorganisms and used as carbon sources for growth. In aerobic environments
the polymers are degraded to carbon dioxide and water.
A number of reports have demonstrated that PHB are compostable over a wide range of
environmental conditions. In one report, the maximum biodegradation rates were observed at
moisture levels of 55% and temperatures of around 60
o
C, conditions similar to those used in
most large-scale composting plants. Up to 85% of the samples degraded within 7 weeks, and
PHB coated paper was rapidly degraded and incorporated into the compost. In another study, the
quality of PHB compost was determined by measuring seedling growth relative to a control.
Seedling growth of around 125% of the control was found for 25% PHB copolymer compost
indicating that the compost can support a relatively high level of growth.
Biodegradation of PHA has also been tested in various aquatic environments. In one
study in Lake Lugano, Switzerland, items were placed at different depths of water as well as on
the sediment surface. A life span of 5-10 years was calculated for bottles under these conditions
(assuming no increase in surface area), while PHA films were completely degraded in the top 20
cm of sediment within 254 days at temperatures not exceeding 6
o
C.
1.1.5 Storage and Handling
PHB is non toxic biopolymer. Therefore, it is biocompatible and hence is suitable
for medical applications. It is important to minimize premature PHB degradation during
fabrication and storage. This is because PHB are biodegradable polymer and its biodegradation is
dependent upon a number of factors such as the microbial activity of the environment and the
exposed surface area. In addition temperature, pH, molecular weight and crystallinity are also
play an important role. In one report, the maximum biodegradation rates were observed at
moisture level of 55% and temperatures of around 60oC. Therefore, it is well advised to packed
PHB in airtight, aluminum-backed, or plastic foil pouches and kept it in the refrigerator.



1.2 Applications of PHB
There are many application of PHB besides it is been used in the production of
biodegradable plastic. PHB have been chosen as petroleum derived plastic replacement because
of its properties that possess high durability and endurance similar like regular plastics but unlike
regular plastics, it can be decomposed to water and carbon dioxide aerobic microorganisms
existing from sewage, sea or soil without forming any toxic products. PHB can be used as
wrapping materials like bags, containers and throwaway items such as cup, plates and diapers.
1.2.1 Medical
Since biodegradability and biocompatibility are properties of PHB, the combination of
PHB with hydroxyapatite (HA) were used as scaffolding material in tissue engineering.
(Christopher J. Brigham, 2012) For instance, the medical practitioners use PHB scaffolding
material to treat bone defects. While the combinations of copolymer of polyglycolic acid (PGA)
and PHB was used to produce pulmonary valve leaflets and pulmonary artery scaffolds in sheep.
PHB also used in medical devices such as for dental, craniomaxillofacial, orthopaedic,
hernioplastic and skin surgery. (A.P. Bonartsev, 2007) The efficiency of these devices in term of
biocompatibility, biodegradation and therapeutic is still in progress.

1.2.2 Aquaculture

PHB was used as a food add-on to aquaculture animals in order to control the enormous
deaths caused by pathogenic contaminations. It was found that this compound might shield
gnotobiotic Artemia franciscana against vibriosis. The release of the PHB monomer -
hydroxybutyric acid was suggested to inhibit the growth and/or the activity of the pathogens.
(Schryver, 2010) By integrating the accumulation of PHB in bio-flocs, this technique can
possibly decrease the rate of death during larval and young stages of aquaculture animals and can
therefore become all the more cost effective. Moreover, the study shows that the fish growth
performance becomes increased.



