Green Chemistry Letters and Reviews

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Bacterial polyhydroxyalkanoates-eco-friendly next

generation plastic: Production, biocompatibility,
biodegradation, physical properties and
applications

Muhammadi, Shabina, Muhammad Afzal & Shafqat Hameed

To cite this article: Muhammadi, Shabina, Muhammad Afzal & Shafqat Hameed (2015)
Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production,
biocompatibility, biodegradation, physical properties and applications, Green Chemistry
Letters and Reviews, 8:3-4, 56-77, DOI: 10.1080/17518253.2015.1109715

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© 2015 Taylor & Francis

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 GREEN CHEMISTRY LETTERS AND REVIEWS, 2015
VOL. 8, NO. 3-4, 56–77
http://dx.doi.org/10.1080/17518253.2015.1109715

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production,

biocompatibility, biodegradation, physical properties and applications
Muhammadi, Shabina, Muhammad Afzal and Shafqat Hameed
Department of Energy and Environmental Management, Islamabad Business School, Islamabad, Pakistan

ABSTRACT ARTICLE HISTORY

Polyhydroxyalkanoates (PHAs) are intracellular aliphatic polyesters synthesized as energy reserves, Received 3 November 2014
in the form of water-insoluble, nano-sized discrete and optically dense granules in cytoplasm by a Accepted 14 October 2015
diverse bacteria and some archae under conditions of limiting nutrients in the presence of excess
KEYWORDS
carbon source. Bacteria synthesize different PHAs from coenzyme A thioesters of respective PHA biosynthesis;
hydroxyalkanoic acid, and degrade intracellularly for reuse and extracellularly in natural biocompatibility;
Downloaded by [Central Michigan University] at 07:42 19 December 2015

environments by other microorganisms. In vivo, PHAs exist as amorphous mobile liquid and biodegradation;
water-insoluble inclusions but in vitro, exhibit material and mechanical properties, ranging from physiochemical properties;
stiff and brittle crystalline to elastomeric and molding, similar to petrochemical thermoplastics. applications
Further, they are hydrophobic, isotactic, biocompatible and exhibit piezoelectric properties. But
as commodity plastics their applications are limited by high production cost, low yield, in vivo
degradation, complexity of technology and difficulty of extraction. Therefore, to replace the
conventional plastic with PHAs, it is prerequisite to standardize the PHA production systems.

Introduction PHAs are a class of polyesters of various hydroxycar-

Expansion of conventional petrochemical plastics pro-
duction and consumption are having a significant
impact both visibly and invisibly on the environment
and society. Improper disposal of plastics has threatened
natural environment worldwide since long time ago (1).
Conventional petrochemical plastics are recalcitrant to
microbial degradation and accumulate in environment
at a rate of 28 million tons per year (2) In 2011, nearly
280 million tons of petrochemicals-based polymers
were produced with expected increased in 4% per
annum to 2016. Production of synthetic polymers is
expected to increase to around 810 million tons by
2050 (3). The challenging problems associated with pet-
roleum-based plastic accumulation in the environment
and rapid depletion of natural resources being used in
their production are motivating factors for research
into sources and tools for alternatives to petroleum-
based polymers. Considering current advances in biopo-
lymers research, bacterial polyhydroxyalkanoates (PHAs)
which are biodegradable thermoplastics have shown
great potential as a replacement for petroleum-based
plastics. Therefore, to overcome these problems, the pro-
duction and applications of eco-friendly PHAs become
inevitable.
boxylic acids, which are produced and accumulated by
a large number of bacteria under unbalanced growth
conditions as intracellular hydrophobic inclusions of
carbon and energy storage compounds in the cytoplasm
to levels as high as 90–97% of the cell dry weight (4–9) or
as an electron sink mechanism for redundant reducing
power under the condition of limiting nutritional
elements such as N, P, S, O or Mg in the presence of
excess carbon source (10–13). They are synthesized as
byproducts not the major ones when there is no suffi-
cient nutrient to promote their growth and do not play
an essential role in development of the producing organ-
isms, but convey survival in the biological community
and environment, therefore, microbial PHAs are second-
ary metabolites (14). The accumulated PHAs are
degraded by intracellular depolymerases and metab-
olized as carbon and energy source as soon as the
supply of the limiting nutrient is restored (15, 16). The
presence of sudanophilic, lipid-like inclusions which
were soluble in chloroform was initially observed in Azo-
tobacter chroococcum in early last century (17). The
chemical composition of similar inclusions in Bacillus
megaterium was later identified as poly(3-hydroxybuty-
ric acid) (P(3HB) or PHB) by Lemoigne (18, 19) and
coworkers (20) During the following 30 years, interest

CONTACT Muhammadi muhammadi12@yahoo.com

© 2015 Taylor & Francis
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

in this material was negligible. The first report on func-
tions of PHB appeared in 1958 by Macrae and Wilkinson
(21). They reported the rapid biodegradability of PHB
produced by B. megaterium, by Bacillus cereus and
B. megaterium itself. From here on, the interest in PHB
increased dramatically. Once PHAs are extracted from
bacterial cells, these polymers show crystalline, flexible,
elastic and thermoplastic properties (22–25). Further,
they are synthesized from renewable carbon resources,
based on agriculture or even on industrial wastes or fer-
mentation feedstocks (25–27) so do not lead to the
depletion of finite resources (28). Bacterial PHAs gained
particular interest since they are completely biodegrad-
able, non-toxic, biocompatible and also sources for com-
mercially useful pool of chiral monomers (29–33).
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Growing concerns about environmental pollution have
renewed interest in the development of PHAs which
are totally degraded by microorganisms present in
most environments and ecologically useful alternatives
to synthetic conventional plastics which are non-degrad-
able, and release harmful chemicals like hydrogen chlor-
ide and hydrogen cyanide during incineration (31, 34,
35). Besides being biodegradable, PHAs are recyclable
like the petrochemical thermoplastics (7). Since their dis-
covery, all these properties have made these microbial
polyesters very attractive as a source of alternative bio-
degradable materials to conventional petrochemical-
based plastics (23, 24, 36). Therefore, they are considered
for several applications in the packaging industry, biome-
dical, medicine, agriculture and food industry, or as raw
materials for the synthesis of enantiomerically pure
chemicals and the production of paints (28, 37–39).
Due to wide industrial application, PHB are considered
as green plastics. Unfortunately, the current production
costs are much higher than for petroleum-based syn-
thetic plastics, which make PHAs substantially more
Figure 1. The general molecular structure of
polyhydroxyalkanoates.
GREEN CHEMISTRY LETTERS AND REVIEWS 57
expensive and hamper the widespread usage of this
high-quality material (30, 40, 41). However, environ-
mental-friendly features such as biodegradability and
biocompatibility, which are lacking in conventional plas-
tics, have continued as the commercial interest in PHAs
(42). In the early 1990s, despite higher costs of pro-
duction, a German company Wella started using PHAs
as packaging material for some of its hair-care products
(43). Recently, PHAs have attracted considerable atten-
tion due to their potential use as biodegradable thermo-
plastics and sources of chiral monomers (34, 44, 45). Thus,
research is currently being performed to improve pro-
ductivity, to reduce production costs and, more impor-
tantly, to produce specific functionalized PHAs (46–48).
Therefore, the aim of the present study is to review the
current advances and progress in biosynthesis, accumu-
lation, extraction, chemical and physical properties, bio-
compatibility, degradation and potential applications of
bacterial thermoplastic PHAs.
Chemical structure
The PHAs that have been identified to date are rather
complex class of primarily aliphatic/linear, head-to-tail
and optically active biopolyoxoesters and composed of
(R)-3-hydroxy fatty acid monomers (Figure 1) (7, 9, 43,
45). In these polymers, the carboxyl group of one
monomer forms an ester bond with the hydroxyl group
of the neighboring monomer (Figure 1) (18, 30). In all
PHAs that have been characterized so far, the hydroxyl-
substituted carbon atom is of the R-configuration due
to the stereo specificity of PHA biosynthetic enzymes,
except in some special cases where there is no chirality
(Figure 1) (26, 43, 49, 50). Because of the chiral (R)
center in the backbone, the chemical synthesis of PHA
is difficult (49). At the same C-3 or β position, an alkyl
group which can vary from methyl to tridecyl is posi-
tioned (Figure 1). However, this alkyl side chain is not
necessarily saturated: aromatic, unsaturated, haloge-
nated, epoxidized and branched monomers have been
reported as well (51–55). The majority of PHAs are com-
posed of R(-)-3-hydroxyalkanoic acid monomers
ranging from C3 to C14 carbon atoms with a variety of
saturated or unsaturated and straight or branched
chain containing aliphatic or aromatic side groups (27,
56). Approximately 150 different hydroxyalkanoic acids
are now known to occur as constituents of PHAs (57)
and this number continues to increase with the introduc-
tion of new types of PHA through the chemical or phys-
ical modification of naturally occurring PHA, or through
the creation of genetically modified organisms to
produce PHA with specialized functional groups (58).
The molecular weight of the PHA is in the range of 2 ×
 58 MUHAMMADI ET AL.
105 to 3 × 106 Da, depending on the number of carbon
atoms constituting monomer units, the type of microor-
ganism and growth conditions (16, 43). Based on the
number of carbon atoms in the monomer units, PHAs
can be divided into four groups. The short chain length
polyhydroxyalkanoates (scl-PHAs), which consist of C3–
C5 atoms, medium chain length polyhydroxyalkanoates
(mcl-PHAs) consisting of C6–C14 atoms and long chain
length polyhydroxyalkanoates (lcl-PHAs) containing
>C14 monomers, and PHAs containing both scl- and
mcl-monomer units have been identified in several bac-
teria (59–64). In addition, the random copolymer poly(3-
hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-3HH)
or P(HB-HH) was found to be accumulated in some Aero-
monas strains (64). This grouping is due to the substrate
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specificity of the PHA synthesis that only accepts 3-
hydroxyalkanoates (3HAs or HAs) of a certain range of
carbon length (26). The PHA synthases of Alcaligenes
eutrophus can only polymerize 3HAs (scl) while that of
Pseudomonas oleovorans only polymerize 3HAs (mcl).
For scl-PHAs, the monomer units are oxidized at pos-
itions other than the third carbons while for mcl-PHAs,
all the monomers units are oxidized at the third position
except in few cases (28). A lot of PHAs (mcl) containing
various functional groups such as olefins, branched
alkyls, halogens, aromatic and cyano have been reported
(28, 53, 55). This flexibility of PHA biosynthesis makes it
possible to design and produce related biopolymers
having useful physical properties ranging from stiff and
brittle plastic to rubbery polymers (26).
Screening of bacteria for PHA production
To date two major methods have described for screening
of PHA-producing natural bacteria from environments,
namely, phenotypic-based screening and genotypic-
based screening. There are many phenotypic detection
methods for detecting intracellular PHA granules,
which are applied to the screening of PHA producers,
including Sudan black B staining (65–67), Nile blue A
staining (68–70) and Nile red (71), which result in dark
blue or fluorescent granules. Alternative staining
methods have been developed for directly staining colo-
nies (72) or growing bacteria on plates containing Nile
blue A or Nile red (68, 73), resulting in fluorescent colo-
nies that can be visualized by UV illumination. PHA-pro-
ducing colonies on plates containing Sudan black B
appear as black-bluish (74). It was reported that PHAs
stained by Nile red show a similar fluorescence behavior,
maximum at an excitation wavelength between 540 and
560 nm and an emission wave length between 570 and
605 nm detected by fluorescence spectroscopy or flow
cytometry (75, 76). This is suitable for the analysis of
granule size distributions in biotechnology (77). The
staining of cell suspensions during cultivation exper-
iments revealed that Nile red has a high potential for
the quantitative determination of hydrophobic bacterial
polyhydroxyalkanoic acids. Such optical methods offer
the advantages of online monitoring in real time with
high specificity (71). Although these staining methods
are quite sensitive, but it is rather time-consuming and
labor-intensive work to screen a large number of
environmental isolates. Moreover, prior to identification
and isolation of PHA-producing bacteria by phenotypic
methods, it is necessary to provide appropriate carbon
sources, nutrient limitation conditions and a long
culture time is required for PHA granule accumulation
to the bacterial cells (78, 79). Sudan black B is non-
specific to PHA as it also stains other lipid bodies. Nile
blue A and Nile red are reported to be more specific
than Sudan black B for detection (79). In addition,
these methods cannot distinguish between bacteria
that accumulate PHA granules and those that accumu-
late lipid compounds (78). Hong et al. developed a
rapid noninvasive technique using Fourier Transformed
Infrared Spectroscopy (FT-IR) which allows detecting
intracellular PHA accumulation in intact cells within a
few seconds. If the PHA content is high in the cells, the
FTIR spectra of PHA can also differentiate the various
structures of PHB (scl), mcl-PHAs and PHA consisting of
HB and mcl-3HA monomers (80). Genotypic-based
screening method is specific, reliable and quick which
circumvents the major drawbacks inherent in phenoty-
pic detection methods (78, 81–84). For genotypic-based
screening, the degenerate or and specific oligo nucleo-
tide primers are designed based on multiple sequence
alignment results and are used as PCR primers to
detect PHA synthase genes (78, 79, 81, 83–85). For
rapid detection, colony PCR technique is employed for
screening PHA producers from the environment (78,
80). The PHA accumulation ability of well-separated colo-
nies isolated from environmental samples can be directly
validated by PCR with no further culturing or chromoso-
mal DNA extraction procedures. In addition to its appli-
cation to the screening of wild-type isolates, the
individual PCR-amplified product is also suitable as a
specific probe for PHA operon detection in producers
and cloning (78).
Diversity among PHA-producing bacteria
PHA producers have been reported to reside at various
ecological niches which are naturally or accidently
exposed to high organic matter or growth-limited con-
ditions such as dairy wastes, hydrocarbon-contaminated
sites, pulp and paper mill wastes, agricultural wastes,

