11

Chapter 2 Review of Literature

12

2.1   Plastic wastes: An aesthetic nuisance 

Increasing amounts of plastic waste are being generated following the rapid

rate of urbanization in India. Today, there is a staggering demand for plastic

products with the rising affluence and public embracement of western

consumerism. However, this expansion of plastic production and consumption is

having a significant impact both visibly and invisibly on the environment and

society in India. The problems with plastic waste may seem surprising in a country

where traditional materials fulfilling the current role of plastics have existed. The

winning factor for plastics is its functional superiority (convenience) and cost

effectiveness. By sheer economies of scale, plastics have eroded the traditional

industries in India and have slowly perpetuated the throwaway culture in the

Indian society. The bottled water, fast food and Pepsi Coke culture in the country

contributes to the increasing plastic waste generation in India. The problem

becomes very visible when there is no effective end of life management to take

care of the litter, and this creates an environmental and social problem. The

widespread use of plastics as a packaging medium has resulted in the landscapes

of India being littered with non-biodegradable plastic bags and PET bottles, with

plastic bags dominating the litter. Much plastic waste has a value, and is

consequently taken care of by the informal recycling sector. Market forces guide

the informal sector, and they contribute to the waste system immensely by

collecting waste material that has a value, thereby taking over a part of the burden

on the municipalities. Despite the attempts from the formal and the informal

sector, significant quantities of the plastic waste remain uncollected. Waste

management is also constrained by the lack of public awareness and low municipal

budgets in the country. Most municipalities are starved of budgets and this impairs

the system of waste collection and disposal in many cities in India. Even when

budgets are adequate for collection, safe disposal remains a major problem. In

essence, inefficient waste management leads to a number of environmental

problems. The situation is more acute in countries such as India where economic

growth as well as urbanization is quite rapid. In view of the limited resources and

availability of land for disposal, especially in the metropolitan cities, there is a

13

need for a concerted effort to develop cost-effective and feasible policy options for

tackling the waste management problems.

Figure 2.1: Linkage between waste prevention and waste minimization

2.2   Plastic Industry in India 

The growth of the Indian plastic industry has been phenomenal - the growth

rate (17%) is higher than for the plastic industry elsewhere in the world. India has a

population of over 1 billion and a plastic consumption of 4 million tonnes. One third

of the population is destitute and may not have the disposable income to consume

much in the way of plastics or other goods. The virgin industry does not target this

population to expand its markets. However, one third of the population is the middle

class whose aspirations could be molded to increase consumption. Plastic

manufacturers create needs for this segment of population. The rising needs of the

middle class, and abilities of plastics to satisfy them at a cheaper price as compared to

other materials like glass and metal, has contributed to an increase in the consumption

of plastics in the last few years. The consumption trends for key commodity plastics

are presented below in Table 2.1.

A total of 36.5 million tonnes/year (36.5 kg/individual) of municipal solid

waste is generated in the country. Considering the fact that the plastic consumption in

the country is 4 million tonnes and 52% of the plastics is used for packaging, and then

we could estimate that the plastic waste generated is at least 2 million tonnes and not

more than 4 million tonnes. Since plastics constitute only between 1-4% of the waste,

and then the total waste generated should be between 50 million tonnes – 400 million

tonnes /year that constitute 50-400 kg/individual, which is higher than the reported

figures.

14

Table 2.1: Total predicted increase in consumption of resins

Polymer 1995-1996 2001-2002 2006-2007

Polyethylene (PE) 823 1835 3267

Polypropylene (PP) 340 885 1790

Polyvinyl chloride (PVC) 489 867 1287

Polyethyleneterephthalate (PET) 34 140 289

Others 203 647 1415

Total 1889 4374 8054

Plastics in Packaging 976 2272 4037

% of Plastics in Packaging 52% 52% 50%

(Source: National Plastic Waste Management Task Force -1997). Figures in thousand tonnes

Table 2.2: Demand scenario for key commodity plastics in India

1995-1996 2001-2002 2006-2007

Total polymers 1889 4374 8054

Process Waste (2%) 38 87 161

Post consumer Waste17 870 (46%) 1966(45%) 3624(45%)

(Source: National Plastic Waste Management Task Force -1997) Figures in thousand tonnes

2.3   Plastic waste generation in India 

Recycling can be done but is very tedious. The sorting of the wide variety

of discarded plastic material is also a very time-consuming process. Moreover, the

presence of a wide variety of additives such as pigments, coatings, fillers, limits

the use of the recycled material. Thus significant step towards, replacement of

non-degradable polymers by degradable polymers and make environment pollution

free is a major interest both to decision-makers and the plastic industry (Anderson

15

and Dawes 1990, Song et al., 1999). In India plastic recycling is a lucrative

business, and its sustainability is maintained by the price difference between

virgin and reprocessed granules. Recycled granules are 60% cheaper than the

virgin granules. Much of this price difference is owing to the poor wages paid in

this sector and pilferage of electricity in the informal sector. As long as there is an

ever-growing demand for cheap plastic items in the country by the poor people,

plastic recycling in this low grade manner (using poor quality plastic waste in low

grade melters and extruders most of which do not have temperature control

mechanisms) with toxic additives and colours will continue. The range of end

products produced from recycling is seemingly endless, and suffices the demand

of a population living below the poverty line, which accepts products of lower

quality. Therefore the structure of the plastic recycling industry indicates that there

are several issues of concern that need immediate attention. Some of the issues

pertain to the health and hygiene of the workers involved in the reprocessing trade,

upgrading of processing equipments used in the recycling of plastics, quality of

the effluent from the recycling plants, and finally, the quality of the products from

recycled plastics waste. Besides the regulatory relationship, the industry has also

come up with voluntary initiatives of awareness generation in the view to protect

the image of the plastic industry. The Indian Centre for Plastics and Environment

(ICPE) has been set up as a result of the recommendations made in the plastic

waste management task force. Responsibility to protect the environment and

enforcing the existing regulation lies within the Ministry of Environment and

Forests (MOEF) in India. The Central Pollution Control Board (CPCB) reporting

to MOEF is an autonomous body, with no bona fide powers to enforce laws, with

its major function to provide advice and technical assistance. In addition, every

state has a State Pollution Control Boards (SPCB), which is an autonomous body

under the State Government that enforces rules and regulations. An increasing

number of environmental legislation requires the SPCB and CPCB to work

together in implementing rules and regulations.

16

Table 2.3: State specific initiatives to address the problem of plastic waste

States Date Action Taken

Himachal Pradesh (Shimla) July 1996

A ban was placed on plastic littering. The act also provides provision for imposing deterrent penalties.

Jammu and Kashmir November 1998

Ban on the use of plastic bags. They have been replaced by bags of alternative material such as jute and paper.

Maharashtra March 1999 Ban on thin plastic bags but failure of the rule due to poor enforcement.

Mumbai 15 August 2000

State law was passed upholding national law prohibiting manufacture of polybags less than 20 microns thick and also mandated names and addresses of manufacturers to be printed on all bags. It also stated that licences would be revoked for all who disobey the rule The Mumbai Municipal Corporation has appointed a team of 97 detectors who monitor the compliance and fine the defaulters. Harsh measures like a fine of Rs 2000 (USD 42) for any shopkeepers using the bags and Rs 500 (USD 10) to the user have been imposed. In fact the anti plastic drive had led to the collection of Rs 100,000 (USD 2127) in fines.

Goa January 1998

15 August 2000 October 2000

Goa Non-Biodegradable Garbage Disposal Act Goa State Government announced a total ban on the use of recycled plastic bags less than 20 microns. Eighty day clean up drive to remove plastic bags from drains, beaches and roads of the city.

2.4   Emergence of substitutes: Bioplastics  

Sustainable product development has attracted a lot of attention in the last few

years, and there has been extensive research looking at ways to provide material needs

using energy efficient, non-toxic and renewable sources rather than finite materials. It

is increasingly being realized that the use of long-lasting polymers for short-lived

applications is not entirely justified, especially when increased concern exists about

the preservation of finite resources. Conventional plastics are persistent in the

environment, if improperly disposed; they are a significant source of environmental

pollution and have a costly impact on waste management. For these reasons,

17

replacement of non-degradable polymers by degradable plastics, particularly for

single-use disposables and packaging applications, is of major interest to decision-

makers. Further, researchers have been very optimistic on the enormous potential of

replacing oil-based plastics with materials from naturally occurring polymers.

2.5   Composition of bioplastics 

Bioplastics are made by the building blocks of complex carbohydrates like

cellulose or starch and reconstructing (polymerizing) them into plastics chemically,

biologically or thermally using microorganisms. The degradation of the polymer may

be caused by naturally occurring micro-organisms, the assistance of UV and heat

radiation, sunlight, hydrolysis by water and oxidation by air. There are four main

types of bioplastics available in the market today:

• Thermoplastic starch

• Cellulose acetates

• Polyhydroxy alkanoates (PHA)

• Polylactides

The three types of biodegradable plastics introduced are photodegradable,

semi-biodegradable, and completely biodegradable.

Photodegradable plastics have light sensitive groups incorporated directly into

the backbone of the polymer as additives. Extensive ultraviolet radiation (several

weeks to months) can disintegrate their polymeric structure rendering them open to

further bacterial degradation (Kalia et al., 2000a,b). However, landfills lack sunlight

and thus they remain non-degraded.

