Bioresource Technology 325 (2021) 124632

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Polyhydroxybutyrate production from ultrasound-aided alkaline pretreated

finger millet straw using Bacillus megaterium strain CAM12
Sivagnanam Silambarasan a, Peter Logeswari a, Ramachandran Sivaramakrishnan b,
Arivalagan Pugazhendhi c, Balu Kamaraj d, Antonieta Ruiz a, e, Govindarajan Ramadoss f,
Pablo Cornejo a, e, *
a
Centro de Investigación en Micorrizas y Sustentabilidad Agroambiental, CIMYSA, Universidad de La Frontera, Avenida Francisco Salazar 01145, Temuco, Chile
b
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
c
Institute of Research and Development, Duy Tan University, Da Nang, Vietnam
d
Department of Neuroscience Technology, College of Applied Medical Science in Jubail, Imam Abdulrahman Bin Faisal University, Jubail, Saudi Arabia
e
Scientific and Technological Bioresource Nucleus, BIOREN-UFRO, Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Avenida
Francisco Salazar 01145, Temuco, Chile
f
School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur 613401, India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Ultrasound aided NaOH pretreatment

showed a greater delignification and
sugar yield.
• Pretreated finger millet straw used for
Polyhydroxybutyrate (PHB) production.
• Strain CAM12 produced 8.31 g L− 1 of
PHB in finger millet straw hydrolysates.
• Structural characterizations ensured the
extracted biopolymer was PHB.

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, finger millet straw (FMS) was utilized for the production of Polyhydroxybutyrate (PHB) by Bacillus
Bacillus megaterium megaterium strain CAM12. Ultrasound-assisted alkaline (NaOH) pretreatment of FMS under optimized conditions
Enzymatic hydrolysis followed by enzymatic saccharification resulted in the maximum delignification (72%), hydrolysis yield (84%),
Finger millet straw
glucose yield (86%) and xylose yield (61%). The effects of different pH, temperature, incubation period, inoc­
Polyhydroxybutyrate
Ultrasound-alkaline pretreatment
ulum concentration, agitation speed and FMS enzymatic hydrolysates concentration were investigated to
improve the PHB production. Under optimized conditions, strain CAM12 used the FMS hydrolysates as the sole
carbon source for their growth and produced 8.31 g L− 1 of PHB. The extracted polymer on Fourier transform
infrared (FTIR), X-ray diffraction (XRD) and Nuclear magnetic resonance (NMR) analyses were confirmed to be
PHB. These results suggest the potential of combined ultrasound and alkaline pretreated FMS hydrolysates as a
promising feedstock for PHB production.

* Corresponding author at: Centro de Investigación en Micorrizas y Sustentabilidad Agroambiental, CIMYSA, Universidad de La Frontera, Avenida Francisco
Salazar 01145, Temuco, Chile.
E-mail address: pablo.cornejo@ufrontera.cl (P. Cornejo).

https://doi.org/10.1016/j.biortech.2020.124632
Received 18 November 2020; Received in revised form 23 December 2020; Accepted 24 December 2020
Available online 8 January 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
S. Silambarasan et al.
1. Introduction
Bioplastics have received great interest as a potential alternative to
conventional petroleum-based plastics that are non-degradable. Bio­
plastics in the form of Polyhydroxybutyrate (PHB) is the most widely
studied polymer in Polyhydroxyalkanoates (PHAs) due to its sustain­
ability, biocompatibility and biodegradability (Venkateswar Reddy
et al., 2017; Anburajan et al., 2019; Arul Manikandan et al., 2020). The
bacterial species such as Alcaligenes latus (Berwig et al., 2016), Bacillus
megaterium and Ralstonia eutropha (Arul Manikandan et al., 2020),
Burkholderia sacchari (Oliveira-Filho et al., 2020), Cupriavidus necator (Li
and Wilkins, 2020) and Iodobacter sp. PCH194 (Kumar et al., 2021) have
been reported as the most significant PHB producers, which are syn­
thesized and stored as the carbon and energy compound within the cell
wall (Saratale and Oh, 2015). However, the raw material cost limits the
commercial production of PHB and it’s accounted for 20–50% of the
overall cost of production (Sathiyanarayanan et al., 2013). Therefore,
researchers are focusing on the use of lignocellulosic biomass as an
inexpensive carbon source for the production of PHB, and studies were
done with rice straw (Sandhya et al., 2013), wheat straw (Soto et al.,
2019) and kenaf biomass (Saratale et al., 2019), etc. Nevertheless,
further exploration of such cost-effective renewable feedstock for the
production of PHB using a proficient bacterial strain is still required.
