Journal of Biotechnology 237 (2016) 13–17

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Production of polyhydroxybutyrate from wheat bran hydrolysate

using Ralstonia eutropha through microbial fermentation
Neelamegam Annamalai a,b,∗ , Nallusamy Sivakumar b
a
Hawaii Natural Energy Institute, University of Hawaii at Manoa, 1680, East-west Road, Honolulu, 96822, HI, USA
b
Department of Biology, College of Science, Sultan Qaboos University, PO Box 36, PC 123, Muscat, Oman

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

Article history: The increasing global demand for sustainable resources necessitates the complete utilization of feed-
Received 14 July 2016 stock. Wheat bran consists of significant amount of cellulose and hemicellulose which can be used as a
Received in revised form 29 August 2016 renewable resource for production of fermentable sugars. In this study, alkaline pretreated wheat bran
Accepted 1 September 2016
was enzymatically hydrolyzed using cellulase of Trichoderma reesei (37 FPU/g) and ␤ - glucosidase of
Available online 3 September 2016
Aspergillus niger (50 CBU/g). Among the nitrogen sources tested, ammonium sulphate was identified as
best nitrogen source for the production of polyhydroxybutyrate (PHB). The overall sugar concentration
Keywords:
was about 62.91 g/L with the corresponding sugar yield of 629.1 mg/g wheat bran and the sugars released
Polyhydroxybutyrate
Wheat bran
were mainly composed of glucose (48.35 g/L) and xylose (14.56 g/L). The PHB producing mutant strain,
Enzymatic hydrolysis Ralstonia eutropha NCIMB 11599 grown in wheat bran hydrolysate produced cell density, PHB and yield
Ralstonia eutropha of 24.5 g/L, 62.5%, and 0.319 g/g sugar respectively, with a productivity of 0. 0.255 g/L/h. Thus, the results
Lignocellulosic biomass suggested that the wheat bran could be a potential alternative feedstock as it does not require any detox-
ification due to less inhibitory compounds for production of high cell density with significant amount of
polyhydroxybutyrate.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Polyhydroxybutyrate (PHB) is an organic polymer with com-
mercial potential as a biodegradable thermoplastic and a
biomaterial (Ramsay et al., 1990). PHB is well known as a car-
bon and energy reserve produced by a variety of microorganisms
and its synthesis is favored by environmental stresses such as
nitrogen, phosphate or oxygen limitations (Steinbuchel, 1991; Lee,
1996). PHBs are synthesized and deposited intracellularly in the
form of granules and might amount up to 90% of the cellular dry
weight (Schlegel et al., 1961). In recent days, the investigations on
production of PHB have received much attention due to its poten-
tial applications in packaging, medical, agricultural and fisheries
(Rehm, 2006). The major limiting factor for its commercial success
is the cost of sugars used for production and it was estimated that
3 tons of glucose are required for the production of a ton of PHB
(Collins, 1987). Moreover, about 45% of the total production cost
is attributed to the raw materials where the carbon source could
account for 70–80% of the total expense (Choi and Sang, 1997;
∗ Corresponding author at: Hawaii Natural Energy Institute, University of Hawaii
at Manoa, 1680, East-west Road, Honolulu, 96822, HI, USA.
E-mail address: annabact@gmail.com (N. Annamalai).
Du et al., 2012). Therefore, several efforts have been made con-
tinuously in search of suitable renewable and inexpensive carbon
sources to reduce the cost of production (Khanna and Srivastava,
2005; Annamalai et al., 2014).
Cellulosic biomass such as agricultural residues are potentially
inexpensive renewable feedstock for the biorefineries of alterna-
tive fuels, chemicals and materials (Wyman, 2001). Cellulose from
lignocellulosic biomass could be hydrolyzed into sugars, such as
glucose, which are potential carbon resources for PHB production
(Rahman et al., 2007; Chen et al., 2007). Bioconversion of cellu-
lose into fermentable sugars is a bio-refining area that has invested
enormous research efforts, as it is a prerequisite for the subsequent
production of bioenergy (Kumar et al., 2008). However, the effi-
cient hydrolysis of cellulose from cellulosic biomass to glucose is
a major bottleneck due to its recalcitrant composite structure of
lignin, hemicellulose and cellulose in plant cell walls which blocks
the hydrolysis of polysaccharides into fermentative sugars (Mosier
et al., 2005; Hall et al., 2010). The enzymatic saccharification is con-
sidered as a promising technology compared to other methods; but
its efficiency and effectiveness are affected by many factors, includ-
ing feedstock characteristics, pretreatment methods and hydrolysis
conditions (Mansfield et al., 1999).
Wheat bran is an important by-product of the cereal industry
produced in vast quantities worldwide (Martel et al., 2010). It con-

