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Current Biotechnology, 2013, 2, 81-88

81

Pseudomonas aeruginosa MTCC 7925: Producer of a Novel SCL-LCLPHA Co-Polymer

Akhilesh Kumar Singh*,1, Ranjana Bhati2,, Shilalipi Samantaray2, and Nirupama Mallick 2
1

Amity Institute of Biotechnology, Amity University Uttar Pradesh Lucknow Campus, Uttar Pradesh, India

Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, West Bengal, India
Abstract: Pseudomonas aeruginosa MTCC 7925, a sludge isolate, was found to synthesize a novel short-chain-lengthlong-chain-length (SCL-LCL) co-polymer with 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), 3hydroxyhexadecanoic acid (3HHD) and 3-hydroxyoctadecanoic acid (3HOD) as constituents. Under batch mode study,
cells harvested at the stationary phase of growth depicted maximum PHAs accumulation, i.e. 24% of dry cell weight
(dcw) at 48 h of incubation. The co-polymer accumulation was raised to 49% (dcw) under 2% ethanol-supplemented
condition. A further, rise up to 78% (dcw) was recorded by optimizing the critical variables by Response Surface
Methodology (RSM). When palm oil and its cakes were used as carbon sources, yield of 5.9 g/l was achieved, which was
almost 85-fold higher against control. The thermal and mechanical properties of the polymer are comparable with
polypropylene and polyethylene, thus opening new possibilities for various industrial applications.

Keywords: Inexpensive carbon sources, N-deficiency, P-deficiency, PHAs, Pseudomonas aeruginosa MTCC 7925, RSM,
SCL-LCL-PHA co-polymer.
1. INTRODUCTION
Plastics are ubiquitous in todays society and
undoubtedly being wonderful materials. The major problem
associated with plastic waste disposal is its extreme
resistance to microbial attack [1]. Thus, several communities
are now sensitive to the impact of the discarded plastic
materials on the environment, including their deleterious
effects on wildlife and on the aesthetic qualities of cities and
forests. Apart from this, the potential hazards from plastic
waste incineration such as dioxin emission from polyvinyl
chloride, makes plastic wastes a deadly evil. Therefore, there
is increasing interest in producing biodegradable polymers
from materials that can be readily eliminated from our
biosphere in an eco-friendly way.
Of the various types of biodegradable polymers reported
so far, the microbially-synthesized polyhydroxyalkanoates
(PHAs) offer much potential as bioplastics. It is not only
the biodegradability that makes the PHAs so fascinating but
these are also derived from renewable sources. Being the
product of renewable carbon sources, its production and uses
follows sustainable closed cycle [2].
PHAs can be divided into three major types based on the
number of carbons in their monomer units, i.e. short-chainlength (SCL), medium-chain-length (MCL) and long-chainlength (LCL) PHAs consisting of hydroxyacids with 3-5, 614 or more than 14 carbon atoms, respectively [3]. The
*Address correspondence to this author at the Amity Institute of
Biotechnology, Amity University Uttar Pradesh Lucknow Campus, Uttar
Pradesh, India; Tel: +91-5222-399593; Fax: +91-5222-721934; E-mails:
akhiliit@yahoo.co.in, akhiliit@gmail.com

Equal contribution.
2211-551X/13 $58.00+.00

majority of bacteria synthesize either SCL- or MCL-PHAs,

but not normally SCL-MCL co-polymers because of the
mutual exclusivity of the SCL- and MCL-PHA synthase
enzymes [4]. A few bacterial species, such as Aeromonas
caviae, Rhodococcus ruber, Nocardia corallina, Bacillus
megaterium and some species of Pseudomonas (P.
fluorescens, Pseudomonas sp. A33, P. marginalis, P.
mendocina and Pseudomonas sp. 61-3) are however, found
to accumulate the co-polymer of SCL-MCL-PHAs with a
maximum carbon skeleton up to C14 [5, 6, 7, 8, 9]. In this
report, we summarize our experience with a sludge-isolated
Pseudomonas aeruginosa MTCC 7925 producing a novel
SCL-LCL co-polymer with carbon skeleton greater than 14,
i.e. C16 and C18 with SCL components of C4 and C5.
2. MATERIALS AND METHODS
2.1. Organisms and Growth Conditions
The bacterial strains isolated from the sludge samples of
municipal waste treatment plant, Titagarh, Kolkata, India,
following the standard microbial techniques were maintained
in the laboratory at 30 2
C and pH 7.0. For culturing the
newly isolated bacterial species/strains mineral salt medium
was used. The mineral salt medium composition was as
follows ( per liter distilled water) 4 g Na2HPO4, 1 g KH2PO4,
0.2 g MgSO47H2O, 0.05 g CaCl22H2O, 3 g NH4NO3, 0.2 g
NaCl, 1 g glucose and 1 ml of trace element solution [10].
The trace element solution contained (per liter 0.5 N HCl)
5.56 g FeSO47H2O, 3.96 g MnCl2 4H2O, 5.62 g CoSO47H2O,
0.34 g CuCl22H2O, 0.58 g ZnSO47H2O, 0.6 g H3BO3, 0.04 g
NiCl26H2O and 0.06 g Na2 MoO42H2O [11]. The medium
was buffered with MES (5 mM) to maintain the pH 7.0. The
axenic cultures were grown in 150 ml Erlenmeyer flasks
containing 50 ml of the medium under agitation of 200 rpm.
2013 Bentham Science Publishers