The production of PHB utilizing inexpensive biomass from agro-
wastes could reduce the overall production cost of PHB and make the
process more eco-friendly and sustainable (Saratale et al., 2020). The
key components of lignocellulosic biomass include cellulose 35–50%,
hemicelluloses 20–35%, lignin 5–30% and 1–10% of other extracted
substances (Menon and Rao, 2012; Sivaramakrishnan et al., 2021).
However, very scarce studies have been conducted to examine the
production of PHB in the lignocellulosic biomass hydrolysates by bac­
terial species. Finger millet (Eleusine coracana) is a drought-resistant
crop grown in rainfed regions of India and accounts for around 85%
of the total production of millets (Sakamma et al., 2018; Jamaldheen
et al., 2019; Israni and Shivakumar, 2020). After finger millet harvest­
ing, husk and straw are obtained as the by-products and they are typi­
cally discarded in open fields. High cellulose and hemicellulose contents
are present in the finger millet straw structure, which is a hopeful and
low-cost source of carbohydrates for use in biotechnological processes to
produce biofuels (Jamaldheen et al., 2019). Hence, this FMS waste can
be used as cost-effective valuable biomass for PHB production. To utilize
FMS as a potential feedstock for the production of PHB, pretreatment of
lignocellulosic biomass is very crucial as to not only release the cellu­
losic and hemicellulosic polymeric chains but also to modify the pores of
the substances (surface area of the biomass) for improving enzymatic
hydrolysis into fermentable sugars (Wang et al., 2017; Chandel et al.,
2018).
Alkaline pretreatment has drawn particular interest among the
different pretreatment strategies since it has a range of attractive ben­
efits. The greater delignification of biomass by eliminating lignin-
carbohydrate linkages and rise the digestibility is the primary role of
alkaline pretreatment (Saratale and Oh, 2015; Wang et al., 2017). On the
other hand, ultrasound has been revealed to be an effective reaction
intensification technique and ultrasound-assisted alkaline pretreatment
of lignocellulosic materials has emerged recently. The combination of
ultrasound and alkaline pretreatment increases the efficiency of hy­
drolysis, resulting in substantial lignocellulosic biomass delignification
and sugar yield (Karimi et al., 2014; Saratale et al., 2020). For instance,
the rice straw pretreated with NaOH and ultrasound achieved the
maximum reducing sugar yield of 2.91 g L− 1 was reported by Wu et al.
(2017). However, there are very limited reports on the ultrasound-aided
alkaline (NaOH) pretreatment of lignocellulosic biomass. The utilization
of ultrasound-assisted alkaline pretreated finger millet straw hydroly­
sates for the production of PHB is not reported so far.
Considering the necessity of efficient PHB producers and economic
production processes, this research aimed to study the utilization of FMS
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Bioresource Technology 325 (2021) 124632
hydrolysates sugars as the sole source of carbon for PHB production by
Bacillus megaterium strain CAM12. Firstly, alkaline and a combination of
ultrasound and alkaline pretreatment approach for the delignification
and enzymatic saccharification of FMS were thoroughly examined.
Subsequently, the optimum condition for the production of PHB from
FMS hydrolysates by strain CAM12 in the batch fermentation process
was investigated. The structural properties of extracted PHB were
analyzed by FTIR, NMR and XRD techniques.
2. Materials and methods
2.1. Lignocellulosic biomass and bacterial strain
The FMS was acquired from a local farmer in the Vellore district,
Tamil Nadu, India. The lignocellulosic material was rinsed with water
and dried in a hot air oven at 60 ◦ C. The dried material was milled to
approximately 0.4–0.6 mm and stored in sealed plastic bags at 4 ◦ C for
further experiments. The B. megaterium strain CAM12 used for PHB
production in this study was previously isolated from rhizosphere soil
(Silambarasan et al., 2019). The strain CAM12 was maintained on Luria-
Bertani (LB) agar slant at 4 ◦ C. PHB producing trait of strain CAM12 was
initially confirmed by the Sudan black B staining plate method
(Mohanrasu et al., 2020).
