Energy Conversion and Management 207 (2020) 112504

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Energy Conversion and Management

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An innovative approach of mixed enzymatic venture for 2G ethanol T

production from lignocellulosic feedstock
⁎
Rajiv Chandra Rajaka, Rintu Banerjeeb,
a
Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur, Kharagpur 721302, West Bengal, India
b
Agricultural & Food Engineering Department, Indian Institute of Technology, Kharagpur, Kharagpur 721302, West Bengal, India

A R T I C LE I N FO A B S T R A C T

Keywords: Time is an important factor for bioenergy and bio-product producing industries. Longer process/fermentation
Crystalline times are the major hurdles in making the process more cost effective for any desired product. Integration of
Energy different process units were usually carried out to minimize the entire processing time. However, apart from
Enzyme consolidation, microbial adaptation and biomass pretreatment methods plays an important role during product
Ethanol
generation. Microorganisms normally take longer time to adapt to the surrounding environment, which certainly
Porosity
affect the entire processing time. Thus, the present work was framed to use mixture of crude and low titre
enzymes for lignin degradation and holocellulose hydrolysis followed by fermentation in a single unit to produce
second generation ethanol from Kans grass. In the present study, an enzymatic venture was attempted to de-
lignify the recalcitrant lignin molecule and simultaneously hydrolyse the biomass for enhanced sugars genera-
tion followed by ethanol production in a common platform. The entire ethanol production process has been
optimized through response surface methodology that resulted in 59.96 g/L of ethanol within 24 h. The biomass
porosity analysis in terms of surface area, pore size and pore volume of the fermented biomass were found to be
decreased that indicates effective action of applied enzymes. Structural characterisation of the fermented bio-
mass showed an extensive surface distortion that revealed the combined action of delignifying and saccharifying
enzymes. X-ray diffraction analysis indicates reduced biomass crystallinity after fermentation, which inferred
that para-crystalline and crystalline cellulose were utilized maximally for ethanol production. Thus, the results
obtained in the present study substantiate the feasibility of the mixed enzymes application in bioprocessing of
Kans grass for second generation ethanol production.

1. Introduction lignocellulosic ethanol generation. Integration or consolidation aims to

The art of technological conversion of forest produce, agricultural
residues and grasses are significantly advancing for biofuels production
[1,2]. Interest has now been directed towards the utilization of second
generation or lignocellulosic biomass for products generation con-
sidering the fact of food, fodder and fuel issue. Second generation
biofuels provide an alternative route to minimize the energy de-
pendency and environmental issues which arise due to reliance on fossil
based fuels [3]. Recent progress on energy sector illustrates a shifting
trend from food crops to lignocellulosic biomass. For second generation
ethanol production, various biomass processing technologies being
implemented namely, separate hydrolysis and fermentation (SHF), si-
multaneous saccharification and fermentation (SSF) and consolidated
bioprocessing (CBP). Delignification, saccharification and fermentation
technologies have been developed in the past few decades for
optimize these single processes on the system level to improve the
techno-economic of the whole process thereby making bioethanol
competitive with the gasoline or petroleum fuels [4]. Generally, pro-
duction of ethanol through SHF was limited due to saccharifying en-
zyme inhibition by the released reducing sugars [5]. Thus, integration
of two processes i.e., simultaneous saccharification and fermentation in
a single vessel (SSF) not only minimizes the cost but at the same time
reduces the enzyme inhibition by simultaneous conversion of released
sugars to ethanol [6]. Though, SSF is the widely used fermentation
mode for bioethanol production, but it suffers with the limitation of
optimum working temperature mis-match of saccharifying enzymes
with the yeast [7].
Consolidated bioprocessing (CBP) is an alternative to the above
ethanol production processes (SHF and SSF), where enzyme production,
biomass pretreatment, polysaccharides hydrolysis, and fermentation of

⁎
Corresponding author.
E-mail address: rb@iitkgp.ac.in (R. Banerjee).