82 Current Biotechnology, 2013, Volume 2, No. 1

2.2. Growth and Dry Weight Measurement

Cell growth was monitored spectrophotometrically
(Specord S 100, Analytic Jena, Germany) at 600 nm as
previously described [12]. For the determination of dry cell
weight (dcw), the cell mass was harvested by centrifugation,
washed thoroughly, transferred to pre-weighed aluminum
cups and dried to a constant mass at 80C following Pal et
al. [13]. The corresponding dcw for optical density of the
culture broth was determined by simple linear correlation.
2.3. Extraction of Polyhydroxyalkanoates (PHAs)
The biomass obtained after centrifugation was dried at
80C and PHAs were extracted in hot chloroform followed
by precipitation with cold diethyl ether. The precipitate was
centrifuged at 11,000 g for 20 min, washed with methanol
and dissolved again in hot chloroform [14]. To verify the
presence of the real co-polymer, fractionation of the polymer
was carried out in boiling acetone following Yao et al. [15].
2.4. Detection and Quantification of PHAs
PHAs were detected and quantified following the
propanolysis method of Riis and Mai [16] using a gas
chromatograph (GC) (Clarus 500, Perkin-Elmer) equipped
with Elite-1 dimethylpolysiloxane capillary column (30 m x
0.25 mm x 0.25
m) and flame ionization detector. Benzoic
acid served as the internal standard. PHB, P(3HB-co-3HV)
co-polymer (Aldrich, USA), 3-hydroxyhexadecanoic acid
and 3-hydroxyoctadecanoic acid (Larodan Fine Chemicals,
Sweden) were used as standards to determine the PHAs
compositions of samples both quantitatively and
qualitatively, based on the areas and retention times of
individual 3-hydroxyalkanoate (3HA) monomers on the
chromatogram. The composition of PHAs was confirmed by
GC-MS study.
2.5. Impact of pH, Temperature and Inoculum Size on
Biomass Yield and PHAs Accumulation
The pH of the medium was adjusted to different values,
ranging from 5.5-11.5 (MES buffer, 5.0 mM for pHs 5.5-7.5
and Tris-buffer, 2.0 mM for pHs 8.5-11.5) before
introducing the cells into it, and grown for the stipulated
time period. A temperature range of 20-40C was selected
with an interval of 5C. Similarly, different inoculum size
was also selected ranging from 20 to 200 mg dcw/l.
2.6. Impact of Carbon Sources on Biomass Yield and
PHAs Accumulation
Effect of different concentrations (0.2-4%) of exogenous
carbon sources such as glucose, maltose, fructose, ethanol,
sodium acetate, tri-sodium citrate, mannitol, sucrose and
butyrate on biomass yield and PHAs accumulation were
studied. PHAs content and biomass yield were analyzed at
regular intervals.
2.7. Studies on Nitrogen and Phosphorus Deficiencies on
Biomass Yield and PHAs Accumulation
PHAs accumulation was also studied under N- and Pdeficient conditions. For nitrogen-deficiency, the medium

Singh et al.

was devoid of NH4NO3. In phosphate-deficiency, the

Na2HPO4 and KH2PO4 of the medium were substituted by
equimolar concentration of Na2SO4 and KCl, respectively.
For nutrient-limited incubation, cells grown in mineral salt
medium were washed with mineral salt medium without the
specific nutrient and transferred to the deficient medium.
The interactive effects of exogenous carbons and nitrogen-/
phosphate-deficiency on PHA yield were also analyzed.
Carbon sources and their concentrations were chosen based
on their performance on biomass yield and PHA
accumulation in the earlier experiments.
2.8. Optimization of PHAs Accumulation by Response
Surface Methodology (RSM)
Central composite rotary design (CCRD) with four
variables (five level of each variable) was used to study the
interactions of the most critical variables to maximize PHAs
accumulation [17]. Experiments were conducted according to
the CCRD design and RSM was applied to the experimental
data using a commercial statistical package, Design Expert
(version-7.1.1, Stat-Ease Inc., Minneapolis, USA).
2.9. Experience with Inexpensive Carbon Sources
As supplementation of exogenous carbons such as
glucose and ethanol was found to have profound impact on
PHA accumulation in the test bacterium, various inexpensive
substrates such as palm, mustard, soybean and coconut oils,
whey, rice and wheat brans, mustard and palm oil cakes were
explored as cheap carbon sources to boost PHA
accumulation. Mustard and palm oil cakes were suspended
in distilled water (5 g/100 ml) and were autoclaved. The
supernatant was poured out and was used as mustard/ palm
oil cakes stock solutions.
2.10. Properties of the Polymer
For analysis of thermal properties, differential scanning
calorimetry (DSC) was performed as per Kato et al. [18].
Thermal stability was determined with a Perkin Elmer
thermogravimetric analyzer (Pyris Diamond TG/DTA)
following Luo et al. [19]. For mechanical tests, the stressstrain curves of the solution-cast films (0.1 mm thickness)
were analysed as previously described [8]. All the
experiments were performed in triplicate to check the
reproducibility. The results were analysed statistically by
Duncans new multiple range test.
3. RESULTS
3.1. Screening of Bacterial Isolates for Accumulation of
PHAs
Batch cultures under shaking mode were used to screen
the ability of various bacterial isolates for presence of PHAs.
Among all the isolates, AKS-1, latter identified as
Pseudomonas aeruginosa with an accession number MTCC
7925 at Microbial Type Culture Collection (MTCC),
Institute of Microbial Technology, Chandigarh, India, was
emerged as the accumulator of a novel SCL-LCL copolymer with composition of 3-hydroxybutyric acid (3HB),
3-hydroxyvaleric acid (3HV), 3-hydroxyhexadecanoic acid
(3HHD) and 3-hydroxyoctadecanoic acid (3HOD) by gas