1.2.3 Pharmaceutical

In pharmaceutical, PHB is applied into slow-released carrier for lasting drug delivery due
to their biocompatibility and biodegradability properties. It is also used as cell and tablet
packaging material. Polyhydroxybutyrate (PHB), co-polyesters of 3-hydroxybutyrate and 3-
hydroxyhexanoate (PHBHHx), and polylactic acid (PLA) were used to study drug sustained
release. The results showed that over a period of at least 20 days for PHB and PHBHHx
nanoparticles, while PLA nanoparticles and free drug lasted only 15 days and a week,
respectively. (Yu-Cui Xiong, 2010)















1.3 Market Survey
1.3.1 Global Market Demand
Table 1 : World Bioplastic Demand for 2005 2015 ( (Mohan, 2011)

In 2015, global demand for biodegradable and bio-based plastics will more than triple to more
than 1 million metric tons (1.1 million tons), valued at $2.9 billion. This is due to customer
demand for more environmentally-sustainable products, development of bio-based feed stocks
for commodity plastic resins, increasing restrictions on the use of non-degradable plastic
products and high rise of crude oil and natural gas prices. Biodegradable plastics accounted for
90% of the world bioplastics market in 2010. Excellent growth is forecast for the two leading
biodegradable plasticsstarch-based resins and polylactic acid (PLA)both of which are
expected to more than double in demand through 2015. The report adds that the fastest gains for
biodegradable plastics, however, will be seen for polyhydroxy-alkanoate (PHA) resins, which are
just entering the commercial market. (Mohan, 2011)






1.3.2 Global Bioplastics Demand by Application


Figure 1 : World bioplastics demand by application for 2016 (marketsandmarkets.com, 2011)
The increasing demand for renewable and bio-based materials and shift in consumer preference
for eco-friendly packaging is driving the market for global biodegradable plastics. The global
biodegradable plastics market in terms of volume (thousand metric tons) is expected to grow
from 664 thousand metric tons in 2010 to 2330 thousand metric tons by 2016, at an estimated
CAGR of 20.24% from 2011 to 2016. Biodegradable plastics offer tremendous potential in
various applications including packaging, electronics, transport, textiles, and medical. Amongst
all market segments, the starch-based plastics market commands the largest share in terms of
volume, while PLA-based plastics lead the market in terms of value. Packaging forms the largest
application market due to increased consumer awareness for sustainable packaging. The
packaging application contributed over 50% of the global biodegradable plastics market in 2010.
In PLA-based plastics, textiles applications are expected to have the highest CAGR of 23.16%
from 2011 to 2016. (marketsandmarkets.com, 2011)



1.3.3 Global Consumption of PHB

Figure 2 : World consumption of biodegradable plastics
In 2012, the two most important commercial, biodegradable polymers were polylactic acid
(PLA) and starch-based polymers, accounting for about 47% and 41%, respectively, of total
biodegradable polymers consumption. Starch sources vary worldwide, but include corn, potatoes,
cassava and sugar beets. In Europe, starch-based biodegradable polymers are the major type
consumed, accounting for 62% of the market, due to Europes large, starch-based capacity and
their use in many applications. This is followed by PLA, with 24% and other biodegradable
polymer types with 14%.
Huge advantages to the waste management effort as food packaging, dishes and cutlery create a
major market for the product as these materials can be degrade with the food waste without
separate them. Encouragement from the government itself as well as consumer pressures, force
the retailers and manufactures to seek out these biodegradable products and materials
(Plastermat.com).




55%
29%
16%
World consumption of biodegradable plastics
Europe
North America
Asia
1.3.4 Global Usage of PHB

Figure 3 : Use of bioplastics by global region
The global use of bioplastics was 0.64 million metric tons in 2010 and 0.85 million metric tons
in 2011. BCC expects that the use of bioplastics will increase up to 3.7 million metric tons by
2016, a compound annual growth rate (CAGR) of 34.3%. The European market segment
accounted for around 34% of the global market in 2010 and was nearly 0.21 million metric tons.
This segment increased to 0.3 million metric tons in 2011 and will further grow to 1.2 million
metric tons in 2016, a CAGR of 32.1%. The American market accounted for around 37.5% of
the total market in 2010 and was 0.24 million metric tons. BCC has forecasted that the American
market will increase from 0.3 million metric tons in 2011 to 1.2 million metric tons by 2016, a
CAGR of 33%. (Research, 2012)





