Process Biochemistry 40 (2005) 607–619

Recent advances in microbial polyhydroxyalkanoates

Shilpi Khanna, Ashok K. Srivastava∗
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India

Received 5 September 2003; accepted 26 January 2004

Abstract

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as an intracellular carbon
and energy storage compound. These are accumulated in the cytoplasm of cells. A number of bacteria including Alcaligenes, Pseudomonas,
recombinant Escherichia coli and methylotrophs have been used for the production of PHAs and high productivities have been achieved.
Production of PHA by transgenic plants has been demonstrated on a laboratory-scale and large-scale production will be feasible in the near
future. By controlling the monomer composition of PHA, it has been indicated that the physical properties of polymers can be regulated
to a great extent. Even though PHAs have been recognised as a good candidate for biodegradable polymers, their high production cost
limits their industrial application. It is hoped that with improvement in fermentation and downstream processing techniques, development of
new recombinant strains and large-scale production by transgenic plants will reduce the cost of production of PHAs thereby making them
competitive with conventional plastics.
© 2004 Elsevier Ltd. All rights reserved.

Keywords: Polyhydroxyalkanoates; Polyhydroxybutyrate; Biodegradable; Fermentation; Transgenic plants; Recombinant E. coli

1. Introduction ditives such as pigments, coatings, fillers, limits the use

of the recycled material. In such a scenario, biodegradable
Plastics have versatile qualities of strength, lightness, plastics offer the best solution to the environmental hazard
durability and resistance to degradation. They have become posed by conventional plastics.
an important commodity to enhance the comfort and quality Biodegradable plastics can be divided into three cate-
of life. Plastics are an essential part of almost all industries gories:
and have replaced glass and paper in packaging, but these
very desirable properties have now become their greatest 1. Chemically synthesised polymers: Polyglycollic acid,
problem. Accumulation of recalcitrant plastics in the en- polylactic acid, poly(ε-caprolactone), polyvinyl alcohol,
vironment has become a world-wide problem. Solutions poly(ethylene oxide) fall into this category. These are
to plastic waste management includes source reduction, susceptible to enzymic or microbial attack. Since they
incineration, recycling and bio- or photo-degradation. How- do not match all the properties of plastics, they are not
ever, most of these have problems associated with them. commercially viable as substitute for plastics.
Incineration of plastics is potentially dangerous and can be 2. Starch-based biodegradable plastics: In this type, starch
expensive. During the combustion of plastic waste, hydro- is added as filler and cross-linking agent to produce
gen cyanide can be formed from acrylonitrile-based plastics a blend of starch and plastic (for example, starch–
and may cause potential health hazards. Recycling can be polyethylene). Soil micro-organisms degrade the starch
done but is very tedious. The sorting of the wide variety easily, thus breaking down the polymer matrix. This
of discarded plastic material is also a very time-consuming results in significant reduction of degradation time.
process. Moreover, the presence of a wide variety of ad- But such plastics are only partially degradable. The
fragments left after starch removal are recalcitrant and
remain in the environment for a long time.
∗ Corresponding author. Tel.: +91-11-26591010; 3. Polyhydroxyalkanoates (PHAs): These are the only
fax: +91-11-26582282. 100% biodegradable polymers. They are polyesters
E-mail address: [email protected] (A.K. Srivastava). of various HAs which are synthesised by numerous

0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.01.053
608 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619

micro-organisms as energy reserve materials when an weight of these compounds range from 2×105 to 3×106 Da,
essential nutrient such as nitrogen or phosphorus is depending on the micro-organism and the growth conditions
available only in limiting concentrations in the presence [6].
of excess carbon source. They possess properties similar PHA exists as discrete inclusions that are typically
to various synthetic thermoplastics like polypropylene 0.2 ± 0.5 mm in diameter localised in the cell cytoplasm
and hence can be used in their place. They are also and can be visualised with a phase contrast light micro-
completely degraded to water and carbon dioxide under scope due to their high refractivity [7]. When thin sections
aerobic conditions and to methane under anaerobic con- of PHA-containing bacteria are observed under transmis-
ditions by micro-organisms in soil, sea, lake water and sion electron microscopy, the PHA inclusions appear as
sewage. electron-dense bodies. Native PHA inclusions can be stained
Polyhydroxybutyrate (PHB) was the first PHA to be with Sudan Black B [8] indicating that they are of a lipid
discovered and is also the most widely studied and best nature [9,10]. Besides Sudan Black B, PHA is more specif-
characterised PHA. It is accumulated as a membrane en- ically stained by the oxazine dye Nile Blue A, exhibiting a
closed inclusion in many bacteria at up to 80% of the dry strong orange fluorescence at an excitation wavelength of
cell weight. It has mechanical properties very similar to 460 nm [11]. While staining methods can be used to identify
conventional plastics like polypropylene or polyethylene the presence of PHA, chemical analysis is often required to
and can be extruded, moulded, spun into fibres, made determine their monomeric compositions.
into films and used to make heteropolymers with other Although the presence of these intracellular granules in
synthetic polymers. In spite of its numerous advantages, bacterial cells was recognised early by many microbiolo-
PHB has yet not been able to replace conventional plas- gists, the composition of these particles was first clarified
tics on a large scale because of its high cost. by Lemoigne in 1923 [12]. It was noticed by him that when
A large number of review papers are available, which cultures of Bacillus subtilis were allowed to autolyse in dis-
give a detailed description about the general features of tilled water, the pH value decreased due to formation of an
PHAs [1–3], characterisation of PHA polymers [4,1] and unknown acid. This unknown acid was found to be identi-
their biodegradation [3,5]. However, there are no recent cal to ␤-hydroxybutyric acid, which is present in urine of
reviews which explain in detail the fermentative advances diabetic patients. Subsequently, PHB was identified as the
in PHA production. This paper is an attempt to summarise monomer component of the granules [12].
recent fermentative advances that have taken place in Depending on the number of carbon atoms in the
PHA production by various bacteria. chain PHAs can be divided into two groups—short-chain
length (SCL), which consist of 3–5 carbon atoms, and
medium-chain length (MCL), which consist of 6–14 carbon
2. Polyhydroxyalkanoates atoms [13]. This difference is mainly due to the substrate
specificity of the PHA synthases that can accept 3HAs of
PHAs are polyesters of HAs with the general structural a certain range of carbon length [13]. The PHA synthase
formula shown in Fig. 1 [6]. Bacteria synthesise PHAs as of Alcaligenes eutrophus can polymerise 3HAs consisting
a carbon and energy source under conditions of limiting of 3–5 carbon atoms whereas that present in Pseudomonas
nutrients in the presence of excess carbon source [3,6]. Once oleovorans can only accept 3HAs of 6–14 carbon atoms.
the limiting nutrient is provided to the cell, these energy
storage compounds are degraded and used. The molecular
3. PHA biosynthesis

