CHAPTER 1

PROCESS BACKGROUND & SELECTION

1.1 Introduction

Polyhydroxyalkanoates (PHAs) are biopolyesters that are stored by various

microorganism within cells as energy storage materials (Poli et al., 2011). Biopolyester can be
referred as a microbial polyester that is biosynthesized by microorganism from sugar and plant
oil and hence the bio- prefix. Polyester refers to polymers that contain ester groups within their
molecular chains. In Chen’s study, it was found that each and every polymer is polymerized in
vitro by chemical reactions excluding polyhydroxyalkanoates that are performed in vivo (Raza,
Abid and Banat, 2018). Fig. 1.1.1 shows the general structure of PHAs. Polyhydroxyalkanoates
(PHAs) are high molecular weight aliphatic bio-polymers, synthesized by microorganisms as
accumulation in granular form inside of the cell for energy storage. Madison and Huisman’s
work had reported that the accumulation could reach up to 90% of cell dry weight under nutrient
deficiency. PHA are produced by plethora of gram-positive and gram-negative bacteria of more
than 75 distinct genera (Reddy et al., 2003). The common PHA in the form of poly (3-
hydroxybutyrate) (PHB) was initially discovered in 1925 by a French scientist, Lemoigne (Chee
et al., 2010).

Figure 1.1.1 General structure of polyhydroxyalkanoates (Rs represent C1-C13 alkyl groups, X
numbered 1–4, n numbered 100–30000) (Akaraonye et al., 2010)

The exceptional resemblance of PHAs physical properties with synthetic polymers such
that polyvinylchloride has lured the commercial appeal of PHAs as plastic materials. The main
edge of PHAs compared to synthetics is that its physical possessions and degradation rate can be
adjusted by altering source of bacterial of polymer and fermentation strategies. The incorporation
of supplementary functional groups to satisfy certain applications is allowed due to specific
growth conditions of bacteria. PHAs are insoluble in water and manifest high polymerization
degree that spans from 105 to roughly 107. Surface modification by mixing PHAs with other
polymers can improve its biocompatibility therefore making them compatible for vast
applications (Kang et al., 2010).

1.1.1 Classifications of PHAs

Different number of carbon atoms in the monomeric units of PHAs can classify them into
three classes. There are short, medium or long chain length (scl, mcl and lcl), respectively (Raza,
Abid & Banat, 2018). PHAs can be classified as short-chain-length PHAs (C3–C5), which
consists of 3–5 carbon monomers, medium-chain-length PHAs (MCL-PHA, C6–C14), which
consists of 6–14 carbon monomers and long-chain-length PHAs (LCL-PHA) which have more
than 14 carbon atoms. For example, poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate)
(PHV) and their copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are typical
examples of short-chain-length PHAs, whereas poly(3-hydroxyoctanoate) (PHO) and poly(3-
hydroxynonanoate) (PHN), which are primarily formed as copolymers with 3-hydroxyhexanoate
(HHx), 3-hydroxyheptanoate (HH) and/or 3-hydroxydecanoate (HD), are typical examples of
MCL-PHAs as shown is Fig. 1.1.2.

Figure 1.1.2 Common molecular building blocks of bio-polymers naturally created via bacterial
fermentation (Raza, Abid & Banat, 2018).
1.1.2 PHAs Properties

In recent years, attention towards polyhydroxyalkanoates is increasing due to its

similarity in properties with petrochemical polymers such as polystyrene and polypropylene
(Mozejko-Ciesielska & Kiewisz, 2016). The properties of PHAs polymers are different
depending on their chemical composition. Some general properties of PHAs are summarized as;
water insoluble and relatively resistant to hydrolytic degradation, good ultra-violet resistance but
poor resistance to acids and bases, soluble in chloroform and other chlorinated hydrocarbons,
biocompatible and hence suitable for medical applications, sinks in water thus facilitates its
anaerobic biodegradation in sediments, non-toxic, and less ‘sticky’ than traditional polymers
when melted (Bugnicourt et al., 2014). A range of typical properties of PHAs are summarized in
Table 1.1.1 below.
Table 1.1.1: Range of typical properties of PHAs