Semi-biodegradable plastics are the starch-linked plastics where starch is

incorporated to hold together short fragments of polyethylene. The idea behind starch-

linked plastics is that once discarded into landfills, bacteria in the soil will attack the

starch and release polymer fragments that can be degraded by other bacteria. Bacteria

indeed attack the starch but are turned off by the polyethylene fragments, which

thereby remain non-degradable (Johnstone, 1990).

The third type of biodegradable plastics rather new and promising because of

its actual utilization by bacteria to form a biopolymer. Included are

18

polyhydroxyalkanoates (PHA), polylactides (PLA), aliphatic polyesters,

polysaccharides, copolymers and/or blends of the above.

2.6  Status of bioplastics in India 

There is a lot of awareness among the research institutions on the potential of

bioplastics in India. However; India appears to be investing only in first generation of

oil based plastics that have starch to impart it with biodegradability. Biodegradation

of polymers can be achieved in two ways. One way is to synthesize polymer like

PLA/PHB, which are biodegradable in nature. The other way is to modify the

properties of non-biodegradable plastics by the incorporation of additives that help in

breaking the molecular chain and permit direct metabolisation by microbes when

disposed in nature. In India the R&D efforts have focused more on the second

approach of biodegradation. The National Research Development Corporation of

India has developed a biodegradable plastic by mixing plastics/ LDPE and starch

made from tapioca and a soluble chemical agent that will soon bring biodegradable

bags in the market. This plastic is found to have adequate mechanical strength, and

has taken only 2 months under soil burial for complete disintegration. The starch

component, being organic, degrades in the soil and once the molecule of the

compound breaks, its vulnerability to bacterial attacks increases, thus resulting in its

disintegration (CDC, 2001). Currently there are two units in India who have opted for

the above-mentioned technology for production of biodegradable plastic. One of these

units located in North India is producing 30-40 tonnes/year of biodegradable plastics

in the granular form. Their products have been quality tested in India’s premiere

research institutions. Their focus has been mainly on the packaging sector (carry bags,

shopping bags, woven sack, and disposable containers. etc. The selling price of the

biodegradable plastic is Rs 80-100/kg and the price of a normal plastic product is Rs

60-70/kg. Further there is an excise duty of 16% on all products. Unfortunately cost

constraints have limited their markets, and thereby production (CDC, 2001). The

company claims its products are biodegradable when buried or under continued

exposure in atmosphere in 4 to 24 weeks thereby avoiding environmental pollution.

The market size of biodegradable plastics is estimated at present to be 46,000

tonnes in India and is likely to go up to 96,000 tonnes in 2006-07 based on a 15%

penetration level of potential segments (CDC, 2001). Product applications for bioplastics

19

will largely depend on its material properties like its strength, lifespan, resistance to heat

and water and the ability to package food items. Further the successful introduction of

these products also depends on their functional advantage, cost, waste management

attributes (ways of dealing with bio waste), ways of marketing and legislation.

2.7   Challenges ahead 

There are number of factors that impede the adoption of bioplastics. A few of

them are mentioned below:

• All bioplastics are relatively more expensive than oil based plastics. There are

high research and development costs associated with them. The scale of

production is low, and hence the price bias exists. There are technical

uncertainties with the right choice of material for selected applications.

• The legal framework for the utilization of biodegradable materials is still very

unclear. Within waste management, local authorities have not treated

bioplastics as compostable material

• Definitions of biodegradability and compostability are unresolved. An

international standard for degradable materials is now being developed, which

is vital for the bioplastic stream to operate successfully.

• The achievement of total biodegradability.

The development of starch-based biodegradable plastics looks very promising

given the fact that starch is inexpensive, available throughout the year, and

biodegradable in various environments. The main drawbacks the industry is running

into are low water-barrier properties of bioplastics and the migration of hydrophilic

plasticizers with consequent ageing phenomena. The bioplastic industry is still in its

infancy and there are still several uncertainties that prevent the large-scale adoption of

bioplastics but continuous research in this direction may ensure that bioplastics of the

future will be produced from renewable sources and will display in-use properties

similar to those of conventional plastics. This in turn may change the scenario of

plastic waste management to a large extent. One has to remember that moving from

one technology to another based on non–toxic renewable input, will not by itself

change the development paradigm. Bioplastics and products made from renewable

sources may address the issues of toxicity and conserving finite oil resources, but

20

bioplastic is just another manufactured material. Introduction of bioplastics may well

solve the waste problems of packaging, and more so single use disposals, but it will

still encourage a more conscience free consume and throwaway culture.

Bio-plastics are a promising technology that can change the scenario of plastic

waste management. There are still several aspects that are unclear about bio-plastics,

but further research may bring to light several features that might aid the replacement of

oil-based plastics. Such a development may of course solve several waste problems

related to packaging, but would probably still propagate a throwaway culture. Plastic

waste is a pressing issue in the country today. A large number of Indians have turned

away from traditional modes of consumption, and are moving towards more wasteful

patterns of resource use. The increasing purchasing power and consumerism of the

burgeoning Indian middle class is moving India into the vicious use-and-discard cycle.

Halting these consumption patterns seems difficult in the light of globalization and

modernity. Ideally, looking at more sustainable means of satisfying need should be the

goal for the near future, but we also have to face the reality and challenges of the

existing situation. The consumption of plastic will double to 8 million tonnes in 2006,

and subsequently, the plastic waste will also escalate. Given this scenario, it is crucial

for India to check the use of plastic in the country. The existing policies have not been

able to provide any respite against littering and its associated problems. Therefore there

is an urgent need to identify policy options that can help in establishing an efficient

waste management process, and ensure efficient resource use in the country. However,

finding solutions to these problems calls for an active involvement from the

stakeholders, particularly the Government, to translate the goals into reality, by taking

the required initiatives. Such a proactive approach by the Government, along with a

clear policy agenda, and the cooperation of all the stakeholders to realize the policy

goals, will only help to ensure sustainable use of plastics in the country.

2.8   Polyhydroxyalkanoates 

2.8.1   History of polyhydroxyalkanoates 

The first of the discovered PHAs was polyhydroxybutyate. Now the number of

identified PHAs exceeds 100. PHAs are stored in the form of granules by bacteria. The

stained granules are distinctly visible under the microscope. The first determination of

P(3HB) had to wait until 1926 by Lemoigne. He extracted two components from Bacillus

21

megaterium, which he considered as a product of P(3HB) hydrolysis. During the following

30 years, interest in PHA was scant. It was concluded that PHB was an intracellular

reserve material. This paper marks the time when interest of microbiologists and

biochemists in PHB began to increase. The following 40 years saw intense research on the

subject and featured important developments in the knowledge about the polymer’s

widespread occurrence in various microorganisms. Until the end of 1973, interest in PHB

had been directed almost solely at its physiological significance in the functioning of

microbes and at the influence of environmental factors on its synthesis and reutilization.

The oil crisis of 1973 and the subsequent increase in the price of oil, and basic material,

cast doubts on the future of petroleum based polymer industry and set the stage for the

search for alternative types of plastic material. In 1976, Imperial Chemical Industries (ICI)

of England started investigating whether PHB could be profitably produced by bacterial

fermentation from photosynthesis-derived carbohydrate feedstocks (Senior and Dawes,

1973). Not only could PHB be synthesized from renewable sources, but some of its

properties resembled those of polypropylene. In the following years, research on PHB and

other forms of PHAs included investigations with other microorganisms and the potential

use of these biopolymers was realized (Braunegg et al., 1998; Volova et al., 2005). Since

mid-1980s, PHB and P(3HB-co-3HV) (marketed under the trademark of BIOPOL) in

agricultural and pharmaceutical industry, Zeneca Ltd (Great Britain, Bellingham) was

established. In 1996, BIOPOL business was acquired by Monsanto. Recently, PHA

production has become the goal of many large companies in the USA, Germany, Italy,

Japan, Scandinavian countries. The cost of the first lots of Biopol was US $16/Kg as

compared to the cost of polyolefins on the world market US $1/Kg. So BIOPOL as a

packaging material is not economically competitive with them. PHAs of such high cost

can be reasonably used for specific purposes, e.g. materials for medical applications. Thus,

to produce and apply PHAs on a large scale, it is necessary first to reduce their cost. Many

firms and industrial companies such as Monsanto, Metabolix Inc., Tepha and Procter and

Gamble, deal with the commercialization of PHAs.

The most widely produced microbial bioplastics are polyhydroxyalkanoates

(PHAs) and their derivatives (Madison and Huisman, 1999). Poly(D-3

hydroxybutyrate) is the most ubiquitous and most intensively studied PHA. PHAs are

the only 100% biodegradable polymers. They are polyesters of various HAs, which

are synthesized by numerous microorganisms under unbalanced growth conditions,

22

such as limitation of an essential nutrient such as nitrogen, oxygen magnesium and

phosphorus and presence of excess carbon source. They possess properties similar to

various synthetic thermoplastics like polypropylene and hence can be used in their

place. They are also completely degraded to water and carbon dioxide under aerobic

conditions and to methane under anaerobic conditions by microorganisms in soil, sea,

lake water and sewage. PHAs are of commercial interest because chemical or

biological hydrolysis of PHAs can yield optically pure (R)-form hydroxycarboxylic

acids (Lee et al., 2000; Park et al., 2002), which are used in the manufacture of

antibiotics, vitamins, perfumes and pheromones. To date, over 150 types of

hydroxycarboxylic acid have been identified as components of PHAs (Steinbuchel

and Valentin, 1995).

