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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
To link to this article: http://dx.doi.org/10.1080/17518253.2015.1109715
© 2015 Taylor & Francis
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Download by: [Central Michigan University] Date: 19 December 2015, At: 07:42
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-

boxylic acids, which are produced and accumulated by
Expansion of conventional petrochemical plastics pro-
a large number of bacteria under unbalanced growth
duction and consumption are having a significant
conditions as intracellular hydrophobic inclusions of
impact both visibly and invisibly on the environment
carbon and energy storage compounds in the cytoplasm
and society. Improper disposal of plastics has threatened
to levels as high as 90–97% of the cell dry weight (4–9) or
natural environment worldwide since long time ago (1). as an electron sink mechanism for redundant reducing
Conventional petrochemical plastics are recalcitrant to power under the condition of limiting nutritional
microbial degradation and accumulate in environment elements such as N, P, S, O or Mg in the presence of
at a rate of 28 million tons per year (2) In 2011, nearly excess carbon source (10–13). They are synthesized as
280 million tons of petrochemicals-based polymers byproducts not the major ones when there is no suffi-
were produced with expected increased in 4% per cient nutrient to promote their growth and do not play
annum to 2016. Production of synthetic polymers is an essential role in development of the producing organ-
expected to increase to around 810 million tons by isms, but convey survival in the biological community
2050 (3). The challenging problems associated with pet- and environment, therefore, microbial PHAs are second-
roleum-based plastic accumulation in the environment ary metabolites (14). The accumulated PHAs are
and rapid depletion of natural resources being used in degraded by intracellular depolymerases and metab-
their production are motivating factors for research olized as carbon and energy source as soon as the
into sources and tools for alternatives to petroleum- supply of the limiting nutrient is restored (15, 16). The
based polymers. Considering current advances in biopo- presence of sudanophilic, lipid-like inclusions which
lymers research, bacterial polyhydroxyalkanoates (PHAs) were soluble in chloroform was initially observed in Azo-
which are biodegradable thermoplastics have shown tobacter chroococcum in early last century (17). The
great potential as a replacement for petroleum-based chemical composition of similar inclusions in Bacillus
plastics. Therefore, to overcome these problems, the pro- megaterium was later identified as poly(3-hydroxybuty-
duction and applications of eco-friendly PHAs become ric acid) (P(3HB) or PHB) by Lemoigne (18, 19) and
inevitable. coworkers (20) During the following 30 years, interest
CONTACT Muhammadi muhammadi12@yahoo.com

