431

Plastic bacteria? Progress

and prospects for
polyhydroxyalkanoate production
in bacteria
SangYup Lee

Polyhydroxyalkanoates (PHAs) are synthesized by numerous bacteria as carbon

and energy storage compounds, and are good candidates for biodegradable
plastic material. With the current advances in PHA research, PHA concentrations
of greater than 80 g l 1 with productivities of greater than 2 g PHA I-lh -~ can be
routinely obtained by fed-batch cultivation of several bacteria. Metabolic
engineering approaches have been used to expand the spectrum of utilizable
substrates and to improve PHA production. These advances will lower the price of
PHA from the current market price of ca. US$16 kg-1, and will allow PHAto become
a leading biodegradable plastic material in the near future.

In response to increasing public concern about the
harmful effects of petrochemical-derived plastic ma-
terials in the environment, many countries are con-
ducting various solid-waste management programs,
including plastic waste reduction by developing
biodegradable plastic materials. These biodegradable
plastic materials nmst retain the desired material prop-
erties of conventional synthetic plastics, and should be
completely degraded without leaving any undesirable
residues when discarded. Polyhydroxyalkanoates
(PHAs) are polyesters of various hydroxyalkanoates
which are synthesized by numerous microorganisms as
an energy reserve material, usually when an essential
nutrient such as nitrogen or phosphorus is limited in
the presence of excess carbon source I 4. PHAs are
considered to be strong candidates for biodegradable
polymer material because they possess material prop-
erties similar to various synthetic thermoplastics and
elastomers currently in use (from polypropylene to
synthetic rubber) and, upon disposal, they are con>
pletely degraded to water and carbon dioxide (and
methane under anaerobic conditions) by microorgan-
isms in various environments such as soil, sea and lake
water and sewage s.('.
Poly(3-hydroxy.butyrate) [P(3HB)] is a homopoly-
mer of 3-hydroxybutyrate and is the most widespread
S. Y. Lee Oeesy(~9"orak.haist.ac.kr) is at the Depamnent qf Chemical
Eny,ineerin£~ and BioProcess Engineerin~ Research Cemer, Kowa
Advanced Institute ~f Science and T,'chnolow , Ta~jotl 305-701,
Korea,
and best characterized member of the PHAs. Many
bacteria can synthesize PHAs consisting of different
monomer units when suitable carbon sources, usually
those which exhibit structures related to the PHA con-
stituents, are provided. The monomer units in PHAs
are all in the D-(-) configuration owing to the stereo-
specifici~" of the biosynthetic enzymes 1,3. More than
90 different monomer units have been identified as
constituents of PHAs in various bacteria 7, including
3-hydroxyalkanoates o f 3-12 carbon atoms with a
large variety of R-pendant groups, 4-hydroxD'alka-
noates of 4-8 carbon atoms, 5-hydroxypentanoate,
5-hydroxyhexanoate and 6-hydroxydodecanoate
(Fig. 1). However, only a few of these PHAs have been
produced in amounts sufficient to enable the charac-
terization of their material properties and to develop
potential applications.
P(3HB) is a partially crystalline polymer which has
material properties similar to polypropylene. However,
industrial application of P(3HB) has been hampered
owing to its low thermal stabilitT and excessive
brittleness upon storage s. The copolymer poly-
(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-
co-3HV), developed by Z E N E C A Bio Products
(formerly ICI; Cleveland, UK) is more flexible and
tougher than P(3HB), and can be used to make vari-
ous products, including films, coated paper and board,
compost bags, disposable food service-ware, and
molded products such as bottles and razors. P(3HB-
co-3HV) is produced on a fairly large-scale and sold
under the tradename of B I O P O L T M at U S $ 1 6 k ~ 1