There are four different pathways for the synthe-

O CH2 O CH2 O CH2
sis of PHAs found to date. These pathways in detail
have been reported elsewhere [3,6]. In A. eutrophus,
CH C CH C CH ␤-ketothiolase carries out the condensation of two molecules
of acetyl-CoA to acetoacetyl-CoA. An NADPH-dependent
CH3 O R O CH3 acetoacetyl-CoA reductase then carries out its conversion
n to 3-hydroxybutyryl-CoA. The third and the final step is the
n varies from 600 to 35000 polymerisation reaction catalysed by PHB synthase (Fig. 2)
[13]. In Rhodopsuedomonas rubrum, the pathway differs
R= hydrogen Poly(3-hydroxypropionate) after the second step where the acetoacetyl-CoA formed by
R=methyl Poly(3-Hydroxybutyrate)
R=ethyl Poly(3-hydroxyvalerate) ␤-ketothiolase is reduced by a NADH dependent reductase
R=propyl Poly(3-hydroxyhexanoate) to l-(+)-3-hydroxybutyryl-CoA which is then converted
R=pentyl Poly(3-hydroxyoctanoate) to d-(−)-3-hydroxybutyryl-CoA by two enoyl-CoA hy-
R=nonyl Poly(3-hydroxydodecanoate)
dratases. A third type of PHA biosynthetic pathway is
Fig. 1. Structure of PHA [109]. found in most Psuedomonas species belonging to rRNA
 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619 609

Acetyl-CoA+Acetyl-CoA

CoA β-ketothiolase
Acetoacetyl-CoA Synthase
Acetoacetyl-CoA
Acetoacetate
NADPH
Reductase
NADP+
NADH
D(-)-hydroxybutyryl-CoA
D(-)-3-hydroxybutyrate
P(3HB)n
NAD+ dehydrogenase
PHA Synthase

P(3HB)n+1 D(-)-3-hydroxybutyrate
PHA Depolymerase

Fig. 2. Metabolic pathway involved in the synthesis and breakdown of PHB in R. eutropha [109].

homology group I. P. oleovorans and other Psuedomonas bacteria belonging to the first group produce PHA more ef-
species accumulate PHA consisting of 3-hydroxyalkanoic ficiently when a nutrient is limited but not completely de-
acid of MCL if cells are cultivated on alkanes, alkanols or pleted. For the cultivation of these bacteria, a mixture of
alkanoic acids [14–16]. The fourth type of PHA biosynthetic carbon source and a nutrient to be limited should be fed at
pathway is present in almost all Psuedomonas species be- an optimal ratio to produce PHA with high productivity.
longing to rRNA homology group II. This pathway involves For the fed-batch culture of bacteria belonging to the sec-
the synthesis of copolyesters consisting of MCL 3HAs from ond group, a nutrient feeding strategy is important to obtain
acetyl-CoA. This pathway has not been studied in detail. a high yield of PHAs. Complex nitrogen sources such as
corn steep liquor, yeast extract or fish peptone can be sup-
plemented to enhance cell growth as well as polymer accu-
4. Fermentation strategies mulation since PHA synthesis is not dependent on nutrient
limitation in these bacteria. Cell growth and PHA accumula-
Bacteria that are used for the production of PHAs can be tion need to be balanced to avoid incomplete accumulation
divided into two groups based on the culture conditions re- of PHA or premature termination of fermentation at low cell
quired for PHA synthesis. The first group of bacteria requires concentration.
the limitation of an essential nutrient such as nitrogen, phos- Selection of micro-organism for the production of PHA
phorous, magnesium or sulphur for the synthesis of PHA should be based on several factors including the cell’s ability
from an excess carbon source. The bacteria included in this to utilise an inexpensive carbon source, growth rate, poly-
group are A. eutrophus, Protomonas extorquens, and Pro- mer synthesis rate, and the maximum extent of polymer ac-
tomonas oleovorans. The second group of bacteria, which cumulation. Preliminary calculation of PHA yields can be
include Alcaligenes latus, a mutant strain of Azotobacter done by using the equation for prediction of overall yields
vinelandii, and recombinant E. coli, do not require nutrient available in literature [18]. Recovery of PHA should also be
limitation for PHA synthesis and can accumulate polymer considered because it significantly affects the overall eco-
during growth. These characteristics have to be taken into nomics. In view of above-mentioned factors, several bacteria
consideration while production of PHA. described further are suitable for the production of PHAs.
High productivity of PHA can be obtained by fed-batch
or continuous fermentation. For fed-batch culture of bacteria 4.1. PHA production in A. eutrophus
belonging to the first group, a two-step cultivation method
is most often employed. A desired concentration of biomass A. eutrophus (now renamed Ralstonia eutropha) has been
is obtained without nutrient limitation in the first stage after studied most extensively due to its ability to accumulate
which an essential nutrient is kept in limiting concentration large amount of PHB from simple carbon sources, for ex-
in the second stage to allow efficient PHA synthesis. Dur- ample, glucose, fructose and acetic acid. Imperial Chemical
ing nutrient limitation (second) stage, the residual cell con- Industries (UK) has been producing PHB on large scale from
centration (defined as the cell concentration minus the PHA glucose, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
concentration) remains almost constant and the cell concen- (P(3HB-co-3HV)) from a mixture of glucose and propionic
tration increases only because of the intracellular accumu- acid by fed-batch culture of A. eutrophus. Initially the cells
lation of PHA. A. eutrophus accumulates a large amount were grown on glucose-salts medium containing only cal-
of polymer (up to 80% dry cell weight) when nitrogen or culated amounts of phosphate to support a desired amount
phosphorous is completely depleted [17]. However, the other of cell growth. Cells encounter phosphate limitation after
610 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619

