ENHANCED PRODUCTION OF POLYHYDROXYBUTYRATE (PHB) FROM AGRO-INDUSTRIAL WASTES; FEDBATCH CULTIVATION AND STATISTICAL MEDIA OPTIMIZATION
Mahmoud M. Berekaa1* and Adil M. Al Issa2
Address(es): Dr. Mahmoud Berekaa,
1
University of Dammam, College of Applied Medical Sciences, Environmental Health Department, Dammam 31441, Saudi Arabia. +966-0133331304.
2
University of Dammam, College of Education, Biology Department, Dammam 31451, Saudi Arabia.
*Corresponding author: mberekaa@uod.edu.sa
doi: 10.15414/jmbfs.2016.5.6.606-611
ARTICLE INFO
ABSTRACT
Received 20. 9. 2014
Revised 7. 2. 2016
Accepted 1. 3. 2016
Published 1. 6. 2016
Bacillus megaterium SW1-2 showed enhanced growth and polyhydroxybutyrate (PHB) production during cultivation on date palm
syrup (DEPS) or sugar cane molasses. FT-IR and NMR spectroscopic analyses of the polymer accumulated during growth on DEPS
revealed specific absorption peaks characteristic for PHB. 1.65 g/L of PHB (56.9% CDW) was produced during growth on medium
supplemented with 2 g/L of DEPS. Approximately, 36.1% CDW of PHB were recorded during growth on sugar cane molasses. Six
runs of different fed-batch cultivation strategies were tested, the optimal run showed approximately 6.87-fold increase. Modified E2
medium was prefered recording 10.11 and 11.34 g/L of total PHB produced for runs 1 and 2, at the end of 96 h incubation period,
respectively. Decrease in PHB was recorded during growth on complex medium (run 3 and run 4). In another independent optimization
strategy, ten variables were concurrently examined for their significance on PHB production by Plackett-Burman statistical design for
the first time. Among variables, DEPS-II and inoculum concentration followed by KH2PO4 and (NH4)2SO4 were found to be the most
significant variables encourage PHB production. Indeed, DEPS-II or Fresh syrup is more significant than commercial syrup DEPS-I (pvalue= 0.05). RPM, incubation period have highly negative effect on PHB production. Role of ago-industrial wastes, especially DEPS,
in enhancement of PHB production was closely discussed.
Regular article
Keywords: Date syrup or DEPS, Polyhydroxybutyrate (PHB), Bacillus megaterium, Plackett-Burman design, fed-batch cultivation,
optimization
INTRODUCTION
Among
the
most
commonly
known
biopolymers,
bacterial
polyhydroxyalkanoates (PHAs) polyesters gain more interest. It is synthesized
and intracellular accumulated as storage materials to be used as energy source
during unbalanced growth (Anderson and Dawes, 1990; Steinbuchel 2001; Du
and Yu, 2002; Ojumu et al., 2004). PHB production by microorganisms
attracted attention of many scientists due to close resemblance to synthetic
petroleum–based plastics such as polypropylene (Mokhtari-Hosseini et al.,
2009), biodegradability and biocompatiblility (Madison and Huisman, 1999;
Ojumu et al., 2004; Khanna and Srivastava, 2005). Several applications in
medicine, veterinary practice, tissue engineering materials, food packaging and
agriculture have been reported (van der Walle et al., 2001; Zinn et al., 2001;
Luengo et al., 2003; Chen and Wu, 2005; Bucci et al., 2005; Urtuvia et al.,
2014). Many bacilli have been reported to accumulate PHA and co-polymers of
3HB during growth on different substrates (Belma et al., 2002; Tajima et al.
2003; Valappil et al., 2007; Adwitiya et al., 2009; Reddy et al., 2009). In spite
the privilege of microbial PHAs production compared with petroleum-derived
plastics, costs of the feedstock are the main limiting factor for their mass
production (Ojumu et al., 2004; Nikel et al., 2005). Economically, the use of
agro-industrial wastes, improved fermentation strategies and downstream
processes have positive contribution on the overall biopolymer production costs
(Liu et al. 1998; Tamer et al. 1998; Kim, 2000; Halami, 2008).