© 2015 Taylor & Francis
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GREEN CHEMISTRY LETTERS AND REVIEWS 57
in this material was negligible. The first report on func- expensive and hamper the widespread usage of this
tions of PHB appeared in 1958 by Macrae and Wilkinson high-quality material (30, 40, 41). However, environ-
(21). They reported the rapid biodegradability of PHB mental-friendly features such as biodegradability and
produced by B. megaterium, by Bacillus cereus and biocompatibility, which are lacking in conventional plas-
B. megaterium itself. From here on, the interest in PHB tics, have continued as the commercial interest in PHAs
increased dramatically. Once PHAs are extracted from (42). In the early 1990s, despite higher costs of pro-
bacterial cells, these polymers show crystalline, flexible, duction, a German company Wella started using PHAs
elastic and thermoplastic properties (22–25). Further, as packaging material for some of its hair-care products
they are synthesized from renewable carbon resources, (43). Recently, PHAs have attracted considerable atten-
based on agriculture or even on industrial wastes or fer- tion due to their potential use as biodegradable thermo-
mentation feedstocks (25–27) so do not lead to the plastics and sources of chiral monomers (34, 44, 45). Thus,
depletion of finite resources (28). Bacterial PHAs gained research is currently being performed to improve pro-
particular interest since they are completely biodegrad- ductivity, to reduce production costs and, more impor-
able, non-toxic, biocompatible and also sources for com- tantly, to produce specific functionalized PHAs (46–48).
mercially useful pool of chiral monomers (29–33). Therefore, the aim of the present study is to review the
Growing concerns about environmental pollution have current advances and progress in biosynthesis, accumu-
renewed interest in the development of PHAs which lation, extraction, chemical and physical properties, bio-
are totally degraded by microorganisms present in compatibility, degradation and potential applications of
most environments and ecologically useful alternatives bacterial thermoplastic PHAs.
to synthetic conventional plastics which are non-degrad-
able, and release harmful chemicals like hydrogen chlor-
Chemical structure
ide and hydrogen cyanide during incineration (31, 34,
35). Besides being biodegradable, PHAs are recyclable The PHAs that have been identified to date are rather
like the petrochemical thermoplastics (7). Since their dis- complex class of primarily aliphatic/linear, head-to-tail
covery, all these properties have made these microbial and optically active biopolyoxoesters and composed of
polyesters very attractive as a source of alternative bio- (R)-3-hydroxy fatty acid monomers (Figure 1) (7, 9, 43,
degradable materials to conventional petrochemical- 45). In these polymers, the carboxyl group of one
based plastics (23, 24, 36). Therefore, they are considered monomer forms an ester bond with the hydroxyl group
for several applications in the packaging industry, biome- of the neighboring monomer (Figure 1) (18, 30). In all
dical, medicine, agriculture and food industry, or as raw PHAs that have been characterized so far, the hydroxyl-
materials for the synthesis of enantiomerically pure substituted carbon atom is of the R-configuration due
chemicals and the production of paints (28, 37–39). to the stereo specificity of PHA biosynthetic enzymes,
Due to wide industrial application, PHB are considered except in some special cases where there is no chirality
as green plastics. Unfortunately, the current production (Figure 1) (26, 43, 49, 50). Because of the chiral (R)
costs are much higher than for petroleum-based syn- center in the backbone, the chemical synthesis of PHA
thetic plastics, which make PHAs substantially more 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
Figure 1. The general molecular structure of produce PHA with specialized functional groups (58).
polyhydroxyalkanoates. 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 granule size distributions in biotechnology (77). The
atoms constituting monomer units, the type of microor- staining of cell suspensions during cultivation exper-
ganism and growth conditions (16, 43). Based on the iments revealed that Nile red has a high potential for
number of carbon atoms in the monomer units, PHAs the quantitative determination of hydrophobic bacterial
can be divided into four groups. The short chain length polyhydroxyalkanoic acids. Such optical methods offer
polyhydroxyalkanoates (scl-PHAs), which consist of C3– the advantages of online monitoring in real time with
C5 atoms, medium chain length polyhydroxyalkanoates high specificity (71). Although these staining methods
(mcl-PHAs) consisting of C6–C14 atoms and long chain are quite sensitive, but it is rather time-consuming and
length polyhydroxyalkanoates (lcl-PHAs) containing labor-intensive work to screen a large number of
>C14 monomers, and PHAs containing both scl- and environmental isolates. Moreover, prior to identification
mcl-monomer units have been identified in several bac- and isolation of PHA-producing bacteria by phenotypic
teria (59–64). In addition, the random copolymer poly(3- methods, it is necessary to provide appropriate carbon
hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-3HH) sources, nutrient limitation conditions and a long
or P(HB-HH) was found to be accumulated in some Aero- culture time is required for PHA granule accumulation
monas strains (64). This grouping is due to the substrate to the bacterial cells (78, 79). Sudan black B is non-
specificity of the PHA synthesis that only accepts 3- specific to PHA as it also stains other lipid bodies. Nile
hydroxyalkanoates (3HAs or HAs) of a certain range of blue A and Nile red are reported to be more specific
carbon length (26). The PHA synthases of Alcaligenes than Sudan black B for detection (79). In addition,
eutrophus can only polymerize 3HAs (scl) while that of these methods cannot distinguish between bacteria
Pseudomonas oleovorans only polymerize 3HAs (mcl). that accumulate PHA granules and those that accumu-
For scl-PHAs, the monomer units are oxidized at pos- late lipid compounds (78). Hong et al. developed a
itions other than the third carbons while for mcl-PHAs, rapid noninvasive technique using Fourier Transformed
all the monomers units are oxidized at the third position Infrared Spectroscopy (FT-IR) which allows detecting
except in few cases (28). A lot of PHAs (mcl) containing intracellular PHA accumulation in intact cells within a
various functional groups such as olefins, branched few seconds. If the PHA content is high in the cells, the
alkyls, halogens, aromatic and cyano have been reported FTIR spectra of PHA can also differentiate the various
(28, 53, 55). This flexibility of PHA biosynthesis makes it structures of PHB (scl), mcl-PHAs and PHA consisting of
possible to design and produce related biopolymers HB and mcl-3HA monomers (80). Genotypic-based
having useful physical properties ranging from stiff and screening method is specific, reliable and quick which
brittle plastic to rubbery polymers (26). circumvents the major drawbacks inherent in phenoty-
pic detection methods (78, 81–84). For genotypic-based
screening, the degenerate or and specific oligo nucleo-
Screening of bacteria for PHA production
tide primers are designed based on multiple sequence
To date two major methods have described for screening alignment results and are used as PCR primers to
of PHA-producing natural bacteria from environments, detect PHA synthase genes (78, 79, 81, 83–85). For
namely, phenotypic-based screening and genotypic- rapid detection, colony PCR technique is employed for
based screening. There are many phenotypic detection screening PHA producers from the environment (78,
methods for detecting intracellular PHA granules, 80). The PHA accumulation ability of well-separated colo-
which are applied to the screening of PHA producers, nies isolated from environmental samples can be directly
including Sudan black B staining (65–67), Nile blue A validated by PCR with no further culturing or chromoso-
staining (68–70) and Nile red (71), which result in dark mal DNA extraction procedures. In addition to its appli-
blue or fluorescent granules. Alternative staining cation to the screening of wild-type isolates, the
methods have been developed for directly staining colo- individual PCR-amplified product is also suitable as a
nies (72) or growing bacteria on plates containing Nile specific probe for PHA operon detection in producers
blue A or Nile red (68, 73), resulting in fluorescent colo- and cloning (78).
nies that can be visualized by UV illumination. PHA-pro-
ducing colonies on plates containing Sudan black B
Diversity among PHA-producing bacteria
appear as black-bluish (74). It was reported that PHAs
stained by Nile red show a similar fluorescence behavior, PHA producers have been reported to reside at various
maximum at an excitation wavelength between 540 and ecological niches which are naturally or accidently
560 nm and an emission wave length between 570 and exposed to high organic matter or growth-limited con-
605 nm detected by fluorescence spectroscopy or flow ditions such as dairy wastes, hydrocarbon-contaminated
cytometry (75, 76). This is suitable for the analysis of sites, pulp and paper mill wastes, agricultural wastes,
activated sludges of treatment plants, rhizosphere and bacteria) also produces copolymers of 3-hydroxybutyric
industrial effluents (86). A wide range of taxonomically acid and 3-hydroxyvaleric acid (HV) P(HB–HV) (90). Copo-
and physiologically different natural bacteria and some lymers of 3-hydroxybutyric acid with 4-hydroxybutyric
archae accumulate PHAs as storage reserve materials and acid can also be produced in A. eutrophus (91). The bac-
deposit them as insoluble granules in the cytoplasm (10, teria Rhodospirillum rubrum (59), Rhodocyclus gelatinosus
11, 69, 70, 83, 84). After the discovery of PHB from the bac- (60) and Rhodococcus sp (59) produced terpolyesters
terium B. megaterium (18), over 300 different bacteria, consisting of 3HA units of C4, C5 and C6 from hexanoate.
including Gram-negative and -positive species, have Aeromonas caviae produced a random copolymer of HB
been reported to accumulate various PHAs (11, 28, 83, and 3-hydroxyhexanoate (HH) (92–94). Pseudomonas
