Research Article

Received: 6 February 2016 Revised: 16 March 2016 Accepted article published: 8 April 2016 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.4993

Conversion of rice husks to

polyhydroxyalkanoates (PHA) via a three-step
process: optimized alkaline pretreatment,
enzymatic hydrolysis, and biosynthesis by
Burkholderia cepacia USM (JCM 15050)
King-Sern Heng,a Rajni Hatti-Kaul,b Farook Adam,c Toshiaki Fukuid and
Kumar Sudesha*

Abstract
BACKGROUND: Rice husks (RH) are agricultural residues with abundant storage of cellulose and hemicellulose, making them
a potential feedstock for polyhydroxyalkanoate (PHA) production. In this study, optimization of pretreatment with alkali
under various conditions was performed before enzymatic hydrolysis using Celluclast 1.5 L (EC 3.2.1.4) and Novozyme 188 (EC
3.2.1.21). The hydrolysate was fed to two strains, Burkholderia cepacia USM (JCM 15050) and Cupriavidus necator NSDG-GG, an
engineered strain of Cupriavidus necator H16, to evaluate their PHA production.

RESULTS: Pretreatment of RH using 1.0 mol L−1 potassium hydroxide (KOH) at high temperature and pressure (HTP) (121 ∘ C, 0.1
MPa) gave maximum sugar yield of up to 87% (per total carbohydrate content) after optimized enzymatic hydrolysis, whereby
the undiluted hydrolysate contained approximately 20 g L−1 total reducing sugars (TRS). B. cepacia USM utilized the hydrolysate
more efficiently compared with C. necator NSDG-GG, with a maximum cell dry weight (CDW) of 4.9 g L−1 and 40 wt% PHA at
shake-flask scale. The CDW and PHA content of B. cepacia USM cultivated in a 5 L fermentor were 7.8 g L−1 and 50%, respectively.
The decrease in total phenolics at the end of fermentation suggested that B. cepacia USM was able to metabolize phenolic
compounds.

CONCLUSION: Through optimized alkali pretreatment and enzymatic hydrolysis, RH has the potential to be converted to PHA by
B. cepacia USM, thus valorizing this agricultural by-product.
© 2016 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: rice husks; alkali pretreatment; Burkholderia cepacia; polyhydroxyalkanoate

INTRODUCTION Agricultural biomass is mainly composed of lignocellulose,

Polyhydroxyalkanoate (PHA) is a type of biodegradable polyester
synthesized within bacterial cells. Under nutrient-limited condi-
tions but with an excess of carbon, these intracellular inclusions
are accumulated as energy and carbon storage for the cells.1 PHAs
have industrial significance owing to their mechanical properties
that are comparable with petrochemically derived plastics such
as polypropylene (PP) and polyethylene (PE). In addition, PHAs
are biodegradable, further highlighting their potential as an alter-
native to synthetic plastics.2 In an effort to make commercializa-
tion of PHAs more viable, researchers have looked towards using
waste products as carbon source. Plant-based sources of sugars
are abundant in nature, however, sugars that are easily available
such as sucrose or fructose are often used in the food industry.
Therefore, utilization of inedible plant materials, such as agricul-
tural biomass, is the more feasible choice of feedstock for PHA
production.
which provides plants mechanical support and protection.3 In
order to perform such functions, lignocellulosic biomass is very
∗ Correspondence to: K Sudesh, School of Biological Sciences, Universiti Sains
Malaysia, 11800 Penang, Malaysia. Email: ksudesh@usm.my
a School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang,
Malaysia
b Biotechnology, Center for Chemistry and Chemical Engineering, Lund Univer-
sity, P.O. Box 124, SE-22100 Lund, Sweden
c School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang,
Malaysia
d Department of Bioengineering, Graduate School of Bioscience and Biotech-
nology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama
226-8501, Japan

J Chem Technol Biotechnol (2016) www.soci.org © 2016 Society of Chemical Industry

