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Ultrasonic induced mechanoacoustic effect on

Cite this: Sustainable Energy Fuels,
2021, 5, 1832
delignification of rice straw for cost effective
biopretreatment and biomethane recovery
Yukesh Kannah R., †a Kavitha S., †a Sivashanmugam P.,b
Gopalakrishnan Kumar c and Rajesh Banu J. *d

Received 9th December 2020
Accepted 10th February 2021
DOI: 10.1039/d0se01814g
rsc.li/sustainable-energy
The mass transfer limitation of biopretreatment (BAC) of rice straw biomass can be improved by
a delignification process prior to BAC for energy efficient biomethane recovery. In the present study, rice
straw biomass was subjected to delignification by ultrasonic homogenization. As a result, a higher lignin
removal efficiency of 70.28% was achieved at an optimal biomass to water medium ratio of 0.02 (w/v)
and a specific energy input of 450 kJ per kg TS. Delignification can increase the accessibility of cellulose
present in the rice straw to biopretreatment. The delignified and biopretreated rice straw (Delign-BAC)
sample was more efficient with a chemical oxygen demand (COD) solubilization of 35% than BAC with
21% solubilization and the control (untreated sample) with 3.2% solubilization. The lignin content of the
Delign-BAC sample was 0.3 g L
1 and it did not lead to the inhibition of methanogenesis with a specific
methane production of 165.5 mL per g VS. Economic analysis exposed that Delign-BAC gained
a profitable net profit (68.62 USD per ton) with a cost benefit ratio (1.23) higher than that of BAC
(
22.912 USD ton and 0.62).

The enormous amount of these components in rice straw

Introduction
The necessity to increase energy security and problems on
climatic alterations are incentives for the progress of novel and
protable energy conversion processes.1 Protable energy
generation from alternative sources is essential to overcome the
greater need for fossil fuels, exhausting petroleum sources,
rising price of oil, and global warming issues. Biomass, being
a cheap and renewable energy source, is gaining more attention
owing to its greater accessibility in India.2 Biomass availability
in India is 150 million MT per annum.3 Lignocellulosic
biomass is regarded as the most abundantly available biomass
throughout the world.4 Agricultural residues are considered as
one of the cheap and sustainable forms of energy sources.5 Rice
straw is one of the abundantly available agricultural residues at
the global level and Asia accounts for 90% of its generation
worldwide yearly.6 The cellulose, hemicellulose and lignin are
considered as the main essential components of rice straw.7,8
a
Department of Civil Engineering, Anna University Regional Campus, Tirunelveli,
Tamil Nadu, India
b
Department of Chemical Engineering, National Institute of Technology,
Tiruchirappalli, Tamil Nadu, India
c
School of Civil and Environmental Engineering, Yonsei University, Seoul 03722,
Republic of Korea
d
Department of Life Sciences, Central University of Tamil Nadu, Neelakudi,
Thiruvarur, Tamil Nadu, 610005, India. E-mail: rajeshces@gmail.com; Tel: +91-
9444215544
† These two authors have contributed equally in this work.
biomass makes it a perfect feedstock for biomethane recovery.9
However, the existence of cellulose, hemicellulose and lignin in
rice straw resists microbial and enzymatic action and is there-
fore regarded as recalcitrant biomass.10 In the lignocellulosic
biomass skeleton, the cellulose, hemicellulose and lignin are
linked by chemical bonds within a complicated matrix and
binding materials provide greater resistance of biomass to
bacterial and enzymatic attack.11 The cellulose compound is
hydrophilic due to the presence of R–OH groups and hydrogen
linkages. Hemicellulose being an amorphous compound
imparts a major role in rming the biomass structure. The
macromolecule lignin being hydrophobic and aromatic
provides a higher extent of resistance.12 Thus, pretreatments are
mandatory prior to biomethane production. Different physical,
chemical, mechanical, and biopretreatments and combinative
treatments are suggested in reports to obtain efficient methane
production.13–15 Among these pretreatments, biopretreatments
(enzymatic or using pure cultures) are preferable due to less
energy consumption and less inhibitory products formation,
mild operation conditions etc.16,17 Among biopretreatments,
using pure cultures is advantageous as it considerably elimi-
nates the cost associated with enzyme dosage since commercial
enzymes are expensive. However, biopretreatment possess few
limitations such as improper mass transfer which leads to
minimal enzyme secretion thus resulting in higher production
cost18 due to which the bio-based pretreatments are not
commercially feasible.19 The possible diffusion of the enzyme