Recently, scientists have been exploring different cultivation strategies involving
inexpensive, renewable carbon substrates in order to reduce production cost and
obtain high productivity (Ojumu, 2004). Many carbon sources derived from
wastes like whey, cane molasses, sugar beet molasses and date syrup can be used
in production (Beaulieu et al., 1995; Lee et al., 1997; Liu et al., 1998; Omar et
al., 2001; Khanafari et al., 2006; Albuquerque et al., 2007; Hamieh et al.,
2013). Nowadays, PHB proved to be produced from relatively cheaper substrates
such as methanol (Kim et al., 2003; Mokhtari-Hosseini et al., 2009), carbon
dioxide (Ishizaki et al., 2001), and several agro-industrial by-product such as rice
bran, pulp, paper and cardboard industry, whey, dairy wastes, sea water and
municipal wastes (Law et al., 2001; Nikel et al., 2005; Nath et al., 2008;
Santimano et al., 2009; Pandian et al., 2010; Bhuwal et al., 2013; Hamieh et
al., 2013; Singh et al., 2013; Watanabe et al., 2014). Furthermore, fed-batch
cultivation in chemically defined medium for optimization of PHB production
has been applied (Wang and Lee., 1997; Liu et al., 1998). Investigations have
also focused on reducing the total cost of PHB through optimizing fermentation
processes. Recently, application of statistical methods has gained a lot of impetus
for medium optimization and understanding the interactions among various
physiochemical parameters involved in biopolymer production (Khanna and
Srivastava, 2005; Nikel et al., 2005; Sharma et al., 2007; Mokhtari-Hosseini
et al., 2009; Pandian et al., 2010; Berekaa and Al Thawadi., 2012; Hamieh et
al., 2013).
The main objective of this study was to investigate the possible production of
PHB from several agro-industrial wastes mainly; date palm syrup (DEPS) and
sugar cane molasses. Produced polymer was identified by chemical
characterization using FT-IR, C13NMR and H1NMR spectroscopy. Furthermore,
optimization of PHB production by application of six different fed-batch
cultivation strategies was closely investigated. Special emphasis was given to the
application of statistical experimental design (Plackett-Burman) for optimization
of PHB production from DEPS for the first time.
MATERIAL AND METHODS
Microorganism
Group of bacilli previously isolated, maintained and screened for PHB
production (Berekaa and Al Thawadi, 2012; Berekaa, 2012), were tested for
PHB production from agro-industrial wastes namely; date syrup (DEPS) and
sugar cane molasses. The potent PHB producing bacterial strain Bacillus
megaterium SW1-2 used in this study was identified by 16S DNA gene analysis
as previously reported (Berekaa and Al Thawadi, 2012).
Growth and production conditions
The bacterium was grown in 50mL aliquot of nutrient broth dispensed in 250 mL
Erlenmeyer flask and incubated at 37°C for 24h or 48h at (150 rpm). 1.5%
inoculums of the overnight culture was used to inoculate modified E2 medium of
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J Microbiol Biotech Food Sci / Berekaa and Issa 2016 : 5 (6) 606-611
the following composition (g/L): ammonium sulfate; 2.5, KH2PO4; 1.5,
Na2HPO4; 3.5, MgSO4.7H2O; 0.2, traces of yeast extract and 1 mL of trace
element solution (FeSO4.4H2O, CaCl2.2H2O, MnSO4.4H2O, ZnCl2 1 mM each) at
37°C. To test the possible production of PHB, date palm syrup (DEPS) or sugar
cane molasses was used as a sole carbon source at different concentrations. Two
types of date syrup (DEPS) were used in this study, the commercial (DEPS-I)
composed mainly of (per 100 g); carbohydrates 55 g (mainly sucrose), vitamin B
complex 0.57 mg, calcium 685 mg, phosphate 75 mg, iron 16 mg and magnesium
258 mg) and Freshly prepared date palm syrup (DEPS-II) as following; 500 g of
date palm (Khalas from Kaseem province) were placed in 1.5 L dist. water and
boiled for 90 min with mixing. At the end of incubation period the extract was
filtered in cloth for 3-4 times. Subsequently, the extract kept in oven at 60oC till
net volume of 200 mL. Finally, the fresh date syrup was kept in refrigerator till
use. Sugar cane molasses used in this study were obtained from an industrial
sugar manufacturing plant, Egypt. Sugar content of molasses was 76% (w/v), and
composed mainly of sucrose (62%), and fructose (38%). At the end of
incubation period, PHB was determined and the cell dry weight was estimated.
