BioEnergy Research (2023) 16:1667–1682

https://doi.org/10.1007/s12155-022-10517-y

Organic Acid Pretreatments and Optimization Techniques for Mixed

Vegetable Waste Biomass Conversion into Biofuel Production
Babu Dharmalingam1 · Prapakorn Tantayotai2 · Elizabeth Jayex Panakkal1 · Kraipat Cheenkachorn3 ·
Suchata Kirdponpattara3 · Marttin Paulraj Gundupalli1 · Yu‑Shen Cheng4 · Malinee Sriariyanun1

Received: 30 June 2022 / Accepted: 20 September 2022 / Published online: 26 September 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Mixed vegetable and food wastes left from consumption and processing were important uncontrolled sources of greenhouse
gas emissions. Biofuel production from mixed vegetable waste is ineffective due to the recalcitrant structure of lignocellu-
lose. In this work, an artificial neural network (ANN) and response surface methodology (RSM) have been used to optimize
the process parameters for the pretreatment of mixed vegetable waste by using organic acid (acetic, citric, oxalic acid) and
inorganic acid (hydrochloric acid). The study revealed that the maximum reducing sugars yield of 314.1 mg ­g−1 biomass
was obtained from acetic acid-pretreated biomass at a time of 68.32 min, a temperature of 140 °C, and an acid concentration
of 10% w/v, which was 1.66 fold higher than the HCl-pretreated sample. The analysis of biomass composition and FTIR
suggested that both inorganic acid and organic acid pretreatment removed hemicellulose and lignin from biomass, and subse-
quently enhanced the cellulase accessibility to biomass during enzymatic saccharification. It was observed that organic acid
pretreatment is more effective to remove lignin compared to inorganic acid. The pretreated samples were converted to ethanol,
and the maximum yield of 7.60 g L ­ −1 was obtained for the oxalic acid-pretreated sample, which was 2.82 fold higher than
the HCl-pretreated biomass. Also, the maximum biogas yield of 110.72 mL g-VS−1 was obtained for citric acid-pretreated
biomass, which was fivefold higher than the untreated biomass. Hence, organic acid pretreatment is highly recommended
for bioethanol and biogas production from mixed vegetable wastes.

Keywords Bioethanol · Biogas · Biorefinery · Organic acid pretreatment · Mixed vegetable · Optimization

Introduction

Global population, industry development, rapid economic

Babu Dharmalingam and Prapakorn Tantayotai contributed equally
to this work.
* Malinee Sriariyanun
malinee.s@tggs.kmutnb.ac.th
1
Biorefinery and Process Automation Engineering Center,
Department of Chemical and Process Engineering, The
Sirindhorn International Thai-German Graduate School
of Engineering, King Mongkut’s University of Technology
North Bangkok, Bangkok, Thailand
2
Department of Microbiology, Faculty of Science,
Srinakharinwirot University, Bangkok, Thailand
3
Department of Chemical Engineering, Faculty
of Engineering, King Mongkut’s University of Technology
North Bangkok, Bangkok, Thailand
4
Department of Chemical and Materials Engineering,
National Yunlin University of Science and Technology,
Douliu, Yunlin, Taiwan
growth, urbanization, and energy demand have resulted in
the rapid depletion of fossil resources. Global warming and
environmental pollution are the major concerns due to the
continuous utilization of fossil fuels in automobile indus-
tries, agriculture, and power machinery [1]. Bioenergy from
lignocellulose biomass is considered a suitable and potential
replacement for fossil fuels due to lower greenhouse gas
(GHGs) emissions. Agriculture, forest, and industrial sectors
are the main contributors to the availability of lignocellulose
biomass, and the most promising feedstocks are agricultural
waste and forest residues [2]. In recent years, lignocellulose
biomass has emerged as a potential source to produce biofu-
els and biochemicals due to its abundance and low cost. Lig-
nocellulose materials are composed of three polymer layers,
including cellulose, hemicellulose, and lignin [3]. Commer-
cial production of biofuels and biochemical products from