2.8.2   Location of polyhydroxyalkanoates in the cell   

In the prokaryotes, PHA occurs either as inclusion bodies or as complexes of

Ca2+ and polyphosphates in the cytoplasmic membranes. It is accumulated as a

membrane enclosed inclusion in many bacteria up to 80% of the dry cell weight.

Discrete granules of PHA generally occur in the cytoplasm as inclusion bodies of

irregular morphology with diameter of about 0.2 to 0.5µm. These granules appear as

refractile inclusion under electron and phase contrast microscope. Light microscopic

investigation of the cells, stained with Sudan black B or more specific with

epiflourescence microscope using Nile Blue A provides easy means for detecting

PHA in cells.

2.8.3   Structure of polyhydroxyalkanoates 

2.8.3.1 Chemical structure  

Polyhydroxyalkanoates are polyester of various hydroxyalkanoates,

hydroxylated at positions 3, 4, 5 and 6, all of which are (R)-form chiral molecules that

are synthesized by many gram-positive and gram-negative bacteria from at least 75

different genera. General structural formula is shown in Figure 2.2 to 2.4 (Park et al.,

2005). PHB has a perfectly isotactic structure with only the (R) - configuration.

P(3HB) isolated from bacteria possesses 55 to 80% crystallinity. Polyhydroxybutyrate

(PHB) was the first PHA to be discovered in Bacillus species by Lemoigne in 1926

and is also the most widely studied and best characterized PHA. The molecular

23

weight of these compounds range from 2×105 to 3×106 Da. Depending on the number

of carbon atoms in the monomer unit, PHAs can be divided into three groups:

(1) Short chain length PHAs (scl- PHAs), which consist of 3-5 carbon atoms.

(2) Medium chain length PHAs (mcl- PHAs), which consist of 6-14 carbon atoms.

(3) Long chain length PHAs (lcl- PHAs), which consist of 17 and 18 carbon atoms.

This division of polymers into groups is based on the substrate specificity of

PHA synthases that can only accept certain hydroxyalkanoic acids in course of

polymerization (Anderson and Dawes, 1990). The PHA synthase of A. eutrophus can

polymerize 3HAs consisting of 3 to 5 carbon atoms whereas that present in

Pseudomonas oleovorans can only accept 3HAs of 6 to14 carbon atoms.

n varies from 600 to 35000 R= hydrogen Poly(3-hydroxypropionate) R=methyl Poly(3-Hydroxybutyrate) R=ethyl Poly(3-hydroxyvalerate) R=propyl Poly(3-hydroxyhexanoate) R=pentyl Poly(3-hydroxyoctanoate) R=nonyl Poly(3-hydroxydodecanoate)

Figure 2.2: Structure of polyhydroxyalkanoates

Figure 2.3: Structure of co polymer 3-HB-3HV

Figure 2.4: Structure of co polymer 3-HB-4HB

24

2.8.3.2 Physical properties  

PHB is similar to polypropylene with three unique features: thermoplastic

processability, 100% resistance to water, and 100% biodegradability (Hrabak, 1992).

PHB is an aliphatic homopolymer with a melting point of 179°C and highly

crystalline (3HB) molecules within bacteria are amorphous (Barnard and Sanders,

1989; Amor, et al., 1991; Kawaguchi and Doi, 1992) and exist as water insoluble

inclusions. Water is a minor component of PHA inclusions and therefore it was

suggested that water could act as plasticizer (Barnard and Sanders, 1989). About 5 to

10% of water was estimated to be present in the nascent PHB inclusions, which upon

removal allows for the polymer chains to rearrange into lamellar crystals.

The densities of crystalline and amorphous PHB are 1.26 and 1.18 g/cm3,

respectively. The Mw of P(3HB) produced from wild-type bacteria is usually in the

range of 1 x 103 to 3 x 106 g/mol. The family of PHAs exhibits a wide variety of

mechanical properties from hard crystalline to elastic, depending on composition of

monomer units which broadens its application area, for example, MCL-PHAs are

semi-crystalline elastomers with low melting point, low tensile strength and units to

form PHA copolymers can also improve other properties such as crystallinity, melting

high elongation to break and can be used as biodegradable rubber after cross linking.

The glass transition temperature of PHB is around 4oC while the melting temperature

is near 180oC. Mechanical properties like Young’s modulus (3.5 Gpa) and the tensile

strength (43Mpa) of P(3HB) material are close to those of isotactic polypropylene.

Bacterially produced polyhydroxybutyrate and other PHAs have sufficiently high

molecular mass to have polymer characteristics that are similar to conventional

plastics such as polypropylene (Madison and Huisman, 1999).

2.9   Factors  affecting  polyhydroxyalkanoate  synthesis  and  its 

composition 

2.9.1   Feed substrate and growth conditions  

The choice of the substrate used to produce PHAs is determined by

physiological-biochemical properties of PHA-producing microorganisms, economic

efficiency of the preferred strategy and the field of application of the ready produce.

The quality and cost of the substrates for different applications can vary. PHAs can be

25

produced from numerous substrates of different degree of reduction, energy content

and cost (Table 2.4).

Table 2.4: Production cost of polyhydroxybutyarate and substrate cost (Choi and Lee, 1997)

Substrate Approximate price, US$/t

PHB yield, t/t substrate

Substate cost, US$/t PHB

Glucose 220-493 0.38 580-1300

Sucrose 290 0.40 720

Methanol 110 0.18 610

Ethanol 440 0.50 880

Acetate 370-595 0.33-0.38 1220-1560

Dextrose 360 0/33 1180

Hydrogen 500 1.0 500

Cane sugar 200 0.33 660

Molasses 220 0.42 520

Cheese Whey 71 0.33 220

Hemicellulose hydrolysate

69 0.20 340

2.9.2   Strategies of polyhydroxyalkanoate production 

Since most PHA-producing microbes actively accumulate PHAs under

unbalanced growth, when the medium is deficient in one of the nutrients (nitrogen,

phosphorus or oxygen), the main problem in the development of production processes

is to find the conditions that would bring about both high yields of polymers and

sufficiently high production of biomass. As a rule, two-stage fed-batch methods are

used. In the first stage cells grow under optimal fermentation conditions and

accumulate biomass, in the second stage PHA is synthesized under unbalanced

conditions, e.g., in a nitrogen-free medium. PHA producing microorganisms are

usually cultivated by one of the two methods- batch (or fed-batch) cultivation or

continuous cultivation. However, whatever fermentation is chosen, high productivity

of the process is the objective.

Bacteria used to produce PHAs can be divided into two groups, depending on

the method of cultivation. The first group includes the organisms that efficiently

synthesize PHAs when energy and carbon sources are abundant in the medium but one

26

of the biogenic elements (Nitrogen, Phosphorus, Sulfur, Potassium, Magnesium or

Oxygen) is deficient. These are A. eutrophus, Azotobacter vinelandii, P. oleovorans,

etc. The second group unites microorganisms that can efficiently synthesize PHAs at

high rates under optimal growth conditions. These are A. latus and recombinant

organisms harbouring the biosynthetic operon of A. eutrophus.

2.9.3   Bacterial strain 

PHAs are produced by many different bacterial cultures. Cupriavidus necator

(formerly known as R. eutropha or A. eutrophus) is the one that has been most

extensively studied. Imperial Chemical Industries (ICI) were the first to use this

bacterial strain for the production of PHBV copolymer under the trade name Biopol.

Recently, Metabolix Inc. (USA) acquired the Biopol patents. At present, bacterial

fermentation of C. necator seems to be the most cost-effective process and even if

production switches to other bacteria or agricultural crops, these processes are likely

to use C. necator genes. A few important other strains that were recently studied

include: Bacillus sp., Alcaligenes sp., Pseudomonas sp., Aeromonas hydrophila,

Rhodopseudomonas palustris, Escherichia coli, Burkholderia sacchari and

Halomonas boliviensis.

PHA has been industrially produced by pure cultures including A. latus,

A. vinelandii, P. oleovorans, recombinant A. eutrophus and recombinant

E. coli (Grothe et al., 1999; Lee and Choi, 1999). With current advances in PHA

research, a PHA concentration of more than 80 g/l and productivity of more than 2 g/l/h

have been obtained in the laboratory using fed-batch cultivation (Lee, 1996b).

Similarly, recovery methods for PHAs of various purities from microorganisms have

received attention. Wider use of PHAs is prevented mainly by their high production

cost compared with the oil-derived plastics (Byrom, 1987; Lee and Yu, 1997). With the

aim of commercializing PHA, a substantial effort has been devoted to reducing the

production cost through the development of bacterial strains and more efficient

fermentation/recovery processes (Lee, 1996b; Grothe et al., 1999). From the literature,

the major cost in the PHA production is the cost of the substrate (Yamane, 1993).

The yields of PHA from the various substrates are similar, with one exception.

Consequently, the price of substrate has the largest influence on the cost of production

of PHA. The cheapest substrate costs $0.22/ kg of PHA compared with the cost of

27

polypropylene of $0.185/ kg. Productivity also has an effect on the production costs.

However, this is relative to the substrate, and downstream processing apparently has a

weak effect on the final cost. When the PHB productivity increased from 1.98 to 3.2

g/l/h, the PHB production cost decreased from $5.37/kg PHB to $4.91/ kg PHB (Lee

and Choi, 1999). In a laboratory fed-batch system using A. latus, the highest reported

productivity was 4.94 g/l/h which would lead to production costs of $2.6/ kg PHB. PHA

content of the produced biomass strongly affects the efficiency of the recovery process.