86, 87). To date, most PHA-producing bacteria were strain GP4BH1 produced PHA containing HB and 3-
found to be Gram-negative. Comparatively, a limited hydroxyoctanoate (HO) from octanoate and PHA con-
number of Gram-positive bacteria has been reported in taining HB, HO, and 3-hydroxydecanoate (HD) from glu-
genera Bacillus, Caryophanon, Clostridium, Corynebacterium, conate (95). Pseudomonas sp. strain 61–3 isolated from
Micrococcus, Microlunatus, Microcystis, Nocardia, Rhodococ- soil was reported to produce a blend of a PHB homopo-
cus, Staphylococcus and Streptomyces (58). As for archaea lymer and a random copolymer (P(HB–HA)) consisting of
is concerned, PHA production to date, however, has HA units of C4 to C12 from sugars and alkanoic acids (62,
been limited to haloarchaeal species, specifically the 63, 96). Boyandin et al. reported the luminescent bacteria
genera Haloferax, Halalkalicoccus, Haloarcula, Halobacter- Photobacterium leiognathi and Vibrio harveyi as new pro-
ium, Halobiforma, Halococcus, Halopiger, Haloquadratum, ducers of two- and three-component heteropolymeric
Halorhabdus, Halorubrum, Halostagnicola, Haloterrigena, PHA containing hydroxybutyric acid as the main
Natrialba, Natrinema, Natronobacterium, Natronococcus, monomer, and hydroxyvaleric and hydroxyhexanoic
Natronomonas and Natronorubrum (88). acids monomers as minor components (97).
Bacteria used for the production of PHAs can be In addition, two different types of polyester granules
divided to two major groups based on the culture con- were formed in the same cell (98). A recombinant
ditions required for PHA synthesis (35). First group of strain of P. oleovorans expressing R. eutropha PHB
bacteria requires the limitation of an essential nutrient biosynthesis genes has been shown to synthesize a
such as nitrogen, phosphorus, magnesium or sulfur for blend of a PHB homopolymer and a copolymer of HHx
the synthesis of PHA from an excess carbon source. and HO units when grown on octanoate (99). Both poly-
The bacteria included in this group are Ralstonia eutro- esters were stored as separated granules within the cells
pha, Protomonas extorquens and P. oleovorans. The (100). Fourth group of bacteria including Fluorescent
second group of bacteria does not require nutrient limit- pseudomonads is capable of synthesizing lcl-PHAs
ation for PHA synthesis and the polymer is accumulated (101). In addition, Pseudomonas fluorescens and several
during growth phase. It includes Alcaligenes latus, a other Pseudomonas strains were found to produce a
mutant strain of Azotobacter vinelandii, and recombinant P(HB-–HA) copolymer consisting of HA units of C4 to
Escherichia coli. These characteristics are important to be C12 from HB and 1,3-butanediol (102). Although Thio-
considered while the production of PHA. capsa pfennigii accumulated only a PHB homopolymer
In another way, PHA-producing bacteria can also from various carbon sources, a recombinant P. putida
broadly be divided into four groups according to the strain harboring the PHA synthesis genes of T. pfennigii
number of carbon atoms in the monomeric units of the produced a P(HB–HHx–HO) terpolymer from octanoate
PHAs produced (39). One group of bacteria, including (103). Interestingly, the occurrence of PHA is not
Ralstonia eutropha (formerly A. eutrophus), produces limited to the intracellular collection in granules. PHB
scl-PHAs, while the other group, including P. oleovorans with lower molecular weight (cPHB; Mw < 14,000 Da)
and most Pseudomonads belonging to the rRNA hom- was also found in B. subtilis, A. vinelandii and Strepto-
ology group I, can accumulate mcl-PHAs using 3-hydro- myces lividans (104–106) in association with polypho-
xyacyl-CoA intermediates of β-oxidation pathway when sphate and calcium ions. In addition, non-storage PHAs
cultured on various alkanes, alkanols or fatty acids (11, that are of low molecular weight, PHBs have also been
26, 83, 84). Although the majority of bacteria accumulate detected in the cytoplasmic membrane and cytoplasm
either scl- or mcl-PHA, several bacteria (third group) have of E. coli (31).
also been found to synthesize PHAs (copolymers) con-
taining both scl- and mcl-HA. This largely depends on
Biosynthesis
the substrates provided and polyester synthases with
different substrate specificities (45, 89). In the presence PHAs are synthesized by several enzymatic reactions
of propionic or valeric acid, A. eutrophus (among other 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
(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
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- A. eutrophus (16, 127). Whereas P. oleovorans is estimated
quently, the osmotic pressure of the cell is not to have about one or two large granules (128). The PHA
changed extensively (Figure 4) (26). The size and granules are mostly spherical and surrounded by a phos-
number per cell varies depending on the different pholipids membrane (129) separating two crystalline
species (Figure 4). About 8–13 granules per cell having protein layers (130, 131) composed of the PHA poly-
a diameter range of 0.2–0.5 µm were observed in 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
granule.
(133), amphipathic phasing proteins (117, 134), PHA- agglutinating with other granules and acting as a
specific regulator proteins (135–137) and additional pro- barrier between the polymer and other cellular com-
teins with unknown function (Figure 5) (128). Granules ponents (123, 124, 145). In phasins mutants of
membrane coat is about 2 nm thick, containing 0.5% R. eutropha, the cells accumulate less PHB, and they
lipid and 2% protein, of the granule weight (138). PHA possess only one single large granule because separate
polymerases are present in the cytosol; however, they granules coagule due to the “nacked” surface if the
are only active when localized on the surface of granules hydrophobic PHA molecules get in contact (Figure 4)
(103, 132, 134). The intracellular depolymerases are (140, 142, 146). It was also speculated that phasins
required for mobilization of the reserve polyester and might have a protective function to reduce the passive
are attached to the granule surface (139). Among the attachment of cytosolic proteins. Simulation of the self-
proteins associated to PHA granules, phasins constitute assembly process showed that phasins might impact
a very diverse group are the most dominant proteins on the kinetics of granule formation by reducing the
of relatively small molecular size (124, 140, 141). A few lag phase (147). Various PHA-specific regulators such as
that have been studied in detail revealed that they are PhbR from C. necator (137, 142). PhaF from Pseudomonads
associated with a structural role of PHA granules and (135, 148, 149) and PhaR from Paracoccus denitrificans
may have several functions such as coating, stabilizing (136, 150) were found to bind non-covalently to the
granules as structural proteins by non-covalently PHA granules. The cytosolic levels of these repressor pro-
attached to the polyester core of granules (Figure 5) teins are supposed to be low when PHA granules are
(30, 135, 142, 143, 144) or activating genes and/or formed. Additional proteins (PhaI, PhaD and PhaS) were
enzymes involved in PHA synthesis (124). Phasins found to be granule-associated and co-regulated in Pseu-
promote PHA biosynthesis and their copy number has domonads, whose function has not been clarified yet
impact on PHA granule size (143, 144). Phasins are inhi- (128, 135, 148, 149).
biting individual granules from coalescing and
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
Figure 5. The ideal PHA granule surrounded by phospholipids or solvent-based extraction can be chosen as isolation
membrane and crystalline protein. methods. For solvent-based extraction of PHA, the
concentrated biomass is dried either by freeze- or spray- salt produced as a by-product and the amount of
drying. PHA is extracted directly from ground biomass by surfactant-containing wastewater generated from the
dissolving it in an organic solvent, usually chloroform, process, potentially resulting in high costs for wastewater
chlorolimited methylene chloride, propylene carbonate treatment. Singh et al. reviewed a novel self-disruption cell
and dichloroethane (70, 151, 152, 153). After removing system for recovery PHA developed in B. megaterium
cellular components (residuals) by filtration and centrifu- (167). In this system, a gene cassette carrying the cell
gation, the PHA is precipitated in a non-PHA-dissolving lysis system (holin and endolysin of B. amyloliquefaciens
solvent such as cold ethanol or methanol (154–158). In phage) (168) was inserted into the E. coli–B. subtilis
contrast to mcl-PHAs, scl-PHAs are not soluble in shuttle vector pX. In this expression system, xylR-xylA’
acetone, thus, enabling a separation of both PHA target genes are induced by xylose but inhibited by
classes (159). Recovery of PHA from bacterial cells using glucose, which acts as an anti-inducer (169). It synchro-
organic solvents is often applied in industrial processes nizes the processes of spontaneous cell lysis and substrate
and in laboratory scale PHA extractions due to the recov- exhaustion, which results in the release of accumulated
ery efficiency of the process, simplicity, rapidity, polymer PHAs. The efficiency of this regulatory process can be
purity obtained and the possible removal of endotoxins enhanced by manipulating the YoeB, a cell wall-associated
from the recovered polymer, which is important for protein, which gets induced in response to antibiotics
medical applications (160–162). Solvents extract the stress. The expression of yoeB in B. subtilis is under a
polymer without degrading it by improving the cellular xylose-inducible promoter. yoeB mutants display an
membrane permeability and subsequent solubilization increased rate of autolysis in response to nutrient
of the PHA (162). But a large amount of hazardous depletion and various cell envelope stress conditions.
solvent is needed to repeat the same process. Thus, The process of autolysis in B. subtilis 168 can be aided
this method is not environmentally friendly and unsuita- by mutating yoeB gene and in the process making it
ble for mass production of bioplastic (22). Excellent independent of xylose regulation (168).
recovery was reported for the extraction in supercritical