2.2. Pretreatment of FMS
2.2.1. Alkaline pretreatment
The FMS biomass (2.5%, w/v) was immersed in a 250 mL Erlen­
meyer flask containing 100 mL of NaOH solution and boiled for 45 min
at 100 ◦ C. The biomass sample was subsequently cooled down and solid
residues were obtained by suction filtration. Thereafter, delignified FMS
was washed properly with water and dried until a steady weight was
attained at 60 ◦ C. The samples of dried FMS biomass were stored at 4 ◦ C
in sealed plastic bags for further chemical composition analysis and
enzymatic saccharification. The cellulose, hemicellulose and lignin
content in FMS biomass were determined following the method
described by Ramadoss and Muthukumar (2015). To obtain the superior
delignification of lignocellulosic biomass, the effects of different con­
centrations of alkali (1–4% of NaOH, w/v) and FMS substrate (5–20%,
w/v) were studied. All the experiments were conducted in triplicates.
2.2.2. Ultrasound-assisted alkaline pretreatment
The optimized concentration of NaOH and FMS substrate load was
used in this ultrasound aided pretreatment. The ultrasound pretreatment
was carried out by using an ultrasonic processor (20 kHz; VCX750,
Sonics & Materials Inc, USA) equipped with a probe. Initially, the FMS
biomass (5%, w/v) was soaked in 100 mL of alkaline solution and an
ultrasound probe was introduced into the solution with a certain power
(1.0 W mL− 1) and pretreatment time (40 min). The same protocol as
specified in the alkaline pretreatment (boiling, filtration, drying, storing
and chemical composition) was then followed. The effects of different
ultrasound power density (1.0–3.0 W mL− 1) and pretreatment time
(20–80 min) on the delignification and hydrolysis of FMS biomass were
determined. All the experiments were conducted in triplicates.
2.3. Enzymatic hydrolysis of pretreated FMS biomass
The pretreated FMS biomass was hydrolysed using cellulase enzyme
according to the protocol described previously (Saratale et al., 2020).
The enzymatic hydrolysis of pretreated FMS biomass (2%, w/v) was
carried out in 20 mL of 50 mM citrate buffer (pH 5.0) containing sodium
azide (0.005%, w/v). Subsequently, the cellulase enzyme (30 FPU/g of
FMS substrate) was added into this mixture and incubated on a rotary
shaker at 150 rpm at 50 ◦ C for 24 h. Reaction mixtures were immediately
heated to inactivate the enzymes at 100 ◦ C for 15 min after enzymatic
hydrolysis, then cooled and centrifuged at 10,000g for 10 min at 4 ◦ C.
S. Silambarasan et al.
The obtained FMS biomass hydrolysates were stored at − 20 ◦ C and later
the reducing sugar content was estimated using the dinitrosalicylic acid
methodology (Miller, 1959). The glucose and xylose content in enzy­
matic hydrolysates of pretreated FMS biomass were analyzed by High
Performance Liquid Chromatography (Agilent 1260) equipped with Hi-
Plex H column and RI detector. Prior to the study, all the samples were
filtered by a 0.45 µm nylon filter membrane. The sulphuric acid (0.01 M)
was used as the mobile phase at a flow rate of 0.6 mL min− 1 (Arul
Manikandan et al., 2020). The yields of hydrolysis, glucose and xylose
were calculated by the formulas reported earlier by Saratale and Oh
(2015).
2.4. Production of PHB from pretreated FMS enzymatic hydrolysates by
B. megaterium strain CAM12
The PHB production was studied in mineral salt medium (MSM)
supplemented with pretreated FMS hydrolysates as the sole carbon
source using B. megaterium strain CAM12. The MSM contained (g L− 1):
NaH2PO4 3.6; Na2HPO4 2.84; NaOH 0.4; K2SO4 3.486; yeast extract 0.2;
CaCl2 0.062; MgSO4⋅7H2O 0.39; (NH4)2SO4 0.1; ZnSO4⋅7H2O 0.024;
CuSO4⋅5H2O 0.005; FeSO4⋅7H2O 0.15 and MnSO4⋅H2O 0.024 (Saratale
et al., 2019) with FMS hydrolysates sugar concentration of 20 g L− 1 and
pH 7.0. The 1% (v/v) of strain CAM12 (108 CFU mL− 1) was inoculated
into the PHB production medium and incubated on a rotary shaker at
120 rpm for 48 h at 28 ± 2 ◦ C. Moreover, the effects of different pH
(6–8), temperature (25–40 ◦ C), the incubation period (12–60 h), inoc­
ulum concentration (1–5%, v/v), agitation speed (80, 100, 120, 140 and
160 rpm) and pretreated FMS enzymatic hydrolysates concentration
(20–60 g L− 1) on the strain CAM12 cell growth and PHB production
were studied. All the experiments were performed in triplicates. After
the cultivation period, the sample was centrifuged at 6000g for 10 min
and the supernatant was discarded. The obtained bacterial biomass
(pellet) was rinsed twice with distilled water, freeze-dried in a lyophi­
lizer and then dry cell weight (DCW) was measured.