Fed-batch cultivation and PHB production strategies
For optimization of PHB production from date syrup (DEPS), fed-batch
cultivation was applied. During fed-batch cultivation, two different feeding
strategies were applied. The first pulsed feeding of DEPS-I was carried out after
24 and 60 h time intervals, while the second was performed after 48 and 84 h,
respectively, with final concentration of 15 g/L. Furthermore, three basal media
were tested in this study namely; modified E2 medium, nutrient broth medium
and a mixture of nutrient broth and modified E2 medium (50% w/w each) with
the sum of six different fed-batch cultivation strategies (run 1 to run 6). In each
experiment, sample was taken during different time intervals, PHB was
determined and the cell dry weight was estimated.
between 400 and 4000 cm−1 were recorded using Nicolet 6700 FTIR
spectrometer from the Nicolet Instrument Corporation, USA.
Characterization of PHB by C13 NMR and H1 NMR analysis
Extracted PHB biopolymer from B. megaterium SW1-2 was characterized by
spectroscopic analysis. H1NMR spectrum was recorded on a JEOL JNM-LA 500
MHz spectrometer at 30oC in CDCl3 as solvent. While, C13 NMR spectral
experiments were performed at 125.65 MHz with the following acquisition
parameters: 32 k data point, 0.967 s acquisition time, recycle delay 1 s and
contact time 4.50 ms.
Fractional factorial design
Plackett-Burman experimental design
Screening design namely; Plackett-Burman experimental design was applied to
investigate the significance of various medium factors on PHB production
(Plackett and Burman, 1946). 10 chemical and environmental independent
variables were examined in two levels: –1 for low and +1 for high level based on
Plackett-Burman design (Table 1). According to the matrix shown in Table 2, the
independent variables were screened in 14 combinations. The main effect of each
variable is the difference between average of measurements at high setting (+1)
and average of measurements observed at low setting (-1) of that factor. PlackettBurman experimental design was based on the first order model (Equation 1):
Y=β0+Σ βiXi (1)
Where, Y is the predicted response, β0 and βi are constant coefficients, and Xi is
the coded independent variables estimates or factors. The quality of fit of the
polynomial model equation was expressed by the coefficient of determination R2.
Extraction of PHB from the isolate
PHB was extracted from the cell masses by using modified hypochlorite method
as previously described (Rawte and Mavinkurve, 2002; Berekaa and Al
Thawadi, 2012).
Table 1 Variables and their settings employed in Plackett-Burman design for
optimization of PHB production by Bacillus megaterium SW1-2 during growth
on date syrup or DEPS.
Levels
Medium Composition
Analytical procedures
DEPS-I (%)
DEPS-II (%)
(NH4)2SO4 (g/L)
Yeast Extract (g/L)
Na2HPO4 (g/L)
KH2PO4 (g/L)
Inoculum conc. (%)
Temperature (oC)
RPM
Incubation Period
Cell dry weight
After centrifugation of the culture medium, supernatant was discarded and cell
pellet was washed with distilled water. The washed pellet was resuspended in 1
mL distilled water, transferred to pre weighed boats and dried to constant weight
at 60oC.