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Paper
during biopretreatment via the brous arrangement of rice
straw is difficult and it is a sluggish process. This diminishes
the pretreatment rate. The existence of multi-layered aromatic,
polymeric, cross linking substances such as lignin wraps the
cellulose and hemicelluloses in the rice straw biomass.20 In
addition, some researchers have suggested that the presence of
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remaining lignin in the disintegrated lignocellulosic biomass
unenviably upsets the biological process by binding with the
enzyme through hydrophobic and electrostatic linkages. For
example, Li et al.21 stated that the tangled lignin present on
disintegrated cellulosic bres harshly inhibits the hydrolysis
process mediated by cellulase enzymes as the accessibility to
the cellulosic surface is greatly reduced. Besides, higher
concentration of silica in rice straw may hamper the biode-
gradability of rice straw in addition to lignin. This effect could
be because of a silicied cuticular layer of rice straw biomass
and this would reduce the accessibility of cellulose.22–24 Hence,
delignication and desilication of rice straw before pretreat-
ment are mandatory. Numerous biobased pretreatments for
pretreating agricultural residues have been stated in reports so
far.1,25 However, most of these reports described are mainly on
fungal pretreatment for enhanced saccharication and bio-
ethanol production. Only few reports deal with biomethanation.
For example, Mustafa et al.26 discussed about fungal pretreat-
ment of rice straw with two fungi (Pleurotus ostreatus and Tri-
choderma reesei) and studied the efficiency of pretreated
biomass on methane production and added that extended
pretreatment is the main drawback of fungal pretreatment. In
this case, the bacteria used for pretreatment are excellent
cellulase secreting bacteria as they considerably solubilize the
rice straw biomass. Many literature studies have reported that
biological optimization inputs have resulted in enhanced
cellulase production but with inadequate success. It seems that
the way forward for biopretreatment (cellulase secreting
bacteria) is to enhance the enzyme production by increasing the
accessibility of cellulose.27 Therefore, in this study, mechanical
delignication was done prior to biopretreatment as a phase
separation process to increase the extent of delignication and
solubilization (soluble organics, cellulose and hemicellulose)
and with less energy consumption. Besides, an energy balance
and cost analysis is done to evaluate the economic viability of
pretreatments.
Experimental section
Collection of rice straw
Rice straw biomass was obtained from a rice eld in Tirunelveli.
The obtained straw was dried and cut manually into small
pieces (<1 cm) using a knife. The pieces were taken in a vessel
and kept airtight at ambient temperature and used for further
experiments. The characteristics of rice straw biomass are
tabulated in Table 1.
Delignication of rice straw
Delignication was done with an ultrasonic homogenizer
(Bandelin, Model: HD 2200, Berlin, Germany). During
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Table 1 Characteristics of rice straw biomass
S. no. Parameters Values
1 Total solids 20  0.54
2 Volatile solids 14.8  0.31
3 Total chemical oxygen demand (g L
1) 19.92  0.52
Soluble chemical oxygen demand (g L
1) 0.05  0.0014
4
5 Total cellulose (g L
1) 7.2  0.19
6 Total hemicellulose (g L
1) 5.5  0.12
7 Total lignin (g L
1) 4.064  0.11
8 Silica content (g L
1) 3.010  0.08
9 Biomass : water ratio (w/v) 0.02
delignication using the ultrasonic homogenizer, the rice straw
with a biomass to water medium ratio of 0.02 (w/v) was sub-
jected to treatment with varying sonic contact time and sonic
power levels (0 to 10 min and 10 to 80%). The samples were
taken at different intervals and estimated. Aer delignication,
the solids and liquid mixture were ltered using vacuum lters.
The collected hydrolyzates were analyzed for soluble lignin
release. The residual solids were cleaned with distilled water to
remove the remaining lignin and used for ensuing
biopretreatment.
Cellulase secreting biopretreatment
For biopretreatment, the excellent cellulase enzyme producing
bacterial strain, (Bacillus sp. – accession number KX373535)
which was isolated and recognized in the previous study,28,29
was used. The bacterial cells were cultured in minimal media
(0.1 percent potassium dihydrogen phosphate, 0.1 percent
dipotassium hydrogen phosphate, 0.04 percent magnesium
sulphate, 0.005 percent sodium chloride, and 0.0001 percent
ferrous sulphate, pH 7.0) supplemented with 1% carboxy
methyl cellulose (CMC) as the carbon source and used for the
cultivation of cellulase secreting bacteria. For the preparation of
the seed inoculum, freshly isolated culture was added in 5 mL
minimal media and kept at 110 rpm at 35  C for 24 h. Then, the
seed inoculum was added into the minimal production media
containing 1% CMC and incubated at 35  C at 110 rpm. The
collected culture at 24 h was used as the inoculum source for
pretreatment. The bacterial cells of 0.5 g L
1 (dry basis) were
utilized for biopretreatment. The lignin removed rice straw with
a biomass to water medium ratio of 0.02 (w/v) (0.5 L) was taken
in a 1 L conical ask. The ask with samples was incubated for
24 h and homogenized at 110 rpm speed and a temperature of
40  C. Two extra asks with the biopretreated sample alone and
the non-treated sample (0.5 L with a biomass to water medium
ratio of 0.02 w/v) were treated under similar operational
conditions to study the delignication effect on
biopretreatment.
Biomethanation experiment
The biomethanation experiment was performed in three labo-
ratory scale reactors (1 L serum bottles) based on the protocol
elaborated in earlier studies.30–33 The experiments were per-
formed at 35  C and neutral pH. Rumen uid rich in anaerobic
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microbes was utilized as the inoculum. The substrates used the gas constant (0.08206 atm L per mole K; and T is the
were delignied and biopretreated rice straw (Delign-BAC), operational temperature of the reactor ( C).
biopretreated alone (BAC) and non-pretreated (control)
samples. The substrate to inoculum ratio was maintained as Mass, energy & economic analysis
0.5 g COD/g COD. Once the inoculum and substrates were
The mass, energy and economic assessment of rice straw was
added to the reactors, all the reactors were purged with nitrogen
performed to analyze the effect of delignication and bio-
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and carbon dioxide for 5 min to maintain a strict anaerobic