activated sludges of treatment plants, rhizosphere and
industrial effluents (86). A wide range of taxonomically
and physiologically different natural bacteria and some
archae accumulate PHAs as storage reserve materials and
deposit them as insoluble granules in the cytoplasm (10,
11, 69, 70, 83, 84). After the discovery of PHB from the bac-
terium B. megaterium (18), over 300 different bacteria,
including Gram-negative and -positive species, have
been reported to accumulate various PHAs (11, 28, 83,
86, 87). To date, most PHA-producing bacteria were
found to be Gram-negative. Comparatively, a limited
number of Gram-positive bacteria has been reported in
genera Bacillus, Caryophanon, Clostridium, Corynebacterium,
Micrococcus, Microlunatus, Microcystis, Nocardia, Rhodococ-
cus, Staphylococcus and Streptomyces (58). As for archaea
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is concerned, PHA production to date, however, has
been limited to haloarchaeal species, specifically the
genera Haloferax, Halalkalicoccus, Haloarcula, Halobacter-
ium, Halobiforma, Halococcus, Halopiger, Haloquadratum,
Halorhabdus, Halorubrum, Halostagnicola, Haloterrigena,
Natrialba, Natrinema, Natronobacterium, Natronococcus,
Natronomonas and Natronorubrum (88).
Bacteria used for the production of PHAs can be
divided to two major groups based on the culture con-
ditions required for PHA synthesis (35). First group of
bacteria requires the limitation of an essential nutrient
such as nitrogen, phosphorus, magnesium or sulfur for
the synthesis of PHA from an excess carbon source.
The bacteria included in this group are Ralstonia eutro-
pha, Protomonas extorquens and P. oleovorans. The
second group of bacteria does not require nutrient limit-
ation for PHA synthesis and the polymer is accumulated
during growth phase. It includes Alcaligenes latus, a
mutant strain of Azotobacter vinelandii, and recombinant
Escherichia coli. These characteristics are important to be
considered while the production of PHA.
In another way, PHA-producing bacteria can also
broadly be divided into four groups according to the
number of carbon atoms in the monomeric units of the
PHAs produced (39). One group of bacteria, including
Ralstonia eutropha (formerly A. eutrophus), produces
scl-PHAs, while the other group, including P. oleovorans
and most Pseudomonads belonging to the rRNA hom-
ology group I, can accumulate mcl-PHAs using 3-hydro-
xyacyl-CoA intermediates of β-oxidation pathway when
cultured on various alkanes, alkanols or fatty acids (11,
26, 83, 84). Although the majority of bacteria accumulate
either scl- or mcl-PHA, several bacteria (third group) have
also been found to synthesize PHAs (copolymers) con-
taining both scl- and mcl-HA. This largely depends on
the substrates provided and polyester synthases with
different substrate specificities (45, 89). In the presence
of propionic or valeric acid, A. eutrophus (among other
GREEN CHEMISTRY LETTERS AND REVIEWS 59
bacteria) also produces copolymers of 3-hydroxybutyric
acid and 3-hydroxyvaleric acid (HV) P(HB–HV) (90). Copo-
lymers of 3-hydroxybutyric acid with 4-hydroxybutyric
acid can also be produced in A. eutrophus (91). The bac-
teria Rhodospirillum rubrum (59), Rhodocyclus gelatinosus
(60) and Rhodococcus sp (59) produced terpolyesters
consisting of 3HA units of C4, C5 and C6 from hexanoate.
Aeromonas caviae produced a random copolymer of HB
and 3-hydroxyhexanoate (HH) (92–94). Pseudomonas
strain GP4BH1 produced PHA containing HB and 3-
hydroxyoctanoate (HO) from octanoate and PHA con-
taining HB, HO, and 3-hydroxydecanoate (HD) from glu-
conate (95). Pseudomonas sp. strain 61–3 isolated from
soil was reported to produce a blend of a PHB homopo-
lymer and a random copolymer (P(HB–HA)) consisting of
HA units of C4 to C12 from sugars and alkanoic acids (62,
63, 96). Boyandin et al. reported the luminescent bacteria
Photobacterium leiognathi and Vibrio harveyi as new pro-
ducers of two- and three-component heteropolymeric
PHA containing hydroxybutyric acid as the main
monomer, and hydroxyvaleric and hydroxyhexanoic
acids monomers as minor components (97).
In addition, two different types of polyester granules
were formed in the same cell (98). A recombinant
strain of P. oleovorans expressing R. eutropha PHB
biosynthesis genes has been shown to synthesize a
blend of a PHB homopolymer and a copolymer of HHx
and HO units when grown on octanoate (99). Both poly-
esters were stored as separated granules within the cells
(100). Fourth group of bacteria including Fluorescent
pseudomonads is capable of synthesizing lcl-PHAs
(101). In addition, Pseudomonas fluorescens and several
other Pseudomonas strains were found to produce a
P(HB-–HA) copolymer consisting of HA units of C4 to
C12 from HB and 1,3-butanediol (102). Although Thio-
capsa pfennigii accumulated only a PHB homopolymer
from various carbon sources, a recombinant P. putida
strain harboring the PHA synthesis genes of T. pfennigii
produced a P(HB–HHx–HO) terpolymer from octanoate
(103). Interestingly, the occurrence of PHA is not
limited to the intracellular collection in granules. PHB
with lower molecular weight (cPHB; Mw < 14,000 Da)
was also found in B. subtilis, A. vinelandii and Strepto-
myces lividans (104–106) in association with polypho-
sphate and calcium ions. In addition, non-storage PHAs
that are of low molecular weight, PHBs have also been
detected in the cytoplasmic membrane and cytoplasm
of E. coli (31).
Biosynthesis
PHAs are synthesized by several enzymatic reactions
from acetyl-CoAs catalyzed by substrate-specific PHA
 60 MUHAMMADI ET AL.