https://doi.org/10.1016/j.enconman.2020.112504
Received 2 November 2019; Received in revised form 11 January 2020; Accepted 13 January 2020
0196-8904/ © 2020 Elsevier Ltd. All rights reserved.
R.C. Rajak and R. Banerjee
the release sugars were accomplished in a single platform or vessel. CBP
holds a good potential for low cost biofuels production because of
simpler biomass processing, reduced energy inputs, and high conver-
sion efficiencies compared to SHF processes [8]. CBP is usually per-
formed through either monoculture or co-culture of microorganisms.
However, increased ethanol yield has been reported by co-cultures of
microorganisms due to the high metabolic capacity of the microbes to
utilize both cellulose and hemicellulose [9]. Lignin degradation or re-
moval from lignocellulosics is usually negligible during consolidated
bioprocessing [10]. Also, biological lignin degradation through en-
zymes such as laccase, manganese peroxidase, lignin peroxidase, and
versatile peroxidase are considered as a promising technique due to
mild process conditions, no inhibitor formation, less energy require-
ment and eco-friendly in nature [11–13]. The major limitations of
biological pretreatment are long incubation periods and low efficiency,
when carried out with whole cell microorganisms [14,15]. Other lignin
removal or degradation modes such as organosolv, and thermochemical
or ionic liquids (ILs) are effective in lignin solubilizing and decrys-
tallizing cellulose. Though, these methods have many advantages, but
suffers from several limitations namely, recovery and recycling of sol-
vents and ILs, requirement of high temperatures and pressures during
thermochemical pretreatment, inhibitor formation, and high cost of ILs
[16].
Acid or alkali pretreated lignocellulosics were generally employed
for ethanol production through consolidated biomass processing that
resulted in moderate ethanol yield [10,17,18]. Furthermore, prolonged
pretreatment time (more than 20 days) was observed upon treating
biomass directly with the microbes followed by fermentable sugars and
ethanol production [19,20]. In addition, genetically engineered mi-
croorganisms were also employed for their potential in ethanol pro-
duction. Metabolically engineered thermophile Caldicellulosiruptor bescii
was used for direct conversion of switchgrass to ethanol without any
biomass pretreatment or lignin removal [21]. Consolidated biomass
processing without lignin removal or degradation is regarded as second
generation CBP but it suffers with the limitations of moderate ethanol
yield [21,22]. Currently, a new CBP based strategy termed as con-
solidated bio-saccharification (CBS) is employed for lignocellulose so-
lubilization that separates fermentation from the integrated CBP pro-
cess and combines hydrolytic enzyme production and saccharification
in one step. Under the optimum process conditions, CBS of wheat straw
was resulted in enhanced sugar yield. However, CBS usually takes long
reaction time to record high sugar yield and saccharification efficiency
[23].
CBP systems always preferred a novel microbial consortia that have
the capability to carry out optimum substrate conversion and sub-
sequent product formation [10]. Although, CBP is a promising tech-
nology for biofuels production but certain limitations are still exist such
as, existence of recalcitrant lignin in the biomass, long fermentation
time, low yield of targeted product due to by-product formation (e.g.
organic acids), low hydrolysis rate, and microorganisms sensitivity to
produced alcohols [24]. Such type of limitations need a significant re-
search on identifying the potent wild microorganisms or enzymes for
lignin and holocellulose degradation, and enhancing the robustness of
microorganisms through recombinant approaches on solventogenic or
cellulolytic organisms [25,26]. In this perspective, an approach of
mixed enzyme system was used to combine the process of delignifica-
tion, and hydrolysis for simultaneously lignin degradation and fer-
mentable sugars generation followed by fermentation in a common
platform. Both the enzymes were produced separately and subsequently
used for biomass delignification and hydrolysis. Since, biological and
enzymatic modes of biomass pretreatment and hydrolysis does not
generate any by-products such as organic acids or fermentation in-
hibitors, the yield of targeted product is expected to be high [27,28].
Laccase was used for lignin degradation, while cellulase-xylanase
cocktail for cellulose and hemicellulose hydrolysis followed by fer-
mentation using yeast in a single vessel. The mode of laccase action
2
Energy Conversion and Management 207 (2020) 112504
consists of oxidation and reduction reactions. The catalytic core of the
laccase consists of four copper atoms. Laccase mainly attacks the phe-
nolic units of lignin that led to the oxidation of Cα, Cα-Cβ and aryl-alkyl
linkages. The mode of laccase action is primarily consists of three steps;
type 1 copper reduction by the reducing substrate followed by internal
electron transfer from type 1 to type 2 and type 3 copper atoms and
finally formation of water via reduction of molecular oxygen at the
trinuclear site [29].
In addition, the present work deals with the selection of different
influential process parameters for ethanol production and their opti-
mization through response surface methodology (RSM) based on cen-
tral composite design (CCD). Energy density, porosity and structural
characterization of the raw and fermented biomass were carried out to
validate the action of enzymes and microbes used during the entire
process of second generation (2G) ethanol production.
2. Materials and methods
2.1. Substrate
The wasteland weed, Saccharum spontaneum (Kans or Sarkanda),
was obtained from campus area of the Indian Institute of Technology
Kharagpur, India. The whole plant, including leaf sheaths and stems
were cut into small pieces by chopper. The small pieces are further sun
dried and pulverized (Pulverizer, Murhopye Scientific Company,
Mysore, India) to a particle size of approximately 0.2 mm and was used
for further studies.
2.2. Biochemical characterization of Kans grass
Cellulose content of the raw biomass was estimated by “semi-micro
determination of cellulose” method whereas; hemicellulose was esti-
mated by anthrone method [30,31]. Lignin content was estimated using
the titrimetric method [32]. Klason lignin was determined according to
the sulphuric acid hydrolysis method [33]. Moisture, total solid, vola-
tile and ash content of the raw biomass were determined according to
the AOAC (Association of Official Analytical Chemists) protocols [34].
2.3. Enzymes and microorganism
Laccase for lignin degradation was produced from Lentinus squar-
rosulus MR13 using rice straw as a substrate through solid state fer-
mentation [35]. Cellulase-xylanase cocktail for cellulose and hemi-
cellulose hydrolysis was obtained from Trichoderma reesei Rut C30 using
wheat bran as a substrate through solid state fermentation [36]. One
unit of laccase activity was defined as the quantity of enzyme required
to oxidize 1 µmole of substrate per minute under the assay conditions