environment. Aer purging, the reactors were covered with an
air tight rubber septum and folded with silver foil to afford
a dark surrounding suitable for the growth of anaerobes. The
reactants were mixed at 150 rpm. A needle was inserted into the
septum of reactors to estimate the biogas. While the gas was
produced, the syringe piston was pushed up owing to the
increase of pressure inside the reactors and the volume of gas
was measured from the syringe displacement. The methane
fraction was analysed using a gas chromatograph (Model No
G1311C, 1260, Baroda). It has been assumed that the obtained
methane data vs. digestion time symbolizes sigmoidal logistic
growth of methanogens and therefore the values were modelled
using a sigmoidal logistic t as shown below:
y ¼ a(1 + exp(
b(X
XC)))
y – cumulative methane production (mL); a – exponential
methane production potential (mL): upper asymptote; b – rate
constant (day
1); XC
lag time (days) and X – digestion period
(days). The theoretical methane production can be calculated
from the consumed COD using the following equation:
VCH4 ¼ CODCH4/K(t)
where, VCH4 is the volume of methane produced (mL); CODCH4
is the load of COD consumed (g); K(t) is the correction factor for
the operational temperature of the reactor (g COD per L)
K(t) ¼ P  K/R(273 + T)
where P is the atmospheric pressure (1 atm); K is the COD
corresponding to one mole of methane (64 g COD per mole); R is
pretreatment on methane generation at a scalable extent.
Energy and economic analysis was estimated as per the
methods described in earlier studies.34 The rice straw taken for
estimation was 1 ton. The parameters included in estimation
are summarized in Table 2.
Statistical methods
All the experiments were done three times and the average of
the three values was taken as optimum data. Signicance of the
data was checked by ANOVA analysis.
Analytical methods
The solids content, total COD and SCOD were determined as per
the standard protocol.35 The cellulase activity was calculated as
(1)
per the procedure followed by Khatiwada et al.36 The silica
content of the rice straw was estimated as per the methods
described by Khaleghian et al.24 The total lignin concentration
in the solid fraction was determined by Klason procedure as per
the method described by Sluiter et al.37 The total lignin
concentration is the summation of acid soluble and acid
insoluble lignin in the soluble fraction. Acid insoluble lignin
(2) (Klason) lignin concentration was measured gravimetrically by
Klason procedure in which the samples were subjected to acid
hydrolysis with 72 percent H2SO4 at a temperature of 30  C for
60 min. Then, the samples were added with 84 mL of distilled
water. The nal hydrolysis was done by subjecting the samples
to autoclaving at 121  C for 60 min. The sample blend was kept
(3) at 30  C for cooing. The hydrolyzed samples were vacuum
ltered with the help of a ltering crucible. Finally, the sample
residues were subjected to drying (105  C) and incinerating (575

C) in a muffle furnace. Then, the acid insoluble content was
measured gravimetrically. The fraction of acid soluble lignin

Table 2 Energy analysis parametersa

Parameter Unit Bac Delign-Bac Reference

Initial solids kg 1000 1000 Kannah et al.20

COD solubilization % 21.09 35.14 This study
SS reduction % 18 33 This study
Pretreatment time h 24 24 Kavitha et al.28

Pretreatment temperature C 40 40 Kavitha et al.29
Pump height m 3 3 Kannah et al.30
Pump efficiency h % 70 70 Metcalf and Eddy69
Pumping time min 10 10 This study
Flow rate m3 s
1 0.0833 0.0833 Calculated
Electricity consumption for AD stirring (3) kW m
3 0.005 0.005 Metcalf and Eddy69

Ambient temperature C 25 25 This study

Digestion temperature C 35 35 Metcalf and Eddy69
Digestion time days 15 15 This study
a
Bac-biopretreatment with cellulase secreting bacteria; Delign-Bac – delignied biopretreatment.