 www.soci.org
stable and recalcitrant, making conversion of biomass to fer-
mentable sugars a challenging endeavor. As such, additional
processes to pretreat the biomass are required to make the sugars
more accessible. Among the many pretreatment methods that
have been tested, alkali pretreatment has been extensively stud-
ied, mostly owing to its ability to remove large quantities of lignin
from biomass.4
Rice husks (RH) are a type of residual agricultural biomass,
obtained from the rice milling process as the paddy is converted
to rice. The RH account for up to 20% of the paddy harvest.5 Rice
is one of the largest food sources in the world. According to the
Food and Agricultural Organization of the United Nations (FAO),
the worldwide paddy production has been increasing at a steady
rate each year since 2002.6 Paddy production in Malaysia was 2.5
million tonnes in 2010 and rose to more than 2.6 million tonnes
in 2014.7 The increasing amount of RH generated presents a
problem in disposal management. Incineration of the rice residues
appeared to be the most common method of disposal in Southeast
Asian countries, with only a small amount being used as fuel.8
Development of a proper and sustainable method of agricultural
waste management is crucial to address the environmental issues
caused by incineration. One possible method is to convert the
polysaccharides present in the RH into fermentable sugars.
The present study was aimed at production of PHA from sugars
present in RH through a systematic three-step process. The RH
were first pretreated with alkali under different conditions to
determine the combination method which results in highest sugar
yield upon enzymatic hydrolysis. The potential of the hydrolysate
as a feedstock for bacterial synthesis of PHA was evaluated based
on cell biomass and PHA accumulation in two selected strains, B.
cepacia USM and C. necator NSDG-GG. The strain showing the most
efficient utilization of the hydrolysate was used for fermentation
to investigate the possibility of scale-up PHA production on this
carbon source.
EXPERIMENTAL
Raw material
The RH used in this study was obtained from Kim Thye Leong
Rice Mill Sdn. Bhd., Kepala Batas, Pulau Pinang, Malaysia. The RH
was washed with tap water, sun-dried, and kept in plastic bags
at room temperature until further use. Compositional analysis of
the untreated RH was carried out following the protocols of the
National Renewable Energy Laboratory (NREL).9
Alkali pretreatment
For all pretreatment experiments, the solid loading of the RH was
kept constant at 6% (w/v). The concentrations of alkali used were
0.1 mol L−1 and 1.0 mol L−1 for both sodium hydroxide (NaOH) and
potassium hydroxide (KOH). Three different pretreatment condi-
tions were tested; room temperature (RT), microwave irradiation
(MI), and high temperature and pressure (HTP). The mixture of
RH and alkali was agitated at a speed of 200 rpm for 24 h at RT
(26 ± 4 ∘ C). Pretreatment using a combination of alkali and MI was
performed by placing a mixture of RH and alkali in a tabletop
microwave oven (LG Electronics, South Korea). The microwave fre-
quency was 2450 MHz and the power was set at 700 W. The pre-
treatment duration was kept constant at 15 mins. Pretreatment
using a combination of alkali and HTP was performed in an auto-
clave unit (Hirayama, Japan), whereby the mixture of RH and alkali
was pretreated at 121 ∘ C and 0.1 MPa for 15 mins. After the set
wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry
K-S Heng et al.
duration for each pretreatment, the mixture was filtered and the
solid fraction was washed thoroughly with distilled water until
the pH was approximately 7.0 ± 0.5. The washed solid fraction
was then oven-dried until all excess moisture was removed. The
efficiencies of each pretreatment were evaluated based on total
reducing sugar yield after enzymatic hydrolysis, expressed based
on the total carbohydrate content in the untreated raw material.
Thermogravimetric analysis (TGA)
Thermogravimetric analysis (TGA) was performed using TGA7
thermoanalyzer (Perkin-Elmer, United States). Approximately
10 mg of dry RH samples were heated from 30 ∘ C to 920 ∘ C under
continuous N2 flow at a heating rate of 20 ∘ C min−1 . The differ-
ences in composition of the RH samples were taken based on
percentage weight loss at specific ranges of temperature.
Enzymatic hydrolysis
The alkaline-pretreated RH were used as substrates for enzymatic
hydrolysis. In the initial evaluation of RH pretreatment under var-
ious conditions, only Celluclast 1.5 L (Novozyme, Denmark) was
used at an enzyme loading of 5.0% (v/v) with substrate loading
of 5.0% (w/v). The RH pretreated under optimum conditions was
then selected as the substrate for further experiments on enzy-
matic hydrolysis. Optimized enzyme loading of Celluclast 1.5 L and
Novozyme 188 (Novozyme, Denmark) was 1.0% (v/v) and 0.25%
(v/v), respectively, with substrate loading of 2.5% (w/v) (refer to
Supporting information). A 0.05 mol L−1 citrate buffer (pH 4.8) was
used for all enzymatic reactions. The reaction mixture was incu-
bated at 50 ∘ C with agitation at 160 rpm for 72 h. At the end of
incubation, the reaction mixture was filtered through a What-
man No.1 filter paper to separate the solid substrate and enzy-
matic hydrolysate. The hydrolysate was then filtered through a
0.2 μm cellulose acetate membrane filter using vacuum filtration
to remove smaller particles. Sterilization of the hydrolysate prior
to use in bacterial cultivation was performed at 121 o C and 0.1 MPa
for 15 mins using hydrolysates with pH 4.8 and pH 7.0 to observe
the effect of pH on the composition of sugars. Based on the results,
the hydrolysate for all subsequent biosynthesis experiments was
autoclaved at pH 4.8.
Microorganisms
The bacterial strains used in this study were B. cepacia USM
(JCM 15050), isolated from wastewater polluted with oil10 and
C. necator NSDG-GG. The strain NSDG-GG is a glucose-utilizable
engineered strain of the wild strain H16, having modifications in
nag operon and replacement of phaC by a mutant gene phaC
derived from Aeromonas caviae on the chromosome.11,12 The cells
were maintained on nutrient rich (NR) medium with 25% glycerol
at −80 ∘ C.
Media
The composition of the NR medium (pH 7.0) was as follows (per
L): 10 g meat extract, 10 g peptone, and 2 g yeast extract. The
mineral salts medium (MM) composition (pH 7.0) for B. cepacia
USM was as follows (per L): 3.32 g Na2 HPO4 , 2.8 g KH2 PO4 , 0.25 g
Mg2 SO4 ·7H2 O, and 1 mL trace elements.13 The trace elements
solution contained (per L): 0.22 g CoCl2 ·6H2 O, 9.7 g FeCl3 , 7.8 g
CaCl2 , 0.12 g NiCl2 ·6H2 O, 0.11 g CrCl3 ·6H2 O, 0.16 g CuSO4 ·5H2 O
in 0.1 N HCl.14 For C. necator NSDG-GG, the composition of MM
