Microbiological Research 192 (2016) 271–282

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Bacterial polyhydroxyalkanoates: Still fabulous?

Justyna Możejko-Ciesielska ∗ , Robert Kiewisz
Department of Microbiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Bacterial polyhydroxyalkanoates (PHA) are polyesters accumulated as carbon and energy storage materi-
Received 20 March 2016 als under limited growth conditions in the presence of excess carbon sources. They have been developed
Received in revised form 19 July 2016 as biomaterials with unique properties for the past many years being considered as a potential substitute
Accepted 28 July 2016
for conventional non-degradable plastics. Due to the increasing concern towards global climate change,
Available online 7 August 2016
depleting petroleum resource and problems with an utilization of a growing number of synthetic plas-
tics, PHAs have gained much more attention from industry and research. These environmentally friendly
Keywords:
microbial polymers have great potential in biomedical, agricultural, and industrial applications. How-
Polyhydroxyalkanoates
Bacterial polyesters
ever, their production on a large scale is still limited. This paper describes the backgrounds of PHAs and
Biopolymers discussed the current state of knowledge on the polyhydroxyalkanoates. Ability of bacteria to convert
Bacteria different carbon sources to PHAs, the opportunities and challenges of their introduction to global market
as valuable renewable products have been also discussed.
© 2016 Elsevier GmbH. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
2. PHAs structure and synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
3. PHAs properties and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4. Fermentation processes using wild type and recombinant bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
5. Problems and challenges in PHAs production on an industrial scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

1. Introduction become problematic. First of all the accumulation of plastic waste

in the environment is commonly known world-wide issue. Post-
Synthetic plastics have comprehensive features of strength, consumer plastics waste ended up in the waste upstream resulting
lightness and durability. Even being non-biodegradable, they have in a significant burden on solid waste management. Moreover, con-
become an important material to improve the quality of human life cern over the rapid depletion of fossil fuels has created a renewed
replacing commodity like glasses or paper in packaging. According interest to develop alternative processes to produce biologically
to the latest report published by the Associations of Plastic Manu- derived polymers that will meet human needs. It is predicted that
facturers (Plastics Europe, 2015) with continuous growth of plastics the global capacity of bio-based polymers production will triple
production for more than 50 years, global production in 2013 rose from 5.1 million tonnes in 2013 to 17 million tonnes in 2020
to 299 million tonnes, meaning a 3.9% increase compared to 2012. In (Aeschelmann and Carus, 2015). According to the Institute for Bio-
spite of many possible applications, the usage of petroleum plastics plastics and Biocomposites production of biopolymers reached in
Europe 1.7 million tonnes and is expected to grow in 2018 to 6.7
milion tonnes (European Bioplastics, 2015).
∗ Corresponding author at: Department of Microbiology, Faculty of Biology and
Among the various groups of biopolymers, polyhydroxyalka-
Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A,
noates (PHAs) are the most well known. They are polyesters of
10-719 Olsztyn, Poland. hydroxyalkanoates (HAs) synthesized by numerous bacteria as
E-mail address: justyna.mozejko@uwm.edu.pl (J. Możejko-Ciesielska). carbon and energy storage compounds. For the first time they

http://dx.doi.org/10.1016/j.micres.2016.07.010
0944-5013/© 2016 Elsevier GmbH. All rights reserved.
272 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Fig. 1. Structure of polyhydroxyalkanoates.
n-varies from 1 to 4, x varies from 100 to 300 000, R-alkyl side chain.
have been observed in 1888 by Beijerincka. However, he could
not define their role and composition. In 1926 French researcher
Lemoigne obtained the poly-3-hydroxybutyric acid (P(3HB)) from
Bacillus megaterium (Lemoigne, 1926). In 1958 Macrae and Wilkin-
son proved that PHAs in bacterial cells play the role as the reserved
materials of carbon and energy, and they are collected only in an
increased carbon to nitrogen ratio. Beginning in the 1959s, many
companies have been set up to commercialize PHAs as environ-
mentally friendly bioplastics, fully independent from petroleum
sources. W.R. Grace & Company was the first company that tried to
produce the poly-3-hydroxybutyric acid (P(3HB)). However, low
synthesis efficiency and problems with PHAs purification forced
it to close the company. Beginning in 1980s, PHAs were pro-
duced under the trade names of BiopolTM , NodaxTM , BiocycleTM ,
BiomerTM , BioGreenTM . Nowadays, the PHAs market is very small.
Recently, the joint venture Telles, set up by Metabolix and ADM in
2006, aimed at big capacity but hardly sold any PHAs and subse-
quently collapsed in 2012. PHAs producers are optimistic and still
see potential in this biomaterials claiming that polyhydroxyalka-
noates are new generation of biopolymers and their market needs
time to develop. It is estimated that demand for PHAs will grow
tenfold by 2020 (Aeschelmann and Carus, 2015)
This paper reviews the key aspects of microbial polyhydrox-
yalkanoates production focused on the recent advances and
highlight the main problems and challenges in the PHAs production
on an industrial scale.
2. PHAs structure and synthesis
Polyhydroxyalkanaotes serve as water insoluble storage com-
pounds which are synthesized by microorganisms as granules
during times of environmental stress conditions. They are biosyn-
thesized under certain conditions, in carbon excess and limiting
concentration of essential growth nutrients such as nitrogen or
phosphate (Schlegel et al., 1961; Ciesielski et al., 2010a). When the
carbon source is exhausted, the collected biopolymers are depoly-
merised, and their degradation products can be used as a source of
carbon and energy materials (Anderson and Dawes, 1990; Ciesielski
et al., 2010b). For a long time PHAs had been an example of biopoly-
mers that was accumulated only in the cytoplasm of bacterial cells.
However, Sabirova et al. (2006) confirmed fascinating information
that PHAs can be also deposited extracellularly by genetically mod-
ified Alcanivorax borkumensis SK2.
Polyhydroxyalkanoates are a class of linear polyesters con-
sisting of hydroxy acid monomers (HA) connected together by
an ester bond (Fig. 1). This bonds is produced by connecting
the carboxylic group of a monomer with the hydroxyl group of
a neighbouring one (Philip et al., 2007). Depending upon the
number of carbon atoms in the monomers, PHAs are classified
mainly into two distinct groups: scl-PHAs (short chain length
PHAs) and mcl-PHAs (medium chain length PHAs). Scl-PHAs
consists of 3–5 carbon atoms and are synthesized by a wide range
of bacteria such as Cupriavidus necator. Mcl-PHAs are composed
of monomers having 6–14 carbon atoms and are accumulated
mainly by Pseudomonas species. Moreover, bacteria are able
to produce copolymers when mixed substrates are used. The
bacteria convert the carbon sources into scl-copolymers e.g.
poly(3-hydroxybutyrate-co-3-hydroxyvalerate (P(3HB-co-3HV))
or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-
4HB)) and mcl-copolymers e.g. poly(3-hydroxyhexanoate-co-3
hydroxyoctanoate) (P(3HHx-co-3HO)). There are also scl-mcl-
copolymers which consists of scl- and mcl-monomers e.g.
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-
3HHx). In addition, Pederson et al. (2006) determined that when
carbon sources are alternated overtime during bacterial fermenta-
tion process, microorganisms synthesize PHAs block copolymers.
The structural composition of PHAs polymers depends on the car-
bon compound supplied as the growth substrate and the bacterial
strain used. The side chain can be saturated or not, and can possess
branched, aromatic, halogenated, and even epoxidized monomers.
For example, PHAs polymers composed of a bromide and aromatic
group were extracted from Pseudomonas putida (Fritzsche et al.,
1990). Furthermore, chemical modifications of the PHAs side
chains can be used to introduce the desired functional group into
natural PHAs influencing the material properties of the polymers
to a great extent (Hartmann et al., 2006). Furthermore, PHAs
research and development enabled to precisely design and isolate
PHA homopolymers, random and block copolymers, functional
PHAs using ␤-oxidation-deleted Pseudomonas putida (Wang et al.,
2011).
However, PHA synthase plays a crucial role in the PHA poly-
merization process using the (R)-3-hydroxyacyl-CoA as a substrate
for PHAs synthesis. Based on the constituent subunits, amino
acid sequence and substrate specifity, polyester synthases can be
divided into four classes. It is known that class I, III and IV poly-
merize short-chain-length (scl) monomers (C3 –C5 ), whereas class
II PHA synthase utilizes medium-chain-length (mcl) monomers
(C6 –C14 ). Class I (e.g. in Ralstonia eutropha) and class II (e.g. in Pseu-
domonas putida) PHA synthases consist of one subunit (PhaC) with
molecular masses between 61 kDa and 73 kDa (Qi and Rehm, 2001).
Whereas, class III (e.g. Allochromatium vinosum) and class IV (e.g.
Bacillus megaterium) require two types of subunits for their activ-
ity, PhaC (40.3 kDa) and PhaE (20 or 40 kDa), PhaC (41.5 kDa) and
PhaR (22 kDa), respectively. The diversity among the class I PHAs
synthases was investigated suggesting that this type shows more
diverse enzymological properties. However, the data indicate that
PHA synthases from all classes have a similar topology. Interest-
ingly, class I and II differed at positions 100–130 or 80–110 (Rehm,
2003). It was confirmed that PhaE and PhaR are involved in PHAs
polymerization, however their exact role is still unknown. Further-
more, PhaCR subunits of class IV can be categorized into two groups,
Bacillus cereus and Bacillus megaterium groups. Recently, Tomizawa
et al. (2011) investigated that class IV PHA synthases from Bacil-
lus cereus decrease the molecular masses of P(3HB). Although PhaC
structures are still unclear, recently published results suggested
that the active site of class III synthases may be more polar than
that of class I synthases and both are sensitive to the modifications
in the alkyl side chain (Jia et al., 2016).
However, there are some exceptions to the above mentioned
classification confirming that PHA synthases show braod sub-
strate specificity. PHA synthases of Thiocapsa pfenningii belong
to class III having the substrate specifity to CoA thioesters of scl
and mcl 3-hydroxy fatty acids. PHA synthases found in Aeromonas
caviae show high similarity to Class I synthesizing a copolymer of
3-hydroxybutyrate and 3-hydroxyhexanoate. Furthermore, Pseu-
domonas sp. 61-3 synthases belongs to class II and polymerize PHAs
comprising a copolyester of 3-hydroxybutyrate and mcl-3-hydroxy
fatty acids. Even, class I PHA synthase of Ralstonia eutropha catal-
yses synthesis of medium-chain-length 3-hydroxy fatty acid-CoA
thioesters (Rehm, 2003).
 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Fig. 2. PHAs biosynthesis pathways.