Copyright © 1996, Elsevier Science LtdI All rights reserved. 0167 7799/96/$15.00. PII: S0167-7799(96)10061-5 TIBTECH NOVEMBER1996 (VOL 14)
 432

reviews

accumulate much polymer. Among the more than 300

different microorganisms that are known to syn-
o\ thesize PHAs, only a few bacteria have been employed
for the production o f PHAs. These include Alcaligenes
eutrolohUS 21-23, Alcaligenes [atus 24 , A zotobacter vinelandii 25,26,
several strains o f methylotrophs 2v-29, Pseudomonas
O / J 1 0 0 - 3 0 000 oleovorans 2°,3°,31 and, recently, recombinant AI. eutro-
phus 32, recombinant Escherichia coli33 36 and recombi-
n=l R= hydrogen Poly(3-hydroxyp ropionate) nant KlebsMla aerogenes 37. These bacteria have been
R= methyl Poly(3-hydroxybutyrate) selected mainly because they can be cultivated effi-
R= ethyl Poly(3-hyd roxyvalerate) ciently to high cell densities with a high PHA content
R= propyl Poly(3-hydroxyhexanoate) (defined as the ratio of PHA concentration to dry cell
R= pentyl Poly(3-hydroxyoctanoate) concentration expressed as a percentage) in a relatively
R= nonyl Poly(3-hydroxydodecanoate) short period of time, resulting in high PHA produc-
tivity (defined as gram PHA produced per liter per
n=2 R = hydrogen Poly(4-hydroxybutyrate) hour) 16. High productivity is one of the most impor-
R = methyl Poly(4-hydroxyvalerate) tant factors for the economical production o f these
biodegradable polymers. The carbon source should be
n=3 R =hydrogen Poly(5-hydroxyvalerate) inexpensive since it is the major contributor to the
R = methyl Poly(5-hydroxyhexanoate) total substrate cost. In the same context, the yield of
PHA with respect to carbon substrate (defined as gram
n=4 R = hexyl Poly(6-hydroxydodecanoate) PHA produced per gram carbon substrate consumed)
should be high so as not to waste substrate to non-
Figure 1 PHA materials. The PHA content can be considered
Generalstructure of polyhydroxyalkanoatesand some representative as the measurement of the cell's ability to accumulate
members3,7,9.
PHA in a given condition. High PHA content often
results in high PHA yield and is beneficial for the
(L. A. Naylor, pers. commun.). Considering that the recovery process.
price ofpolypropylene is less than US$1 k ~ 1, one of
the major problems that prevents the commercial Fermentation strategies
application of PHAs is their high price. Much effort Bacteria that are used for the production o f PHAs
has been devoted to reduce the price o f PHAs by the can be divided into two groups based on the culture
development of better bacterial strains, more efficient conditions required for PHA synthesis. The first group
fermentation and more economical recovery of bacteria requires the limitation of an essential nutri-
processes. A number of recent review articles are avail- ent such as N, R Mg, K, O or S for the efficient
able for further details: on general features o f synthesis of PHA from an excess carbon source. The
PHAs 1-4,9-12, physiological and genetic aspects o f PHA second group of bacteria does not require nutrient
biosynthesis >4,13, physical and chemical properties of limitation for PHA synthesis, and can accumulate
PHAs 8,t4,15, production and recovery ofPHAs 4,9,16and polymer during growth. Alcaligenes eutrophus,
biodegradation o f PHAs s,<17. In this paper, recent Protomonas exwrquens, Ps. oleovorans and many other
advances in the development of processes for the bacteria belong to the first group, while some bacteria
production of PHAs employing several bacteria are such as Al. latus, a mutant strain o f A z . vinelandii, and
reviewed and their prospects are discussed. The cur- recombinant E. coli harboring the A1. eutrophus PHA
rent status and future prospects o f metabolic engi- biosynthesis operon belong to the second group.
neering for strain improvement and synthesis of novel Therefore, these characteristics should be considered
polymers are also discussed. in developing cultivation methods for the efficient
production o f PHAs. Either fed-batch or continuous
Production strategies cultivation techniques can be used for the production
Even though many different PHAs have been of PHA with high productivity.
described in the literature, only a few have been pro- For the fed-batch culture of bacteria belonging to
duced in quantities suitable for material characteriz- the first group, a two-step cultivation method (but not
ation and development o f applications. These are necessarily requiring two fermentor vessels) is most
P(3HB) (Ref. 16), P(3HB-co-3HV) (P, ef. 16), often employed. Cells are first grown to a desired con-
P(3HV) (P,.ef. 18), poly(4-hydroxybutyrate)[P(4HB)] centration without nutrient limitation, after which an
(Ref. 19) and poly(3-hydroxyhexanoate-co-3- essential nutrient is limited to allow efficient PHA syn-
hydroxyoctanoate) [P(3HHx-co-3HO)] (Ref. 20). thesis. During this nutrient limitation stage the resid-
The other known PHAs have been produced in rather ual cell concentration (defined as the cell concen-
low amounts either because the substrates (often the tration minus the PHA concentration) remains almost
analogs of the unusual monomer units in the polymer) constant, and the cell concentration increases only
are expensive or toxic to ceils, or because bacteria syn- because o f the intracellular accumulation o f PHA.
thesizing these PHAs are difficult to cultivate or do not Alcali2enes eutrophus, the bacterimn currently employed

