International Journal of Biological Macromolecules 70 (2014) 208213

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

Bacterial production of the biodegradable plastics

polyhydroxyalkanoates
Viviana Urtuvia, Pamela Villegas, Myriam Gonzlez, Michael Seeger
Laboratorio de Microbiologa Molecular y Biotecnologa Ambiental, Departamento de Qumica & Center for Nanotechnology and Systems Biology & Centro
de Biotecnologa, Universidad Tcnica Federico Santa Mara, Avenida Espana 1680, Valparaso, Chile

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

Article history: Petroleum-based plastics constitute a major environmental problem due to their low biodegradabil-
Received 3 March 2014 ity and accumulation in various environments. Therefore, searching for novel biodegradable plastics is
Received in revised form 13 May 2014 of increasing interest. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are biodegradable
Accepted 3 June 2014
plastics. Life cycle assessment indicates that PHB is more benecial than petroleum-based plastics. In this
Available online 26 June 2014
report, bacterial production of PHAs and their industrial applications are reviewed and the synthesis of
PHAs in Burkholderia xenovorans LB400 is described. PHAs are synthesized by a large number of microor-
Keywords:
ganisms during unbalanced nutritional conditions. These polymers are accumulated as carbon and energy
Polyhydroxyalkanoate
Biodegradable plastic reserve in discrete granules in the bacterial cytoplasm. 3-hydroxybutyrate and 3-hydroxyvalerate are two
Polyhydroxybutyrate main PHA units among 150 monomers that have been reported. B. xenovorans LB400 is a model bacterium
Burkholderia xenovorans for the degradation of polychlorobiphenyls and a wide range of aromatic compounds. A bioinformatic
pha gene analysis of LB400 genome indicated the presence of pha genes encoding enzymes of pathways for PHA
synthesis. This study showed that B. xenovorans LB400 synthesize PHAs under nutrient limitation. Stain-
ing with Sudan Black B indicated the production of PHAs by B. xenovorans LB400 colonies. The PHAs
produced were characterized by GCMS. Diverse substrates for the production of PHAs in strain LB400
were analyzed.
2014 Elsevier B.V. All rights reserved.

1. Introduction companies in America, Asia and Europe produce PHAs at industrial

The highly versatile petroleum-based plastics have made them
valuable materials for modern life with a wide range of applica-
tions. However, due to their low biodegradability and accumulation
in various environmental compartments they are major environ-
mental pollutants. Searching for novel biodegradable polymers
that possess similar physicochemical properties as conventional
plastics is of increasing interest. PHAs are biodegradable plastics
synthesized by diverse bacteria from a range of substrates including
sugars and fatty acids. These biopolymers are stored as intracellular
carbon and energy reserve granules by Gram-negative and Gram-
positive bacteria under nutrient limitation. 3-hydroxybutyrate
(3HB) and 3-hydroxyvalerate (3HV) are two common and widely
studied PHA monomers. However, 150 different PHA monomers
have been reported. Depending on the microorganism, the car-
bon source and culture conditions different PHA homopolymers,
copolymers and combination thereof are synthesized. A number of
Corresponding author. Tel.: +56 32 2654236; fax: +56 32 2654782.
E-mail address: michael.seeger@usm.cl (M. Seeger).
scale.
In this report bacterial production of PHAs and their industrial
applications are reviewed and the synthesis of PHAs in the model
aromatic-degrading bacterium Burkholderia xenovorans LB400 is
described.
2. Petroleum-derived plastics: Applications and pollution
The versatility and low production costs of plastics derived
from petroleum have allowed them to substitute other materials
such as paper, glass and wood. Plastics are important for various
applications in different industrial sectors. During the year 2008,
245 million tons were consumed at global level and a 9% annual
growth in the plastic consumption was forecasted. Fig. 1 illus-
trates that in Chile, plastic consumption is mainly concentrated
in the areas of packaging, mining, construction and agroindustry
[1]. The remaining 9% is distributed among household, agricul-
ture and transport. 844,000 t of plastic consumption in Chile for
2012 was expected, with 51 kg plastic consumption per capita, pla-
cing Chile as the second largest plastic-consuming country in Latin