Precursors for PHAs synthesis are thought to be derived from
fatty acid de novo biosynthesis when the microorganism is grown
on unrelated carbon sources, such as glucose, gluconate or acetate
and ␤-oxidation when the microorganism is grown on related car-
bon sources, such as fatty acids (Huijberts et al., 1992). In the first
case, the resulting PHA composition depends on the carbon source,
whereas in the second case, no relationship between the carbohy-
drates used as carbon sources and the resulting PHA composition
exists (Fig. 2). In the scl-PHAs biosynthesis pathway a key role play
three enzymes, ␤-ketothiolase, acetoacetyl-CoA reductase and PHA
synthase encoded by phaA, phaB and phaC, respectively. This path-
way was found in Cupriavidus necator, Aeromonas hydrophila or
Pseudomonas stutzeri. There are three kinds of known pathways
associated with mcl-PHAs synthesis. In the first pathway carbon
source is metabolized via ␤-oxidation to produce 3-hydroxyacyl-
CoA. The current supposition is that the (R)-specific enoyl-CoA
hydratase PhaJ is an enzyme that could potentially provide 3-
hydroxyacyl-CoA precursors for PHA synthesis derived via this
pathway when the organism is grown on fatty acids (Fiedler
et al., 2002). In the second pathway non-related substrates gen-
erates 3-hydroxyacyl-CoA precursors. The third one is a chain
elongation reaction, in which acetyl-CoA moieties are extended
to 3-hydroxyacyl-CoA. For PHAs production from unrelated car-
bon sources, a protein PhaG was identified to be a key link
between fatty acid biosynthesis and mcl-PHA biosynthesis. PhaG
was first reported as a 3-hydroxyacyl-acyl carrier protein (ACP)-
CoA transferase and was thought to transfer the 3-hydroxyacyl
group from the ACP moiety to the CoA moiety (Rehm et al.,
2001). However, the results of Wang et al. (2012) suggest that
PhaG functions as a 3-hydroxyacyl-ACP thioesterase to produce
3-hydroxy fatty acids. Therefore, at least one 3-hydroxyacyl-CoA
ligase is hypothesized to be necessary to provide the PHA pre-
cursor (R)-3-hydroxyacyl-CoA. Furthermore, Chohan and Copeland
(1998) revealed that some bacteria could use NADH-dependent
acetoacetylo-CoA reductase to oxidize (S)-3-hydroxybutyryl-CoA.
273
It was also reported that Clostridium kluyveri could involve succinic
semialdehyde dehydrogenase (SucD), 4-hydroxybutyrate dehy-
drogenase (4hbD) and 4-hydroxybutyrate-CoA:CoA transferase
(OrfZ) to generate 4-hydroxybutyryl-CoA for polymerizing 4-
hydroxybutyrate (Valentin and Dennis, 1997). Some bacteria are
able to engage lactonase and hydroxyacyl-CoA synthase to form
4,5-alkanolactone into 4,5-hydroxyacyl-CoA for polyhydroxyalka-
noates synthesis. Xie and Chen (2008) found that Aeomonas
hydrophila 4Ak4 uses pathway based on the putative alcohol dehy-
drogenase for 4-hydroxybutyrate synthesis.
Although the technological aspects of PHAs production,
including bacterial fermentation, isolation and physicochemical
characterization of the polymer have been extensively studied
during the past few decades, knowledge on the regulatory mech-
anisms at the molecular level is relatively limited especially in a
case of medium-chain-length PHAs synthesis. Six proteins directly
involved in the biosynthesis and degradation of mcl-PHAs have
been already characterized at the molecular level: two poly-
merases, PhaC1 and PhaC2; a depolymerase, PhaZ; two phasins,
PhaI and PhaF; regulatory protein, PhaD and PhaG. PhaC1, PhaC2,
PhaZ, and PhaD are encoded by genes transcribed in the same direc-
tion, whereas PhaF and PhaI are transcribed in the opposite way
(Sandoval et al., 2007). PhaG encodes transacylase, which is essen-
tial for PHAs synthesis from non-related carbon sources and is not
co-localised with the PHA biosynthesis gene cluster (Wang et al.,
2012). It is well known that phaC1 and phaC2 genes are responsi-
ble for mcl-PHAs synthesis, and that the phaZ gene encodes a PHA
depolymerase that allows the hydrolysis of accumulated biopoly-
mers and the products can then be fed into central metabolism.
PHAs granules are covered with a single layer of phospholipids on
the surface of which is found PHA polymerase, PHA depolymerase,
the phasins PhaI and PhaF, and other proteins related to monomer
synthesis. PhaD is the only known protein expressed by the pha
cluster that is not found on the PHA granule surface. PhaF and PhaI
play a structural role in granule formation. They serve as an inter-
274 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Table 1
Comparison of properties of scl-PHAs, mcl-PHAs and their copolymers with polypropylene.
Homopolymer Homopolymer
scl-PHAs mcl-PHAs
Melting temperature (◦ C) 179 80
Glass transition temperature (◦ C) 4 −40
Young’s modulus (GPa) 3.5 – 0.7–2.9
Elongation to break (%) 40 300
Tensile strength (Mpa) 5 20
Scl-PHAs – short-chain-length polyhydroxyalkanaotes; mcl-PHAs – medium-chain-length polyhydroxyalkanoates; P(3HB-co-6%3HD) – poly(3-hydroxybutyrate-co-6% 3-
hydroxydecanoate).
face between the granule and the cytoplasm, and are believed to
facilitate a segregation of the granules during cell division (Galán
et al., 2011). Recently, Obeso et al. (2015) reported the role played
by phasin PhaF in the control of bacterial shape and size. Sierro
(2005) revealed that the activity of phaF gene is reinforced by the
presence of PhaD and higher amounts of PhaF and PhaI phasins are
produced. These authors have also investigated that PhaI appears
to block the Pc1, Pc2 i Pi promoters driven expression, whereas
PhaD is the inducer of both phasins (PhaF and PhaI). The numerous
affects of Pha proteins show that the regulation of the PHA accu-
mulation process seems to complex. It is likely that this complexity
does not only result from the effects of PhaD, PhaF or PhaI but also
from other proteins affecting the activity of promoters of the pha
gene cluster.
3. PHAs properties and applications
Polyhydroxyalkanoates have attracted much attention in recent
years because of their similarities in properties with petrochem-
ical polymers like polypropylene or polystyrene. The properties
of mcl-PHAs are quite different compared to scl-PHAs (Table 1).
However, the features of PHAs are dependent on bacterial host
of this biopolymer and the fermentation conditions used towards
their production. Scl-PHAs are highly crystalline (typically 55–80%)
being a completely stereoregular polyester with high melting and
low glass transition temperatures. The high crystallinity makes
them relatively stiff and brittle. The melting point (Tm ) ranges from
173 to 180 ◦ C whereas the glass transition temperature (Tg ) lies
between 5 and 9 ◦ C. Glass and melting transition temperatures are
Fig. 3. Potential applications of PHAs.
copolymer Copolymer Polypropylene
P(3HB-co-3HV) P(3HB-co-
6%3HD)
137–170 130 176
10 to −6 −8 −10
– 1.7
30–38 680 38
up to 690 17 400
important parameters relative to in-service applications of PHAs.
They define lower and upper temperature limits for numerous
applications. The scl-copolymers was reported to have better prop-
erties. Short-chain-length copolymers such as P(3HB-co-3HV) are
known as more desirable than scl-homopolymers because their
melting point is much lower, and they are less crystalline, easier to
mold and tougher (Luzier, 1992). Moreover, these thermomechan-
ical properties can be widely varied by the percentage composition
of P(3HB-co-3HV) (Braunegg et al., 1998).
Mcl-PHAs act as elastomers within a very narrow temperature
range due to their low melting temperature. Their melting point
ranges from 39 to 61 ◦ C and are strongly dependent on the thermal
history of the material. Their glass transition temperature is usually
below room temperature, ranging from −43 to −25 ◦ C, and they are
about 25% crystalline. These characteristics make mcl-PHAs more
flexible and elastic materials than scl-PHAs (Ciesielski et al., 2015).
For the reason that PHAs have useful properties such as:
biodegradability, thermoplasticity, biocompatibility, non-toxicity,
they are considered a replacement for petrochemical polymers. In
recent years companies have been interested in the use of PHAs in
packaging, biomedical, agricultural applications (Fig. 3). It is well
known that PHAs were initially used for manufacturing cosmetic
containers such as shampoo bottles (Hocking and Marchessault,
1994), moisture barriers in sanitary products (Lauzier et al., 1993)
or pure chemicals as raw materials for the production of latex paints
(Scholz, 2000). Also, they can be used as carriers for long term
release of herbicides or insecticides (Galego et al., 2000). Ultra-
high molecular weight of PHAs can be useful to produce ultrastrong
fibers for fisheries industry (Bugnicourt et al., 2014). However,
 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
in view of their properties PHAs are promising materials espe-
cially in a biomedical field. Especially, P(3HB) homopolymer and
copolyester of P(3HB-co-3HV) are the most studied PHAs for med-
ical applications. In recent years they are considered as materials
in the fabrication of cardiovascular products (heart valves, stents,
vascular grafts), in drug delivery system (tablets, microcarriers for
anticancer therapy), in wound management (sutures, nerve cuffs,
swabs, straples), in orthopaedy (bone plates, spinal cages). High
immunotolerance, low toxicity, and biodegradability are the ben-
efits associated with polyhydroxyalkanoates in tissue engineering.
In the study carried out by Lomas et al. (2013) the suitability of
P(3HB-co-3HHx) and collagen hybrid constructs with a view to cre-
ating a future tissue-engineered product was investigated. Here,
human embryonic stem cells (hESCs), spontaneously differenti-
ated hESCs (SDhESCs), and mesenchymal stem cells (hMSCs) were
tested. It was shown that undifferentiated hESCs are not viable
after 20 days of culture within a PHBHHx/collagen gel hybrid scaf-
fold, whereas hMSCs and SDhESCs demonstrated good viability
over the long-term with an overall lack of stimulus independent
differentiation. Also, unmodified/raw mcl-PHAs, without any co-
polymerization, was tested as a novel scaffold for tissue engineering
applications using mesenchymal stromal cell (MSCs). The data
revealed that this biomaterial supports cell attachment what was
further confirmed through cell viability assay (Naveen et al., 2015).
However, to improve properties and to match biological
requirements of human tissues, PHAs have been blended with
hydroxyapatite (HA) and polymers such as gelatin, silk, collagen.
Composite nano scaffolds consisting of PHAs and ceramics or nat-
ural polymers seem to be more efficient substrates for bone tissue
(Goonoo et al., 2016). Lu et al. (2013) evaluated the effect of HA and
orientation of fibers on cell proliferation and differentiation in vitro.
Mesenchymal stem cells (MSCs) were seeded on scaffolds made
from blends of P(3HB-co-HV) and hydroxyapatite. The results con-
firmed that the MSCs attached and proliferated more favorably on
random-oriented P(3HB-co-3HV) nanofibrous meshes without HA.