synthases located in the cytosol of the cell where PHA In vivo formation of PHA granules
accumulation takes place (12, 13, 26, 28, 107, 108). The
Two models of PHA granule formation have been
majority of bacteria synthesize scl-PHA, PHB and the
described: (i) the micelle model and (ii) the budding
second major class of PHA is composed of mcl-(R)-3-
model (Figure 3). Both models consider the defined
hydroxyfatty acids (45). The biosynthetic pathway of
location of the polyester synthase and to some extent
PHB consists of three enzymatic reactions catalyzed by
the phasin protein on the surface of the granule. The
three different enzymes (Figure 2). The first reaction con-
micelle model is certainly supported by PHA granule for-
sists of the condensation of two acetyl coenzyme A
mation in vitro and in the absence of membranes (Figure
(acetyl-CoA) molecules into acetoacetyl-CoA by β-ketoa-
3) (45, 117). However, electron microscopy studies
cylCoA thiolase (phbA). The second reaction is the
showing membrane-like material surrounding PHA gran-
reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA
ules in intact cells (118) or isolated granules (119) pro-
by an NADPH-dependent, (R)-specific acetoacetyl-CoA
vided evidence for the budding model. Tian et al.
dehydrogenase/reductase (phbB). Lastly, the (R)-3-hydro-
showed that early stage granules are not randomly dis-
xybutyryl-CoA monomers the direct precursor of PHB are
tributed in the cytoplasm and close to the inner cell
polymerized into PHB by P(3HB) polymerase (phbC)
membrane (120), as would be anticipated from the two
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(Figure 2) (28, 45, 109). PHB can also be synthesized

models of granule formation (121).They found that emer-
through de novo fatty acid biosynthesis and β-oxidation
ging granules arose from only the center of the cell at
pathways from sugars and fatty acids (110). In contrast,
unknown mediation elements suggesting a new model
PHAs composed of mcl-(R)-3-hydroxyfatty acids are syn-
for PHA granule formation (Figure 3) (120). Dennis et al.
thesized converting intermediates of fatty acid metab-
observed large structures (35 nm) on the surface of
olism to (R)-3-hydroxyacyl-CoA (Figure 2). If the carbon
PHB-containing granules from C. necator cells using
source is oxidized to acetyl-CoA excluding the fatty acid
atomic force microscopy which might function as syn-
β-oxidation pathway, then fatty acid de novo biosynthesis
thesis–degradation centers (121). Recent fluorescence
intermediates are diverted towards PHA biosynthesis
microscopic studies employing green fluorescent
catalyzed by the transacylase (PhaG). This specific transa-
protein (GFP)-labeled PHA synthase, that is, GFP, were
cylase catalyses the transfer of the (R)-3-hydroxyacyl
fused to the N-terminus of class I and class II PHA
moiety of the respective acyl carrier protein (ACP) thioester
synthases, without affecting PHA particle formation,
to CoA (Figure 2) (111, 112). If the carbon source is oxidized
enabled in vivo monitoring of PHA granule formation
through the fatty acid β-oxidation pathway, then the
as well as subcellular localization (122). In this study,
(R)-specific enoyl-CoA hydratase (PhaJ) catalyses the
early stage granules were found to be localized at the
oxidation of enoyl-CoA to (R)-3-hydroxyacyl-CoA (98).
cell poles suggesting that granule formation starts at
(R)-3-hydroxyacyl-CoA is a substrate for the PHA synthase
the cell poles according to the budding model. Localiz-
(PhaC) and the direct precursor of PHA biosynthesis (45)
ation of granule formation was found to be dependent
(Figure 2).
on nucleoid structure suggesting that nucleoid occlusion
occurred (122). It remains unclear whether PHA chain
synthesis is required for subcellular localization of
In vitro formation of PHA granules granule formation. Interestingly, this study led to the
observation that small emerging granules are rapidly
Gerngross and Martin were the first to demonstrate in
oscillating between the cell poles. This might play a
vitro synthesis of PHB and self-assembly of spherical
role in equal distribution of storage materials between
granules by only using purified polyester synthase and
the daughter cells (122). Overall, these in vivo studies
substrate (Figure 3) (113). This study clearly defined
using GFP-labeled polyester synthase supported the
that the PHA synthases possessed all the features
budding model by localizing granule formation close to
required for self-organization into spherical particles.
the cytoplasmic membrane sat the cell poles (Figure 3).
This was further supported by establishment of in vitro
PHA synthesis using purified PHA synthases from other
microorganisms, such as for example from Cupriavidus
Structure of PHA granules
necator, Allochromatioum vinosum, Pseudomonas aerugi-
nosa (PhaC1 and PhaC2) and P. oleovorans (PhaC1) Generally PHAs are accumulated as light-refracting dis-
(114–116). The class II PHA synthases were only recently crete granules inside the cell (123, 124). These PHA gran-
purified and provision of 3-hydroxydecanoyl-CoA as a ules can be stained specifically with Sudan black B or
substrate was sufficient for in vitro synthesis of poly(3- light fluorescent stains Nile blue A and Nile red (Figure
hydroxydecanoate) (115, 116). 4) (65, 68, 71, 125, 126). This accumulation in granules
 GREEN CHEMISTRY LETTERS AND REVIEWS 61
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Figure 2. Anabolic pathways towards PHA (scl and mcl) synthesis in bacteria. Dashed lines represent engineered biosynthesis pathways.
Triangles depict targets for inhibitors enabling PHA synthesis. PhaA/PhbA (β-Ketothiolase), PhaB/PhbB (NADPH-dependent acetoacetyl-
CoA reductase), PhaC/PhbC (PHA synthase), PhaG (Transacylase) and PhaJ (R-specific enoyl-CoA hydratase).

Figure 3. Models for PHA granule self-assembly. (a) In vitro assembly process, (b) in vivo assembly depicting two possible routes 1 and
2. CM, cytoplasmic membrane.

separates the polymers from the cell lumen and, conse-
quently, the osmotic pressure of the cell is not
changed extensively (Figure 4) (26). The size and
number per cell varies depending on the different
species (Figure 4). About 8–13 granules per cell having
a diameter range of 0.2–0.5 µm were observed in
A. eutrophus (16, 127). Whereas P. oleovorans is estimated
to have about one or two large granules (128). The PHA
granules are mostly spherical and surrounded by a phos-
pholipids membrane (129) separating two crystalline
protein layers (130, 131) composed of the PHA poly-
merases (132), the intracellular PHA depolymerase
 62 MUHAMMADI ET AL.
Figure 4. Transmission electron micrograph of bacterial cells with intracellular PHA granules (arrows). (a) and (b) Bacillus sp. strain CL1
and R. eutropha, respectively, contained PHA granules within cell and (c) Phasin mutant R. eutropha cell containing a single large PHA
Downloaded by [Central Michigan University] at 07:42 19 December 2015
granule.
(133), amphipathic phasing proteins (117, 134), PHA-
specific regulator proteins (135–137) and additional pro-
teins with unknown function (Figure 5) (128). Granules
membrane coat is about 2 nm thick, containing 0.5%
lipid and 2% protein, of the granule weight (138). PHA
polymerases are present in the cytosol; however, they
are only active when localized on the surface of granules
(103, 132, 134). The intracellular depolymerases are
required for mobilization of the reserve polyester and
are attached to the granule surface (139). Among the
proteins associated to PHA granules, phasins constitute
a very diverse group are the most dominant proteins
of relatively small molecular size (124, 140, 141). A few
that have been studied in detail revealed that they are
associated with a structural role of PHA granules and
may have several functions such as coating, stabilizing
granules as structural proteins by non-covalently
attached to the polyester core of granules (Figure 5)
(30, 135, 142, 143, 144) or activating genes and/or
enzymes involved in PHA synthesis (124). Phasins
promote PHA biosynthesis and their copy number has
impact on PHA granule size (143, 144). Phasins are inhi-
biting individual granules from coalescing and
Figure 5. The ideal PHA granule surrounded by phospholipids
membrane and crystalline protein.
agglutinating with other granules and acting as a
barrier between the polymer and other cellular com-
ponents (123, 124, 145). In phasins mutants of
R. eutropha, the cells accumulate less PHB, and they
possess only one single large granule because separate
granules coagule due to the “nacked” surface if the
hydrophobic PHA molecules get in contact (Figure 4)
(140, 142, 146). It was also speculated that phasins
might have a protective function to reduce the passive
attachment of cytosolic proteins. Simulation of the self-
assembly process showed that phasins might impact
on the kinetics of granule formation by reducing the
lag phase (147). Various PHA-specific regulators such as
PhbR from C. necator (137, 142). PhaF from Pseudomonads
(135, 148, 149) and PhaR from Paracoccus denitrificans
(136, 150) were found to bind non-covalently to the
PHA granules. The cytosolic levels of these repressor pro-
teins are supposed to be low when PHA granules are
formed. Additional proteins (PhaI, PhaD and PhaS) were
found to be granule-associated and co-regulated in Pseu-
domonads, whose function has not been clarified yet
(128, 135, 148, 149).
Methods for extraction
Efficient recovery and purification of PHA from cells is
required for its cost-efficient industrial production but
the extraction of PHA from cells poses yet another chal-
lenge. Several protocols have been described and
patented over the past 35 years. In all methods, it is
essential to concentrate cells by centrifugation, cross-
flow filtration and flocculation which are the methods
of choice. Subsequently, either water-based separation
or solvent-based extraction can be chosen as isolation
methods. For solvent-based extraction of PHA, the