For example, a relatively low PHB content of 50% results in a high recovery cost of

$4.8 /kg PHB. On the other hand, the recovery cost for a process with 88% PHB

content was only $0.92/ kg PHB (Lee and Choi, 1999). A lower PHB content clearly

results in a high recovery cost. This is mainly due to the use of large amounts of

digesting agents for breaking the cell walls and to the increased cost of waste disposal.

Table 2.5: Overview of bacterial strains used to produce polyhydroxyalkanoates

Bacterial strain Carbon source (s) Polymer (s) produced Reference

Aeromonas hydrophila

Lauric acid, oleic acid mcl-PHAs Lee et al. 2000; Han et

al. 2004

Alcaligenes latus Malt, soy waste, milk waste, vinegar waste, sesame oil

PHB Wong et al. 2004, 2005

Bacillus cereus

Glucose, e-caprolactone, sugarbeet molasses

PHB, terpolymer

Labuzek and Radecka 2001; Yilmaz and Beyatli 2005; Valappil et al. 2007

Bacillus sp.

Nutrient broth, glucose, alkanoates, e-caprolactone, soy molasses

PHB, PHBV, copolymers

Katircioglu et al. 2003; Shamala et al. 2003; Tajima et al. 2003; Yilmaz et al. 2005; Full et al. 2006

Burkholderia cepacia

Palm olein, palm stearin, crude palm oil, palm kernel oil, oleic acid, xylose, levulinic acid, sugarbeet molasses

PHB, PHBV

Keenan et al. 2004; Nakas et al. 2004; Alias and Tan 2005; Elik et al. 2005

Caulobacter crescentus

Caulobacter medium, glucose PHB Qi and Rehm 2001

28

Bacterial strain Carbon source (s) Polymer (s) produced Reference

Escherichia coli mutants

Glucose, glycerol, palm oil, ethanol, sucrose, molasses

(UHMW)PHB

Mahishi et al. 2003; Kahar et al. 2005; Park et al. 2005; Nikel et al. 2006a; Sujatha and Shenbagarathai 2006

Halomonas boliviensis

Starch hydolysate, maltose, maltotetraose and maltohexaose

PHB Quillaguaman et al. 2005, 2006

Legionella pneumophila Nutrient broth PHB James et al. 1999

Methylocystis sp. Methane PHB Wendlandt et al. 2005

Microlunatus phosphovorus Glucose, acetate PHB Akar et al. 2006

Pseudomonas aeruginosa

Glucose, technical oleic acid, waste free fatty acids, waste free frying oil

mcl-PHAs Hoffmann and Rehm 2004

P. oleovorans Octanoic acid mcl-PHAs Durner et al. 2000; Foster et al. 2005

P. putida Glucose, octanoic acid, undecenoic acid

mcl-PHAs Tobin and O’Connor 2005; Hartmann et al. 2006

P. putida P. fluorescens, P. jessenii

Glucose, aromatic monomers

aromatic polymers

Tobin and O’Connor 2005; Ward and O’Connor 2005.

P. stutzeri Glucose, soybean oil, alcohols, alkanoates

mcl-PHAs Xu et al. 2005

Rhizobium meliloti, R. viciae, Bradyrhizobium japonicum

Glucose, sucrose, galactose, mannitol, trehalose, xylose, raffinose, maltose, dextrose, lactose, pyruvate, sugar beet molasses, whey

PHB Mercan and Beyatli 2005

29

Bacterial strain Carbon source (s) Polymer (s) produced Reference

Rhodopseudomonas palustris

Acetate, malate, fumarate, succinate, propionate, malonate, gluconate, butyrate, glycerol, citrate

PHB, PHBV Mukhopadhyay et al. 2005

Spirulina platensis (cyanobacterium) Carbon dioxide PHB Jau et al. 2005

Staphylococcus epidermidis

Malt, soy waste, milk waste, vinegar waste, sesame oil

PHB Wong et al. 2004, 2005

Cupriavidus necator

Glucose, sucrose, fructose, valerate, octanoate, lactic acid, soybean oil

PHB, copolymers

Kichise et al. 1999; Taguchi et al. 2003; Kahar et al. 2004; Khanna and Srivastava 2005a; Kim et al. 2005; Volova and Kalacheva 2005; Volova et al. 2005

C. necator H16 Hydrogen, carbon dioxide PHB Pohlmann et al. 2006

mcl-PHAs: medium-chain-length polyhydroxyalkanoates, PHB: poly(3-hydroxybutyrate), PHBV: poly(3-hydroxybutyrate-co-valerate), UHMW: ultra high molecular weight

2.9.4   Substrate  

One of the problems preventing the commercial application of P(3HB) is its

high production cost. From an economical point of view, the cost of substrate (mainly

carbon source) contributes most significantly to the overall production cost of P(3HB)

(Choi and Lee 1997; Lee and Choi, 1999). To reduce the substrate cost, recombinant

strains utilizing a cheap carbon source and corresponding fermentation strategies have

been developed (Lee, 1996a). There have been several reports on the production of

P(3HB) from cheap carbon sources by wild-type P(3HB) producers (Kim and Chang,

1995). However, the P(3HB) concentration and P(3HB) content obtained were

considerably lower than those obtained using purified carbon substrates. Therefore,

more efficient fermentation strategies should be developed for efficient production of

30

P(3HB) from a cheap carbon source. Several bacterial strains can produce P(3HB)

from waste products (Steinbüchel and Valentin, 1995; Cho et al., 1997; Song et al.,

1999). If waste product:stream can be used as a substrate for the production of

P(3HB), combined advantages of reducing disposal cost and production of value-

added products can be realized. However, so far, only low P(3HB) content and

productivity were achieved from waste products. Since PHA production from glucose

or sucrose already had been optimized, development of fermentation technology to

use cheaper carbon sources would be a key factor in reducing the PHA production

cost.

The production of PHA by different microorganisms under different growth

conditions is summarized in Table 2.6.

Table 2.6: Polyhydroxybutyrate production from biowastes by diverse microorganisms PHB

Microorganism Substrate Culture mode

Time (h) Yield

(%) Conc. (g/l)

Reference

Archaea Extruded rice bran 55.6 77.8

Haloferax mediterranei Extruded

corn starch

Fed batch 120 38.7 24.2

Huang et al., 2006

Actinobacteria

Rhodococcus ruber

Low- rank coal liquefaction products

Batch 120 6 0.118

Füchtenbusch and Steinbüchel, 1991

Firmicutes

Bacillus megaterium

Date syrup/ Beet molasses

Batch 48 52 1.76 Omar et al., 2001

Staphylococcus epidermidis Malt wastes Batch 48 0.121 6.93 Wong et al.,

2000 α-Proteobacteria Methylobacterium sp. ZP24 Whey Batch 48 59.6 5.9 Yellore and

Desai, 1998

M. extorquens Methanol Fed batch 186 40 114 Bourque et al., 1995

M. rhodesianum Glycerol and Casein hydrolysates

Batch 45 50 11 Bormann and Roth, 1999

Sinorhizobium meliloti

Cheese Whey permeate

Batch 96 35 169 Povolo and Casella, 2003

31

PHB Microorganism Substrate Culture

mode Time (h) Yield

(%) Conc. (g/l)

Reference

β-Proteobacteria Alcaligenes eutrophaa

Starchy waste water Batch 48 34.1 1.2 Yu, 2001

A. eutropha DSM545

Potato processing wastes

Batch 120 77 5 Rusendi and Sheppard, 1995

A. eutropha strain H16 Plant oils Batch 72 79-82 2.9-3.3 Fukui and

Doi, 1998 A. eutropha NCIMP11599

Pulp fiber sludge Batch 30 78 2.5 Zhang et al.,

2004 Malt waste 32.4 18.4 A. latus

DSM1124 Soya waste Fed batch 69

22.7 6.0 Yu et al., 1999

Burkholderia cepacia

Palm oil Mill effluent Fed batch 672 57.4 2.46 Alias and

Tan, 2005 Comamonas testosterone

Vegetable oils Batch 48 53-58 41-50 Thakor et al.,

2005

Hydrogenophaga pseudoflava

Cheese Whey permeate

Batch 96 44 165 Povolo and Casella, 2003

Ralstonia eutrophaa

Topioca hydrolysate Fed batch 59 58 61 Kim and

Chang, 1995

R. eutropha DSM11348

Glycerol and Casein hydrolysates

Batch 67 47 15 Bormann and Roth, 1999

R. eutropha Food scraps Batch 80 72.6 11.3 Du and Yu, 2002

R. eutropha H16 Soybean oil Batch 96 76 0.76 Kahar et al., 2004

γ-Proteobacteria

Azotobacter vinelandiiUWD

Beet molasses fractions

Fed batch 24 70 7.8 Page, 1992

A. vinelandiiUWD Molasses Fed batch 36 66 22

Page and Cornish, 1993

Azotobacter vinelandiiUWD

Swine waste liquor Batch 18 58.3 9.4 Cho et al.,

1997 Fed batch 70 46 25 Kim, 2000 A. chroococcum

Starch Batch 58 73.9 0.864 Kim, 2000

Pseudomonas oleovorans

Low- rank coal liquefaction products

Batch 120 8 0.363 Steinbüchel and Hein, 2001

P. putida PGA1 Saponified Palm Kernel Oil

Batch 48 19-37 0.5-1.1 Tan et al., 1997

32

PHB

Microorganism Substrate Culture mode

Time (h) Yield

(%) Conc. (g/l)