Production of a Novel SCL-LCL-PHA Co-Polymer by Pseudomonas aeruginosa MTCC 7925

chromatographic analysis. Chemical proof for the presence

of 3HB, 3HV, 3HHD and 3HOD units was obtained by GCMS analysis [20].
3.2. Accumulation of PHAs in Relation to Growth
The time-course of growth along with accumulation of
PHAs in Pseudomonas aeruginosa MTCC 7925 under batch
mode was studied. Growth of the test bacterium showed a
lag phase of 8 h followed by logarithmic phase and attained
the stationary phase at 32 h. Maximum accumulation of
PHAs, i.e. 24% of dry cell weight (dcw) was recorded at 48
h of incubation.
3.3. Impact of pH, Temperature and Inoculum Size
PHAs accumulation was found to be maximum at pH 7.5
(22.1% dcw) followed by pHs 8.5 (18%) and 6.5 (11.8%) at
48 h of incubation (Table 1). Acidic pHs were not found
suitable for PHAs accumulation. Temperature range of 2535
C was found to be ideal for PHAs accumulation (Table
2). However, maximum PHAs accumulation of 22.1% (dcw)
was found at 30
C followed by 35
C (20.5%) and 25
C
(19%) at 48 h of incubation. The maximum PHAs
accumulation of 24% (dcw) was observed at 48 h with
inoculum size of 80 mg dcw/l (Table 3). In all the cases the
polymer was found to be dominated by 3HV fraction,
ranging from 68-78 mol%.
Table 1.

Accumulation of PHAs in P. aeruginosa MTCC 7925

at Different pH Values
Polymer Composition (mol%)

Current Biotechnology, 2013, Volume 2, No. 1

83

mannitol, sucrose and butyrate were used to boost PHA

accumulation in P. aeruginosa MTCC 7925. Maximum
accumulation of 48.8% (dcw) was recorded under 2%
ethanol supplementation followed by 2% glucose (43.1%
dcw). Furthermore, a significant rise in 3HB unit vis--vis
drastic reduction in 3HV fraction was observed under
glucose and ethanol supplementation (Fig. 1).
Table 2.

Accumulation of PHAs in P. aeruginosa MTCC 7925

at Different Temperature
Polymer Composition (mol%)

Temperature
(
C)

PHAs Content
(% dcw)

3HB

3HV

3HHD

3HOD

15

11.7 0.12a

28.0

69.1

0.9

2.0

20

28.3

68.2

1.3

2.2

25.2

69.7

1.7

3.4

23.9

70.9

1.8

3.4

dc

25.6

69.4

1.8

3.2

26.7

69.7

1.4

2.2

23.1

75.4

0.5

1.0

14.6 0.53

25

19.0 0.65

30

22.1 0.79

35

20.5 0.29

40

16.3 0.37

45

9.9 0.67

Incubation period: 48 h.
PHAs content: mean SE, n = 3.
Values in the column superscripted by different letters are significantly different from
each other (P < 0.05, Duncans new multiple range test).

Table 3.

PHAs Accumulation in P. aeruginosa MTCC 7925 at

Various Inoculum Size
Polymer Composition (mol%)

3HB

3HV

3HHD

3HOD

Inoculum Size
(mg dcw/l)

PHAs Content
(% dcw)

3HB

3HV

3HHD

3HOD

3.03 0.13

20.6

78.4

0.3

0.7

20

9.8 0.98a

6.5

11.8 0.06

20.8

77.1

0.8

1.3

7.5

22.1 0.86c

23.9

71.0

1.8

3.3

8.5

18.0 0.47

21.4

72.2

2.4

4.0

10.4 0.48

20.7

77.8

0.6

0.9

26.9

71.5

0.6

1.0

28.4

69.8

0.7

1.1

pH
5.5

9.5

PHAs Content (% dcw)

10.5

7.2 0.49

11.5

4.5 0.2a

29.0

69.0

0.9

1.1

40

12.5 1.19

28.3

68.5

1.4

1.8

60

20.1 0.90b

25.6

69.5

1.7

3.2

80

24.0 0.73

23.9

70.9

1.8

3.4

23.7 1.30

25.7

69.2

1.8

3.3

23.5 1.21

25.9

70.7

1.2

2.2

cb

24.1

74.4

0.4

1.1

100
150
200

22.8 1.19

Incubation period: 48 h.
PHAs content: mean SE, n = 3.
Values in the column superscripted by different letters are significantly different from
each other (P < 0.05, Duncans new multiple range test).

Incubation period: 48 h.
PHAs content: mean SE, n = 3.
Values in the column superscripted by different letters are significantly different from
each other (P < 0.05, Duncans new multiple range test).

3.4. Impact of N-/P-Deficiency

3.6. Optimization of
Accumulation by RSM

Stimulatory effects were observed on the co-polymer

accumulation under nitrogen and phosphorus deficiencies. P.
aeruginosa MTCC 7925 registered PHAs pool up to 31.3
and 27.8% of dcw, respectively under N- and P-deficient
medium for 48 h [20].
3.5. Impact of Exogenous Carbon Supplementation on
Biomass Yield and PHAs Accumulation
Various exogenous carbon sources, i.e. glucose, maltose,
fructose, ethanol, sodium acetate, tri-sodium citrate,

SCL-LCL-PHA

Co-Polymer

The critical variables influencing the co-polymer

accumulation in P. aeruginosa MTCC 7925 were
concentrations of glucose (A), ethanol (B), phosphate (C)
and nitrate (D). Thus, nitrogen and phosphate deficiencies
along with ethanol and glucose were selected as the
independent variables for further study by central composite
rotary design (CCRD) [35]. Applying multiple regression
analysis, the mathematical regression model for the copolymer accumulation fitted in terms of coded factors was as
follows:

84 Current Biotechnology, 2013, Volume 2, No. 1

Singh et al.