whereas, cellulase-xylanase activity defined as the amount of enzyme to
produce 1 μmol of reducing sugars per minute under the assay condi-
tions. The crude laccase activity was recorded to be 21,000 IU/g dry
substrate and for cellulase-xylanase, 120 and 1040 IU/g dry substrate,
respectively. The fermenting microorganism, Saccharomyces cerevisiae
was used for bioethanol production in the present study. S. cerevisiae
was maintained in a medium consisting of glucose (2%, w/v) and yeast
extract (0.5%, w/v), pH 6.5 and incubated at 37 °C. S. cerevisiae sus-
pension culture of 24 h (2.5 × 108 cells/ml) was inoculated into the
reaction mixture for fermentation.
2.4. Methodology for bio-processing of Kans grass for ethanol production
Bioprocessing Kans grass includes all the three steps namely, de-
lignification, saccharification and fermentation in a single flask. In a
conical flask, Kans grass, laccase, cellulase-xylanase concoction and
yeast were added simultaneously and covered with parafilm for main-
taining facultative anaerobic environment for yeast. The flasks were
then incubated for various time intervals at 37 °C. Aliquot of samples
R.C. Rajak and R. Banerjee
were withdrawn at regular time interval and solid was separated from
the liquid through cheesecloth. The solid biomass was dried for residual
lignin estimation and the liquid was centrifuged at 2000 rpm for 5 min.
The resultant liquid was used for ethanol estimation. All the trials were
carried out in triplicates to calculate the mean values and standard
deviation.
2.5. Ethanol estimation
Ethanol content (%, v/v) was estimated by potassium dichromate
method [37]. Ethanol concentration (g/L), productivity rate (g/L/h),
conversion efficiency (%) and yield of the process has been calculated
by the standard methods [38]. The following equations were used to
calculate the conversion efficiency and yield of the process are given
below:
Experimental Ethanol(g)
Ethanol Conversion Efficiency(%) = × 100
Theoritical Ethanol(g)
Experimental Ethanol(g/L)
Ethanol Yield(g/g) =
Solid Loading(g/L)
2.6. Statistical model and optimization
Optimization of bioprocessing of raw Kans grass was performed by
considering five factors at three levels (−1, 0, +1) that constitutes 25
full factorial Central Composite Design (CCD) followed by analysis and
point prediction using RSM. Five different influential process para-
meters namely, solid loading, incubation time, temperature, inoculum
volume and laccase to cellulase ratio were selected to study their effect
on ethanol production utilizing raw Kans grass. The boundary levels
(−1, 0, +1) of each parameter for bioprocessing of raw Kans grass was
selected based on one variable at a time approach such as, solid loading
(15–25%, w/v), incubation time (12–36 h), temperature (35–40 °C),
inoculum volume (8–12%, v/v) and laccase to cellulase ratio (1:4–1:8).
The activity of laccase (3125 IU/g), and cellulase-xylanase (75 IU/g and
650 IU/g) were maintained throughout the experiments and are re-
ported to be optimum for lignin degradation and holocellulose hydro-
lysis from our previous reports [7,39] (Figs. 1–5). The experimental
designs were carried out in triplicates, and the design of experiment for
bioprocessing was shown in table (Table 1). The data obtained was
further analysed by second order polynomial regression equation (Eq.
(3)).
Y= β0 + ∑ βi Xi+ ∑ βii X 2i + 4 ∑ β kijXiXj
i= 1 i= 1 j=i+1
Fig. 1. Effect of laccase: cellulase on bioprocessing of Kans grass.
Energy Conversion and Management 207 (2020) 112504
Where, Y indicates the predicted response (ethanol %, v/v) whereas,
βm0, βmi, βmii and βmij stands for coefficients and Xi and Xj represents
independent coded variables that affects the response variable Y.
Minitab 16 software was employed for analysis of the experimental data
followed by analysis of variance (ANOVA) and to study the regression
equation. Accordingly, the above given fitted regression equation may
be further interpreted in the form of response surface plots to visualize
the correlations and interactions between coded experimental and
predicted variables to conclude the optimized process conditions.
Coefficient of determination (R2) was used to evaluate the desirability
and accuracy of the fitted polynomial regression equation. F-value was
employed to check the significance of the regression coefficient.
2.7. Energy density of the raw and fermented biomass
The energy density of the samples was calculated according to the
protocol described in ASTM (American Society for Testing and
Materials) manual [40]. The solid biomass recovered after fermentation
(1) was calculated on wet weight basis. Energy density of the biomass was
measured by using standard bomb calorimeter (Oxygen Bomb Calori-
meter, Eastern Instruments, Kolkata, India). The solid biomass was
(2) dried overnight at 60 °C to remove the moisture content and com-
pressed to form pellets. The weight of the biomass was taken before
being compressed to pellet. In case of fermented broth sample, a fixed
weight of broth along with crucible was taken and kept in a calorimeter
for energy density measurement. The energy density of the biomass and
broth sample was determined based on the temperature difference in
the presence of oxygen with excess pressure of 400 psi.
2.8. Porosity analysis of raw and fermented biomass
Biomass porosity of the samples were analysed through
Quantachrome® ASiQwin™ pore and surface area analyser. The biomass
samples were kept in the degassing module and then vacuum degassed
for 3 h at room temperature prior to analysis. The degassed biomass
samples were then kept in the multipoint Brunauer–Emmett–Teller
(BET) analyser for analysis. Surface area of the pores was calculated
according to the BET method [41]. Pore diameter and volume were
determined based on the Barrett-Joyner-Halenda (BJH) method [42].
2.9. Structural characterization of raw and fermented biomass
2.9.1. Scanning electron microscopy (SEM)
The alteration in structural architecture of the fermented biomass in
comparison to raw substrate was studied by mounting dried samples on
metallic stub with an adhesive coated with gold and viewed under JEOL
(3) JSM 5800 SEM (Jeol Ltd., Tokyo, Japan).
3
R.C. Rajak and R. Banerjee
Fig. 2. Effect of solid loading on bioprocessing of Kans grass.
2.9.2. X-ray diffraction (XRD)
The alteration in degree of crystallinity of the biomass before and
after fermentation was determined through XRD using CoKα radiation
(α = 0.179 nm) at 20 mA and 40 kV. The samples were scanned in the
range of 2θ = 10° to 70° with a speed of 3°/min and the percentage
crystallinity was calculated from [(I002 − Iam)/I002] × 100, where
I002 = maximum peak intensity (2θ) between 22° and 23° and
Iam = minimum peak intensity (2θ) between 18° and 19° for cellulose I
(% crystallinity) [43].
3. Results and discussion
3.1. Biochemical characterization of raw Kans grass
Biomass composition is the key deciding factor from which one can
select or decide the feedstock for biofuels production. The composi-
tional analysis of Kans grass in the current work showed high cellulose