CO2, which may be especially applicable to reduce the
Biodegradability of PHA
endotoxins contamination from genetically engineered
E. coli strains (29). The second protocol is digestion- The biodegradation of PHAs in natural environments
based extraction strategies that utilize enzymatic treat- (river and sea waters, soil, sludge and compost) is one
ment of cellular components to release PHA and of the commercially attractive features which distin-
design to avoid the use of organic solvents. In this guishe the PHAs from petroleum-based plastics (170–
method, the cells need to be broken up and various 175). The main advantage of PHAs over other types of
chemical additives added to digest non-PHA material. biodegradable plastic is that they do not require
Bacterial cells debris is treated with a cocktail of special environmental conditions and can degrade in
enzymes (including proteases, nucleases and lysozymes) either aerobic or anaerobic environments through
and detergents (5% sodium dodecylsulfate) to remove thermal degradation or enzymatic hydrolysis (176, 177).
proteins, nucleic acids and cell walls, leaving the PHA Biodegradation of PHAs is performed by microorgan-
intact. Finally, the PHA is concentrated through cross- isms, which inhabit a specific natural environment
flow filtration and obtained as white latex of 95% (176). But in the absence of biological agents (bacteria,
purity, ready to be used as coating material (22, 25, algae and fungi) PHAs are practically not subject to
158, 163). Compared to solvent-extracted PHA, the mol- mass lost under normal conditions (178) and they are
ecular weight may be lower following enzymatic recov- degraded in biological media to form products innocu-
ery methods despite the mild reaction conditions (164). ous to the environment. PHA biodegradation is per-
One critical consideration of these chemical-based formed by microorganisms that secrete intra- or
methods is that harsh chemical treatment to achieve extracellular PHA depolymerases, which differ in their
high purities may lead to a reduction of the molecular molecular organization and substrate specificity (179).
weight of the polymer (165). Yang et al. developed an While intracellular PHA depolymerases are synthesized
strategy for poly(hydroxybutyrate-co-hydroxyvalerate) by PHA-producing bacteria and are used by them to
(P(HB-co-HV)) recovery using linear alkylbenzene sulfonic hydrolyze their own PHA storages, extracellular
acid LAS-99 as an alternative to the commonly used enzymes are produced by other microorganisms to
sodium dodecyl sulfate. In this method, only 21% of utilize PHAs usually released into environment after
the surfactant is required (166), compared to previous death and cell lysis of PHA-accumulating cells (180).
SDS-based methods. The main disadvantages of The first microorganisms degrading poly-3-HB were iso-
chemical-based strategies are the large amount of lated over 50 years ago (181). Six hundred PHA
64 MUHAMMADI ET AL.
depolymerases from various microorganisms have been depolymerases, physical and chemical properties of the
identified by now; comparison of their amino acid polymer (stereo-configuration of its molecules, compo-
sequences provided a basis for uniting them in 8 super- sition, shape of specimen, crystallinity and molecular
families including 38 families (179). The same strain can weight, polydispersity), surface area, microbial activity
contain several genes encoding PHA depolymerases of the disposal environment, the pressure of other nutri-
with different specificities. The ability to degrade extra- ent materials, physicochemical conditions (pH, tempera-
cellular PHAs is determined by the activity and type of ture, oxygen availability, moisture, salinity, acidity of the
PHA depolymerases, which hydrolyze the polymer by environment, etc.), the shape and size of PHA-based
surface erosion to water-soluble monomers and/or oligo- devices, and weather and climate in different regions
mers a substrate for microorganisms. All PHA-degrading (28, 176, 180, 189). It has been reported that poly(3-
bacteria, algae and fungi use extracellular enzymes to hydroxybutyrate-co-4-hydroxybutyrate) had a much
depolymerize, or break down, PHAs and, through a faster degradation rate than homopolymer PHB (178).
process known as mineralization, absorb the remaining While according to the current investigation of Boyandin
fragments for use as minerals (182). The end products et al., the homopolymer of 3-hydroxybutyric acid is
of PHA degradation in both aerobic and anaerobic degraded at higher rates than the P(HB-HV) (176). Simi-
environments are carbon dioxide and water, while in larly, the average rates of mass loss were 0.04–0.33%
anaerobic conditions methane is also produced (28, per day for films and 0.02–0.18% for compact pellets,
175). If the limiting growth factor is supplied again, the suggesting preferential degradation of the amorphous
accumulated PHAs can be also intracellularly depolymer- phase over pellet. The differences in degree of degra-
ized by degradation enzymes via hydroxycarboxylic dation are due to differences in the kinetics and mechan-
acids to acetyl-CoA and reused in metabolism (16, 28). ism of PHA biodegradation is caused by qualitative and
All microorganisms that synthesize PHAs contain intra- quantitative dissimilarities between microbial commu-
cellular depolymerase system for this purpose. Studies nities of different habitats and other environmental
using R. eutropha showed that the intracellular degra- and specimen-related factors (176).
dation of PHB inclusions is a very slow process (183). In microbially active environments, PHAs are initially
The rate of PHB degradation was calculated to be viewed by microorganisms as energy sources and then
about 10 times slower than the rate of its synthesis start to degrade (107, 190). Microorganisms colonize on
(184, 185). It may be because that PHA inclusions are pro- the surface of the polymer and secrete exodepoly-
tected by granule-associated proteins on the surface merases which degrade P(HB–HV) into HB and HV
from attack by the depolymerase (186). But, Merrick units. These units are then used up by the cell as a
et al. (187) proposed that an activator acts to modify an carbon source for biomass growth (15). P(HB–HV) is
inhibitor present on native PHB inclusions, thereby allow- water insoluble and is not affected by moisture, does
ing the intracellular depolymerase to access its substrate not degrade under normal conditions of storage and is
(188). Based on the finding that the number of polymer stable indefinitely in air (107, 171). The effect of different
chains was almost constant during the PHB degradation environments on the degradation rate of PHB and P(HB–
process, it was suggested that the intracellular depoly- HV) has been studied by several workers (171, 184).
merase is an exotype hydrolase acting at the carbonyl Degradation occurs most rapidly in anaerobic sewage
terminus of the polymer chain (184). Low concentrations and slowest in seawater. Lee showed that P(HB–HV) com-
of diisopropyl fluorophosphate inhibit the depolymerase pletely degraded after 6, 75 and 350 weeks in anaerobic
activity leading to the suggestion that this enzyme is a sewage, soil and sea water, respectively (107, 191). Effec-
serine esterase (188). In comparison to the extracellular tive PHA destructors include various bacteria from wide-
depolymerase which attacks crystalline PHA, the mech- spread soil and water genera (Pseudomonas, Alcaligenes,
anism for intracellular depolymerase is presumed to be Comamonas, Streptomyces, Ilyobacter) (192), as well as
different because of the amorphous nature of the intra- fungi (Ascomycetes, Basidiomycetes, Deuteromycetes, Mas-
cellular PHA inclusions. The end products of PHA degra- tigiomycetes and Myxomycetes) (180). Potential microbial
dation in aerobic environments are carbon dioxide and PHA destructors are generally isolated by inoculation of
water, while methane is also produced in anaerobic con- microbiological samples on agar plates or latex
ditions (28, 175). In vivo and in vitro PHA degradation is medium based on PHA particles or granules as the only
estimated in terms of weight loss (surface erosion), mol- carbon and energy source. Microbial exodepolymerases
ecular weight decrease, increase in the degree of crystal- hydrolyze PHA to soluble products forming lysis zones
linity and loss of mechanical properties (171). PHA on surface of the plate (175). The ability to degrade extra-
degradability is influenced by many factors, including cellular PHA depends on the secretion of specific PHA
the specificity and activity of extracellular depolymerases which hydrolyze the polymer to water-
soluble products (181, 192) and is widely distributed mesenchymal stem cells were shown to adhere and pro-
among bacteria (171, 193). Aerobic and anaerobic PHA- liferate on several PHA substrates, with a terpolymer poly
degrading bacteria of many taxa were isolated from (hydroxybutyrate-cohydroxyvalerate-co-hydroxyhexano-
various ecosystems, and several PHA depolymerases ate) (P(HB-co-HVco-HHx)) yielding the optimum results
were isolated and characterized (185, 194, 195). All puri- (212, 213). Further, Sevastianov et al. have also tested
fied depolymerases were specific for PHB and/or other the PHA matrices for hemocompatibility by inspecting
scl-PHAs, such as the PHB depolymerases of A. faecalis, the response of mammalian blood when incubated
Comamonas sp., or P. lemoignei (196–198) or for mcl- with polymer films (161). It was shown that PHB or P
PHAs, such as the poly(3-hydroxyoctanoate) (P(HO)) (HB-co-HV), when in contact with blood, did not affect
depolymerase of P. fluorescens GK13 (194). Whereas platelet responses, nor did the polymer activate comp-
most PHA-degrading bacteria analyzed so far apparently lement system (214). However, more involved polymer
produced only one PHA depolymerase, purification procedures had to be followed to signifi-
Pseudomonas lemoignei was found to have at least five cantly reduce the amount of bacterial cell wall material
PHA depolymerases (199). The structural genes of associated with the purified PHA (161).
several PHA depolymerases have been cloned and Biocompatibility of PHB has been demonstrated in
vivo under subcutaneous implantation of PHB films
sequenced (197, 200–202). Biochemical analysis of the