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content was estimated by gauging the absorbance of the
hydrolyzate at 320 nm.
The extractable lignin content from the liquid fraction was
determined calorimetrically by the modied Pearl and Benson
method (Nitrosation method).38 A 50 mL liquid fraction of the
pretreated sample (settled or ltered) was taken in a glass
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ask. 1 mL of acetic acid and 1 mL sodium nitrite solutions
were added into the ask containing the liquid sample. The
solution was mixed manually and kept at 30  C for 15 min.
Then, 2 mL of ammonium hydroxide solution was added to the
sample and mixed, and then, the sample was kept at 30  C for
10 min. The absorbance was read spectrophotometrically at
430 nm.
The concentration of cellulose and hemicellulose was
determined spectrophotometrically by the anthrone method26,39
and para-bromoaniline method.40
For cellulose determination, the samples were added with
3 mL acetic/nitric reagent in a test tube and the contents were
mixed well (cellulose is the polysaccharide made up of glucose
units and it undergoes acetolysis reaction with acetic/nitric
reagents and form cellodextrins). The test tube was placed in
a water bath for 30 min at 100  C. The samples were cooled and
then the contents were centrifuged for 15–20 min. The super-
natant was discarded. The residues were washed with distilled
water. 10 mL of 67% sulphuric acid was added to the contents
and the test tube was incubated for 60 min (the cellodextrins
was solubilized and hydrolyzed to form glucose molecules on
reacting with sulphuric acid). 1 mL of this solution was diluted
to 100 mL. To 1 mL of this diluted solution, 10 mL of anthrone
reagent was added and mixed well. Then, the tubes were boiled
for 10 min in a water bath (the glucose molecule undergoes
dehydration and form hydroxymethyl furfural and this in turn
forms a green color product when reacting with anthrone). The
contents were cooled and the absorbance was measured at
630 nm.
For hemicellulose determination, samples are added with
3% sulphuric acid in the ratio of 1 : 10. Then, the samples were
autoclaved at 121  C, 15 psi. Then, the samples were cooled and
added with distilled water to make up to 100 mL. The pH of the
sample was adjusted to 7–7.5. To 1 mL of this diluted solution,
5 mL of para-bromoaniline reagent was added and incubated
for 10 min at 70  C. (hemicellulose consists of pentose sugars
such as xylans, mannans, glucomannans, galactans and arabi-
nogalactans. The pentose sugars in the hemicellulose get con-
verted into furfural when heated with parabromoaniline and
form pink coloured compound). Then, the samples were incu-
bated for 70 min in dark at room temperature and the absor-
bance was measured at 540 nm.
Results and discussion
Delignication of rice straw via ultrasonic homogenization
Delignication involves liquefaction or exclusion of lignin and
deaggregation of microbrils from lignocellulosic biomass.
This study involves the removal of lignin from rice straw
(delignication) with an ultrasonic homogenizer and subse-
quent biopretreatment with cellulase secreting bacteria. In the
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delignication process, the effect of ultrasonic factors such as
sonication contact time, power% and specic energy on lignin
removal was studied, optimized and evaluated.
Optimization of sonication contact time and power for
delignication
Optimization of sonication contact time and power is essential
since they inuence the sternness and prevalence of cavity
generation during ultrasonic delignication of rice straw. Fig. 1
represents the inuence of sonication contact time and power
on the removal of total lignin of the solid fraction and extract-
able lignin release of the liquid fraction. From the gure, it was
clearly identied that at a sonication contact time of 5 min, the
total lignin concentration showed a decrease from 1016 mg L
1
to 648.94, 625.51, 600.59, 574.07, 545.86, 462.89, 440.77 and
417.76 mg L
1, respectively, for power % ranging from 10 to
80%. In the present study, the lignin concentration represents
20.04% of the COD, which was consistent with the study of
Ghorbani et al.25 The two crucial mechanisms that are involved
in effective delignication were increased accessibility of
cellulose via surface erosion and removal of lignin. Removal of
lignin via sonication happens through cleavage of linkages
connecting lignin with cellulose and hemicellulose.41 It has
been hypothetically assumed that the removal of lignin can be
achieved via sonochemical effects and mechanoacoustic effects.
The radicals or oxidising species generated during sonochem-
ical reactions may react with the rice straw biomass and
delignify them.41 The hydromechanical shear generated in the
medium as a result of cavitation during mechanoacoustic
reactions may enhance the mass transfer of the solid to the
liquid phase boundary.41 This eventually may cause the
mechanical forces to separate the twisting of lignin with cellu-
lose and hemicellulose of rice straw and solubilize the lignin
material.42,43 In addition, application of mechanical homoge-
nization may improve the porosity and permeability of
biomass.28 This in turn may increases the surface area contact
and enhance the accessibility of cellulose to subsequent cellu-
lase secreting bacterial pretreatment. The reduction in total
lignin concentration from the solid fraction implied that the
lignin content from the solid fraction of rice straw biomass was
removed and shied to the liquid phase and this could be
attributable to the impact of ultrasonic homogenization.
Several literature studies have reported that sonication
enhanced the removal and extraction of lignin and on the other
hand, the sonic contact time showed varying impacts on lignin
removal, and oen, extended treatment times are not reported
to be appropriate for delignication44,45 and it has been proved
in this case. When the sonic contact time was increased further
to 6 min from 5 min, no substantial increase was observed
(Fig. 1). A similar shorter contact time was suggested by other
researchers while delignifying various lignocellulosic biomass.
For instance, Garcı́a et al.44 have reported that during deligni-
cation of olive tree residues, a shorter contact time of 15 min
was found to be sufficient and it was reported that increase in
contact time in excess of 15 min decreased the efficiency of
delignication. Therefore, in this study, a lesser sonic contact
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Fig. 1 Effect of ultrasonic contact time and power on (a) total lignin reduction from solid fraction and (b) extractable lignin release in the liquid
fraction.
time of 5 min was regarded as optimal for effective
delignication.
Akin to contact time, sonic power percentage also impacts
the efficiency of delignication. On increasing the power
percentage from 10% to 80%, a drastic reduction in total lignin
concentration of the solids fraction from 648.94 to
462.89 mg L
1 at a sonic contact time of 5 min was obtained,
whereas further increase in sonic power percentage greater than
60% resulted in no signicant decrease in total lignin content.
The extractable lignin release in the liquid phase showed
a rapid increase from 0 to 533.11 mg L
1 while increasing the
power percentage from 10 to 80%. At a sonic contact time of
5 min and a power percentage of 60%, a delignication of
54.44% was obtained.
Specic energy input at different sonic power levels
Optimization of power levels and specic energy input has
a predominant role in mechanical mediated delignication.
The effect of sonic power levels and specic energy input on
delignication of lignin is depicted in Fig. 2a. This optimization
experiment was carried out with a xed biomass to water ratio of
0.005 w/v. It has been clearly seen that delignication has direct
correlation with that of the specic energy input (Fig. 2a). The
delignication trend was categorized as three patterns such as
trend 1, trend 2 and trend 3. Trend 1 symbolizes a slower
delignication trend and that was mainly attributable to lesser
power levels ranging from 0.01 kW to 0.05 kW, respectively. In
trend 1, the achieved delignication was found to be less in the
range of 36.13 to 46.27%, respectively. The specic energy range
spent to achieve these levels of delignication was calculated to
1836 | Sustainable Energy Fuels, 2021, 5, 1832–1844
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be 300 to 1500 kJ per kg TS, respectively. Trend 2 symbolizes
a peak delignication at a power level of 0.06 kW. A hike
increase in delignication of 54.4% was achieved and at the
same time, the specic energy spent to achieve this range of
delignication was calculated to be very less as 1800 kJ per kg
TS, respectively. For sonic power levels 0.01, 0.02, 0.03, 0.04 and
0.05 kW (at an optimal treatment of 5 min), the achieved
delignication (%) was observed to be 36.13%, 38.43%, 40.89%,
43.5% and 46.27%, respectively. The difference in delignica-
tion (%) between these sonic power levels was observed to be
2%. However, further increase of sonic power levels from 0.05 to
0.06 kW (at optimal treatment time of 5 min) i.e. from 46.27% to
54.4% resulted in nearly 6% difference. Therefore, trend 1 was
considered as the slower delignication trend and trend 2 was
considered as the peak delignication trend. The obtained
delignication (54.4%) was comparable with the result of Zhang
et al.46 where the authors achieved a comparable state of
delignication (53.22%) during ultrasound assisted ionic liquid
and acid pretreatment of rice straw biomass. Trend 3 represents
a mere delignication trend and that represents power levels
ranging from 0.07 kW to 0.08 kW, respectively. In trend 3,
meagre increase in delignication was noted and this was in the
range of 56.62–58.88%. However, the specic energy spent
(2100–2400 kJ per kg TS) to achieve this range of delignication
was higher when compared to trend 2. Thus, it has been proved
that increasing the sonic power level or contact time leads to
only marginal rise in delignication with tremendous wastage
of energy in delignication.
The impact of specic energy on cellulose, hemicellulose
and SCOD at optimal sonic power (0.06 kW) and sonic contact
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Fig. 2 (a) Effect of specific energy input on delignification at varying power input, (b) effect of specific energy input on cellulose and hemi-
cellulose at an optimal power input of (0.06 kW), (c) SCOD and silica removal at an optimal power input of (0.06 kW), and (d) effect of the biomass
to waster medium ratio on delignification with respect to specific energy input.
time (5 min) is represented in Fig. 2b and c. In this study, the
biomass lysis indexes employed were cellulose, hemicellulose
and SCOD. From Fig. 2b, it was evidently seen that the cellulose
and hemicellulose concentrations remain zero until a specic
energy input of 1800 kJ per kg TS specifying no biomass lysis. At
the same time, for the same specic energy input of 1800 kJ per
kg TS, a higher delignication of 54.4% was achieved, which
was evident in Fig. 2a. Zheng et al.47 and Zou et al.48 have stated
that selection of pretreatment relies upon the biomass type and
it was categorized based on its biodegradation enhancement,
minimal inhibitor formation and lesser energy consumption.
The ultrasonic pretreatment showed that it could effectively
remove lignin, increasing the porosity without cellulose degra-
dation as depicted in Fig. 2b. Enhanced delignication of rice
straw can be achieved at very less ultrasonic power and specic
energy input (450 kJ per kg TS), whereas in the case of other crop
residues, ultrasonication demands 4839 kJ per kg TS to treat
wheat straw.49 However, with further increase of specic energy
>1800 kJ per kg TS (For instance, 2160 kJ per kg TS), both the
cellulose and hemicellulose concentration began to increase
(4.6 mg L
1 and 3.3 mg L
1, respectively), specifying biomass
lysis. In the case of SCOD release, the release was lesser up to
a specic energy input of 1800 kJ per kg TS, implying partial
release of organic matter into the aqueous phase. However,
upon further increase in specic energy by 2160 kJ per kg TS,
a drastic increase in SCOD (320 mg L
1) was noted, implying
that harsh treatment conditions may disrupt the biomass
structure and promote the release of more organics into the
liquid phase. Therefore, it was conrmed that a specic energy
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input of 1800 kJ per kg TS was optimal for effective deligni-
cation without biomass lysis.
Optimization of the biomass to water ratio for delignication
In any delignication process, the biomass to water ratio is
a crucial parameter to be optimized to make the process energy
efficient (Fig. 2d). Fig. 2d clearly depicts that the delignication
percentage showed a marked increase of 70.28% up to
a biomass to water ratio of 0.02 w/v. A specic energy input of
450 kJ per kg TS was spent to obtain this state of delignication.
Enhanced delignication of rice straw can be achieved at very
less ultrasonic power and specic energy input (450 kJ per kg
TS), whereas in the case of other crop residues, ultrasonication
demands a higher specic energy of 4839 kJ per kg TS to
pretreat wheat straw.49 However, for pretreating silage, an
ultrasonic specic energy input of 407  67 kJ per kg TS was
spent to achieve effective delignication.50 Furthermore, the
delignication showed decrease on increasing the biomass to
water ratio. For example, when the biomass to water ratio was
increased to 0.025 w/v, a decrease in delignication (61.42%)
was noted. On the other hand, no considerable reduction in
specic energy input (360 kJ per kg TS) was noted. This specied
that adding a higher concentration of biomass with low water
content might upset the homogenizing and mechanoacoustic
potential of ultrasonic homogenization. Accordingly, the
surface area contact for the lignin with ultrasonic wave cavita-
tion was improved only at a lower biomass to water ratio. This
was consistent with the study of Bussemaker et al.41 Issues
related with mixing and mass transfer may also arise while
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working with high biomass content which would decrease the
extent of delignication and potential of enzymes production
during cellulase secreting bacterial pretreatment. At the same
time, limited availability of carbon sources in the medium is an
additional important issue for the start of the enzyme produc-
tion by bacteria in subsequent bacterial pretreatment.25 Still,
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Therefore, in the present study, ultrasonication was chosen as
the best pretreatment to remove the higher accumulation of
silica in rice straw than other crop residues such as wheat straw
and barley straw. Similar to delignication, a higher desilica-
tion (silica removal) of 73.2% was achieved at a biomass to
liquid ratio of 0.02 (w/v) (Fig. 2d). Hence, further experimenta-