about 60 h and accumulate PHB during the next 40–60 h thetic pathway [31]. R. eutropha H16 and P. oleovorans were
from supplied glucose. By controlling the glucose concen- able to use the residual oil from rhamnose production as the
tration at 10–20 g/l during fed-batch culture the final cell sole C source for growth and for the accumulation of PHA.
mass, PHB concentration and PHB content of 164, 121 g/l, Approximately 20–25% of the components of the residual
and 76%, respectively, was obtained in 50 h, resulting in oil were converted into PHA. The corresponding yields were
the highest productivity of 2.42 g/(l h) [17]. Several carbon 41.3 and 38.9% of the cell dry mass for R. eutropha and
sources other than glucose have also been used as the sub- P. oleovorans, respectively [32]. PHB production has also
strates for PHA production by A. eutrophus. An amount been carried out from a mixture of l-lactic acid and acetic
equal to 7.1 g/l of PHB got accumulated when lactic acid acid by using R. eutropha ATCC 17697. The concentrations
was fed periodically as a carbon source [19] whereas when of lactic acid and acetic acid were controlled and the pH-
it was fed in a two-stage fed-batch culture system PHB yield increasing rate (measured by a pH meter linked computer)
was around 59 g/l [20]. The production of PHB from batch in the culture medium was used as an indicator of feed
cultures of R. eutropha ATCC 17699 has been studied using control. When pH-increasing rate decreased to 5% of the
acetic acid as the sole carbon source [21]. Continuous pro- maximum increasing rate, acidic substrate solution was fed.
duction of PHB by R. eutropha WSH3 in a two-stage cul- This was continued until the pH decreased to 7. Cell con-
ture system has also been demonstrated [22]. The first-stage centration and PHB content after 42 h were 75 g/l and 73.1%
cultivation on glucose produced maximum cell dry weight (w/w), respectively resulting in high PHB productivity of
of 27.1 g/l at a dilution rate of 0.21 h−1 while in the second 1.30 g/(l h) [33]. PHB has also been produced by mixed cul-
stage, 47.6 g/l PHB was accumulated at a dilution rate of ture cultivation in which sugars such as glucose were con-
0.14 h−1 which peaked at lower value of dilution rate under verted to lactic acid by Lactobacillus delbrueckii IAM1928,
nitrogen-limited conditions. Overall, at a dilution rate of and the lactic acid was in turn converted to PHB by R. eu-
0.075 h−1 , PHB productivity was 1.23 g/(l h), yield of PHB tropha H16 in the same fermentor [34,35]. Few mathemat-
to glucose was 0.36 g/g and PHB content was 72.1% [22]. ical models have also been proposed for PHB biosynthesis
It has also been reported that R. eutropha can utilise carbon which can describe the dynamic behaviour of the mixed
dioxide under autotrophic conditions and lead to the pro- culture for PHB production. These models may be used for
duction of PHB. Plant oils have also been found to be an several purposes such as control, optimisation, and under-
excellent substrate [23]. Production of 32.5 g/l of PHB in standing process dynamics particularly under mixed culture
60 h by fed-batch culture of A. eutrophus using oleic acid as conditions [36,37].
a carbon source has also been reported [24]. PHB has also PHB is a highly stiff, crystalline and relatively brittle ther-
been produced from R. eutropha DSM 11348 using me- moplastic. Its melting point (Tm = 175 ◦ C) is only slightly
dia containing glycerol and casein hydrolysate as C and N lower than the temperature at which it starts degrading to
sources, respectively [25]. In addition to the carbon sources crotonic acid (approximately 185 ◦ C), making processing
mentioned earlier, R. eutropha can also use specialised car- difficult. Due to relatively poor physical properties of PHB
bon sources like 4-hydroxybutyric acid, ␥-butyrolactone and homopolymer, extensive efforts are being directed towards
1,4-butanediol and can give rise to 4HB monomers along the synthesis of copolymers that have better properties
with 3HB [26]. However, in one experimental study it was than PHAs. Incorporation of 3- or 5-carbon monomers
also found that MCL-fatty acids, such as nonanoic (9:0) and into a polymer consisting mainly of 3-hydroxybutyrate
octanoic (8:0) acids were more toxic to R. eutropha than leads to a decrease in crystallinity and melting point com-
volatile fatty acids, such as acetic acid, propionic acid and pared to PHB homopolymer for example in the case of
butyric acid [27]. Utilisation of even numbered n-alkanoates P(3HB-co-3HV). Addition of propionic acid or valeric acid
resulted in production of PHB homopolymer whereas to the growth media containing glucose leads to the pro-
odd numbered n-alkanoates produced copolymers of 3HB duction of P(3HB-co-3HV). The feeding of propionic acid
and 3-hydroxyvalerate (3HV) [28]. Production of terpoly- for production of P(3HB-co-3HV) by A. eutrophus ATCC
mers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co- 17697 has been optimised using a fed-batch culture system
3-hydroxyheptanoate) has been carried out from alkanoic [38]. P(3HB-co-3HV) production has also been reported
acids of odd carbon numbers by recombinant strain of A. from cottonseed oil and valeric acid in batch culture of
eutrophus harbouring the PHA synthase gene of Aeromonas Ralstonia sp. strain JC-64 [39].
caviae [29]. Large-scale fermentative production of poly(3-
hydroxybutyrate-co-5 mol% 3-hydroxyhexanoate) (P(3HB- 4.2. PHA production in A. latus
co-5 mol% 3HHx)) from soybean oil as sole carbon source
has been synthesised using the above-mentioned recombi- A. latus produces PHB from cheap C sources such as
nant strain [30]. R. eutropha H16 has been shown to enhance glucose and sucrose at a rate comparable to that of A. eu-
the production of PHB and its copolymer P(3HB-co-3HV) trophus H16. A. latus DSM 1123 is able both to grow and
when given oxidative growth conditions. Cultivation in the produce PHB at 35 ◦ C, thus lowering cooling demands.
presence of hydrogen peroxide and methyl viologen was This strain requires only 5 h to reach an intracellular PHB
shown to stimulate the flux of NADPH to the PHA biosyn- concentration of 80% of dry biomass, and synthesis of PHB
 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619 611