(pH 6.8) was as follows (per L): 4.0 g NaH2 PO4 , 4.6 g Na2 HPO4 , 0.45 g
J Chem Technol Biotechnol (2016)
Conversion of rice husks to PHA via a three-step process
K2 SO4 , 0.39 g Mg2 SO4 , 0.062 g CaCl2 , and 1 mL trace elements.
The trace elements were composed of (per L): 15 g FeSO4 ·6H2 O,
2.4 g MnSO4 ·H2 O, 2.4 g ZnSO4 ·7H2 O, and 0.48 g CuSO4 ·5H2 O in
0.1 N HCl.15 Ammonium chloride (NH4 Cl) and urea were used as
nitrogen sources and tested at varying concentrations. In control
experiments, pure glucose was used as carbon source and was
fed at a concentration of 20 g L−1 . For biosynthesis experiments
using RH hydrolysate, the hydrolysates containing approximately
20 g L−1 total reducing sugars (TRS) were used in place of MM and
either NH4 Cl or urea was added as nitrogen source, and adjusted
to pH 7.0 for B. cepacia USM and pH 6.8 for C. necator NSDG-GG
prior to inoculation. The pH for all media was adjusted using
hydrochloric acid (HCl) or sodium hydroxide (NaOH).
Cultivation of cells in shake flasks
Microbial culture was carried out at shake flask scale using a
one-stage cultivation strategy. Pre-inoculation was done by cul-
tivating the cells in (NR) medium at 37 ∘ C for B. cepacia USM
and 30 ∘ C for C. necator NSDG-GG accompanied by agitation at
200 rpm. When OD600 nm = 4.0, 3% (v/v) of inoculum was transferred
to MM or hydrolysates to induce PHA production.
Cultivation of B. cepacia USM in fermentor
From the results obtained at shake-flask scale, B. cepacia USM was
selected for further fermentation experiments. Batch fermentation
of B. cepacia USM was performed in a 5 L fermentor (Infors HT,
Switzerland) with a 40% working volume. The experimental run
was performed using hydrolysate and supplemented with urea at
0.54 g L−1 . As a comparison, MM was used with the addition of
20 g L−1 glucose as carbon source with the addition of 0.54 g L−1
urea. The inoculum was prepared by growing the cells in NR broth
for 24 h, as carried out for shake flask cultivation, and added at
10% of the working volume. The pH of the medium was set at 7
and regulated by addition of NaOH or HCl. The temperature was
maintained at 37 ∘ C. The airflow rate was set at 1 vvm. Impeller
speed was set on cascade mode to allow an air saturation level of
40% dissolved oxygen. Fermentation was performed over a period
of 48 h.
ANALYTICAL METHODS
Determination of total reducing sugars (TRS)
The TRS content in the hydrolysate was determined by
3,5-dinitrosalicylic acid (DNS) assay.16 The TRS yields were
expressed based on the total carbohydrate content in the
untreated RH, which was approximately 52% as determined
by compositional analysis in this study.
Determination of total phenolics
The amount of total phenolics in the hydrolysate was determined
using the Folin–Ciocalteu assay.17 Total phenolics in the samples
were expressed as milligrams of gallic acid equivalents (GAE) per
liter based on the standard curve of gallic acid.
Determination of monosaccharides
Determination of monosaccharides in the hydrolysate was per-
formed using high performance anion-exchange chromatogra-
phy (HPAEC) equipped with a GP50 gradient pump and AS50
autosampler. Detection was performed using an ED40 electro-
chemical detector. The monosaccharides were quantified with a
J Chem Technol Biotechnol (2016) © 2016 Society of Chemical Industry
www.soci.org
Dionex CarboPac PA-20 analytical column attached to a Dionex
CarboPac PA-20 guard column (Thermo Fisher Scientific, United
States). The mobile phase was 0.75 mmol L−1 NaOH with the flow
rate of 0.5 mL min−1 . The column was regenerated at the end of
each cycle with 200 mmol L−1 of NaOH for 4 min. The flow rate was
the same for regeneration.
Determination of cell dry weight (CDW)
The cells were harvested by centrifugation at 8000 rpm for 10 min
at 4 ∘ C. The cells were washed twice with distilled water and frozen
overnight at −20 ∘ C. The cells were then lyophilized and the weight
of the dried cells was determined.
Gas chromatography (GC) analysis for PHA content
GC analysis was performed to determine the PHA content in the
cells. Samples were prepared as described previously.18 Briefly,
2 mL methanol acidified with 15% (v/v) H2 SO4 and 2 mL chlo-
roform were added to approximately 10 mg of lyophilized cells.
Methanolysis was carried out at 100 ∘ C for 140 mins to allow the
conversion of PHA monomers to their corresponding hydroxyacyl
methyl esters. Upon completion of methanolysis, the mixture was
cooled to room temperature and 1 mL distilled water was added.
The mixture was vortexed until two phases were formed, a lower
chloroform phase and an upper aqueous phase. Samples for GC
analysis were taken from the lower chloroform phase. Caprylic
methyl ester (CME) was added to the samples as an internal stan-
dard at a ratio of 1:1. Analysis was carried out using a GC-2010 Gas
Chromatograph (Shimadzu, Japan) equipped with a Supelco SPB-1
column (Sigma-Aldrich, United States) operated at a temperature
of 280 ∘ C. An AOC-20i autoinjector (Shimadzu, Japan) was used
with a temperature of 270 ∘ C. The carrier gas was N2 with a flowrate
of 14 mL min−1 . Detection was performed using a flame ionization
detector (FID) with a temperature of 280 ∘ C. Data from the analy-
sis were obtained from GC Solution Version 2.30.00 SU3 software
(Shimadzu, Japan).
RESULTS
The composition of the RH used in this study is shown in Table 1.
The total carbohydrate content in the RH was 52% (35% cellulose
and 17% hemicellulose) and the lignin content was 26% on an
extractives-free basis. The ash content was determined to be 15%.
Based on Fig. 1, under RT conditions, the pretreatment of NaOH
at 1.0 mol L−1 resulted in total reducing sugar yield of 28% per
total carbohydrate content in the RH. This was two-fold the yield
obtained from using 0.1 mol L−1 NaOH at RT, which was only 13%.
The use of NaOH with MI for 0.1 mol L−1 and 1.0 mol L−1 resulted
in 24% and 35% sugar yield, respectively. Further increase in
sugar yield from the RH was observed when HTP was used in
combination with NaOH, and was 34% for 0.1 mol L−1 and 62% for
1.0 mol L−1 . At RT, the use of KOH resulted in higher sugar yield
than NaOH, with a maximum of 22% and 32% for 0.1 mol L−1 and
1.0 mol L−1 , respectively. Pretreatment with 1.0 mol L−1 KOH and
MI resulted in 42% yield of sugars, which was 12% more than the
yield obtained with 0.1 mol L−1 KOH and MI, i.e. 30%. Pretreatment
using 1.0 mol L−1 KOH with HTP resulted in the highest sugar yield,
i.e. 70% of total carbohydrate content. It is also interesting to note
that the use of 0.1 mol L−1 KOH and HTP resulted in 60% sugar
yield, which is comparable with the yield from 1.0 mol L−1 NaOH
and HTP, thus indicating the higher efficiency of KOH in pretreating
the RH.
wileyonlinelibrary.com/jctb
 www.soci.org K-S Heng et al.