purified depolymerase proteins and analysis of the (161). Tissue reaction to films from PHB of different Mw
DNA-deduced amino acid sequences revealed that PHA (300, 450, 1000 and 1500 kDa) implanted subcutaneously
depolymerases apparently possess a catalytic triad con- was relatively low and did not change from tissue reac-
sisting of serine, histidine and aspartate residues which tion to control glass plate. The low tissue reaction to
have been demonstrated to form the active site in bac- implanted PHB films indicates the high biocompatibility
terial lipases (203). In addition, the hydrolysis of several of PHB in vivo (161, 214). This high biocompatibility of
scl-PHAs by lipases has also been reported (204). There- PHB is due to the presence of natural PHB oligomers
fore, the commercial production of PHA as a biological and HB, the intermediate product of PHB degradation,
source will bring down the use of expensive chemicals in animal tissues at normal conditions in comparison
and also reduce the load on pollution. with chemically synthesized biodegradable polymers,
for example, polylactides and polyglycolides. Therefore,
Toxicological certificate of Institute of Medical Technique
In vivo biocompatibility
(Ministry of Health, Russia) approves of PHB for appli-
PHAs such as PHB and their breakdown products 3- cation in medicine as non-toxic and biocompatible
hydroxyacids have been found in a wide range of organ- mate (214). Given these findings, it is highly understand-
isms, from bacteria to higher mammals. A number of able that this family of polymers is biocompatible and
studies have shown that PHB take part in formation of ideal material for biomedical applications.
transmembrane ion channel complexes in eukaryotic
and prokaryotic cell membranes (31, 205–207). Interest-
Physical properties
ingly, degradation of PHB polymer results ultimately in
the production of, R-3-hydroxybutyric acid which also is Within the cell, PHAs exist as mobile liquid, amorphous
a normal constituent of blood at concentrations (26, 83, 87, 89, 91) and water-insoluble inclusions (43,
between 0.3 and 1.3 mM and known to be associated 22). However, after extraction from the cell with
with ketone body formation. There is a strong likelihood organic solvents, PHAs become highly crystalline and
that surgical implants, sutures, etc. produced from PHA show material properties that are similar to conventional
will not result in an immune response in the host organ- commodity plastics such as polypropylene, polyethylene
ism. Furthermore, sterilization of PHA-based materials and polystyrene (Table 1) (22, 215–217). Because, in vivo,
does not appear to affect the average molecular non-covalently attached phasins as structural proteins
weight (Mw), tensile strength or other properties (208, stabilizing the PHA granules (30, 141) and inhibiting indi-
209). Surface properties of PHA films have been shown vidual granules from coalescing and agglutinating with
to be favorable for proliferation and attachment of other granules (123, 145). In addition, water is a minor
tissue culture cells (210, 211), suggesting that PHA is suit- component of PHA inclusions and therefore it was
able for scaffolding material in tissue engineering. The P suggested that water could act as a plasticizer (218,
(HBco-HV-co-HHx) polymer exhibits the greatest surface 219). About 5–10% of water was estimated to be
roughness, as well as the highest water contact angle present in the nascent PHB inclusions which upon
characteristics which are important for adherence and removal allows for the polymer chains to rearrange
proliferation of cells on PHA surfaces. For example, 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)
Polypropylene 170 – 34 400 (44, 233) decrease in stiffness (Young’s modulus) and an increase
Polystrene 110 – 50 – (44, 221) in toughness, producing more desirable properties for
Polyethylene 130 −10 500 (221)
HDPE 135 29 (220, 221) commercial application. Consequently, a range of prop-
LDPE 130 –30 10 620 (44) erties is feasible from the hard and brittle homopolymer
PET 262 56 7300 (44, 233) via a balance of stiffness and toughness to soft and
Note: Tm: melting temperature; Tg: glass-transition temperature; HB: 3-hydro-
xybutyrate; HV: 3-hydroxyvalerate; HHx: 3-hydroxyhexanoate; LDPE: low-
tough for copolymers with a high incorporation of HV
density polyethylene; HDPE: high-density polyethylene; PET: poly(ethylene (26). Besides the mechanical properties, various HA
terephthalate) monomer structures affect the PHA degradation rates.
It has been reported that poly(3-hydroxybutyrate-co-4-
suggested that water molecules could form hydrogen hydroxybutyrate) had a much faster degradation rate
bonds with the carbonyl groups of the polyester back- than homopolymer PHB (178). mcl-PHAs are rubbery
bone to form “pseudo cross-links” between adjacent and flexible materials with low crystallinity and can be
polymer chains. This type of molecular arrangement used in a wide range of applications which cannot be ful-
may also explain the mobile amorphous nature of PHA filled by PHB and other scl-PHAs (223) but these physical
inclusions as well as the plastic deformation phenom- properties of mcl-PHAs are considered unsuitable for
enon (94, 118, 220) observed in freeze-fracture exper- applications due to their low Tm and Tg (226, 227). In con-
iments (221). But for extraction, when PHA inclusions trast to PHB and P(HB–HV), mcl-PHAs have a much lower
are subjected to physical treatments such as centrifu- level of crystallinity and are more elastic (226, 228) but
gation, they readily coalesce into larger masses and not strong enough for applications. These mcl-PHAs
this can lead to the apparent acceleration of crystalliza- potentially have a different range of applications than
tion (222). Furthermore, any damage to the surface the scl-PHAs. PHA with monomer chain lengths to 12
coating of the inclusion will allow heterogeneous nuclea- carbon atoms or more occur as sticky, more elastic and
tion, that is, crystallization induced by external molecules liquid (178, 229). However, in general, all the bacterial
other than PHA, further accelerating crystallization (223). PHAs are (i) biodegradable thermoplastic and/or elasto-
The definition of their thermal and mechanical proper- meric compounds which can be processed with appar-
ties is normally expressed in terms of the glass-to- atus used by the plastic manufacturing industry
rubber transition temperature (Tg) of the amorphous without losing biodegradability. In addition, they are (ii)
phase and the melting temperature (Tm) of the crystalline insoluble in water and resistance to hydrolytic degra-
phase (Table 1) (43, 217). The mechanical properties of dation, (iii) highly crystalline, (iv) exhibit a rather high
PHA range from brittle to flexible and elastic, depending degree of polymerization ranging from 105 to almost
on the branched length of the HAs or from the distance 107 Da, (v) they are optically active and (vi) isotactic
between the ester linkages in the polymer backbones. (enantiomerically pure chemicals consisting, in general,
Typically, PHAs with short pendant groups are hard crys- only of the R-stereoisomers). They are (vii) non-toxic,
talline materials, whereas PHAs with longer pendant (viii) biocompatible and (ix) exhibit piezoelectric proper-
groups are elastomeric (Table 1) (29). The scl-PHA such ties as revealed (at least) for poly(3HB) and poly(3HB-co-
as PHB is stiffer and more brittle than polypropylene 3HV) (7, 30, 50). These features make them highly com-
phase (217, 224). Because of its brittleness, PHB is not petitive with polypropylene, the petrochemical-derived
very stress-resistant. Also, the relatively high Tm of PHB plastic (31, 216, 217, 230).
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.
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). PHA). Nodak™ has already been made into a variety of
For these different reasons, reaching the conditions of different prototype objects such as plastic fiber or
conventional plastic replacements by degradable poly- twine and molded plasticware such as plates and cups.
mers, particularly for short-term applications (packaging, Metabolix is already producing preliminary PHA
agriculture … ), is of major interest to the society as a materials, but is teaming up with BP for two years to
whole, from the plastic industries to the citizens. produce bioplastics (243). In response to an increased
Although, microbial PHAs like conventional plastics awareness of global environmental problems, PHA is
are also thermoplastics, moldable, naturally UV-resistant gaining serious attention as a potential substitute for
and could be tailor-made for numerous applications non-biodegradable polymers. Now, the advances in
ranging from stiff packaging goods to highly elastic PHA production have opened up the potential opportu-
materials for coatings. But their complete biodegradabil- nities to a number of applications (29). The production of
ity in the environment and naturalness make them quite PHA is intended to replace synthetic non-degradable
beneficial in certain short-lifespan applications for single- polymers for applications especially in packaging, agri-
use packaging, catering, surgery, hygiene, drug delivery culture, leisure, fast-food, hygiene as well as medicine
and agriculture (239). But according to Beucker and and biomedical since PHA is biocompatible and
Marscheider-Weidemann bioplastic can have a short or degradable.