Y = + 79.04 + 1.22A + 5.30B - 0.70C - 2.35D - 0.89AB 0.10AC + 2.60AD - 0.14BC - 1.33BD - 6.99CD - 5.25A2 2.02B2 - 6.71C2 - 7.09D2
where, Y was the response, i.e. the P(3HB-co-3HV-co3HHD-co-3HOD) content, and A, B, C and D were the
coded terms for the four independent variables.

400
60
300
40
200
20

100

0
CONTROL
3HV

3HHD

GLUCOSE
3HOD

PHA (% dcw)

ETHANOL
Biomass

PHA (mg/l))

As found in this study, supplementation of glucose and

ethanol boost PHA accumulation in P. aeruginosa MTCC
7925. This stimulates us to focus on inexpensive carbon
sources for PHA production. Various inexpensive carbon
sources depicted that plant oils (palm, coconut, mustard and
soybean oils) had stimulatory effects on growth as well as
PHAs accumulation. Cakes of mustard and palm oils also
exhibited positive effects on growth. PHAs accumulation
however, not registered any significant rise in those vessels.
A maximum rise in PHA pool up to 33% (dcw), dominating
with 3HB units was recorded in 0.3% palm oil-supplemented
vessels [36]. Therefore, impact of various concentrations of
palm oil (0.2-2%) on PHAs accumulation was studied (Fig.
2). PHA yield was boosted maximum up to 62.2% (dcw)
under 0.7% palm oil at 48 h of incubation.
5000

Fig. (1). Biomass and polymer yield with polymer composition in

P. aeruginosa MTCC 7925 under 2% glucose and ethanol
supplementation after 48 h of incubation.

Table 4.

60
4000
50

Biomass yield (mg/l)

PHA yield (mg/l)

The regression analysis of the experimental design

demonstrated that the interactive model terms (AB, AD, BD
and CD) were significant (P < 0.05), whereas AC and BC
were found to be insignificant (P > 0.05). Response surface
analysis further demonstrated the optimal values of the
independent variables and the interaction between each
independent variable pairs for maximum response [35]. After
knowing the possible direction for maximizing P(3HB-co3HV-co-3HHD-co-3HOD) co-polymer accumulation from
the response surface analysis, optimization was done using
point optimization technique with the help of the same
software. A maximum polymer accumulation of 80.1%
(dcw) was predicted at 1.5% (v/v) ethanol, 1.1% (w/v)
glucose, 2.97 g/l KH2PO4 and 1.86 g/l NH4NO3 (Table 4).
Experiments were performed in triplicate using the
optimized conditions to verify the model. It could be
visualized from Table 4 that an experimental value of 77.6%
(dcw) was clearly evident. The co-polymer content before
optimization was 68.7% (dcw) with the variables, viz.
ethanol level of 3% (v/v), KH2PO4 of 2 g/l, Na2HPO4 of 8 g/l

70

3000

40
30

2000

20

Biom ass

PHA content (% dcw)

500

80

3HB

3.7. Experience with Various Inexpensive Carbon

Sources

600

Biomass yield (mg/l)

PHA yield (mg/l)

Polymer composition (mol%)

PHA content (%dcw)

100

and 3 g/l NH4NO3 were optimized to get an accumulation of

77.6% for ethanol, glucose, KH2PO4 and NH4NO3
concentration of 1.5% (v/v), 1.1% (w/v), 2.97 and 1.86 g/l,
respectively. The polymer was dominated with 3HB fraction
(95.7 mol%).

PHA (m g/l)

1000

10

PHA (% dcw )

0
0

0.4

0.8

1.2

1.6

Concentration (% v/v)

Fig. (2). Effect of palm oil supplementation on biomass and SCLLCL-PHA co-polymer accumulation in P. aeruginosa MTCC 7925
after 48 h of incubation (modified from Singh and Mallick [36]).

Fig. (3) shows the maxima of PHAs content along with

its composition for various concentrations of palm oil (0.21%) when supplemented with the extract of palm oil cake
(0.2-1%). PHAs content reached up to 61.1% (dcw) under

Optimum Conditions of the Critical Variables for Maximum Co-Polymer Accumulation (Modified from Singh and
Mallick [35])

Variable

Lower
Limit

Upper
Limit

Optimum
Value Obtained

Glucose (% w/v)

1.50

Ethanol (% v/v)

1.10

KH2PO 4 (g/l)

2.97

NH4NO3 (g/l)

1.86

Predicted Co-Polymer
Content (% dcw)

Actual Co-Polymer
Content (% dcw)

80.1

77.6

Coefficient of determination (R2) = 0.9928; adjusted R2 = 0.9861; Adequate precision = 40.768; coefficient of variation (CV) = 2.35%.

Production of a Novel SCL-LCL-PHA Co-Polymer by Pseudomonas aeruginosa MTCC 7925

supplementation of 0.6% palm oil + 0.6% palm oil cake. The

polymer composition exhibited significant rise in 3HB and
reduction in 3HV units (Fig. 3B).
(A)
70

5000

60
50
3000

40
30

2000

20

Biom ass

PHA content (% dcw)

Biomass yield (mg/l)

PHA yield (mg/l)

4000

10

PHA (% dcw )

0
0

0.2

0.4

0.6

0.8

Concentration (% v/v)

Polymer composition (mol %)

(B)

100

80
3HOD

60

3HHD
3HV

40

3HB

20

0
0

0.2

0.4

0.6

0.8

85

(Td(5%)) was studied with respect to thermal stability of the

polymer. The Td(5%) of the isolated polymer was ranged from
248-262C. The tensile strength was within the range of 1719 MPa. The elongation to break values were found to be
significantly higher, i.e. 682-723%. The Youngs modulus
was within the range of 0.2-0.3 GPa. Table 6 compares the
material properties of this novel co-polymer with the
common conventional polymers such as polypropylene (PP)
and low-density polyethylene (LDPE), which depicts its
comparable properties with PP and LDPE.
4. DISCUSSION

PHA (mg/l)

1000

Current Biotechnology, 2013, Volume 2, No. 1

Concentration (% v/v)

Fig. (3). Effect of various concentrations of palm oil + palm oil

cake on (A) biomass yield and SCL-LCL-PHA co-polymer
accumulation, and (B) polymer composition in P. aeruginosa
MTCC 7925 after 48 h of incubation.