TIBTECH NOVEMBER1996 (VOL 14)

 433
reviews
for the commercial production of P(3HB) and
P(3HB-co-3HV), accumulates a large amount of
polymer (up to 80% of dry cell weight) when nitro-
gen or phosphorus is completely depleted 21 23. H o w -
ever, most other bacteria belonging to the first group,
such as Pr. extorquens 27 and Ps. oleovorans2°, produce
PHA more efficiently when a nutrient is limited
but not completely depleted. For the cultivation
of these bacteria, a mixture o f carbon source and a
nutrient to be limited at an optimal ratio should be
fed to produce PHA with high productivity. Since the
cell concentration at which a nutrient is initially
limited significantly affects the final PHA concen-
tration obtainable, it should be optimized with each
bacterial strain to be employed. A premature limi-
tation of nutrient will result in low final cell and PHA
concentrations, resulting in low PHA productivity,
even though high PHA contents may be obtained.
If application of nutrient limitation is delayed too
long, cells are not able to accumulate much polymer,
resulting in a low PHA content and a low PHA pro-
ductivity even though high cell concentrations can
be achieved. This has been demonstrated experimen-
tally with AI. eutrophus using nitrogen as a limiting
nutrient 22. P(3HB) concentrations of 71 and 92 g F 1
were obtained by limiting nitrogen at cell concen-
trations of 30 and 5 5 g F 1, respectively. By further
delaying the nitrogen limitation, until the cell con-
centration reached 70 g 1<, a final P(3HB) concen-
tration of 121 g F ~ could be obtained (Fig. 2). How-
ever, delaying nitrogen limitation, until the cell con-
centration reached 9 0 g F 1 resulted in lower PHA
concentration and productivity2=.
For the fed-batch culture of bacteria belonging to
the second group, the development of a nutrient feed-
ing strategy is crucial to the success of the fermen-
tation. Complex nitrogen sources such as corn steep
liquor, yeast extract or fish peptone can be supple-
mented to enhance cell growth as well as polymer
accunmlation since PHA synthesis is not dependent
on nutrient limitation in these bacteria 2<3s. Cell
growth and PHA accumulation need to be balanced
to avoid incomplete accumulation of PHA or prema-
ture termination of fermentation at low cell concen-
tration. There is an interesting relationship between
the residual cell concentration and PHA content.
Since PHA is accumulated in the cytoplasm, the resid-
ual cell concentration will determine how much PHA
can potentially be produced. A high PHA content
with a low residual cell concentration will result in a
low PHA concentration and productivity. A high
residual cell concentration with a low PHA content
will also reduce the final PHA concentration, prod-
uctivity and yield. A high residual cell concentration
with a high PHA content will give the best results but
there exists an upper limit of PHA concentration that
can be obtained owing to the maximum cell concen-