starvation of carbon sources can aid in the earlier production of tion was proceeded with a biomass to water ratio of 0.02 (w/v).
cellulase enzymes into the medium and their subsequent
cellulose hydrolysis;25 less content of the carbon source can Delignication induced biopretreatment
deter the bacterial growth and decrease their enzyme produc-
Biopretreatment was performed with cellulase secreting bacte-
tion, which revealed that there must be an optimal biomass to
rial strain, Bacillus sp. Several literature studies have reported
water ratio value for the carbon source content to enhance
on the production of cellulase enzyme by this bacterial
subsequent bacterial pretreatment.25 In addition, it has been
strain.54,55 As cellulose and hemicellulose are the principal
reported that the very high biomass to liquid ratio reduces the
compounds of rice straw biomass, cellulase secreting bio-
specic energy input51 but it may increase the medium viscosity
pretreatment was preferred in order to disintegrate the cellu-
hindering effective mixing and mass transfer. At the same time,
losic rich biomass. The application of biobased bacterial
a very low biomass to water ratio increases the water usage and
pretreatment substitutes the necessity on the commercial
resulted in high specic energy input. This may affect the
enzyme and decreases the total cost of process. The efficiencies
economic feasibility of the process. Therefore, it is essential to
of biopretreatment of delignied rice straw were evaluated by
select an optimal biomass to water ratio in order to sustain an
assessing the SCOD, cellulose and hemicellulose released from
equilibrium between the specic energy input and ultra-
Delign-BAC (delignication with sonication followed by cellu-
sonication performance.52 Therefore, from an economic point
lase secreting bacterial pretreatment), BAC (bacterial pretreat-
of view and delignication extent, it can be concluded that
ment alone) and control (untreated) samples.
a biomass to water ratio of 0.02 w/v was considered to be
optimal for effective delignication and for subsequent bacte-
rial pretreatment. In addition to lignin, silica, the cuticular SCOD release and COD solubilization
layer, also hampers the biodegradation of rice straw as indi- The outcome of SCOD release and COD solubilization of Delign-
cated in the Introduction section. Ultrasonic homogenizers are BAC, BAC and control samples achieved during biopretreat-
considered as the more efficient pretreatment process for ment is depicted in Fig. 3a and b. A clear existence of two trends
dispersing silica than other high-shear homogenizers.53 was seen for SCOD release in Fig. 3a. An increase phase (from