http://dx.doi.org/10.1016/j.jbiotec.2016.09.001
0168-1656/© 2016 Elsevier B.V. All rights reserved.
14 N. Annamalai, N. Sivakumar / Journal of Biotechnology 237 (2016) 13–17
sists of non-starch polysaccharides [cellulose, arabinoxylans and ␤
- (1,3)/␤ - (1, 4) - glucan (38%)], starch (19%), protein (18%) and
lignin (6%) (Bergmans et al., 1996). Hence, it is assumed that the
industrial wheat bran has the potential to serve as a low-cost feed-
stock for the production of renewable energy or chemicals and its
utilization would greatly facilitate potential applications in a biore-
finery concept. However, enzymatic hydrolysis of wheat bran is
not adequate to liberate sufficient fermentable sugars; therefore,
a range of physical or chemical pretreatments are required (Yang
and Wyman, 2008). Though several attempts have been made on
enzymatic hydrolysis of lignocellulosic biomass for production of
biofuels and other chemicals, no attempts were made on its uti-
lization for production of bioplastics. Hence, the present study was
made on pretreatment, enzymatic hydrolysis of wheat bran into
fermentable sugars and production of PHB through microbial fer-
mentation using glucose utilizing mutant strain R. eutropha NCIMB
11599 in order to develop a feasible process for sustainable utiliza-
tion of this lignocellulosic biomass into bioplastics.
2. Materials and methods
2.1. Materials and microorganism
Wheat bran (WB) purchased from local market was destarched
by the method of Zhou et al. (2010). Cellulase from Trichoderma ree-
sei ATCC 26921 (C2730) and ␤ - glucosidase from Aspergillus niger
(49291) were purchased from Sigma-Aldrich (MO, USA). The activ-
ity of cellulase and ␤ - glucosidase was estimated as 185 FPU/mL
and 518 CBU/mL respectively. The cellulase activity was deter-
mined by the standard filter paper assay (Ghose, 1987). One unit of
enzyme activity (FPU) is defined as the amount of enzyme required
to liberate 1 ␮mol of glucose from filter paper in 60 min at 50 ◦ C
and pH 4.8. The ␤ - glucosidase activity was determined by mea-
suring the amount of p-nitrophenol released from p-nitrophenyl –
␤ – D - glucopyranoside (pNPG). One unit of enzyme activity (CBU)
is defined as the amount of enzyme required to produce 1 ␮mol of
p-nitrophenol from p -nitrophenyl- ␤-d-glucopyranoside (pNPG)
per minute at 50 ◦ C and pH 5. Other chemicals and materials used
in this study were purchased from Sigma-Aldrich (St. Louis, MO,
USA) or as indicated.
2.2. Pretreatment of wheat bran
For pretreatment, destarched wheat bran (WB) was added with
1% NaOH at the ratio of 1:10 (w/v) in a 500 mL screw cap bottle
and then autoclaved at 121 ◦ C for 30 min. The residue collected
by centrifugation at 5000 xg for 10 min was washed several times
with distilled water till obtain pH between 7 and 8, dried at 50 ◦ C
and used for further studies. The moisture and ash contents were
determined using NREL/TP – 510 - 42621 (Sluiter et al., 2008a) and
NREL/TP – 510 - 42622 (Sluiter et al., 2008b) methods, respectively.
The structural carbohydrates and acid soluble and insoluble lignin
of both untreated and pretreated wheat bran were determined by
NREL/TP – 510 - 42618 method (Sluiter et al., 2012). The cellu-
lose and hemicellulose content was analyzed by HPLC (Shimadzu;
LC10AD) equipped with aminex HPX - 87H (Bio-Rad) column at
65 ◦ C using 5 mM sulfuric acid as mobile phase (0.6 mL/min) with
refractive index detector (Shimadzu; RID10A) and the sugars (glu-
cose and xylose) were quantified by external calibration with
standards.
2.3. Enzymatic hydrolysis of pretreated wheat bran
The enzymatic hydrolysis was carried out in 250 mL conical flask
containing 10% (w/v) of wheat bran in 50 mM citrate buffer (pH 4.8),
incubated at 50 ◦ C and 150 rpm for 96 h and the enzyme loading
was 37 FPU/g and 50 CBU/g of substrate. Samples were withdrawn
at regular interval of 12 h, centrifuged at 10,000 xg for 10 min and
the supernatant was subjected to sugar analysis. The percentage of
hydrolysis was calculated based on the amount of glucan (cellulose)