tration practically achievable in a fermentor (e.g.
ca. 280 gl -< for A1. eutrophus and 200 gl < for recombi-
nant E. coil>). This can be better understood by the
following simple equations:
x = R + p (1)
f= e x, (2)
from Eqns 1 and 2,
P = R f (1 _ f ) - I (3)
since
X = P f - ' <_X~I.... P,,~a_*-< X~..... ~,,ax (4)
where 2(, P and R are the concentrations of cell, PHA
and residual cell, respectively, fi is the PHA content,
and P, .... .X~,a.~ and fnax are the maximum attainable
PHA concentration, cell concentration and PHA
content, respectively.
It is important to decide when to stop the culti-
vation. In most cases, fermentation should be stopped
when the productivity is highest (point PI in Fig. 2).
Cells can be cultivated further to obtain a higher PHA
concentration (point P2 in Fig. 2), but this may result
in lowered overall productivity. Prolonged cultivation
to achieve higher PHA concentration with a slightly
lower productivity will be advantageous only if the
PHA content is also increased, thus allowing easier
recovery and purification of the polymer.
Theoretically, continuous cultivation should give
highest PHA productivities. To date, however, AI. latus
and Ps. oleovorans are the only bacteria that have been
successfully cultivated continuously for the production
o f P H A (Refs 30,31). A steady-state P(3HB) concen-
tration of 16.2 g1-1 with a productivity of 2.55 g F 1h <
could be obtained by continuous cultivation of
A1. latus from sucrose (T. Yamane, pers. commun.).
Pseudomonas oleovorans was cultivated at varying dilution
rates in a two-liquid phase medium containing 15%
(v/v) octane with ammonium as the limiting nutrient.
Cell concentration and PHA content decreased from
2.25gl -1 and 46.7% to 1.32gl -1 and 8.3%, by increas-
ing the dilution rate from 0.09 to 0.46h <, respec-
tively31. A steady-state cell concentration of 11.6 gl <
and a PHA productivity of 0 . 5 8 g l < h 1 could be
obtained by optimizing the feeding strategy and
increasing the oxygen transfer rate > . Even though
continuous cultivation of other PHA producers has
not been reported, a general strategy can be suggested
as follows. A two-stage chemostat should be employed
for most of the bacteria belonging to the first group.
For those bacteria in the first group that do not require
a great change in the concentration of a limiting nutri-
ent between the cell growth phase and the polymer
accumulation phase, such as Ps. oleovorans, a one-stage
chemostat may be used, as previously demonstrated 3°.
However, two-stage chemostats will also definitely
result in higher PHA productivities for these bacteria.
For the continuous cultivation of bacteria belonging
to the second group, a one-stage chemostat can be
employed.
Recently, a simulation study on the two-stage con-
tinuous cultiwttion o f A l . eutrophus has been reported,
in which it was established how the PHA productiv-
ity might be maximized 4°. Under optimized condi-
tions, cell and PHA concentrations in the second
-TIBTECH NOVEMBER 1996 (VOL 14)