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lignocellulose materials requires a high cellulose content
and a low lignin content because lignin is composed of com-
plex aromatic polymers that hinder the process efficiency
during biofuel and biochemical production [4].
The susceptibility of cellulose to hydrolysis is a key com-
ponent that is limited due to the hindrance caused by lignin
and hemicellulose that surround the cellulose microfibrils in
the plant cell wall [5]. Therefore, pretreatment is important
in improving the process efficiency by solubilizing hemicel-
lulose and lignin. Post-pretreatment, lignocellulose biomass
is subjected to saccharification and fermentation to produce
biofuel. The pretreatment efficiency is evaluated through
glucose and ethanol yield during the saccharification and
fermentation process [6]. Different conventional pretreat-
ment techniques are available, such as acid hydrolysis, steam
explosion, ammonia fiber expansion, wet alkaline oxidation,
and hot water pretreatment which exist to liberate cellulose
from the lignin and hemicellulose [7]. Hydrochloric acid,
sulfuric acid, nitric acid, and acetic acid are the most often
used chemicals in the pretreatment of lignocellulose bio-
mass. However, utilizing these acids can lead to equipment
corrosion and produce chemical inhibitors as byproducts [8].
Physical pretreatment has several disadvantages, including
high processing, operating cost, and instrument corrosion.
Furthermore, the use of alkaline pretreatment can cause
condensation and redistribution of lignin, making the cel-
lulose more resistant than the native cellulose [9]. In addi-
tion, physicochemical pretreatment requires high tempera-
ture, specialized reactor, and expensive instruments, whereas
organic acid is a weak acid and produces less amount of
chemical inhibitors as compared to other pretreatments [10].
The effectiveness of pretreatment is dependent upon the con-
ditions, such as type of biomass, biomass loading, tempera-
ture, reaction time, catalyst concentration, and mixing [11].
Among various methods, chemical pretreatment has been
demonstrated and selected for different types of biomasses
due to its simplicity, cost-effectiveness, and efficiency.
Ahmad et al. [12] investigated the effect of sulfuric acid-
assisted hydrothermal pretreatment on methane produc-
tion from sugarcane bagasse using the RSM approach. The
experimental studies revealed that the lowest solid yield
of 13% was reported at 200 °C, 19 min, and 3% ­H2SO4,
whereas maximum methane recovery of 160 NmL (g TVS)−1
was recorded at 180 °C, 12 min, and 2% ­H2SO4 suggesting
the importance of pretreatment optimization. Thulluri et al.
[13] examined the effect of acid pretreatment on the cellu-
lose of vegetable waste and enzymatic hydrolysis efficiency.
The study revealed that acid pretreatment enhanced the
sugar, phenol, and cellulose yields. Another study reported
the enhancement of biogas and bioethanol production from
rice straw extractives using water and ethanol. The study
observed that the addition of rice straw water extractives
improved the ethanol yield to 95%, whereas the addition
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BioEnergy Research (2023) 16:1667–1682
of rice straw water extractives resulted in a significant drop
in biogas yield and a shift of the biogas composition from
74% to less than 1% methane. By increasing the ethanol
extractives concentration of rice straw, the biogas yield was
increased from 167 to 524 mL ­g−1 [14]. Bittencourt et al.
[15] investigated the fractionation of sugarcane bagasse by
hydrothermal and alkaline pretreatments for the production
of bioethanol and biogas. The enzymatic hydrolysate was
fermented with Saccharomyces cerevisiae, yielding 190.8
L ethanol ton-SB−1. Anaerobic digestion of liquid streams
from the pretreatment processes produced 27.46 ­NLCH4
kg-SB−1 of biogas. However, furfural and formic acid are
formed during chemical pretreatment due to the degrada-
tion of xylan monomers caused by severe pretreatment
conditions, such as high temperature and longer reaction
time [16]. These sugar degradation compounds (furfural,
5-hydroxymethylfurfural, formic acid, acetic acid, and lev-
ulinic acid) inhibit the activity and metabolism of enzymes
and microorganisms. Therefore, the selection of the pre-
