International Journal of Biological Macromolecules 160 (2020) 446–455

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Acid soaking followed by steam flash-explosion pretreatment to enhance

saccharification of rice husk for poly(3-hydroxybutyrate) production
Youwei Zhang a,⁎, Li Wang b, Tingting Li c, Yingbin Shen d, Jianguang Luo a
a
School of Food Science and Technology, Jiangsu Food and Pharmaceutical Science College, 4 Meicheng Road, Huai'an 223003, China
b
School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
c
Department of Food Science and Technology, College of Light Industry and Food Engineer, Nanjing Forestry University, Nanjing 210037, China
d
School of Life Science, Guangzhou University, Guangzhou 510006, China

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

Article history: In order to exploit an abundant and cheap carbon source for poly(3-hydroxybutyrate) (PHB) production, the rice
Received 21 March 2020 husk was pretreated with dilute sulfuric acid and steam flash-explosion to enhance the enzymatic saccharifica-
Received in revised form 15 May 2020 tion. The yield of reducing sugars of pretreated rice husk hydrolysate came up to 266.5 mg/g rice husk, much
Accepted 26 May 2020
higher than that of untreated rice husk hydrolysate (72.67 mg/g rice husk). This result indicated that the pretreat-
Available online 30 May 2020
ment can significantly enhance the yield of reducing sugars. Then the hydrolysate was used as a sole carbon
Keywords:
source for PHB production by using Cupriavidus necator. Response surface method was used to optimize the
Rice husk fermentation conditions. Under optimum fermentation conditions (carbon source, 31.81 gL−1; nitrogen source,
Steam flash-explosion 1.8 gL−1; pH-value, 7.0; temperature, 27.1 °C), the PHB yield was 5.0 gL−1, which was in good agreement with the
Reducing sugars predicted value.
Hydrolysate © 2020 Elsevier B.V. All rights reserved.
Cupriavidus necator

1. Introduction
Since the full-synthetic, commercially-successful plastic was
invented by Leo Hendrik Baekeland, N100 years has passed. Nowadays,
a variety of plastics have been invented for different purposes. Plastics
and plastic products have permeated through every corner of human
life [1–3], such as furniture, decoration materials, packing material,
communication equipment, domestic appliance, automotive [4], under-
ground pipe network system, office equipment, outdoor equipment,
body building apparatus, children's toys, medical devices and equip-
ment [5], clothing zippers and buttons, and so on. In a word, human
clothing, food (food packaging material), housing, work and entertain-
ment cannot be separated from plastics and plastic products [6,7]. Plas-
tics and their products greatly improve and facilitate human life due to
their light weight, versatility, durability, low production cost and other
suitable properties [8–10]. However, the environmental problems
caused by waste plastics have been perplexing human society since
the birth of plastics. According to research statistics, about 400 million
tonnes of plastics are produced per annum worldwide. The production
and use of such a large amount of plastics by human being society
every year puts great pressure on the environment [11–13]. The waste
plastic will pollute the land, water and air, and affect the agricultural ac-
tivities and cause the animal and plant to die [14]. Moreover, the natural
⁎ Corresponding author.
E-mail address: garfield201507@126.com (Y. Zhang).
breakdown of plastic material often takes over more than hundreds of
years in the wild. Therefore, searching for a new type of material
which is environmentally friendly and easily biodegradable to replace
plastic material has become an impatient need.
Polyhydroxyalkanoates (PHA) are polyesters which can be produced
by many microorganisms from renewable resources in a sustainable
manner and have gained more and more attention because of their bio-
degradability and biocompatibility [15–18]. Poly(3-hydroxybutyrate)
(PHB), one of the PHA family members, is an intracellular biopolyester
which is often synthesized by some mircoorganisms as storage sub-
stance of carbon source and energy in case of extreme environment of
energy scarcity [19]. Given its thermoplastic properties similar to poly-
ethylene and polypropylene, PHB has been recognized as the most
promising material to substitute conventional plastics in many applica-
tion fields [19–21]. Especially the biodegradability and biocompatibility,
make PHB take possession of advantaged superiority than any other
material in many application fields, including pharmaceuticals, biomed-
ical products, food packaging material, food additives, and agriculture
[19,22,23]. From the perspective of environmental protection and sus-
tainable development, PHB has more advantages compared with con-
ventional fossil fuel-based plastics. However, high production cost of
PHB compared to low cost of conventional plastics retards its large-
scale production and commercial application [24,25]. According to pre-
vious literature, the production cost of PHB is 4 to 9 times higher than
that of polyethylene, and the cost of raw material accounts for 50% of
PHB production cost [25]. And it is calculated that N70% of the raw

https://doi.org/10.1016/j.ijbiomac.2020.05.218
0141-8130/© 2020 Elsevier B.V. All rights reserved.
 Y. Zhang et al. / International Journal of Biological Macromolecules 160 (2020) 446–455 447

material cost for PHB production is the cost of carbon source [26]. Cur- Table 1
rently, pure fructose and glucose are frequently-used carbon sources Chemicals used in the study.