is growth-associated. The PHB concentration in this strain oleovorans grown in media containing n-alkanoic acids,
has been increased from 50 to 87% in fed-batch cultivation from formate to decanoate as carbon source. However, for-
by induction of nitrogen limitation [40]. mation of PHA was observed only for hexanoate and higher
Strains of A. latus can be used to produce PHB from n-alkanoic acids [15]. The same culture has been reported
molasses or sugar syrup. Batch culture of A. latus ATCC to utilise chemically heterogeneous low-rank coal liquefac-
29713 was reported to produce PHB up to 63% of dry tion products as complex C sources for growth and leads
cell mass after 93 h of culture. The average biomass yield to production of various PHAs like 3-hydroxyhexanoate,
coefficient on sucrose was about 0.4 kg/kg. Four nitrogen 3-hydroxydecanoate and 3-hydroxydodecanoate [47]. Pseu-
sources––ammonium chloride, ammonium sulphate, ammo- domonas stutzeri 1317 was found to synthesise a variety of
nium nitrate, and urea—were used and it was reported that PHAs when grown in glucose and/or fatty acids [48]. It was
only the first two supported the culture growth and PHB found that the monomeric structure of the PHAs was depen-
production [41]. Fed-batch (pH-stat feeding profile) fermen- dent on the structures of related fatty acids taken as carbon
tation with the same strain resulted in maximum specific source. Monomer percentage content in PHA was adjusted
growth rate of 0.265 h−1 , compared to 0.075 h−1 achieved by regulating ratios of fatty acids in growth substrates.
with batch cultures. The maximum PHB productivity was Thus, the PHA structures can be tailor made by using
1.15 g/(l h) [42]. structure-related fatty acids and varying ratios of fatty acids.
Acetoacetyl-CoA reductase and the PHA synthase
4.3. PHA production by methylotrophs genes from R. eutropha were expressed in E. coli and
PHA-negative mutants of Pseudomonas putida. E. coli
The cost of producing biodegradable plastic may be re- strains accumulated PHB when grown on fatty acids
duced by the use of methanol as substrate for growth and as carbon substrate [49]. However, P. putida accumu-
PHB production. Fermentor studies using Methylobacterium lated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
extorquens in a continuously stirred baffled fermentor (3.5 l) (P(3HB-co-3HHx)) when even chain fatty acids were pro-
in a discontinuous fed-batch mode for methanol addition was vided as carbon source, and P(3HB-co-3HV) when odd
carried out [43]. Growth was significantly affected above chain fatty acids were provided as carbon source. This sug-
8 g/l methanol and the optimal methanol concentration was gests that fatty acid degradation can be directly accessed by
estimated as 1.7 g/l. Biomass levels of 9–10 g/l containing employing only acetoacetyl-CoA reductase and the PHA
30–33% PHB were obtained when methanol concentration synthase.
was controlled using an open-loop configuration.
The use of methylotrophs was considered by ICI for the 4.5. PHA production by recombinant E. coli
production of PHB from methanol, which is one of the cheap
carbon sources available. However, accumulation of less E. coli has proven to be a versatile performer not only
than satisfactory amounts of PHA made the recovery pro- in expediting the molecular analysis of PHA biosynthesis,
cess difficult, and the company abandoned this process [2]. but also in: (a) synthesising the biopolymer to extremely
It was demonstrated that a very high concentration (149 g/l) high intracellular levels; (b) being amenable to specific ge-
of PHB could be obtained by fully automatic fed-batch cul- netic strategies such as genetically mediated lysis; and (c)
ture of P. extorquens using methanol as a carbon source. the utilisation of mutants to metabolically engineer strain
However, it took 170 h to reach this concentration, result- that produces P(3HB-co-3HV) copolymer. Synthesis of PHB
ing in low productivity of 0.88 g/(l h) [44]. Thus, the ben- by recombinant E. coli does not require limitation of spe-
efit of producing a high concentration of PHA from cheap cific nutrient, but is dependent on the amount of acetyl-CoA
methanol will be fully appreciated when the productivity available. Advantages of employing recombinant E. coli for
can be enhanced at least by two-fold. the production of PHA include fast growth, high cell den-
sity, ability to use several inexpensive carbon sources and
4.4. PHA production by Pseudomonas species easy purification [50,51]. Given the vast amount of knowl-
edge concerning E. coli genetics and the increasing amount
Production of MCL-PHA by fed-batch and continuous of knowledge concerning the metabolic events necessary for
culture of P. oleovorans has been investigated to achieve PHA synthesis, it is fully expected that E. coli will continue
high concentration of PHA and high productivity. With to play a significant part in the determination of the mecha-
optimisation of culture conditions the steady state cell con- nisms of PHA synthesis and, possibly, the commercial pro-
centration and productivity of 11.6 g/l and 0.58 g/(l h), re- duction of PHAs.
spectively, were reached in a continuous mode of operation Production of PHA by recombinant E. coli harbouring A.
[45]. P. oleovorans NRRL B-778 was shown to accumulate eutrophus PHA biosynthesis genes has been investigated.
mixture of PHB and MCL-PHAs when grown on glucose The accumulation of PHB in recombinant E. coli could
and octanoic acid whereas copolymers of P(3HB-co-3HV) reach 80–90% of dry cell weight at the end of cultivation.
were produced during grown on nonanoic acid [46]. For- A PHB concentration of higher than 80 g/l with produc-
mation of intracellular PHA has been demonstrated in P. tivity greater than 2 g/(l h) has been obtained by pH-stat
612 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619