(38.70% ± 0.30, w/w), hemicellulose (29.00% ± 0.26, w/w) along
with moisture (4.95% ± 0.06, w/w) and ash (3.00% ± 0.22, w/w),
respectively. From the compositional analysis it can be deciphered that
an efficient delignification process was indeed essential to degrade the
high lignin content (17.46% ± 0.34, w/w) in Kans grass to utilize
cellulose and hemicellulose further. The sulphuric acid hydrolysis
method of lignin resulted in Klason lignin content of 18.20% ± 0.35
(w/w) of Kans grass. The variation observed in the lignin content of
Kans grass might be due to the sensitiveness shown by different ana-
lytical methods towards a specific phenolic compound. Cellulose,
hemicellulose, and lignin content were found to be higher compared to
date palm fronds, agricultural residues such as prairie grasses and corn
stover [44,45]. Further, the reported monosaccharide profile that in-
cludes glucan (36.81%, w/w), xylan (21.53%, w/w), galactan (0.72%,
Fig. 3. Effect of incubation time on bioprocessing of Kans grass.
4
Energy Conversion and Management 207 (2020) 112504
w/w), arabinan (2.16%, w/w), mannan (0.16%, w/w) and rhamnan
(0.14%, w/w) of raw Kans grass reflects their potential as a viable
lignocellulosic feedstock for biochemical and biofuels generation [46].
The total and volatile solid content of the raw Kans grass were found to
be 95.05% (w/w of wet weight) and 97% (w/w of dry weight), re-
spectively. Thus, due to relatively high content of holocellulose (cel-
lulose and hemicellulose) and volatile solids, Kans grass can be con-
sidered as a futuristic energy feedstock for bioprocessing that is
presently unutilized.
3.2. Selection of process parameter for bioprocessing of Kans grass
3.2.1. Effect of laccase: cellulase ratio
The main objective of process integration is to combine all the in-
dividual units in a common platform that subsequently reduce the
number of steps and total process time for biofuel and bio-products
generation. Maintenance of proper solid to liquid ratio or proportions of
different enzymes play an important role to achieve the goal of process
integration or consolidation. In the present work, laccase to cellulase
ratio was maintained at different proportions (1:1, 1:2, 1:4, 1:6, 1:8,
and 1:10). The other factors which are fixed during the experiment
were solid loading 20% (w/v), incubation time 72 h, 37 °C, and in-
oculum volume 8% (v/v), respectively. The laccase to cellulase ratio of
1:6 was obtained as optimum because at higher ratio not much sig-
nificant increase was observed. Ethanol concentration of 3.09% (v/v) or
24.38 g/L was recorded at laccase to cellulase ratio of 1:6 (Fig. 1).
3.2.2. Effect of solid loading
Solid loading usually decides the economic viability of the process
as it quantifies the amount of solid and liquid to be used in the process.
In addition, it minimizes the waste water treatment and distillation cost
R.C. Rajak and R. Banerjee
Fig. 4. Effect of temperature on bioprocessing of Kans grass.
besides reduced water usage. Hence, solid loading at various ranges
(5–30%, w/v) was selected to study its effect on ethanol production
(Fig. 2). The other parameter that are fixed during the experiment were
laccase to cellulase 1:6, incubation time 72 h, temperature 37 °C, and
inoculum volume 8% (v/v), respectively. Maximum ethanol con-
centration of 4.07% (v/v) or 32.11 g/L was observed at 25% (w/v) solid
loading. Significant increase in ethanol concentration was not observed
at higher solid loading that might be due to less availability of enzyme
active sites to convert holocellulose to reducing sugars and in turn to
ethanol. Low hydrolysis and fermentation yield was reported at high
solid loading because of increase in viscosity of the fermentation broth
and due to mass transfer restrictions [47].
3.2.3. Effect of incubation time
Incubation time plays a crucial role in yeast adaptation, its growth
and in turn during ethanol production. Usually, longer incubation times
causes toxicity to the fermenting organisms due to high ethanol con-
centration whereas, low incubation time resulted in inefficient fer-
mentation due to improper utilization of fermentable sugars. In the
present work, incubation time was varied from 0 to 72 h at laccase to
cellulase 1:6, solid loading 25% (w/v), 37 °C, and inoculum volume 8%
(v/v), respectively to study its effect on ethanol production from raw
Kans grass (Fig. 3). Ethanol concentration of 4.65% (v/v) or 36.68 g/L
was recorded in 24 h. A significant increase in ethanol concentration
was not observed at higher incubation time. Alkali pretreated empty
palm fruit bunch fiber as a lignocellulosic biomass has recorded max-
imum ethanol yield of 21 g/L within 28 h of incubation time upon
fermentation [48].
3.2.4. Effect of temperature
The outer layer of the yeast cell wall is primarily made up of gly-
cosylated mannoproteins which protect the inner cell wall layer
Fig. 5. Effect of inoculum volume on bioprocessing of Kans grass.
5
Energy Conversion and Management 207 (2020) 112504
consisting of glucan chains. The proteins present in the outer cell wall
layer are highly sensitive to the temperature fluctuations. High tem-
perature usually denatures the proteins that results in cell lysis whereas,
low temperature is not able to maintain the membrane turgidity due to
loss of functional proteins [49]. Maximum ethanol concentration of
4.88% (v/v) or 38.50 g/L was recorded at 37 °C where, other fixed
parameter are namely, laccase to cellulase 1:6, solid loading 25% (w/
v), incubation time 24 h, and inoculum volume 8% (v/v), respectively
(Fig. 4). At high temperature, reduced ethanol generation was observed
that might be due to cell lysis because of denaturation of the proteins
present in the outer layer of yeast cell wall. High ethanol yield was
reported from steam exploded wheat straw followed by laccase (Cor-
iolopsis rigida) and S. cerevisiae treatment when fermentation was car-
ried out at 37 °C [50].
3.2.5. Effect of inoculum volume
Fermentation is directly influenced by the quantity of viable yeast
cells present in the reaction mixture. Low inoculum volume of yeast
cells in a fermentation broth results in reduced yield of ethanol due to
less conversion of sugars to ethanol whereas, high inoculum volume
leads to reduction in ethanol concentration because the unutilized yeast
cells consumes the produced ethanol as carbon source for their growth.
Ethanol concentration of 5.42% (v/v) or 42.76 g/L was obtained at 10%
(v/v) upon varying the inoculum volume from 1 to 12 % (v/v). The
other fixed parameter were laccase to cellulase 1:6, solid loading 25%
(w/v), incubation time 24 h, and 37 °C, respectively. Significant im-
provement at higher inoculum volume was not observed (Fig. 5). The
result obtained in the present work is in accordance with the reported
results where, 10% (v/v) and 15% (v/v) inoculum volume were found
to results maximum ethanol generation from laccase delignified mix-
ture of lignocelluloiscs and pineapple leaf waste upon partial con-
solidated bioprocessing (P-CBP) and SSF [51,78].
R.C. Rajak and R. Banerjee Energy Conversion and Management 207 (2020) 112504