a long lifespan depending on the material or the com-
pound used to produce them (240). Degradation of com- (1) Packaging, molding and coating
mercially available shampoo bottles and film made from
PHA (Biopol@ bottles; molecular composition: 92% 3-HB, PHAs are natural and biodegradable thermoplastic
8% 3-HV, the inner and outer surface areas of the bottles polyesters, and hence the majority of their applications
= 336 and 356 cm2, respectively, the average mass = are as replacements for petrochemical polymers cur-
31.90 g (n = 13) in depth of 85 m of an aquatic ecosystem rently in use for packaging and coating applications
(Lake Lugano, Switzerland) under natural conditions for (26, 31, 230, 244). They can be used in a wide variety of
250 days has measured the degradation rates of 10–20 products including bags, containers, paper coatings,
mg/d giving an average lifespan of 5–10 years for the pens, golf tees, razors, feminine hygiene products,
specific bottle type (241). The degradation rates vary diaper backsheets, utensils, cosmetic containers,
with the water depth, that is, with the spatial heterogen- bottles, cups and materials for food packaging (26, 28,
eity of the ecosystem. Bottles made from polyethylene– 31, 38, 190). The latex of PHAs can be used to produce
starch (PE + S; 9% and 13% starch, respectively) blends a water-resistant layer for paper, film or cardboard (190,
showed no degradation at all. In order to reduce the 245). Manufacturers in the USA used PHB and copolymer
challenging burdens of currently being used plastic on P(HB–HV) as water-proof films on the back of diaper
health and environment, it is needed to determine the sheets (31, 246). This copolymer P(HB–HV), with flexibility
lifespan of plastic products in the real usage conditions. 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
Applications of PHA
polymers in toner and developer compositions or as
Bacterial PHA first received commercial attention way ion-conducting polymers. PHAs can be used as a latex,
back in the early 1960s from an American company, W. for instance for paper-coating applications (26). New
R. Grace, but apparently was discontinued due to some plastic properties can be achieved by blending PHA
technical problems (242). Although patents were orig- with other polymers and there is much activity in this
inally filed in the USA by Baptist J.N. in 1962, but first field (28, 30, 247, 248). Often the mixture changes the
industrial production of PHB and PHA did not occur crystallinity of the plastic, and crystallization rate and
until 1982. The major petroleum crisis of the 1970s finally also the mechanical properties of the material.
then motivated a British company, ICI Bioproducts Thus, a mixture of 40–60% of PCL in PHB improved the
(now known as Zeneca Bio Products) to get involved mechanical properties over P(HB–HV). A 40% PCL and
with this bacterial polymer and marketed them under 60% PHB mixture had a decreased oxygen permeability
the trade name Biopol (26). Besides, currently a number that was only 5% of that of polyethylene (249).
of companies are developing PHAs for use in plastics Maekawa and coworkers reported that blends of PHB
worldwide including Kaneka in Japan, and P&G Chemi- and cellulose propionate were completely miscible
cal, and BP and Metabolix in the USA. Kaneka and P&G since they had a single glass transition, a depression in
Chemical have teamed up to commercialize a product the equilibrium melting temperature of PHB and a
“Nodax” (also known as Nodak™, which is a specialized decrease in spherulitic growth rate of the PHB
component. Also tensile strength was better for the properties, in bone plates and blood vessel replacements
blend of PHB/cellulose propionate than for PHB only (31). PHA can be easily depolymerized to a rich source of
(250). However, not all blends are compatible. optically active, pure, bi-functional hydroxy acids. PHB for
instance is readily hydrolyzed to R-3-hydroxybutyric acid
(2) Medical and pharmaceutical applications and used in the synthesis of Merck’s anti-glaucoma drug
“Truspot”. In tandem with R-1, 3-butanediol, it is also
In addition to their biodegradability, many PHAs are used in the synthesis of β-lactams (31). However, cur-
also biocompatible. Their breakdown products are 3- rently, the medical and pharmaceutical applications of
hydroxyacids, which are naturally found in animals. PHAs are limited due to the slow biodegradation and
Such as PHB is biocompatible, which is not surprising high hydraulic stability in sterile tissues (259). The pres-
when considering the fact that R-3-hydroxybutyric acid ence of 150 different monomers identified yet and new
is a normal constituent of blood at concentrations types of PHA through the chemical or physical modifi-
between 0.3 and 1.3 mM (31, 205, 206) and is also cation of naturally occurring PHA gave rise to diverse
found in the cell membrane of eukaryotes (207). These properties. With these features PHA is also considered
PHAs have the potential to become important and very as pharmaceutically active compound and currently
useful compounds for medical applications such in investigated as potential anti-HIV drugs, anti-cancer
wound management used as surgical suture, skin substi- drugs, antibiotics, etc. (58).
tutes, nerve cuffs, surgical meshes, implants, gauzes, PHAs appear to be potentially useful in controlling
staples, gauzes, swab, lubricating powders (29, 30, 38), bacterial pathogens in certain aquaculture applications
blood vessels, tissue scaffolds and bone fracture fixation (260). For example, administration in the feed of 1000
plates (3, 251, 252). On account of their varied and mg/l of PHB particles of an average diameter of 30 μm,
diverse properties, the PHA biopolymers provide a or addition of inactivated cells (107 cells ml−1) of PHB-
three-dimensional scaffold means to support the cell containing Brachymonas bacteria (equivalent to 10 mg
growth and then degrade away leaving viable tissue, L−1 PHB) to the culture water of brine shrimp (Artemia
with potential product applications for the cardiovascu- nauplii) larvae, conferred a complete protection from a
lar system, cornea, pancreas, gastrointestinal system, virulent strain of the intestinal pathogen Vibrio campbellii
kidney and genitourinary system, musculoskeletal (261). Other similar reports have claimed an inhibitory
system, nervous system, teeth and oral cavity, skin and effect of PHB on certain gut microflora of the giant fresh-
so forth (29, 32). In orthopaedy, PHAs can also be used water prawn (Macrobrachium rosenbergii) larvae (260).
as scaffolds for cartilage engineering, bone graft substi- Administration of PHB in the feed significantly increased
tutes and spinal cages. PHAs can be used to engineer the survival of the prawn larvae and improved their
the heart valves, cardiovascular fabrics, pericardial development. The total bacterial counts and Vibrio spp.
patches and vascular grafts (3, 30, 32, 253). PHAs are also counts were significantly reduced in PHB-fed larvae com-
useful as stereoregular compounds which can serve as pared to the control larvae (3).
chiral precursors for the chemical synthesis of optically
active compounds (32, 254). Such compounds are particu- (3) Denitrification in water and wastewater treatment
larly used to synthesize the micro- and nanosphere of
PHAs as biodegradable carriers or drug delivery systems A promising application of PHAs is as the solid sub-
for controlled release of therapeutics such as medicines, strate for denitrification of water and wastewater. This
hormones, insecticides and herbicides into systemic circu- type of denitrification, termed here “solid-phase denitri-
lation are receiving attention (30, 31, 253, 255, 256). fication”, has several advantages over the conventional
According to Sudesh et al., PHAs have the oil absorption system supplemented with liquid organic substrate.
and retention capacities which offer commercial appli- PHAs serve not only as constant sources of reducing
cation value in the cosmetics and skin care industries power for denitrification but also as solid matrices favor-
(257). Voinova et al. have investigated the possibility of able for development of microbial films (189). In addition,
use of PHAs as a biodegradable carrier for pesticides (α- in contrast to conventional processes, the use of PHAs
hexachlorocyclohexane and lindane) for targeted and has no potential risk of release of dissolved organic
controlled delivery of these compounds to soil. According carbon with the resultant deterioration of effluent
to their investigation, pesticides embedded in a PHA water quality. Denitrification processes using PHAs actu-
carrier are released gradually and slowly, without surges, ally give high rates of nitrogen removal (262). Due to its
as the polymer is degraded by the soil microflora (258). oil absorption capacities, PHB has been successfully
They are also used as osteosynthetic materials in the tested for removing lipid-soluble organic pollutants
stimulation of bone growth owing to their piezoelectric from water (257, 263).
70 MUHAMMADI ET AL.
(4) Miscellaneous applications groundwater sediments and engineered ecosystems