fed-batch culture of recombinant E. coli [52]. However, the In addition to the above-mentioned micro-organisms,
problem of high oxygen demand during the fermentation PHB production has also been demonstrated in Thiosphaera
need to be addressed to allow this process to be useful pantotropha [60], Caulobacter crescentus DSM 4727 [61],
and economical alternative to the ones currently in use. Azotobacter sp. strain FA8 [62], Synechococcus sp. MA19
Molasses has also been used for the production of PHB [63,64], Synechocystis sp. PCC6803 [65], Rhodopseu-
from recombinant E. coli strain HMS174/pTZ18u-PHB domonas palustris 42OL [66], Bacillus cereus UW85 [67],
containing plasmid TZ18u-PHB (which carries A. eutro- Zoogloea sp. (Z5-GII) [68], M. extorquens strain P14 [69],
phus PHB biosynthetic genes and ampicillin resistance) Methylobacterium rhodesianum MB 126 [25], Bacillus
[53]. The final dry cell weight and PHB content was mycoides RLJ B-017 [70], Azospirillum brasilense [71],
39.5 g/l and 80% (w/w) after 35 h of fermentation. Recom- Americoccus kaplicenses [72]. However, no reports are
binant E. coli XL1-Blue harbouring plasmid pKSSE5.3 available for large-scale PHB production from any of the
was used to produce poly(4-hydroxybutyric acid) ho- above cultures.
mopolyester using mineral medium containing glucose and
4-hydroxybutyric acid as carbon source [54]. The final 4.6. PHA production from cheap substrates
cell dry weight and P(4HB) concentration was found to be
12.6 and 4.4 g/l, respectively, after 60 h of fed-batch fer- Despite its basic attractiveness as a substitute for
mentation at constant pH. Recombinant E. coli containing petroleum-derived polymers, the major hurdle facing com-
Aeromonas PHA biosynthetic genes was shown to produce mercial production and application of PHA in consumer
a terpolymer of 3-hydroxybutyrate and 3-hydroxyvalerate products is the high cost of bacterial fermentation, mak-
and 3-hydroxyhexanoate (P(3HB-co-3HV-co-3HHX)) ing bacterial PHA 5–10 times more expensive than the
from dodecanoic acid plus odd carbon number fatty acid petroleum-derived polymers, such as polypropylene and
[55]. A recombinant E. coli strain has been reported that polyethylene, which costs approximately US$ 0.25–0.5 kg−1
produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [73]. The most significant factor for increasing the produc-
when grown in complex medium containing glucose. tion cost of PHA is the cost of substrate (mainly carbon
This was accomplished by introducing into E. coli source). In order to reduce this cost, several recombinant
DH5␣, separate plasmids harbouring the PHA biosyn- strains utilising a cheap carbon source and corresponding
thesis genes from R. eutropha and the succinate degra- fermentation strategies have been developed. There have
dation genes from Clostridium kluyveri, respectively. also been several reports on the production of PHAs from
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) levels up cheap carbon sources by wild-type PHA producers. How-
to 50% of the cell dry weight containing up to 2.8 mol% ever, the polymer concentration and content obtained were
4-hydroxybutyrate has been obtained [56]. considerably lower than those obtained using purified car-
A genomic DNA fragment from Streptomyces aureofa- bon substrates. Therefore, there is a need for development
ciens NRRL 2209 was cloned in a plasmid vector and in- of more efficient fermentation strategies for production of
troduced into E. coli. The recombinant E. coli accumulated these polymers from a cheap carbon source.
PHB as cytoplasmic inclusions with a molecular weight of Pseudomonas cepacia ATCC 17759 grown on xylose pro-
2.85 × 105 [57]. Recombinant E. coli cells accumulated duced 2.6 g/l biomass with 60% (w/w) PHB in shake flasks
25–28 times more PHB than the native S. aureofaciens when on ammonium-limited mineral salts culture medium with
grown on glycerol as the carbon source. Concentrated whey 10 g/l xylose. Substrate cost (in terms of hydrolysed hemi-
solution has also been used for the production of PHB by cellulose) for PHB production was comparable to that of
using recombinant E. coli (containing A. latus PHA biosyn- cane molasses and half that of bulk glucose [74]. PHB and
thesis gene). Final cell and PHB concentrations of 119.5 P(3HB-co-3HV) have also been produced from activated
and 96.2 g/l, respectively, were obtained in 37.5 h, which re- sludge. Addition of valerate to the medium lead to the pro-
sulted in PHB productivity of 2.57 g/(l h) [58]. duction of copolymer whereas when butyric acid was added
A novel recombinant E. coli strain VG1 (pTU14) was only PHB was produced [75]. A recombinant E. coli strain
obtained by cloning the Vitreoscilla globin gene (vgb) of has been reported to carry out the production of PHB on
Vitreoscilla into E. coli. Introduction of this gene led to molasses as carbon source. The final dry cell weight, PHB
decrease not only in the critical oxygen concentration, but content and PHB productivity were 39.5 g/l, 80% (w/w) and
also affected the volumetric oxygen transfer coefficient of 1 g/(l h), respectively [53]. PHB was produced by R. eu-
the recombinant strain. Initially, the kLa was very high and tropha DSM 11348 when it was grown in a culture medium
then decreased quickly with growth of VG1 (pTU14) cells. containing 20–30 g/l casein peptone or casamino acids as a
This decrease ceased and was unchanged for a very long sole N source [76]. Residual oil from biotechnological rham-
time when the DO was lower than about 50%, however, nose production was used as a C-source for growth and for
a slight increase appeared when DO decreased to about accumulation of PHA by R. eutropha H16 and P. oleovorans.
10%. Thus, high cell density and PHB accumulation at Approximately 20–25% of the components of the residual
low production cost was obtained at low DO concentrations oil were converted into PHA [32]. Bacillus megaterium was
[59]. grown on various carbon sources such as date syrup, beet
 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619 613