Table 1
Central composite design based experimental designs (variables and responses) for bioprocessing.
Run order Solid Loading Incubation Temperature Inoculum Laccase: Ethanol (%, v/v)
(%) Time (h) (°C) Volume Cellulase Experimental Predicted
(%, v/v)

1 25 36 40 12 1:8 2.06 2.04

2 25 12 40 8 1:8 4.87 4.78
3 20 24 37.5 12 1:6 5.01 5.08
4 15 36 40 8 1:8 3.21 3.20
5 25 12 40 12 1:4 4.83 4.74
6 20 24 37.5 10 1:8 5.21 5.25
7 20 24 37.5 10 1:6 6.58 6.50
8 20 24 37.5 8 1:6 5.56 5.97
9 20 24 37.5 10 1:6 6.58 6.50
10 25 36 35 12 1:4 2.47 2.45
11 20 24 37.5 10 1:6 6.58 6.50
12 25 24 37.5 10 1:6 5.41 5.85
13 15 36 40 12 1:4 2.32 2.31
14 15 12 40 8 1:4 6.13 6.04
15 25 12 35 8 1:4 6.22 6.13
16 15 24 37.5 10 1:6 5.28 5.21
17 20 24 40 10 1:6 4.41 4.86
18 15 12 40 12 1:8 4.74 4.64
19 20 24 37.5 10 1:6 6.58 6.50
20 15 36 35 8 1:4 1.88 1.87
21 25 36 35 8 1:8 3.53 3.52
22 15 36 35 12 1:8 2.91 2.89
23 20 24 37.5 10 1:6 6.58 6.50
24 15 12 35 8 1:8 5.83 5.74
25 25 12 35 12 1:8 6.27 6.17
26 20 24 37.5 10 1:6 6.58 6.50
27 20 24 37.5 10 1:4 4.85 4.87
28 20 36 37.5 10 1:6 4.41 4.53
29 25 36 40 8 1:4 2.19 2.18
30 20 12 37.5 10 1:6 6.52 6.58
31 20 24 35 10 1:6 4.81 4.90
32 15 12 35 12 1:4 4.29 4.19