Reference

49 80 69 Wong and Lee, 1998

36.5 87 168 Ahn et al., 2001

Fed batch

26 70 35.7 Park et al., 2002

Fed batch with O2 limitation

52 80 25

Recombinant Escherichia coli Whey

Fed batch without O2 limitation

35 57 32

Kim, 2000

Recombinant E. coli

Agro-industrial byproducts

Fed batch 24 72.9 51.1 Nikel et al., 2006b

Recombinant Klebsiella aerogenes

Molasses Fed batch 32 65 24 Zhang et al., 1994

Others Raw rice grain- based distillery spent wash

Batch 96 40 2.7 Khardenavis et al., 2007

Malt and Soya waste Batch 2 70 22.4 Wang et al.,

2007

Activated Sludge

Mixed liquor Batch 30 62 1.5 Satoh et al., 1998

(a: Renamed as Cupriavidus necator)

2.10   Polyhydroxyalkanoate biosynthesis pathways 

2.10.1  R. eutropha polyhydroxyalkanoate biosynthetic pathway 

Most of the organisms synthesize PHA using this pathway. The biosynthesis

pathways of R. eutropha, Zoogloea ramigera and A. beijerinckii are well established

(Doi et al., 1990). Firstly, a substrate is condensed to acetyl-coenzyme A (acetyl-

CoA). Two moles of acetyl-CoA are then used to synthesize one mole of PHB.

Acetyl-CoA is subjected to a sequence of three enzymatic reactions for PHB

synthesis.

When propionic acid is used as a sole substrate, PHB-PHV copolymer is

formed. Acetyl-CoA is formed by the elimination of carbonyl carbon from propionyl-

33

CoA. Two moles of acetyl-CoA are used to form a HB unit of the copolymer, while a

HV unit is formed by the reaction of acetyl CoA and propionyl-CoA. Figure 2.5 shows

the biosynthesis pathway of PHB-PHV copolymer by R. eutropha (Doi et al., 1990).

According to Doi et al., 1990, the degradation of PHA by R. eutropha can

occur simultaneously with its biosynthesis under nitrogen limitation. This observation

is called “a cyclic nature of PHA metabolism”. The author reported that the

composition of polymer was changed from PHB homopolymer to PHB-49%PHV

copolymer when the substrate was changed from butyric acid to pentanoic acid after

96 hours of nitrogen limitation accumulation period, i.e., there was a replacement of

PHB by PHB-PHV. Likewise, when R. eutropha with a PHV fraction of 56% of its

PHA content was fed with butyric acid as a sole substrate under nitrogen limitation,

the PHA composition changed markedly, i.e., the fraction of PHV decreased from

56% to 19% after 48 hours. These findings show the simultaneous synthesis and

degradation of PHA, i.e., the cyclic nature of PHA metabolism (Doi et al., 1990).

2.10.2  Rhodospirillum rubrum polyhydroxyalkanoate biosynthetic pathway 

This pathway is similar to the R. eutropha pathway but two enoyl-CoA

hydratases are also involved in the second step of catalyzing the conversion of L-3-

hydroxybutyryl-CoA to D-3- hydroxybutyryl-CoA via crotonyl-CoA (Anderson and

Dawes, 1990; Doi, et al., 1990; Lee, 1996a). A simple schematic of this pathway is

shown as:

Acetate acetyl CoA aceto-acetyl-CoA L - 3- hydroxybutyryl-CoA

crotonyl CoA D-3-hydroxybutyryl-CoA PHB.

2.10.3  Pseudomonas oleovorans polyhydroxyalkanoate biosynthetic pathway 

This biosynthesis pathway is found in P. oleovorans and most pseudomonads

from the rRNA homology group I (Lee, 1996a). These organisms produce medium-

chain-length (MCL) PHAs (from C6-C9) from MCL-alkanes, alcohols, or alkanoates.

According to Doi et al., 1990, productions of short-chain-length (SCL) PHAs, i.e.,

PHB homopolymer and PHB-PHV copolymer, cab be produced by these organisms

but the productions were less than 1.5%. This PHA biosynthesis involves the cyclic-

β-oxidation and thiolytic cleavage of fatty acids, i.e., 3- hydroxyacyl-CoA, and

intermediates of the β-oxidation pathways, are used for PHA biosynthesis.

34

2.10.4  Pseudomonas aeruginosa polyhydroxyalkanoate biosynthetic pathway 

Most pseudomonads from the rRNA homology group I except P. oleororans

also produce MCL PHAs using this pathway. The pathway used in these organisms is

called the P. aeruginosa PHA biosynthetic pathway. Steinbuchel, 1991 stated that

MCL-PHAs produced by this pathway are from unrelated substrates, e.g., gluconate

or acetate. PHA is synthesized from acetyl-CoA via fatty acid synthetic pathways.

2.11   Genes and enzymes involved in polyhydroxyalkanoate synthesis  

The organization of PHB metabolic genes in R. eutropha is shown in

Figure 2.5. The phaCBA cluster encodes three proteins: PhaA (β-ketothiolase), which

catalyses the synthesis of acetoacetyl–CoA from acetyl– CoA; PhaB (NADPH-

oxidoreductase), which stereospecifically reduces acetoacetyl–CoA to (R)-3-

hydroxybutyryl– CoA; and PhaC (PHB polymerase), which promotes the

incorporation of (R)-3-hydroxybutyryl– CoA enantiomers in the growing polymer

(Figure 2.5). PhaC is very active towards monomers containing less than five carbon

atoms, although it also synthesizes polymers containing small quantities of higher-

length monomers (C6–C8) (Sudesh et al., 2000a; Zinn et al., 2001; Salehizadeh et al.,

2004). The regulation of the PHB pathway seems to be complex. An excess of acetyl–

CoA reduces the synthesis of PHBs, whereas all the metabolic or environmental

conditions that cause a reduction in the pool of acetyl–CoA start, or restore, PHB

synthesis (Steinbu¨chel and Schegel, 1991; Zinn et al., 2001). Furthermore, B.

megaterium PhaC is synthesised as an inactive protein that requires a different

polypeptide (PhaR) to be converted into a functional enzyme (McCool and Cannon,

2001), suggesting that PHB regulation involves different environmental, metabolic

and genetic signals (Madison and Huisman, 1999, Luengo et al., 2003). Two

additional proteins, PhaZ and PhaP (phaZ and phaP gene products, Figure 2.5), also

participate either in the catabolism (PhaZ) or in the stabilization (PhaP) of the PHB

granule. PhaZ is a depolymerase (structurally related to esterases) that catalyses the

release of (R)-3- hydroxybutyrate from the polymer (or from oligomers longer than

dimers) (Figure 2.6) (Saegusa et al., 2001, Jendrossek and Handrick, 2002). In the

absence of PHB, PhaZ is produced as an inactive protein that requires PHB and an

activator (which could be replaced by trypsin) to be transformed into an active

35

enzyme. These observations suggest that either PhaZ is synthesized as a proenzyme,

or the attack of PhaZ to the granule surface requires the participation of a proteolytic

enzyme (Zinn et al., 2001). Recent studies have shown that the degradation of PHBs

is a complex mechanism that requires several depolymerases (PhaZ1, PhaZ2 and

PhaZ3) together with other as yet uncharacterized enzymes (Saegusa et al., 2001).

PhaP (phasin) is a low-molecular-weight protein (accumulated to high levels during

PHB synthesis) that enhances PHB production by binding to the granules (it regulates

the size, number and surface to volume ratio of PHB inclusions) (Madison and

Huisman, 1999; Sudesh et al., 2000b; Zinn et al., 2001). Recently, it has been reported

that the synthesis and accumulation of PhaP is a PHB-dependent mechanism

involving the participation of PhaR (an autoregulated repressor) (York et al., 2001).

However, regulation of the size and number of PHB inclusions is not only modulated

by PhaP but also by the quantity of PhaC present in the cells. PHAs are polyesters

containing monomers of medium chain length (mclPHAs, C5–C14) or long-chain

length (lclPHAs, >C14). Although PHAs are structurally related to PHBs (short-chain

length, sclPHAs) (Lageveen et al., 1998), the microbes that synthesize PHBs usually

fail to make PHAs. However, recombinant organisms containing mixed catabolic

pathways are able to synthesize either polymers (or co-polymers) containing scl, mcl

monomers, or both (Lee, 1996a; Matsusaki et al., 1998; Sudesh et al., 2000b; Zinn et

al., 2001). The organization of the mclPHA biosynthetic genes in P. oleovorans and

in P. putida is shown in Figure 2.6. The phaC1ZC2D operon encodes two

polymerases (PhaC1 and PhaC2), a depolymerase (PhaZ) and the PhaD protein

(Huisman et al., 1991; Steinbu¨chel and Hein, 2001). The two polymerases, which are

members of the a/b hydrolase subfamily, catalyze the condensation into PHAs of

several (R)-3- hydroxy-acyl–CoA derivatives (saturated, unsaturated, linear, cyclic,

branched or substituted with different functions such as halogen atoms, hydroxy,

cyano, carboxy, or phenyl groups) whose side chains range between C5 and C14

atoms (Lageveen et al., 1998; Sudesh et al., 2000b; Steinbu¨chel and Hein, 2001).