molasses, fructose, lactose, sucrose, glucose in mineral salts a significant reduction in growth and seed production rel-
medium. Best results with regard to growth and PHB pro- ative to wild-type. The apparent problems associated with
duction were obtained in the cheaper carbon sources also, accumulation of PHB granules in various organelles and
for example, in date syrup and beet molasses [77]. the reduced growth of transgenic plants may be alleviated
by regulating the tissue specificity, timing of expression,
4.7. PHA production by transgenic plants and cellular localisation of the enzymes involved in PHB
synthesis.
Synthesis of PHA in plants was first demonstrated in PHB synthesis has been demonstrated in the cells of cot-
1992 by the accumulation of PHB in the cytoplasm of ton fibres [79]. In this case, the PHA produced was used
cells of Arabidopsis thaliana [78]. Since then, a range of as an intracellular agent that modifies the heat exchange
different PHAs had been synthesised in various species properties of the fibre. The phaA, phaB and phaC genes
through the creation of novel metabolic pathways either in from R. eutropha were expressed in transgenic cotton under
the cytoplasm, plastid or peroxisome. Although the initial the control of a fibre specific promoter [79]. In Nicotiana
driving force behind synthesis of PHA in plants was for tabacum PHB was synthesised through the co-expression of
the biotechnological production of biodegradable polymers, the phaB gene from R. eutropha and the PHA synthase from
PHA synthesis in plants has more recently emerged as a A. caviae [80]. By targeting the three enzymes of the entire
useful and novel tool to study fundamental aspects of plant PHB synthetic pathway to plastids, the PHB yield in leaves
metabolism. It was known that PHB was synthesised in bac- of transgenic Arabidopsis has been increased to 140 mg/g
teria from acetyl-CoA. Since acetyl-CoA is present in plant dry weight without much disturbance of plant growth and
cells in the cytosol, plastid, mitochondrion and peroxisome, development [81]. Different crop species have been used for
the synthesis of PHB in plants could, in theory, be achieved transformation with PHA genes, but formation of significant
in any of these sub-cellular compartments. However, the amounts (>1%) of PHAs has only been seen in oil seed rape
cytoplasm was targeted as the first site for PHB synthesis (Brassica napus), with a maximum storage capacity of up
because it had the advantage that the bacterial enzymes to 7.7% (fresh weight) in the seeds [82].
could be directly expressed in this compartment without Plastics produced in plants would be a renewable re-
any modification of the proteins. Moreover, the enzyme source and would have prices comparable to that of
3-ketothiolase was present in the cytoplasm of higher plants, non-biodegradable plastics produced from oil. There is a
where it is involved in the synthesis of mevalonate, the pre- complete range of genes that are available from various
cursor to isoprenoids. The same enzyme is involved in the micro-organisms and can be used for metabolic engineer-
synthesis of PHB in R. eutropha. Thus, the production of ing of plants. All of these achievements indicate that it is
PHB in the cytoplasm of plants required the expression of possible to direct the synthesis of PHA to a specified loca-
two additional enzymes, reductase and synthase. So the R. tion in transgenic plants. For large-scale production, it has
eutropha phbB and phbC genes, encoding acetoacetyl-CoA been suggested that oilseed crops such as rapeseed would
reductase and PHB synthase, respectively, were expressed appear to be a particularly good target crop since results
into plants to complete the PHB biosynthetic pathway un- obtained with PHA production in the closely related species
der the control of the cauliflower mosaic virus (CaMV) A. thaliana could be directly applied to rapeseed [73]. The
35S promoter, allowing a relatively high expression of the recent discoveries of metabolic links to PHA from fatty
enzymes in a broad range of tissues [78]. PHA granules, acid biosynthesis [83] and ␤-oxidation [23] in bacteria will
which had accumulated in plant cells, were visualised by be additional advantages since in plants the PHA produc-
both epifluorescence microscopy of tissues stained with tion pathway would eventually have to be metabolically
Nile Blue A and by transmission electron microscopy [78]. engineered from unrelated carbon sources [84].
Both of these methods revealed the presence of granules in
all organelles of the hybrid transgenic plants, including root,
leaf, cotyledon, and seed. In addition to cytoplasm, PHB 5. Properties and applications
granules were found in cytosol, vacuole and nucleus [78].
However, no PHB inclusions were found in plastids and PHAs are partially crystalline polymers with a degree
mitochondria. From these results, it was assumed that the of crystallinity in the range of 60–80% [85]. Within the
hydrophobic nature of PHB inclusions allows them to pass bacterial cell they exist as amorphous and water-soluble
through the single membrane of the vacuole but not through inclusions [86]. Upon disruption of cells, when the poly-
the double membrane of organelles such as the plastid and mer is extracted rapid crystallisation occurs. This may be
mitochondrion. Their presence in the nucleus may be due explained by a kinetic nucleation mechanism. This implies
to some affinity of the inclusions for nuclear constituents. that polymer granules within cells are very small and that
However, a major problem for the production of PHA probability of a nucleation event triggering crystallisation
in plants is the adverse effect of the expression of the so low. It is only when the cell is disrupted and granules
phb genes on plant growth. Expression of high amounts allowed to coalesce that rapid heterogeneous nucleation is
of acetoacetyl-CoA reductase in transgenic plants caused possible.
614 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619

Table 1
Comparison of polymer properties [6,110–112]
Polymer Melting point (◦ C) Young’s modulus Tensile strength Elongation to Glass transition
(GPa) (MPa) break (%) temperature (◦ C)
P(3HB) 179 3.5 40 5 4
P(3HB-co-3HV)
3 mol% 3HV 170 2.9 38 – –
9 mol% 3HV 162 1.9 37 – –
14 mol% 3HV 150 1.5 35 – –
20 mol% 3HV 145 1.2 32 – –
25 mol% 3HV 137 0.7 30 – –
P(3HB-co-4HB)
3 mol% 4HB 166 – 28 45 –
10 mol% 4HB 159 – 24 242 –
16 mol% 4HB – – 26 444 –
64 mol% 4HB 50 30 17 591 –
90 mol% 4HB 50 100 65 1080 –
P(4HB) 53 149 104 1000 –
P(3HHx-co-3HO) 61 – 10 300 –
P(3HB-co-6 mol% 3HA) 133 0.2 17 680 −8
P(3HB-co-67 mol% HP) 44 – – – −19
P(3HB-co-3HHx) 52 – 20 850 −4
Polypropylene 170 1.7 34.5 400 45
Polyethylene–terepthalate 262 2.2 56 7300 3400
polystyrene 110 3.1 50 – 21
LDPE 130 0.2 10 620 −30