3.3. Optimization and evaluation of bioprocessing of Kans grass relation between the ethanol concentration and the independent vari-
ables such as solid loading, incubation time, temperature, inoculum
Optimization and evaluation of the bioethanol production was volume and laccase to cellulase ratio. The variables such as solid
carried out by considering the five process parameters namely, solid loading, incubation time, temperature and inoculum volume were re-
loading (15–25%, w/v), incubation time (12–36 h), temperature presented as % (w/v), (h), (°C), and % (v/v), respectively.
(35–40 °C), inoculum volume (8–12%, v/v), and laccase to cellulase Analysis of variance (ANOVA) is used to determine the significance
ratio (1:4–1:8) according to the above single parameter selection. The and adequacy of the model for ethanol production. The summary of
design of experiment and the ethanol concentration were tabulated in ANOVA for ethanol generation was given in Table 2. The value of
Table 1. coefficient of determination (R2) was found to be 0.94 which is close to
The result obtained from the experiments performed based on the 1 that indicates the data obtained were well fitted onto the regression
CCD was regressed onto a second order polynomial equation. The re- equation. Moreover, a significant difference was not observed between
gression equation for ethanol generation from Kans grass was re- R2 (0.94) and adjusted R2 (0.86) values that validate the fitness of the
presented as Eq. (4). regression model for ethanol production from Kans grass. Residual error
to pure error comparison from design points was carried out through
Ethanol (%, v/v) = −266.42 + 1.491 × solid loading Lack of Fit Test. The Lack of Fit value obtained was 1.11 that is found to
− 0.253 × incubation time + 12.506 × temperature be non-significant as the P value was observed to be > 0.05. The non-
+ 1.207 × inoculum volume + 4.571 × laccase
: cellulase ratio − 0.009 × solid loading Table 2
ANOVA analysis for regression coefficients and corresponding F and P values.
× solid loading − 0.001×incubation
Source DFa Seq SSb Adj SSb Adj MSc F P
time× incubation time − 0.152 × temperature × temperature
Regression 20 68.86 68.86 3.44 8.93 < 0.001
− 0.069 × inoculum volume × inoculum volume
Linear 5 36.54 8.84 1.76 4.59 0.017
− 0.133 × laccase: cellulase ratio × laccase Square 5 27.72 27.72 5.34 14.39 0.000
: cellulase ratio + 0.007 × laccase Interaction 10 4.59 4.59 0.459 1.19 0.387
Residual Error 11 4.23 4.23 0.385 – –
: cellulase ratio×incubationtime Lack-of-Fit 6 4.23 4.23 0.706 1.11 0.195
− 0.007 × solid loading × laccase: cellulase ratio + 0.016 × laccase Pure Error 5 0.100 0.100 0.02 – –
Total 31 73.10 – – – –
: cellulase ratio×inoculum R2 = 94.20% R2 (adj) = 86.82%
volume+solid loading×inoculum volume
a
(4) Degrees of Freedom.
b
Sum of Squares.
c
The second order regression equation (Eq. (4)) represents the Mean Square.

6
R.C. Rajak and R. Banerjee
Fig. 6. Response surface plots of Kans grass bioprocessing for (A) laccase to cellulase ratio and temperature, (B) temperature and inoculum volume and (C) laccase to
cellulase ratio and solid loading.
significant Lack of Fit value signified that the model was valid for the
present work.
The interactions between different variables were illustrated in
terms of 3D response surface plots. The effects of two variables on
ethanol production were represented by each surface plots. The surface
plots between laccase to cellulase ratio and temperature, temperature
and inoculum volume and laccase to cellulase ratio and solid loading
for ethanol generation were given in Fig. 6(A, B and C, respectively).
Increase in the concentration of ethanol was observed up to optimum
laccase to cellulase ratio (1:6) and temperature (37 °C) (Fig. 6A). Si-
milarly, maximum ethanol was recorded up to optimum inoculum vo-
lume (8.8%, v/v) and temperature (37 °C) (Fig. 6B). Further increase in
inoculum volume does not show any significant increase in ethanol
concentration.
An increase in ethanol production up to optimum solid loading of
24% (w/v) and laccase to cellulase ratio (1:6) was illustrated through
Fig. 6(C). It was observed that upon increasing the solid loading beyond
the optimum conditions does not improve the ethanol yield that may be
due to mass transfer limitations because of improper mixing. In addi-
tion, increase in laccase to cellulase ratio further does not have sig-
nificant effect on ethanol production.
3.4. Model validation
The optimized conditions for Kans grass bioprocessing were solid
loading (24%, w/v), incubation time (24 h), temperature (37 °C), in-
oculum volume (8.8%, v/v) and laccase to cellulase ratio (1:6) re-
spectively. The regression model based on the above optimized
7
Energy Conversion and Management 207 (2020) 112504
conditions predicted an optimum ethanol concentration of 8.10% (v/v).
To confirm the obtained optimized conditions and model fitness further
experiments were carried out. The experimental ethanol obtained was
7.62% (v/v) or 59.96 g/L (approximately, 60.00 g/L) under optimized
fermentation conditions which is in close proximity to the predicted
ethanol of 8.10% (v/v). Further, increase in ethanol concentration is
might be due to uptake of hexose sugars released from hemicellulose
moieties after complete consumption of the total sugars. The pro-
ductivity rate and ethanol conversion efficiency was found to be 2.50 g/
L/h and 65.01% whereas, yield was observed to be 0.249 g/g. This
mode of ethanol production (ethanol yield, 0.249 g/g) from lig-
nocellulosic biomass can compete with the expensive hydrothermal and
twin-gear reactor pre-treatment process followed by ethanol generation
from aquatic weeds (ethanol yield, 0.231 g/g) and rice straw (ethanol
yield, 0.032 g/g) [52,53].
It is worth mentioning that enzymes increases the porosity of bio-
mass that is easily accessible by cellulose and hemicellulose degrading
enzymes which ultimately reduce the overall process time. Maximum
ethanol production was reported from acid and alkali pre-treated rice
straw at 96 h incubation upon consolidated bioprocessing by utilizing
anaerobic bacteria (Clostridium thermocellum DSM 1313) [17]. Simi-
larly, consolidated bioprocessing of alkali treated sugarcane bagasse
resulted in low ethanol concentration of 5.825 g/L after 24 h incubation
time [54]. In another study, ionic liquid pretreated pine needle was
used as a feedstock for one pot consolidated bioprocess that results in
ethanol concentration of 7.4 g/L after 72 h of fermentation [55]. Also,
consolidated bioprocessing of ramon flour obtained from Brosimum
alicastrum by using native strain of Trametes hirsuta Bm-2 recorded
R.C. Rajak and R. Banerjee Energy Conversion and Management 207 (2020) 112504