reviews
200
---(3-- Cell
~-- 150 ! ~0~ P(3HB) / O ~ ~ L
~~- ~ Residualcell // ~ of starch (corn and tapioca), cellulose and hemicellu-
.o P2~
~ 100
{3
0 by recombinant K. aero~enes from molasses has been
o 50
0 - i i i i /
0 20 30 40 50 60
Time (h)
Figure2
Time profiles of the concentrations of cell, P(3HB) and residual cell during the fed-
batch culture of Alcahgeneseutrophus (redrawn from Kim et al.22). Line L was drawn
to find the point of maximum productivity from the time profile of P(3HB) concen-
tration. The highest productivity is at P1 even though the highest P(3HB) concentration
isat P2.
fermentor were 75 and 44.8 gl t, respectively, at a dilu-
tion rate of 0.064 h 1, resulting in a PHA productivity
of 2.86 gl l h 1. The productivity of the two-stage
chemostats was 1.7 times higher than that of the one-
stage chemostats. More experiments need to be
carried out to see if continuous cultivation can truly
give higher productivity than fed-batch cultures, with-
out any process problems such as culture instability and
contamination. In particular, AI. latus, recombinant
E. coli and several other bacteria which produce PHA
in a growth-associated manner seem to be good
candidates for application in chemostats. However, it
should be mentioned that PHA yields are generally
lower in continuous cultures and, therefore, a careful
economic analysis has to be performed by consider-
ing both productivity and yield.
Carbon substrate and yield
Excluding the recovery process, the economics of
PHA production are largely determined by substrate
cost and PHA yield. The efficiency of substrate con-
version is important, and can be predicted from the
physiology and biochemistry involved in PHA syn-
thesis. Among the various nutrients in the fermen-
tation medium, the carbon source contributes most
significantly to the overall substrate cost in PHA pro-
duction. A number of carbon sources, including
carbohydrates, oils, alcohols, acids and hydrocarbons,
can be used by various bacteria. Recently, the theo-
retical yield (the yield based on the reaction stoi-
chiometry) of P(3HB) has been estimated for several
of these carbon substrates 41. Regeneration of nico-
tinamide nucleotides, which are used as cotqtctors for
PHA synthesis, has been taken into account in this
analysis. It was also suggested that the overall yield,
which is the yield in actual fermentation, would be
TIBTECH NOVEMBER1996 (VOL 14)
434
roughly proportional to the theoretical yield and PHA
content 41. Table 1 summarizes the cost of carbon sub-
strate based on the theoretical yield. Because of their
low price, crude carbon substrates such as cane and
beet molasses, cheese whey, plant oils and hydrolysates
lose can be excellent substrates to several bacteria uti-
lizing them. Production of PHA by AI. eutrophus from
plant oil or tapioca hydrolysate, by Az. vitwlandii
from molasses, by recombinant E. coli from whey and
demonstrated ~6,25,3(,,43
Production of PHA
At present, Z E N E C A Bio Products is the only com-
/ mercial producer of PHA by fed-batch culture of
J AL eutrophus*. The current production of P(3HB-co-
3HV) is about 1000 tonnes per year 12. Various bacteria
have been employed in different fed-batch processes
for the production of PHA, and the details of these
processes can be found elsewhere I(~. Alcaligenes eutro-
phus has most often been employed for the production
of P(3HB) and P(3HB-co-3HV), and the high prod-
uctivities of 2.42 and 2.55 gl / h l , respectively, have
been achieved22,23. Propionic acid has been used as a
co-substrate for the production o f the copolymer
P(3HB-co-3HV). Since propionic acid is about twice
as expensive as glucose, the price of P(3HB-co-3HV)
is inevitably higher than that of P(3HB). Fed-batch
cultures of AI. eutrophus using other carbon sources
such as ethanol, oleic acid and even carbon dioxide
(with hydrogen) have also been reported I(~. Alcaligenes
lares is also a good candidate for the production of
P(3HB) since it grows fast and accumulates a large
amount of polymer during growth. It also utilizes
sucrose most efficiently, and so cheap molasses can be
used to lower the production cost of PHA. The
biotechnological research unit Biotechnologisch
Forschungsgesellschaft (btE Austria) had developed a
method of producing 1 tonne of P(3HB) in a week in
a 15m 3 fermentor 24. Even though this process is no
longer operational, there has been continued research
interest in A1. latus in academia. It was recently
reported that cell and P(3HB) concentrations of 142
and 68.4gl 1, respectively, were obtained in 18h by
the pH-stat fed-batch culture of AI. latus using high
inoculum size (13.7g I)CW1 1), resulting in a high
PHA productivity of 3.97 gl--1 h I (lZef. 51). However,
the PHA content was rather low' (50%).
Owing to the low price of methanol, methylotrophs
have drawn much attention for the production o f
PHA. A very high concentration (149 g 1-1) of P(3HB)
could be produced by a fully automated fed-batch cul-
ture of P extorquens by feeding nutrients (carbon and
nitrogen source) at an optimal ratio 27. However, a
relatively low productivity of 0.88 gl 1h 1 was ob-
tained because of the long cultivation time of 170 h.
Recently, fed-batch cultures of Methylobacterimt~
* Z E N E C A Bio Products no longer produces PHA since it has sold these
activities to Monsanto (USA).
 435