After one, two and four weeks of cell seeding, osteogenic markers
including alkaline phosphate (ALP), osteocalcin (OCN), and min-
eralized matrix deposits were detected, respectively. The results
thus confirmed that the introduction of HA could induce MSCs
to differentiate into osteoblasts. Moreover, 3D P(3HB-co-3HV)/HA
scaffolds made from aligned and random-oriented nanofibers were
implanted into critical-sized rabbit radius defects and exhibited
significant effects on the repair of critical bone defects. Lizarraga-
Valderrama et al. (2015) confirmed that PHAs blend films with
varying ratios of
poly(3-hydroxyoctanoate)/poly(3-hydroxybutyrate)
(P(3HO)/P(3HB)) can serve as base material for the manufac-
ture of nerve guidance conduits. In the conducted study the
NG108-15 neuronal cells were seeded on 25:75, 50:50, and 75:25
blend films of P(3HO)/P(3HB). Although all of the blends were
biocompatible, the 25:75 P(3HO)/P(3HB) blend supported the cells
growth and differentiation significantly better. The mechanical
properties of PHA blends correspond to the reported properties
of peripheral nerves.These results thus confirmed the fact that
P(3HO)/P(3HB) blends can be used as scaffolds for nerve tissue
engineering.
Polyhydroxyalknaotes are a promising biomaterials in phar-
macy. Recently, Chung et al. (2013) produced 3-hydroxyalkanoic
acids (3HA) by genetically modified Pseudomonas entomophila as
precious precursors for synthesis of value added chemicals includ-
ing pharmaceuticals, antibiotics, food additive, fragrances, and
vitamins. Ruth et al. (2007) shown that pure medium-chain-length
homopolymer, 3-hydroxyoctanoic acid (3HO), exhibits poten-
tial antimicrobial activities. Furthermore, Faveau et al. (2006)
demostrated that 3-hydroxyhexanoic acid (3HHx) can be used as
intermediate for synthesizing analogs of laulimalide that is an
275
anti-cancer chemical. In recent years polyhydroxyalkanoates have
gained much interest in research on the development of novel
drug-loaded nanoparticles for pharmaceutical industry. Recently,
polyhydroxyalkanoates have been claimed to provide a multi-
functional platform for the sustained release of drugs based on
microencapsulation technology. They have been demonstrated
for preparation of nanoparticles (NPs) that can be used in con-
trolled release of various drugs. Ceftiofur-loaded P(3HB-co-3HV)
microparticles (PHBV-CEF) have been formulated and successfully
developed as a drug delivery system to treat infectious diseases.
It was confirmed that the PHBV-CEF particles have a potential
application for improving the treatment of infectious diseases in
livestock compared to current practices in the breeding industry.
The in vitro release experiment showed a sustained release profile
of ceftiofur during 7 days, and the antimicrobial test demonstrated
that ceftiofur kept its antibiotic activity after the encapsulation
process, and the cell proliferation assay showed a very low cyto-
toxicity of PHBV-CEF on Hep-G2 cells with an IC50 > 10 mg/mL
(Vilos et al., 2012). Also, a model anticancer drug ellipticine was
successfully encapsulated in P(3HB-co-3HV) confirming a poten-
tial of this system to increase the cytotoxic effect of ellipticine by
increasing its bioavailibility (Masood et al., 2013). Furthermore,
Pramual et al. (2016) suggested that nanoparticles of the same
copolymer have the ability as carriers of a hydrophobic photosen-
sitizer for the treatment of cancers. The authors showed that the
pTHPP-loaded PHAs nanoparticles exhibited high photocytotoxic-
ity against HT-29 cancer cells and may be an attractive biopolymer
in photodynamic therapy and can be an alternative to other con-
ventional cancer treatments. It was speculated that PHAs could
be used as an alternative to conventional drugs with decreased
potential for bacterial resistance development. Dinjaski et al. (2014)
reported that the recently synthesized a naturally functionalized
bacterial polyhydroxyalkanoates (PHACOS) inhibited the growth
of methicillin-resistant Staphylococcus aureus. The authors revealed
that the more antimicrobial thioester ligands linked to the polyester
back bone, the more effective biocide activity of PHACOS.
4. Fermentation processes using wild type and
recombinant bacteria
So far, poly-3-hydrobutyric acid (P(3HB), poly(3-
hydroxybutyrate-co-3-hydroxyvalerate (P(3HB-co-3HV)) or
poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)),
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-
3HHx) and medium-chain-length PHAs are produced on a large
scale. Table 2 presents wild type and recombinant bacteria used
for pilot and large scale production of polyhydroxyalkanoates.
It is well known that the most important factor to effectively
produce PHAs is a biopolymer producer. In recent years, hun-
dreds of microorganisms possessing the ability to produce different
types of PHAs have been studied. They have been recognized in
a wide range of gram-positive and gram-negative bacteria and
in archea. Most of them can not be considered as the hosts in
the industrial production because their ability to synthesize PHAs
is insufficient. One of the bacterial species that can accumulate
PHAs in satisfactory amounts is Cupriavidus necator. It is a com-
mon species that synthesized P(3HB) in the process regulated
by three enzymes: ␤-ketatiolaza (PhaA), acetoacetyl-CoA reduc-
tase NADPH depended (PhaB), and PHA polymerases (PhaC) (Chen,
2010). Cupriavidus necator was the first species that was used for
the production of P(3HB-co-3HV) copolymer by Imperial Chemi-
cal Industries under the trade name BiopolTM . The fermentation
process with this bacteria is one of the most effective. Cupriavidus
necator was able to synthesize P(3HB) in the amount of 76% in
the fed-batch fermentation process maintaining glucose concen-
276 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Table 2
Commercialized PHAs with their fermentation data, trade mark and manufacturers (Chen, 2009; Bugnicourt et al., 2014).
Manufacturer Trade mark Strain
Biomer, Germany Biomer Alcaligenes latus
PHB Industrial S.A., Brazil Biocycle Bacillus sp.
Kaneka Corporation, Japan Kaneka PHBH Ralstonia eutropha
Metabolix, USA Mirel Ralstonia eutropha
Tianan Biologic Material Enmat Ralstonia Entropha
Co., Ltd. Ningbo, China
Tianjin GreenBio Materials, GreenBio Escherichia coli
China
Jiang Su Nan Tian, China Jiangsu Nantian Escherichia coli
ETH, Switzerland PHA Pseudomonas putida
phaCAc – PHA synthase gene phaC from Aeromonas caviae; phbCAB – PHB synthesis genes encoding ␤-ketothiolase, acetyloacetylo-CoA reductase and PHB synthase; vgb –
gene encoding Vitreoscilla hemoglobin.
trations at 10–20 g/L during 50 h of the culture (Kim et al., 1994).
The same authors obtained about 110 g/L of P(3HB-co-3HV) copoly-
mer using the same culture strategy. Recently, oleic acid as the main
carbon source and alcohol-based 3HV precursor, 1-pentanol, have
been used to produce 66% P(3HB-co-3HV) of CDW from newly iso-
lated strain of Cupriavidus sp. Furthermore, it was shown that the
consideration of oxygen uptake rate (OUR) and oxygen transfer
rate (OTR) seems to be a preeminent strategy in improving this
copolymer production. By comparing with the batch fermentation,
fed-batch fermentation has resulted in 200% increment in produc-
tivity (Shantini et al., 2015). Higher P(3HB-co-3HV) copolyester
concentration
(83.3% of CDW) was obtained by applying pH-stat fed-batch cul-
ture technique, using organic acids as the main carbon source in
combination with an additional pO2 -dependent feed for deliver-
ing organic acid salts (Huschner et al., 2015). Moreover, for the
first time, P(3HB) has been recently successfully produced under
autotrophic growth conditions by Cupriavidus eutrophus B-1064
(Volova et al., 2013). This strain was able to synthesize up to 85% of
CDW grown on the CO2 + O2 + H2 gaseous mixture with CO2 as the
main carbon substrate and organic precursor substrates added in
small amounts.
Also, Burkholderia sacchari has been reported as a potential
PHAs producer. Mendonça et al. (2014) evaluated in the cultiva-
tion of this species thirty different carbon sources as cosubstrates
to incorporate different monomers into PHAs. The highest P(3HB)
concentration (51.5% of CDW) was observed in the culture sup-
plemented with hexanoic acid. The authors for the first time
demonstrated the modulation of the composition of P(3HB-co-
3HHx) using mixtures of carbohydrate and hexanoic acid as carbon
sources. It was also revealed that bacteria belong to Methylobac-
terium species synthesized scl-PHAs from the cheap substrate,
methanol. Studies applying cultivations of Methylobacterium sp.
GW2 using this carbon source as a nutrient supplement have
demonstrated that this bacteria was capable to accumulate up to
40% of P(3HB) (Yezza et al., 2006). These authors reported that
this strain produced copolyester poly-3-hydroxybutyrate-poly-3-
hydroxyvalerate when valeric acid was supplied as an auxiliary
carbon source to methanol. After 66 h of shake-flasks cultivation,
a copolymer content of 30% (w/w) was achieved with a P(3HB) to
P(3HV) ratio of 1:2.
Several researchers were focused on the PHAs production in the
cultivations supplemented with oily substrates. Chee et al. (2010)
demonstrated that Burkholderia sp. USM (JCM15050) was identi-
fied as an efficient P(3HB) producer and was able to synthesize
70% of P(3HB) using crude palm kernel oil. Promising results have
been published by Chen et al. (2014) in which newly isolated strain
belong to Pseudomonas mosselli cultivated on palm kernel and soy-
bean oil was able to accumulate mcl-PHAs in the concentration of
DNA manipulation Carbon source PHA type Cell dry weight (g/L)
final content (%
CDW)
No sucrose P(3HB), >75% >60
No sugar cane P(3HB), >90% >90
phaCAc fatty acids P(3HB-co-HHx), >80% >100
No glucose P(3HB-co-4HB), >75% >100
No glucose + propionate P(3HB-co-3HV), >75% >160
phbCAB glucose + 1,4-butanodiol P(3HB-co-4HB), >75% >100
phbCAB + vgb glucose P(3HB), >80% >150
No Fatty acids mcl-PHAs, about 60% about 45
50% of CDW. Furthermore, it was revealed that glycerol could be
successfully utilised for the production of PHAs. Hermann-Krauss
et al. (2013) applying pure glycerol in the cultivation of Haloferax
mediterranei obtained 75.4% of P(3HB-co-3HV) copolyester. Fur-
thermore, supplying??-butyrolactone as 4-hydroxybutyrate (4HB)
precursor this bacteria was able to biosynthesized P(3HB-co-3HV-
co-4HB) terpolyester synthesis in the same concentration. Ibrahim
and Steinbuchel (2009) employed Zobellella denitrificans strain
MW1 for the production of polyhydrxyalkanoates using glycerol
that resulted in the P(3HB) concentration at the level of 80.4%
CDW. Furthermore, it was evaluated that this bacterium is a good
producer of P(3HB-co-3HV) copolymer during sodium gluconate
co-feeding together with glycerol as the sole substrate.