1.3.5 Global Production

Figure 4 : Production capacity of biopolymers in 2010
From (Shen, 2011) , it shows that Europe and North America remain as location for research and
development and also important as sales markets. However, the establishment of new production
capacities is favoured in South America and Asia. (Shen, 2011)

1.3.6 Local Demand
Malaysias first fully automated PHA Bioplastics Pilot Plant was launched by Science,
Technology and Innovation Minister Datuk Seri Dr. Maximus Johnity Ongkili at Jalan
Beremban. Scaled-up to 2,000 L, the bioreactor facilities and integrated manufacturing process
of the plant are able to produce various options of PHA materials from crude palm kernel oil and
palm oil mill effluent.



1.3.8 Prices of Products, Raw Materials and Chemicals

Market prices for PHB, raw materials of culture medium in PHB production and chemicals are
shown in Table 1.1:
Table 1: Media for the production of PHB
Materials Amount (g/L)
Jatropha oil 20 g/L
Urea

1 g/L
E85 Ethanol 1.5 % v/v

Table 2: The prices of the material components
Materials Prices (USD/kg)
Jatropha oil 0.39
Urea

0.34
E85 Ethanol 0.69 USD/L
PHB 277.30
Biomass (for fuel) 0.50







1.4 Screening of Synthesis Routes
PHB have a great potential as a biodegradable bio plastic. However, the major drawback
to the commercialisation of PHB is their high cost of production compared with conventional
petrochemical based plastic materials. The cost of carbon feed stocks or raw materials required
can significantly affect the PHB production cost in large production scale. Therefore, the
production cost can be considerably lowered when alternative cost-effective carbon feedstock,
type of microbial production strain, nutrient supply as well as the process parameters during the
biosynthesis for the commercialisation of bio plastics are identified.
1.4.1 Availability of Raw Materials
The most crucial factor in order to run a plant is the availability of raw materials. Various
substrates especially plant oils have been evaluated as an excellent carbon source in PHA
production. Examples given are soybean oil (Kahar et al., 2004), palm oil (Loo et al., 2005), and
sunflower oil and coconut oil (Lee et al. 2008). Due to their high carbon content, plant oils yield
almost two-fold higher than from glucose (Akiyama et al., 2003). However, the use of edible oils
in production of bio plastics may cause depletion of global food supply and sources.
As the world is in a state of biofuels fever, many countries have started planting jatropha
plant as this non-edible plant has promising future as biofuels. Approximately 900,000 hectares
of jatropha have already been planted throughout the world. Although the industry is in its early
stages, it is identified 242 jatropha plantation projects, totalling approximately 900,000 hectares.
More than 85% of the land cultivated is located in Asia. Africa counts for approximately 120,000
hectares followed by Latin America with approximately 20,000 hectares. Jatropha saw enormous
growth: 5 million hectares were expected by 2010. The number and size of jatropha projects
currently being developed is increasing sharply. This is the case in almost all regions of the
world which are suitable for jatropha cultivation. It is predicted that each year for the next 5-7
years approximately 1.5 to 2 million hectares of Jatropha will be planted. This will result in a
total of approximately 5 million hectares by 2010 and approximately 13 million hectares by
2015.

Figure 1 shows the total acreage of jatropha oil plantations in selected countries.