100
Table 1. Composition of the RH used in this study
90 (A) Untreated
Component Composition (%) (B) 1 M NaOH/HTP
80
(C) 1 M KOH/HTP
Total solids* 92.6 70
Structural carbohydrates* Cellulose 34.7

Weight (%)
60
Hemicellulose 17.4
Total 52.1 50
Lignin* Acid soluble lignin 2.3 40
Acid insoluble lignin 23.2 30 (A)
Total 25.5
20 (B)
Ash* 15.0
10 (C)
Moisture* 7.4
Extractives Water extractives 7.2 0
Alcohol extractives 2.0 30 100 200 300 400 500 600 700 800 900
Total 9.2 Temperature (°C)

*Values for each of the components were calculated on an Figure 2. TGA thermograms of untreated RH, RH pretreated with
extractives-free basis. 1.0 mol L−1 NaOH/HTP, and RH pretreated with 1.0 mol L−1 KOH/HTP. The
temperature was increased from 30 ∘ C to 900 ∘ C at a heating rate of
20 ∘ C min−1 .
80
h
RT
Yield of total reducing sugars (%)

70 Table 2. TRS content in RH hydrolysate before and after autoclaving

MI g g
60 HTP at different pH

50 RH hydrolysate pH TRS (g L−1 )* Total phenolics (mg L−1 GAE)**

f
40 e e Before autoclaving 4.8 20.0 ± 0.3 91.3 ± 0.4
c c d
30 b b After autoclaving 4.8 19.5 ± 0.4 94.1 ± 0.1
After autoclaving 7.0 10.0 ± 0.2 147.3 ± 0.6
20 a
RH, rice husk; TRS, total reducing sugars.
10
*TRS determined from DNS assay expressed as an average of three
0 replicates.
0.1 M NaOH 1.0 M NaOH 0.1 M KOH 1.0 M KOH **Total phenolics expressed as milligrams gallic acid equivalents (GAE)
Pretreatment combinations per L, average of three replicates.

Figure 1. Effect of different NaOH and KOH pretreatment conditions on

yield of total reducing sugars after 72 h of enzymatic hydrolysis. Yields of
total reducing sugars were obtained from mean sugar concentrations of
triplicate samples and calculated based on total structural carbohydrates
in the untreated raw material. Mean values indicated by different letters
are significantly different. Error bars indicate standard deviation.
To evaluate the effects of the two alkalis on the compositional
changes of the RH, TGA was performed and the resulting ther-
mograms are illustrated in Fig. 2. After initial weight loss due to
moisture, the untreated RH samples witnessed a weight loss of
50% at 370 ∘ C. This percentage weight loss was in agreement with
the total structural carbohydrates determined by compositional
analysis, which was 52%. At this stage of decomposition for the
pretreated samples, the RH pretreated with NaOH/HTP showed a
weight loss of 60%, while the RH pretreated with KOH/HTP showed
a weight loss of 70%. Above 370 ∘ C, the weight loss was 20% for
RH pretreated with NaOH/HTP compared with 30% for untreated
RH. Only 10% weight loss was observed for RH pretreated with
KOH/HTP at this stage, signifying that this pretreatment was able
to remove higher amounts of lignin. At the end of heating, the
percentage of remaining solid material in untreated RH samples
was approximately 20%, while for NaOH/HTP pretreated samples,
only 12% of material remained undegraded. Meanwhile, the RH
pretreated with KOH/HTP had approximately 5% solid material
remaining.
Based on analysis of TRS, the RH hydrolysates contained approx-
imately 20 g L−1 sugars (Table 2). Sterilization of the hydrolysates
by autoclaving resulted in about 50% sugar loss when the pH of
the hydrolysate was 7.0. No significant loss of sugars was observed
when the hydrolysate with pH 4.8 was autoclaved.
From HPAEC analysis, the monosaccharides present in the RH
hydrolysate at pH 4.8 were determined to be glucose, xylose, and
arabinose (Fig. 3). Glucose was present in the highest proportion,
which was approximately 80%, followed by 15% xylose and 5%
arabinose. The loss of sugars observed when the hydrolysate was
autoclaved at pH 7 was also reflected in the chromatogram. It
was also interesting to note that the formation of other monosac-
charides, i.e. fructose and mannose, was observed. However, the
peaks corresponding to these sugars were low, indicating that
their presence was negligible. Thus, the hydrolysates were steril-
ized at pH 4.8 for all subsequent biosynthesis experiments to main-
tain the sugar content.
Table 3 shows the CDW and PHA content of B. cepacia USM and
C. necator NSDG-GG cultivated on hydrolysate. As a large propor-
tion of the TRS in the hydrolysate was glucose, pure glucose was
used as a comparison. It was found that B. cepacia USM had higher
CDW when cultivated on hydrolysate, which was 4.9 g L−1 com-
pared with 4.7 g L−1 when glucose was used, although the PHA
content was lower (40 wt %) when grown on the hydrolysate. C.
necator NSDG-GG showed high CDW (10.4 g L−1 ) and PHA content
(70 wt%) when grown on pure glucose, but showed significantly
lower growth on the hydrolysate, i.e. 3.7 g L−1 CDW and 38 wt%
PHA content.

wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry J Chem Technol Biotechnol (2016)