Chemical analysis of polymer
Characterization of PHB by FT-IR
The presence and characterization of PHB in dry cell matter was confirmed by
Fourier Transform Infrared Spectroscopy (FT-IR) (Hong et al., 1999). Dry PHB
polymer from B. megaterium SW1-2 was used to prepare KBr discs. Spectra
-1
1
2
2
1.5
0.5
2
1
1
28
60
48
4
4
3
1
4
2
2
37
120
72
Table 2 Effect of different environmental conditions on PHB production from Bacillus megaterium SW1-2 during growth on date palm syrup or DEPS
Exp.
No.
1
DEPS-I
(%)
DEPS-II
(%)
(NH4)2SO4
(g/L)
Yeast
Extract
(g/L)
Na2HPO4
(g/L)
KH2PO4
(g/L)
Inoculum
(%)
Temperature
(oC)
RPM
Incubation
Period (h)
PHB
(% CDW)
4
4
3
0.5
4
2
1
37
60
48
46
9.8
2
4
2
3
1
2
2
1
28
60
72
3
1.5
1.5
2.5
0.5
3.5
1.5
1.5
37
120
48
31
4
4
2
3
0.5
2
1
2
37
120
48
27.4
5
4
4
1.5
1
2
1
1
37
120
72
11.5
6
2
2
1.5
1
4
2
1
37
120
48
7.1
7
2
2
3
1
4
1
2
37
60
72
32.7
8
2
4
3
1
2
2
2
28
120
48
53.6
9
2
4
3
0.5
4
1
1
28
120
72
10.6
10
4
2
1.5
0.5
4
2
2
28
120
72
21.3
11
4
4
1.5
1
4
1
2
28
60
48
42.4
12
2
2
1.5
0.5
2
1
1
28
60
48
23.8
13
1.5
1.5
2.5
0.5
3.5
1.5
1.5
37
120
48
28.6
14
2
4
1.5
0.5
2
2
2
28
60
72
46.1
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Data of the PHB production were subjected to multiple linear regressions using
MICROSOFT EXCEL 97 to estimate t-value, P-value and confidence level. The
significance level (P-value) was determined using the Student’s t-test. Factors
having highest t-value and confidence level over 95% were considered to be
highly significant on PHB production. Data presented in this study measured in
duplicate.
RESULTS AND DISCUSSION
Growth of Bacillus megaterium SW1-2 on date syrup (DEPS)
Among a group of polyhydroxybutyrate-producing bacilli explored for potential
production of PHB biopolymer from an agro-industrial waste namely; date palm
syrup (DEPS), Bacillus megaterium SW1-2 showed potent growth on DEPS or
date syrup as a sole source of carbon. Elemental analysis revealed that 70% of the
polymer accumulated during growth on date syrup or DEPS was carbon (data not
shown).
reported by Liu et al. (1998). Belal (2013) successfully used simple carbon
sources as well as molasses as a source of carbon for PHB production. It is
assumed that higher concentrations of sugars in cane molasses namely; sucrose
and fructose contribute to enhanced production of PHB in B. megaterium SW1-2.
CDW 24 (g/L)
CDW 48 (g/L)
CDW (g/L) & PHB (g/L)
4
PHB 24 (g/L)
PHB 48 (g/L)
3,5
3
2,5
2
PHB 24 (g/L)
PHB 48 (g/L)
2.5
2
1.5
1
0.5
PHB accumulation by Bacillus megaterium SW1-2 cultivated on DEPS-I
For economic visibility of PHB biopolymer production by B. megaterium SW1-2
date syrup or DEPS was used as a natural carbon source. Monitoring of PHB
accumulation in presence of different DEPS concentrations was carried out. Data
shown in Figure 1 indicated that the agro-industrial date palm syrup or DEPS can
be used as a suitable renewable carbon source during PHB production. Maximum
PHB production was recorded at the end of log phase and approximately 1.65 g/L
of PHB was accumulated after 48 h during cultivation on modified E2 medium
supplemented with 2 g/L of DEPS-I or date syrup. Furthermore, lower
concentration of DEPS-I enhanced PHB accumulation (average of 42% CDW as
well as 52.65% CDW of PHB accumulated after 24 and 48 h at concentration of
2 and 2.5 (w/v) of DEPS-I, respectively). Omar et al. (2001) successfully used
date palm syrup or DEPS as carbon source during PHB production. Page (1992)
reported that the polymer production greatly enhanced in presence of unrefined
sugars as well as complex nitrogen sources. It is assumed that various sugars
namely; sucrose, glucose and fructose in date syrup or DEPs can be used as a
carbon source by the B. megaterium SW1-2 cells during PHB production and
may contribute to enhanced production. As previously recorded, glucose or
sucrose can be used as carbon source during biopolymer production by many
bacteria (Zhang et al., 1994, Valappil et al., 2007; Adwitiya et al., 2009; Reddy
et al., 2009, Wang, 2011, Belal, 2013), supporting this conclusion.