treatment method and the optimization of the pretreatment
conditions are important steps to maximize the enzymatic
saccharification and fermentation efficiency [10].
Organic acid pretreatment has been proposed as an alter-
native to conventional acid pretreatment methods due to
lower sugar degradation and less formation of inhibitory
byproducts. Organic acids are non-toxic and environmentally
friendly compared to mineral acids, such as sulphuric acid
­(H2SO4) and hydrochloric acid (HCl) [17]. Different organic
acids have been explored for various lignocellulose biomass.
However, fewer studies exist on the pretreatment of vegeta-
ble waste using organic acids to produce ethanol and biogas
via fermentation and anaerobic digestion (AD) process.
Vegetable waste, as a type of food waste, is obtained during
processing and cutting before handling by consumers. Food
waste is one of the major sources to generate the uncon-
trolled released greenhouse gas, about 10%, in the form of
methane from landfills (Food and Agriculture Organization
of the UN). In 2020, it was estimated that only in the USA,
170 million metric tons of carbon dioxide (­ CO2) equivalent
to GHG emissions were produced from food waste (The US
Environmental Protection Agency (EPA) report). Therefore,
it is important to develop a controlled process to convert
vegetable waste to value-added products, such as biogas and
bioethanol to reduce the devastation of uncontrolled GHG
emissions to the environment.
Several studies have focused on employing various
approaches to produce biofuel production. However, these
approaches have several disadvantages, including processing
complexity, low accuracy, and higher error rate [1]. The arti-
ficial neural network approach has been used in the optimi-
zation of biofuel production worldwide due to its higher pre-
cision and low error rate. Pomeroy et al. [18] reviewed the
importance of the ANN approach on chemical production
BioEnergy Research (2023) 16:1667–1682
via a biorefinery. It was found that machine learning and
ANN have emerged as important methods for predicting
chemical behavior in many of the most well-known biomass
valorization processes. RSM is another approach to be used
as a cost-effective tool in research and industry, as it saves
time and money by reducing the numbers of experiments. It
has been applied in different scientific research areas, such as
biofuel production, C­ O2 capture, food engineering, chemical
engineering, and the petroleum refinery industry [19]. Das
et al. [20] used both RSM and ANN techniques for optimiz-
ing the process parameters affecting enzymatic hydrolysis.
This study observed that fermentation of hydrolysate yielded
a maximum ethanol concentration of 10.44 g L ­ −1 using
−1
Pichia stripes followed by 8.24 and 6.76 g ­L for Candida
shehata and Saccharomyces cerevisiae, respectively. The
average errors for ANN and RSM were 3% and 4.8%, and the
predicted errors for ANN and RSM were 0.95 and 1.41%,
respectively, which highlighted the significance in selecting
the appropriate approach for the process optimization.
The above literature studies exposed that fermentation
and AD are the biological processes for the conversion of
a wide range of biomass feedstock to produce bioethanol
and biogas at a cheaper cost. Even though a variety of pre-
treatment procedures have been tested on a variety of feed-
stock, only a few reported techniques achieved high biogas
and bioethanol yield at low enough prices. The successful
pretreatment should be able to improve the digestibility,
avoid product degradation, require low energy, and be cost-
effective and environmentally friendly. However, the type,
quality, and property of feedstock have a significant impact
on fermentation and AD efficiency. To achieve the feasible
process for biorefinery of lignocellulose biomass, optimiza-
tion experiment and modeling are necessary to maximize the
process efficiency for each type of biomass. Therefore, most
research works and industrial applications, especially in the
production of 2nd generation ethanol, have selected and
utilized a single type of lignocellulose for their processes
based on the empirical concept in scientific works. Practi-
cally, natural agricultural wastes, including vegetable waste
obtained from cutting and processing found in dumpsters