Property [units] Values

Tg [ºC] 2

Tm [ºC] 160-175

Xcr [%] 40-60

E [GPa] 1-2

σ [MPa] 15-40

ε [%] 1-15

WVTR [g.mm/m2.day] 2.36

OTR [cc.mm/m2.day] 55.12

Tg: glass transition temperature, Tm: melting temperature, Xcr: crystallinity degree, E: Young’s
*

modulus, σ: tensile strength, ε: elongation at break, WVTR: water vapour transmission rate,
OTR: oxygen transmission rate.
 Crystalline polymers such as scl-PHAs, P(3HB) and P(3HB-co-3HV) possess high
melting points and are rather brittle and rigid. Nevertheless, scl-PHA such that P(4HB) is a
sturdy, workable polyester that has rather simple formation in defiance of its complex
biosynthesis (Martin et al., 2007). Apart from that, mcl-PHAs are low crystalline thermoplastic
elastomers with low tensile strength but high fracture strain. However, they hold relatively lower
melting points to scl-PHAs.

1.1.3 Properties of P(3HB)

PHB is the most extensively explored biopolymer. It has high crystallinity due to its high
degree of successive configurations. PHB is insoluble in water and has high hydrolytic
degradation resistance. It has quite low tensile strength and Young’s modulus in comparison to
synthetic polymers like polypropylene as well as poor permeability to O2 (Sudesh et al., 2000).
Respectively, amorphous and crystalline PHB have densities of 1.18 and 1.26 g/cm3. The poly
diversity of P(3HB) that has molecular weight between 10,000 to approximately 3,000,000 Da is
about 2. According to Philip et al. (2007), osteogenesis of PHB is assisted by its pure optical and
piezo electrical possession. However, PHB was found to be quite brittle with high elongation to
break. Table 1.1.2 below breaks down PHB properties in better picture.

Table 1.1.2: Properties and features of Poly(3-hydroxybutyrate), P(3HB) (Akaraonye et

al., 2010)
1. Melting point is 180 ◦C
2. High crystallinity
3. Water-insoluble
4. Possesses high polymerization degree
5. Osteoinductive due to piezoelectricity
6. Optical isomer
7. Stereochemical arrangement of atoms
8. Poor elongation to break (6%)
9. Resembles the tensile strength of polyethylene
10. Ductile and pliable (ability to be moulded)
1.1.4 Uses and Applications of PHA
Nowadays, PHAs have gain much interests in a wide range of applications in various
industries such as material industry, medical industry, fuel industry and industrial microbiology,
since it has a potential to replace petroleum-derived plastics in diverse applications (Mozejko-
Ciesielska & Kiewisz, 2016). The applications of PHAs in various industries are summarized in
the Table 1.1.3 below.

Table 1.1.3: Applications of PHAs in various industries

Industry Applications

Material  Packaging materials, daily consumables

 Smart materials
 Paper coatings
 Feminine hygiene products

Medical  Bio-implant materials

 Stents, sutures, orthopedic pins
 Drug delivery carriers, controlled drug released
 Tissue engineering materials
 Nutritional and energy supplements

Fuel  Biofuels additives

Industrial  PHAs biosynthesis as a metabolic regulator

microbiology  PHAs biosynthesis to increase robustness of industrial
microbiology
1.1.5 Biosynthesis Pathways
Biosynthetic pathways of PHA are divided into three customary routes (Fig. 1.1.3). The
utilization of carbon source usually determine the composition of monomer. The most popular of
the paths is Pathway I that is used by C. necator. Through this, two acetyl-CoA molecules that
come from tricarboxylic acid (TCA) cycle go through condensation to fashion acetoacetyl-CoA
by the enzyme B-ketothiolase. Acetoacetyl-CoA forms 3-hydroxybutyryl-CoA through the
assistance of acetoacetyl CoA reductase. Lastly, esterification process of 3 hydroxybutyryl-CoA
conducted by PHA synthase enzyme converts the structure into poly(3-hydroxybutyrate)
(P(3HB)).

Figure 1.1.3 Biosynthesis Pathways (Philip et al., 2007)

 While Pathway II shows that fatty acid degradation develops substrate for polymerization
of PHA by PHA synthases such that the ribosomal RNA-homology group I like Pseudomonas
aeruginosa. Trans-2-enoyl-CoA that is B-oxidation intermediate changed form into (R)-
hydroxyacyl-CoA. General scheme for synthesis of PHAs intracellularly by utilization of varying
types of carbon source is shown in Fig. 1.1.4.