http://dx.doi.org/10.1016/j.ijbiomac.2014.06.001
0141-8130/ 2014 Elsevier B.V. All rights reserved.
 V. Urtuvia et al. / International Journal of Biological Macromolecules 70 (2014) 208213
Fig. 1. Plastic consumption in Chile in different areas. Adapted from Ref. [1].
America [2]. The plastic consumption per capita in European
countries exceeds 100 kg per year [3].
Chemical and physical properties of plastics provide them with
high durability and versatility for a wide range of applications.
However, their properties convert plastics in pollutants when they
are disposed in the environment. The plastics present very low
degradation rates in the environment, with a half-life up to > 500
years. The low degradability causes, among other drawbacks, a
high accumulation of plastics in sanitary landlls, decreasing the
life thereof. The plastic wastes are about 8% in weight and 25% in
volume of the total solid urban wastes. The low degradability com-
bined with the inadequate waste disposal and low environmental
education have caused that plastics are accumulated in differ-
ent environmental compartments. An example is the presence of
plastic fragments in almost all oceans, which causes plastic inges-
tion by marine organisms with largely unknown consequences
[4]. The implementation of environmental management politics to
reduce the plastic wastes is crucial for the conservation of natu-
ral resources and the reduction of environmental pollution. The
research and development of new biodegradable materials is of
increasing importance. In recent decades plastic manufacturing
using renewable feedstock has been promoted and the productions
of biologically based plastics known as biopolymers, which could
be biodegradable, are of increasing interest [5].
3. Biopolymers and polyhydroxyalkanoates
The term biopolymer includes polymers of natural origin, but
also high molecular mass molecules that have been polymerized
by chemical and biological methods. Not all biopolymers are
biodegradable [5]. The biodegradable plastics primarily are pro-
duced from renewable raw materials by aerobic synthesis and from
waste management by composting and anaerobic digestion [6].
The biopolymers are classied according to their biodegradability
or composition. PHAs and polylactic acid (PLA) are well-studied
biopolymers due to their biodegradability and physicochemical
properties [5,7].
PHAs are thermoplastic polyesters of R-hydroxyalkanoic acids.
PHAs are synthesized and stored as intracellular carbon and
energy reserve in Gram-negative and Gram-positive bacteria under
209
nutrient limitation of nitrogen, phosphorus, oxygen or after pH
shifts [3,8,9]. PHAs accumulate in cytoplasmic granules that typ-
ically have a diameter from 0.2 to 0.5 m. These granules can be
visualized by phase contrast microscopy due to their high refrac-
tivity or using staining dyes such as Sudan Black B and Nile red. Once
the limiting nutrient is provided to the cell, these energy storage
compounds are degraded and used as carbon source for bacterial
growth [10].
Polyhydroxybutyrate (PHB) was discovered in 1926 by Maurice
Lemoige in the bacterium Bacillus megaterium, which showed intra-
cellular granules [11]. PHB is the most common and the best studied