or municipal landfills, are mixtures of various types of lig-
nocellulose biomass. Therefore, using mixed lignocellulose
biomass as a substrate for biorefining process is more practi-
cal and it helps to secure the dependency on one single type
of biomass in the situations that our world is facing global
disasters, e.g., flooding and severe drought. Hence, in this
present work, an attempt has been made to investigate and
compare the effect of acid pretreatment on mixed vegeta-
ble lignocellulose biomass using organic acids (i.e., acetic
acid, citric acid, oxalic acid) and inorganic acid (i.e., HCl)
to improve the enzymatic saccharification, fermentation, and
anaerobic digestion process. The pretreatment parameters,
including time, temperature, and acid concentration, are
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optimized using the response surface methodology (RSM)-
based Box-Behnken design (BBD) and artificial neural net-
work (ANN) to improve the process efficiency of vegetable
waste conversion. The mechanisms of inorganic and organic
acids to improve enzymatic saccharification, and the pro-
duction of bioethanol and biogas were deciphered based on
analysis of composition analysis and chemical modification
of biomass. The optimized process in this work could be fur-
ther applied with the management of mixed vegetable wastes
in controlled processes and facilities to reduce greenhouse
gas emissions and increase energy security at the same time.
Materials and Methods
Sample Preparation and Characterization of Mixed
Vegetable Wastes
In this study, vegetable wastes, including Chinese broccoli
(CB), cauliflower, and Napa cabbage (NC), obtained after
product processing and cutting were collected from the local
market located in the central part of Thailand. The size of
each vegetable waste was reduced to about 1-cm size by
manually cutting. In addition to that, the dried sample size
was reduced by grinding, and sieving through a 20-mesh
aluminium sieve (~ 0.841 mm sizing) to achieve consistency
in sample size. Then, three types of dried vegetable wastes
were mixed in one big batch with the ratio of 1:1:1 by dried
weight and stored in a sealed plastic bag until used for all
experiments in this study. The samples were characterized
for the contents of cellulose, hemicellulose, and lignin by
Van Soest and Wine method as used in the previous study
[21]. The volatile solids (VS) and total solids (TS) of the
mixed vegetables were measured by the American Public
Health Association (APHA) standard water and wastewater
examination methods. The contents of TS and VS in mixed
vegetables were 9.11 ± 0.58% and 8.01 ± 0.23%, respectively.
The compositions of cellulose, hemicellulose, and lignin
contents of mixed vegetable biomass were 20.67 ± 0.94,
16.97 ± 2.54, and 31.70 ± 4.88, respectively.
Design of Experiment for Process Optimization
of Pretreatment
Design expert software (version 10.0.3, Stat-Ease, USA) has
been chosen in this study for experimental design, modeling,
and optimization. This work used the Box-Behnken design
to determine the relationship between the response (reduc-
ing sugar yield) and pretreatment parameters (reaction time,
reaction temperature, and acid concentration). The Box-
Behnken design is a three-level incomplete factorial design
that is described as a cube, with center points and middle
points of the edges, and necessitates a certain number of
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1670
experiments [5]. In this work, time (X1, 30–90 min), tem-
perature (X2, 100–140 °C), and acid concentration (X3,
2–10% w/v) were selected as tested parameters because they
have been demonstrated in previous studies to influence the
reducing sugar yield (Y, mg g­ −1 biomass) and they are eas-
ily adjustable parameters during the process operation [21].
Table 1 depicts the independent variables assigned with
coded and un-coded values. Equation 1 shows the generic
expression for the second-order model with three independ-
ent variables.
3 3 3
∑ ∑ ∑ method [25]. This study used the backpropagation learn-
Y = b0 + bj xj + bij xi xj + bjj xi2
j=1 ij=1 j=1
where Y is the expected reducing sugar yield, xi and xj are
the parameters, bo is the constant, bj is the linear effect, bij
is the interaction effect, and bjj is the square effect of the