Polyhydroxyalkanoates are expensive materials when com-
pared to synthetic polymers. To solve this problem, alternative
synthesis routes that employ cheaper carbon substrates are
required to decrease costs and make it feasible to cost-effectively
produce PHAs. Renewable carbon sources seems to be economi-
cally reasonable and excellent potential feedstocks for industrial
PHAs production. Several waste carbon sources were tested as
substrates for PHA production. Especially crude glycerol, the main
by-product of biodiesel manufacturing, has become a very attrac-
tive substrate for PHA production (Ciesielski et al., 2015). It is well
known that due to higher reduced carbon atoms in glycerol com-
pared to carbohydrates, bacterial cells using glycerol are in a more
reduced physiological state, stimulating intracellular polymer syn-
thesis (Hermann-Krauss et al., 2013). Accordingly to Cavalheiro
et al. (2009) DSM 545 strain of Cupriavidus necator is able to effec-
tively synthesized P(3HB) at the level of 50% of CDW with the
productivity of 1.1 g/L/h when waste glycerol was supplied as the
carbon source. This bacterium is capable to utilize other by-product
from biodiesel industry, rapeseed meal hydrolyzates. García et al.
(2014) conducting fed-batch fermentation obtained up to 55.6% of
P(3HB-co-3HV).
It has been also demonstrated that the application of other
waste oils could improve PHAs production. Waste rapeseed oil was
found to enhance P(3HB-co-3HV) copolymer production by adding
precursors such as propanol, propionate and valerate during cul-
tivation of Cupriavidus necator H16. Obruca (2010) demonstrated
that propanol improved biopolymer synthesis, up to 80% of CDW
was obtained. The feeding of valeric acid influenced positively on
3HV molar fraction resulting in a concentration of 18 mol%. Further-
more, Cupriavidus necator is able to incorporate 4-hydroxybutyrate
monomer to form P(3HB-co-4HB) copolymer grown on plant oils.
This bacterium was found to synthesize up 83% of P(3HB-co-4HB)
with 4HB molar fraction in the range from 6 to 10 mol% grow-
ing on soybean oil (Park and Kim, 2011). Also, the use of waste
plant oils seems to be profitable during the fermentations of Pseu-
domonas species. Możejko and Ciesielski (2013) reported that the
 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282 277

Table 3
Comparison of several PHAs biosynthesis processes in recombinant bacteria.

Strain Cultivation PHAs biosynthesis Type of PHAs Carbon sources Biomass PHA PHA yield References
mode genes source (g/L) content (%) (g/L)

Short-chain-length PHAs
Escherichia coli Fed-batch Cupriavidus necator P(3HB) Glycerol 41.9 63 26.4 Nikel et al. (2010)
K24KL
Escherichia coli Aerobic batch Azotobacter sp. P(3HB) Glucose 9.43 37.2 3.5 Almeida et al.
K24KP (FA8) (2010)
P(3HB) Glycerol 4.75 30.1 1.4
Escherichia coli Batch Azotobacter sp. P(3HB) Glycerol 12.23 27 3.3 Almeida et al.
K24KP (FA8) (2011)
Escherichia coli Batch Bacillus megaterium P(3HB) Glucose 9.3 80 7.4 Tomizawa et al.
JM109 NBRC15308 T (2011)
Bacillus cereus YB-4
Escherichia coli Batch Ralstonia eutropha P(3HP) Glucose 5.35 18.41 1.0 Meng et al. (2015)

Medium-chain-length PHAs
Escherichia coli Batch Pseudomonas mcl-PHA Molasses 4.05 75.5 3.06 Saranya and
sp.LDC-5 Shenbagarathai
(2011)
Escherichia coli Batch Pseudomonas mcl-PHA Glucose – 15 – Agnew et al. (2012)
aeruginosa PAO1

Copolymers
Escherichia coli Batch Comamonas P(3HB-co-3HV) Glucose 1.6 46.1 0.7 Yee et al. (2012)
JM109 sp. EB172
Burkholderia sp. Fed-batch Aeromonas caviae P(3HB-co-3HHx) Crude palm kernel 1.7 66 1.1 Chee et al. (2012)
USM (JCM 15050) oil
Cupriavidus necator Fed-batch Burkholderia sp. P(3HB-co-4HB) Crude palm kernel 2.4 66 1.6 Lau and Sudesh
USM (JCM 15050) oil (2012)
Shimwellia blatae Two step Ralstonia eutropha P(3HB-co-3HP) Glycerol 23.2 30.7 7.1 Sato et al. (2015)
fed-batch

Terpolymers
Cupriavidus necator Batch Aeromonas caviae P(3HB-co-3HV- Palm kernel oil 7.9 79 6.2 Bhubalan et al.
co-3HHx) (2008)
Cupriavidus necator Fed-batch Chromobacterium P(3HB-co-3HV- Sodium valerate 9.4 86 8.1 Bhubalan et al.
sp. USM2. co-3HHx) (2010)
Delftia acidovorans Batch Pseudomonas P(3HB-co-3HV- Lard – 39.33 – Romanelli et al.
DSM39 stutzeri BT3 co-4HB) (2014)

newly isolated strain Gl01 which belongs to Pseudomonas species
was capable of producing up to 48% of mcl-PHAs at 17 h of the cul-
tivation using saponified waste palm oil as the only carbon source.
High PHAs concentration has been reached by Możejko et al. (2011,
2012) during a cultivation of the above mentioned strain using
waste rapeseed oil that was supplied as the sole substrate into the
bioreactor.
Furthermore, the use of digestate liquor as a nutrient medium
has been determined to be an ideal substrate for PHAs produc-
tion by Cupriavidus necator. Passanha et al. (2013) showed that a
mixture of micro-filtered liquors from food waste and from wheat
feed digesters stimulated PHAs accumulation up to the level of
90% of CDW. Interestingly, the data revealed that ammonia, potas-
sium, magnesium, sulfate and phosphate provided in the digestate
liquors were vital for the initial growth of C. necator whereas copper,
iron and nickel have played a significant role in PHA accumulation.
Also, grass biomass has been tested for biopolymers production.
Davis et al. (2013) reported that Pseudomonas fluorescens 555 grow-
ing on hydrolysates (3.6 g/L of total sugar) as the sole carbon source
could produce 32.7% of PHAs.
However, there are still many potential polyhydroxyalkanoates
producing bacteria that remain undiscovered. In the past three
years, several researchers have attempted to isolate new PHAs pro-
ducing bacteria from various environments in order to produce
novel PHAs constituents with potential applications. Han et al.
(2014) isolated Massilia sp. UMI-21 from a green algae. The authors
described the accumulation by this species of about 45.5% P(3HB)
suggesting that this strain could produce higher PHAs productivity
when starch is used as the carbon source in the growth medium
instead of glucose. Also, Aquitela sp. USM4 from the unique envi-
ronment in Malaysia has been proposed as a novel PHAs producer
(Ng and Sudesh, 2016). Based on the obtained results, the authors
showed that wild-type Aquitalea sp. USM4 was able to accumulate
up to 1.5 g/L of poly(3-hydroxybutyrate). However, much higher
yield of PHAs was evaluated by Li et al. (2013) who tested a novel
facultative psychrotroph Pseudomonas mandelli CBS-1. Accordingly
to the authors, this strain is able to accumulate up to 22.3 g/L
of P(3HB) growing on sucrose under low temperature conditions
being more promising strain for industrial use.
In addition to the wild strains that produce PHAs, efforts have
been made to use genetically recombined strains to produce PHAs
(Table 3). For example, Escherichia coli does not normally produce
PHAs, but genetic engineering has made it possible to introduce
changes that enable the bacteria to produce biopolymers becom-
ing the most promising host microorganisms for PHAs. So, the
available comprehensive knowledge about its molecular genetics
and physiology made E. coli the pioneer organisms in the research
concerning PHAs biosynthesis. It was reported that genetically
modified E. coli is able to accumulate up to 90% of P(3HB) (Wang
et al., 2009). Schubert et al. (1988) established the first metabolic
pathway towards scl-PHAs synthesis by cloning the whole phb gene
operon into E. coli. After that, many research groups identified
and functionally expressed in E. coli PHAs biosynthesis genes (Li
et al., 2007). Genes responsible for PHAs biosynthesis from a num-
ber of microorganisms, such as Cupriavidus necator (Vandamme
and Coenye, 2004; Horng et al., 2010); Pseudomonas aeruginosa
(Lagenbach et al., 1997); Alcaligenes latus (Choi et al., 1998; Seo et al.,
2004); Thiocapsa pfennigii (Liu and Steinbüchel, 2000) and Strepto-
myces aureofaciens (Mahishi et al., 2003), have been introduced into
E. coli.
278 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Nikel et al. (2006) has proved that genetically modified E. coli
can utilize a wide range of waste substrates such as whey and
corn steep liquor as main carbon and nitrogen sources. After 24 h
of the cultivation process, a recombinant E. coli strain (K24K) that
bears the pha biosynthetic genes from Azotobacter sp. strain FA8,
accumulated P(3HB) up to 72.9% of CDW, reaching a volumetric
productivity of 2.13 g/L/h. Also, it was shown that by-products from
sugar cane industry could be considered as ideal substrate. Accod-
ingly to Saranya and Shenbagarathai (2011) a recombinant E.coli
harbouring phaC1 gene of Pseudomonas sp.LDC-5 was able to syn-
thesized PHAs at the level of 3.06 g/L on a molasses-based medium.
Recently, recombinant E. coli was used to enhanced PHAs produc-
tion from beechwood xylan (Salamanca-Cardona et al., 2014). The
authors successfully engineered a ␤-xylosidase and an endoxy-
lanase into the Escherichia coli strain to obtain a xylanolytic strain
which PHAs production yields increased up to 18-fold when com-
pared to xylan-based production demonstrating the importance of
the accumulation of biomass-derived sugars in the media prior
to their uptake by the cells. Furthermore, Wang et al. (2014)
demonstrated the ability of the filamentary recombinant E.coli to
accumulate more P(3HB-co-3HV) from glucose in comparison to a
wild type. The overexpression of sulA gene that inhibits the cell divi-
sion FtsZ ring assembly resulted in the larger E.coli cellular space
leading to higher PHAs concentrations up to 78% of CDW.