concentrated biomass is dried either by freeze- or spray-
drying. PHA is extracted directly from ground biomass by
dissolving it in an organic solvent, usually chloroform,
chlorolimited methylene chloride, propylene carbonate
and dichloroethane (70, 151, 152, 153). After removing
cellular components (residuals) by filtration and centrifu-
gation, the PHA is precipitated in a non-PHA-dissolving
solvent such as cold ethanol or methanol (154–158). In
contrast to mcl-PHAs, scl-PHAs are not soluble in
acetone, thus, enabling a separation of both PHA
classes (159). Recovery of PHA from bacterial cells using
organic solvents is often applied in industrial processes
and in laboratory scale PHA extractions due to the recov-
ery efficiency of the process, simplicity, rapidity, polymer
purity obtained and the possible removal of endotoxins
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from the recovered polymer, which is important for
medical applications (160–162). Solvents extract the
polymer without degrading it by improving the cellular
membrane permeability and subsequent solubilization
of the PHA (162). But a large amount of hazardous
solvent is needed to repeat the same process. Thus,
this method is not environmentally friendly and unsuita-
ble for mass production of bioplastic (22). Excellent
recovery was reported for the extraction in supercritical
CO2, which may be especially applicable to reduce the
endotoxins contamination from genetically engineered
E. coli strains (29). The second protocol is digestion-
based extraction strategies that utilize enzymatic treat-
ment of cellular components to release PHA and
design to avoid the use of organic solvents. In this
method, the cells need to be broken up and various
chemical additives added to digest non-PHA material.
Bacterial cells debris is treated with a cocktail of
enzymes (including proteases, nucleases and lysozymes)
and detergents (5% sodium dodecylsulfate) to remove
proteins, nucleic acids and cell walls, leaving the PHA
intact. Finally, the PHA is concentrated through cross-
flow filtration and obtained as white latex of 95%
purity, ready to be used as coating material (22, 25,
158, 163). Compared to solvent-extracted PHA, the mol-
ecular weight may be lower following enzymatic recov-
ery methods despite the mild reaction conditions (164).
One critical consideration of these chemical-based
methods is that harsh chemical treatment to achieve
high purities may lead to a reduction of the molecular
weight of the polymer (165). Yang et al. developed an
strategy for poly(hydroxybutyrate-co-hydroxyvalerate)
(P(HB-co-HV)) recovery using linear alkylbenzene sulfonic
acid LAS-99 as an alternative to the commonly used
sodium dodecyl sulfate. In this method, only 21% of
the surfactant is required (166), compared to previous
SDS-based methods. The main disadvantages of
chemical-based strategies are the large amount of
GREEN CHEMISTRY LETTERS AND REVIEWS 63
salt produced as a by-product and the amount of
surfactant-containing wastewater generated from the
process, potentially resulting in high costs for wastewater
treatment. Singh et al. reviewed a novel self-disruption cell
system for recovery PHA developed in B. megaterium
(167). In this system, a gene cassette carrying the cell
lysis system (holin and endolysin of B. amyloliquefaciens
phage) (168) was inserted into the E. coli–B. subtilis
shuttle vector pX. In this expression system, xylR-xylA’
target genes are induced by xylose but inhibited by
glucose, which acts as an anti-inducer (169). It synchro-
nizes the processes of spontaneous cell lysis and substrate
exhaustion, which results in the release of accumulated
PHAs. The efficiency of this regulatory process can be
enhanced by manipulating the YoeB, a cell wall-associated
protein, which gets induced in response to antibiotics
stress. The expression of yoeB in B. subtilis is under a
xylose-inducible promoter. yoeB mutants display an
increased rate of autolysis in response to nutrient
depletion and various cell envelope stress conditions.
The process of autolysis in B. subtilis 168 can be aided
by mutating yoeB gene and in the process making it
independent of xylose regulation (168).
Biodegradability of PHA
The biodegradation of PHAs in natural environments
(river and sea waters, soil, sludge and compost) is one
of the commercially attractive features which distin-
guishe the PHAs from petroleum-based plastics (170–
175). The main advantage of PHAs over other types of
biodegradable plastic is that they do not require
special environmental conditions and can degrade in
either aerobic or anaerobic environments through
thermal degradation or enzymatic hydrolysis (176, 177).
Biodegradation of PHAs is performed by microorgan-
isms, which inhabit a specific natural environment
(176). But in the absence of biological agents (bacteria,
algae and fungi) PHAs are practically not subject to
mass lost under normal conditions (178) and they are
degraded in biological media to form products innocu-
ous to the environment. PHA biodegradation is per-
formed by microorganisms that secrete intra- or
extracellular PHA depolymerases, which differ in their
molecular organization and substrate specificity (179).
While intracellular PHA depolymerases are synthesized
by PHA-producing bacteria and are used by them to
hydrolyze their own PHA storages, extracellular
enzymes are produced by other microorganisms to
utilize PHAs usually released into environment after
death and cell lysis of PHA-accumulating cells (180).
The first microorganisms degrading poly-3-HB were iso-
lated over 50 years ago (181). Six hundred PHA
 64 MUHAMMADI ET AL.
depolymerases from various microorganisms have been
identified by now; comparison of their amino acid
sequences provided a basis for uniting them in 8 super-
families including 38 families (179). The same strain can
contain several genes encoding PHA depolymerases
with different specificities. The ability to degrade extra-
cellular PHAs is determined by the activity and type of
PHA depolymerases, which hydrolyze the polymer by
surface erosion to water-soluble monomers and/or oligo-
mers a substrate for microorganisms. All PHA-degrading
bacteria, algae and fungi use extracellular enzymes to
depolymerize, or break down, PHAs and, through a
process known as mineralization, absorb the remaining
fragments for use as minerals (182). The end products
of PHA degradation in both aerobic and anaerobic
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environments are carbon dioxide and water, while in
anaerobic conditions methane is also produced (28,
175). If the limiting growth factor is supplied again, the
accumulated PHAs can be also intracellularly depolymer-
ized by degradation enzymes via hydroxycarboxylic
acids to acetyl-CoA and reused in metabolism (16, 28).
All microorganisms that synthesize PHAs contain intra-
cellular depolymerase system for this purpose. Studies
using R. eutropha showed that the intracellular degra-
dation of PHB inclusions is a very slow process (183).
The rate of PHB degradation was calculated to be
about 10 times slower than the rate of its synthesis
(184, 185). It may be because that PHA inclusions are pro-
tected by granule-associated proteins on the surface
from attack by the depolymerase (186). But, Merrick
et al. (187) proposed that an activator acts to modify an
inhibitor present on native PHB inclusions, thereby allow-
ing the intracellular depolymerase to access its substrate
(188). Based on the finding that the number of polymer
chains was almost constant during the PHB degradation
process, it was suggested that the intracellular depoly-
merase is an exotype hydrolase acting at the carbonyl
terminus of the polymer chain (184). Low concentrations
of diisopropyl fluorophosphate inhibit the depolymerase
activity leading to the suggestion that this enzyme is a
serine esterase (188). In comparison to the extracellular
depolymerase which attacks crystalline PHA, the mech-
anism for intracellular depolymerase is presumed to be
different because of the amorphous nature of the intra-
cellular PHA inclusions. The end products of PHA degra-
dation in aerobic environments are carbon dioxide and
water, while methane is also produced in anaerobic con-
ditions (28, 175). In vivo and in vitro PHA degradation is
estimated in terms of weight loss (surface erosion), mol-
ecular weight decrease, increase in the degree of crystal-
linity and loss of mechanical properties (171). PHA
degradability is influenced by many factors, including
the specificity and activity of extracellular
depolymerases, physical and chemical properties of the
polymer (stereo-configuration of its molecules, compo-
sition, shape of specimen, crystallinity and molecular
weight, polydispersity), surface area, microbial activity
of the disposal environment, the pressure of other nutri-
ent materials, physicochemical conditions (pH, tempera-
ture, oxygen availability, moisture, salinity, acidity of the
environment, etc.), the shape and size of PHA-based
devices, and weather and climate in different regions
(28, 176, 180, 189). It has been reported that poly(3-
hydroxybutyrate-co-4-hydroxybutyrate) had a much
faster degradation rate than homopolymer PHB (178).
While according to the current investigation of Boyandin
et al., the homopolymer of 3-hydroxybutyric acid is
degraded at higher rates than the P(HB-HV) (176). Simi-
larly, the average rates of mass loss were 0.04–0.33%
per day for films and 0.02–0.18% for compact pellets,
suggesting preferential degradation of the amorphous
phase over pellet. The differences in degree of degra-
dation are due to differences in the kinetics and mechan-
ism of PHA biodegradation is caused by qualitative and
quantitative dissimilarities between microbial commu-
nities of different habitats and other environmental
and specimen-related factors (176).
In microbially active environments, PHAs are initially
viewed by microorganisms as energy sources and then
start to degrade (107, 190). Microorganisms colonize on
the surface of the polymer and secrete exodepoly-
merases which degrade P(HB–HV) into HB and HV
units. These units are then used up by the cell as a
carbon source for biomass growth (15). P(HB–HV) is
water insoluble and is not affected by moisture, does
not degrade under normal conditions of storage and is
stable indefinitely in air (107, 171). The effect of different
environments on the degradation rate of PHB and P(HB–
HV) has been studied by several workers (171, 184).
Degradation occurs most rapidly in anaerobic sewage
and slowest in seawater. Lee showed that P(HB–HV) com-
pletely degraded after 6, 75 and 350 weeks in anaerobic
sewage, soil and sea water, respectively (107, 191). Effec-
tive PHA destructors include various bacteria from wide-
spread soil and water genera (Pseudomonas, Alcaligenes,
Comamonas, Streptomyces, Ilyobacter) (192), as well as
fungi (Ascomycetes, Basidiomycetes, Deuteromycetes, Mas-
tigiomycetes and Myxomycetes) (180). Potential microbial
PHA destructors are generally isolated by inoculation of
microbiological samples on agar plates or latex
medium based on PHA particles or granules as the only
carbon and energy source. Microbial exodepolymerases
hydrolyze PHA to soluble products forming lysis zones
on surface of the plate (175). The ability to degrade extra-
cellular PHA depends on the secretion of specific PHA
depolymerases which hydrolyze the polymer to water-