parameters. Multiple analyses was performed to calculate
the coefficient of variation, and the resulting equation was
used to predict the response parameters. The response was
statistically analyzed using analysis of variance (ANOVA).
Table 1  Independent variables along with un-coded values
Independent variable Units Uncoded values
Time X1 min 30 60
Temperature X2 °C 100 120
Organic acid concentration X3 % w/v 2 7
A point prediction approach was used to predict the optimal
pretreatment condition for lignocellulose biomass. Table 2
shows the BBD matrix for three independent variables,
which consisted of 17 experimental runs.
Modeling of an Artificial Neural Network
for Pretreatment of Biomass
An artificial neural network (ANN) is a mathematical or
computational model working based on the functional
aspects of a biological neural network. The ANN approach is
a nonlinear statistical data modeling tool that is used to rep-
resent complex input–output relationships. One input layer
and an output layer with one or more hidden layers make
up the ANN architecture. Each layer is made up of neurons,
which comprise the ANN processing elements [22]. There
is no method for determining the number of neurons in a
hidden layer or the number of hidden layers [1]. Therefore,
the ANN network is trained by selecting the number of hid-
den layers and neurons randomly. As a result, trial and error
approaches were used to determine the number of neurons
and hidden layers in it [23]. The optimal network was chosen
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BioEnergy Research (2023) 16:1667–1682
based on the number of neurons and hidden layers with an
R-value close to unity.
Backpropagation multilayer feed-forward neural network
(BPNN) is the most widely used in engineering applications
to predict the output parameters among the other kinds of
ANN architecture [24]. For ANN modeling, the backpropa-
gation training technique is the most extensively used. The
Levenberg Marquart (LM) uses second-order derivatives,
which results in better convergent behavior, whereas ordi-
nary gradient descent focuses only on first-order derivatives.
In addition, the LM is a close approximation to the Newton’s
(1)
ing approach to train the ANN model. To achieve the best
ANN model, the Trainlm training function was used, and
the transfer function for the hidden layer was the logistic
sigmoid. An ANN with several architectures was trained and
assessed using experimental data to obtain the best predic-
tion. The ANN modeling was done by the MATLAB 10.3
neural network toolbox.
Organic Acid and Inorganic Acid Pretreatment
of Lignocellulose Biomass
Acetic acid (AA), citric acid (CA), oxalic acid (OA), and
hydrochloric acid (HCl) pretreatments were performed for
90 mixed vegetable wastes according to the condition listed in
Table 2. Five grams of dried vegetable samples were mixed
140
with acid solutions (with different concentrations of 2–10%
12
w/v) in the screw cap bottle with the volume to prepare the
solid–liquid ratio of 1:10% w/v. Each sample was placed
in a hot-air oven that was pre-heated to the temperature
at the designed pretreatment temperature (100–140 °C)
with the retention time of the designed pretreatment time
(30–90 min) (Table 2). After the pretreatment process, the
sample was allowed to cool in an iced bath to room tempera-
ture to stop the progress of the pretreatment. The solid and
the liquid hydrolysate were separated by using centrifugation
at 8000 × g for 10 min. The residual solids were repeatedly
washed with deionized water to neutralize the pH and dried
in a hot air oven at 60 °C for 24 h until the dried weight
is constant. The dried solid fraction was subsequently sub-
jected to enzymatic saccharification.
Fourier Transform Infrared Spectroscopy Analysis
Fourier transform infrared spectroscopy (FTIR) analysis
was performed for treated mixed vegetable lignocellulose
biomass to understand the changes in the functional groups
before and after organic acid pretreatment [21]. An FTIR
spectrometer was used to conduct the analysis (model: Spec-
trum 2000; make: Perklin Elmer, USA). The IR spectra were
collected with a resolution of 4 ­cm−1 from 400 to 4000 ­cm−1.
 Table 2  Design matrix for pretreatment of mixed vegetable wastes
BioEnergy Research (2023) 16:1667–1682