It was also reported that Cupriavidus necator seems to be an
ideal host for the PHA biosynthesis. Wong et al. (2012) demon-
strated that Cupriavidus necator Re2160/pCB113 harboring PHA
synthase gene from Ralstonia aetherivorans can produce P(3HB-co-
3HHx) copolymer on crude palm kernel oil. Recently, the ability
to convert waste plant oils into above mentioned copolymer has
been proven by Kamilah et al. (2012). This transformant was
found to produce up to 85% of CDW of P(3HB-co-3HHx) copolymer
grown on waste cooking oil resulting in the biomass concentra-
tion of 22.3 g/L. Crude palm kernel oil was successfully employed
for P(3HB-co-3HHx) synthesis by Cupriavidus necator H16 harbor-
ing Aeromonas caviae PHA synthase gene. This recombinant was
shown to accumulate 78% of above mentioned copolyester in the
culture supplemented with butyrate as a cosubstrate (Sato et al.,
2015). Moreover, Tsuge et al. (2009) working with C. necator har-
boring the wild-type Pseudomonas sp. 61-3 PHA synthase 1 grown
on soybean oil obtained successfully terpolymer P(3HB-co-3HO-
co-3HD). Recently, Ralstonia eutropha was developed to produce
PHAs consisting of 2-hydroxyacid monomers such as lactate and
2-hydroxyacids. Park et al. (2013) engineered R. eutropha by replac-
ing the R. eutropha phaC gene in the chromosome with either the R.
eutropha phaC S506G A510K gene or the Pseudomonas sp. MBEL6-
19 phaC1437 gene. The mutants were capable of synthesizing up to
48.7% of PHAs growing on glucose and lactate. Also, engineered Ral-
stonia eutropha strains containing deletions of the acetoacetyl-CoA
reductase (phaB) genes and replacing the native PHA synthase with
phaC2 from Rhodococcus aetherivorans I24 was reported to accumu-
late significant amounts of PHAs. Jeon et al. (2014) using, for the
first time, butyrate as the sole carbon source, demonstrated that
above mentioned recombinant was able to biosynthesize 40% of
P(3HB-co-3HV). The authors proved that PHAs copolyesters accu-
mulation is related to a marked decrease in the major precursor
supply pathway from acetoacetyl-CoA to 3-hydroxybutyl-CoA by
acetoacetyl-CoA reductase (PhaB).
Pathways containing engineered PHA synthases have been
proven to be useful for enhanced mcl-PHAs production with high-
productivity fermentations. Yuan et al. (2008) demonstrated for
the first time that medium-chain-length 3-hydroxyalkanoic acids
(mcl-3HA) could be produced using an overexpression of fadL
genes from P. putida KT2442 and fadD gene from Escherichia
coli MG1655 combined with the polyhydroxyalkanoates depoly-
merase gene phaZ from Pseudomonas stutzeri. P. putida KT2442
(pYZPst06) harboring all above mentioned genes achieved up to
5.8 g/L of extracellular 3HHx and 3HO monomers in the fed-
batch fermentation. Also, some strategies were applied to obtain
PHAs homopolymers at a high level which are required for pure
3HA production. It was shown that after deletion of fadB gene
(encoding 3-ketoacyl-CoA thiolase) and fadA genes (encoding
3-hydroxyacyl-CoA dehydrogenase) P. putida was able to synthe-
sized increased 3-hydroxydodecanoate (3HDD) monomer fraction
(Ouyang et al., 2007). The production of pure extracellular 3HA
can be also achieved by fadBA knockout mutant of P. putida
KT2442 harboring tesB gene when a growth medium was sup-
plemented with dodecanoic acid (Chung et al., 2013). ␤-oxidation
pathway inhibition in L48 strain of Pseudomonas entomophila
proved to be a good approach to produce mcl-PHAs homopoly-
mer. The highest 3HDD concentration was reached by Chung
et al. (2011) who have engineered P. entomophila by deleting
3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase and
acetyl-CoA acetyltransferase. It was demostrated that this mutant
is capable of producing 90% PHAs with 3HDD fraction at the level
of 98% comapred to all monomers. Zhuang et al. (2014) reported
that by engineering the reversed fatty acid ␤-oxidation cycle,
Escherichia coli was able to synthesisze mcl-PHAs directly from
glucose. The recombinant produced 6.62% CDW of mcl-PHAs het-
eropolymers.
Recently, Rhodospirillum rubrum was bioengineered and tested
towards the effect of systematically overexpressing of PHAs genes
on the biopolymers production (Jin and Nikolau, 2014). The result
confirmed that PHA polymerase gene indicates that phaC1 and
phaC2 are significant contributors to PHA productivity, whereas
phaC3 has little impact on biopolymer production. The mutants
biosynthesized up to 30% of PHAs, which is approximately 2.5-fold
higher than the wild type of this strain, indicating the feasi-
bility of using this approach to produce value added bio-based
products. Interestinlgy, also clustered regularly interspaced short
palindromic repeats interference (CRISPRi) was successfully used
to control P(3HB-co-4HB) copolyester synthesis. Lv et al. (2015)
constructed the pathway in Escherichia coli by the expression of
gene sad encoding E. coli succinate semi-aldehyde dehydrogenase
under CRIPSPi control. The created system allowed to obtained up
to 71.8% of P(3HB-co-3HV) copolymer. The results suggest that this
method is useful for multiple genes manipulation in E.coli and could
enhance the efficiency of PHAs synthesis.
5. Problems and challenges in PHAs production on an
industrial scale
Polyhydroxyalkanoates have gained much attention in recent
years both in research and industry. Without any doubts, they are
still valuable materials with versatile properties.The major draw-
back of these useful biopolymers is their high production cost. Singh
et al. (2009) estimated that they are about 15 times more expensive
than the petroleum derived polymers such as polypropylene. Thus,
researchers are trying to undertaken some crucial steps to improve
PHAs synthesis process and make it more feasible.
The final costs of PHAs mainly depend on the price of substrates
added as a carbon source for microbial growth. Furthermore, the
PHAs yield on carbon source, PHAs productivity and downstream
costs determine their introduction into global market (Choi and
Lee, 1999). The costs of carbon sources was reported to constitute
about 50% of the final production cost. Analysis and economical
evaluation confirmed that large-scale production
of PHAs from octane would cost about US$ 5–10 per kilogram
(Hazenberg and Witholt, 1997). Obruca (2010) calculated that the
theoretical price of PHAs produced in fed-batch mode using waste
materials could reach up to 3.51 Eur/kg PHAs, whereas synthetic
 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
alternatives like polypropylene and polyethylene cost 1.47 and 1.15
Eur/kg respectively. The conversion of raw products into biopoly-
mers seems to be a major point in the development of sustainable
biotechnological process and a solution to the cost limitations.
However, the bioprocesses costs depend on the price of feedstock
such as waste glycerol, vegetable oils starch, molasses. Further-
more, the down-stream processes influence on the price of the
whole process. The separation processes are especially challeng-
ing. As previously reported, final calculations was based on the
presumption that the price of the PHAs recovery after fermenta-
tion represent 30% of the overall production costs (Sun et al., 2007).
Several factors should be taken into considerations while selecting
the PHAs recovery method e.g. the PHAs producer, type and com-
position of biopolymers, product purity requirements, impact on
the PHAs properties (Koller et al., 2013). Several strategies of PHAs
isolation and purification have been investigated. Downstream pro-
cessing involves the microbial mass separation and supernatant
usually by filtration followed by chemical or enzymatic digestion,
precipitation or chromatography. The widely used strategy is PHAs
extraction with a solvent, such as chloroform or acetone. These sep-
aration methods are expensive involving extra costs for disposal
of waste solvents. Furthermore, PHAs are considered as environ-
mental friendly polymers, solvent extraction of PHAs from biomass
creates an additional harmful waste to the environment. Other
methods involving enzyme digestion are too expensive. Therefore,
there is a need to optimize extraction processes based on recyclable
solutions that do not create any health risk, for example bio-based
lactic acid esters can be regarded as a promising method.
It was reported that large amounts of PHAs in the bacterial cells
make them fragile and biopolymer could be efficiently purified by
treatment for example with mild alkaline solution (Koller et al.,
2013). The extraction processes are challenging especially when
PHAs are considered to be used in the biomedical field where a
high product purity is a crucial factor. Bearing in mind a PHAs
production scale ranging from kilograms to tonnes, manufacturing
and bacterial fermentations experience is essential and requires an
investment into capital equipment.
Recently published scientific data about a new secretion mecha-
nism of PHAs in Alcanivorax borkumensis give us an information that
PHAs could be deposit in the extracellular environment (Sabirova
et al., 2006). Without any doubts, this finding opens new ways
for scientists. The main disadvantage for intracellulary accumula-
tion of this biopolymers is the cytoplasm capacity and downstream
processing for PHAs recovery from biomass. Whereas, the extracel-
lularly PHAs production is not limited by the cell space and does
not need to apply any cell breakage procedures making this process
less complex.
One of the major limitation of the industrial production of PHAs
is to maintain the optimal bacterial growth conditions. To date,
most fermantations processes do not allow maximum synthesis
of PHAs granules at the end of the cultivation. The accumulation
process occurs too early and in consequences it lowers the produc-
tivity of the process (Lee and Na, 2013). On the other hand, many
strategies do not imply the optimization of the growth rate and
PHAs concentration at the same time. It is well-known that intra-
cellularly PHAs can occupy the cytosol as a result the biopolymers
accumulation at a high level could influence on the cell by inhibit-
ing their growth and affects their metabolism (Lee, 1996). In other
words, the PHAs productivity could decrease because of too fast
PHAs biosynthesis. It is well known that too slow PHAs accumula-
tion process could cause less PHAs concentration in inside the cells
by wasting carbon sources to bacterial growth.
Metabolically engineered strains seems to successfully synthe-
sized PHAs at the high level. The above described scientific data
clearly confirm that metabolic engineering of pathways involved in
PHAs biosynthesis could enhance the final productivity of this bio-
279
process. However, the instability of the introduced genes occurs
being one of the major drawback (Suriyamongkol et al., 2007).
Andreeßen et al. (2010) suggested to use chromosomal inser-
tion and plasmid addiction system. However, due to single-cell
transcriptional heterogeneity, hypersensitivity to concentration of
inducer and expensive inducible promoter and antibiotics, this sys-
tem can not be applied on a large scale (Leong et al., 2014). New
strategies have been developed but they are still uneconomic for
industrial applications. Furthermore, little is known about mech-
anisms of granule formation, regulatory mechanisms and links
between cells in high density populations. The full knowledge about
pha biosynthesis genes and a better understanding of their regula-
tions will be helpful to improve PHAs production and also to allow
obtain new, tailor-made polymers useful in many applications.
6. Conclusions
PHAs are still fabulous being one of the most potential biopoly-
mers that could replace conventional plastics in the future. Much
more effort should be done to improve microbial biomass and
PHAs formation substantially. Further research are essential to con-
duct high-cell-density cultivation of newly isolated strains that
have potential to be effective PHAs producers. On the other side,
industry demand the optimization of fermentation processes for
well-known microbes to improve biomass concentration and PHAs
content with desired properties. The productivity should be fur-
ther improved using advances strategies of cultivation control. To
decrease the costs of this bioproduct further work should be done
especially on high cell density cultivation method together with
PHAs bioproduction using cheap carbon sources and easier, non-
harmful isolation and purification of PHAs polymers.