ethanol concentration of 13 g/L at 244 h of incubation time [56]. In the Table 3

present study, maximum ethanol was obtained at 24 h that includes Delignification of Kans grass during bioprocessing.
delignification, saccharification and fermentation because of robust Incubation Time (h) % Delignification Residual Lignin (%, w/w)
delignifying enzyme, which has resulted in higher biomass porosity and
ultimately increase the yield of ethanol. 3 31.83 ± 0.78 11.91
6 70.98 ± 1.24 5.07
Globally, the production rate of plant biomass is approximately,
12 71.12 ± 1.16 5.05
200 × 109 tons/year. The primary biomass remains accessible for the 18 71.80 ± 1.92 4.93
production of biofuels is nearly 8–20 × 109 tons [38]. It has been es- 24 72.20 ± 2.10 4.86
timated that around 442 billion litres of ethanol can be produced every
year by utilizing the primary biomasses such as agricultural and forest
residues [57,58]. The production cost of lignocellulosic ethanol in 2020 Table 4
are anticipated to be 25% less than corn and wheat ethanol because of Porosity analysis of Kans grass before and after fermentation.
exploitation of low cost abundantly available agro residues [59]. Bio- Sample Surface Area Pore Volume Pore Diameter
mass pretreatment for lignin removal or degradation is considered as (m2/g) (mL/g) (nm)
the core area which is capital intensive and requires an investment that
Raw biomass 1.38 0.0042 10.19
constitutes 30–50% of the total equipment cost [60]. High biomass
Treated biomassa 0.63 0.0034 9.10
pretreatment costs is might be due to harsh process conditions or use of
acids and other corrosive reagents being employed for reduction of the a
Biomass represents solid residual biomass after fermentation.
intrinsic recalcitrance of the lignocellulosic biomass [61]. POET-DSM
Advanced Biofuels LLC (South Dakota, United States of America) has 70% lignin removal was reported by utilizing eucalyptus woody bio-
reported 70 gallons of bioethanol per ton of lignocellulosics with their mass [73]. It is worth mentioning that lignin degradation of 20–65% is
novel biomass pretreatment technology and on-site enzyme production significant enough to improve the release of reducing sugars from the
facility [62]. The cost of enzyme based bioethanol production has been pre-treated biomass [74].
reduced from $1.22/L to $0.32/L due to improvements in biomass
pretreatment methods, hydrolytic enzymes, and fermentative organ-
3.6. Porosity analysis of Kans grass
isms [63–65]. Furthermore, the above bioethanol production cost is
based on a biomass cost of $46/T, which represents $0.12/L of the total
The surface area, pore volume and pore diameter of the untreated
product cost. Therefore, the total production cost would further reduce
substrate was found to be higher compared to residual biomass of Kans
if a low cost agricultural residues or waste feedstock is used [65]. Apart
grass bioprocessing (Table 4). Effective release of sugars from the bio-
from cheap biomass feedstock, cost of enzymes also serve as important
mass resulted in partial collapse or breakdown of pore wall that led to
cost component of the production process and reducing the cost is a key
decrease in porosity of fermented biomass. The decrease in biomass
to make the lignocellulosic bioethanol commercially viable [66,67].
porosity in terms of surface area, pore volume and diameter indicates
In the present study, Kans grass has been used a biomass feedstock
the effective action of laccase for lignin degradation and cellulase-xy-
which is a perennial C4 energy crop that grows well in marginal land,
lanase for maximum release of reducing sugars into the reaction mix-
requires less water for growth and holds good potential for energy
ture. Similar kind of observation was reported where reducing sugars
generation [68]. The regeneration ability of the plant is very high and
release was found to be increased from pineapple leaf wastes after
once it is planted can supply biomass over the years. It gives approxi-
laccase and cellulase-xylanase treatment [75]. Thus, the study of bio-
mately 10 ton/hectare of biomass compared to other crops which gives
mass porosity highlighted the effective role of enzymes for second
only 5–6 ton of biomass/hectare/year [69]. In addition, crude enzymes
generation ethanol production.
produced from fungal strains by utilizing low cost feedstocks have been
used for lignin degradation and polysaccharides hydrolysis that might
help in cost reduction of the bioethanol production process. Moreover, 3.7. Energy density estimation
adopted process conditions for bioethanol production are mild in nature
that do not require much solid-liquid handling processing steps which The energy density of raw biomass was found to higher
might further reduce the cost of the entire production process. (12.10 ± 0.08 KJ/g) compared to the residual biomass obtained after
fermentation (5.90 ± 0.42 KJ/g). The plausible reason for increase in
3.5. Delignification of Kans grass at various time intervals during Kans grass energy density of the raw Kans grass was due to presence of high
bioprocessing quantity of lignin. Being rich in carbon and nitrogen, lignin has high
heating value than cellulose and hemicellulose [76]. Similar types of
The biological or eco-friendly route from lignocellulose to the final results were reported in case of laccase and cellulase-xylanase treated
product comprised of three main steps: biomass delignification, enzy- pineapple leaf waste and mixture of lignocellulosics [75,77]. The dif-
matic hydrolysis of holocellulose and fermentation of the obtained su- ference in the energy density value of raw and fermented Kans grass is
gars. Bioprocessing in a single unit is an integration of all these three might be due to the presence of residual lignin, unutilized reducing
steps where delignifying enzyme, saccharifying enzymes and yeast were sugars and ethanol, in the case of fermented broth.
simultaneously employed for ethanol generation in a single reactor
under optimized environmental conditions. In the present study, lignin 3.8. Structural characterization of biomass
degradation from Kans grass was monitored at different time intervals
during bioprocessing to study its effect on ethanol production. It was 3.8.1. Scanning electron microscopy
observed that the extent of delignification was increased up to 6 h The structural changes before and after bioprocessing of the biomass
(70.98%) and thereafter significant increase in delignification was not was examined. The SEM analysis of the fermented Kans grass indicates
observed that might be saturation of all the active sites of laccase at disruption of the surface structure compared to the rigid, flat and intact
longer time interval (Table 3). This is in accordance with the results surface observed in the case of raw Kans grass. The surface distortion
reported in the case of laccase delignified Bambusa bamboos, Napier indicates the degradation of cell wall materials, specifically lignin that
grass and Eucalyptus globules [70-72]. Also, the amount of lignin de- might results in exposure of hidden cellulose microfibrils. Similar kind
gradation (70.98%) through eco-friendly mode is quite competitive to of surface distortion was observed after steam explosion pre-treated
the two-stage alkali and steam pre-treatment method where around birchwood followed by SSF and in the case of partially consolidated