reviews

organophilum were carried out under potassium-

limited conditions to obtain 130 gF 1 of P(3HB) with Table 1. Effect of substrate cost and P(3HB) yield on the
production cost of P(3HB}
a high productivity of 1.86 gl < h 1 (Ref. 29). However,
the PHA content was only 52%, resulting in a PHA Substrate
yield of 0.19 gram of P(3HB) per gram of methanol, cost
which is much lower than the theoretical yield Approximate P(3HB) yield {US$
(Table 1). This is one of the major disadvantages of price [g P(3HB) [kg
employing methylotrophs since a low PHA content Substrate {US$ kgq) (g substrate)q] P(3HB)]-1}
not only lowers the PHA yield but also makes poly-
mer recovery difficult. Therefore, a method of increas- Glucose 0.493 a 0.38 b 1.30
ing PHA contents needs to be developed to employ (0.220 c) (0.58)
methylotrophs for the production of PHA from Sucrose 0.290 d 0.40 b 0.72
inexpensive methanol. Methanol 0.180 e 0.43 b 0.42
A mutant strain o f A z . vinelandii which accumulates Acetic acid 0.595 e 0.38 b 1.56
P(3HB) during growth has been developed, and was Ethanol 0.502 a 0.50 b 1.00
used to produce P(3HB) from glucose in a fed-batch Cane molasses 0.220 a 0.42 a 0.52
culture. A P(3HB) concentration of 32 g1-1 could be Cheese whey 0.071 a 0.33 ~ 0.22
obtained in 47h with a relatively high yield of Hemicellulose
0.34 gram of P(3HB) per gram of glucose by supple- hydrolysate 0.069 ~ 0.20 a 0.34
menting the medium with a small amount of fish
peptone 26. However, the productivity was low at aData taken from Hocking and Marchessault15.
0.34 gl < h 1, and needs to be increased by developing bCalculated by multiplying the theoretical yield41 by 0.8 (assuming
a better fermentation strategy. Pseudomonas oleovorans 80% of polymer accumulation).
is the only bacterium that has been employed for the CEstimate of the value of hydrolyzed corn starch.
production of medium-chain-length (C6-C14) PHA dlnternational market price of raw sugar.
to a relatively high concentration. Two liquid phase elnternational market price from Chem.J. (Korea).
fed-batch cultures of Ps. oleovorans using octane as a
carbon source have been carried out and produced
12.1 gl < of P(3HHx-co-3HO) in 38 h, resulting in a a further cost reduction of PHA is possible by using
low productivity of 0,32 g F 1h < (Ref. 20). A summary cheap substrates such as molasses, whey and hemicel-
of PHA production by various bacteria is presented in lulose hydrolysateu'. A recombinant K. aeroyenes har-
Table 2. boring the A1. eutrophus PHA biosynthesis genes was
also developed, and used for the production of P(3HB)
Metabolic engineering from molasses. These are good examples of the intro-
Recombinant DNA techniques can be used to duction of the heterologous PHA biosynthetic path-
modify or to introduce new metabolic pathways way for PHA production in bacteria which would not
(metabolic engineering) to broaden the utilizable sub- normally produce PHA. This strategy can be extended
strate range, to enhance PHA synthetic capacity and to virtually any bacterium if it possesses metabolic
to produce novel PHAs. Recent advances in our advantages over those currently in use.
understanding of the biochemistry of PHA biosyn- Homologous amplification of enzyme activities
thesis and cloning of PHA biosynthesis genes from involved in PHA biosynthesis has been demonstrated.
more than 24 different bacteria 44 have allowed the When a broad-host-range plasmid containing the
development of various recombinant bacteria that can AI. eutrophus PHA synthase gene was introduced into
produce PHA more efficiently or synthesize unusual the same AI. eutrophus strain, the activity o f P H A syn-
PHAs. thase could be considerably increased. In flask cultures,
Recombinant E. coli strains harboring the AI. eutro- the PHA production rate of the recombinant AI. eutro-
phus PHA biosynthesis genes in a stable high-copy- phus was 1.24 times higher than that of the parent
number plasmid have been developed, and were used strain32. It will be interesting to see if this recombinant
for the production of a high concentration ofP(3HB), AI. eutrophus allows more rapid production of PHA in
with a high PHA content and high productivity33 36,38. fed-batch cultures. Similar strategies can be employed
Since E. coli does not utilize propionic acid efficiently, to develop other recombinant bacteria for the
the production of P(3HB-co-3HV) in recombinant enhanced production of PHAs. Production of P(4HB)
E. coli had been considered difficult. However, recent by the application of mutagenesis and recombinant
developments have allowed the production of P(3HB- D N A techniques 19 is a good example of producing
co-3HV) by employing the mutant strain @dR atoC) novel polyesters by metabolic engineering.
of E. coli45 or by appropriately inducing the culture Heterologous expression of PHA biosynthesis genes
with acetic or oleic acid46. Ceils of recombinant E. coli in various bacteria which naturally produce PHAs has
accumulating a large amount of polymer became frag- been reported. Expression of AI. eutrophus PHA
ile, which is an advantage in the subsequent recovery biosynthesis genes in Ps. oleovorans, which only syn-
process 3s,36. Since E. coli can utilize various carbon thesizes medium-chain-length PHAs, has allowed the
sources, including glucose, sucrose, lactose and xylose, production of a blend of P(3HB) and of medium-

TIBTECH NOVEMBER1996 (VOL14)

 436

reviews

Table 2. Production of PHA by various bacteria

Major Cell PHA PHA

carbon Culture conc, conc, content Productivity
Bacterium PHA a source time (h) (g 1-1) (g 1-1) (%) (g 1-1 h -1) Refs