CDW 24 (g/L)
CDW 48 (g/L)
3
CDW (g/L) & PHB (g/L)
Statistical analysis of the data
0
2
2.5
5
7.5
Molasses Conc. (w/v)
Figure 2 Effect of different sugar cane molasses on growth and PHB production
by B. megaterium SW1-2.
FT-IR spectroscopy analysis
FT-IR Spectroscopic analysis of the polymer extracted from B. megaterium cells
grown on modified E2 medium with date syrup or DEPS-I as a sole carbon
source revealed a clear C–H and carbonyl stretching bands highly characteristic
to PHB. Two absorption bands occurring at 2921.36 and 2859.92 cm -1 indicated
the presence of aliphatic group -CH3 and -CH2. The two other absorption bands
at 1718.26 and 1267 cm-1 in the PHB sample conforming the presence of C=O
and C–O stretching groups and were identical to PHB from some bacilli (Hong et
al., 1999, Pandian et al., 2010; Valappil et al., 2007).
H1NMR and C13NMR spectroscopy
Furthermore, analysis of extracted polymer by H1NMR revealed three groups of
distinctive signals of the PHB polymer. A doublet at 1.22 and 1.25 ppm represent
methyl group (-CH3) coupled to one proton and 2.28 ppm resulted from
methylene group (-CH2) adjacent to an asymmetric carbon atom. The third signal
was at 5.2 ppm attributed to a methyne group (-CH). Furthermore, C13NMR
analysis was used to determine the structure of the isolated polymer from B.
megaterium SW1-2 grown on the modified E2 medium. Four narrow lines
appeared which were identical to the C13NMR spectra of PHB (Doi and Abe,
1990). The four peaks assigned for methyl (CH3; 21.2 ppm), methylene (CH2;
42.7 ppm), methine (CH; 68.5 ppm) and carbonyl (C=O; 169.7 ppm) carbon
resonance of PHB (Doi and Abe, 1990). Analysis collectively confirmed the
molecular composition of the polymer to be PHB.
Fed-batch cultivation and application of different pulsed feeding strategies
1,5
1
0,5
0
2
2,5
5
7,5
DEPS Conc. (w/v)
Figure 1 Effect of different date palm syrup or DEPS on growth and PHB
production by B. megaterium SW1-2.
PHB accumulation by Bacillus megaterium SW1-2 cultivated on Molasses
In a trial to use other low-cost substrates for PHB production, B. megaterium
SW1-2 allowed to grow on sugar cane molasses. Results revealed that B.
megaterium SW1-2 showed clear growth and PHB accumulation during
cultivation on modified E2 medium supplemented with sugar cane molasses as a
sole carbon source (Figure 2). It revealed that 5 g/L of sugar cane molasses was
the optimal concnetration for PHB production by B. megaterium cells.
Approximately, 29.24% and 26.1% CDW of PHB accumulated after 24 and 48 h,
respectively. Lower concentrations of molasses resulted in approximately 50%
reduction in PHB, while clearly enhanced cell biomass. In concordance with our
results, Beaulieu et al. (1995) used cane molasses together with ammonium salt
for growth of Alcaligenes eutrophaus and PHB production. Recombinant
Escherichia coli strain (HMS174/pTZ18u-PHB) capable of PHB production was
Since PHB is a carbon-based biopolymer, it depends mainly on the nature and
concentration of carbon source during synthesis and accumulation.