2.5. Extraction, purification and characterization of PHB
The extraction and purification of PHB from lyophilized cell pellets
were carried out according to the protocol reported earlier with slight
modification (Saratale and Oh, 2015). Briefly, the lyophilized cell pellet
(4 g) was blended with 50 mL mixture of an equal volume of NaClO
(30%, w/v) solution and CHCl3, and incubated at 28 ± 2 ◦ C for 2.5 h.
Subsequently, the suspension was centrifuged at 8000g for 10 min. The
bottom phase of chloroform containing PHB was precipitated with a
threefold volume of cold methanol. The precipitated biopolymer was
collected by centrifugation at 8000g for 10 min and resuspended in
chloroform to attain a greatly purified biopolymer. The obtained PHB
polymer was air-dried and used for further analysis. The PHB production
was quantified by the crotonic acid method (Law and Slepecky, 1961).
In this assay, extracted PHB was treated with concentrated sulfuric acid
(98%) and heated at 100 ◦ C for 10 min in a water bath (this reaction
converts PHB to crotonic acid). The optical density of the sample was
measured at a wavelength of 235 nm using a UV spectrophotometer
(Shimadzu, model UV-1800). The PHB was quantified by means of a
calibration curve using commercially available PHB as the standard. The
extracted PHB was mixed with KBr and functional groups were deter­
mined in the spectral range of 500–4000 cm− 1 using FT-IR instrument
(Perkin Elmer, USA). The structural characterization of PHB was carried
out in Bruker Avance III, 400 MHz NMR spectrophotometer. For this, 10
mg of purified PHB sample were dissolved in 1 mL of deuterated chlo­
roform (CDCl3) and the obtained solution was subjected to 13C NMR
spectrum analysis. XRD analysis of PHB was performed using D2 Phaser
(Bruker, Germany) at a voltage of 30 kV and Cu-K-α (1.54 Å) beam.
3
Bioresource Technology 325 (2021) 124632
3. Results and discussion
3.1. Effects of alkaline pretreatment on hydrolysis of FMS
The major chemical compositions of FMS used in this study con­
tained 38.57% cellulose, 29.60% hemicellulose and 15.32% lignin. The
pretreatment of lignocellulosic biomass with alkaline (NaOH) is regar­
ded as a promising method to remove the lignin by hydrolyzing covalent
linkages between lignin and hemicellulose ensuing in an increase in the
holocellulose surface area and enzymatic accessibility of biomass (Sar­
atale et al., 2020). The effects of different concentrations of NaOH
(1–4%) on delignification and hydrolysis yield of FMS (2.5%, w/v) were
evaluated (Fig. 1a). It was witnessed that the increase of NaOH con­
centrations significantly increased the delignification and hydrolysis
yield of FMS by 38–51% and 47–63%, respectively. A Higher rate of
delignification with a rise in alkali concentration could be associated
with that hydroxyl ion catalysing the cleavage of ether and ester bonds
in the lignin-carbohydrate structure (Subhedar and Gogate, 2014; Kim
et al., 2016b). The NaOH is dissociated into hydroxide ion (OH–) and
sodium ion (Na+) during the pretreatment process and therefore, the
rate of the hydrolysis reaction increases accordingly as the concentra­
tion of OH– increases (Kim et al., 2016b). Notably, FMS treated with 2%
NaOH resulted in greater glucose (66%) and xylose (48%) yields
compared to other NaOH (1%, 3% and 4%) treated FMS. Considering the
glucose and xylose yields, a NaOH concentration of 2% was selected for
further studies. The observed results are in consistent with the findings
of Saratale et al. (2020), who reported that the wheat waste biomass
pretreated with 2% NaOH showed 50%, 65%, 76% and 58% of
delignification, hydrolysis yield, glucose yield and xylose yield,
respectively. Saratale and Oh (2015) also found that the alkaline pre­
treatment of paddy straw caused maximal yields of hydrolysis (84.19%)
and reducing sugar (703 mg g− 1 of biomass).