Alcaligenes P(3HB) Glucose 50 164 121 76 2.42 22

eutrophus
AI. eutrophus P(3HB) CO2 40 91.3 61.9 67.8 1.55 42

AI. eutrophus P(3HB) Tapioca 59 106 61.9 57.5 1.03 43

hydrolysate

AI. eutrophus P(3HB-co- Glucose + 46 158 117 74 2.55 23

3HV) propionic acid

Alcaligenes P(3HB) Sucrose 18 143 71.4 50 3.97 b 51

latus
AI. latus P(3HB) Sucrose D = 0.16 hq _c 16.2 - 2.6 Yamaned

Azotobacter P(3HB) Glucosee 47 40.1 32 79.8 0.68 26

vinelandii

Chromobacterium P(3HV) Valeric acid 39.5 24.5 62 - 18

violaceum
Methylobacterium P(3HB) Methanol 70 250 130 52 1.86 29
organophilum
Protomonas P(3HB) Methanol 170 233 149 64 0.88 27
extorquens
Pseudomonas P(3HHx-co- n-Octane D = 0.2 h 1 11.6 2.9 25 0.58 30
oleovorans 3HO)

Ps. oleovorans P(3HHx-co- n-Octane 38 37.1 12.1 33 0.32 20

3HO)

Recombinant P(3HB) Glucose e 39 101.4 81.2 80.1 2.08 33

Eschenchia coli
Recombinant P(3HB) Molasses 32 37 24 65 0.75 37
Klebsiella
aerogenes
aAbbreviations: P(3HB), poly(3-hydroxybutyrate);P(3HB-co-3HV),poly{3-hydroxybutyrate-co-3-hydroxyvalerate);P(3HV),poly(3-hydroxyvalerate);
P(3HHx-co-3HO),poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate).
bin this experiment, high inoculum size of 13.7 g DCW 1-1 was used.
eData not available.
dPers, commun, from T. Yamane (Nagoya University, Japan).
eComplexnitrogen sources were supplementedin these experiments.

chain-length PHAs 52. In another study, an increase in
P(3HB) synthesis by transferring the Al. eutrophus P H A
biosynthesis genes into Mycoplana rubra has been
reported 47. In addition, unusual PHAs can be pro-
duced by heterologous expression of P H A biosynthesis
genes, as seen in recombinant Pseudomonas putida
harboring the PHA biosynthesis genes o f Thiocapsa
pfennigii. This recombinant strain was able to accumu-
late a polyester consisting of 3-hydroxybutyrate,
3-hydroxyhexanoate and 3-hydroxyoctanoate when
cultivated on octanoate 9.
Attempts to broaden the utilizable substrate range
have been less successful. Introduction of the [3-galac-
tosidase gene and the E. coli gal operon into AI. eutro-
phus has allowed only slow growth of this strain on lac-
tose 48. In another study, a recombinant AI. eutrophus
harboring the Bacillus subtih's levanase gene was devel-
oped, but it grew poorly on sucrose 49. Two approaches
can be taken in the development of bacterial strains
that produce PHA from inexpensive carbon substrates.
First, substrate utilization genes can be introduced
into the P H A producers as above. Second, P H A
biosynthesis genes can be introduced into the non-
P H A producers which can utilize cheap substrates. At
present, the second approach seems to be more
promising.