Figure 1.1.4 General scheme for synthesis of PHAs intracellularly by utilization of varying types
of carbon source (Sudesh et al., 2000)

In similar fashion, Pathway III is the most important amongst three pathways due to
employment cheap carbon sources like glucose and fructose in formation of PHAs. Enzyme acyl-
ACP-CoA transacylase convert the (R)-3-hydroxyacyl intermediates that are the acyl carrier
protein (ACP) form to their CoA form. The significance of this particular pathway is because the
PHAs are generated by bacteria by using unrelated and easily available raw material. This builds
foundation to PHA production in commercial scale such as this project. The PHAs production
routes are generally illustrated in Fig. 1.1.5 below.
 Figure 1.1.5 General illustration of PHAs production (Chen et al., 2009)

1.2 Design Constraints and Goals

The suitability for large scale production of PHA is always a major concern. The costs of
production with the support of realistically low PHA yield by bacteria are some of the reasons
why the design of the plant has to be well-planned, efficient and effective.

1.2.1 The difficulty in maintaining condition

Bacteria cells are living organisms usually used in the intracellular accumulation of PHA.
However, due to their complicated nature. Certain environmental conditions need to be
maintained during the process of fermentation to allow the maximum PHA production by
bacteria. Therefore, the right nutrients have to be supplied and operational conditions such as the
temperature, pressure and pH of media are retained.
1.2.2 Acquirement of bacteria cells
It is important to identify the best possible bacteria strain for PHA production based on the types
of substrate used. It is known that acquirement of bact
1.3 Process Selection

Substrates or raw materials are the most important factor for high production cost in most
production processes. This is particularly true for large scale of PHA production. Overwhelming
interest in the use of attractively cheap carbon substrates for PHA production is developing since
the cost of the carbon source regards for about half of the production costs (Gholami et al., 2016).
We have considered three sources of raw materials for our production of PHA which are:

1) Palm Oil Mill Effluent (POME)

2) Oil Palm Empty Fruit Bunches (OPEFB)
3) Paddy Straw

The selection of process and raw material were made based on many criteria but most
critically the cost and availability of material. Accordingly, works have been developed in such
to improve on production cost by utilizing abundant renewable and cheap carbon sources
(Khanna and Srivastava, 2005). With Malaysia having a lot of rain and sunlight intensity all year
around in its tropical climate, it has assisted the agricultural industries to prosper, substantially,
the oil yield from oil palm industry. This correspondingly resulted in generation of large amount
of oil palm wastes through mills processing. These wastes have credible potential to be exploited
in PHA production, rather than being disposed to the environment and ultimately raise many
safety issues to many.
Likewise, the conception of biodegradable polymer is far more for than just the product
being environmentally friendly as opposed to petroleum based polymer, in fact the whole
approach for producing it has to be taken into account. The processes involved in PHA
production has to fit the objective of preserving nature.
1.3.1 Process Overview and Comparisons

Raw Materials Palm Oil Mill Effluent (POME) OPEFB (Oil Palm Empty Fruit Paddy Straw
Bunch)

Pre-treatments Acidogenic fermentation Delignification via steam explosion Alkali-Acidic treatment (Sandhya et al.,
Used (Setiadi et al., 2015) and enzymatic hydrolysis processes 2013)

Fermentation Aerobic fed batch (Setiadi et al., Batch Batch

Type 2015)

Possible Ralstonia eutropha (Setiadi et Bacillus megaterium (Zhang et al., Ralstonia eutropha (Sandhya et al.,
Bacterial al., 2015) 2013) 2013)
Strain(s)

Availability of -Abundant, large amount of -Oil palm biomass is the largest -3.2 million tonnes paddy straw
raw material biomass residue produced yearly biomass in Malaysia (Hassan et al., produced annually in Malaysia (Shafie,
by oil palm industry in Malaysia 2013) 2015)
-50 million tonnes of POME -The annual quantity of OPEFB
was discharged annually generated in Malaysia is
(Hassan, 2008) approximately 16 million tonnes
(Rahman, Choudhury and Ahmad,
 2006)

Cost of Material RM 40-400 per tonne RM 100 per tonne (from Kedah farmer)
[Utusan]

PHA Yield 37.55 % of CDW PHA accumulation

Product Type 3HB PHB PHB

Operating -Anaerobic acidogenic - Fermentation at 35 oC, agitation speed

Conditions fermentation at 27 oC. of 150 rpm for 4 days (Yamanaka et al.,
-PHA accumulation at 30 oC, 2010)
agitated at a speed of 150 rpm
(Setiadi et al., 2015)

Advantages -Readily available with -Less competitor for other -Abundantly available in Malaysia
carbon/nitrogen ratio merits its production -Cheap carbon source
use as feedstock for PHA -Easy to obtain and cheap as they are
production. seen as wastes in palm oil industry
-Supply of POME in Malaysia is -Cellulase enzyme pretreatment
continuous and never be over leads to greater conversion of the
and done with. cellulose in OPEFB into sugars
(Hassan et al., 2013)
Environmental The use of large amount of toxic
Issues chemical used in pre-treatment stage
may endanger environment.