Fig. 3 Effect of cellulase secreting bacterial pretreatment, (a) SCOD release of rice straw biomass, (b) COD solubilization (control-untreated,
BAC-biopretreatment alone with cellulase secreting bacteria, Delign-BAC – delignification followed by cellulase secreting biopretreatment) and
(c) cellulase enzyme activity.

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0 to 24 h) and a decline phase (30 to 72 h) were observed. The
SCOD release increased from a value of 0 to 629.5 mg L
1,
4201.1 mg L
1 and 6999.9 mg L
1 during the increase phase in
control, BAC and Delign-BAC samples. This kind of rise in
SCOD release obtained in the present study was comparable
with the outcome of Shetty et al.56 where they achieved a similar
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peak increase in SCOD release from alkali pretreated rice straw
biomass. The solubilizing efficiency of biopretreatment was
considerably raised because of the cellulolytic potential of the
bacterial strain to hydrolyze the rice straw biomass and its
components. 0.5 g L
1 cellulase secreting bacteria of (dry basis)
was added at the start of the experiment. During the course of
pretreatment, the cells increased with increase in dry cell weight
and at 24 h, it reaches a dry cell weight of 1.2 g L
1 (dry basis). In
addition, when compared to BAC and control samples, Delign-
BAC showed higher SCOD release due to delignication and
this contributed to the increase in SCOD release. In addition,
a trivial increase in SCOD release was noted in the control
implying the impact of homogenization (i.e. the stirring
provided for mixing the samples). In the decline phase, the
SCOD release in Delign-BAC and BAC samples reduced appar-
ently owing to the consumption of released sugars by the
inoculated cellulase secreting bacteria. An akin effect was also
suggested by several researchers in bacterial pretreatment of
Fig. 4 (a) Effect of cellulase secreting bacterial pretreatment on soluble components of rice straw biomass and (b) statistical analysis of soluble
components of rice straw biomass (control-untreated, BAC-biopretreatment alone with cellulase secreting bacteria, Delign-BAC – delignifi-
cation followed by cellulase secreting bacterial pretreatment).
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different organic biomass.57,58 The respective COD solubiliza-
tion during the increase phase at 24 h peak pretreatment time
was found to be 3.2%, 21% and 35% for the control, BAC and
Delign-BAC, respectively, as shown in Fig. 3b. The values of
SCOD obtained in Delign-BAC showed a marked difference
when compared to BAC and the values are calculated to be
statistically signicant with p values of 0.0002. During bio-
pretreatment, the cellulase enzyme activity increased with
increase in pretreatment time up to 24 h (Fig. 3c). At 24 h, the
enzyme activity in BAC and Delign-BAC was observed to be 0.4
and 0.6 U mL
1. When compared to BAC, Delign-BAC showed
higher enzyme activity and this could be due to the effect of
delignication prior to cellulase secreting bacterial pretreat-
ment. Aer 24 h, the activity of enzyme in both the samples
reaches stabilization owing to cellulose hydrolysis (Fig. 3c and
4c) and reduction in availability of carbon sources, as shown by
Ghorbani et al.25 Even though carbon starvation stimulates the
production of enzymes and their subsequent cellulose hydro-
lysis, lesser content of carbon sources may hamper the bacterial
growth and decrease their enzyme production which implies
that there must be an optimal carbon source content. In addi-
tion, aer 24 h, the hydrolysis of cellulose to reducing sugars
reduces the availability of cellulose content for enhanced bio-
fuel production as observed by Ghorbani et al.25 Based on the
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results mentioned above, it has been concluded that 24 h
pretreatment time was found to be optimal for cellulase enzyme
production and delignication was essential prior to bacterial
pretreatment for enhanced enzyme activity.
Cellulose and hemicellulose release
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the distribution of cellulose and hemicellulose values obtained
from the control, BAC and Delign-BAC samples, box plot
interpretation (to prove statistically) has been done (Fig. 4b). It
is assumed that in control samples, 25% of the cellulose and
hemicellulose data (lower quartile) were less than 41.58 mg L
1
and 30.03 mg L
1 respectively. Similarly, 75% of the cellulose