and xylan (xylose) in the initial substrates and sugars released from
hydrolysis.
2.4. Effect of nitrogen source and C/N ratio for cell growth and
PHB production
To study the effect of nitrogen source, the glucose utilizing
mutant strain R. eutropha NCIMB 11599 was grown in mineral salt
medium (MSM) [2.4 - KH2 PO4 ; 2.5 - Na2 HPO4 ; 0.5 - MgSO4 and
0.1 - Ferric ammonium citrate (g/L) (pH - 6.8) containing glucose
(10 g/L) was supplemented with both inorganic and organic nitro-
gen sources to be investigated such as NH4 Cl, NH4 NO3 , (NH4 )2 SO4 ,
yeast extract, beef extract, peptone and tryptone (2 g/L) and incu-
bated (150 rpm) at 30 ◦ C for 72 h. The effect of C/N ratio on growth as
well as PHB production was investigated by cultivating R. eutropha
NCIMB 11599 in MSM (pH - 6.8) with various C/N ratio (20:1, 20:2,
20:3, 20:4 and 20:5) with glucose (10 g/L).
2.5. Production of polyhydroxybutyrate
The strain, R. eutropha NCIMB 11599 was precultured in a nutri-
ent medium containing 10 g/L of yeast extract, 10 g/L of peptone,
5 g/L of meat extract, and 5 g/L of (NH4 )2 SO4 at 30 ◦ C for 48 h. The
cells with low PHB content were harvested by centrifugation at
5000 xg for 10 min, resuspended in sterile distilled water and used
as inoculum for PHB production. After enzymatic hydrolysis, the
hydrolysate was collected by centrifugation at 10,000 xg for 10 min,
adjusted to pH - 6.8 and used directly for PHB production without
detoxification. Further, 2.4 - KH2 PO4 ; 2.5 - Na2 HPO4 ; 0.5 - MgSO4 ;
0.1- Ferric ammonium citrate and 4.8 - NH2 SO4 (g/L) were added to
the hydrolysate (C/N - 20) and filtered through pre-sterilized mem-
brane filter (0.2 ␮m), seeded with 1% (v/v) inoculum and incubated
in a rotary shaker at 150 rpm for 72 h at 30 ◦ C. Cell growth was mon-
itored by measuring optical density at 600 nm. The cell dry weight
(DCW) was estimated by centrifuging the culture broth (5000 xg for
10 min at 4 ◦ C) and washed twice with deionized water and dried
at 70 ◦ C (g/L).
2.6. Extraction and quantification of PHB
PHB from the harvested cells was extracted by adding 6% sodium
hypochlorite and chloroform (1:1) at 37 ◦ C at 300 rpm for 2 h. The
chloroform solutions were centrifuged (5000 xg, 10 min) to remove
cell debris and concentrated by rotary evaporation. The PHB poly-
mer was precipitated using chilled methanol (1:9), separated by
centrifugation at 3000 xg and the methanol-chloroform mixture
was decanted. The precipitated polymer was once again dissolved
in chloroform and precipitated with methanol to obtain highly puri-
fied polymer. Finally, the pellets were vacuum dried and weight of
the pellet was measured.
PHB content was estimated by gas chromatography (GC) anal-
ysis through methanolysis of the polymers in sulfuric acid and
methanol as described in previous studies (Wei et al., 2011;
Zhang et al., 2013). Briefly, 30 mg of freeze dried cells were added
with 2 mL chloroform and 2 mL acidic methanol (2.8 M H2 SO4 in
methanol) and heated at 100 ◦ C for 4 h in a water bath. Octanoic
acid (10 mL/L) was dissolved in acidic methanol and used as an
internal standard. After cooling, 1 mL of distilled water was added
to the mixture, shaken vigorously for 3 min till separation of clear
phase and the organic phase was then filtered and subjected
to GC analysis (Varian 450). The gas chromatographic separa-
tion was performed using an Agilent J&W HP-5 capillary column
 N. Annamalai, N. Sivakumar / Journal of Biotechnology 237 (2016) 13–17
Table 1
The composition of untreated and alkaline pretreated wheat bran.
Raw Material Cellulose as Hemicellulose as
glucose (%) xylose (%)
Untreated 32.43 ± 0.34 26.34 ± 0.25
Pretreated 54.84 ± 0.57 20.25 ± 0.66
(30 × 0.32 mm × 0.25 ␮m). The temperature of the injector was
kept at 250 ◦ C. One ␮L of the sample was injected and helium
(99.999%) was used as the carrier gas with a constant flow rate of
2 mL/min. The oven temperature was programmed to increase from
100 ◦ C (1 min) to 250 ◦ C (4 min) at a heating rate of 15 ◦ C/min. The