TIBTECHNOVEMBER1996(VOL14)
 437
reviews
Prospects
At present, P(3HB) and P(3HB-co-3HV) are the
only members of PHAs that are produced on a com-
mercial scale. Even though the price of PHAs is still
too high, current advances in fermentation and purifi-
cation technology as well as the development of su-
perior bacterial strains by recombinant D N A tech-
niques are likely to lower the price of PHA to
US$4kg < (S. Y. Lee, unpublished), which will be
close to that of other biodegradable plastic materials
such as polylactide and aliphatic polyesters. The iso-
lation and development of bacterial strains that can uti-
lize cheap carbon substrates will be pursued intensively.
The isolation of bacterial strains that can synthesize
P(3HB-co-3HV) from simple sugar substrates s° is a
good example of reducing the substrate cost by elimi-
nating the necessity of supplementing more expensive
propionic acid. Several of the bacteria described
earlier will be employed for the production of PHAs
over the next few years, but, eventually, metabolically
engineered bacterial strains will likely surpass the wild-
type bacteria currently in use. In this case, the stabil-
ity of recombinant bacteria will become important to
successful PHA production and, therefore, much effort
will be exerted to develop stable plasmids and to
understand the host-plasmid interactions for a given
bacterium. With these advances, the production of a
high concentration of PHAs with high productivity
and a PHA yield close to the theoretical limit will be
possible. The PHA content will be 80% or higher for
the sake of efficient recovery, and we may call these
polymer-filled bacteria 'Plastic Bacteria'.
Recently, transgenic plants harboring the AI. eutro-
phus PHA biosynthesis genes have been developed 4,
with the aim of ultimately reducing the price of PHA
to close to that of starch (20 US cents kg-l). Up to
10 mg [g fresh weight ofP(3HB)] -1, which represents
ca, 14% of dry cell weight, could be produced in the
model transgenic plant Arabidopsis thaliana 4. The con-
centration and yield of PHA will increase as our under-
standing of plant biochemistry and genetics improves.
Production of other PHAs in transgenic plants may also
be possible by metabolic engineering. However, until
the early 21st century, PHA production will rely on
efficient bacterial fermentation. With all these advances,
there is little doubt that PHAs will become a leading
biodegradable plastic material in the next decade.
Acknowledgements
I would like to thank Dr H. N. Chang and Dr
Y. K. Chang for their continued interest and support.
The research of the author on polyhydroxyalkanoates
has been supported by grants provided by the Korea
Advanced Institute of Science and Technology, the
Korea Science and Engineering Foundation, the
Ministry of Science and Technology and by LG
Chemical Ltd.
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Modelling the morphology of

filamentous microorganisms
Jens Nielsen

The rapid development in image analysis techniques has made it possible to study
the growth kinetics of filamentous microorganisms in more detail than previously.
However, owing to the many different processes that influence the morphology it is
important to apply mathematical models to extract information about the underlying
mechanisms from experimental data. This review is concerned with the development
and application of such models.

Filamentous fungi and Actinomycetes form two
groups of industrially very important microorganisms.
Owing to their ability to secrete large amounts of pro-
tein, filamentous fungi are used extensively for the
production of industrial enzymes - a continuously
increasing market - but they are also very important
in the production of various secondary metabolites,
including [3-1actams, which are by far the largest
group of antibiotics. Actinomycetes (mostly species of
Streptomyces) are mainly of industrial interest for their
production of secondary metabolites, which includes
both classic antibiotics of the ~-lactam group and new"
pharmaceuticals. Despite the large differences between
these two groups of microorganisms they have many
similarities with respect to their growth mechanisms.
They both grow fdamentously, and in submerged cul-
tures they may both form pellets. Their growth ki-
netics can, therefore, often be expressed using the same
mathematical equations. Furthermore, the effect of
environmental conditions on the development of their
morphology is, in many cases, similar. Thus the effects
of hydrodynamic forces on fragmentation of both
hyphal elements and pellets are the same for filamentous
fungi and Actinomycetes.
J. Nielsen (jn@ibt.dtu.dk) is at the Center for Process Biotechtwlow,
Technical University of Denmark, Building 223, DK-2800 Ll,~by,
Denmark.
With the recent development of new analytical tech-
niques based on image analysis for characterization of
the morphology of filamentous microorganisms it has
become possible to study the growth kinetics of these
microorganisms in much more detail than previously.
It is in this light that the state of the art of kinetic
modelling of morphological development is reviewed
and that pitfalls in connection with interpretation
of morphological data from submerged cultivations
are discussed.
Morphology of filamentous microorganisms in
submerged cultures
Vegetative cells of filamentous microorganisms are
connected in multicellular structures called hyphal el-
ements, which make up a so-called mycelium. A hyphal
element arises from the outgrowth of a single spore.
Upon germination, the growth of the spore rapidly
becomes polarized, resulting in the formation of a
germ tube which extends its length by growth at the
tip. Eventually, the germ tube develops into a hypha,
and when the hypha has exceeded a certain length new
tips (or branches) are formed along the hypha, result-
ing in a hyphal element consisting of a branched
network of hyphae (Fig. 1). Quantification of the
morphology of the individual hyphal elements and the
population distribution in submerged cultures using
image analysis has been a major research topic within

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