8
R.C. Rajak and R. Banerjee
Table 5
XRD values of raw and fermented substrate in terms of % crystallinity and re-
lative % crystallinity.
Biomass % Crystallinity Relative % increase/decrease in
crystallinity
Raw 51.49 –
Treated substrate 39.34 −23.60
bioprocessing of mixture of lignocellulosics [77,78].
3.8.2. X-ray diffraction analysis of fermented biomass
Biomass crystallinity plays an important role in biomass conversion
and is the major factor in lignocellulose digestibility [79]. Because of
the presence of lignin in the raw Kans grass, which covers the major
portion of cellulose showed less % crystallinity compared to fermented
substrate where the lignin content was drastically reduced upon enzy-
matic action (Table 5). The raw substrate showed relative % crystal-
linity of 51.49%, which was reduced to 20.15% that might be due to
combined action of both laccase and cellulase and utilization of para-
crystalline and crystalline cellulose. The results obtained were corro-
borated with the reported results of consolidated bioprocessing of
wheat straw where % crystallinity was reduced to 23.6% after fer-
mentation [80].
4. Conclusion
Process integration aims to consolidation of single units and has
been considered as a feasible technology for value added products
generation in a single common platform. The present work dealt with
the bioprocessing of Kans grass where mixture of crude robust de-
lignifying and saccharifying enzymes were added for lignin decon-
struction and holocellulose hydrolysis followed by fermentation to
bioethanol. The advantage of mix enzyme application is to selectively
degrade the lignin while maintaining the high biomass porosity for
cellulolytic enzyme action to release more sugars and in turn results
high yield of ethanol. Maximum ethanol concentration of 59.96 g/L was
recorded after 24 h fermentation with conversion efficiency of 60.05%.
The efficacy of the entire enzymatic venture of bioethanol production
was substantiated through biomass porosity, energy analysis and
structural characterization of the raw and fermented biomass. Biomass
porosity analysis showed partial collapse or breakdown of pore wall,
which was evident through SEM studies. Moreover, biomass crystal-
linity was also reduced due to combined action of laccase and cellulase-
xylanase which resulted in improved ethanol production. In addition,
the entire process conditions are mild in nature that reflects the eco-
friendly behaviour of this process. Thus, the present attempt of bio-
processing of Kans grass through mix enzyme application for ethanol
production provides a feasible technology for energy generation in a
cost effective manner.
CRediT authorship contribution statement
Rajiv Chandra Rajak: Methodology, Formal analysis, Validation,
Investigation, Writing - original draft. Rintu Banerjee:
Conceptualization, Resources, Writing - review & editing, Supervision,
Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
9
Energy Conversion and Management 207 (2020) 112504
Acknowledgements
We are thankful to the University Grants Commission, New Delhi,
India, for providing a scholarship to Mr. Rajiv Chandra Rajak to carry
out this study.
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