The family of PHAs exhibits a wide variety of mechan- by using a simple annealing treatment after initial crystalli-
ical properties from hard crystalline to elastic, depending sation [91].
on composition of monomer units which broadens its appli- In addition to improvement in physical properties due
cation area, for example, MCL-PHAs are semi-crystalline to increased molecular weight incorporation of other
elastomers with low melting point, low tensile strength and hydroxy-acid units to form PHA copolymers can also im-
high elongation to break [87] and can be used as biodegrad- prove other properties such as crystallinity, melting point,
able rubber after cross linking. stiffness and toughness. Random copolymers containing
Since PHB is the most widely studied polymer so the 3HB as a constituent along with other HA units of chain
properties of PHAs have been explained by taking it lengths ranging from 3 to 14 carbon atoms have been pro-
into consideration. The molecular weight of PHB pro- duced from various carbon substrates by a variety of bacte-
duced from wild-type bacteria is usually in the range of ria. PHA composition produced by bacteria is dependent on
10,000–3,000,000 Da with a polydispersity of around 2 the substrate specificities of enzymes in the PHA biosynthe-
[88]. The glass transition temperature is near 180 ◦ C as mea- sis pathway. Typical PHA copolymer and micro-organisms
sured by calorimetric analysis. The densities of crystalline producing them are shown in Table 2.
and amorphous PHB are 1.26 and 1.18 g/cm3 , respectively. The structure and properties of random copolymers of
Mechanical properties like Young’s modulus and tensile 3HB and 3HV have been investigated most extensively.
strength are close to that of polypropylene though exten- P(3HB-co-3HV) copolymers have approximately the same
sion to break is markedly lower than that of polypropylene. degrees of crystallinity as homopolymer (50–70%) [92].
Hence, PHB is stiffer and becomes brittle over a period of Copolymers of 3HB and 3HV are isodimorphic, i.e. they dis-
several days upon storage under ambient conditions [89]. play a melting point minimum at 3HV content of 30 mol%
Comparison of properties of PHB with other plastics is and cocrystallisation of the two monomer units occurs in
given in Table 1. Much work has been carried out to under- either of the homopolymer crystal lattices of PHB and PHV
stand the reasons behind the brittle nature of PHB and to depending on whether the 3HV component is above or be-
improve the polymer’s physical properties. It has been re- low 40 mol%. As the fraction of 3HV increases the copoly-
ported that embrittlement of PHB materials occurs during mer becomes tougher (increase in impact strength) and more
storage after their initial crystallisation from the melt [90]. flexible (decrease in Young’s modulus) as can be seen from
This secondary crystallisation results in reorganisation of Table 1. The elongation to break also increases. Further-
lamellar crystals formed during the initial crystallisation pro- more, the decrease of melting temperature with increasing
cess which tightly constrains the amorphous chains between 3HV fraction without affecting degradation temperature al-
crystals. This can be prevented and PHB can be toughened lows thermal processing of copolymer melts without thermal
 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619 615

Table 2
Copolymers produced by various micro-organisms [85]
Micro-organism Carbon substrate Copolymer

R. eutropha Propionic acid P(3HB-co-3HV)

R. eutropha Pentanoic acid, 3-hydroxypropionic acid P(3HB-3HP)
A. latus, Aeromonas cavie 1,5-Pentadiol, plant oils P(3HB-3HHx)
Pseudomonas sp., R. eutropha Sugars, 4-hydroxybutyric acid P(3HB-3HD)
A. latus, Comamonas acidovorans -Butyrolactone, 1,4-butanediol, 1,6-hexanediol P(3HB-4HB)

degradation. Thus, material properties can be controlled by 5.75 Å) [96]. A detailed description for the extraction of
adjusting the fraction of 3HV during the fermentation. PHB from biomass with solvents has been summarised else-
Introduction of co-monomers other than 3HV such a where [97].
3-hydroxyhexanoate, 3-hydroxypropionate, 4-hydroxybuty- The above-mentioned methods are time-consuming and
rate into PHB chain also give copolymers of improved not accurate particularly for low PHB concentrations [98].
mechanical properties as shown in Table 1. The physical Large sample volumes and a high degree of replicates are
and thermal properties can be regulated by varying the required to minimise errors. Furthermore, cell material can
copolymer compositions. interfere with PHB extraction [99]. These methods allow for
PHAs have a wide range of applications owing to their the quantification of total PHA but not the individual PHA
novel features. Initially, they were used in packaging films components. The use of gas chromatography (GC) for the
mainly in bags, containers and paper coatings. Similar ap- identification of PHA components was then proposed. This
plications as conventional commodity plastics include the method involves simultaneous extraction and methanolysis
disposable items, such as razors, utensils, diapers, feminine of PHA, in mild acid or alkaline conditions, to form hy-
hygiene products, cosmetic containers—shampoo bottles droxyalkanoate methylesters which are then analysed by GC
and cups. In addition to potential as a plastic material, PHA [99]. This method is rapid (4 h), sensitive, reproducible and
are also useful as stereo regular compounds which can serve requires only small samples. It features reaction and extrac-
as chiral precursors for the chemical synthesis of optically tion in the same screw-capped tube.
active compounds [93,94]. Such compounds are particularly Another GC method for increased PHA recovery was pro-
used as biodegradable carriers for long-term dosage of drugs, posed by carrying out propanolysis in HCl [100] rather than
medicines, hormones, insecticides and herbicides. They are acidic methanolysis in sulphuric acid.
also used as osteosynthetic materials in the stimulation of Other recent methods for PHA quantification include
bone growth owing to their piezoelectric properties, in bone HPLC [101], ionic chromatography, and enzymic determi-
plates, surgical sutures and blood vessel replacements. nation [102]. HPLC measures only PHB and is based on
conversion of PHB to crotonic acid followed by UV detec-
tion at 210 nm. This method features 84% recovery of PHB.
6. Quantification of polyhydroxyalkanoates PHA detection by ionic chromatography is based on
the conversion of monomers to alkanoic acids. The de-
After the fermentation is over, the cells containing PHB termination involves acid propanolysis followed by an
are separated from fermentation broth by centrifugation. The alkaline hydrolysis with Ca(OH)2 or acidic hydrolysis
harvested cells are then lysed for recovery of PHAs. A num- with concentrated H2 SO4 . After centrifugation, the sam-
ber of different methods are available in literature for recov- ple is injected into an anionic column with a conductivity
ery of PHAs. Most of the methods have been standardised detector. An enzymic estimation method was developed
for PHB production by R. eutropha. Until the end of the by Roche Molecular Biochemicals, USA (No. 127833)
1950s, the most common analytical technique used for PHA [102]. 3HB is enzymically oxidised and the NADH pro-
estimation was a gravimetric method [12], which consisted duced from NAD+ was reoxidised in the presence of
of PHB extraction from lyophilised biomass with chloro- iodonitro-tetrazoliumchloride to produce formazan, which
form followed by precipitation with diethyl ether or ace- was spectrophotometerically measured at 492 nm.
tone. In 1958, Williamson and Wilkinson showed that under The determination of PHA inside intact cells by two-
controlled conditions of time and temperature all cell ma- dimensional fluorescence spectroscopy and flow cytometry
terial, except PHB granules, dissolved in alkaline sodium has also been proposed recently [103,104]. Cells stained
hypochlorite solution [9]. with Nile Blue, show a clear fluorescence maximum between
Further developments on this method were introduced 570 and 605 nm when excited between 540 and 560 nm. A
[95] wherein the extracted PHB was converted (with concen- good correlation between fluorescence intensity and PHB
trated sulphuric acid) to crotonic acid, and estimated spec- concentration was obtained. However, differentiation of
trophotometerically at 235 nm. Detection of PHA extracted PHA composition was not possible with this method [104].
with chloroform can also be done by IR spectroscopy (at Ease of recovery of PHA is a very important parameter
616 S. Khanna, A.K. Srivastava / Process Biochemistry 40 (2005) 607–619