Jatropha has been commercially farmed in Malaysia specifically Sarawak owned by Bio
Oil National Group Malaysia (The Star, 2011). A few local private companies also have engaged
in jatropha cultivation scaling from 400 ha to 1000 ha. It has been identified that total current
acreage of Jatropha plantation projects is 1,712 ha. Project owners state plans to increase the
cultivation scale to a total of 57,601 ha by 2015. The Ministry of Plantation of Industries and
Commodities is undertaking a jatropha pilot research project for which 300 ha have been
allocated.
Using other plant oils such as soybean oil is not in consideration as it is unethical as it is
wasteful to convert food to bio plastics. Additionally, Ng et al. (2010) also stated that the plant
oils price has increased drastically because of recent crisis of food shortage and increase of food
demand. As a result of evaluation of using edible plant oils, jatropha oil, as a non-edible one,
would not affect the global food chain crisis and has potential as a renewable resource can be the
alternative substrate for bioplastic production. Jatropha oil, derived from Jatropha curcas seed,
also yield high amounts of oil, yielding almost four times than the soybean and ten times from
the maize (Fitzgerald, 2006). It is also relatively cheap, costing less than soybean oil as the
fertilizer and pesticide requirement of jatropha is lower (Gui et al, 2008).

1.4.1.1 Utilization of jatropha oil
The operational and maintenance costs for the jatropha oil extraction are very
minimal, estimated at approximately 10 15% of the capital cost per year. In Ghana, for
instance, in 2010, whilst the cost of jatropha oil and kerosene were estimated to be
US$0.085/liter and US$1.23/liter respectively, the cost of biodiesel from jatropha oil and
petroleum diesel were also estimated at US$0.99/liter and US$1.21/liter respectively (Ofori-
Boateng and Teong). On the other hand, the seeds from the northern part of Malaysia contain
high lipid content of 60% of oil (Salimon and Abdullah). Using jatropha as carbon source in
PHB production can reduce the recovery cost (Choi and Lee) as it yields high PHB content.

1.4.2. Type of Microbial Production Strain
Several bacteria strains have been studied in accumulation of PHB. Researches are
conducted and microorganisms such as Alcaligenes latus (Yamane et al., 1996), Alcaligenes
eutrophus now is known as Cupriavidus necator (Kim et al., 1994), Azotobacter vinelandii (Page
and Knosp, 1989), Pseudomonas oleovorans (Brandl et al. 1988), and recombinant Escherichia
coli (Lee and Chang, 1994; Lee et al., 1994 have showed some promising high yield of PHB
production. E. coli strains are considered as impractical in large scale production of PHB as it is
expensive. They require expensive Luria-Bertani (LB) medium, ampicillin and pure O
2
(Liu et
al., 1998). E.coli is also unable of producing PHAs, however it can utilise several carbon sources
including some substrates that cannot be easily used by most of the microorganisms, such as
lactose (Lee et al., 1997). Table 1.4.1 shows comparison between promising microorganisms in
PHB cultivation, an analysis from Choi and Lee (1997). It is shown that PHB concentration,
PHB productivity and PHB yield are higher in C. necator.
Bacterium C. necator A. latus
Recombinant
E.coli
Carbon source Glucose Sucrose Glucose
Limiting nutrient Nitrogen None None
Fermentation method
Glucose
concentration
control
pH-stat pH-stat
Culture time (h) 50 28.45 39
Cell concentration (g/L) 164 143 110
PHB concentration (g/L) 121 71.4 85
PHB content (%) 76 50 77.3
PHB productivity (g/L.h) 2.42 2.5 2.18
PHB yield (g PHB/ g
substrate)
0.3 0.17 0.29
kg substrate/kg PHB 3.33 5.88 3.5
Reference Kim et al. 1994
Yamane et al.
1996
Lee and Chang
1994

Cupriviadus necator use up palmitic acids, oleic acids and linoleic acids contained in
jatropha oil. Freitas et al. (2009) reported that C. necator has been proven to accumulate PHB up
to 80% of its cell dry weight. Whilst Khan et al. (2013) reported that cell growth curve of C.
necator H16 has a classical pattern with an exponential phase up to 50 hours followed by
stationary phase that lasted until 65 hours. They also stated that the highest cell dry weight of
11.6 g/L was obtained at 55 hour followed by highest PHB concentration of 8.6 g/L at 61.5 hour.
Kadouri et al. (2005) reported that C. necator can tolerate adverse stress conditions such as heat,
osmotic pressure, UV radiation and toxins such as ethanol and hydrogen peroxide. It should be
clear that C. necator is the most suitable microorganism to be used with jatropha oil in PHB
production.