for PHB production by microbial fermentation. Therefore, the search Chemicals Supplier Purity (%)
for a carbon source substitute for fructose and glucose is a key factor Cellobiases Novozymes 99
to reduce the production cost of PHB, which will help to accelerate the Endoglucanase Novozymes 99
large-scale production and widespread application of PHB. In order to Hemicellulase Novozymes 98
reduce the production cost, the production of PHB by using industrial Citric acid Tianjin Zhiyuan Chemical Reagent Co., Ltd. 99.5
Sodium citrate Tianjin Zhiyuan Chemical Reagent Co., Ltd. 99
and agricultural by-products and waste as an alternative carbon source
Benzene Shanghai Runjie Chemical Reagent Co., Ltd. 99.5
has caused extensive attention because of their abundance, renewabil- Ethanol Tianjin Zhiyuan Chemical Reagent Co., Ltd. 99.5
ity, sustainability and low cost [27–29]. NaOH Tianjin Zhiyuan Chemical Reagent Co., Ltd. 97
Rice (Oryza sativa L.) is one of the most important crops in the world H2SO4 Tianjin Zhiyuan Chemical Reagent Co., Ltd. 98
due to its huge output and huge consumer population. The Food and Ag- BaSO4 Tianjin Zhiyuan Chemical Reagent Co., Ltd. 97
Glucose Tianjin Zhiyuan Chemical Reagent Co., Ltd. 98
ricultural Organization of the United Nations (FAO) has stated that a Xylose Shanghai Runjie Chemical Reagent Co., Ltd. 99
total of 700 million tons of rice was produced worldwide in 2010 [30]. Arabinose Shanghai Runjie Chemical Reagent Co., Ltd. 99
China is the world's largest producer and consumer of rice. In 2010,
the output of rice in China was about 200 million tons, accounting for
N25% of world rice yield. Rice husk is a by-product of rice processing, ac-
counting for about 20% of rice quality. According to the data above, it can 2.2. Microorganism strain
be calculated that there was about 40 million tons of rice husk produced
in 2010 in China. The rice husk yield is increasing due to the increase of The C. necator strain was purchased from China General Microbio-
rice production in China year by year. Rice husk is rich in cellulose, logical Culture Collection Center (CGMCC). Then the strain was
hemicellulose, and lignin and has been regarded as an abundant bio- reactivated and maintained on nutrient agar slope at 4 °C recommended
mass feedstock with low cost [31]. However, the complexity and recal- by CGMCC. The inoculum for PHB production was prepared by inoculat-
citrance of rice husk compositions constitutes a natural barrier against ing C. necator bacteria to 250 mL flask containing 100 mL of sterile nutri-
enzymatic attack and hydrolysis [32,33]. Therefore, the first step for uti- ent broth (NB) medium. The components of the NB medium are as
lization of lignocellulosic biomass of rice husk is to liberate cellulose and follows: 10.0 gL − 1 peptone, 3.0 gL − 1 beef extract, 5.0 gL − 1 NaCl,
hemicellulose from rice husk. At present, various pretreatments have and 1.0 L distilled water. The pH-value was adjusted to pH-value 7.0. Af-
been adopted to enhance enzymatic hydrolysis of rice husk in order to terwards, the NB was incubated in an air bath shaker at 30 °C for 72 h.
obtain more fermentable sugars which can be used for production of The cells from actively growing C. necator were collected by centrifuga-
chemical products and microbial products [30,34–36]. tion at 6000g for 10 min at room temperature, and resuspended in ster-
Cupriavidus necator (formerly called Ralstonia eutropha, Wautersia ilized saline. After adjusting to a concentration of 108 colony-forming
eutropha, Alcaligenes eutrophus, etc.) is an aerobic gram-negative bacte- units (cfu)/mL, the cell suspension was to inoculate the PHB production
rium, which has been studied and exploited as a promising PHA produc- medium. The PHB production medium and their compositions were
ing strain [37–39]. As we know, N300 microorganisms can produce PHB prepared according to the previous literature [49].
but due to their low productivity and yields [40,41], only a few strains