flame ionization detector (FID) was kept at 270 ◦ C (air 300 mL/min,
hydrogen 30 mL/min, nitrogen 30 mL/min). The PHB content was
expressed as percentage of cell dry weight (%).
3. Results and discussion
3.1. Effect of pretreatments on composition of raw materials
The composition of the untreated wheat bran (%w/w) was
as follows: cellulose, as glucose, 32.43 ± 0.34; hemicellulose, as
xylose, 26.34 ± 0.25; lignin, 20.15 ± 0.28; ash, 8.32 ± 0.12; mois-
ture, 7.32 ± 0.24. After pretreatment, the wheat bran consists
of 54.84 ± 0.57% cellulose, 20.25 ± 0.66% xylose and 8.93 ± 0.40%
lignin (Table 1). The results suggested that the pretreatment with
1% NaOH decreased the lignin and hemicellulose content from
20.15 to 8.93% and 26.34 to 20.25% respectively and also increased
cellulose content from 32.43 to 54.84%. It is evidenced that the
pretreatment at 121 ◦ C for 30 min with 1% NaOH removed high
amount of lignin and hemicellulose from wheat bran. Likewise,
several reports were also reported that the pretreatment with 1%
NaOH significantly increased the cellulose content of wheat straw
in addition to the higher removal of lignin (Toquero and Bolado,
2014; Steffien et al., 2014).
3.2. Enzymatic hydrolysis of pretreated wheat bran
The enzymatic hydrolysis of pretreated wheat bran was per-
formed using cellulase from Trichoderma reesei (37 FPU/g) and ␤
- glucosidase from Aspergillus niger (50 U/g) for 96 h. The result
suggests that the rate of hydrolysis was comparatively higher at
the initial 36 h and started decresing afterwards in both treated
and untreated wheat bran. However, the percentage hydrolysis of
pretreated wheat bran (83.75%) was 4 fold higher than untreated
wheat bran (26.80%) (Fig. 1). It is also evidenced that production of
Fig. 1. The percentage hydrolysis of untreated and pretreated wheat bran. The
results were presented in mean ± SD, n = 3.
15
Lignin (%)
20.15 ± 0.28
08.93 ± 0.40
Fig. 2. The amount of sugars released from (a) untreated and (b) pretreated wheat
bran through enzymatic hydrolysis by cellulase of T. reesei. The results were pre-
sented in mean ± SD, n = 3.
sugars reached maximum (15.8 g/L) at 48 h with untreated wheat
ban, however it was increased afterwards and reached maximum
(63 g/L) at 72 h with pretreated wheat bran (Fig. 2). The overall
sugar concentration was about 15. 9 and 62.91 g/L corresponding to
the sugar yield of 159 and 629.1 mg/g of untreated and pretreated
wheat bran respectively. However, the sugars produced from both
untreated and pretreated wheat bran were mainly composed of
glucose (10.57 and 48.35 g/L) and xylose (5.28 and 14.56 g/L)
respectively. Likewise, Zhang et al. (2013) obtained 48.3 g/L of glu-
cose and 29.8 g/L of xylose from alkaline pretreated oil palm empty
fruit bunch (OPEFB) hydrolysate with overall sugar concentration
of 78.1 g/L corresponding to a sugar yield of 520 mg/g OPEFB. Cao
et al. (2012) yielded about 12.55 g/L of glucose and 4.98 g/L of xylose
from alkali pretreated sweet sorghum bagasse.
3.3. Effect of nitrogen source and C/N ratio on cell growth and
PHB production
The effect of nitrogen source on growth (DCW) and production
of PHB by R. eutropha NCIMB 11599 was determined using vari-
ous organic and inorganic nitrogen sources and the results were
presented in Table 2. The results revealed that the ammonium sul-
phate produced higher amount of DCW (6.14 g/L), PHB production
(2.69 g/L) and PHB content (43.7%) followed by ammonium chloride
(5.85 g/L, 2.44 g/L and 41.7%). Though peptone enhanced the cell-
growth (5.73 g/L), PHB content (36.34%) was comparatively lower
than inorganic nitrogen sources. Further, there was no much varia-
tion in PHB content with different nitrogen sources suggested that
the cell growth was highly influenced than accumulation of PHB.
It is clearly evidenced that the ammonium sulphate seems to be
a best nitrogen source than other organic and inorganic nitrogen
sources for the growth as well as production of PHB. The results
16 N. Annamalai, N. Sivakumar / Journal of Biotechnology 237 (2016) 13–17