The substrate concentration is essential for an effective alkaline
pretreatment process. Considering this, optimum substrate loading
concentration (5–20%, w/v) was determined by treating the FMS in 2%
NaOH solution at 100 ◦ C for 45 min. The 5% FMS substrate treated with
NaOH favoured the maximum delignification (Fig. 1b). This may be
owing to the better interaction of NaOH with FMS at lower concentra­
tions, which leads to break the linkage between lignin and hemicellulose
in lignin-carbohydrate complexes (Kim et al., 2016b; Wang et al., 2017).
The maximum delignification (69%), hydrolysis yield (72.5%), glucose
yield (76%) and xylose yield (58%) were observed at 5% FMS substrate
concentration (Fig. 1b). However, a further increase in FMS concen­
trations (>5%, w/v) generated a reduction in delignification and sugar
yields. Considering the results, 5% FMS concentration was found to be
optimal for better interaction with alkaline treatment and enzymatic
digestibility. Similar results have been reported in ultrasound-aided
alkaline pretreatment of wheat waste (Saratale et al., 2020) and carrot
grass (Singh et al., 2014).
3.2. Ultrasound-assisted alkaline pretreatment on hydrolysis of FMS
It has been reported that ultrasound-assisted Ca(OH)2 or NaOH
pretreatment had a superior effect on eliminating hemicellulose and
lignin from lignocellulosic biomass than alkaline pretreatment (Wang
et al., 2017; Saratale et al., 2020). In this study, the combined ultrasound
and alkaline (NaOH) pretreatment process was employed to maximize
the saccharification yield; i.e., glucose and xylose. The effects of ultra­
sound power and ultrasound pretreatment time on the delignification
and hydrolysis of FMS biomass were presented in Fig. 1c, d. The
delignification rate (53–58%), hydrolysis yield (61–71%), glucose yield
(66–78%) and xylose yield (54–59%) were increased with the rise of
ultrasound power from 1.0 to 2.5 W mL− 1 (Fig. 1c). The further increase
in ultrasound power to 3.0 W mL− 1 showed a reduction of saccharifi­
cation yield (data not shown). Moreover, the ultrasound pretreatment
time increased from 20 to 60 min were showed the increase of
S. Silambarasan et al.
Fig. 1. Effects of different (a) NaOH concentrations, (b) FMS concentrations, (c) ultrasound power output and (d) ultrasound pretreatment time on delignification,
hydrolysis yield, glucose yield and xylose yield of FMS biomass. Each value represents the mean ± SD of three replicates per treatment.
delignification, hydrolysis yield, glucose yield and xylose yield by
57–65%, 72–80%, 77–83% and 51–58%, respectively (Fig. 1d). Notably,
a higher yield of sugars was obtained at the ultrasound pretreatment
time of 60 min. Further, an extension of ultrasound pretreatment time
>80 min was hindered the sugar yields (data not shown). Therefore, the
suitable ultrasound power and ultrasound pretreatment time for FMS
biomass was 2.0 W mL− 1 and 60 min, respectively (Fig. 1c and d).