can be cultivated at a commercial scale for PHB production [42,43]. 2.3. Pretreatment of RH
Among the PHB producing bacteria, C. necator has been the most widely
studied strain because of its completely sequenced genome and high ca- The rice husk powder (1 g) was pretreated with 0.4% of 10 mL di-
pacity to accumulate PHB [44,45]. Besides, C. necator can synthesize PHA luted sulfuric acid (pH-value, 1.10), they were stirred and mixed thor-
by using various and structurally completely different carbon sources, oughly. The mixture was boiling and kept warm (100 °C) for 30 min.
such as fructose, glucose, glycerol, plant oil, palm oil, biodiesel, CO2, Then the mixture was filtrated with four-layer gauze. The filtrates
and so on [46,47], and a wide range of carbon sources can reduce the were collected for further analysis. Subsequently, the acid pretreated
production cost of PHB. Therefore, C. necator has gained more and rice husk was washed for four times with distilled water, drained until
more interest for PHB production. In our study, in order to find cheap al- there was no water dripping. The presoaked rice husk (sample size
ternative carbon source for PHB production, acid and steam flash- b0.4 mm) was transferred to a reaction kettle of steam flash-explosion
explosion pretreatment methods were used to treat rice husk before en- (SFE) equipment (QBS-200B steam explosion process test bed, Fig. 1),
zymatic hydrolysis, and the enzymatic hydrolysate was used as a sole open the intake valve and let the high-pressure steam flow into the re-
carbon source to culture C. necator for PHB production. action kettle. When the steam reached 1.8 MPa, maintained this pres-
sure for 5 min. Then the intake valve was shut off and the piston
2. Materials and methods device was triggered. The steam explosion was completed in millisec-
ond. After that, the pretreated rice husk sample was oven-dried at
2.1. Materials and chemical agents 60 °C. The sample was ground and pass through 40 mesh, then the pow-
der was collected and stored at room temperature.
Rice husk (RH) was purchased from local rice factory, and then the
RH was washed thrice with running water to remove dust. Afterwards, 2.4. Scanning electron microscopy (SEM)
the RH was dried at 105 °C for 24 h, the dried RH was ground to fine
powder which passed through 40 mesh. The RH powder was collected In order to investigate the efficiency of the pretreatment on rice husk
and stored in a plastic bag for use. The cellulose, hemicellulose and lig- sample, QUANTA-200 SEM (FEI Company) was used to detect the mor-
nin contents of rice husk are determined according to the standard pro- phological differences of unpretreated and pretreated rice husk
cedure outlined by Association of Official Analytical Chemists [48]. The samples.
work enzymes, cellobiase, endoglucanases and hemicellulase were
purchased from Novozymes (China) Biotechnology Co., Ltd. (Tianjin, 2.5. Enzyme activity assay and enzymatic hydrolysis
China).
All other chemical agents used were of analytic grade made in China. The cellobiase activity was assayed according to the method de-
More detailed information about chemicals used in this work is shown scribed by Ghorai et al. [50], the endoglucancase activity was measured
in Table 1. by the method of Xue et al. [51], and the hemicellulase activity was
448 Y. Zhang et al. / International Journal of Biological Macromolecules 160 (2020) 446–455
Fig. 1. Steam flash-explosion equipment.
determined based on the method of Shamala and Sreekantiah [52]. The
cellobiase activity 20.15 U/mg, the endoglucancase activity was
180.21 U/mg, and the hemicellulase activity was 5.00 U/mg. The
cellobiase, also known as β-glucosidase (β-glucosidase EC 3.2.1.21), is
abbreviated as BGL, belonging to hydrolase, also known as glucoside
hydrolase, gentil disaccharide enzyme, taking with strong trans-
glycosidase activity, which is obtained by deep liquid fermentation of
fungal strains. The endoglucancase (EC 3.2.1.4) is one of cellulases,