Both enzymes are quite similar in their amino acid sequence (about 50%) and

substrate specificity (3-OH-acyl–CoA derivatives and 4-, 5- or 6-OH-acyl-CoAs)

(Madison and Huisman, 1999), although when expressed in foreign hosts, they are

also able to polymerize other monomers (Lee, 1996b; Madison and Huisman, 1999;

Antonio et al., 2000; Sudesh et al., 2000a; Steinbu¨chel and Hein, 2001).

Identification of the amino acid residues required for catalysis (Rehm et al., 2001) has

36

allowed modification of the catalytic rates in several PHA syntheses (Taguchi et al.,

2002). Most mclPHA intermediates are obtained through fatty acid b-oxidation,

although other monomers (synthesized from different carbon sources) can also be

obtained via different pathways (Figure 2.6). The use of one or the other seems to be a

strain-specific trait. A different type of polymerase (type III) exists in Chromatium

vinosum, Thiocystis violacea, Thiocapsa pfennigii and Synechocystis sp. PCC 6803

(Sudesh et al., 2000b). This is formed by PhaC and PhaE, which mainly synthesise

sclPHAs but which also polymerise scl and mcl monomers (Sudesh et al., 2000b;

Steinbu¨chel and Hein, 2001). The existence of two polymerases in the same

microorganism (probably as a consequence of gene duplication) represents an

interesting evolutionary event that could have contributed to the biochemical

transition from PHBs (which only require a single polymerase) to PHAs (where two

enzymes are involved). Further studies are needed to confirm this hypothesis. phaC1

and phaC2 are separated by a third gene that encodes the PhaZ product (Figure 2.6).

This protein, containing a conventional lipase box (Madison and Huisman, 1999;

Sudesh et al., 2000a; Zinn et al., 2001; Williams and Martin, 2002), shows a certain

homology with depolymerases (enzymes involved in the mobilization of sclPHAs)

and with many hydrolytic enzymes, suggesting that it participates in the release of

hydroxyacyl–CoA derivatives from PHAs. The topological localization of PhaZ

(granule surface) (Sudesh et al., 2000b; Zinn et al., 2001) and the inability of certain

bacteria to mobilize mclPHAs when phaZ is mutated strongly supports its

physiological function (Madison and Huisman, 1999). Expression of the phaC1ZC2D

cluster in P. putida U must be under the control of promoter sequences located

upstream of phaC; otherwise, it would not be possible to account for the drastic

reduction in PHA synthesis that occurs when phaZ is disrupted, or when a plasmid is

introduced between the duplicated copies of phaC1 (Figure 2.6) (Zinn et al., 2001).

An additional cluster (phaFI), also involved in the biosynthesis of PHAs, is located

downstream from the phaC1ZC2D operon. These genes encode phasins PhaF and

PhaI. PhaF, a histone-H1-like protein, plays a dual function: it is involved in the

stabilization of the granule, and it acts as a regulator (Prieto et al., 1999). PhaF is a

granule-associated protein that represses phaC1ZC2D and phaI and contributes to the

stabilization of PHA granules; whereas PhaI, another granule-associated protein, only

participates in the formation and stabilization of PHA inclusions (Prieto et al., 1999).

In the absence of PHAs precursor, phaC1ZC2D and phaI are not expressed, whereas

37

when mcl monomers are synthesized, PhaF is removed from the DNA, initiating (or

restoring) PHAs production. Under these new conditions, PhaF and PhaI interact with

the hydrophobic nascent polymeric chains, contributing (in an isolated fashion or as a

complex) to granule formation (Prieto et al., 1999). The physiological role of PhaD

remains obscure. It is not a granule-associated protein, although it seems to be

required for PHA formation (Klinke et al., 2000). Very recently, we have observed

that the deletion of phaDFI in the overproducing mutant P. putida U DfadBA causes a

considerable reduction (>70%) in the synthesis of aliphatic PHAs, whereas poly(3-

hydroxyphenylalkanoates) are not produced. Furthermore, the restoration of PHA

synthesis in this double-deleted mutant (DfadBAD phaDFI) requires the expression of

phaF, whereas in its absence, even when phaD and phaI are expressed, this effect was

not reversed.

Figure 2.5: Organization of the genes and enzymes involved in the biosynthesis of bioplastics. (a) Biosynthesis of PHBs in Ralstonia eutropha (formerly Alcaligenes eutrophus) (b) PHAs in Pseudomonas oleovorans (c, d) PHAs in different mutants of Pseudomonas putida U designed to prove the existence of promoters downstream from phaC1 (c) Pseudomonas putida U mutant disrupted by the insertion of the integrative plasmid pK18::mob into the depolymerase gene (d) Pseudomonas putida U mutant in which the phaC1 gene has been duplicated and a new cluster phaC1ZC2DFI, without the promoter region (P1) located upstream from phaC1, has been generated. (Luengo et al., 2003)

38

Figure 2.6: Structural organization of a polyhydroxyalkanoates granule and metabolic interconnections between the different pathways involved in the biosynthesis and catabolism of polyhydroxybutyrates and polyhydroxyalkanoates. (a) Alkane oxidation pathway. (1) Alkane 1-monooxygenase, (2) alcohol dehydrogenase, (3) aldehyde dehydrogenase. (b) Fatty-acid b-oxidation. (4) acyl–CoA ligase, (5) acyl–CoA dehydrogenase, (6) enoyl–CoA hydratase, (7) 3-hydroxyacyl–CoA dehydrogenase, (8) 3-ketothiolase, (9) (R)-enoyl–CoA hydratase, (10) 3-ketoacyl–CoA reductase. (c) Biosynthesis from carbohydrates. (11) b-ketothiolase, (12) NADPH-dependent acetoacetyl–CoA reductase. (d) De novo fatty acid synthesis. (13) acetyl–CoA carboxylase, (14) ACP-malonyltransferase (15) 3-ketoacyl-ACP synthase, (16) 3- etoacyl-ACP reductase, (17) 3-hydroxyacyl-ACP reductase, (18) enoyl-ACP reductase, (19) 3-hydroxyacyl-ACP–CoA transacylase. (Luengo et al., 2003)

2.11.1  Polyhydroxyalkanoate production in recombinant Escherichia coli  

In recent years, a combination of genetic engineering and molecular

microbiology techniques has been applied to enhance PHA production in

microorganisms. Several mutants with phenotypes in PHA synthesis were

characterized in order to develop optimal recombinant host strains. Over-expression

39

of pha genes in the natural PHA producer, however, resulted in little difference in

polymer accumulation. Natural producers, such as R. eutropha, are well adapted to

PHA accumulation in their cells. R. eutropha can store up to 90% of its dry weight

(dwt) in PHA granules. Most natural producers, however, take a long time to grow

during fermentation and extraction of polymers from their cells is difficult. Therefore,

these PHA producers are not suitable for industrial production of the biopolymer. On

the other hand, although E. coli does not naturally produce PHA, this bacterium is

considered to be appropriate host for generating higher yields of the biopolymer

because of its fast growth and the ease with which it can be lysed. Even after

extensive attempts at maximizing PHB production in non-PHB producing

microorganisms, the PHB accumulation level was not as high as what could be

obtained with the natural producers of the biopolymer. One of the major obstacles in

producing PHB in recombinant organisms is associated with the instability of the

introduced pha genes. Loss of the plasmid due to metabolic load often limits high

yields of the biopolymer (Steinbüchel and Pieper, 1992; Lee et al., 1996b; Madison

and Huisman, 1999). Other parameters have been adjusted to enhance PHB

production including increased carbon supply, changes in fermentation temperature,

changes in the number of plasmid copies and choice of bacterial strains (Kim et al.,

1994; Lee et al., 1996b, Nikel et al., 2006b). Growth of the recombinant cells was

impaired in many of these studies, especially in nutrient-rich medium (Lee, 1996a;

Wang and Lee, 1997). Recombinant E. coli cultured under optimal conditions has

been shown to accumulate PHB up to 85% of the cell dwt. PHB formed in these E.

coli, however, were of higher molecular weight than PHB produced by natural

producers (Zhang et al., 2004). The molecular mass of the PHB produced in E. coli

cells depended strongly on culture condition. In higher glucose concentration (20 g/l),

37 °C and pH 6.0, cells produced PHB with highest molecular mass value (20 MDa).

It has been suggested that a chain-transfer agent is generated in E. coli cells during the

accumulation of PHB. After the cloning of the R. eutropha PHA biosynthesis genes in

E. coli, recombinant E. coli has been investigated for the production of P(3HB)

because it has several advantages over other bacteria (Lee, 1996b). There have been a

series of papers that described the development of host–plasmid systems and the

strategies for producing a high concentration of P(3HB) with high productivity (Wang

and Yu, 2000). By the pH-stat fed-batch culture of recombinant E. coli harboring the

R. eutropha PHA biosynthesis genes, a cell dry weight of 206 g:l, P(3HB)

40

concentration of 149.7 g:l, and P(3HB) content of 73% were achieved in a chemically

defined medium, resulting in the P(3HB) productivity of 3.4 g:l per h (Wang and Yu,

2001). During the fed-batch culture of recombinant E. coli, a large amount of oxygen

was necessary to maintain the dissolved oxygen concentration above 10% of air

saturation. Since the use of a large amount the efficient production of P(3HB) by E.

coli. Recombinant E. coli strains harboring the initially cloned 6.4-kb DNA fragment

of A. latus produced P(3HB) to 50% of dry cell weight. A higher P(3HB)

concentration and P(3HB) content could be obtained by deleting the unnecessary

DNA fragment upstream of the PHA biosynthesis operon. By the pH-stat fed-batch

culture of recombinant E. coli harboring an optimally designed plasmid containing the

A. latus PHA biosynthesis genes in a chemically defined medium, final cell and

P(3HB) concentrations of 194.1 and 141.6 g:l, respectively, were obtained in 30.6 h,

resulting in a much higher productivity of 4.63 g P(3HB)/l/h. This should allow more

economical production of P(3HB) by recombinant E. coli.