Unfortunately, longer incubation period during batch cultivation leads to nutrient
limitations especially for C-source, hence fed-batch cultivation may provide
promising solution. In this study commercial date syrup or DEPS-I was added in
two pulsed feeding experiment. Results presented in figure 3 indicated that fedbatch cultivation can be used as successful strategy to optimize PHB production
by Bacillus megaterium SW1-2 especially when using 48 h (runs 6 and 4) and 84
h (runs 2 and 4) pulsed feeding process. Modified E2 medium was preferred for
PHB accumulation, recording total amount of 10.11 and 11.34% CDW of PHB
for run1 and 2 after 96 h cultivation, respectively. On the other hand, the lowest
amount of PHB accumulated, 8.93% and 9% CDW of PHB during use of
complex nutrient broth medium and applying the two different pulsed feeding
processes run 3 and run 4, respectively. Surprisingly, there was general trend of
increase in PHB production after feeding process and the total amount of PHB
after fed-batch cultivation for 96 h recorded 6.86-fold increase in comparison to
batch cultivation. Albuquerque et al. (2007) developed several strategies for
polyhydroxyalkanoate (PHA) production from sugar cane molasses. They
reported that beet molasses successfully replaced glucose as sole carbon source to
produce poly-b-hydroxybutyrate by a recombinant Escherichia coli strain
(HMS174/pTZ18u-PHB). Also, the positive impact of fed-batch cultivation on
PHB production was reported by Hameih et al., (2013).
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J Microbiol Biotech Food Sci / Berekaa and Issa 2016 : 5 (6) 606-611
Run 1
Run 3
Run 2
Run 4
30
Per cent (%)
PHB content (g/L)
1,8
25
1,6
1,4
20
1,2
15
1
10
0,8
0,6
5
0,4
12
Legend:
24
36
48
60
Incubation Period (h)
72
84
0
96
Runs 1,3,5 = Feeding after 24 and 60 h & Runs 2,4,6 =
Feeding after 48 and 84 h
Runs 1,2 = MSM & Runs 3,4 = NB (conc.= 50% ) & Runs
5,6 = MSM+NB (50% NB+50% MSM)
Arrows, pulsed feeding: straight = 24 and 60 h & dashed =
48 and 84 h
Figure 3 Strategies for fed-batch cultivation and PHB production by B.
megaterium SW1-2 using two pulsed feeding processes.
Figure 4B Pareto plot for Plackett-Burman parameter estimates of PHB
production by Bacillus megaterium SW1-2.
Table 3 Statistical analysis of Plackett-Burman design showing coefficient
values, t- and P-values for each variable.
Variable
Evaluation of factors affecting PHB production
Optimization of PHB production by application of Plackett-Burman design
For optimization studies a series of 14 experiments, with two central points (3
and 13) having the same concentrations used in basal medium, were carried out
to screen for the most significant variables affecting PHB production. As
presented in Table 1, ten crucial variables; covering biochemical and cultivation
parameters were prescribed into 2 levels, coded -1, and +1. The design of the
optimization experiment is given in Table 2 together with the experimental
results (% CDW). Regression analysis was performed to fit the response function
(PHB production) with the experimental data. Analysis of variance indicated that
PHB production can be well described by a polynomial model with a relatively
high coefficient of determination (R2 = 0.93).