In this study, the delignification and saccharification yield was found
to be higher in ultrasound + NaOH pretreatment when compared to
NaOH pretreatment alone. This might be owing to the minor shock and
cavitation deterioration caused by ultrasound that improved the alka­
line and lignocellulose interaction, thus improving the removal of lignin
and hemicellulose degradation. The results obtained from the present
study was similar to Wang et al. (2017), who stated that the decompo­
sition rate of grass clipping pretreated by ultrasound + Ca(OH)2 and
ultrasound + NaOH was higher than that of individual alkaline treat­
ment. Zhang et al. (2008) also found that the lignin degradation (43.3%)
and saccharification rate (45.6%) of corn stover pretreated with
ultrasound-assisted alkaline was greater than that of alkaline treatment
alone. Saratale et al. (2020) reported that the wheat waste pretreated
with combined ultrasound and NaOH exhibited a delignification of 63%,
and a saccharification yield of 86% glucose and 62% xylose, which were
in good agreement with the present findings. Based on the above find­
ings, the optimum ultrasound aided NaOH pretreatment conditions were
ultrasound settings of 20 kHz, 2.0 W mL− 1 and 60 min, and 2% NaOH
with 5% FMS substrate concentration. The FMS pretreated under these
conditions showed lower hemicellulose (10.53%) and lignin (4.28%)
content, and higher cellulose (66.40%) content compared to untreated
FMS (described in Section 3.1). This increased content of cellulose
4
Bioresource Technology 325 (2021) 124632
revealed the solubilization of hemicellulose and lignin during the NaOH
+ ultrasound pretreatment. The maximum delignification of 72%, hy­
drolysis yield of 84%, glucose yield of 86% and xylose yield of 61% were
recorded from the substrate under these optimized conditions. This is
the first report of ultrasound aided alkaline pretreated FMS biomass
used for the production of PHB by B. megaterium.
3.3. Production of PHB using pretreated FMS enzymatic hydrolysates by
B. megaterium CAM12
3.3.1. Optimization of PHB production conditions
The optimum conditions of pH, temperature, incubation period,
inoculum concentration, agitation speed and various concentrations of
pretreated FMS enzymatic hydrolysates were determined for the
enhanced production of PHB by strain CAM12 (Fig. 2). For this analysis,
hydrolysates attained from the enzymatic saccharification of the ultra­
sound + NaOH pretreated FMS were utilized as reducing sugar at a
concentration of 20 g L− 1. It was observed that strain CAM12 was able to
produce PHB over an extensive range of pH 6–8 (Fig. 2a). The increase of
medium pH from 6 to 7 improved both the bacterial growth and PHB
production. The higher sugar consumption (70%), CAM12 growth (8.25
g L− 1 DCW) and PHB production (4.81 g L− 1) were obtained at pH 7.
However, at higher pH (7.5 and 8) was exhibited a sharp decrease in
CAM12 growth and PHB production. These results revealed that the
initial pH considerably impacted the bacterial growth and their pro­
ductivity of PHB (Mohanrasu et al., 2020; Mostafa et al., 2020). The
results of the current study are in accordance with a previous reports
that showed an optimal pH 7 for PHB production by B. megaterium
(Rodríguez-Contreras et al., 2013; Arul Manikandan et al., 2020). In this
S. Silambarasan et al. Bioresource Technology 325 (2021) 124632

Fig. 2. Effects of different parameters including (a) pH, (b) incubation temperature, (c) incubation period, (d) inoculum concentration, (e) agitation speed and (f)
pretreated FMS enzymatic hydrolysates concentration on PHB production from pretreated FMS hydrolysates using B. megaterium strain CAM12. Each value represents
the mean ± SD of three replicates per treatment.

study, the maximum sugar consumption, DCW and PHB production was
observed at initial pH 7.0 of the medium and thus it was selected as the
optimal for PHB production in the following experiments. Temperature
is another important environmental factor that impacts the growth of
bacteria. Fig. 2b shows that the PHB production differed in response to
various incubation temperatures. Especially, the maximum assimilation
of sugar (73%), DCW (9.21 g L− 1) and PHB concentration (5.49 g L− 1) by
strain CAM12 was produced at 37 ◦ C. The increase of temperature
beyond 37 ◦ C has a negative impact on CAM12 cell growth and PHB
production. This result is similar to Mohanrasu et al. (2020), who found
the maximal production of PHB by the B. megaterium at 37 ◦ C. The
decrease in PHB production at high temperature (40 ◦ C) may be due to
the cell growth inhibition and denaturation of the enzyme system
required for PHB production (Getachew and Woldesenbet, 2016;
Mohanrasu et al., 2020). The effects of different incubation periods on
PHB production were shown in Fig. 2c. It was indicated that the maximal
PHB production (5.88 g L− 1) was achieved at 48 h of incubation. The
PHB production was declined slightly when the incubation period was
above 48 h, which was in accordance with the observation by Saratale
and Oh (2015). This may be due to the lack of essential nutrient sources
for bacterial growth and PHB production, and the decaying activity of
the enzyme for PHB biosynthesis (Bhagowati et al., 2015; Mohanrasu

5
S. Silambarasan et al. Bioresource Technology 325 (2021) 124632

et al., 2020; Mostafa et al., 2020). The condition of nutrient depletion

may also instigate the bacteria to use the accumulated PHB as an energy
source (Getachew and Woldesenbet, 2016). Previously, there are several
reports on the microbes needing an incubation time of a minimum of
30–64 h for the production of PHB in pretreated lignocellulosic biomass
hydrolysates or medium containing commercial sugars (Saratale et al.,
2019, 2020; Li and Wilkins, 2020; Arul Manikandan et al., 2020;
Mohanrasu et al., 2020). The effects of various inoculum concentrations
of CAM12 (1–5%) were shown good results but the greater bacterial
growth biomass (12.72 g L− 1 DCW), sugar consumption (83%) and PHB
production (6.93 g L− 1) were observed with 3% inoculum (Fig. 2d).