It is anticipated that there will be a growing demand for bacterial
polymers with special properties that are tailored for applica-
tions in many fields. Using current knowledge and progress in
genetic engineering, and synthetic biology it seems to be possi-
ble to construct an ideal PHA producer which can biosynthesize
new biopolymers and could be used cost-effectively for industrial
production. But the development of genetically modified strains,
fermentations and recovery processes should be integrated. Being
able to control PHAs structures and forms (such as homopolymers,
block or random copolymers) their introduction in the high end
markets as high value added materials has a chance to come true.
Acknowledgements
This study was financially supported by the National Science
Center (Poland), Project No. 2014/15/D/NZ9/04255. The authors
have declared no conflict of interest.
References
Aeschelmann, F., Carus, M., 2015. Bio-based Building Blocks and Polymers in the
World – Capacities, Production and Applications: Status Quo and Trends
Toward, http://www.bio-based.eu/market study/media/files/15-05-13
Biobased Polymers and Building Blocks in the World-nova Booklet.pdf
(accessed 20.03.2016).
Agnew, D.E., Stevermer, A.K., Youngquist, J.T., Pfleger, B.F., 2012. Engineering
Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose.
Metab. Eng. 14, 705–713.
Almeida, A., Giordano, A.M., Nikel, P.I., Pettinari, M.J., 2010. Effects of aeration on
the synthesis of poly(3-Hydroxybutyrate) from glycerol and glucose in
recombinant Escherichia coli. Appl. Environ. Microbiol. 76, 2036–2040.
Almeida, A., Catone, M.V., Rhodius, V.A., Gross, C.A., Pettinari, M.J., 2011.
Unexpected stress-reducing effect of PhaP, a poly(3-hydroxybutyrate)
granule-associated protein, in Escherichia coli. Appl. Environ. Microbiol. 77,
6622–6629.
Anderson, A.J., Dawes, E.A., 1990. Occurrence, metabolism, metabolic role, and
industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54,
450–472.
280 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Andreeßen, B., Lange, A.B., Robenek, H., Steinbüchel, A., 2010. Conversion of
glycerol to poly(3-Hydroxypropionate) in recombinant Escherichia coli. Appl.
Environ. Microbiol. 76, 622–626.
Bhubalan, K., Lee, W.-H., Loo, C.-Y., Yamamoto, T., Tsuge, T., Doi, Y., Sudesh, K.,
2008. Controlled biosynthesis and characterization of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) from
mixtures of palm kernel oil and 3HV-precursors. Polym. Degrad. Stab. 93,
17–23.
Bhubalan, K., Rathi, D.N., Abe, H., Iwata, T., Sudesh, K., 2010. Improved synthesis of
P(3HB-co-3HV-co-3HHx) terpolymers by mutant Cupriavidus necator using the
PHA synthase gene of Chromobacterium sp. USM2 with high affinity towards
3HV. Polym. Degrad. Stab. 95, 1436–1442.
Braunegg, G., Lefebvre, G., Genser, K.F., 1998. Polyhydroxyalkanoates,
biopolyesters from renewable resources: physiological and engineering
aspects. J. Biotechnol. 65, 127–161.
Bugnicourt, E., Cinelli, P., Lazzeri, A., Alvarez, V., 2014. Polyhydroxyalkanoate
(PHA): review of synthesis, characteristics, processing and potential
applications in packaging. eXPRESS Polym. Lett. 8, 791–808.
Cavalheiro, J.M.B.T., de Almeida, M.C.M.D., Grandfils, C., da Fonseca, M.M.R., 2009.
Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste
glycerol. Process Biochem. 44, 509–515.
Chee, J.Y., Yoga, S.S., Lau, N.S., Ling, S.C., Abed, R.M., Sudesh, K., 2010. Bacterially
produced polyhydroxyalkanoate (PHA): converting renewable resources into
bioplastics. In: Méndez-Vilas, A. (Ed.), Current Research, Technology and
Education Topics in Applied Microbiology and Microbial Biotechnology, vol. 2.
Formatex, Badajoz, pp. 1395–1404.
Chee, J.Y., Lau, N.S., Samian, M.R., Tsuge, T., Sudesh, K., 2012. Expression of
Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia. sp
USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil
products. J. Appl. Microbiol. 112, 45–54.
Chen, Y.J., Huang, Y.C., Lee, C.Y., 2014. Production and characterization of
medium-chain-length polyhydroxyalkanoates by Pseudomonas mosselii TO7. J.
Biosci. Bioeng. 118, 145–152.
Chen, G.Q., 2009. A microbial polyhydroxyalkanoates (PHA) based bio- and
materials industry. Chem. Soc. Rev. 38, 2434–2446.
Chen, G.Q., 2010. Plastics completely synthesized by bacteria:
polyhydroxyalkanoates. In: Plastics from Bacteria. Springer, Berlin, Heidelberg,
pp. 17–37.
Chohan, S.N., Copeland, L., 1998. Acetoacetyl coenzyme a reductase and
polyhydroxybutyrate synthesis in Rhizobium (Cicer) sp. strain CC 1192. Appl.
Environ. Microbiol. 64, 2859–2863.
Choi, J., Lee, S.Y., 1999. Factors affecting the economics of polyhydroxyalkanoate
production by bacterial fermentation. Appl. Microbiol. Biotechnol. 51, 13–21.
Choi, J.I., Lee, S.Y., Han, K., 1998. Cloning of the Alcaligenes latus
polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced
production of Poly(3-hydroxybutyrate) in Escherichia coli. Appl. Environ.
Microbiol. 64, 489–4903.
Chung, A.L., Jin, H.L., Huang, L.J., Ye, H.M., Chen, J.C., Wu, Q., Chen, G.Q., 2011.
Biosynthesis and characterization of poly(3-hydroxydodecanoate) by
␤-oxidation inhibited mutant of Pseudomonas entomophila L48.
Biomacromolecules 12, 3559–3566.
Chung, A.L., Zeng, G.D., Jin, H.L., Wu, Q., Chen, J.C., Chen, G.Q., 2013. Production of
medium-chain-length 3-hydroxyalkanoic acids by ␤-oxidation and phaC
operon deleted Pseudomonas entomophila harboring thioesterase gene. Metab.
Eng. 17, 23–29.
Ciesielski, S., Możejko, J., Przybylek, G., 2010a. The influence of nitrogen limitation
on mcl-PHA synthesis by two newly isolated strains of Pseudomonas sp. J. Ind.
Microbiol. Biotechnol. 37, 511–520.
Ciesielski, S., Pokój, T., Klimiuk, E., 2010b. Cultivation-dependent and -independent
characterization of microbial community producing polyhydroxyalkanoates
from raw glycerol. J. Microb. Biot. 20, 853–861.
Ciesielski, S., Możejko, J., Pisutpaisal, N., 2015. Plant oils as promising substrates for
polyhydroxyalkanoates production. J. Cleaner Prod. 106, 408–421.
Davis, R., Kataria, R., Cerrone, F., Woods, T., Kenny, S., O’Donovan, A., Guzik, M.,
Shaikh, H., Duane, G., Gupta, V.K., Tuohy, M.G., Padamatti, R.B., Casey, E.,
O’Connor, K.E., 2013. Conversion of grass biomass into fermentable sugars and
its utilization for medium chain length polyhydroxyalkanoate (mcl-PHA)
production by Pseudomonas strains. Bioresour. Technol. 150, 202–209.
Dinjaski, N., Fernández-Gutiérrez, M., Selvam, S., Parra-Ruiz, F., Lehman, S.M., San
Román, J., García, E., García, J.L., García, A.J., Prieto, M.A., 2014. PHACOS, a
functionalized bacterial polyester with bactericidal activity against
methicillin-resistant Staphylococcus aureus. Biomaterials 35, 14–24.
European Bioplastics, 2015. Biosplastics Facts and Figs. 2015, http://en.european-
bioplastics.org/wpcontent/uploads/2013/publications/EuBP FactsFigures
bioplastics 2013.pdf (accessed 29.11.16).
Faveau, C., Mondon, M., Gesson, J.P., Mahnke, T., Gebhardt, S., Koert, U., 2006.
Synthetic studies on a phenyl-laulimalide analogue. Tetrahedron Lett. 47,
8305–8308.
Fiedler, S., Steinbuchel, A., Rehm, B.H., 2002. The role of the fatty acid
beta-oxidation multienzyme complex from Pseudomonas oleovorans in
polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA
operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P.
oleovorans and Pseudomonas putida. Arch. Microbiol. 178, 149–160.
Fritzsche, K., Lenz, R.W., Fuller, R.C., 1990. An unusual bacterial polyester with a
phenyl pendant group. Macromol. Chem. 191, 1957–1965.
Galán, B., Dinjaski, N., Maestro, B., de Eugenio, L.I., Escapa, I.F., Sanz, J.M., García, J.L.,
Prieto, M.A., 2011. Nucleoid-associated PhaF phasin drives intracellular
location and segregation of polyhydroxyalkanoate granules in Pseudomonas
putida KT2442. Mol. Microbiol. 79, 402–418.
Galego, N., Rozsa, C., Sánchez, R., Fung, J., Vázquez, A., Tomás, J.S., 2000.
Characterization and application of poly(␤-hydroxyalkanoates) family as
composite biomaterials. Polym. Test. 19, 485–492.
García, A., Segura, D., Espín, G., Galindo, E., Castillo, T., Pena, C., 2014. High
production of poly-␤-hydroxybutyrate (PHB) by an Azotobacter vinelandii
mutant altered in PHB regulation using a fed-batch fermentation process.
Biochem. Eng. J. 82, 117–123.
Goonoo, N., Bhaw-Luximon, A., Passanha, P., Esteves, S.R., Jhurry, D., 2016. Third
generation poly(hydroxyacid) composite scaffolds for tissue engineering. J.
Biomed. Mater. Res. B Appl. Biomater., http://dx.doi.org/10.1002/jbm.b.33674.
Han, X., Satoh, Y., Kuriki, Y., Seino, T., Fujita, S., Suda, T., Kobayashi, T., Tajima, K.,
2014. Polyhydroxyalkanoate production by a novel bacterium Massilia sp
UMI-21 isolated from seaweed, and molecular cloning of its
polyhydroxyalkanoate synthase gene. J. Biosci. Bioeng. 118, 514–519.
Hartmann, R., Hany, R., Pletscher, E., Ritter, A., Witholt, B., Zinn, M., 2006.
Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] by
Pseudomonas putida GPo1: batch versus chemostat production. Biotechnol.
Bioeng. 93, 737–746.
Hazenberg, W., Witholt, B., 1997. Efficient production of medium chain-length
poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: economic
considerations. Appl. Microbiol. Biotechnol. 48, 588–596.
Hermann-Krauss, C., Koller, M., Muhr, A., Hubert, F., Stelzer, F., Braunegg, G., 2013.
Archaeal production of Polyhydroxyalkanoate (PHA) co- and terpolyester from
biodiesel industry-derived by-products. Archaea 2013, 1–10.