which can bind to the inside of the cellulose chain randomly and cut
β − 1,4- glycosidic bond to break the cellulose long chain for further ac-
tion by other enzymes. Hemicellulase is a complex enzyme system,
mainly including xylanase and mannanase, and its function is to
degrade hemicellulose and eliminate anti-nutritional properties. The
rice husk sample pretreated with diluted sulfuric acid and SFE was
saccharified by the work enzymes. Briefly, rice husk sample (20 g) and
distilled water (200 mL) was placed into a 500-mL-capacity screw-
capped bottle, adjusted pH-value to pH-value 4.8 by adding sodium ac-
etate buffer. After this, the bottle was kept warm at 50 °C in a water-bath
shaker with 150 rpm of agitation. Then cellobiase (240 units per g sam-
ple or 34.03 mg protein/g-cellulose), endoglucancase (240 units per g
sample or 3.81 mg protein/g-cellulose), and hemicellulase (500 units
per g sample or 285 mg protein/g-cellulose) dissolved in citrate buffer
(0.01 M, pH 4.8) were added into the flask. The enzymatic reaction
was kept at 50 °C for 48 h, and every 8 h interval the yield of reducing
sugars was assayed. The unpretreated rice husk sample was also hydro-
lyzed under the same conditions as a control.
2.6. Analytical methods
The yield of reducing sugars released was determined according to
dinitrosalicylic method [53]. The concentrations of glucose, xylose and
arabinose were quantified by a high performance liquid chromatogra-
phy (HPLC, Waters system), using a silica gel column bonded with
amide group (Innovation HP amide, 250 mm × 4.6 mm, 5 μm,
CHROM-MATRIX BIO). Acetonitrile-water (65:35) was used as mobile
phase, detection with refractive index and diode array detectors, and
the flow rate was 0.8 mL/min. The injection volume was 10 μL, and
the column temperature was set at 40 °C. The total phenolic content
was determined according to the method described by Singleton and
Rossi [54]. Furfural and hydroxymethylfurfural content was determined
according to the method described by Dong et al. [55]. In order to
remove these fermentation inhibitors, 3% activated carbon was used
to detoxify the enzymatic hydrolysate.
2.7. Fermentation for PHB production
According to previous medium components for PHB production de-
scribed by Beaulieu et al. [49], briefly, a 500-mL-capacity flask contain-
ing 200 mL of sterile medium was inoculated with C. necator, and
incubated at 140 rpm of agitation in an air bath shaker for PHB produc-
tion. In the study, the effect of pH-value, temperature, nitrogen source
and carbon source on PHB production was investigated and they were
optimized using response surface methodology. On the basis of single-
factor experiments, a three-level-four-factor BBD (Box-Behnken de-
sign) was designed to optimize PHB production. The factors and levels
were shown in Table 2. The four variables, carbon source (reducing
sugars from rice husk hydrolysate), nitrogen source [(NH4)2SO4], pH-
value, and temperature, were coded as A, B, C, and D, respectively.
Each variable was prescribed into three levels, called high, middle, and
low, coded as +1, 0, −1, respectively. In total, 29 runs based on BBD
with five central points are listed in Table 3.
2.8. Extraction and purification of PHB
After fermentation, microorganism cells were harvested by centrifu-
gation at 10000g for 10 min and followed by washing twice with dis-
tilled water to remove debris. Then the precipitate was subjected to
lyophilization at vacuum on a freeze dryer. PHB extraction from the
dry cell mass was performed according to the method described by
Dong and Sun [56]. Briefly, dry cell mass (0.5 g) was mixed with 40% so-
dium dodecyl sulfate (SDS) solution (5 mL) at 50 °C for 10 min,
Table 2
Independent variables and their levels in the Box-Behnken design.
Independent variables Symbols Coded levels
−1 0 +1
Carbon source (gL−1) A 20 35 50
Nitrogen source (gL−1) B 1 2 3
pH C 6 7 8
Temperature (°C) D 24 27 30
 Y. Zhang et al. / International Journal of Biological Macromolecules 160 (2020) 446–455 449