and hemicellulose data (upper quartile) were less than

During biopretreatment, the changes in cellulose and hemi-
139.10 mg L
1 and 100.46 mg L
1 respectively. For BAC, 25% of
cellulose concentration were essential to be noted as they are
the cellulose and hemicellulose data (lower quartile) were less
the major components present in the rice straw biomass.
than 265.32 mg L
1 and 191.62 mg L
1 respectively, whereas
Similar to SCOD release, the cellulose and hemicellulose also
75% of the cellulose and hemicellulose data (upper quartile)
showed a marked increase up to a pretreatment time of 24 h
were less than 931.54 mg L
1 and 672.78 mg L
1 respectively.
(Fig. 4a). The cellulose and hemicellulose concentrations
Regarding the Delign-BAC sample, 25% of the cellulose and
during 24 h of pretreatment were observed to be 226.62,
hemicellulose data (lower quartile) were less than
1512.39, 2519.96 and 163.67, 1092.28, 1819.97 mg L
1, respec-
441.76 mg L
1 and 319.04 mg L
1 respectively, whereas 75% of
tively, for the control, BAC and Delign-BAC samples. The
the cellulose and hemicellulose data (upper quartile) were less
cellulase secreting biopretreatment targeted deconstruction of
than 1550.41 mg L
1 and 1119.74 mg L
1 respectively. Fig. 4b
the lignocellulosic structure of rice straw biomass via cellulase
clearly depicts the absence of outliers since the data of the plot
mediated action. Aer 24 h, the solubilized or released cellulose
lies within the whiskers of the box plot. Based on the shape of
and hemicellulose into the liquid phase got decreased, implying
the box plot, the skewness of the data is analyzed. The shape of
that the solubilized cellulosic materials were hydrolyzed to
all the box plots appeared to be symmetrical, specifying that the
reducing sugars by the cellulolytic activity of inoculated bacte-
data of cellulose and hemicellulose for the control, BAC and
rial strain. This observation was noted by Tsegaye et al.54 during
Delign-BAC samples followed a normal type distribution. Thus,
biopretreatment of rice straw biomass. To verify and nd out

Fig. 5 Effect of cellulase secreting bacterial pretreatment on specific methane production of (a) control, (b) BAC-biopretreatment alone with
cellulase secreting bacteria and (c) Delign-BAC – delignification followed by cellulase secreting bacterial pretreatment. (d) Effect of lignin on
methane production.

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statistically, it was evident that the greater sample value indi-
cates the optimal point. Besides, the data sets were not skewed
and it showed normal type distribution.
Specic methane generation
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Fig. 5a–c represents the specic methane generation of the
control, BAC and Delign-BAC rice straw samples. The inoculum
used in these experiments took some time to get acclimatized
and therefore this was the reason behind the lower amount of
methane generation during the beginning of experiments.59 On
the 5th day of biodegradation, the obtained specic methane
generation in the control, BAC and Delign-BAC samples was
7.5 mL per g VS, 50.5 mL per g VS and 84.3 mL g
1 VS respec-
tively. Even though the methane production was very low at start
of the experiments, it began to augment with increase in diges-
tion time and achieved a maximum value on the 15th day of
biodegradation. The specic methane generated from the
control, BAC and Delign-BAC samples was found to be 14.8 mL
per g VS, 99.35 mL per g VS and 165.5 mL per g VS respectively.
The results were comparable with the results of Amnuaycheewa
et al.60 where the authors achieved 148 mL per g VS of biogas from
hydrochloric acid pretreated rice straw. Teghammar et al.61 have
achieved 157 mL per g raw material of methane from rice straw
pretreated with N-methylmorpholine-N-oxide prior to bio-
methanation. On the other hand, when the cumulative methane
generation values of BAC and Delign-BAC are compared,
evidently, Delign-BAC showed nearly 66.6% percentage increase
in methane generation when compared to BAC. This state of
percentage increase was found to be higher than in the study of
Chen et al.16 where the authors have stated that extruded rice
straw biomass showed an increased methane yield of nearly 32%
in comparison with milled rice straw biomass. Likewise, Bauer
et al.58 and Zhao et al.62 have achieved a methane yield increase
over 20% and 35% from steam exploded and mild acid pretreated
rice straw. The variation in percentage increase in methane of
present study when compared to other researchers lies in the fact
that it could be owing to different pretreatments and varying
operational conditions. The greater accessibility of cellulose to
methanogens was the reason behind the greater methane yield in
Delign-BAC in comparison to BAC. Overall, the control sample
showed lesser methane production due to lower availability of
the substrate without any treatment. The values of specic
methane generation were well tted with the logistic model (eqn
(1)) and estimated kinetic parameters are tabulated within
Fig. 5a–c. The derived kinetic parameters imply that a higher
specic methane production potential and rate constant of
167.04  1.58 mL per g VS and 0.5243  0.02 mL per g VS per day
was obtained for Delign-BAC when compared to BAC (100.25 
0.95 mL per g VS and 0.4094  0.02 mL per g VS per day) and the
control (14.88  0.14 mL per g VS and 0.302  0.012 mL per g VS
per day) respectively. The model has a reasonable t with the
obtained data values with a correlation coefficient which lies
within (0.95–0.99) for all the three samples.
Fig. 5d shows the impact of lignin inhibition towards
cumulative methane production for the control, BAC and
Delign-BAC samples and the experimental and theoretical
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methane production of the control, BAC and Delign-BAC
samples. The experimental methane values were very closer to
the theoretical values calculated for all the three samples.
Experimental values are 65–85% of the theoretical methane
yield. Koyama et al.63 reported that a concentration of lignin of
more than 1 g L
1 will inhibit both hydrolysis and methano-
genesis. As it was seen in Fig. 5d, the remaining lignin present
in the control, BAC and Delign-BAC rice straw samples is
observed to be 1.095 g L
1, 1.06 g L
1 and 0.3 g L
1 respectively.
Thus, it was clearly proved that in Delign-BAC, most of the
lignin was removed from rice straw samples, enhancing the
methane production. This does not inhibit methane produc-
tion, whereas in BAC and control, the lignin concentration was
found to be in excess of 1 g L
1, implying inhibition of
methanogenesis.
Mass, energy balance and economic analysis
Fig. 6 presents the outcome obtained from the mass balance
analysis using 1000 kg of rice straw (1 ton) for BAC and Delign-
BAC samples. In Delign-BAC and BAC samples, 1000 kg of solids
was decreased to 670 kg and 820 kg owing to 33% and 18%
solids reduction during pretreatment. A tremendous amount of
solids were reduced in Delign-BAC when compared to BAC
alone. This was due to the enhanced delignication which
paved the way for effective bacterial pretreatment. The solids
were reduced further to 402 kg and 574 kg in Delign-BAC and
BAC, respectively, during AD. The enhanced biomass liquefac-
tion obtained by ultrasonic assisted biopretreatment resulted in
40% solids reduction in Delign-BAC during AD respectively.
When compared to Delign-BAC, BAC achieved lesser solid
reduction of around 30% during AD. Therefore, the nal solids
reduction was found to be 598 kg and 426 kg in Delign-BAC and
BAC samples, respectively. The solids that remained to be
disposed in landlls from Delign-BAC and BAC were calculated
to be 402 and 574 kg respectively.
In the case of energy balance analysis, the energy input
should be compensated for by the energy output to achieve the
net energy yield.64 The input energy covers the energy consumed
for ultrasonic homogenization (for delignication alone),
bacterial pretreatment stirring energy, bacterial culturing
energy, rise of temperature, anaerobic digestion stirring energy,
heat loss and pumping energy. The total input energies in
Delign-BAC and BAC were calculated as 384.39 kW h per ton and
259.39 kW h per ton, respectively. The slight rise of input energy
in Delign-BAC was attributable to energy spent for delignica-
tion (i.e. the energy spent for ultrasonic homogenization). The
two benets associated with pretreatments were as follows:
output energy was obtained as methane and reduction in solids
to be disposed. The output energy recovered as methane for
Delign-BAC and BAC was estimated to be 879.06 and 339.95
kW h respectively. The obtained net energy of Delign-BAC and
BAC was calculated to be 494.67 kW h per ton and 80.557 kW h
per ton respectively. The net energy of the present study was
found to be comparatively lesser than in the study of Nguyen
et al.65 where the authors achieved a relatively higher net energy
of
3744 MJ per mg rice straw (equivalent to 938.61 kW h per
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Fig. 6 Mass, energy and economic balance analysis of DeligBAC (delignification followed by cellulase secreting bacterial pretreatment) and
BAC-(biopretreatment alone with cellulase secreting bacteria).
ton). This variation could be due to the mode of pretreatment,
composition of biomass etc. From the above mentioned results,
it has been conrmed that a great deal of energy was saved by
Delign-BAC. Even though Delign-BAC demands some amount
of extra energy for delignication, the output energy obtained as
methane compensated for it. This revealed that delignication
prior to bacterial pretreatment enhanced the solubilization of
cellulosic biomass and provides an energy efficient bio-
methanation on a large-scale extent.
Likewise, the outcome of economic analysis exposed that the
total input cost for Delign-BAC and BAC was
301.69 USD per ton
and
320.38 USD per ton respectively. Vasco-Correa and Shah66
investigated the economic viability of biological pretreatment (by
fungi) for four lignocellulosic biomass, namely—perennial
grasses, corn stover, agricultural residues and hardwood—and
they estimated an input cost of 1.7, 1.6, 2.0, and 2.8 USD per kg
(equivalent to 1700, 1600, 2000 and 2800 USD per ton) for
perennial grasses, corn stover, agricultural residues and hard-
wood. This was observed to be relatively higher than the cost
spent in the present study. Similarly, in another study, Baral and
Shah67 evaluated the economic viability of steam explosion, dilute
sulfuric acid, ammonia ber explosion, and enzymatic pretreat-
ments of corn stover. The outcome implied that the input costs
for steam explosion, dilute sulfuric acid, ammonia ber explo-
sion, and biological (enzymatic hydrolysis) pretreatments were
calculated to be 0.43, 0.42, 0.65, and 1.41 (USD per kg) (430, 420,
650 and 1410 USD per ton). This was observed to be compara-
tively higher than the input cost of the present study.
The total output cost of Delign-BAC and BAC was calculated
to be 369.62 USD per ton and 197.47 USD per ton respectively.
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The net prot achieved in Delign-BAC and BAC was and