Hocking, P.J., Marchessault, R.H., 1994. Biopolyesters. In: in: Griffin, G.J.L. (Ed.),
Chemistry, Technology of Biodegradable Polymers. Blackie Academic &
Professional, pp, pp. 48–96.
Horng, Y.T., Chang, K.C., Chien, C.C., Wei, Y.H., Sun, Y.M., Soo, P.C., 2010. Enhanced
polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb
genes controlled by arabinose PBAD promoter in Escherichia coli. Lett. Appl.
Microbiol. 50, 158–167.
Huijberts, G.N.M., Eggink, G., De Waard, P., Huisman, G.W., Witholt, B., 1992.
Pseudomonas putida KT2442 cultivated on glucose accumulates
poly(3-hydroxyalkanoates) consisting of saturated and unsaturated
monomers. Appl. Environ. Microbiol. 58, 536–544.
Huschner, F., Grousseau, E., Brigham, C.J., Plassmeier, J., Popovic, M., Rha, C.,
Sinskey, A.J., 2015. Development of a feeding strategy for high cell and PHA
density fed-batch fermentation of Ralstonia eutropha H16 from organic acids
and their salts. Process Biochem. 50, 165–172.
Ibrahim, M.H., Steinbuchel, A., 2009. Poly(3-Hydroxybutyrate) production from
glycerol by Zobellella denitrificans MW1 via high-cell-density fed-batch
fermentation and simplified solvent extraction. Appl. Environ. Microbiol. 75,
6222–6231.
Jeon, J.M., Brigham, C.J., Kim, Y.H., Kim, H.J., Yi, D.H., Kim, H., Rha, C., Sinskey, A.J.,
Yang, Y.H., 2014. Biosynthesis of
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from
butyrate using engineered Ralstonia eutropha. Appl. Microbiol. Biotechnol. 98,
5461–5469.
Jia, K., Cao, R., Hua, D.H., Li, P., 2016. Study of class I and class III
polyhydroxyalkanoate (PHA) synthases with substrates containing a modified
side chain. Biomacromolecules 17, 1477–1485.
Jin, H., Nikolau, B.J., 2014. Evaluating PHA productivity of bioengineered
Rhodosprillum rubrum. PLoS One 9, e96621.
Kamilah, H., Tsuge, T., Yang, T.A., Sudesh, K., 2012. Waste cooking oil as substrate
for biosynthesis of poly(3-hydroxybutyrate) and
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate): turning waste into a
value-added product. Malaysian J. Microbiol. 9, 51–59.
Kim, B.S., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K., Woo, S.I., 1994. Production of
poly-(3-hydroxybutyric-co-3-hydroxyvaleric acid) by fed-batch culture of
Alcaligenes eutrophus with substrate feeding using on-line glucose analyser.
Enzyme Microbiol. Technol. 16, 556–561.
Koller, M., Niebelschütz, H., Braunegg, G., 2013. Strategies for recovery and
purification of poly[(R)-3-hydroxyalkanoates] (PHA) biopolyesters from
surrounding biomass. Eng. Life Sci. 13, 549–562.
Lagenbach, S., Rehm, B.H., Steinbüchel, A., 1997. Functional expression of the PHA
synthase gene phaC1 from Pseudomonas aeruginosa in Escherichia coli results in
poly(3-hydroxyalkanoate) synthesis. FEMS Microbiol. Lett. 150, 303–309.
Lauzier, C.A., Monasterios, C.J., Saracovan, I., Marchessault, R.H., Ramsay, B.A., 1993.
Film formation and paper coating with poly(b-hydroxyalkanoate), a
biodegradable latex. Tappi J. 76, 71–77.
Lau, N.S., Sudesh, K., 2012. Revelation of the ability of Burkholderia sp. USM (JCM
15050) PHA synthase to polymerize 4-hydroxybutyrate monomer. AMB
Express 2, 41.
Lee, G.N., Na, J., 2013. Future of microbial polyesters. Microb. Cell Fact. 12, 54–57.
Lee, S.Y., 1996. Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49, 1–14.
Lemoigne, M., 1926. Produits de deshydratation et de polymersation de la
acideoxybutyrique. Bull. Soc. Chim. Biol. 8, 770–782.
Leong, Y.K., Show, P.L., Ooi, C.W., Ling, T.C., Lan, J.C., 2014. Current trends in
polyhydroxyalkanoates (PHAs) biosynthesis: insights from the recombinant
Escherichia coli. J. Biotechnol. 180, 52–65.
Li, R., Zhang, H., Qi, Q., 2007. The production of polyhydroxyalkanoates in
recombinant Escherichia coli. Bioresour. Technol. 98, 2313–2320.
 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Li, R., Jiang, Y., Wang, X., Yang, J., Gao, Y., Zi, X., Zhang, X., Gao, H., Hu, N., 2013.
Psychrotrophic Pseudomonas mandelii CBS-1 produces high levels of
poly-␤-hydroxybutyrate. Springerplus 2, 335–342.
Liu, S.J., Steinbüchel, A., 2000. A novel genetically engineered pathway for
synthesis of poly(hydroxyalkanoic acids) in Escherichia coli. Appl. Environ.
Microbiol. 66, 739–743.
Lizarraga-Valderrama, L.R., Nigmatullin, R., Taylor, C., Haycock, J.W., Claeyssens, F.,
Knowles, J.C., Roy, I., 2015. Nerve tissue engineering using blends of
poly(3-hydroxyalkanoates) for peripheral nerve regeneration. Eng. Life Sci. 15,
612–621.
Lomas, A.J., Webb, W.R., Han, J., Chen, G.Q., Sun, X., Zhang, Z., Haj, A.J., Forsyth, N.R.,
2013. Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagen hybrid
scaffolds for tissue engineering applications. Tissue Eng. Part C Methods 19,
577–585.
Lu, L.X., Zhang, X.F., Wang, Y.Y., Ortiz, L., Mao, X., Jiang, Z.L., Xiao, Z.D., Huang, N.P.,
2013. Effects of hydroxyapatite-containing composite nanofibers on
osteogenesis of mesenchymal stem cells in vitro and bone regeneration in
vivo. ACS Appl. Mater. Interfaces 5, 319–330.
Luzier, W.D., 1992. Materials derived from biomass/biodegradable materials. Proc.
Natl. Acad. Sci. 89, 839–842.
Lv, L., Ren, Y.L., Chen, J.C., Wu, Q., Chen, G.Q., 2015. Application of CRISPRi for
prokaryotic metabolic engineering involving multiple genes, a case study:
controllable P(3HB-co-4HB) biosynthesis. Metab. Eng. 29, 160–168.
Mahishi, L.H., Tripathi, G., Rawal, S.K., 2003. Poly(3-hydroxybutyrate) (PHB)
synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens
PHB biosynthesis genes: effect of various carbon and nitrogen sources.
Microbiol. Res. 158, 19–27.
Masood, F., Chen, P., Yasin, T., Fatima, N., Hasan, F., Hameed, A., 2013.
Encapsulation of Ellipticine in poly-(3-hydroxybutyrate-co-3-hydroxyvalerate)
based nanoparticles and its in vitro application. Mater. Sci. Eng. C Mater. Biol.
Appl. 33, 1054–1060.
Mendonça, T.T., Gomez, J.G., Buffoni, E., Sánchez Rodriguez, R.J., Schripsema, J.,
Lopes, M.S., Silva, L.F., 2014. Exploring the potential of Burkholderia sacchari to
produce polyhydroxyalkanoates. J. Appl. Microbiol. 116, 815–829.
Meng, D.C., Wang, Y., Wu, L.P., Shen, R., Chen, J.C., Wu, Q., Chen, G.Q., 2015.
Production of poly(3-hydroxypropionate) and
poly(3-hydroxybutyrate-co-3-hydroxypropionate) from glucose by
engineering Escherichia coli. Metab. Eng. 29, 189–195.
Możejko, J., Ciesielski, S., 2013. Saponified waste palm oil as an attractive
renewable resource for mcl-polyhydroxyalkanoate synthesis. J. Biosci. Bioeng.
116, 485–492.
Możejko, J., Przybyłek, G., Ciesielski, S., 2011. Waste rapeseed oil as a substrate for
mcl-PHAs production. Eur. J. Lipid Sci. Technol. 113, 1550–1557.
Możejko, J., Wilke, A., Przybyłek, G., Ciesielski, S., 2012. Mcl-PHAs Produced by
Pseudomonas sp. Gl01 using fed-batch cultivation with waste rapeseed oil as
carbon source. J. Microbiol. Biotechnol. 22, 371–377.
Naveen, S.V., Tan, I.K.P., Goh, Y.S., Raghavendran, H.R.B., Murali, M.R., Kamarul, T.,
2015. Unmodified medium chain length polyhydroxyalkanoate (uMCL-PHA) as
a thin film for tissue engineering application – characterization and in vitro
biocompatibility. Mater. Lett. 141, 55–58.
Ng, L.M., Sudesh, K., 2016. Identification of a new polyhydroxyalkanoate (PHA)
producer Aquitalea sp. USM4 (JCM 19919) and characterization of its PHA
synthase. J. Biosci. Bioeng., http://dx.doi.org/10.1016/j.jbiosc.2016.03.024.
Nikel, P.I., Almeida De, A., Melillo, E.C., Galvagno, M.A., Pettinari, M.J., 2006. New
recombinant Escherichia coli strain tailored for the production of
poly(3-hydroxybutyrate) from agroindustrial by-products. Appl. Environ.
Microbiol. 72, 3949–3954.
Nikel, P.I., Giordano, A.M., Almeida, A., Godoy, M.S., Pettinari, M.J., 2010.
Elimination of d-Lactate Synthesis Increases Poly(3-Hydroxybutyrate) and
ethanol synthesis from glycerol and affects cofactor distribution in
recombinant Escherichia coli. Appl. Environ. Microbiol. 76, 7400–7406.
Obeso, J.I., Gómez-Botrán, J.L., Olivera, E.R., Luengo, J.M., 2015. The phasin PhaF
controls bacterial shape and size in a network-forming strain of Pseudomonas
putida. J. Biotechnol. 199, 17–20.
Obruca, S., 2010. Controlled Production and Degradation of Selected Biomaterials.
Doctoral Thesis. Brno University of Technology.
Ouyang, S.P., Luo, R.C., Chen, S.S., Liu, Q., Chung, A., Wu, Q., Chen, G.Q., 2007.
Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate
monomer content by fadB and fadA knockout mutant of Pseudomonas putida
KT2442. Biomacromolecules 8, 2504–2511.
Park, D.H., Kim, B.S., 2011. Production of poly(3-hydroxybutyrate) and
poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from
soybean oil. N. Biotechnol. 28, 719–724.
Park, S.J., Jang, Y.A., Lee, H., Park, A.R., Yang, J.E., Shin, J., Oh, Y.H., Song, B.K., Jegal, J.,
Lee, S.H., Lee, S.Y., 2013. Metabolic engineering of Ralstonia eutropha for the
biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates. Metab. Eng.