with fluctuating nutrient contents support the popu-
PHAs with long side chain hydroxyacids have been lation actively involved in PHA accumulation to meet
used in pressure-sensitive adhesive formulations. PHAs the metabolic energy requirements during starvation
can be used to produce dairy cream substitutes or period and this concept can be implemented industrially
flavor delivery agents in foods. Moreover, PHAs are also to reduce the cost of biopolymers commercially with sus-
used to produce fiber materials, such as non-woven tainable production processes (268). Therefore, it is econ-
fabrics (7). The PHAs are considered as a source for the omical to use naturally producing microorganism due to
synthesis of chiral compounds (enantiometrically pure safety and constant productivity rate, and there is great
chemicals) and are raw materials for the production of potential for producing PHAs in low-cost substrates,
paints (31, 32, 264). In addition to its range of material which can reduce PHAs production costs, because the
properties and resulting applications, PHAs promise to cost of substrate is the most important factor for PHAs
be a new source of small molecules. PHA can be hydro- production (269–271). Moreover, novel-constituent poly-
lyzed chemically, and the monomers can be converted to mers will reduce the unfavorable characteristics of PHA.
commercially attractive molecules such as β-hydroxy The cost of raw material itself accounts for 40–50% of
acids, 2-alkenoic acids, β-hydroxyalkanols, β-acyllac-
the total production cost. Recovery of polymer from