soluble products (181, 192) and is widely distributed
among bacteria (171, 193). Aerobic and anaerobic PHA-
degrading bacteria of many taxa were isolated from
various ecosystems, and several PHA depolymerases
were isolated and characterized (185, 194, 195). All puri-
fied depolymerases were specific for PHB and/or other
scl-PHAs, such as the PHB depolymerases of A. faecalis,
Comamonas sp., or P. lemoignei (196–198) or for mcl-
PHAs, such as the poly(3-hydroxyoctanoate) (P(HO))
depolymerase of P. fluorescens GK13 (194). Whereas
most PHA-degrading bacteria analyzed so far apparently
produced only one PHA depolymerase,
Pseudomonas lemoignei was found to have at least five
PHA depolymerases (199). The structural genes of
several PHA depolymerases have been cloned and
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sequenced (197, 200–202). Biochemical analysis of the
purified depolymerase proteins and analysis of the
DNA-deduced amino acid sequences revealed that PHA
depolymerases apparently possess a catalytic triad con-
sisting of serine, histidine and aspartate residues which
have been demonstrated to form the active site in bac-
terial lipases (203). In addition, the hydrolysis of several
scl-PHAs by lipases has also been reported (204). There-
fore, the commercial production of PHA as a biological
source will bring down the use of expensive chemicals
and also reduce the load on pollution.
In vivo biocompatibility
PHAs such as PHB and their breakdown products 3-
hydroxyacids have been found in a wide range of organ-
isms, from bacteria to higher mammals. A number of
studies have shown that PHB take part in formation of
transmembrane ion channel complexes in eukaryotic
and prokaryotic cell membranes (31, 205–207). Interest-
ingly, degradation of PHB polymer results ultimately in
the production of, R-3-hydroxybutyric acid which also is
a normal constituent of blood at concentrations
between 0.3 and 1.3 mM and known to be associated
with ketone body formation. There is a strong likelihood
that surgical implants, sutures, etc. produced from PHA
will not result in an immune response in the host organ-
ism. Furthermore, sterilization of PHA-based materials
does not appear to affect the average molecular
weight (Mw), tensile strength or other properties (208,
209). Surface properties of PHA films have been shown
to be favorable for proliferation and attachment of
tissue culture cells (210, 211), suggesting that PHA is suit-
able for scaffolding material in tissue engineering. The P
(HBco-HV-co-HHx) polymer exhibits the greatest surface
roughness, as well as the highest water contact angle
characteristics which are important for adherence and
proliferation of cells on PHA surfaces. For example,
GREEN CHEMISTRY LETTERS AND REVIEWS 65
mesenchymal stem cells were shown to adhere and pro-
liferate on several PHA substrates, with a terpolymer poly
(hydroxybutyrate-cohydroxyvalerate-co-hydroxyhexano-
ate) (P(HB-co-HVco-HHx)) yielding the optimum results
(212, 213). Further, Sevastianov et al. have also tested
the PHA matrices for hemocompatibility by inspecting
the response of mammalian blood when incubated
with polymer films (161). It was shown that PHB or P
(HB-co-HV), when in contact with blood, did not affect
platelet responses, nor did the polymer activate comp-
lement system (214). However, more involved polymer
purification procedures had to be followed to signifi-
cantly reduce the amount of bacterial cell wall material
associated with the purified PHA (161).
Biocompatibility of PHB has been demonstrated in
vivo under subcutaneous implantation of PHB films
(161). Tissue reaction to films from PHB of different Mw
(300, 450, 1000 and 1500 kDa) implanted subcutaneously
was relatively low and did not change from tissue reac-
tion to control glass plate. The low tissue reaction to
implanted PHB films indicates the high biocompatibility
of PHB in vivo (161, 214). This high biocompatibility of
PHB is due to the presence of natural PHB oligomers
and HB, the intermediate product of PHB degradation,
in animal tissues at normal conditions in comparison
with chemically synthesized biodegradable polymers,
for example, polylactides and polyglycolides. Therefore,
Toxicological certificate of Institute of Medical Technique
(Ministry of Health, Russia) approves of PHB for appli-
cation in medicine as non-toxic and biocompatible
mate (214). Given these findings, it is highly understand-
able that this family of polymers is biocompatible and
ideal material for biomedical applications.
Physical properties
Within the cell, PHAs exist as mobile liquid, amorphous
(26, 83, 87, 89, 91) and water-insoluble inclusions (43,
22). However, after extraction from the cell with
organic solvents, PHAs become highly crystalline and
show material properties that are similar to conventional
commodity plastics such as polypropylene, polyethylene
and polystyrene (Table 1) (22, 215–217). Because, in vivo,
non-covalently attached phasins as structural proteins
stabilizing the PHA granules (30, 141) and inhibiting indi-
vidual granules from coalescing and agglutinating with
other granules (123, 145). In addition, water is a minor
component of PHA inclusions and therefore it was
suggested that water could act as a plasticizer (218,
219). About 5–10% 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 (220). Based on this finding, it was
 66 MUHAMMADI ET AL.

Table 1. Comparison of physical properties of various (around 170°C) is close to the temperature where this
polyhydroxyalkanoates with conventional plastics. polymer decomposes thermally and thus limits the
Tensile Elongation ability to process the homopolymer. However, copoly-
Tm Tg strength at break
Samples (°C) (°C) (Mpa) (%) Reference mers consisting of HB and HV P(HB–HV) have better
PHB 177 4 43 5 (44, 220, mechanical properties than homopolymer PHB (Table
233) 1) (10, 11, 216). Copolymers consisting of HB and HH
P(3HB-co-20% 145 –1 20 50 (44, 221)
3HV) are less stiff and brittle than PHB, thus improve the
P(3HB-co-16% 150 –7 26 444 (233) mechanical properties of P(HB–HV) even further (225),
4HB)
P(3HB-co-15% 115 23 760 (44, 221) making them comparable with conventional plastics
3HHx) such as polypropylene, polystyrene, polyethylene ter-
P(HB-co-10% 150 – 25 20 (221) ephthalate and high-density polyethylene (Table 1).
HV)
P(HB-co-20% 135 – 20 100 (44, 233) Actually, when copolymer formation occurs with HB
HV) and HV monomer units, the properties of the material
P(HB-co-10% 127 –1 21 400 (220, 233)
HHx) (Biopol; ICI) alter as a consequence of decreased crystal-
P(HB-co-17% 120 –2 20 850 (221) linity and Tm. These result, in mechanical terms, in a
HHx)
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Polypropylene 170 – 34 400 (44, 233)
Polystrene 110 – 50 – (44, 221)
Polyethylene 130 −10 500 (221)
HDPE 135 29 (220, 221)
LDPE 130 –30 10 620 (44)
PET 262 56 7300 (44, 233)
Note: Tm: melting temperature; Tg: glass-transition temperature; HB: 3-hydro-
xybutyrate; HV: 3-hydroxyvalerate; HHx: 3-hydroxyhexanoate; LDPE: low-
density polyethylene; HDPE: high-density polyethylene; PET: poly(ethylene
terephthalate)
suggested that water molecules could form hydrogen
bonds with the carbonyl groups of the polyester back-
bone to form “pseudo cross-links” between adjacent
polymer chains. This type of molecular arrangement
may also explain the mobile amorphous nature of PHA
inclusions as well as the plastic deformation phenom-
enon (94, 118, 220) observed in freeze-fracture exper-
iments (221). But for extraction, when PHA inclusions
are subjected to physical treatments such as centrifu-
gation, they readily coalesce into larger masses and
this can lead to the apparent acceleration of crystalliza-
tion (222). Furthermore, any damage to the surface
coating of the inclusion will allow heterogeneous nuclea-
tion, that is, crystallization induced by external molecules
other than PHA, further accelerating crystallization (223).
The definition of their thermal and mechanical proper-
ties is normally expressed in terms of the glass-to-
rubber transition temperature (Tg) of the amorphous
phase and the melting temperature (Tm) of the crystalline
phase (Table 1) (43, 217). The mechanical properties of
PHA range from brittle to flexible and elastic, depending
on the branched length of the HAs or from the distance
between the ester linkages in the polymer backbones.
Typically, PHAs with short pendant groups are hard crys-
talline materials, whereas PHAs with longer pendant
groups are elastomeric (Table 1) (29). The scl-PHA such
as PHB is stiffer and more brittle than polypropylene
phase (217, 224). Because of its brittleness, PHB is not
very stress-resistant. Also, the relatively high Tm of PHB
decrease in stiffness (Young’s modulus) and an increase
in toughness, producing more desirable properties for
commercial application. Consequently, a range of prop-
erties is feasible from the hard and brittle homopolymer
via a balance of stiffness and toughness to soft and
tough for copolymers with a high incorporation of HV
(26). Besides the mechanical properties, various HA
monomer structures affect the PHA degradation rates.
It has been reported that poly(3-hydroxybutyrate-co-4-
hydroxybutyrate) had a much faster degradation rate
than homopolymer PHB (178). mcl-PHAs are rubbery
and flexible materials with low crystallinity and can be
used in a wide range of applications which cannot be ful-
filled by PHB and other scl-PHAs (223) but these physical
properties of mcl-PHAs are considered unsuitable for
applications due to their low Tm and Tg (226, 227). In con-
trast to PHB and P(HB–HV), mcl-PHAs have a much lower
level of crystallinity and are more elastic (226, 228) but
not strong enough for applications. These mcl-PHAs
potentially have a different range of applications than
the scl-PHAs. PHA with monomer chain lengths to 12
carbon atoms or more occur as sticky, more elastic and
liquid (178, 229). However, in general, all the bacterial
PHAs are (i) biodegradable thermoplastic and/or elasto-
meric compounds which can be processed with appar-
atus used by the plastic manufacturing industry
without losing biodegradability. In addition, they are (ii)
insoluble in water and resistance to hydrolytic degra-
dation, (iii) highly crystalline, (iv) exhibit a rather high
degree of polymerization ranging from 105 to almost
107 Da, (v) they are optically active and (vi) isotactic
(enantiomerically pure chemicals consisting, in general,
only of the R-stereoisomers). They are (vii) non-toxic,
(viii) biocompatible and (ix) exhibit piezoelectric proper-
ties as revealed (at least) for poly(3HB) and poly(3HB-co-
3HV) (7, 30, 50). These features make them highly com-
petitive with polypropylene, the petrochemical-derived
plastic (31, 216, 217, 230).
 GREEN CHEMISTRY LETTERS AND REVIEWS 67