Table 3 eluent with flow rate at 1.0 mL/min, and the narrow distribution poly-
Response and experiment design of PHB production. styrene standards were used employed for calibration. The temperature
Run no. A B C D PHB (gL−1) of the column and detector was set at 30 °C.
1 0 0 −1 −1 4
2 1 0 −1 0 3.8 3. Results and discussion
3 −1 1 0 0 3.9
4 0 0 1 −1 2.6
5 0 0 −1 1 2.8 3.1. Chemical compositions of rice husk
6 1 0 0 1 2.8
7 0 1 0 1 3.5 The chemical compositions of rice husk are subjected to analysis and
8 1 0 1 0 2.8
the results are listed in Table 4. The rice husk contained 34.15 ± 1.73% of
9 0 0 0 0 4.9
10 −1 0 1 0 4 cellulose, 14.36 ± 2.37% of hemicellulose, and 19.81 ± 3.88% of lignin.
11 −1 0 0 −1 3.5 The total structural carbohydrate content (cellulose and hemicellulose)
12 0 1 −1 0 3.6 wad 48.51 ± 4.1%. This value is very close to previous descriptions
13 0 0 0 0 5.1 [35,36]. These structural carbohydrates are the main objects of enzy-
14 0 0 0 0 5
15 0 −1 −1 0 4.3
matic hydrolysis in this study.
16 0 0 0 0 5
17 0 −1 0 −1 3.7
18 1 −1 0 0 3.7
3.2. Pretreatment of RH
19 0 1 1 0 3.8
20 1 0 0 −1 3.5 The composition of biomass is complex and stable, the tiny constitu-
21 0 −1 1 0 3.6 ent units of plant fibers are surrounded by hemicellulose, followed by
22 0 −1 0 1 4
sheath shrinkage of lignin layer [57,58]. Although lignin has no hin-
23 0 0 1 1 3.8
24 −1 −1 0 0 4.4 drance to the decomposition of cellulose, it prevents the contact be-
25 0 0 0 0 5 tween cellulose and cellulase [59]. Therefore, it is necessary to pretreat
26 −1 0 −1 0 3.7 the rice husk, separate cellulose from lignin and hemicellulose, break
27 1 1 0 0 3.6 the hydrogen bonds inside the cellulose, and converse crystalline cellu-
28 −1 0 0 1 4
lose to amorphous cellulose in order to improve enzymatic hydrolytic
29 0 1 0 −1 3.7
effects [60]. In the study, the rice husk was pretreated with 0.4% sulfuric
acid at 100 °C for 30 min, subsequently subjected to SFE pretreatment.
After acid pretreatment, the solid yield was 87.31%. The sugar in the fil-
subsequently PHB was obtained by centrifugation and washed with dis- trates was analyzed and the results are listed in Table 5. The pretreated
tilled water. The resulting precipitate of PHB was dried in air, weighed, rice husk was subjected to SEM analysis and the results are showed in
and used for various analytical characterizations. Fig. 2.
The results in Table 5 show that there was a few arabinose and glu-
2.9. Description of produced PHB cose dissolved in the filtrate during the course of sulfuric acid pretreat-
ment. Fig. 2 shows the differences between untreated rice husk and
2.9.1. Thermal analysis pretreated rice husk. Fig. 2a shows that the untreated rice husk has com-
Thermogravimetric analysis (TGA) is an effective means to explore pact structure, the surface is rough and uneven, and there are many
the stability of materials, this method can detect the changes of PHB folds. Fig. 2b shows that the structure of the pretreated rice husk by sul-
sample mass with the increasing temperature. In this study, the PHB furic acid became loose, there was breakage to some extent, and the sur-
sample was subjected to TGA on a thermogravimetric analyzer (Q600, face became smooth. It is probably that some soluble sugars and other
TA, USA) in order to evaluate its thermal stability. Briefly, PHB sample impurities are removed from the rice husk surface because of dilute sul-
(5 mg) was placed in to an aluminium box and subjected to a heating furic acid immersion. Previous literature have proved that the surface
rate of 10 °C/min from 40 °C to 800 °C under nitrogen atmosphere characteristic of lignocellulosic biomasss treated by acid was referring
(20 mL/min). The melting temperature of PHB sample was determined to dissolution of molecule especially organic molecule mostly monosac-
by differential scanning calorimetry (DSC) on a differential scanning cal- charide which had deposited at epidermis [61–63]. And it has also been
orimeter (Q20, TA, USA). PHB sample (8 mg) was placed into an alu- reported that diluted acid treatment is one of the most effective pre-
mina crucible and then heated from 20 °C to 300 °C at a heating rate treatment for lignocellulosic biomass [64], which can hydrolyze hemi-
of 10 °C/min under nitrogen environment (50 mL/min). cellulose to sugars with high output, change the structure of lignin,
and broaden surface area of the cellulose [65,66]. Furthermore, the deg-
2.9.2. Fourier-transform infrared (FTIR) analysis radation and removal of hemicellulose can increase porosity and im-
In order to reveal the functional groups in the produced PHB, the prove accessibility of enzymatic hydrolysis, with maximum enzymatic
PHB sample was pretreated and subjected to analysis on a FTIR-4100 digestibility usually coinciding with complete hemicellulose removal
LE, JASCO, Japan. The PHB sample was pretreated according the method [64,67]. Besides, according to Fig. 2b, it can be inferred that the changes
described by Azizi et al. [27]. Briefly, weighed 2 mg of PHB sample, dis- of rice husk microstructure caused by diluted acid soaking is beneficial
solved in 0.5 mL of chloroform and was layered on KBr pellet. After
evaporation of chloroform, the PHB sample was analyzed by FTIR spec-
trum. The Fourier-transform infrared spectrum was recorded against Table 4
the wavelength of absorption ranging from 4000 to 400 cm−1. Chemical components of rice hull (% dry weight).