member of PHAs [8]. The presence of 3HB as a PHA monomer
was reported in activated sludge [12]. The presence of 3HV as
a major constituent and 3-hydroxyhexanoate (3HHx) as a minor
constituent were described [13]. The oil crisis of the early 1970s
prompted the search for new materials to replace petroleum-based
plastics [9]. Fifty years after its discovery, PHB production was car-
ried out at commercial scale.
PHAs could have different composition depending on the
microorganism, the carbon source and culture conditions (Table 1).
The most common polymers are the short-chain-length PHA
(PHASCL ), which contain from 3 to 5 carbons atoms. The struc-
tural formulas of PHASCL are depicted in Fig. 2. These biopolymers
are stiff, brittle and possess a high degree of crystallinity. Middle-
chain-length PHA (PHAMCL ) contain from 6 to 14 carbons atoms
and are exible and possess low crystallinity, tensile strength and
high melting point [9,10,14,15]. Currently 150 different monomer
constituents of PHAs that could be homopolymers, copolymers and
combination thereof have been reported [10,16,17].
PHB is produced by bacteria from a wide range of substrates
(Fig. 3). In contrast, specic substrates such as propionic acid,
valeric acid and other organic acids are required for the synthe-
sis of the PHV monomer [17,24]. The metabolic pathways for the
synthesis of both monomers have been reported in the model
PHA-producing bacterium Ralstonia eutropha strain H16, which
synthesizes PHASCL [17,2729]. PHAs could reach up to 80% of the
bacterial cell dry biomass.
Life cycle assessment (LCA) indicates that the energy require-
ments for PHB production are lower than the energy requirements
for conventional high density plastics polyethylene and polypro-
pylene [30]. In addition, the production of PHB is more benecial for
ozone layer protection, and reduces toxicity levels, abiotic deple-
tion and global warming. However, PHB showed higher impacts
than polyethylene on acidication and eutrophication. On a cradle-
to-factory gate LCA, PHB is more benecial than conventional
polymers such as polypropylene [30].
4. Substrates for bacterial PHA production and PHA
production cost
PHAs are produced by bacteria from a wide range of substrates
such as renewable sources (e.g., sucrose, starch, cellulose, trigly-
cerides, hemicellulose, wheat), sub-products (e.g., molasses, whey,
glycerol, corn steep liquor, rice bran), organic acids (e.g., propionic
acid, 4-hydroxybutyric acid), fossil resources (e.g., methane, min-
eral oil, lignite, hard coal) and wastes (e.g., wastewater, palm oil
mill and activated sludge efuents) [15,24,27,3133].
Substrates such as glucose and sucrose have been used for
industrial PHA production. The raw materials showed a high
impact on the PHA production cost. Therefore, low cost raw
materials are attractive for industrial PHA production [8]. Xylose
could be an interesting alternative substrate for PHA production
(Fig. 3). Xylose is the main sugar component of hemicellulose in
Gramineae plants and hardwood. Hemicellulose is the third most
abundant polymer in nature that is converted into fermentable
210 V. Urtuvia et al. / International Journal of Biological Macromolecules 70 (2014) 208213