Conversion of rice husks to PHA via a three-step process
Figure 3. HPAEC chromatogram of monosaccharides detected in the RH hydrolysate before and after heat sterilization at pH 7.0.
Table 3. Growth and PHA accumulation of Burkholderia cepacia USM (JCM 15050) and Cupriavidus necator NSDG-GG on glucose or RH hydrolysate
supplemented with 0.54 g L−1 urea
B. cepacia USM
Carbon source* CDW (g L−1 ) PHA content (wt%)
Glucose 4.66a ± 0.05 48x ±3
Hydrolysate 4.85c ± 0.06 40x ±2
*Carbon sources were added at a concentration of 20 g L−1 TRS.
Mean values indicated by different superscript letters are significantly different.
To determine the effect of different nitrogen sources on B.
cepacia USM, NH4 Cl and urea were supplemented at concen-
trations of 0.5 g L−1 and 0.27 g L−1 , respectively, equivalent to
0.009 mol N. Based on Table 4 it was determined that urea was
a better nitrogen source than NH4 Cl for growth, with increases
in CDW when supplemented for both glucose and hydrolysate.
However, the PHA content was reduced from 72 wt% to 62 wt%
when urea was added to the hydrolysate compared with NH4 Cl.
Similar to the results in Table 3 it was found that cultivation
of B. cepacia USM on hydrolysate improved the growth and
PHA accumulation of the cells compared with glucose. From GC
analysis, it was found that only P(3HB) was synthesized by the
cells.
Fermentation in a 5 L fermentor using RH hydrolysate and urea
(0.54 g L−1 ) resulted in significant improvements in growth and
PHA content compared with shake-flask results (Fig. 4(a)). The
maximum CDW increased almost 2-fold to 7.8 g L−1 , with PHA
content of 50 wt%. The TRS was completely consumed at 36 h,
proving that B. cepacia USM was able to efficiently consume all
the sugars present in the hydrolysate. In contrast, fermentation
using pure glucose resulted in a maximum CDW of 4.2 g L−1 at
24 h, while maximum PHA content was 49 wt% at 36 h (Fig. 4(b)).
Notably, the total phenolics remaining in the hydrolysate at the
end of fermentation witnessed a 50% decrease (Fig. 4(a)). No
phenolic content was detected in the fermentation broth when
pure glucose was used.
J Chem Technol Biotechnol (2016) © 2016 Society of Chemical Industry
www.soci.org
C. necator NSDG-GG
CDW (g L−1 ) PHA content (wt%)
10.38b ± 0.12 70y ± 3
3.68d ± 0.06 38x ± 7
DISCUSSION
The conversion of lignocellulosic biomass to fermentable sugars
offers a promising alternative to reduce the cost of carbon feed-
stock in the quest to make PHA production viable at a commercial
level. The abundance of RH as residues of the rice milling industry
has spurred interest in evaluating its potential as a carbon source
for PHA production. The RH composition reported in other studies
had a cellulose content ranging from 34 to 40% and hemicellu-
lose content from 12 to 18%, while the lignin content ranged from
15 to 25%.19 – 22 Variations in the RH composition may be due to
many factors such as the physico-chemical properties of the soil
where the paddy is planted or the variety of the paddy. In this
study, the yield of sugars from enzymatic hydrolysis was calculated
based on the total carbohydrate content, which indicated the max-
imum theoretical amount of sugars that can ideally be obtained
from the RH.
To make the cellulosic components in the RH more accessi-
ble to enzymatic hydrolysis, the alkali pretreatment was per-
formed under highly pressurized thermal conditions. This enabled
removal of significant amounts of lignin and rapid decompression
of the cellulose fibers. Alkali pretreatment is less severe than other
chemical methods and is sometimes performed at ambient tem-
perature. However, this results in longer reaction periods, which
may require several days when done without the addition of any
physical or thermal methods.4,23 Such processes may not be time
wileyonlinelibrary.com/jctb
 www.soci.org K-S Heng et al.

Table 4. Growth and PHA accumulation of Burkholderia cepacia USM (JCM 15050) on glucose and RH hydrolysate supplemented with different
nitrogen sources

Carbon source* Nitrogen source** CDW (g L−1 ) PHA content (wt%) Total PHA (g L−1 )

Glucose NH4 Cl 1.71a ± 0.07 49x ± 2 0.85 ± 0.04

Hydrolysate NH4 Cl 2.66b ± 0.16 72y ± 3 1.92 ± 0.12
Glucose Urea 2.15c ± 0.02 50x ± 4 1.07 ± 0.07
Hydrolysate Urea 3.80d ± 0.06 62x ± 1 2.35 ± 0.11

*Carbon sources were added at a concentration of 20 g L−1 TRS.

**Nitrogen sources were added at a concentration of 0.5 g L−1 NH4 Cl and 0.27 g L−1 urea, equivalent to 0.009 mol N.
Mean values indicated by different superscript letters are significantly different.

(a) swelling refers to penetration that occurs in the crystalline

(b)
Figure 4. Batch fermentation of B. cepacia USM in (a) RH hydrolysate
autoclaved at pH 4.8 (equivalent to 20.0 g L−1 TRS), and (b) pure glucose
(20.0 g L−1 ). Error bars indicate standard deviation.
or cost effective. Thus, an additional thermal method was supple-
mented in this study to increase the efficiency of the pretreatment
process. The use of NaOH in pretreatment has been extensively
studied on a variety of biomasses. Nonetheless, KOH has been
gaining attention as another alkali for efficient pretreatment of lig-
nocelluloses including rice straw.24,25
It has been suggested that alkalis act by degrading the bonds
between hemicellulose and lignin, thus destroying the native
lignin structure.26 Furthermore, alkalis are able to chemically
modify cellulose by ionizing the hydroxyl groups, resulting in a
‘swelling’ effect that increases the surface area for enzymes to bind
and initiate hydrolysis. Interfibrillar swelling refers to penetration
of aqueous alkali in the amorphous region, while intrafibrillar
region.27 Although hemicellulose is also solubilized by alkaline
treatment, retention of a large fraction of hemicellulose in the
solid residue has been reported in several studies. In addition,
alkaline pretreatment is able to remove side chain groups of
hemicellulose, thus increasing the accessibility of enzymes to
the polysaccharide chains.28,29 However, the use of alkali alone
without any physical methods did not appear to be efficient in
pretreating the RH, as the maximum sugar yield obtained was less
than half of the available carbohydrates.
Investigations into the effect of irradiation as a form of biomass
pretreatment was reported in an early study by Ooshima and
colleagues whereby rice straw and bagasse were irradiated using
microwaves without the addition of any chemicals apart from
water.30 From their study, it was suggested that irradiation was
superior in improving digestibility of biomass because the material
was able to absorb kinetic energy directly, compared with other
thermal methods like steam pretreatment. A study by Li and col-
leagues found that microwave pretreatment was able to decrease
the crystallinity of cellulose.31 Thus, the increase in sugar yield
from RH when a combination of alkali and MI was applied could be
due to the breakdown of the rigid crystalline structure, compared
with the alkali pretreatment at room temperature. Although both
methods provide heating, improvements using the HTP method
over MI could be related to the rapid compression and decom-
pression of the fibers in biomass caused by the highly pressurized
environment. Thus, this method not only enhances leaching of
silica and delignification in the rice husks, it also loosens the cellu-
lose micro- and macro-fibrils. Autoclave usage was also reported
in studies involving pretreatment of rice husks with dilute acids19
and lime32 to achieve a temperature of 121 ∘ C. In their work with
woody biomass, Bozell and coworkers33 used autoclaving as a
means of fractionation of the main lignocellulosic components.
Furthermore, Gabriele et al. coined the term ‘DiCoDe’ process,
which stands for digestion, compression, and decompression,
to describe a novel method for extracting cellulose fibers from
Spartium junceum L. using an autoclave unit.34
The TGA thermograms also indicate the efficiency of the pre-
treatment based on the compositional percentage of the RH. Over-
all, three main stages of decomposition were observed. The weight
loss in the first stage, from 30 ∘ C to 150 ∘ C, was attributed to mois-
ture. The second stage, from approximately 150 ∘ C to 370 ∘ C, was
due to structural carbohydrates (cellulose and hemicellulose). The
third stage of decomposition occurred from 370 ∘ C onwards and
was attributed mostly to the lignin content.35 The residue remain-
ing at the end of heating was believed to be composed of char and
ash, which is mostly made up of silica.36 The decomposition pro-
files observed for RH pretreated with alkali and MI (data not shown)

wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry J Chem Technol Biotechnol (2016)