When the inoculum concentration was increased to 4–5% displayed a
reduction in the DCW and PHB production. This may be due to a nutrient
deficit or consumption of accumulated polymer at high cell counts
(Mohandas et al., 2017; Mohanrasu et al., 2020). Based on the results
obtained, 3% inoculum concentration was found to be optimal for PHB
production. Agitation also could be necessary to scatter the microbial
cells in the medium to improve their interactions with the substrate and
thus to achieve faster cell growth (Saratale and Oh, 2015). In the present
Fig. 3. PHB production from pretreated FMS hydrolysates (30 g L− 1 sugar
study, the effects of various agitation speeds were also evaluated to
concentration) using B. megaterium strain CAM12 under optimized conditions.
achieve maximum production of PHB (Fig. 2e). It was found that strain Each value represents the mean ± SD of three replicates per treatment.
CAM12 showed the finest performance of sugar consumption (82.8%),
biomass (12.9 g L− 1 DCW) and PHB production (6.78 g L− 1) at 140 rpm
B. megaterium strain CAM12 was shown in Supplementary data. The
(Fig. 2e). Besides, the effects of different concentrations of pretreated
intense peak observed at 1721 cm− 1 indicated the presence of a carbonyl
FMS hydrolysates on the CAM12 growth and PHB production were
bond (C– – O), which is a characteristic peak of PHB. The absorbance
carried out (Fig. 2f). The FMS hydrolysates concentration used from 20
appeared at 1378 cm− 1 corresponds to the methyl group (CH3). The
to 30 g L− 1 revealed the enhanced CAM12 biomass (9.73–14.27 g L− 1
peaks at 1236 to 1045 cm− 1 are related to the stretching of the ester
DCW) and PHB production (5.9–7.3 g L− 1), and the subsequent increase
group carbonyl (C–O–C) and C–O bonds. Moreover, peaks between 937
of hydrolysates concentration (40 and 60 g L− 1) reduced the sugar
and 609 cm− 1 also confirmed the existence of C–O or C–O–C stretching
consumption as well as the production of PHB (Fig. 2f). Incomplete
bonds that are generally present in the PHB molecules. These FTIR re­
utilization of hydrolysates by the CAM12 strain and a limited number of
sults are in good consonance with the earlier studies (Arul Manikandan
fermentation inhibitors that may exist in the FMS hydrolysates were the
et al., 2020; Anburajan et al., 2019; Mohanrasu et al., 2020; Saratale
key reason behind the decrease of PHB at higher hydrolysates concen­
et al., 2020). The XRD analysis of PHB showed different peaks at (2θ)
tration (Sindhu et al., 2016; Saratale et al., 2019, 2020). In this study,
13.4◦ , 16.7◦ , 22.3◦ , 25.5◦ and 26.8◦ (Supplementary data). The high
the maximum sugar consumption (85%), DCW (14.27 g L− 1) and PHB
intense peak at 13.4◦ denoted the crystalline nature of PHB (Saratale
production (7.3 g L− 1) were observed with 30 g L− 1 of pretreated FMS
et al., 2019; Mohanrasu et al., 2020). The 13C NMR spectrum of PHB
hydrolysates (Fig. 2f), and thus it was selected as an optimal concen­
showed the peaks at 169.06 ppm, 67.69 ppm, 40.86 ppm and 19.74 ppm
tration for PHB production in the following experiments.