The interactive effects of N-/P- limitation in the presence

0.6% palm oil + extract of 0.6% palm oil cakes were also
investigated. PHAs content of 76.6% (dcw) was recorded
with a mol fraction of 90.0:4.8:2.2:3.0 of 3HB:3HV:
3HHD:3HOD units, respectively when Pseudomonas
aeruginosa MTCC 7925 cells pre-grown in 0.6% palm oil +
the extract of 0.6% palm oil cakes-supplemented mineral salt
medium transferred to N-limited medium in presence of
0.6% palm oil + the extract of 0.6% palm oil cakes for 48 h
(Table 5). However, the PHAs content registered up to
70.4% (dcw) with a mol fraction of 84.8:7.2:3.1:4.9 in the
above order under P-limitation (Table 5).
3.8. Properties of the Polymer
The composition of the polymer was found to vary with
treatment. Therefore, the polymers obtained from the
optimized condition and palm oil supplementation (Tables 4
and 5) were taken for characterization of the physical
properties such as differential scanning calorimetry (DSC),
thermogravimetric analysis (TGA) and mechanical property
studies and presented in Table 6. The melting temperature
(Tm) of this novel polymer was ranged between 115-131C,
while the glass transition temperature (Tg) and enthalpy of
fusion (
Hm) were found to vary between -8 to -14C and
26 to 44 J/g, respectively. The temperature at 5% weight loss

In P. aeruginosa MTCC 7925, P(3HB-co-3HV-co3HHD-co-3HOD) co-polymer accumulated during the

logarithmic growth phase, reached the maximum when the
cultures become stationary [20]. This is in well agreement
with the reports of Sun et al. [21], Mccool et al. [22], Pal et
al. [13] and Potter et al. [23] where maximum accumulation
of PHA was observed at the stationary phase of the
bacterium such as Vibrio harvey, Bacillus megaterium,
Actinobacillus sp. EL-9, Azotobacter chroococcum and
Ralstonia eutropha H16, respectively. The decrease in the
co-polymer level at the latter phase may possibly be due to
intracellular utilization of the polymer as energy and carbon
[24, 25, 26, 27].
Temperature and pH were found to be the most important
physical factors affecting the microorganisms. Enzymatic
reactions proceed at maximum speed and efficiency at
optimum temperature, which varies with the organism. The
range of temperature preferred by bacteria is genetically
determined, resulting in enzymes with different temperature
requirements. High temperature is harmful to the
microorganisms because it denatures proteins, causing
irreversible changes and total enzyme destruction, thus
resulting in the death of the cell while low temperatures also
have an inactivating effect on the enzymes. However, pH is
the measure of the relative acidity or alkalinity of a solution.
pH changes of the environment has a profound influence on
the growth, activities and survival of the microorganisms as
enzyme system of an organism has a particular pH range in
which it can function. Although specific pH range for
bacteria is between 4 and 9, the optimum growth usually
occurs between 6.5-7.5. Therefore, to optimize the maximum
concentration of cell mass and co-polymer accumulation, the
test organism was grown at different pH (Table 1) and
temperature (Table 2). The optimal pH and temperature for
the maximal co-polymer accumulation were 7.5 and 30C,
respectively. This is quite in tune with Du and Yu [28],
where maximum PHA yield was obtained at pH 7.4 and
temperature 30C for Pseudomonas oleovorans. The
optimum inoculum size for maximum accumulation of PHAs
was observed at 80 mg dcw/l (Table 3). Further, increasing
the size of the inoculum had no significant effects on PHAs
accumulation.
Nitrogen and phosphorus deficiencies were found to raise
the PHA pool marginally [20]. The possible explanation for
this rise could be that under nitrogen deficiency, reduced cofactor NADPH consumption was reduced owing to
inaccessibility of nitrogen pool, which prevents the amino
acid synthesis pathways, particularly the reaction from
ketoglutarate to glutamate, therefore resulting into buildup of

86 Current Biotechnology, 2013, Volume 2, No. 1

Table 5.

Singh et al.

Maximum Yield and Composition of the Polymer in P. aeruginosa MTCC 7925 Pre-Grown in 0.6% Palm Oil + the
Extract of 0.6% (v/v) Palm Oil Cakes Supplemented-Medium, when Subjected to Nitrogen and Phosphorus Limitations
for 48 h

Condition

Biomass Yield (dcw, mg/l)

Untreated control
N+ + P+- medium

PHA Accumulation

Polymer Composition (mol%)

(mg/l)

(% dcw)

3HB

3HV

3HHD

3HOD

289.9 1.6a

70.4 2.0a

24.3 0.9a

24.6

70.6

1.6

3.2

8691.9 9.1b

5400.9 7.8b

62.1 3.1b

85.4

6.8

3.1

4.7

84.8

7.2

3.1

4.9

90.0

4.8

2.2

3.0

P-limitation
N-limitation

8011.3 8.2
7671.4 7.4

5644.7 7.1

5873.4 8.6

70.4 6.6
76.6 8.6

Values are mean SE, n = 3.

Values in the column superscripted by different letters are significantly different from each other (P < 0.05, Duncans new multiple range test).
Separate analysis was done for each column.

Table 6.