Table 2
Effect of various nitrogen sources on cell growth (DCW), PHB production (g//L) and
PHB content (%) of R. eutropha.

Nitrogen Sources DCW(g/L) PHB production(g/L) PHB content(%)

NH4 Cl 5.85 ± 0.34 2.44 ± 0.24 41.72 ± 1.49

NH4 NO3 5.30 ± 0.28 2.15 ± 0.32 40.54 ± 2.47
(NH4 )2 SO4 6.14 ± 0.19 2.69 ± 0.32 43.77 ± 2.49
Yeast Extract 5.52 ± 0.21 1.93 ± 0.14 34.87 ± 2.38
Beef Extract 4.34 ± 0.22 1.64 ± 0.07 37.87 ± 2.06
Peptone 5.73 ± 0.21 2.08 ± 0.28 36.34 ± 3.17
Tryptone 4.75 ± 0.26 1.93 ± 0.32 40.63 ± 2.47

Fig. 4. Residual glucose and xylose, Cell density (DCW), PHB production, PHB con-
tent produced from enzymatic hydrolysis of pretreated wheat bran using R. eutropha.
The results were presented in mean ± SD, n = 3.

and it was being maximum at the C/N ratio of 15:2 and no significant
variation between 15:1 and 15:3.

3.4. PHB production from enzymatic hydrolysis of pretreated

wheat bran

Enzymatic saccharification of biomass is an essential and most

Fig. 3. The effect of C/N ratio on (a) cell growth (DCW) and (b) PHB content of R.
eutropha. The results were presented in mean ± SD, n = 3.
were comparable with earlier reports, where ammonium sulphate
and ammonium nitrate (inorganic) was identified as suitable nitro-
gen sources for growth and PHB production (Gouda et al., 2001;
Sreekanth et al., 2013). Contrarily, Zhang et al. (2013) and Pal et al.
(2009) were identified tryptone (organic) as a suitable nitrogen
source for higher yield of PHB.
Several reports suggested that the C/N ratio plays an impor-
tant role in accumulation of PHB with minimal cell growth (Gouda
et al., 2001; Sreekanth et al., 2013; Kulpreecha et al., 2009; Johnson
et al., 2010). In the present study, the effect of different C/N ratio
(20:1–20:5) on cell growth (DCW) and PHB content (%) was investi-
gated and the results were presented in Fig. 3. The result suggested
that the PHB content was found to be higher at C/N ratio of 20:1
(62.5%) and lower at 20:5 (46.6%), whereas cell growth (DCW) was
found to be minimum at 20:1 (3.63 g/L) and maximum at 20:5
(6.23 g/L). It clearly evidenced that the nitrogen limitation (C/N
ratio) was not suitable for cell growth and most favorable for accu-
mulation of PHB which is comparable with several earlier reports
(Kulpreecha et al., 2009; Johnson et al., 2010; Lee et al., 2008). Con-
tarrily, Zhang et al. (2013) suggested that the strain B. megaterium
does not require nitrogen limitation for higher PHB accumulation
expensive step in conversion of lignocellulosic biomass into fer-
mentable sugars for the production of value added chemicals
(Klein-Marcuschamer et al., 2012). The strain, Ralstonia eutropha
does not utilize xylose, arabinose and oligosaccharides in the
hydrolysate solution (Yu and Stahl, 2008). Hence, the initial sugar
concentration of wheat bran hydrolysate was considered as 48 g/L
(glucose) and 4.8 g/L ammonium sulphate (C:N ratio - 20:1) was
added along with other nutrients as indicated in material and
methods. The growth (DCW), production of PHB (g/L) and PHB
content (%) was determined and the results were presented in
Fig. 4. The results suggested that the cell growth and PHB produc-