were attributed to the C– – O, C–H, –CH2 and –CH3, respectively (Sup­
plementary data). The obtained 13C NMR results are in strong compli­
3.3.2. PHB production under optimized conditions
ance with the previous reports of PHB produced from R. eutropha
The PHB production was carried out in 30 g L− 1 of pretreated FMS
(Saratale et al., 2020), A. junii BP25 and A. hydrophila ATCC 7966
hydrolysates as the sole carbon source under optimized conditions such
(Anburajan et al., 2019) and Bacillus cereus VIT-SSR1 (Evangeline and
as pH 7, 3% inoculum, incubation temperature at 37 ◦ C, agitation speed
Sridharan, 2019). Hence, the FTIR, XRD and NMR analyses were
at 140 rpm and incubation period of 48 h. Under these conditions, the
ensured that the extracted biopolymer was PHB.
production of PHB by strain CAM12 was increased from 1.4 g L− 1 at 12 h
to 8.31 g L− 1 at 48 h and dropped as 7.73 g L− 1 in 60 h (Fig. 3).
4. Conclusion
Moreover, the maximum dry cell weight of 16.23 g L− 1 and sugar uti­
lization of 87% was also observed at 48 h of incubation (Fig. 3). Simi­
The ultrasound-aided alkaline pretreatment was effective in
larly, a recent study of pretreated wheat waste biomass hydrolysates
improving the enzymatic hydrolysis of FMS. This study is the first report
employed for the synthesis of PHB using Ralstonia eutropha displayed the
of ultrasound + NaOH pretreated FMS as a cheap lignocellulosic feed­
greatest PHB production of 7.85 g L− 1 at 36 h (Saratale et al., 2020). The
stock for PHB production. Strain CAM12 grown in 30 g L− 1 of pretreated
lignocellulosic biomass of carob pod and synthetic medium were used
lignocellulosic biomass hydrolysates exhibited the higher PHB produc­
for PHB production by B. megaterium resulted in the maximum PHB
tion (8.31 g L− 1) with DCW (16.23 g L− 1) under the optimized condi­
production value of 5.7–6.6 g L− 1 at 60–64 h (Mohanrasu et al., 2020;
tions of temperature at 37 ◦ C, agitation speed at 140 rpm and incubation
Arul Manikandan et al., 2020). Nevertheless, the PHB production ob­
period of 48 h. Hence, the ultrasound + NaOH pretreated FMS is an
tained in this research is considerably higher than the previous studies,
economically feasible carbon substrate for PHB production.
where Ralstonia eutropha and Bacillus megaterium produced 3.93–7.85 g
L− 1 of PHB from lignocellulosic biomass (Sandhya et al., 2013; Kim
CRediT authorship contribution statement
et al., 2016a; Azizi et al., 2017; Annamalai et al., 2018; Saratale et al.,
2020; Arul Manikandan et al., 2020). Thus, it indicates that Ultrasound
Sivagnanam Silambarasan: Conceptualization, Methodology,
+ NaOH pretreated FMS biomass is a promising feedstock for PHB
Formal analysis, Investigation, Writing - original draft. Peter Loges­
production using B. megaterium CAM12.
wari: Investigation, Data curation, Writing - original draft. Ram­
achandran Sivaramakrishnan: Conceptualization, Writing - original
3.4. PHB characterization draft. Arivalagan Pugazhendhi: Writing - review & editing. Balu
Kamaraj: Writing - review & editing. Antonieta Ruiz: Validation,
The FTIR spectra of PHB produced in the FMS hydrolysates by

6
S. Silambarasan et al.
Writing - review & editing. Govindarajan Ramadoss: Data curation.
Pablo Cornejo: Conceptualization, Supervision, Project administration,
Funding acquisition, Validation, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
S. Silambarasan thankfully acknowledges the “Vicerrectoría de
Investigación y Postgrado, Universidad de La Frontera, Chile” for
providing financial support. P. Cornejo acknowledges the “Agencia
Nacional de Investigación y Desarrollo” (ANID-Chile) for ANID/FON­
DAP/15130015 grant. R. Sivaramakrishnan is grateful to the Graduate
School and Faculty of Science, Chulalongkorn University (CU) for a se­
nior post-doctoral fellowship from CU Ratchadaphiseksomphot
Endowment Fund.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2020.124632.
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