Conversion of rice husks to PHA via a three-step process
were similar to that of RH pretreated with alkali and HTP. Thus, the
thermogram of RH pretreated with HTP was used as a represen-
tation. It was interesting to note that the residue from KOH/HTP
pretreated RH was the lowest, which indicated that more lignin
and silica were removed with this combination of pretreatment
compared with NaOH/HTP. This could explain the increase in sugar
yield from RH pretreated with KOH/HTP, as the removal of higher
amounts of lignin and ash could have enhanced the accessibility of
enzymes to the carbohydrates. Thus, the results suggest that KOH
is more effective in delignification compared with NaOH.
Characterization of the hydrolysate was an important aspect of
this study because the hydrolysate contained a mixture of sugars
in an organic buffer solution. Culture media used for bacterial
culture are typically sterilized by autoclaving, which is simple and
practical. However, many chemical reactions could occur during
thermal processing owing to the complex composition of the
hydrolysate. Thus, it is essential to determine the ability of the
hydrolysate to withstand autoclaving without undergoing drastic
changes in its composition as a means of assessing its potential
for large-scale application. Sugars are known to be heat-sensitive,
which was reported in a study as early as the 1930s.37 The enzymes
used for hydrolysis of the pretreated RH were not removed at
the end of hydrolysis, and remained in the hydrolysate. At pH
of 7.0, the breakdown of the enzymes into amino acids during
the sterilization process provide substrates for Maillard reaction
to occur, resulting in the loss of sugars. Further reactions may
take place and give rise to formation of fermentation inhibitors
such as furfural and 5-hydroxymethylfurfural (5-HMF).38 Based on
thermal degradation studies by Qi et al., degradation of xylose
at high temperatures occurs more rapidly than glucose.39 Similar
to the conversion of glucose to 5-HMF, xylose also undergoes a
dehydration reaction to form furfural. However, this occurs without
the need for the formation of an intermediate product, unlike
glucose, which must first be converted to fructose before 5-HMF
can be formed. The increase of total phenolics in the hydrolysate
after autoclaving at pH 7.0 (Table 2) provided further evidence that
inhibitory compounds were formed.
The formation of other monosaccharides upon heating could
be related to the degradation processes. Isomerization of glucose
to fructose occurs naturally by the action of glucose isomerase.
Generally, this reaction involves a proton transfer that is mediated
by a base, followed by an acid-catalyzed hydrogen shift.40 There-
fore, a system that contains both a Lewis acid and Brønsted base
enables this reaction to occur.41 One possibility is that the citrate
salts present in the buffer solution had acted either as a Lewis acid
or Brønsted base, thus facilitating isomerization. The formation of
mannose could be attributed to an epimerization reaction, as man-
nose is an epimer of glucose. Previously, the epimerization of glu-
cose to mannose has been reported using metal ion complexes
or molybdate catalysts.42,43 However, in this study, no loss of sug-
ars was observed in the hydrolysate when it was heat-sterilized at
pH 4.8, and no significant increase in total phenolics were detected
(Table 2). The prevention of Maillard reaction by lowering the
pH of the reaction system has also been reported in previous
studies.44,45
The use of crude sugars obtained from hydrolysis or extraction
of plant biomass may be beneficial for the purpose of microbial
cultivation. The unrefined and unpurified sugar solutions often
contain additional compounds, such as organic acids or nitroge-
nous compounds, which could enhance growth of the bacteria
and improve biosynthesis yields. Although the presence of these
chemical components could potentially inhibit bacterial growth,
J Chem Technol Biotechnol (2016) © 2016 Society of Chemical Industry
www.soci.org
this problem can be overcome by using robust strains that are
able to utilize a wide range of compounds. Burkholderia cepa-
cia USM has been reported to be a nutritionally-versatile strain,
as it is able to grow on a variety of sugars and oils.10 In the
present study, the CDW increased when the cells were cultivated
on RH hydrolysate compared with pure glucose, indicating that
other components in the hydrolysate such as citrate were metab-
olized by B. cepacia USM and were capable of promoting cell
growth.
The phenolic compounds detected in the hydrolysate could have
a negative effect on the growth of C. necator NSDG-GG, which
would explain its low CDW and PHA content. Wild-type C. necator
H16 has been proven to grow on hydroxybenzoic acids, which is a
lignin derivative, with PHA accumulation of 65 wt% and 63 wt% on
3-hydroxybenzoic acid and 4-hydroxybenzoic acid, respectively.46
However, it did not grow in the presence of other intermediates
of hydroxybenzoic acid, such as vanillic acid or syringic acid, which
could have been present in the hydrolysate. Therefore, it is possible
that the poor growth of C. necator NSDG-GG on the hydrolysate
was due to low tolerance towards these phenolic compounds.
These results highlight the importance of strain selection for the
utilization of biomass-derived feedstocks.
The marked improvements in CDW of B. cepacia USM when urea
was used instead of NH4 Cl were in agreement with previous stud-
ies, which reported increased growth and PHA accumulation when
complex nitrogen sources were used.47 Furthermore, the ability of
B. cepacia USM to utilize urea is economically advantageous due
to its lower price.48 However, as each mole of urea contributes one
mole of carbon, the use of urea also increased the amount of car-
bon in the RH hydrolysate, leading to a lower C/N ratio and sub-