22.912 USD per ton respectively. Although the cost spent
towards delignication was added in Delign-BAC, it did not
cause any loss in the net prot of Delign-BAC. The greater net
prot (achieved as increased methane energy and reduction in
solids disposal) balanced the cost spent towards delignication
in Delign-BAC.
The cost benet ratio is an essential factor that assesses the
economic viability of pretreatment. A cost benet ratio of more
than 1 designates net prot. The cost benet ratio of Delign-
BAC achieved in this study was calculated to be 1.23, repre-
senting net prot when compared to BAC (0.62). On the basis of
the above results, Delign-BAC was regarded as the viable
process for large scale applications. It has been reported in the
literature that nearly 731 million tons of rice straw was
produced globally whereas 28.7% of its production was covered
by India.68 Therefore, the annual production of rice straw in
India will be nearly 209.797 million tons. Methane production
of approximately 173 and 50 billion m3 was achieved globally,
and in India, a considerable amount of rice straw was utilized.
The calculated methane corresponds to a net annual energy of
21.35 Mtoe in India. It covers nearly 8–10% of the energy need of
India annually.
Conclusion
To increase the accessibility of cellulose from rice straw
biomass for biopretreatment, delignication (through ultra-
sonic homogenization) is regarded as an imperative process to
obtain energy efficient biomethane production. A higher lignin
This journal is © The Royal Society of Chemistry 2021

Paper
removal (70.28%) was obtained at an optimal sonic energy input
of 450 kJ per kg TS. The delignication by ultrasonic homoge-
nization paved the way for enhanced pretreatment of rice straw
biomass with a higher COD solubilization of 35% when
compared to biopretreatment alone (21%). The scalable energy
and economic assessment showed that delignication before
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biopretreatment saved enormous energy with a cost benet
ratio of 1.23.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work is supported by the Department of Biotechnology,
India, under its initiative Mission innovation Challenge Scheme
(IC4). The grant from the project entitled “A novel integrated
biorenery for conversion of lignocellulosic agro waste into
value added products and bioenergy (BT/PR31054/PBD/26/763/
2019) is utilized for this study.
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