1.4.3 Feeding Source of Nutrient Supply

Lee et al. (2008) found that different nitrogen sources affected both cell biomass and
PHA biosynthesis. It is discovered that both urea and sodium nitrate resulted in better biomass
production compared to other nutrient supply. Ng et al. (2010) stated that urea is the most
suitable nitrogen souce to pair up with jatropha oil as carbon source. Besides, urea costs much
lower price and has high productivity of PHB (Kek et al., 2008). Khanna and Srivastava as well
as Sabra and Abu Zeid discovered that urea can yield high cell biomass and PHA production
significantly. Ng et al. (2010) also reported that based on their analysis both CDW and PHB
accumulation increased when the urea concentration increased. They also stated that the optimal
concentration of urea is 0.54 g/L as CDW remained constant while PHB accumulation decreased
significantly after 0.54 g/L of urea.









1.4.4 PHB synthesis
There are four methods identified to cultivate PHAs which are; in vitro, via PHA-
polymerase catalysed polymerization; and in vivo with batch, fed-batch and continuous cultures
(Zinn et al., 2001). However, the fed-batch mode is the most used for PHAs production to
achieve high cell density, which often crucial for the high productivity and yield for the desired
product. Fed-batch mode is chosen as it has a lot of advantages in production of PHB.
Heuristically, fed-batch can maintain the carbon-source concentration at very low concentration,
to maximise the biomass yield. Ng et al. (2010) presented jatropha oil support the cell growth
and PHB production in fed-batch fermentation and high yield of product per jatropha oil was
obtained.
The fermentation process is relatively simple with multistaging from the petri dish to a
shaker flask to a small fermenter which is then used to inoculate the production reactor. In
fermentation process, cells were maintained and pre-cultivated in 2 g/L yeast extract, 10 g/L
meat extract and 10g/L peptone (Khan et al., 2013).

1.4.5 Downstream Process

For subsequent process, several methods have been developed for the recovery of PHAs,
mostly PHB from the cells. Solvents such as chloroform, methylene chloride, propylene
carbonate and dichloroethane have been used for the extraction of PHB (Ramsay et al., 1994).
However, it was difficult to remove the cell residues due to viscosity of 5% (w/v) PHB from
extracted polymer solution (Choi and Lee, 1997). Hahn et al. 1994 suggested that PHB can be
recovered using a dispersion of chloroform and sodium hypochlorite solution. PHB recovery by
this method is more efficient with less polymer degradation.
During recovery of PHB, the harvested cell pellets are treated with chloroform and
sodium hypochlorite. The cells that contain PHB together with culture broth are then separated
by using continuous centrifugal system. The liquid broth is then recycled to the feed of
bioreactor to minimize the cost and the heavy chloroform phase containing PHB was filtered.
The filtrate is precipitated by methanol and water, and the precipitate is filtered. Chloroform and
methanol are recovered to reduce more cost.

1.4.6 Preliminary safety and environmental issues
The main objective is to transform unproductive lands into profitable systems. The main
objective is to transform unproductive lands into profitable systems.
However PRA indicated that most of Jatropha plantations were establish in productive land
previously occupied with staple (mainly maize and beans) and cash crops (peanuts). Forest land
has been rarely transformed into Jatropha culture.

1.5 Objectives

The objective of this project is to develop a new poly(3-hydroxybutyrate) (PHB) plant at
Kuala Ketil industrial estate (phase 2), Kedah. The proposed production amount would be 500
MTPA. The choice of opening a new plant at Kedah is because the price for land at Kuala Ketil
is cheapest among Kapar industrial park and Nilai 3 industrial park. The distance between Kuala
Ketil and Butterworth approximately takes less than one hour which make transportation cost of
raw material cheaper.





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