sequently a lower PHA content in the cells compared with when
NH4 Cl was used.
Improvements in CDW and PHA accumulation when B. cepa-
cia USM was cultivated in a fermentor could be attributed to
the provision of more controlled conditions. In addition, the
hydrolysate was able to sustain growth of the cells up to 48 h,
despite the depletion of sugars by 24 h (Fig. 4(a)). As mentioned
earlier, this could be owing to the presence of additional nitroge-
nous compounds, which could help to promote growth. These
results imply that the additional non-sugar components present in
the RH hydrolysate provide an enriched environment to support
cell growth and at the same time induce sufficient accumulation
of PHA.
Burkholderia strains have been known to utilize compounds that
are typically considered toxic, such as polychlorinated biphenyls
and aromatic compounds.49,50 It has been reported that Burkholde-
ria cepacia ATCC 17759 could tolerate and catabolize low con-
centrations of certain inhibitory compounds present in biomass
hydrolysates.51 Based on the results in this study, it was postulated
that B. cepacia USM could also possess similar metabolic pathways
that enable it to not only survive in the presence of inhibitory
compounds, but also utilize them for cellular activities. This study
represents a significant effort in managing agricultural waste by
channeling RH into processes for obtaining PHA, a value-added
product. As a multi-step process, conversion of a biomass such
as RH involves input of energy and chemicals at each step, which
could increase the overall production costs at industrial level. To
make such a process economically feasible, improvements from
many aspects need to be considered. Nevertheless, with the con-
tinuous emergence of new biotechnological tools, the develop-
ment of cost-efficient biorefineries may be possible in the near
future.
wileyonlinelibrary.com/jctb
 www.soci.org
ACKNOWLEDGEMENTS
The authors thank the Swedish Research Council (Swedish
Research Links grant, 348-2012-6169) for supporting this work.
HKS thanks the Malaysian Ministry of Higher Education for pro-
viding financial support through the MyBrain15 scholarship
programme.
Supporting Information
Supporting information may be found in the online version of this
article.
REFERENCES
1 Macrae RM and Wilkinson JF, Poly-𝛽-hydroxybutyrate metabolism in
washed suspensions of Bacillus cereus and Bacillus megaterium. J Gen
Microbiol 19:210–222 (1958).
2 Sudesh K, Polyhydroxyalkanoates from Palm Oil: Biodegradable Plastics.
Springer Briefs in Microbiology, Germany (2013).
3 Vanholme R, Demedts B, Morreel K, Ralph J and Boerjan W, Lignin
biosynthesis and structure. Plant Physiol 153:895–905 (2010).
4 Chang VS and Holtzapple MT, Fundamental factors affecting biomass
enzymatic reactivity. Appl Biochem Biotechnol 84–86:5–37 (2000).
5 Bansal V, Ahmad A and Sastry M, Fungus-mediated biotransformation
of amorphous silica in rice husk to nanocrystalline silica. J Am Chem
Soc 128:14059–14066 (2006).
6 FAOSTAT, Food and Agriculture Organization of The United Nations Statis-
tics Division 2014. http://faostat3.fao.org [accessed 9 November
2015].
7 Department of Statistics Malaysia, Selected Agricultural Indicators.
http://statistics.gov.my [accessed 3 November 2015].
8 Yevich R and Logan JA, An assessment of biofuel use and burning of
agricultural waste in the developing world. Global Biogeochem Cycles
17:1095–1137 (2003).
9 Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D and Crocker
D, Determination of Structural Carbohydrates and Lignin in Biomass.
National Renewable Energy Laboratory, Colorado (2008).
10 Chee JY, Tan Y, Samian MR and Sudesh K, Isolation and characterization
of a Burkholderia sp. USM (JCM 15050) capable of producing polyhy-
droxyalkanoate (PHA) from triglycerides, fatty acids, and glycerols. J
Polym Environ 18:584–592 (2010).
11 Mifune J, Nakamura S and Fukui T, Targeted engineering
of Cupriavidus necator chromosome for biosynthesis of
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable
oil. Can J Chem 86:621–627 (2008).
12 Orita I, Iwazawa R, Nakamura S and Fukui T, Identification of mutation
points in Cupriavidus necator NCIMB 11599 and genetic reconstitu-
tion of glucose-utilization ability in wild strain H16 for polyhydrox-
yalkanoate production. J Biosci Bioeng 113:63–69 (2012).
13 Doi Y, Kitamura S and Abe H, Microbial synthesis and characteri-
zation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macro-
molecules 12:106–111 (1995).
14 Kahar P, Tsuge T, Taguchi K and Doi Y, High yield production of
polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and
its recombinant strain. Polym Degrad Stabil 83:79–86 (2004).
15 Budde CF, Mahan AE, Lu J, Rha C and Sinskey AJ, Roles of multiple
acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosyn-
thesis in Ralstonia eutropha H16. J Bacteriol 192:5319–5328 (2010).
16 Miller GL, Use of dinitrosalicylic acid reagent for determination of
reducing sugar. Anal Chem 31:426–428 (1959).
17 Singleton VL and Rossi JA, Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic
16:144–158 (1965).
18 Braunegg G, Sonnleitner B and Lafferty RM, A rapid method for the
determination of poly-𝛽-hydroxybutyric acid in microbial biomass.
Eur J Appl Microbiol Biotechnol 6:29–37 (1978).
19 Saha BC, Iten LB, Cotta MA and Wu YV, Dilute acid pretreatment,
enzymatic saccharification, and fermentation of rice hulls to ethanol.
Biotechnol Prog 21:816–822 (2005).
20 Garrote G, Falque E, Dominguez H and Parajo JC, Autohydrolysis
of agricultural residues: study of reaction byproducts. Bioresource
Technol 98:1951–1957 (2007).
21 Vegas R, Kabel M, Schols HA, Alonso JL and Parajo JC, Hydrothermal
processing of rice husks: effects of severity on product distribution.
J Chem Technol Biotechnol 83:965–972 (2008).
wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry
K-S Heng et al.