Run Pretreatment parameters Reducing sugar yield ­(mgg−1 biomass)

Time (min) Temp (°C) Acid concentration Acetic acid Citric acid Oxalic acid HCl acid
(% w/v)

1 90 140 6 224.9 155.2 159.6 186.7

2 60 120 6 266.3 128.0 177.4 145.5
3 90 120 10 248.0 105.7 212.7 198.0
4 60 140 10 314.1 128.0 172.4 113.1
5 30 120 2 180.2 161.9 133.1 632.0
6 60 140 2 251.4 160.2 128.0 118.1
7 90 100 6 263.6 163.3 161.3 123.0
8 60 100 10 193.7 129.0 220.9 145.6
9 90 120 2 224.2 192.8 162.6 125.3
10 60 100 2 292.0 167.0 183.6 127.0
11 60 120 6 220.2 117.5 204.6 150.5
12 30 120 10 170.0 211.4 270.3 118.9
13 30 100 6 214.1 133.5 299.5 144.1
14 60 120 6 270.3 118.2 149.0 148.3
15 60 120 6 229.0 111.6 158.9 118.9
16 60 120 6 279.5 135.5 180.2 137.0
17 30 140 6 214.7 110.7 151.1 109.1

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Enzymatic Saccharification of Lignocellulose
Biomass
Based on the reducing sugar yield obtained from enzymatic
saccharification, the efficiency of the pretreatment and the
optimum settings for the independent variables were cal-
culated. The remaining solid sample produced after pre-
treatment were enzymatically saccharified with a cellu-
lase enzyme mixture containing cellulase (CelluClast1.5L
(Sigma-Aldrich, USA), 20 FPU ­g−1 of dry biomass), and
β-glucosidase (Novozyme 188 (Sigma-Aldrich, USA), 100
CBU ­g−1 of dry biomass) [21]. Each saccharification reac-
tion was carried out in a 50-mL centrifuge tube containing
0.5 g of residual solid, 20-mL citrate buffer (50 mM; pH
4.7), and 200-µL sodium azide (2 M) [26]. The tubes were
kept in a shaking incubator for 72 h with the temperature
and mixing set at 50 °C and 200 rpm. After the incubation
time, the liquid hydrolysate samples were collected by cen-
trifugation at 8000 × g for 10 min, and the concentrations
of reducing sugars in hydrolysate samples were analyzed
using the 3,5-DNS technique [27]. Furthermore, a hydro-
lysate sample for ethanol fermentation was prepared without
the addition of sodium azide. To limit the variance between
the experimental data, all of the studies were repeated three
times. The reducing sugar yield was calculated by the fol-
lowing equation,
Reducing sugar yield g g−1 = m M−1
( )
where m is the mass of reducing sugar (g) in enzymatic
hydrolysis and M is the mass of biomass (g) in enzymatic
hydrolysis.
a local alcohol factory in Thailand, were courtesy provided
by the Thailand Institute of Scientific and Technological
Research (TISTR) to be used in the fermentation process.
The fermentation reaction took place in a yeast culture con-
taining 19 mL of liquid hydrolysate and 1 mL yeast inocu-
lum ­(A600 equal to 1.0). As a carbon and nitrogen source,
glucose (1% w/v) and yeast extract (1% w/v) was added to
the yeast culture to allow acclimatization of yeast culture [5].
The medium’s pH was adjusted to 5.0, and it was incubated
in a temperature-controlled orbital shaker at a temperature
of 32 °C, a stirrer speed of 100 RPM, and a reaction time of
48 h. Then, the liquid fraction (supernatant) of yeast culture
was collected by centrifugation at 8000 × g for 10 min. The
ethanol fermentation experiments were conducted in trip-
licate. The ethanol concentration was measured with a gas
chromatography equipped with a flame ionization detector
(GC-FID) (Model: GC-2010, Make: Shimadzu, Japan). GC-
FID was equipped with a DB-wax column (30 m × 0.25 mm,
0.25 m). An oven program, (a) 40 °C for 4 min, (b) 100 °C
ramp (5 °C ­min−1), and (c) 200 °C ramp (10 °C ­min−1), was
used to analyze the ethanol. The injector volume for ethanol
analysis in the sample was set at 1 μl in split mode (1:20)
[28]. The concentration of ethanol in the sample was calcu-
lated based on the standard curve of absolute ethanol solu-
tion (99.8%). The measurement of ethanol concentration was
conducted in triplicate. The ethanol yield was calculated by
the following equation,
Ethanol yield gg−1 = c ∗ V∕M
( )
where c is the concentration of ethanol in grams per liter, V
is the total volume of fermentation liquid in liter, and M is
the mass of biomass in grams.