Tunable hydrophilicity, hydrolytic stability internalization inside the core of the particles. Third
and controlled degradation method has been investigated to increase degradation
by blending with other polymers or plasticizers which
All PHAs are susceptible to degrade by hydrolysis to
disrupt the polymer crystallinity, improve processability
some extent and under normal conditions they are
by lowering the processing temperature and conse-
water stable (231). But the high degree of polymerization,
quently accelerated hydrolysis (231, 233). According to
high crystallinity, isotacticity (only the enantiomer of
the presence of PEG or PLA in blend of PHB/HV with a
absolute configuration R is present in these polymers)
hydrophilic polymer, PEG or a hydrolysable polyester,
and hydrophobicity are among the factors limiting the
racemic poly(-lactic acid) PLA50 could increase the
bioavailability of PHAs which can affect the rate of biode-
hydrolytic rate (233). From these reports, it is concluded
gradation in natural environment (231, 232). PHA biode-
that the combination of bioconversion and organic
gradation corresponds to hydrolysis involving endo- or
chemistry allows modulating more precisely the physical
exo-enzymatic systems in the breaking cleavage of
properties of these bacterial polymers such as solubility,
esters bonds. This type of degradation is needed for
hydrophilic/hydrophobic balance and bioavailability.
environmental applications. In the case of therapeutic
and biomedical uses, a simple hydrolysis is required.
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Hydrolytic degradation of PHAs is not evident and is

depending on the structure particularly on the nature Lifespan of PHA compared to conventional
of the side chains and materials crystallinity (231). There- plastics
fore, PHAs need to have tunable hydrophilicity, chemical
It is widely accepted that the use of long-lasting poly-
functionalities and appropriate hydrolytic stability to
mers for short-lived applications (engineering, packa-
expand their therapeutic applications towards more
ging, catering, surgery, hygiene) is not entirely
advanced areas. To overcome this issue, three methods
adequate. Therefore, selection of type of plastic for
have been reported so far. The first way to obtain more
certain manufacturing should be made considering the
hydrophilic PHAs is synthetic strategies which consist
lifespan of plastic commodity on the environmental con-
in the preparation of unsaturated PHAs with polar func-
ditions. So that increasing environmental problems
tional groups such as carboxylic, hydroxyl or epoxy
associated with discarded plastics can be controlled.
groups. The functionalization turns the reactive
These petroleum-based conventional plastics are very
pendant double bonds into hydrophilic functions (231,
stable in harsh conditions especially against the attack
232). Functional PHAs with hydrophilic groups such as
of chemical degradation and microbial decomposition,
carboxylic, amine or hydroxyl on the side chains were
thus rendering them durable, highly resistant and
prepared by biotechnological syntheses but the fermen-
blessed with a very long lifespan in the environment
tation conditions are difficult and generally polymers are
(234), since the synthetic plastics are designed in a way
obtained with very low yields (231, 87). The most suitable
to suit the constant performance and trustable qualities
products in regard to hydrolysis concern are PHAs con-
that are used for long life span, therefore causing them
taining carboxylic groups in side chains. Because, car-
to be inert to natural and chemical breakdown (235).
boxylic groups promote water penetration into the
Further, the first development of plastics at the begin-
polymer and participate in hydrolysis ester groups
ning of this century has been the goal of industry and
through better water penetration and catalysis (231).
science to improve the resistance of these materials to
The second method is preparation of PHA-based water-
microbial attack. Therefore, conventional plastic
soluble polymers by the block/graft copolymerization
materials have, in contrast to microbial PHAs, almost
of PHAs with hydrophilic components in various poly-
unlimited lifespan (236). Generally, natural degradation
meric architectures (232). In this case, these hydrophilic
of conventional plastics begins with photodegradation,
moieties/reactive functions are used for grafting oligo-
which leads to thermooxidative degradation (237). Ultra-
mers of hydrolysable polylactic acid (PLA) or hydrophilic
violet light from the sun provides activation energy
polyethylene glycol (PEG). Otherwise block copolymers
required to initiate incorporation of oxygen atoms into
with more crystalline polycaprolactone (PCL) are pre-
the polymer. This causes the plastic to become brittle
pared, aiming at nanoparticles formation in the view of
and to break into smaller and smaller pieces, until the
drug release. Therefore, the hydrophilic/hydrophobic
polymer chains reach sufficiently low molecular weight
balance of these materials is controlled by chemical
to be metabolized by microorganisms (237). The
modification and their stability/hydrolysis. The hydrophi-
microbes either convert the carbon in the polymer
lic PEG could increase the particles hydrolysis whereas
chains to CO2 or incorporate it into biomolecules.
the hydrophobic PLA could increase their stability by
However, this entire process is very slow, and it can
 68 MUHAMMADI ET AL.
take 50 or more years for plastic to fully degrade (238).
For these different reasons, reaching the conditions of
conventional plastic replacements by degradable poly-
mers, particularly for short-term applications (packaging,
agriculture … ), is of major interest to the society as a
whole, from the plastic industries to the citizens.
Although, microbial PHAs like conventional plastics
are also thermoplastics, moldable, naturally UV-resistant
and could be tailor-made for numerous applications
ranging from stiff packaging goods to highly elastic
materials for coatings. But their complete biodegradabil-
ity in the environment and naturalness make them quite
beneficial in certain short-lifespan applications for single-
use packaging, catering, surgery, hygiene, drug delivery
and agriculture (239). But according to Beucker and
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Marscheider-Weidemann bioplastic can have a short or
a long lifespan depending on the material or the com-
pound used to produce them (240). Degradation of com-
mercially available shampoo bottles and film made from
PHA (Biopol@ bottles; molecular composition: 92% 3-HB,
8% 3-HV, the inner and outer surface areas of the bottles
= 336 and 356 cm2, respectively, the average mass =
31.90 g (n = 13) in depth of 85 m of an aquatic ecosystem
(Lake Lugano, Switzerland) under natural conditions for
250 days has measured the degradation rates of 10–20
mg/d giving an average lifespan of 5–10 years for the
specific bottle type (241). The degradation rates vary
with the water depth, that is, with the spatial heterogen-
eity of the ecosystem. Bottles made from polyethylene–
starch (PE + S; 9% and 13% starch, respectively) blends
showed no degradation at all. In order to reduce the
challenging burdens of currently being used plastic on
health and environment, it is needed to determine the
lifespan of plastic products in the real usage conditions.
Applications of PHA
Bacterial PHA first received commercial attention way
back in the early 1960s from an American company, W.
R. Grace, but apparently was discontinued due to some
technical problems (242). Although patents were orig-
inally filed in the USA by Baptist J.N. in 1962, but first
industrial production of PHB and PHA did not occur
until 1982. The major petroleum crisis of the 1970s
then motivated a British company, ICI Bioproducts
(now known as Zeneca Bio Products) to get involved
with this bacterial polymer and marketed them under
the trade name Biopol (26). Besides, currently a number
of companies are developing PHAs for use in plastics
worldwide including Kaneka in Japan, and P&G Chemi-
cal, and BP and Metabolix in the USA. Kaneka and P&G
Chemical have teamed up to commercialize a product
“Nodax” (also known as Nodak™, which is a specialized
PHA). Nodak™ has already been made into a variety of
different prototype objects such as plastic fiber or
twine and molded plasticware such as plates and cups.
Metabolix is already producing preliminary PHA
materials, but is teaming up with BP for two years to
produce bioplastics (243). In response to an increased
awareness of global environmental problems, PHA is
gaining serious attention as a potential substitute for
non-biodegradable polymers. Now, the advances in
PHA production have opened up the potential opportu-
nities to a number of applications (29). The production of
PHA is intended to replace synthetic non-degradable
polymers for applications especially in packaging, agri-
culture, leisure, fast-food, hygiene as well as medicine
and biomedical since PHA is biocompatible and
degradable.
(1) Packaging, molding and coating
PHAs are natural and biodegradable thermoplastic
polyesters, and hence the majority of their applications
are as replacements for petrochemical polymers cur-
rently in use for packaging and coating applications
(26, 31, 230, 244). They can be used in a wide variety of
products including bags, containers, paper coatings,
pens, golf tees, razors, feminine hygiene products,
diaper backsheets, utensils, cosmetic containers,
bottles, cups and materials for food packaging (26, 28,
31, 38, 190). The latex of PHAs can be used to produce
a water-resistant layer for paper, film or cardboard (190,
245). Manufacturers in the USA used PHB and copolymer
P(HB–HV) as water-proof films on the back of diaper
sheets (31, 246). This copolymer P(HB–HV), with flexibility
and impact resistance, was marketed under the trade
name Biopol™ by ICI/Zeneca and later by Monsanto till
1995. PHAs can also be used to replace petrochemical
polymers in toner and developer compositions or as
ion-conducting polymers. PHAs can be used as a latex,
for instance for paper-coating applications (26). New
plastic properties can be achieved by blending PHA
with other polymers and there is much activity in this
field (28, 30, 247, 248). Often the mixture changes the
crystallinity of the plastic, and crystallization rate and
finally also the mechanical properties of the material.
Thus, a mixture of 40–60% of PCL in PHB improved the
mechanical properties over P(HB–HV). A 40% PCL and
60% PHB mixture had a decreased oxygen permeability
that was only 5% of that of polyethylene (249).
Maekawa and coworkers reported that blends of PHB
and cellulose propionate were completely miscible
since they had a single glass transition, a depression in
the equilibrium melting temperature of PHB and a
decrease in spherulitic growth rate of the PHB