2.11.2  Polyhydroxyalkanoate production in eukaryotic cells 

The production of bioplastic in bacteria is limited by its high cost compared to

the costs associated with petroleum-derived plastics production. This aspect has been

one of the driving forces in exploring eukaryotic systems, especially crops, as

production hosts. Studies of PHA formation in yeast and insect cells can provide

valuable information about how these pathways can be incorporated into plants.

Synthesis of PHB has been demonstrated in Saccharomyces cerevisiae by expressing

the PHB synthase gene from R. eutropha. PHB accumulation, however, as very low

(0.5% of cell dwt), possibly because of insufficient endogenous β-ketoacyl- CoA-

thiolase and acetoacetyl- CoA reductase activities. To improve the yield and to

synthesize copolymers of PHAs, studies have focused on channeling the intermediates

of β-oxidation pathway into PHA assembly. Poirier et al., 1995, introduced a modified

phaC1 gene from P. aeruginosa into S. cerevisiae. Peroxisomal targeting (PTS1) of

the gene product was achieved by developing a construct which resulted in the

addition of a 34 amino acid stretch from the carboxylic end of Brassica napus

isocitrate lyase. When the recombinant yeast cells were grown in media containing

fatty acids, they accumulated mcl-PHAs demonstrating that peroxisomal PHA

synthase produces PHA in the peroxisomes using 3-hydroxyacyl- CoA intermediates

of fatty acid oxidation. In contrast to S. cerevisiae, Pichia pastoris grows vigorously

41

on fatty acids as a carbon source. Poirier et al., 1992, introduced the above PTS1-

modified P. aeruginosa phaC1 gene into P. pastoris and achieved mcl-PHA synthesis

in this yeast system with fatty acids in the growth medium. The yield of PHA in the

two described studies with yeast systems, however, was low, with accumulations

lower than 1% cell dwt. Possibilities have been explored of changing monomer

composition of PHA in recombinant yeast cells. The investigators demonstrated that it

was possible to alter the PHA monomer composition of mcl-PHAs produced in yeast

from the intermediates of the β-oxidation of fatty acids by using a modified form of

the peroxisomal multifunctional enzyme 2 (MFE-2, encoded by the fox2 gene). They

transformed yeast cells with genes coding for two mutant forms of the 3-hydroxyacyl-

CoA dehydrogenase domain of the MFE-2 of S. cerevisiae. The mutant MFE-2(aΔ)

retain a broad activity towards short-, medium- and long-chain (R)-3-hydroxyacyl-

CoAs, while the mutant MFE-2(bΔ), did not accept shortchain (R)-3-hydroxyacyl-

CoAs. Expression of MFE-2 (bΔ), along with PHA synthase, resulted in a substantial

increase in the proportion of the short-chain 3-hydroxyacid monomers at the expense

of longer monomers. These transformant yeast cells were inefficient at using short-

chain (R)-3-hydroxyacyl-CoAs generated by the β-oxidation cycle, leading to higher

levels of these intermediates available to the PHA synthase. Zhang et al., 2004,

engineered the synthesis of PHA polymers composed of monomers ranging from 4 to

14 carbon atoms in either the cytosol or the peroxisome of S. cerevisiae by harnessing

intermediates of fatty acid metabolism and achieved accumulation of PHA up to

approximately 7% of its cell dry weight. Insect cells have also been studied as a

model for PHA production in eukaryotes. The phaC gene from R. eutropha was

successfully expressed in cabbage looper cells and a soluble form of PHB synthase

that could be rapidly purified was obtained (Williams and Martin, 2002). In a separate

attempt, Williams and Martin, 2002 transfected fall armyworm cells with a modified

eukaryotic fatty acid synthase, which did not extend fatty acids beyond HB, along

with the phaC gene from R. eutropha. PHB production was achieved in the

transfected cells, although the yield was very low (% of cell dry weight).

2.11.3  Polyhydroxyalkanoate synthesis in transgenic plants 

Polyhydroxyalkanoate production in bacteria and yeast requires growth under

sterile condition in a costly fermentation process with an external energy source such

as electricity. In contrast, PHA production in plant systems is considerably less

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expensive because the system only relies on water, soil nutrients, atmospheric CO2

and sunlight. In addition, a plant production system is much more environmentally

friendly. Plants use photosynthetically fixed CO2 and water to generate the bioplastic,

which after disposal is degraded back to CO2 and water. Synthesis of PHAs in crops is

also an excellent way of increasing the value of the crops (Poirier et al., 1995). Since

starch and sugar are produced in plants at costs below the cost of commodity plastics,

it might be possible to produce PHA at a similar low cost. Unlike the bacterial cell,

the plant cell has different subcellular compartments in which PHA synthesis can be

metabolically localized. As mentioned earlier, PHB is synthesized in bacteria from

acetyl-CoA. This thioester is present in plant cells in the cytosol, plastids,

mitochondria and peroxisomes. Therefore, it should be possible to produce PHB in

any of these subcellular compartments.

2.12  Polyhydroxyalkanoate recovery processes 

In addition to the costs of maintaining pure cultures and the high costs of

organic substrates, polymer recovery process is another factor that contributes to the

high overall cost of PHA production. In the past 2 decades, several recovery processes

have been investigated and studied in order find an economic way to isolate and

purify PHA. According to Doi et al., 1990; Lee, 1996a and Braunegg, 1998, several

methods have been used as a recovery process for PHA. These methods include

solvent extraction; the most common analytical technique used for PHA estimation

was a gravimetric method (Lemoigne, 1926), which consisted of PHB extraction from

lyophilized biomass with chloroform followed by precipitation with diethyl ether or

acetone. In 1958, Williamson and Wilkinson showed that under controlled conditions

of time and temperature all cell material, except PHB granules, dissolved in alkaline

sodium hypochlorite solution (Williamson and Wilkinson, 1958). Further

developments on this method were introduced (Law and Slepecky, 1969) wherein the

extracted PHB was converted (with concentrated sulphuric acid) to crotonic acid, and

estimated spectrophotometerically at 235 nm. Detection of PHA extracted with

chloroform can also be done by IR spectroscopy (at 5.75Å) (Jüttner et al., 1975), and

enzymatic digestion. Details of each method as well as their advantages and

disadvantages will be discussed and summarized here. In most cases, bacterial

biomass is separated from substrate medium by centrifugation, filtration or

43

flocculation. Then, the biomass is freeze dried (lyophilized). Basically, mild polar

compounds, e.g., acetone and alcohols, solubilize non-PHA cellular materials whereas

PHA granules remain intact. Non-PHA cellular materials are nucleic acids, lipids,

phospholipids, peptidoglycan and proteinaceous materials. On the other hands,

chloroform and other chlorinated hydrocarbons solubilize all PHAs. Therefore, both

types of solvents are usually applied during recovery process. Finally, evaporation or

precipitation with acetone or alcohol can be used to separate the dissolved polymer

from the solvent.

2.12.1  Solvent extraction 

This method is used on a small scale for laboratory experiments as well as on

a large scale for commercial production. This method is a widely used method

because it is applicable to many PHA producing microorganisms. However, a large

amount of solvent is employed because PHA solution is highly viscous. According to

Lee, 1996b, approximately 20 parts of solvent is employed to extract 1 part of

polymer. This requirement makes solvent extraction a costly method. PHAs are

soluble in solvents, such as chloroform, methylene chloride or 1,2- dicholoroethane.

These 3 solvents can be used to extract PHA from bacterial biomass. In addition,

other solvents were also reported to be used to extract PHA, e.g., ethylene carbonate,

1,2-propylene carbonate, mixtures of 1,2-trichloroethane with water, and mixtures of

chloroform with methanol, ethanol, acetone or hexane. Doi et al., 1990, described a

chloroform extraction method. PHA is extracted with hot chloroform in a soxhlet

apparatus for over 1 hour. Then, PHA extracted is separated from lipids by

precipitating with diethyl ether, hexane, methanol, or ethanol. Finally, PHA is

redissolved in chloroform and further purified by precipitation with hexane. Ramsay

et al., 1995, examined the recovery of PHA from three different chlorinated solvents

(chloroform, methylene chloride, and 1,2-dichloroethane). They obtained the best

recovery and purity when biomass was pretreated with acetone. The optimum

digestion time for all three solvents was 15 minutes. Further digestion resulted in

degradation in the weight molecular weight (MW) of PHA. The degree of recovery

when the biomass were pretreated with acetone were 70, 24, and 66% when reflux for

15 minutes with chloroform, methylene chloride, and 1,2-dichloroethane,

respectively. Whereas the level of purity of these 3 solvents under these optimum

conditions were 96, 95, and 93%, respectively. Temperatures used of these three

44

solvents were 61, 40, and 83°C, respectively. The authors emphasized that extraction

conditions have a great impact on the degradation of PHA during the recovery

process.