Component names Content (% dry weight)

2.9.3. Molecular mass analysis
Cellulose 34.15 ± 1.73
Gel permeation chromatography (GPC) system (Waters 1515GPC) Hemicellulose 14.36 ± 2.37
was used to determine the molecular mass and polydispersity of PHB Lignin 19.81 ± 3.88
sample. The PHB sample was dissolved in chloroform (1.0 mg/mL), Extractives 1.88 ± 0.11
and was filtrated with 0.2 μm membrane. The filtrates (50 μL) were col- Water 8.32 ± 0.33
Mineral ash 16.17 ± 2.17
lected and injected to the GPC system, the chloroform was used as
450 Y. Zhang et al. / International Journal of Biological Macromolecules 160 (2020) 446–455
Table 5
Chemical compositions of filtrate after acid pretreatment of rice hull.
Name Content (mg/g rice hull)
Arabinose 6.07 ± 0.24
Glucose 47.65 ± 0.15
Xylose 12.35 ± 0.13
to the high pressure steam entering the interior of rice husk, thus en-
hancing the SFE pretreatment effect. Fig. 2c shows that the structure
of rice husk became much looser under the action of mechanical shear
force of high-pressure steam after SFE pretreatment, and the degree of
structure fracture is also more obvious. The changes on structure of
rice husk were probably attributed to that the flash steam evaporation
of water exerted the thermal-mechanical force to destroy the texture
of biomass [68]. Meanwhile, there are a large number of holes appearing
on the rice husk surface. This is mainly due to the self-hydrolysis of
hemicellulose from rice husk under the action of high-pressure steam
[69], thereby causing a large amount of sugars to be dissolved out and
forming the porous structure. The formation of the porous structure of
the pretreated rice husk increases the specific surface area, and is bene-
ficial to the full contact of the enzyme and substrate, so that the sacchar-
ification degree of the rice husk will be enhanced. It has been reported
that the pore size of the substrate, which is in relation to the accessibility
of the enzyme, is the main constraint in the enzymatic hydrolysis of lig-
nocellulosic biomass [70]. Increasing porosity in pretreatment processes
means increasing effect of enzymatic hydrolysis [71]. Then Sun et al.
[72] have further confirmed that lignocellulose porosity caused by SFE
pretreatment had positive impacts on biomass saccharification.
3.3. Enzymatic hydrolysis of pretreated rice husk
The rice husk sample after pretreatment with sulfuric acid and SFE
was subjected to enzymatic hydrolysis by a mixture of cellobiase,
endoglucancase, and hemicellulase. For comparison purposes, the raw
material of rice husk which was not subjected to any pretreatment
was also enzymatically hydrolyzed at the same condition. The process
of enzymatic hydrolysis was monitored by measuring the yield of re-
ducing sugars (glucose, xylose, and arabinose). The yield of reducing
sugars was expressed as mg/g rice husk. The results were showed in
Fig. 3. As shown in Fig. 3a, the yield of reducing sugars for untreated
rice husk was 72.67 mg/g rice husk when the enzymatic reaction termi-
nated. The yield of reducing sugars contained 42.3, 23.5, and 6.87 mg of
glucose, xylose, and arabinose, respectively. The yield of reducing sugars
for pretreated rice husk shown in Fig. 3b was 266.5 mg/g rice husk, and
Fig. 2. SEM photographs of untreated and pretreated rice hull samples: a. untreated rice hull sample; b. pretreated rice hull using diluted sulfuric acid; c. pretreated rice hull sample using
diluted sulfuric acid and SFE.
this yield contained 163.4, 87.5, and 15.6 mg of glucose, xylose, and
arabinose, respectively. The reducing sugars released from pretreated
rice husk are more than three times of the reducing sugars from un-
treated rice husk. If combine the reducing sugar in the filtrate with the
reducing sugar from enzymatic hydrolysate, and yield of reducing
sugar of per gram pretreated rice husk will come up to 332.57 mg,
which is close to five times of the yield of reducing sugars for untreated
rice husk. The results suggested that the pretreatment of diluted sulfuric
acid and SFE had significantly improved the yield of reducing sugars of
rice husk hydrolysates. Dilute acid treatment can hydrolyze hemicellu-
lose, destroy the crystal structure of cellulose, and increase the surface
area and gap of substrate. Dilute sulfuric acid treatment can partly hy-
drolyze hemicellulose, destroy the crystal structure of cellulose, and in-
crease the surface area and pore volume of raw material [73]. Besides,
dilute sulfuric acid treatment is beneficial to degradation of hemicellu-
lose and breaking of lignin ester bond during the course of subsequent
SFE treatment. The influence of SFE treatment on raw materials is the
result of combined action of multiple physical and chemical factors. It
is considered that SFE treatment has the following effect of several as-
pects for lignocellulose materials, acid-like hydrolysis, thermal degrada-
tion, mechanical fracture, breakage of hydrogen bond, and destruction
and reorganization of the cellulose structure [63,74,75]. The results in
Fig. 2c can confirm the viewpoint above. This destruction on rice husk
microscopic structure can improve the effect of enzymatic hydrolysis.
Many research works have proved that SFE pretreatment can damage
hemicellulose and lignin structure, lower degree of polymerization of
cellulose, increase lignocellulose porosity, and enhance the sugar yield
of enzymatic hydrolysis [72,76,77]. All this above gave a reasonable ex-
planation why the yield of reducing sugars of pretreated rice husk hy-
drolysates was much higher than that of untreated rice husk.
The enzymatic hydrolysates consisted of 26.65 g/L of reducing sugar,
0.18 g/L of furfural, 0.1 g/L of hydroxymethylfurfural, and 1.5 g/L of total
phenol. After detoxification with 3% activated carbon, the enzymatic
hydrolysated contained 25.13 g/L reducing sugar, 0.05 g/L of furfural,
0.02 g/L of hydroxymethylfurfural, and 0.3 g/L of total phenol.
3.4. Optimization of fermentation conditions for PHB production
In the study, the effect of factors such as pH value, substrate concen-
trations and culture temperature on the PHB production were consid-
ered by BBD methods. In total, 29 experimental points given by the
model were performed regularly to determine their interactions on
PHB production, and analysis of variance (ANOVA) for PHB production
is presented in Table 6. The p-values were used to check the significance
of each coefficient and indicated the pattern of interactions between
variables. The model F-value of 208.52 implies the model is significant.
 Y. Zhang et al. / International Journal of Biological Macromolecules 160 (2020) 446–455 451