22 Yu J, Zhang J, He J, Liu Z and Yu Z, Combinations of mild physical or
chemical pretreatment with biological pretreatment for enzymatic
hydrolysis of rice hull. Bioresource Technol 100:903–908 (2009).
23 de Jong E and Gosselink RJA, Lignocellulose-based chemical products,
in Bioenergy Research: Advances and Applications, ed by Gupta VK,
Kubicek CP, Saddler J, Xu F and Tuohy MG. Elsevier, Oxford, 277–314
(2014).
24 Ong LGA, Chuah C and Chew AL, Comparison of sodium hydroxide
and potassium hydroxide followed by heat treatment on rice straw
for cellulase production under solid state fermentation. J Appl Sci
10:2608–2612 (2010).
25 Remli NAM, Shah UKM, Mohamad R and Abd-Aziz S, Effects of chemical
and thermal pretreatments on the enzymatic saccharification of rice
straw for sugars production. BioResources 9:510–522 (2014).
26 Menon V and Rao M, Trends in bioconversion of lignocellulose: biofu-
els, platform chemicals and biorefinery concept. Prog Energy Com-
bust 38:522–550 (2012).
27 Wüstenberg T, Cellulose and Cellulose Derivatives in the Food Industry.
Wiley-VCH, Weinheim (2015).
28 Monsier N, Wyman CE, Dale B, Elander R, Lee YY, Holtzapple M et al.,
Features of promising technologies for pretreatment of lignocellu-
losic biomass. Bioresource Technol 96:673–686 (2005).
29 Sills DL and Gossett JM, Assessment of commercial hemicellulases
for saccharification of alkaline pretreated perennial biomass. Biore-
source Technol 102:1389–1398 (2011).
30 Ooshima H, Aso K and Harano Y, Microwave treatment of cellulosic
materials for their enzymatic hydrolysis. Biotechnol Lett 6:289–294
(1984).
31 Li L, Yu ST, Liu FS, Xie CX and Xu CZ, Efficient enzymatic in situ saccha-
rification of cellulose in aqueous-ionic liquid media by microwave
pretreatment. BioResources 6:4494–4504 (2011).
32 Saha BC and Cotta MA, Lime pretreatment, enzymatic saccharifica-
tion and fermentation of rice hulls to ethanol. Biomass Bioenergy
32:971–977 (2008).
33 Bozell JJ, Black SK, Myers M, Cahill D, Miller WP and Park S, Solvent frac-
tionation of renewable woody feedstocks: organosolv generation of
biorefinery process streams for the production of biobased chemi-
cals. Biomass Bioenergy 35:4197–208 (2011).
34 Gabriele B, Cerchiara T, Salerno G, Chidichimo G, Vetere MV, Alampi C
et al., A new physical-chemical process for the efficient production
of cellulose fibers from Spanish broom (Sparticum junceum L.).
Bioresource Technol 101:724–729 (2010).
35 Liou TH, Evolution of chemistry and morphology during the carboniza-
tion and combustion of rice husk. Carbon 42:785–794 (2004).
36 Ndazi BS, Nyahumwa C and Tesha J, Chemical and thermal stability
of rice husks against alkali treatment. BioResources 3:1267–1277
(2007).
37 Smith ML. The effect of heat on sugar solutions used for culture media.
Biochem J 26:1467–1472 (1932).
38 Martins SIFS, Jongen WMF and van Boekel MAJS, A review of Maillard
reaction in food and implications to kinetic modelling. Trends Food
Sci Technol 11:364–373 (2001).
39 Qi X, Watanabe M, Aida TM and Smith Jr RL, Catalytical conversion
of fructose and glucose into 5-hydroxymethylfurfural in hot com-
pressed water by microwave heating. Catal Comm 9:2244–2249
(2008).
40 Fenn TD, Ringe D and Petsko GA, Xylose isomerase in substrate
and inhibitor michaelis states: atomic resolution studies of a
metal-mediated hydride shift. Biochemistry 43:6464–7644 (2004).
41 Choudary V, Mushrif SH, Ho C, Anderko A, Sandler SI and Vlachos DG,
Insights into the interplay of Lewis and Brønsted acid catalysts in
glucose and fructose conversion to 5-(hydroxymethyl)furfural and
levulinic acid in aqueous media. J Am Chem Soc 135:3997–4006
(2013).
42 Brunner H and Opitz D, Enantioselective catalysis. Part 102. Epimeriza-
tion of glucose and mannose in the presence of nickel (II) complexes
of optically active ligands. J Mol Catal A Chem 118:273–282 (1997).
43 Köckritz A, Kant M, Walter M and Martin A, Rearrangement of glucose
to mannose catalyzed by polymer supported molybdate catalysts in
the liquid phase. Appl Catal A Gen 334:112–118 (2008).
44 Pacheco MD, Christian JI and Feng B, Study of Maillard reaction
inhibitors for the sugar cane processing. Am J Food Technol
7:470–478 (2012).
45 Follonier S, Goyder MS, Silvestri AC, Crelier S, Kalman F, Riesen R et al.,
Fruit pomace and waste frying oil as sustainable resources for the
J Chem Technol Biotechnol (2016)
Conversion of rice husks to PHA via a three-step process
bioproduction of medium-chain-length polyhydroxyalkanoates. Int
J Biol Macromol 71:42–52 (2014).
46 Tomizawa S, Chuah JA, Matsumoto K, Doi Y and Numata K, Under-
standing the limitations in the biosynthesis of polyhydroxyalka-
noate (PHA) from lignin derivatives. ACS Sust Chem Eng 2:1106–1113
(2014).
47 Khanna S and Srivastava AK, Recent advances in microbial polyhydrox-
yalkanoates. Process Biochem 40:607–619 (2005).
48 Ng KS, Wong YM, Tsuge T and Sudesh K, Biosynthesis and charac-
terization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymers
using jatropha oil as the main carbon source. Process Biochem
46:1572–1578 (2011).
J Chem Technol Biotechnol (2016) © 2016 Society of Chemical Industry
www.soci.org
49 Mitsui R, Kusano Y, Yurimoto H, Sakai Y, Kato N and Tanaka M,
Formaldehyde fixation contributes to detoxification for growth of
a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid. Appl
Environ Microbiol 69:6128–6132 (2003).
50 Goris J, De Vos P, Caballero-Mellado J, Park J, Falsen E, Quensen
JF 3rd et al., Classification of the biphenyl- and polychlorinated
biphenyl-degrading strain LB400T and relatives as Burkholderia xen-
ovorans sp. nov. Int J Syst Evol Microbiol 54:1677–1681 (2004).
51 Pan W, Perrotta JA, Stipanovic AJ, Nomura CT and Nakas JP, Production
of polyhydroxyalkanoates by Burkholderia cepacia ATCC 17759 using
a detoxified sugar maple hemicellulosic hydrolysate. J Ind Microbiol
Biotechnol 39:459–469 (2012).
wileyonlinelibrary.com/jctb