tones, β-amino acids and β-hydroxyacid esters (265). the biomass is a vital stage of the process. Large-scale
The last class of chemicals is currently receiving attention solvent extraction, although giving high recovery, is an
because of potential applications as biodegradable sol- expensive technique and involves large volumes of
vents (7). Besides helping to replace the existing solvents, solvent and high capital investment in the solvent recov-
β-hydroxy acid esters and derivatives are likely to find ery plant (26). From an economical point of view, the
growing use as “green solvents” similar to lactic acid price of the product ultimately depends on the substrate
esters. The conversion of hydroxy acids into crotonic (mainly carbon source) cost, PHA yield on the substrate
acids such as 1,3-butanediol, lactones, etc. would help and the efficiency of product formulation in the down-
improve the market value as they have an existing stream processing (31, 107). It is a prerequisite to standar-
market demand in thousands of tones (31). dize all the fermentation conditions, and physical and
chemical parameters for the successful implementation
of commercial PHA production systems (269–273).
Economical aspects
PHAs production costs can be reduced by several
One of the problems preventing the pilot-scale pro- means, including the use of cheap substrates, such as
duction, commercial application and wide use of PHA whey, favored in countries with important dairy indus-
as commodity plastics is its high production cost. High tries, or the enhancement of product yield, for example
production cost has hindered the use of PHAs, since by using recombinant E. coli (7, 35, 267, 274). Molasses,
their final price is considerably much higher than that from either sugarcane or beet, is one potential cheap
of petrochemical-based synthetic plastic materials (30, carbon source for PHA production. B. cereus grown in
40, 41, 158, 266, 267). Factors involved include the 1% (w/v) beet molasses produced P(3HB) concentration
product yield, complexity of the technology and hence of 0.1 g/l and a polymer content of 73.8%, whereas in
the capital cost of the plant, and the ease or difficulty 3% molasses P(3HB) was increased by 70% but the
of product separation. Consequently, selection of the polymer content was halved (275). Another sucrose-rich
organism and substrate can critically influence costs inexpensive substrate is sugarcane liquor, which was
(26, 267). For example, PHB from Gram-negative organ- used by Jiang et al. to produce P(3HB) using
isms are widely used in a variety of products but due P. fluorescens (276). In a 5-l batch bioreactor, Castilho
to endotoxin in the outer membrane lipopolysaccharides et al. obtained 22 g/l of P(3HB), at a 70% polymer
(LPS), Bacillus species which lack LPS are extensively used content (277). Santimano et al. reported a comparative
in biopharmaceutical applications (70) but compared to use of low-cost agro-industrial wastes (rice chaff, citrus
Gram-negative bacteria, Gram-positive bacteria were pulp, coconut oil cake, cotton seed cake, cane molasses,
mostly found to produce scl-PHA and at lower PHA con- bakery waste) for the production of PHA by Bacillus sp.
tents between about 2% and 50% CDM, that is why strain COL1/A6. The highest PHA content was obtained
Gram-positive bacteria have yet to be adopted for com- from hydrolyzed wafer residues (62.41 ± 1.04% of dry
mercial PHA production (58). Further, Koller et al. have cell wt.), followed by cane molasses (54.68 ± 1.36% of
suggested to link ecology with economy, because bac- dry cell wt.) and hydrolyzed citrus pulp 47.5 ± 1.01% of
teria residing at ecological niches like estuarine sedi- dry cell wt.) (278). Using organic matter from wastes
ments, marine microbial mats, rhizosphere, and wastewaters can be used as a substrate for the
production of PHB, with combined advantages of redu- from fatty acids, while E. coli grows poorly on fatty
cing disposal cost and production of value-added pro- acid culture medium (289, 290). Therefore, application
ducts. A hydrolysis step is commonly needed before of E. coli as the host to produce PHA has not been
inoculation with pure cultures (279, 280). However, so the best option.
far, only low PHB content and productivity were At present, the application of defined co-cultures for
achieved from waste products (264). Since, the harsh PHA production is still in its infancy. For single-stage
conditions like nutrient limitation and stress are necess- co-culture fermentation systems, one of the main chal-
ary to trigger PHA production and intracellular accumu- lenges is providing cultivation parameters for efficient
lation, therefore, environmental parameters such as pH and effective bioconversion of carbon substrates into
and temperature have significant effects on microbial PHA. Parameters such as inoculum concentration, dis-
growth and PHA production (281). Further, there is diversity solved oxygen, pH, temperature, cultivation time,
among bacteria from different ecological habitats in their carbon and nutrients feed rate, and secondary metab-
PHA production, modes of growth, type of microorganisms olites production rate would need to be fine-tuned in
and media ingredients. But, the most of standardization order to maximize the bioconversion process. Another
studies on PHA-producing bacteria, including Gram-nega- challenge is the harvesting and separation of PHA-con-
tive and Gram-positive species, have reported the taining biomass from non-PHA-containing biomass, par-
optimum pH 7 and incubation at 30–37°C for maximum ticularly for co-cultures comprising PHA-accumulating
yield (282–287). and non-PHA-forming microorganisms as the presence
Additionally, the biochemical and metabolic activities of non-PHA-containing biomass would increase the
including the ability to utilize different carbon and nitro- extraction cost of PHA. Compared to single-stage fer-
gen sources, and hydrolytic enzyme (amylase, lipase and mentation approach, two-stage fermentation approach
protease) activities of bacterial candidates were also may be more advantageous as it enables finer control
reported to play an important role in PHA synthesis. over cultivation parameters and harvesting of PHA-
Therefore, it is economical to use microbes which accumulating biomass. However, higher capital and
produce a variety of enzymes to solubilize waste compo- operation cost are associated with two-stage fermenta-
sites and cheap substrates. According to Sangkharak and tion system. Ultimately, the type of systems chosen
Prasertsan, bacteria belonging to genera Bacillus, Aero- would greatly depend on the microbial characteristics
monas sp. and Alcaligenes sp. had ability to produce of the co-culture as well as the economic viability of
high amounts (2–6.58 g/l) of PHAs and high levels of the bioprocess (58).
hydrolytic enzyme activities which may result in efficient The cost of PHA using the natural producer
utilization of municipal wastewater, palm oil mill effluent, A. eutrophus is US$15–30 per Kg (30, 291) which is 18
glycerol and molasses, and its conversion into PHAs with times more expensive than polypropylene. With recom-
a variety of PHA monomers (270). binant E. coli as a producer of PHA, price can be
To reduce the substrate cost, recombinant strains reduced to US$4 per Kg, which is close to other biode-
utilizing a cheap carbon source and corresponding fer- gradable plastic materials such as PLA and aliphatic poly-
mentation strategies have also been developed (177, esters. The commercially viable price should come to US
266). E. coli harboring phaABC and phaP of Azotobacter $3–5 per Kg (266). Although there are presently econ-
sp. is a suitable host as a heterologous expression back- omic disadvantages and limited uses of bacterial plastics,
ground for foreign genes that can be easily manipu- there is currently a high amount of research devoted to
lated and improved by means of recombinant DNA improve productivity, to reduce production costs, and,
methodologies. Also, high-cell-density cultivation strat- more importantly, to produce specific functionalized
egies for numerous E. coli strains are well established PHAs.
(41, 288). E. coli cells that accumulate large amounts
of PHB become fragile, facilitating the isolation and
Conclusion and future prospects
purification of the biopolymer, and the bacterium
does not express PHA-degrading enzymes (34, 110). The intracellular accumulation of PHAs as hydrophobic
However, there are at least two disadvantages in granules of energy/or carbon storage materials under
using E. coli. First, E. coli does not contain a PHA precur- stress conditions and its mobilization on return of con-
sor-providing pathway, so that a new pathway has to ditions for normal growth is a most widely spread
be constructed to provide precursors for PHA synthesis. phenomenon among bacterial flora. PHAs are degrad-
Second, the metabolic background is quite different able within host cells and also biologically degraded
between E. coli and native PHA production strains. under both aerobic and anaerobic conditions by natu-
Many PHA-producing strains can accumulate PHA rally existing microorganisms in natural environment
72 MUHAMMADI ET AL.
into carbon dioxide and water, which return to the Disclosure statement
environment. Bacteria produce a wide range of different
No potential conflict of interest was reported by the authors.
PHAs with varying monomer compositions depending
on the substrate specificities of PHA synthases, carbon
sources and the metabolic pathways. All PHAs extracted
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Green Chemistry Letters and Reviews

ISSN: 1751-8253 (Print) 1751-7192 (Online) Journal homepage: http://www.tandfonline.com/loi/tgcl20

Bacterial polyhydroxyalkanoates-eco-friendly next

Muhammadi, Shabina, Muhammad Afzal & Shafqat Hameed

To link to this article: http://dx.doi.org/10.1080/17518253.2015.1109715

© 2015 Taylor & Francis

Published online: 17 Nov 2015.

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

ABSTRACT ARTICLE HISTORY

Introduction PHAs are a class of polyesters of various hydroxycar-

CONTACT Muhammadi muhammadi12@yahoo.com

(Figure 2) (28, 45, 109). PHB can also be synthesized

Methods for extraction

sequenced (197, 200–202). Biochemical analysis of the

Hydrolytic degradation of PHAs is not evident and is

Marscheider-Weidemann bioplastic can have a short or degradable.

(4) Miscellaneous applications groundwater sediments and engineered ecosystems

the total production cost. Recovery of polymer from

duction cost which has restricted its potential appli-

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