Table 1
Production of polyhydroxyalkanoates by diverse bacteria.

Bacterial strain Carbon source PHA PHA content References

(%)

Ralstonia eutropha H16 Glucosa, PHB 80 [14,18]

fructose, acetic PHV 90
acid, valeric
acid
Burkholderia sp. DSMZ 9243 Sucrose or PHB 45 [19,20]
gluconate P(3HPEa )
Burkholderia cepacia ATCC 17759 Xylose:levulinic P(3HB-co-3HV) 49 [21,22]
acidb
Burkholderia sacchari IPT189 Sucrose:propionic P(3HB-co-3HV) 30 [23,24]
acidc
Burkholderia xenovorans LB400 Glucose PHB ND [25,26]

ND: not determined.

a
3-Hydroxy-4-pentanoic acid.
b
Ratio (w/v in %) 2.2:(0.070.52).
c
Ratio (w/w) 10:1, 19:1, 30:1, 61.5:1.

sugars by chemical or enzymatic hydrolysis [31]. Hemicellulose
hydrolysis releases xylose, mannose, galactose, arabinose and glu-
cose. PHB production from xylose by Pseudomonas pseudoava
ATCC 33668, Burkholderia cepacia ATCC 17759 and recombinant
Escherichia coli strains TG1(pSYL105) and W3110(pSYL107) has
been reported [3437]. The forestry, which produces high amounts
of hemicellulose-containing sub-products, is an important activity
in diverse countries (e.g., in Canada, Chile, Brazil and USA). There-
fore, PHA production from sub-products containing xylose may be
an attractive alternative.
One of the most signicant barriers for an increased industrial
PHAs production is its high cost. The product price depends up
to 50% on the raw material cost, mainly the carbon source. The
biopolymer recovery process also contributes to higher PHA pro-
duction cost [10,15,35]. PHAs are still produced on a small-scale
production volume from 1000 to 20,000 t per year, which also
increases the cost. In contrast, polyethylene is produced in quanti-
ties exceeding 300,000 t per year, with an important benet in the
nal price due to the economy of scale [7,38]. PHA price varies from
1.5 to 5 D per kg, whereas polypropylene price varies from 0.2 to
0.4 D per kg [7,10]. However, the versatility and biocompatibility
of PHAs have made them interesting candidates for low volume
products of high value, especially for biomedical applications [39].
For example, PHA nanoparticles for drug delivery and biocompati-
ble porous implants made from poly-4-hydroxybutyrate has been
reported [8,39].
Various small and middle-sized enterprises around the world
produce PHAs at small-scale. For example, P&G (USA), Biomer Inc.
(Germany), Tianan Biologic (China) and PHB Industrial (Brazil) [7].
Bags, pencils, plates, glasses and various plastic parts are manufac-
tured from these polymers. PHA resins are used for the manufacture
of sustained release drugs, bone implants and sutures [39]. In addi-
tion, PHAs can be used as latex (for example, for paper-coating
applications) or in foods [40]. However, to enhance the PHA pro-
duction, the production cost should be decreased. Therefore, novel
approaches for PHA production have to be established [5].
5. PHA synthesis by Burkholderia xenovorans strain LB400
The genus Burkholderia possesses high metabolic versatility and
is adapted to different ecological niches including soil and aqueous
environments. B. xenovorans LB400 is a model aerobic bacterium for

Fig. 2. Structural formulas of PHAs. General PHA structural formula (a). R derived from radical represents different substituents. PHB structural formula (b). Structural formulas
of short-chain-length PHA monomers (c).
 V. Urtuvia et al. / International Journal of Biological Macromolecules 70 (2014) 208213 211

Fig. 3. Biosynthesis of 3-hydroxybutyrate and 3-hydroxyvalerate from different substrates. 3HB is synthesized from sugars via the Entner-Doudoroff pathway and from few
fatty acids. 3HV is synthesized from diverse fatty acids. Abbreviations: 3-hydroxyvalerate (3HV), 3-hydroxybutyrate (3HB), phosphate (P); Coenzyme A (CoA) and 2-keto-3-
deoxy-6-phosphogluconate (KDPG).

the degradation of polychlorobiphenyls (PCBs) and a wide range
of aromatic compounds. PCBs have been used as complex mix-
tures for diverse industrial applications. These compounds are toxic
and tend to accumulate as persistent organic pollutants in soil and
water sediment [41,42].
The large genome from B. xenovorans strain LB400 (9.73 Mbp)
has been sequenced and characterized [43]. Strain LB400 possesses
genes encoding enzymes of eleven central and more than twenty
peripheral pathways for the degradation of aromatic compounds.
For example, the hmgABC gene cluster encoding the homogenti-
sate central pathway and the hpaG1G2EDFHI gene cluster encoding
the homoprotocatechuate central pathway are located at chro-
mosome 1 (C1) and chromosome 2 (C2), respectively. Additional
hmg gene copies were identied within the LB400 genome [43,44].
The functionality of diverse catabolic pathways such as biphenyl,
homogentisate, homoprotocatechuate, gentisate, protocatechuate
and 2-aminophenol pathways has been reported [42,4446]. B. xen-
ovorans LB400 is able to grow on 3-hydroxyphenylacetate (3-HPA)