Y% yield= 27.99 -1.29X1 +7.34X2 +2.33X3 -1.51X4 - 1.01X5 + 2.96X6
+9.56X7 + 0.77X8 -5.78X9 – 5.69X10
One of the advantages of the Plackett-Burman design is to rank the effect of
different variables on the measured response independent on its nature (either
nutritional or physical factor) or sign (contributes positively or negatively) as in
(Figure 4A). Ranking of factor estimates in a Pareto chart (Figure 4B) benefits in
displays the magnitude of each factor estimate and is a convenient way to view
the results of Plackett-Burman design (Strobel and Sullivan, 1999). It can be
seen that, among those variables DEPS-II and inoculum concentration followed
by KH2PO4 and (NH4)2SO4 found to be the most significant variables that
encourage PHB production. Interestingly, Beaulieu et al. (1995) reported that
the best growth and PHB production were obtained with ammonium sulfate and
sugar cane molasses as the growth activator. Indeed, DEPS-II or date syrup-II
(Fresh syrup) is more significant than DEPS-I (commercial syrup) as indicated by
the lower p-value (0.05) (Table 3).
25
20
Main Effect
15
10
Coefficient
t Stat
P-value
Date syrup-I
-1.29
-0.57
0.61
Date syrup-II
7.34
3.26
0.05
(NH4)2SO4
2.33
1.03
0.38
Yeast Extract
-1.51
-0.67
0.55
Na2HPO4
-1.01
-0.45
0.68
KH2PO4
2.96
1.31
0.28
Inoculum conc.
9.56
4.24
0.02
Temperature
0.77
0.34
0.75
RPM
-5.78
-2.56
0.08
Incubation period
-5.69
-2.52
0.09
Interestingly, cane molasses contains many trace elements and vitamins such as
thiamine, riboflavin, pyridoxine, and niacinamide (Crueger and Crueger, 1984)
thus can be used as growth activators and help in enhancement of PHB
production (Beaulieu et al., 1995). On the other hand, agitation (RPM) and
incubation period have highly negative effect on PHB production. While, other
factors namely; DEPS-I, yeast extract and Na2HPO4 still showed negative effect.
Interestingly, the presence of growth activators in fresh DEPS-II as well as the
absence of any preservative materials may explain its positive effect in
comparison with the commercial DEPS-I. Furthermore, higher content of growth
activators in fresh date syrup or DEPS-II makes it suitable substitutes for yeast
extract that showed negative effect.
Results revealed that PHB production yield by B. megaterium SW1-2 was 53.6%
CDW when cultivated on optimized medium developed by Plackett-Burman.
Therefore, the statistical experimental design proved to be a powerful and useful
tool for enhancing PHB production and confirm the necessity of the optimization
process. In concordance with the obtained results in this work, enhanced PHB
production by Lactobacillus acidophilus using statistical experimental design was
reported by Hamieh et al., (2013). Interestingly, results reported in this study
represent the first investigation on optimization of PHB production by application
of statistical experimental design using date syrup or DEPS.
5
CONCLUSION
0
One of the most crucial variables affecting PHB production-economy is the
nature of carbon source. B. megaterium SW1-2 stain exhibited nutritional
versality in terms of varied growth and PHB accumulation during cultivatrion on
different concentrations of the carbon-based agro-industrial wasts namely; date
palm syrup (DEPS) or sugar cane molasses. PHB was successfully synthesized
and intracellular accumulated in B. megaterium SW1-2 in presence of any of the
used agro-industrial wastes as proven by FT-IR and NMR spectroscopy.
Application of six different fed-batch cultivation strategies provides promising
solution for nutritional limitation problems and clearly showed improved PHB
production. Fed-batch cultivation recorded 6.87-fold increase in total amount of
PHB produced after 96 h as compared with the amount produced under batch
-5
-10
-15
Variable
Figure 4A Effect of different environmental conditions on PHB production from
Bacillus megaterium SW1-2.
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cultivation. Experimental design namely; Plackett-Burman proved to be a
powerful and useful tool for enhancing PHB production (53.6% CDW). The use
of cheaper carbon sources such as date syrup (DEPS) or sugar cane molasses
rather than glucose or sucrose greatly lower process economy and increase
promises for biotechnological production of PHB biopolymer on industrial scale.
Acknowledgement: Authors are grateful for the financial support of this work by
the Deanship of Scientific Research, Dammam University (Grant Application
No-2012052).
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