Fig. 3. Sugar concentrations in enzymatic hydrolysates of untreated and pretreated rice hull samples: a. untreated rice hull samples; b. pretreated rice hull samples using diluted sulfuric
acid and SFE.

There is only a 0.01% chance that a model F-value that large could occur
due to noise. And the results also showed that the R2 values were 0.9952
indicating that 99.52% of the variabilities can be explained by the fitted
model. The values of R2adj were 0.9905 which also confirmed that the
model was highly significant. At the same time, the “Lack of fit F-
Values” of 0.82 implied the Lack of fit was not significant. Non-
significant lack of fit is good. The fitted model for PHB to predict the re-
lationships between the independent variables and the dependent var-
iables can be expressed as follows:
PHB production were explored in Fig. 4 which showed the effects of the
two factors on the response. According to the 3D surface, the optimum
conditions for PHB production are as follows: carbon source, 31.81
gL−1; nitrogen source, 1.8 gL−1; pH-value, 7.0; temperature, 27.1 °C. Ac-
cordingly, the theoretical highest yield of PHB was predicted as 5.05
gL−1 at the optimum conditions. Verification experiments were per-
formed for three replicates, and the average yield of PHB was 5.0 gL−1.
These experimental results were in good agreement with the predicted
values by the model.

 
PHB gL−1 ¼ 5−0:27A−0:13B−0:13C þ 0:1AB−0:32AC−0:30AD 3.5. Characterization of PHB sample
þ 0:22BC−0:13BD
3.5.1. Thermal analysis
þ 0:60CD−0:67A2 −0:4B2 −0:78C2 −0:89D2
In the case of a thermoplastic material, it is important to maintain
the stability of its chemical structure within the processing temperature
In this case, A, B, C, AB, AC, AD, BC, BD, CD, A2, B2, C2, and D2 for PHB range. Thermogravimetric analysis was carried out to study the thermal
are significant model terms (p b 0.05). The 3D response surface plots for stability of the PHB sample, and the results were showed in Fig. 5a. From
the TGA curve, it is observed that the PHB sample was thermally stable
until 240 °C. When the temperature is higher than 240 °C, the PHB sam-
Table 6 ple begins to degrade and the ester bond breaks. When the temperature
ANOVA for the regression model of PHB production. comes up to 280 °C, the sample degrades sharply, and is converted into
carbon dioxide and water. Finally, the sample completely decomposed
Source Sum of df Mean F value p-Value
squares square prob N F at 397 °C.
The PHB sample was subjected to differential scanning calorimetry
Model 12.68 14 0.91 208.52 b0.0001a
A-Carbon source 0.91 1 0.91 208.85 b0.0001a (DSC) analysis to detect the melting temperature (Tm). The temperature
B-Nitrogen source 0.21 1 0.21 49.10 b0.0001a for DSC varied from room temperature to 300 °C at a heat flow of 10 °C/
C-pH 0.21 1 0.21 49.10 b0.0001a min, and the results were showed in Fig. 5b (the second heating scan).
D-Temperature 0.0008333 1 0.0008333 0.19 0.6681ns According to the DSC curve, it is observed that the Tm for PHB sample
AB 0.040 1 0.040 9.21 0.0089b
AC 0.42 1 0.42 97.23 b0.0001a
is 175.11 °C.
AD 0.36 1 0.36 82.85 b0.0001a
BC 0.20 1 0.20 46.60 b0.0001a 3.5.2. Fourier-transform infrared (FTIR) analysis
BD 0.063 1 0.063 14.38 0.0020b The FTIR analysis results of the PHB sample is shown in Fig. 5c. This
CD 1.44 1 1.44 331.40 b0.0001a
figure showed peak at 1726.1 cm−1, corresponding to carbonyl group (-
A2 2.88 1 2.88 663.46 b0.0001a
B2 1.06 1 1.06 243.85 b0.0001a C=O). The peaks at 1454.4 and 1379.6 relate to methyl groups (-CH3)
C2 3.94 1 3.94 906.27 b0.0001a [78,79]. The peaks at 2933.6, 2875.5, and 1454.6 cm−1, correspond to
D2 5.16 1 5.16 1186.86 b0.0001a methylene groups (-CH2). The bands found at 1284.7, 1219.5, 1184.3,
Residual 0.061 14 0.004354 1132.6, and 1056.9 cm−1 showed the (C\\O) stretching ester of the
Lack of fit 0.041 10 0.004083 0.82 0.6398ns
Pure error 0.020 4 0.005
polymer [80].
Cor total 12.75 28
ns
Not significant.
3.5.3. Molecular mass
a
Significant at p b 0.001. The molecular weight of polymer is an important factor which af-
b
Significant at p b 0.05. fects the processing and molding of materials. Therefore, gel permeation
452 Y. Zhang et al. / International Journal of Biological Macromolecules 160 (2020) 446–455