2.12.2  Sodium hypochlorite digestion 

Sodium hypochlorite solubilizes non-PHA cellular materials and leaves PHA

intact. Then, PHA can be separated from the solution by centrifugation. A severe

degradation of polymers during sodium hypochlorite digestion is frequently reported.

Because sodium hypochlorite is a strong oxidant, care has to be taken to select for

suitable digestion conditions in order to maintain a high molecular weight of the

polymers. Ramsay et al., 1995, examined the PHA recovery process from R. eutropha

using hypochlorite digestion with surfactant pretreatment. Two different surfactants

were investigated: Triton 100 X and sodium dodecyl sulfate (SDS). Improvements in

purity and molecular weight can be obtained by pretreating with surfactant prior to the

extraction with sodium hypochlorite. They reported that surfactant removed

approximately 85% of the total protein and additional protein (10%) was further

removed by sodium hypochlorite digestion. They also stated that this method resulted

in a high MW of extracted PHA and the recovery time was reduced when compared

to surfactant-enzymatic treatment or solvent extraction. In addition, a native PHA

granule could be maintained during this treatment, which allows PHA to be used for

more diverse applications in comparison to solvent extraction method.

The above-mentioned methods are time-consuming and not accurate

particularly for low PHB concentrations (Lafferty et al., 1988). Large sample volumes

and a high degree of replicates are required to minimize errors. Furthermore, cell

material can interfere with PHB extraction (Braunegg et al., 1978). These methods

allow for the quantification of total PHA but not the individual PHA components.

2.12.3  Gas chromatography method 

The use of gas chromatography (GC) for the identification of PHA

components was then proposed. This method involves simultaneous extraction and

methanolysis of PHA, in mild acid or alkaline conditions, to form hydroxyalkanoates

methyl esters, which are then analyzed by GC (Braunegg et al., 1978). This method is

rapid (4 h), sensitive, reproducible and requires only small samples. It features

reaction and extraction in the same screw-capped tube. Another GC method for

45

increased PHA recovery was proposed by carrying out propanolysis in HCl (Riis and

Mai, 1988) rather than acidic methanolysis in sulphuric acid.

Other recent methods for PHA quantification include HPLC (Karr et al.,

1983), ionic chromatography, and enzymatic determination (Hesselmann et al., 1999).

HPLC measures only PHB and is based on conversion of PHB to crotonic acid

followed by UV detection at 210 nm. This method features 84% recovery of PHB.

PHA detection by ionic chromatography is based on the conversion of monomers to

alkanoic acids. The determination involves acid propanolysis followed by an alkaline

hydrolysis with Ca(OH)2 or acidic hydrolysis with concentrated H2SO4. After

centrifugation, the sample is injected into an anionic column with a conductivity

detector. 3HB is enzymically oxidized and the NADH produced from NAD+ was

reoxidised in the presence of iodonitro-tetrazoliumchloride to produce formazan,

which was spectrophotometerically measured at 492 nm. The determination of PHA

inside intact cells by two-dimensional fluorescence spectroscopy and flow cytometry

has also been proposed recently. Cells stained with Nile Blue, show a clear

fluorescence maximum between 570 and 605 nm when excited between 540 and 560

nm. A good correlation between fluorescence intensity and PHB concentration was

obtained. However, differentiation of PHA composition was not possible with this

method. Ease of recovery of PHA is a very important parameter in their economical

production. Thus, there is a need for development of methods for extraction of PHB

so that the overall process could be made much simpler and cheaper.

2.13   Applications of polyhydroxyalkanoates  

2.13.1 Medical and pharmaceutical 

The degradation product of P(3HB), D-3-hydroxybutyric acid, is a common

intermediate metabolic compound in all higher organisms (Lee, 1996a). Therefore, it

is plausible that it is biocompatible to animal tissues and P(3HB) can be implanted in

animal tissues without any toxic. Some possible applications of bacterial PHAs in the

medical and pharmaceutical applications include: biodegradable carrier for long term

dosage of drugs inside the body, surgical pins, sutures, and swabs, wound dressing,

bone replacements and plates, blood vessel replacements, and stimulation of bone

growth and healing by piezoelectric properties. The advantage of using biodegradable

46

plastics during implantation is that it will be biodegraded, i.e., the need for surgical

removal is not necessary.

2.13.2  Agricultural 

PHAs are biodegraded in soil. Therefore, the use of PHAs in agriculture is

very promising. They can be used as biodegradable carrier for long-term dosage of

insecticides, herbicides, or fertilizers, seedling containers and plastic sheaths

protecting saplings, biodegradable matrix for drug release in veterinary medicine, and

tubing for crop irrigation. Here again, it is not necessary to remove biodegradable

items at the end of the harvesting season.

2.13.3  Biodegradable commodity packaging 

PHA has a wide range of applications owing to their novel features. Initially,

PHA was used in packaging films mainly in bags, containers and paper coatings.

Similar applications as conventional commodity plastics include the disposable items,

such as razors, utensils, diapers, feminine hygiene products, cosmetic containers––

shampoo bottles and cups. In addition to potential as a plasticmaterial , PHA are also

useful as stereoregular compounds which can serve as chiral precursors for the

chemical synthesis of optically active compounds (Oeding and Schlegel, 1973; Senior

and Dawes, 1973). Such compounds are particularly used as biodegradable carriers

for long term dosage of drugs, medicines, hormones, insecticides and herbicides.

They are also used as osteosyntheticmaterial s in the stimulation of bone growth

owing to their piezoelectric properties, in bone plates, surgical sutures and blood

vessel replacements. However, the medical and pharmaceutical applications are

limited due to the slow biodegradation and high hydraulic stability in sterile tissues

(Wang and Bakken, 1998). PHA is considered as a source for the synthesis of chiral

compounds (enantiometrically pure chemicals) and is raw material for the production

of paints. PHA can be easily depolymerised to a rich source of optically active, pure,

bi-functional hydroxy acids. PHB for instance is readily hydrolyzed to R-3-

hydroxybutyric acid and used in the synthesis of Merck anti-glaucoma drug

“Truspot”. In tandem with R-1,3- butanediol, it is also used in the synthesis of β

lactams. Plant derived PHA can be depolymerized and used, directly or following

esterification, in the manufacture of bulk chemical. Besides helping to replace the

existing solvents, β-hydroxy acid esters and derivatives are likely to find growing use

47

as green solvents similar to lactic acid esters. The conversion of hydroxy acids into

crotonic acids such as 1,3-butanediol, lactones, etc. would help improve the market

value as they have an existing market demand in thousands of tonnes.

2.14  Economics of polyhydroxyalkanoate production 

It is a prerequisite to standardize all the fermentation conditions for the

successful implementation of commercial PHA production systems. The price of the

product ultimately depends on the substrate cost, PHA yield on the substrate, and the

efficiency of product formulation in the downstream processing (Lee, 1996b). This

implies high levels of PHA as a percentage of cell dry weight and high productivity in

terms of gram of product per unit volume and time (de Koning and Witholt, 1997; de

Koning et al., 1997). Commercial applications and wide use of PHA is hampered due

to its price. The cost of PHA using the natural producer A. eutrophus is US$16 per Kg

which is 18 times more expensive than polypropylene. With recombinant E. coli as

producer of PHA, price can be reduced to US$4 per Kg, which is close to other

biodegradable plastic materials such as PLA and aliphatic polyesters. The

commercially viable price should come to US$3–5 per Kg (Lee, 1996b). The effect of

various substrate costs and the yield on the P(3HB) production cost are described in

Table 2.6. The development of PHB was begun by Imperial Chemical Industries (ICI)

in 1975–76 as a response to the increase in oil prices. ICI started making “Biopol” as

early as in 1982 from A. eutrophus (H16). Cargill Dow Polymers is marketing its

“EcoPla” resins in Japan, Novamont_s starch-based material, “Mater-Bi”, is marketed

in Japan by Nippon Gohsei. Mater-Bi has been used in transport packaging for

electrical goods, agricultural mulch film and in composting trials. Mitsubishi and

Nippon Shokubai under the trade names “LUNARE ZT” and “Lunare SE” market

“EnviroPlastic”. “Bionolle”, thermoplastic aliphatic polyester, is manufactured by

Showa Highpolymer and Denko of Japan. It is produced by the polycondensation of

glycol with dicarboxylic acids. “Lacea” is another type of bioplastic manufactured by

Mitsui Chemicals, Japan, from fermented starch, derived from a variety of renewable

resources, such as corn, beet, cane, and tapioca. Lacea is comparable to polyethylene

in terms of transparency and similar to polystyrene or polyethylene in terms of

processability. It also claims good mould resistance, low heat of combustion which is

similar to that of paper, superior stability in processing use and biodegradability

48

superior to that of earlier polylactic acid-based materials. Daicel Chemical Industries,

Japan has developed biodegradable blends of two different kinds of material,

polycaprolactone and acetyl cellulose resin with the brand name “Celgreen”.

Shimadzu developed a fermentation process for lactic acid and collaborated with

Mitsubishi Plastics Industries to develop poly-L-lactic acid. The resins are marketed

under the trade name “Lacty”. The other bioplastics manufactured by Japan based

firms are “Eco-Ware” and “Eco-Foam” (which are starch-based) and Cardoran and

Pulluran (which are based on polysaccharides).