Disadvantages -Long fermentation period -Acid treatment, which may convert the
cellulose into other products such as
furfural that can be fermentation
inhibitor (Aziz et al., 2002)
-High cost of corrosive-resistant
equipment and optimization of pre-
treatment due to nature of solvent
(Badiei et al., 2013)
1.3.2 PHA Production using POME as Substrate
The aptness of using palm oil mill effluent (POME) as raw material for PHA production
has been disclosed by Hassan et al., (1997). Organic acids attained from POME fermentation are
used in the production of PHAs. The process is acquired in two stages, with the first being
anaerobic acidogenic fermentation and followed by PHA accumulation in aerobic condition as
shown in Figure 1.3.1 (Salmiati et al., 2007). The suitability of it to be used as carbon substrate is
due to its high organic acids content. Though, the complex and complicated form of POME
inhibits the PHA synthesis by specific species of PHA-accumulating bacteria and so require pre-
treatments to break down the structure. For this reason, anaerobic treatment has been suggested
to simplify POME to its functional compound. The first step is converting POME to short-chain
VFAs expressly butyric, acetic and propionic acids by hydrolysis and acidogenesis (See
Fig.1.3.1) Next, VFAs produced are used by PHA producing bacteria for accumulation of PHA
(Lee and Yu, 1997).

Figure 1.3.1 Two-step PHA production process from POME by Ralstonia eutropha
(Setiadi et al., 2015)
 The VFAs could be either utilized in mixed culture for direct PHA generation (Salmiati et
al., 2007) or recovered primarily before being used in a pure culture (Reddy and Mohan, 2012).
However, the application of fermented POME in mixed culture has its low in terms of products
stability as well as productivity in comparison to pure.

Figure 1.3.2 Anaerobic and aerobic reactors used for the PHA production POME (Salmiati et al.,
2007)
1.3.3 PHA Production using Paddy Straw as Carbon Source
Paddy straw which is a complex lignocellulosic material has to go through hydrolysis to
be used as a competent carbon source (Sandhya et al., 2013). Its structure consists of cellulose,
hemicellulose and lignin (see Figure 1.3.3). Lignocellulosic substrates are built to be stubbornly
resistant towards enzymatic degradation. The compact form of its crystalline order is necessary
to be disrupted for chemical or microbial accessibility. Sandhya et al., (2013) reported on
delignification and hydrolysis of rice straw via chemical treatment to release its content into
simple sugars. Dried straw was ground prior to a multiple alkali-acid treatment at elevated
temperature. Straw was initially boiled with sodium hydroxide solution before similarly treated
with hydrochloric acid. The slurry composition was filtered and then washed with water. The
remaining was treated with zinc chloride solution, washed completely and resulted to hydrolyzed
straw. The white mass acquired at the end of pre-treatment was used as substrate for PHA
accumulation.

Figure 1.3.3 Composition of Rice Straw in Weight Percent (Sari and Budiyono, 2014)

The subsequent was fixed into nutrient medium containing PHA producer Ralstonia
eutropha for PHA accumulation. The culture was fermentation at temperature of 35 oC, agitation
speed of 150 rpm for 4 days (Yamanaka et al., 2010). The bacteria was able to accumulate PHA
up to 37.55 % of cell dry weight (Sandhya et al., 2013). However, the alkali-acidic pre-treatment
used to break the lignocelluloses down has its own drawbacks. The acid recovery employed by
the process is costly and due to the corrosive nature of acid, it will require an expensive
corrosion-resistant equipment that will be very hard to maintain (Badiei et al., 2013). Not only
that, the acidic treatment resulted in inhibitory by-products formation that will affect subsequent
enzymatic hydrolysis. Compounds such as furans and phenylics will be formed in such big
amount, therefore inhibit biochemical process (Jonnson and Martin, 2016). Utilization of strong
acid like hydrochloric acid will create environmental issues while it is used in large industrial
volume.