Comparative Account on the Properties of the Co-Polymer of P. aeruginosa MTCC 7925 with Common Plastics [37, 38]
*P(3HB-co-3HV-co-3HHD-co-3HOD)
(mol Fraction 84.8:7.2:3.1:4.9 95.7:1.0:1.8:1.5)

PP

LDPE

115 to131

176

130

Glass transition temperature, C

-8 to -14

-10

-36

Tensile strength (Mpa)

17 to 19

38

10

Elongation to break (%)

682 to 723

400

620

Youngs modulus (GPa)

0.2 to 0.3

1.7

0.2

Thermal stability, Td(5%)

248 to 262

338

387

Enthalpy of fusion (
Hm), J /g

26 to 44

nr

nr

Property
Melting temperature, C


nr = not reported.
*Polymer from this study.

excess NADPH in the cells [29]. This excess NADPH might

be accountable for the increased PHA synthesis in nitrogendeficient cells. The rise of PHA pool under P-deficiency
could be due to surplus reducing power and diminished ATP
production with the commencement of phosphate limitation
[30].
Under glucose and ethanol supplementation a significant
rise in polymer content with high 3HB fraction was clearly
evident. The positive effect of glucose on PHA production
could be attributable to the increased supply of acetyl Co-A
and NADPH [31], which are prerequisite for the activity of
the enzyme acetoacetyl-CoA reductase for conversion of
acetoacetyl-CoA to
-hydroxybutyryl-CoA. During ethanol
metabolisation, in which alcohol dehydrogenase is involved,
acetyl-CoA and reduced coenzymes NADPH are also
formed. These metabolites could slightly inhibit TCA cycle
while flux of acetyl-CoA into PHA biosynthetic pathway is
likely to be supported [32].
For industrial uses, it is crucial to optimize the significant
factors affecting the novel P(3HB-co-3HV-co-3HHD-co3HOD) co-polymer accumulation in P. aeruginosa MTCC
7925. The model so obtained for the co-polymer
accumulation was adequate enough as depicted from the
high F-value (148.5), insignificant lack of fit
(Probability > F = 0.22) and R2 close to 1.0 (0.99) (Table 4).
The polymer content reached up to 77.6%, which is the
highest value reported so far amongst co-polymer producer.
Moreover, the test organism exhibits capability of

incorporating the novel 3HHD and 3HOD units as

constituents.
The major bottleneck for commercialization of PHA is
the high cost of the carbon substrates. The simplest approach
is to choose the cheapest and most readily available carbon
substrates that could support the microbial growth
efficiently. Amongst different inexpensive carbon sources,
palm oil depicted maximum stimulatory impact on the copolymer accumulation; PHAs pool was enhanced up to 62%
(dcw) in the presence of 0.7% palm oil supplementation at
48 in P. aeruginosa MTCC 7925. Intriguingly, PHAs
content was also boosted up to 61.1% (dcw) under
supplementation of 0.6% palm oil + 0.6% of the extract of
palm oil cakes, which could be comparable with the
maximum PHAs yield of 0.7% palm oil-supplemented
cultures (Figs. 4, 5). Thus, supplementation of the extract of
0.6% palm oil cake in combination with 0.6% palm oil
resulted not only into higher PHA yield, the composition of
the polymer and biomass yield were also at par with the
yield of 0.7% palm oil supplemented-cultures. Moreover,
reduction of palm oil concentration from 0.7 to 0.6%
resulted into further reduction in the cost of PHAs
production as palm oil cake is a cheap and plentily available
waste material. A further rise of PHAs content up to 76.6%
boosted when the above culture was subjected to Ndeficiency. Apart from this, it is also evident that the test
organism is capable of synthesizing the co-polymer with
high mol% of SCL 3HB monomer and a low mol% of LCL
3HHD and 3HOD units, which is desirable for commercial

Production of a Novel SCL-LCL-PHA Co-Polymer by Pseudomonas aeruginosa MTCC 7925

applications as the properties of the polymer are greatly

influenced by their monomer composition [9].
DSC analysis showed that this new co-polymer with
small amount of 3HV, 3HHD and 3HOD monomer units to
3HB dramatically reduced the glass transition temperature
(Tg) and melting (Tm) temperature compared to those of PHB
and P(3HB-co-3HA) (Table 6). Apart from this, the
incorporation of 3HV, 3HHD and 3HOD monomer units into
PHB backbone also influenced the enthalpy of fusion (
Hm)
that agrees well with the earlier reports of Luo et al. [19],
where the
Hm for SCL-MCL-PHA co-polymer was lower
than that of PHB for Ralstonia eutropha PHB-4. The lower