Fig. 4. The pha gene cluster organization in B. xenovorans strain LB400 and R. eutropha strain H16. The arrows indicate the PHA metabolic pathway genes wherein the black
arrows indicate the PHA polymerase genes.
212 V. Urtuvia et al. / International Journal of Biological Macromolecules 70 (2014) 208213
Fig. 5. Synthesis of polyhdroxyalkanoates by B. xenovorans strain LB400. LB400 colonies grown on M9 medium with mannitol (10 g L1 ) as sole carbon source and 10% N (NH4 Cl
0.1 g L1 ) were stained with Sudan Black B dye (a). Magnication of the image (b).
and 4-hydroxyphenylacetate (4-HPA) isomers as sole carbon and
energy sources, indicating active peripheral and central catabolic
pathways. The homogentisate and homoprotocatechuate central
pathways are involved in 3-HPA and 4-HPA degradation in strain
LB400. Both ring-cleavage pathways are used in aromatic amino
acid metabolism in Bacteria and Eucarya.
A number of bioinformatic tools are useful to reconstruct
metabolic pathways in bacteria. The genes of the PHA anabolic
pathway from the model PHA-producing bacterium R. eutropha
strain H16 have been characterized. The genes for the synthesis
and degradation of PHA were found in B. xenovorans strain LB400.
Strain LB400 has two PHA anabolic pathways: (i) the pathway that
uses sugars as carbon sources and, (ii) the pathway associated to
fatty acids catabolic pathway (-oxidation). In B. xenovorans strain
LB400, the pha genes for the synthesis of PHAs are arranged in a
gene cluster at the major chromosome [47]. The pha gene clusters
from R. eutropha H16 and B. xenovorans LB400 have a similar orga-
nization, which are shown in Fig. 4. B. xenovorans strain LB400 has
three copies of the phaC gene distributed in the major chromosome,
minor chromosome and the megaplasmid. The PHA polymerase
PhaC from strain H16 possesses 15 functionally key amino acids
[48,49].These amino acids are conserved in PhaC polymerase from
strain LB400 [47].
Different compounds including sugars and alkanoates are sub-
strates for the bacterial production of PHAs. B. xenovorans LB400
is able to grow on glucose, mannitol and xylose as sole carbon
source. In contrast, strain LB400 was not able to grow on lactose
and sucrose. To study the PHA synthesis by strain LB400 in a rst
approach staining of bacterial colonies with Sudan Black B dye
was analyzed [10,50]. LB400 colonies grown on mannitol (10 g L1 )
were stained with Sudan Black B, indicating the intracellular accu-
mulation of PHAs (Fig. 5).
The synthesis of PHAs by strain LB400 was studied during
growth on various sugars and fatty acids. PHAs were identied
by gas chromatography and mass spectrometry. During growth
on glucose the synthesis of the homopolymer PHB was observed.
Searching for low cost renewable materials will be useful to
increase the production of these biopolymers.
6. Conclusions and future prospects
The global trends of plastic consumption promote the produc-
tion of novel biodegradable materials, replacing traditional plastics
and increasing environmental protection and sustainable develop-
ment. PHAs are attractive biopolymers due to their biodegradability
and physicochemical properties. PHAs are synthesized and stored
as intracellular carbon and energy reserve by bacteria under nutri-
ent limitation. Diverse bacteria are able to synthesize PHAs from a
wide range of substrates. The carbon sources have a high impact
on the PHA production cost. Low cost raw materials are attractive
to increase industrial PHAs production, which are currently pro-
duced by small and middle-sized companies. LCA indicates that
PHB is environmentally more benecial than conventional plastics
such as polypropylene. Bioinformatics tools have been used for the
reconstruction of metabolic pathways from bacteria. Our genome
analyses have shown that B. xenovorans strain LB400 possesses
the genes for the synthesis and degradation of polyhydroxyalka-
noates. The pha gene cluster encoding enzymes for PHA synthesis
in strain LB400 has the same gene organization as the pha genes in R.
eutropha strain H16. PhaC polymerase from strain LB400 is closely
related with class I type PhaC polymerase from other bacteria. B.
xenovorans LB400 cultivated on sugars such as glucose and manni-
tol synthesizes PHAs. Xylose is a sugar released from hemicellulose,
which is available in high amounts as by-products of forestry. This
might enable in the future the use of xylose as a low cost substrate
for large-scale production of polyhydroxyalkanoates.
Acknowledgments
The authors gratefully acknowledge Gregorio Gomez and
Luiziana Ferreira da Silva for their generous support. The authors
acknowledge nancial support from CONICYT PhD, Mecesup CD
FSM1204 and Cyted PRIBOP fellowships (VU) and FONDECYT
1110992 (MS), USM 131109 and 131342 (MS, MG), Pie > A (PV,VU),
CN&SB (MS) and Cyted PRIBOP (MS) grants. The funders had no role
in study design, data collection and analyses, decision to publish,
or preparation of the manuscript.
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