Figure 1.3.3 Degradation of lignocellulosic materials due to acidic pre-treatment (Jonnson and
Martin, 2016)

The same thing could be said about alkali pretreatment. According to Brodeur et al.,
(2011), it is unfavorable in industrial scale on account of the expensive cost of solvent and
alkaline catalyst. They also found that the conditions for alkali-acidic pre-treatment are difficult
to optimize and are important to achieve for best treatment outcome.
1.3.4 PHA production using OPEFB as Carbon Source
The Malaysian Government has initiated The 2020 National Biomass Strategy that
includes the conversion of useful biomass such as wastes from palm oil industry to products of
greater benefits (AIM, 2011). The utilization of Oil Palm Empty Fruit Bunch (OPEFB) in PHA
production is an exciting one and proving quite useful as shown by older research (Umikalsom et
al., 1997). The OPEFB, a lignocellulose constitutes of 50 % cellulose, and 25 % each of
hemicellulose and lignin (Hassan et al., 2013). It can be transformed into fermented sugars that
are a suitable carbon source from intracellular PHA accumulation by bacteria. However, due to
recalcitrant structure of OPEFB, it has to go through pre-treatment to open up the lignin prior to
enzymatic hydrolysis.

Figure 1.3.4 Breaking Down of Lignocellulosic Structure due to pre-treatment (Sari and
Budiyono, 2014)

Biomass has to go through reduction of size for better digestibility. This process includes
shredding and grinding material in order to break down its crystalline structure for better surface
area exposure (Sanchez and Cardona, 2008). Researchers also claimed that reducing size of
biomass prior to pretreatment resulted to zero production of inhibitory by-products (Tayyab et al.,
2018). Following that, small-sizes biomass will subjected to steam explosion. Steam explosion
has been successfully utilized as a pretreatment method. It requires heating at high temperature,
usually at 160 to 260 °C of lignocellulosic material such that OPEFB with high-pressure steam
for short period followed by rapid decompression which causes disruption of fibrils. This offers
better contact between enzymes and cellulose. It is a promising pretreatment due to its benefits –
being environmental friendly with little use of chemical and zero recycle.
1.3.4 Process Screening and Process Scoring
Selection of process are done by comparing a number of criteria according to their
importance. Ratings are set from either negative (unfavorable) to positive (favorable) or based on
cumulative score which will reveal higher ratings for better solution.

1.3.4.1 Process Screening

Criteria of the process are rated as follows:
Ratings Sign
Better than +
Relatively moderate 0
Worse than -

Table 1.3.1 Process Screening Matrix

Potential Raw Materials
Selection Criteria POME OPEFB Paddy Straw
Cost of Raw Material + + 0
Environmental Issues + + -
Pretreatment of Raw Material 0 + -
Product Yield 0 0 0
Fermentation Period 0 0 0
Operational Conditions 0 0 0
Sum +’s 2 3 0
Sum 0’s 4 3 4
Sum –‘s 0 0 2
Net Score 2 3 -2
Rank 2 1 3

The scores are not that far off each other relatively. All processes will be resolved and re-
evaluated in the process scoring method.
1.3.4.2 Process Scoring
The score for each process will determine their importance. Score were made according
to the accumulation of points gathered by criteria due to the weightage (of significance) times by
the raw score. Process with highest accumulated score will be chosen essentially. The point
scores were assigned as:
Relative Performance Rating
Least favorable 1
Worse than moderate 2
Comparatively moderate 3
Better than moderate 4
Most favorable 5

Whilst, the weightages for each criterion were distinguished as more important factor to
be ones with higher weightage:
Table 1.3.2 Process Scoring
Potential Raw Material
POME OPEFB Paddy Straw
Selection Criteria Weight Rating Weighted Weighted Weighted
Score Score Score
Cost of Raw material 30% 4 1.2 5 1.5 4 1.2
Environmental issues 5% 4 0.2 4 0.2 3 0.15
Pretreatment of raw material 20% 3 0.6 3 0.6 2 0.4

Product yield 25% 3 0.75 3 0.75 3 0.75

Fermentation Period 10% 3 0.3 3 0.3 3 0.3
Operational conditions 10% 3 0.3 3 0.3 3 0.3
Total 3.35 3.65 3.10
score
Rank 2 1 3
Continue? No DEVELOP No
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