Hm suggests low crystalline nature of the polymer [5].
Thermal stability is of particular importance for PHAs in
processing. It is well known that typical PHAs suffer thermal
decomposition at a temperature above 170C, which is near
the Tm of PHB and P(3HB-co-3HV) [33]. Therefore, it is
desirable to keep the Tm of PHA well below the thermal
decomposition temperature. Thus, the depression of Tm of
P(3HB-co-3HV-co-3HHD-co-3HOD) by the incorporation
of 3HV, 3HHD and 3HOD units could be highly beneficial.
Thermogravimetric analysis showed alteration of thermal
stability of the co-polymers with the incorporation of 3HV,
3HHD and 3HOD units (Table 6), which is agreeing with the
reports of Luo et al. [19], where the temperature at 5%
weight loss (Td(5%)) for SCL-MCL-PHA co-polymer was
higher (Td(5%) = 256.2C) than that of homopolymer PHB
(Td(5%) = 235C) for Ralstonia eutropha PHB-4, reflecting
better thermal stability of P(3HB-co-3HV-co-3HHD-co3HOD) co-polymer than that of PHB. Thermal degradation
of PHB is known to occur by random chain scission reaction
through a
-elimination mechanism, where the degradation
starts with the formation of six-member ring intermediates
[34]. The improved stability of P(3HB-co-3HV-co-3HHDco-3HOD) co-polymer could be explained on the basis of the
earlier reports of Asrar et al. [33], where increases in thermal
stability of SCL-MCL PHA co-polymers may be a result of
steric hindrance to the formation of six-member ring created
by the longer side chains of the MCL-3HA units.
The mechanical properties of P(3HB-co-3HV-co-3HHDco-3HOD) samples were studied using stress-strain
measurement, which showed that the P(3HB-co-3HV-co3HHD-co-3HOD) co-polymer exhibited material properties
comparable to PP and LDPE (Table 6). This is in well
agreement with the earlier report of Doi et al. [8], where the
incorporation of 3HHx units improved the brittle feature of
PHB and the increase of 3HHx unit made the material more
ductile, i.e. soft and flexible. Thus, P. aeruginosa MTCC
7925 emerging as an novel organism in the production of
SCL-LCL-PHA co-polymer for various industrial
applications with 3HB, 3HV, 3HHD and 3HOD units as
components from conventional unrelated carbon sources
such as ethanol, glucose and various plant oils such as palm
oil with its cakes under the same growth conditions under
which other Pseudomonads including P. aeruginosa
accumulate MCL-PHAs units only.

87

synthesizing a novel SCL-LCL-PHA co-polymer from

unrelated carbon sources such as glucose, acetate, maltose,
ethanol etc. Application of response surface methodology for
optimization of process variables resulted into a yield of
78% (dcw). The polymer yield reached up to 5.9 g/l under
the supplementation of palm oil and its waste cakes. This
novel co-polymer exhibited good thermal and mechanical
properties, which could be comparable with polypropylene
(PP) and low-density polyethylene (LDPE).
CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
CONFLICT OF INTEREST
Authors do not have any conflict of interest.
REFERENCES
[1]
[2]
[3]
[4]

[5]

[6]

[7]
[8]
[9]

[10]
[11]

[12]

[13]
[14]

5. CONCLUSION
In conclusion, the sludge-isolated Pseudomonas
aeruginosa MTCC 7925 demonstrated good capability of

Current Biotechnology, 2013, Volume 2, No. 1

[15]

Alexander M. Biodegradation of chemicals of environmental

concern. Science 1981; 211: 132-8.
Braunegg G, Gilles L, Klaus F. Polyhydroxyalkanoates
biopolyesters from renewable resources: physiological and
engineering aspects. J Biotechnol 1998; 65: 127-61.
Steinbuchel A. Biodegradable plastics. Curr Opin Biotechnol 1992;
3: 291-7.
Ashby RD, Solaiman DKY, Foglia TA. The synthesis of short and
medium chain-length poly(hydroxyalkanoate) mixtures from
glucose- or alkanoic acid-grown pseudomonas oleovorans. J Ind
Microbiol Biotechnol 2002; 28: 147-53.
Haywood GW, Anderson AJ, Williams DR, Dawes EA.
Accumulation of a poly(hydroxyalkanoate) co-polymer containing
primarily 3-hydroxyvalerate from simple carbohydrate substrates
by Rhodococcus sp. NCIMB 40126. Int J Biol Macromol 1991; 13:
83-8.
Lee EY, Jendrossek D, Schirmer A, Choi CY, Steinbuchel A.
Biosynthesis of copolyesters consisting of 3-hydroxybutyric acid
and medium-chain-length 3-hydroxyalkanoic acids from 1,3butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33.
Appl Microbiol Biotechnol 1995; 42: 901-9.
Caballero KP, Karel SF, Register RA. Biosynthesis and
characterization of hydroxybutyrate-hydroxycaproate co-polymers.
Int J Biol Macromol 1995; 17: 86-92.
Doi Y, Kitamura S, Abe H. Microbial synthesis and characterization
of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules
1995; 28: 4822-8.
Nomura CT, Taguchi K, Gan Z, et al. Expression of 3-ketoayl-acyl
carrier protein reductase (fabG) genes enhanced production of
polyhydroxyalkanoates co-polymer from glucose in recombinant
Escherichia coli JM109. Appl Environ Microbiol 2005; 71: 4297-6.
Muheim A, Lerch K. Towards a high-yield bioconversion of ferulic
acid to vanillin. Appl Microbiol Biotechnol 1999; 51: 456-61.
Kim DY, Kim YB, Rhee YH. Evaluation of various carbon
substrates for the biosynthesis of polyhydroxyalkanoates bearing
functional group by Pseudomonas putida. Int J Biol Macromol
2000; 28: 23-9.
Kim GJ, Lee Y, Yoon SC, Shin YC, Park YH. Enhanced yield and
a
high
production
of
medium-chain-length
poly(3hydroxyalkanoates) in a two-step fed-batch cultivation of
Pseudomonas putida by combined use of glucose and octanoate.
Enz Microbial Technol 1997; 20: 500-2.
Pal S, Manna A, Paul AK. Nutritional and cultural condition for
production of poly-3-hydroxybutyric acid by Azotobacter
chroococcum. Folia Microbiol 1998; 43: 177-81.
Yellore V, Desia A. Production of poly-
-hydroxybutyrate from
lactose and whey by methylobacterium sp. ZP24. Lett Appl
Microbiol 1998; 26: 391-4.
Yao J, Zhang G, Wu Q, Chen G-Q, Zhang R. Production of
polyhydroxyalkanoates by Pseudomonas nitroreducens. Antonie
Van Leeuwenhoek 1999; 75345-9.