component. Also tensile strength was better for the
blend of PHB/cellulose propionate than for PHB only
(250). However, not all blends are compatible.
(2) Medical and pharmaceutical applications
In addition to their biodegradability, many PHAs are
also biocompatible. Their breakdown products are 3-
hydroxyacids, which are naturally found in animals.
Such as PHB is biocompatible, which is not surprising
when considering the fact that R-3-hydroxybutyric acid
is a normal constituent of blood at concentrations
between 0.3 and 1.3 mM (31, 205, 206) and is also
found in the cell membrane of eukaryotes (207). These
PHAs have the potential to become important and very
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useful compounds for medical applications such in
wound management used as surgical suture, skin substi-
tutes, nerve cuffs, surgical meshes, implants, gauzes,
staples, gauzes, swab, lubricating powders (29, 30, 38),
blood vessels, tissue scaffolds and bone fracture fixation
plates (3, 251, 252). On account of their varied and
diverse properties, the PHA biopolymers provide a
three-dimensional scaffold means to support the cell
growth and then degrade away leaving viable tissue,
with potential product applications for the cardiovascu-
lar system, cornea, pancreas, gastrointestinal system,
kidney and genitourinary system, musculoskeletal
system, nervous system, teeth and oral cavity, skin and
so forth (29, 32). In orthopaedy, PHAs can also be used
as scaffolds for cartilage engineering, bone graft substi-
tutes and spinal cages. PHAs can be used to engineer
the heart valves, cardiovascular fabrics, pericardial
patches and vascular grafts (3, 30, 32, 253). PHAs are also
useful as stereoregular compounds which can serve as
chiral precursors for the chemical synthesis of optically
active compounds (32, 254). Such compounds are particu-
larly used to synthesize the micro- and nanosphere of
PHAs as biodegradable carriers or drug delivery systems
for controlled release of therapeutics such as medicines,
hormones, insecticides and herbicides into systemic circu-
lation are receiving attention (30, 31, 253, 255, 256).
According to Sudesh et al., PHAs have the oil absorption
and retention capacities which offer commercial appli-
cation value in the cosmetics and skin care industries
(257). Voinova et al. have investigated the possibility of
use of PHAs as a biodegradable carrier for pesticides (α-
hexachlorocyclohexane and lindane) for targeted and
controlled delivery of these compounds to soil. According
to their investigation, pesticides embedded in a PHA
carrier are released gradually and slowly, without surges,
as the polymer is degraded by the soil microflora (258).
They are also used as osteosynthetic materials in the
stimulation of bone growth owing to their piezoelectric
GREEN CHEMISTRY LETTERS AND REVIEWS 69
properties, in bone plates and blood vessel replacements
(31). PHA can be easily depolymerized 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’s anti-glaucoma drug
“Truspot”. In tandem with R-1, 3-butanediol, it is also
used in the synthesis of β-lactams (31). However, cur-
rently, the medical and pharmaceutical applications of
PHAs are limited due to the slow biodegradation and
high hydraulic stability in sterile tissues (259). The pres-
ence of 150 different monomers identified yet and new
types of PHA through the chemical or physical modifi-
cation of naturally occurring PHA gave rise to diverse
properties. With these features PHA is also considered
as pharmaceutically active compound and currently
investigated as potential anti-HIV drugs, anti-cancer
drugs, antibiotics, etc. (58).
PHAs appear to be potentially useful in controlling
bacterial pathogens in certain aquaculture applications
(260). For example, administration in the feed of 1000
mg/l of PHB particles of an average diameter of 30 μm,
or addition of inactivated cells (107 cells ml−1) of PHB-
containing Brachymonas bacteria (equivalent to 10 mg
L−1 PHB) to the culture water of brine shrimp (Artemia
nauplii) larvae, conferred a complete protection from a
virulent strain of the intestinal pathogen Vibrio campbellii
(261). Other similar reports have claimed an inhibitory
effect of PHB on certain gut microflora of the giant fresh-
water prawn (Macrobrachium rosenbergii) larvae (260).
Administration of PHB in the feed significantly increased
the survival of the prawn larvae and improved their
development. The total bacterial counts and Vibrio spp.
counts were significantly reduced in PHB-fed larvae com-
pared to the control larvae (3).
(3) Denitrification in water and wastewater treatment
A promising application of PHAs is as the solid sub-
strate for denitrification of water and wastewater. This
type of denitrification, termed here “solid-phase denitri-
fication”, has several advantages over the conventional
system supplemented with liquid organic substrate.
PHAs serve not only as constant sources of reducing
power for denitrification but also as solid matrices favor-
able for development of microbial films (189). In addition,
in contrast to conventional processes, the use of PHAs
has no potential risk of release of dissolved organic
carbon with the resultant deterioration of effluent
water quality. Denitrification processes using PHAs actu-
ally give high rates of nitrogen removal (262). Due to its
oil absorption capacities, PHB has been successfully
tested for removing lipid-soluble organic pollutants
from water (257, 263).
 70 MUHAMMADI ET AL.
(4) Miscellaneous applications
PHAs with long side chain hydroxyacids have been
used in pressure-sensitive adhesive formulations. PHAs
can be used to produce dairy cream substitutes or
flavor delivery agents in foods. Moreover, PHAs are also
used to produce fiber materials, such as non-woven
fabrics (7). The PHAs are considered as a source for the
synthesis of chiral compounds (enantiometrically pure
chemicals) and are raw materials for the production of
paints (31, 32, 264). In addition to its range of material
properties and resulting applications, PHAs promise to
be a new source of small molecules. PHA can be hydro-
lyzed chemically, and the monomers can be converted to
commercially attractive molecules such as β-hydroxy
acids, 2-alkenoic acids, β-hydroxyalkanols, β-acyllac-
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tones, β-amino acids and β-hydroxyacid esters (265).
The last class of chemicals is currently receiving attention
because of potential applications as biodegradable sol-
vents (7). Besides helping to replace the existing solvents,
β-hydroxy acid esters and derivatives are likely to find
growing use 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 tones (31).
Economical aspects
One of the problems preventing the pilot-scale pro-
duction, commercial application and wide use of PHA
as commodity plastics is its high production cost. High
production cost has hindered the use of PHAs, since
their final price is considerably much higher than that
of petrochemical-based synthetic plastic materials (30,
40, 41, 158, 266, 267). Factors involved include the
product yield, complexity of the technology and hence
the capital cost of the plant, and the ease or difficulty
of product separation. Consequently, selection of the
organism and substrate can critically influence costs
(26, 267). For example, PHB from Gram-negative organ-
isms are widely used in a variety of products but due
to endotoxin in the outer membrane lipopolysaccharides
(LPS), Bacillus species which lack LPS are extensively used
in biopharmaceutical applications (70) but compared to
Gram-negative bacteria, Gram-positive bacteria were
mostly found to produce scl-PHA and at lower PHA con-
tents between about 2% and 50% CDM, that is why
Gram-positive bacteria have yet to be adopted for com-
mercial PHA production (58). Further, Koller et al. have
suggested to link ecology with economy, because bac-
teria residing at ecological niches like estuarine sedi-
ments, marine microbial mats, rhizosphere,
groundwater sediments and engineered ecosystems
with fluctuating nutrient contents support the popu-
lation actively involved in PHA accumulation to meet
the metabolic energy requirements during starvation
period and this concept can be implemented industrially
to reduce the cost of biopolymers commercially with sus-
tainable production processes (268). Therefore, it is econ-
omical to use naturally producing microorganism due to
safety and constant productivity rate, and there is great
potential for producing PHAs in low-cost substrates,
which can reduce PHAs production costs, because the
cost of substrate is the most important factor for PHAs
production (269–271). Moreover, novel-constituent poly-
mers will reduce the unfavorable characteristics of PHA.
The cost of raw material itself accounts for 40–50% of
the total production cost. Recovery of polymer from
the biomass is a vital stage of the process. Large-scale
solvent extraction, although giving high recovery, is an
expensive technique and involves large volumes of
solvent and high capital investment in the solvent recov-
ery plant (26). From an economical point of view, the
price of the product ultimately depends on the substrate
(mainly carbon source) cost, PHA yield on the substrate
and the efficiency of product formulation in the down-
stream processing (31, 107). It is a prerequisite to standar-
dize all the fermentation conditions, and physical and
chemical parameters for the successful implementation
of commercial PHA production systems (269–273).
PHAs production costs can be reduced by several
means, including the use of cheap substrates, such as
whey, favored in countries with important dairy indus-
tries, or the enhancement of product yield, for example
by using recombinant E. coli (7, 35, 267, 274). Molasses,
from either sugarcane or beet, is one potential cheap
carbon source for PHA production. B. cereus grown in
1% (w/v) beet molasses produced P(3HB) concentration
of 0.1 g/l and a polymer content of 73.8%, whereas in
3% molasses P(3HB) was increased by 70% but the
polymer content was halved (275). Another sucrose-rich
inexpensive substrate is sugarcane liquor, which was
used by Jiang et al. to produce P(3HB) using
P. fluorescens (276). In a 5-l batch bioreactor, Castilho
et al. obtained 22 g/l of P(3HB), at a 70% polymer
content (277). Santimano et al. reported a comparative
use of low-cost agro-industrial wastes (rice chaff, citrus
pulp, coconut oil cake, cotton seed cake, cane molasses,
bakery waste) for the production of PHA by Bacillus sp.
strain COL1/A6. The highest PHA content was obtained
from hydrolyzed wafer residues (62.41 ± 1.04% of dry
cell wt.), followed by cane molasses (54.68 ± 1.36% of
dry cell wt.) and hydrolyzed citrus pulp 47.5 ± 1.01% of
dry cell wt.) (278). Using organic matter from wastes
and wastewaters can be used as a substrate for the