Fig. 4. Response surface and contour plots showing interaction effects of nitrogen and carbon source (A), pH and carbon source (B), temperature and carbon source (C), pH and nitrogen
(D), temperature and nitrogen (E), temperature and pH (F) on PHB production.

chromatography (GPC) was used to determine the molecular mass of
the PHB sample. In order to calculate the polydispersity index (PDI,
Mw/Mn) of polymer, both weight average molecular mass (Mw) and
number average molecular mass (Mn) are analyzed. Mw and Mn of
the PHB sample was 135 kDa and 124 kDa, respectively. And the PDI
was 1.09. Previous literature stated that the PHB sample produced by
C. necator had relatively low molecular mass (177 kDa) and low PDI
(1.9) [26]. Besides, it has also been reported that the molecular mass
of the final PHB products depended on many factors, such as the me-
dium components [26], culture conditions, the method of PHB extrac-
tion and isolation [81], and so on. Therefore, in the follow-up studies,
the components of substrate, culture conditions, and the methods of ex-
traction and isolation for PHB will be further improved in order to get
the PHB products with high molecular mass for practical applications.
In the study, it was obtained that the yield of PHB based on the
starting rice husk was 1 g PHB/26.72 g of rice husk, amounting to
0.16 g PHB/g reducing sugar. This PHB yield is lower than that (0.3 g
PHB/g sucrose) of previous literature [42] in which the pure sucrose
was used as carbon source for PHB production. In the rice husk hydroly-
sate, furfural, hydroxymethylfurfural, and total phenol were detected,
these compounds are toxic to microorganisms, and may have negative
impact on strain growth and PHB production [26]. This may explain
the reason why the PHB production in our study is lower than that of
other literature. Based on the results of thermal analysis in Section 3.5.1,
the PHB exhibits similar thermal properties with petroleum-derived poly-
mers, thus, it may potentially replace conventional thermoplastics in
many fields [17]. Besides, the produced PHB was subjected to FTIR
analysis, the results were compared with other PHB products or
even commercial PHB products [78–80], and characteristic groups
of PHB were detected in the produced PHB which may confirm the
produced PHB in our study has similar properties to commercial
PHB products.
 Y. Zhang et al. / International Journal of Biological Macromolecules 160 (2020) 446–455
Fig. 5. Photographs of TGA, DSC, and FTIR of the produced PHB: a. Thermogravimetric analysis (TGA) of PHB sample; b. Differential scanning calorimetry (DSC) of PHB sample; c. FTIR
analysis photograph of PHB sample.
In this work, rice husk has been developed and utilized as a source of
fermentable sugars, subsequently the resulting sugars have been used
as carbon source by C. necator for PHB production. This work can reduce
the environmental pressure caused by agricultural waste rice husk, de-
crease the production cost of PHB, and mitigate the environmental pol-
lution caused by synthetic plastics, which is of great significance to save
resources, protect the environment and promote the sustainable devel-
opment of national economy.
4. Conclusions
As environmental pollution and energy crises intensify, the demand
for inexpensive and natural degradable materials is growing. In the
study, in order to exploit abundant and cheap carbon source for PHB
production, the rice husk was pretreated by combination of dilute sulfur
acid and steam flash-explosion to enhance the effect of enzymatic hy-
drolysis. Then the hydrolysate was used as a sole carbon source for
PHB production, and the fermentation conditions for PHB were opti-
mized by using RSM. Finally, after experiments, some conclusions
were concluded. The combination of dilute sulfur acid and steam
flash-explosion to pretreat rice husk for improving saccharification is
feasible and practical, moreover, it can significantly improve the reduc-
ing sugar yields than that of unpretreated rice husk. The optimum con-
ditions for PHB production were obtained, and they were as the
following: carbon source, 31.81 gL−1; nitrogen source, 1.8 gL−1; pH-
453
value, 7.0; temperature, 27.1 °C. Under the conditions, the PHB yield
was 5.0 gL−1.
Authors' statement
Youwei Zhang, Tingting Li, and Yingbin Shen conceived and de-
signed the study. Li Wang and Yingbin Shen performed the experi-
ments. Youwei Zhang wrote the paper. Li Wang reviewed and edited
the manuscript. Youwei Zhang and Jianguang Luo revised the manu-
script according to the reviewers and editors' suggestion with the help
of other co-authors. All authors have read and approved the manuscript.
Acknowledgements
This work has been supported by Natural Science Foundation of
Jiangsu Province (Grants No. BK20150883), Youth Science and Technol-
ogy. And the work has also been supported by Huai'an Key R & D Plan
(Agriculture and Social Department, HAN201902).
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