in their economical production. Thus, there is a need for enzymic hydrolysis of PHB by depolymerase produces
development of methods for extraction of PHB so that the a 3HB dimer as a major product and small amounts of
overall process could be made much simpler and cheaper. 3HB monomer. Hydrolysis of end labelled 3HB oligomers
by PHB depolymerase indicated that the enzyme mainly
cleaved the second and third ester linkage from the hydroxyl
7. Biodegradability of PHAs terminus [108]. Copolymers containing 4HB monomer
units degraded more rapidly than PHB or P(3HB-co-3HV)
One of the unique properties of biological PHA materi- copolymers [9]. Crystallinity and size of PHB crystal also
als is their biodegradability in various environments. The influenced the degradation rates. For melt crystallised PHB
biodegradability of PHA materials in natural environments films rate of enzymic hydrolysis decreased with increase in
such as soil, sea water and lake water has been evaluated crystallinity and average size of PHB crystal [85].
and it has been found that rate of biodegradation was influ-
enced by a number of factors such as microbial population
in a given environment, temperature, moisture level, pH, nu- 8. Prospects
trient supply as well as composition, crystallinity, additives
and surface area of PHA itself. At present PHB and P(3HB-co-3HV) are the only mem-
Since PHA is a solid polymer with a high molecular bers of PHAs that are produced on a commercial scale. Even
weight and is incapable of being transported through the though the price of PHAs is still too high, current advances
cell wall various micro-organisms excrete extracellular in fermentation and purification technology as well as the
PHA depolymerases that hydrolyse PHA into water-soluble development of superior bacterial strains by recombinant
oligomers and monomers and subsequently utilise these DNA technology are likely to lower the price of PHA so
resulting products as nutrients within the cells. Many such that it is close to that of other biodegradable plastic mate-
micro-organisms including bacteria and fungi have been rials such as polylactide and aliphatic polyesters. The iso-
isolated from various environments as shown in Table 3. lation and development of bacterial strains that can utilise
The biochemical and molecular characterisation of PHB cheap carbon substrates should be pursued intensively. Sev-
depolymerases has shown that all enzymes are comprised of eral bacterial strains described earlier should be employed
N-terminal catalytic domain, a C-terminal substrate binding for the production of PHAs over the next few years but even-
domain which is hydrophobic and a linker region connecting tually metabolically engineered bacterial strains may likely
the two domains [105]. surpass the wild-type bacteria currently in use. Recently,
Solid PHB polymer is a water-insoluble substrate while transgenic plants cells harbouring A. eutrophus genes have
PHB depolymerases are soluble in water. The enzymic also been developed with the aim of ultimately reducing the
degradation is therefore a heterogeneous reaction involving price of PHA. The concentration and yield of PHA will in-
two steps, namely, adsorption of enzyme onto the surface crease as the understanding of plant biochemistry and genet-
of PHB material, and hydrolysis of polymer chains by ac- ics improves. With all these advances, it is likely that PHAs
tive site of the enzyme. The adsorption isotherms of PHB will become a major biodegradable plastic in a wide range
depolymerase onto the surface of PHB are expressed by of applications in the near future and will eliminate the dis-
Langmuir adsorption equation [106]. The hydrolysis step posal problems and environmental hazard as are prevalent
takes place by the surface reaction between adsorbed en- with conventional plastic materials.
zymes and free adsorption points on the surface [107]. The
properties of an extracellular PHB depolymerase from Al-
caligenes faecalis have been extensively investigated. The Acknowledgements

The Junior Research Fellowship (JRF) award by Council

Table 3 of Scientific and Industrial Research (CSIR), Govt. of India,
PHA degrading micro-organisms isolated from various environments
New Delhi for the execution of project on PHB production
Source from which isolated Micro-organism Reference is gratefully acknowledged by one of the authors (Ms. Shilpi
Soil Aspergillus fumigatus [113] Khanna).
Acidovorax faecalis [113]
Comamonas sp. [114]
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