production of PHB, with combined advantages of redu-
cing disposal cost and production of value-added pro-
ducts. A hydrolysis step is commonly needed before
inoculation with pure cultures (279, 280). However, so
far, only low PHB content and productivity were
achieved from waste products (264). Since, the harsh
conditions like nutrient limitation and stress are necess-
ary to trigger PHA production and intracellular accumu-
lation, therefore, environmental parameters such as pH
and temperature have significant effects on microbial
growth and PHA production (281). Further, there is diversity
among bacteria from different ecological habitats in their
PHA production, modes of growth, type of microorganisms
and media ingredients. But, the most of standardization
studies on PHA-producing bacteria, including Gram-nega-
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tive and Gram-positive species, have reported the
optimum pH 7 and incubation at 30–37°C for maximum
yield (282–287).
Additionally, the biochemical and metabolic activities
including the ability to utilize different carbon and nitro-
gen sources, and hydrolytic enzyme (amylase, lipase and
protease) activities of bacterial candidates were also
reported to play an important role in PHA synthesis.
Therefore, it is economical to use microbes which
produce a variety of enzymes to solubilize waste compo-
sites and cheap substrates. According to Sangkharak and
Prasertsan, bacteria belonging to genera Bacillus, Aero-
monas sp. and Alcaligenes sp. had ability to produce
high amounts (2–6.58 g/l) of PHAs and high levels of
hydrolytic enzyme activities which may result in efficient
utilization of municipal wastewater, palm oil mill effluent,
glycerol and molasses, and its conversion into PHAs with
a variety of PHA monomers (270).
To reduce the substrate cost, recombinant strains
utilizing a cheap carbon source and corresponding fer-
mentation strategies have also been developed (177,
266). E. coli harboring phaABC and phaP of Azotobacter
sp. is a suitable host as a heterologous expression back-
ground for foreign genes that can be easily manipu-
lated and improved by means of recombinant DNA
methodologies. Also, high-cell-density cultivation strat-
egies for numerous E. coli strains are well established
(41, 288). E. coli cells that accumulate large amounts
of PHB become fragile, facilitating the isolation and
purification of the biopolymer, and the bacterium
does not express PHA-degrading enzymes (34, 110).
However, there are at least two disadvantages in
using E. coli. First, E. coli does not contain a PHA precur-
sor-providing pathway, so that a new pathway has to
be constructed to provide precursors for PHA synthesis.
Second, the metabolic background is quite different
between E. coli and native PHA production strains.
Many PHA-producing strains can accumulate PHA
GREEN CHEMISTRY LETTERS AND REVIEWS 71
from fatty acids, while E. coli grows poorly on fatty
acid culture medium (289, 290). Therefore, application
of E. coli as the host to produce PHA has not been
the best option.
At present, the application of defined co-cultures for
PHA production is still in its infancy. For single-stage
co-culture fermentation systems, one of the main chal-
lenges is providing cultivation parameters for efficient
and effective bioconversion of carbon substrates into
PHA. Parameters such as inoculum concentration, dis-
solved oxygen, pH, temperature, cultivation time,
carbon and nutrients feed rate, and secondary metab-
olites production rate would need to be fine-tuned in
order to maximize the bioconversion process. Another
challenge is the harvesting and separation of PHA-con-
taining biomass from non-PHA-containing biomass, par-
ticularly for co-cultures comprising PHA-accumulating
and non-PHA-forming microorganisms as the presence
of non-PHA-containing biomass would increase the
extraction cost of PHA. Compared to single-stage fer-
mentation approach, two-stage fermentation approach
may be more advantageous as it enables finer control
over cultivation parameters and harvesting of PHA-
accumulating biomass. However, higher capital and
operation cost are associated with two-stage fermenta-
tion system. Ultimately, the type of systems chosen
would greatly depend on the microbial characteristics
of the co-culture as well as the economic viability of
the bioprocess (58).
The cost of PHA using the natural producer
A. eutrophus is US$15–30 per Kg (30, 291) which is 18
times more expensive than polypropylene. With recom-
binant E. coli as a producer of PHA, price can be
reduced to US$4 per Kg, which is close to other biode-
gradable plastic materials such as PLA and aliphatic poly-
esters. The commercially viable price should come to US
$3–5 per Kg (266). Although there are presently econ-
omic disadvantages and limited uses of bacterial plastics,
there is currently a high amount of research devoted to
improve productivity, to reduce production costs, and,
more importantly, to produce specific functionalized
PHAs.
Conclusion and future prospects
The intracellular accumulation of PHAs as hydrophobic
granules of energy/or carbon storage materials under
stress conditions and its mobilization on return of con-
ditions for normal growth is a most widely spread
phenomenon among bacterial flora. PHAs are degrad-
able within host cells and also biologically degraded
under both aerobic and anaerobic conditions by natu-
rally existing microorganisms in natural environment
 72 MUHAMMADI ET AL.
into carbon dioxide and water, which return to the
environment. Bacteria produce a wide range of different
PHAs with varying monomer compositions depending
on the substrate specificities of PHA synthases, carbon
sources and the metabolic pathways. All PHAs extracted
from bacterial cells exhibit varying physical properties
range from brittle to flexible to elastic which make
PHAs attractive for substitution to petrochemical ther-
moplastics. Due to production from renewable
resources, a wide range of mechanical properties, bio-
compatibility, complete biodegradability and potential
chemical pools for useful chiral compounds, PHAs have
been drawn much attention for broad commercial appli-
cations. But a major problem in the commercialization of
PHAs in a wide range of applications is their high pro-
Downloaded by [Central Michigan University] at 07:42 19 December 2015
duction cost which has restricted its potential appli-
cations. So, it is desirable to find out new and low-cost
ways to produce PHAs with superior qualities and quan-
tities. Also in a world with shrinking petroleum reserves
and increasing environmental issues, PHAs are definitely
potential candidates that deserve further exploration.
The challenge for the future application of the PHA poly-
mers depends mostly on the increase in the production
levels of these polymers with the desired various proper-
ties in an economical fashion. This needs improvement
of the current technology for the whole process from
the start to the final step. This suggests the selection
and development of bacterial strains that are capable
of efficient consumption and transformation of various
substrates into a range of PHAs with different properties,
at high yield and productivity; high performance fermen-
tations, and efficient extraction and purification to lower
the price. The utilization of local cheap substrates such as
agri-waste, the cultivation processes combining batch
and fed-batch fermentations might give the highest pro-
ductivity compared to the other reported methods.
However, considering the controllable nature of chemo-
stat, it has the greatest potential to provide higher pro-
ductivities. Therefore, currently much research effort
has been devoted to lower the production cost and
meet the target pricing by developing more efficient fer-
mentation routes, recovery processes and recombinant
organisms. This field of research requires further investi-
gation in future to enhance the productivity and lower
the production costs to make it more competitive. All
efforts at laboratory scale will need to be validated at
pilot scale for future industrial production. The chal-
lenges of scale-up process might put a question mark
against those procedures and processes that have
been proposed to be promising. So, it is expected that
the ongoing research all over the world soon shall
achieve potential PHAs with acceptable prices in near
future.
Disclosure statement
No potential conflict of interest was reported by the authors.
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