tion were increased steadily and reached maximum (24.43 ± 0.60
and 14.82 g/L) at 60 h; however, the PHB content was maximum at
48 h (62.55 ± 0.85%) and decreased further upto 72 h. It may due to
the activation of depolymerase system because of carbon strav-
tion/lack of C:N ratio in the fermentation medium. Zhang et al.
(2012) obtained about 24.19 and 12.48 g/L of cell density and PHB
production respectively when 60 g/L of sugars from OPEFB. The
PHB content of R. eutropha NCIMB 11599 (62.5% DCW) obtained
from the present study was comparable with the earlier studies on
utilizing lignocellulosic biomass (Yu and Stahl, 2008; Silva et al.,
2007). However, Van-Thuoc et al., (2008) obtained about 8 g/L of
biomass and 50% of PHB content through enzymatic hydrolysis of
wheat bran with yeast extract as nitrogen source and Gouda et al.
(2001) reported that 3.6 g/L of DCW, 2.2 g/L of PHB production and
59.4% of the PHB content from Bacillus megaterium using sugar-
cane molasses and corn steep liquor as sole carbon and nitrogen
sources. The earlier reports suggested that detoxification of the
hydrolysate is essentially needed prior to the production of PHB as
it may contains several fermentation inhibitory compounds such as
furfural, hydroxylmethylfurfural, acetic acid, phenolic compounds,
and water soluble lignin (Yu and Stahl, 2008; Silva et al., 2004; Pan
et al., 2012). But, the hydrolysate obtained from the present study
was used for production of PHB without any detoxification and no
significant reduction in PHB content suggested that the hydrolysate
contains less fermentation inhibitory compounds.
 N. Annamalai, N. Sivakumar / Journal of Biotechnology 237 (2016) 13–17
The productivity and yield obtained in the present study from
pretreated wheat bran was about 0.255 g/L/h and 0.319 g/g sugar
respectively which is higher than the yield of B. megaterium
(0.267 g/g) using OPEFB hydrolysate (Zhang et al., 2013) and lower
than the xylose utilizing bacterium B. cepacia IPT 048PHB (0.39 g/g)
from sugarcane bagasse (Silva et al., 2004). Further, the results sug-
gested that the glucose in the hydrolysate was completely utilized
by R. eutropha and xylose remains unconsumed, however, no sig-
nificant reduction was observed in PHB production (Fig. 4). Gouda
et al. (2001) and Zhang et al. (2012) observed that the PHB produc-
tion was significantly affected due to the osmotic pressure from
high concentration of sugar.
4. Conclusions
The results reveled that the cell density (DCW) and PHB pro-
duced by R.eutropha NCIMB 11599 from wheat bran hydrolysate
in this study was comparatively higher than other reports utiliz-
ing various lignicellulosic biomass. Furthermore, no detoxification
required for production of PHB suggested that the hydrolysate of
wheat bran comprises of less fermentation inhibitory compounds
and thus it could be a potential feedstock/biobased renewable car-
bon source for synthesis of bioplastics. The production of PHB using
different strain and/or genetically engineered R.eutropha able to
utilize both glucose and xylose is in progress.
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