20, 20–28.
Passanha, P., Esteves, S.R., Kedia, G., Dinsdale, R.M., Guwy, A.J., 2013. Increasing
polyhydroxyalkanoate (PHA) yields from Cupriavidus necator by using filtered
digestate liquors. Bioresour. Technol. 147, 345–352.
Pederson, E.N., McChalicher, C.W.J., Srienc, F., 2006. Bacterial synthesis of PHA
block copolymers. Biomacromolecules 7, 1904–1911.
Philip, S., Keshavarz, R., Roy, I., 2007. Polyhydroxyalkanoates: biodegradable
polymers with a range of applications. J. Chem. Technol. Biotechnol. 82,
233–247.
281
Plastics Europe, 2015. Plastics – the Facts 2014/2015 An Analysis of European
Plastics Production, Demand and Waste Data, http://www.plasticseurope.org/
documents/document/20150227150049-final plastics the facts 2014 2015
260215.pdf (accessed 01.03.2016).
Pramual, S., Assavanig, A., Bergkvist, M., Batt, C.A., Sunintaboon, P.,
Lirdprapamongkol, K., Svasti, J., Niamsiri, N., 2016. Development and
characterization of bio-derived polyhydroxyalkanoate nanoparticles as a
delivery system for hydrophobic photodynamic therapy agents. J. Mater. Sci.
Mater. Med. 27, 40.
Qi, Q., Rehm, B.H., 2001. Polyhydroxybutyrate biosynthesis in Caulobacter
crescentus: molecular characterization of the polyhydroxybutyrate synthase.
Microbiology 147, 3353–3358.
Rehm, B.H., Mitsky, T.A., Steinbüchel, A., 2001. Role of fatty acid de novo
biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by
pseudomonads: establishment of the transacylase (PhaG)-mediated pathway
for PHA biosynthesis in Escherichia coli. Appl. Environ. Microbiol. 67,
3102–3109.
Rehm, B.H., 2003. Polyester synthases: natural catalysts for plastics. Biochem. J. 37,
15–33.
Romanelli, M.G., Povolo, S., Favaro, L., Fontana, F., Basaglia, M., Casella, S., 2014.
Engineering Delftia acidovorans DSM39 to produce polyhydroxyalkanoates
from slaughterhouse waste. Int. J. Biol. Macromol. 71, 21–27.
Ruth, K., Grubelnik, A., Hartmann, R., Egli, T., Zinn, M., Ren, Q., 2007. Efficient
production of (R)-3-hydroxycarboxylic acids by biotechnological conversion of
polyhydroxyalkanoates and their purification. Biomacromolecules 8, 279–286.
Sabirova, J.S., Ferrer, M., Lünsdorf, H., Wray, V., Kalscheuer, R., Steinbüchel, A.,
Timmis, K.N., Golyshin, P.N., 2006. Mutation in a tesB-Like
hydroxyacyl-Coenzyme a-specific thioesterase gene causes hyperproduction
of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2. J.
Bacteriol. 188, 8452–8459.
Salamanca-Cardona, L., Ashe, C.S., Stipanovic, A.J., Nomura, C.T., 2014. Enhanced
production of polyhydroxyalkanoates (PHAs) from beechwood xylan by
recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 98, 831–842.
Sandoval, A., Arias-Barrau, E., Arcos, M., Naharro, G., Olivera, E.R., Luengo, J.M.,
2007. Genetic and ultrastructural analysis if different mutants of Pseudomonas
putida affected in the poly-3-hydroxy-n-alkanoate gene cluster. Environ.
Microbiol. 9, 737–751.
Saranya, V., Shenbagarathai, R., 2011. Production and characterization of PHA from
recombinant E. coli harbouring phaC1 gene of indigenous Pseudomonas sp.
LDC-5 using molasses. Braz. J. Microbiol. 42, 1109–1118.
Sato, S., Maruyama, H., Fujiki, T., Matsumoto, K., 2015. Regulation of
3-hydroxyhexanoate composition in PHBH synthesized by recombinant
Cupriavidus necator H16 from plant oil by using butyrate as a co-substrate. J.
Biosci. Bioeng. 120, 246–251.
Schlegel, H.G., Gottschalk, G., von Bartha, R., 1961. Formation and utilization of
poly-␤-hydroxybutyric acid by knallgas bacteria (Hydrogenomonas). Nature
191, 463–465.
Scholz, C., 2000. Poly(␤-hydroxyalkanoates) as potential biomedical materials: an
overview. ACS Ser. 764, 328–334.
Schubert, P., Steinbüchel, A., Schlegel, H.G., 1988. Cloning of the Alcaligenes
eutrophus genes for synthesis of poly-␤-hydroxybutyric acid (PHB) and
synthesis of PHB in Escherichia coli. J. Bacteriol. 170, 5837–5847.
Seo, M.C., Shin, H.D., Lee, Y.H., 2004. Transcription level of granule-associated phaP
and phaR genes and granular morphogenesis of poly-␤-hydroxyalkanoate
granules in Ralstonia eutropha. Biotechnol. Lett. 26, 617–622.
Shantini, K., Yahya, A.R., Amirul, A.A., 2015. Influence of Feeding and Controlled
Dissolved Oxygen Level on the Production of
Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) copolymer by Cupriavidus sp.
USMAA2-4 and its characterization. Appl. Biochem. Biotechnol. 176,
1315–1334.
Sierro, N.J.M., 2005. Bi-functionality of the PhaF Protein of Pseudomonas Putida in
the Polyhydroxyalkanoate Production Process. Swiss Federal Institute of
Technology, Zurich (Doctoral thesis).
Singh, M., Patel, S.K., Kalia, V.C., 2009. Bacillus subtilis as potential producer for
polyhydroxyalkanoates. Microb. Cell Fact. 8, 38–49.
Sun, Z.Y., Ramsay, J.A., Guay, M., Ramsay, B.A., 2007. Fermentation process
development for the production of medium-chain-length
poly-3-hyroxyalkanoates. Appl. Microbiol. Biotechnol. 75, 475–485.
Suriyamongkol, P., Weselake, R., Narine, S., Moloney, M., Shah, S., 2007.
Biotechnological approaches for the production of polyhydroxyalkanoates in
microorganisms and plants – a review. Biotechnol. Adv. 25, 148–175.
Tomizawa, S., Hyakutake, M., Saito, Y., Agus, J., Mizuno, K., Abe, H., Tsuge, T., 2011.
Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC
subunit of PHA synthase from Bacillus cereus YB-4 in recombinant
Escherichia coli. Biomacromolecules 12, 2660–2666.
Tsuge, T., Yamamoto, T., Yano, K., Abe, H., Doi, Y., Taguchi, S., 2009. Evaluating the
ability of polyhydroxyalkanoate synthase mutants to produce P(3HB-co-3HA)
from soybean oil. Macromol. Biosci. 9, 71–78.
Valentin, H.E., Dennis, D., 1997. Production of
poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant
Escherichia coli grown on glucose. J. Biotechnol. 58, 33–38.
Vandamme, P., Coenye, T., 2004. Taxonomy of the genus Cupriavidus: a tale of lost
and found. Int. J. Syst. Evol. Microbiol. 54, 2285–2289.
Vilos, C., Constandil, L., Herrera, N., Solar, P., Escobar-Fica, J., Velasquez, L.A., 2012.
Ceftiofur-loaded PHBV microparticles: a potential formulation for a long-acting
antibiotic to treat animal infections. Electron. J. Biotechnol. 15, 1–13.
282 J. Możejko-Ciesielska, R. Kiewisz / Microbiological Research 192 (2016) 271–282
Volova, T.G., Kiselev, E.G., Shishatskaya, E.I., Zhila, N.O., Boyandin, A.N., Syrvacheva,
D.A., Vinogradova, O.N., Kalacheva, G.S., Vasiliev, A.D., Peterson, I.V., 2013. Cell
growth and accumulation of polyhydroxyalkanoates from CO2 and H2 of a
hydrogen-oxidizing bacterium, Cupriavidus eutrophus B-10646. Bioresour.
Technol. 146, 215–222.
Wang, Q., Yu, H., Xia, Y., Kang, Z., Qi, Q., 2009. Complete PHB mobilization in
Escherichia coli enhances the stress tolerance: a potential biotechnological
application. Microb. Cell Fact. 8, 47.
Wang, H.H., Zhou, X.R., Liu, Q., Chen, G.Q., 2011. Biosynthesis of
polyhydroxyalkanoate homopolymers by Pseudomonas putida. Appl. Microbiol.
Biotechnol. 89, 1497–1507.
Wang, Q., Tappel, R.C., Zhu, C., Nomura, C.T., 2012. Development of a new strategy
for production of medium-chain-length polyhydroxyalkanoates by
recombinant Escherichia coli via inexpensive non-fatty acid feedstocks. Appl.
Environ. Microbiol. 78, 519–527.
Wang, Y., Wu, H., Jiang, X., Chen, G.Q., 2014. Engineering Escherichia coli for
enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in
larger cellular space. Metab. Eng. 25, 183–193.
Wong, Y.M., Brigham, C.J., Rha, C., Sinskey, A.J., Sudesh, K., 2012. Biosynthesis and
characterization of polyhydroxyalkanoate containing high
3-hydroxyhexanoate monomer fraction from crude palm kernel oil by
recombinant Cupriavidus necator. Bioresour. Technol. 121, 320–327.
Xie, W.P., Chen, G.Q., 2008. Production and characterization of terpolyester
poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) by
recombinant Aeromonas hydrophila 4AK4 harboring genes phaPCJ. Biochem.
Eng. J. 38, 4823–4830 ().
Yee, L.N., Mumtaz, T., Mohammadi, M., Hassan, M.A., Zakaria, M.R., 2012.
Polyhydroxyalkanoate synthesis by recombinant Escherichia coli JM109
expressing PHA biosynthesis genes from Comamonas sp. EB172. J. Microb.
Biochem. Technol. 4, 103–110.
Yezza, A., Fournier, D., Halasz, A., Hawari, J., 2006. Production of
polyhydroxyalkanoates from methanol by new methylotrophic bacterium
Methylobacterium sp. GW2. Appl. Microbiol. Biotech. 73, 211–218.
Yuan, M.Q., Shi, Z.Y., Wei, X.X., Wu, Q., Chen, S.F., Chen, G.Q., 2008. Microbial
production of medium-chain-length 3-hydroxyalkanoic acids by recombinant
Pseudomonas putida KT2442 harboring genes fadL, fadD and phaZ. FEMS
Microbiol. Lett. 4, 167–175.
Zhuang, Q., Wang, Q., Liang, Q., Qi, Q., 2014. Synthesis of polyhydroxyalkanoates
from glucose that contain medium-chain-length monomers via the reversed
fatty acid ␤-oxidation cycle in Escherichia coli. Metab. Eng. 24, 78–86.