88 Current Biotechnology, 2013, Volume 2, No. 1

[16]
[17]
[18]

[19]

[20]
[21]

[22]
[23]

[24]
[25]
[26]

[27]

Singh et al.

Riis V, Mai W. Gas chromatographic determination of poly-
hydroxybutyric acid in microbial biomass after hydrochloride acid
propanolysis. J Chromatogr 1988; 445: 285-9.
Montgomery DC. (2001) Design and analysis of experiments.
Wiley, New York 2001; p. 492.
Kato M, Bao HJ, Kang C-K, Fukui T, Doi Y. Production of novel
copolyester of 3-hydroxybutyric acid and medium-chain-length 3hydroxyalkanoic acids by Pseudomonas sp. 61-3 from sugars. Appl
Microbiol Biotechnol 1996; 45: 363-70.
Luo R, Chen J, Zhang L, Chen G. Polyhydroxyalkanoate
copolyesters produced by Ralstonia eutropha PHB
4 harboring a
low-substrate-specificity
PHA
synthase
PhaC2Ps
from
Pseudomonas stutzeri 1317. Biochem Eng J 2006; 32: 218-25.
Singh AK, Mallick N. Enhanced production of SCL-LCL-PHA copolymer by sludge-isolated Pseudomonas aeruginosa MTCC 7925.
Lett Appl Microbiol 2008; 46: 350 -7.
Sun W, Cao JG, Teng K, Meighen EA. Biosynthesis of poly-3hydroxybutyrate in the luminescent bacterium, Vibrio harveyi, and
regulation by the lux autoinducer, N-(3-hydroxybutanoyl)
homoserine lactone. J Biol Chem 1994; 269: 20785- 90.
Mccool GJ, Fernandez T, Li N, Cannon MC. Polyhydroxyalkonoate
inclusion body growth and proliferation in Bacillus megaterium.
FEMS Microbiol Lett 1996;138: 41-8.
Potter M, Muller H, Steinbuchel A. Influence of homologous
phasins (PhaP) on PHA accumulation and regulation of their
expression by the transcriptional repressor PhaR in Ralstonia
eutropha H16. Microbiology 2005; 151: 825-33.
Steinbuchel A, Hein S. Biochemical and molecular basis of
polyhydroxyalkanoic acids in microorganisms. Adv Biochem Eng
Biotechnol 2001; 71: 81-123.
Mercan N, Aslim B, Yuksegdag ZN, Beyatli Y. Production of poly
-hydroxybutyrate (PHB) by some Rhizobium bacteria. Turk J Biol
2002; 26: 215-9.
Vazquez GJ, Pettinari MJ, Mendez BS. Evidence of an association
between
poly(3-hydroxybutyrate)
accumulation
and
phosphotransbutyrylase expression in Bacillus megaterium. Int
Microbiol 2003; 6: 127-29.
Yuksekdag ZN, Aslim B, Beyatli Y, Mercan N. Effect of carbon
and nitrogen sources and incubation times on poly-beta-

Received: July 12, 2012

[28]
[29]
[30]

[31]

[32]

[33]

[34]
[35]
[36]

[37]
[38]

hydroxybutyrate (PHB) synthesis by Bacillus subtilis 25 and

Bacillus megaterium 12. Afr J Biotechnol 2004; 3: 63-6.
Lee SY, Park SJ. In: Biosynthesis and fermentative production of
SCL-MCL- PHAs; Doi Y, Steinbuchel A, Eds. Wiley/VCH:
Weinheim, Biopolymers 2002; 3a: 317-336.
Du G, Yu J. Green technology for the conversion of food scraps to
biodegradable thermoplastic polyhydroxybutyrates. Environ Sci
Technol 2002; 36: 5511-6.
Konopka A, Schnur M. Biochemical composition and
photosynthetic carbon metabolism of nutrient limited cultures of
Merismopedia tenuissima (Cyanophyceae). J Phycol 1981, 17: 11822.
Lee EY, Jendrossek D, Schirmer A, Choi CY, Steinbuchel A.
Biosynthesis of co-polyesters consisting of 3-hydroxybutyric acid
and medium-chain-length 3-hydroxyalkanoic acids from 1,3butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33.
Appl Microbiol Biotechnol 1995; 42: 901-9.
Obruca S, Marova I, Stankova M, Mravcova L, Svoboda Z. Effect
of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate)
biosynthetic pathway in Cupriavidus necator H16. World J
Microbiol Biotechnol 2010; 26: 1261-7.
Asrar J, Valentin HE, Berger PA, Tran M, Padgette SR, Garbow
JR. Biosynthesis and properties of poly(3-hydroxybutyrate-co-3hydroxyhexanoate) polymers. Biomacromolecules 2002, 3: 100612.
Noda I, Green PR, Satkowski MM, Schechtman LA. Preparation
and properties of a novel class of polyhydroxyalkanoate copolymers. Biomacromolecules 2005, 6: 580-6.
Singh AK, Mallick N. SCL-LCL-PHA co-polymer production by a
local isolate, Pseudomonas aeruginosa MTCC 7925. Biotechnol J
2009; 4: 703-11.
Singh AK, Mallick N. Exploitation of inexpensive substrates for
production of a novel SCL-LCL-PHA co-polymer by Pseudomonas
aeruginosa MTCC 7925. J Ind Microbiol Biotechnol 2009; 36:
347-54.
Niu P, Liu B, Wei X, Wang X, Yang J. Study on mechanical
properties and thermal stability of polypropylene/hemp fiber
composites. J Reinf Plast Compos 2010; 0: 1-9.
Xia X, Cai S, Xie C. Preparation, structure and thermal stability of
Cu/LDPE nanocomposites. Mater Chem Phys 2006; 95: 122-9.

Revised: January 10, 2013

Accepted: January 15, 2013