Bioethanol Production and Quantification

A fermentation reaction was performed in a batch opera-
tion for bioethanol production from untreated and pretreated
mixed vegetable samples. Two types of yeast strains (Sac-
charomyces cerevisiae TISTR 5606 and Saccharomyces cer-
evisiae TISTR 5596), which were originally obtained from
Biogas Production from Mixed Vegetable
Lignocellulose Biomass
The biogas is produced from a mixed vegetable sample
pretreated with acids (acetic acid, citric acid, oxalic acid,
and HCl acid). The anaerobic sludge of seed inoculum was

Table 3  ANOVA for acetic Source Sum of squares df Mean square Fvalue p-value Prob >F
acid pretreated mixed vegetable
lignocellulose biomass Model 189.93 5 37.99 5.43 0.009 Significant
A-Time 41.27 1 41.27 5.90 0.033
B-Temperature 3.41 1 3.41 0.49 0.499
C-Concentration 0.17 1 0.17 0.02 0.880
BC 73.56 1 73.56 10.51 0.007
A2 71.52 1 71.52 10.22 0.008
Residual 76.99 11 7.0 -
Lack of fit 48.66 7 6.95 0.98 0.540 Not significant

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BioEnergy Research (2023) 16:1667–1682
obtained from anaerobic digesters at the municipal waste-
water treatment plant, located in Bangkok, Thailand. The
seed inoculum was characterized to determine the pH, TS,
and VS and to be found as 7.3 ± 0.05, 65.8 ± 0.66 g L­ −1, and
−1
38.6 ± 0.45 g ­L , respectively. The seed inoculum was kept
to starve in the incubator at 35 °C and stirred at 100 RPM
for 48 h before adding the anaerobic sludge into the anaer-
obic digester of the treated biomass. Biogas productions
from untreated and treated mixed vegetable lignocellulose
solids were carried out in a batch-type sealed Erlenmeyer
flask with a 500-mL working volume containing 3% of TS
(anaerobic sludge), 3% w/v treated biomass, 10 mL of the
Nutrient Broth (NB, HiMedia, India) media, and 15 mL of
the 50 mM phosphate buffer (pH 7.0). The pH of the mix-
ture was adjusted to 7; then, the reactor was purged with
nitrogen gas to maintain an anaerobic environment. The AD
process was carried out for 45 days at a temperature of 35 °C
[21]. Biogas production was observed by using the water
displacement method. After 45 days, the biogas yield for
each day was determined and calculated for accumulated
biogas yields. The biogas production of each sample was
conducted in triplicates.
Statistical Analysis
The statistical significance for the changes in the experimen-
tal data for yields of reducing sugar, ethanol, and accumu-
lated biogas produced from untreated and pretreated samples
was performed through ANOVA and Tukey’s post hoc test
(p < 0.05) by using the SPSS software (version 28.0.0).
Results and discussion
Influence of Acid Pretreatment on Enzymatic
Saccharification of Mixed Vegetable Waste
The mixed vegetable waste samples were pretreated with
organic acids (acetic acid, citric acid, oxalic acid, and inor-
ganic acid (HCl) based on conditions designed and listed
in Table 2. After pretreatment, the solid fraction was sepa-
rated and enzymatically hydrolyzed by a mixed cellulase
enzyme. Reducing sugar concentration in the biomass
hydrolysate was measured as the indicator to determine the
pretreatment efficiency. The reducing sugar yield obtained
from organic acid pretreated samples (AA, CA, OA, HCl)
varied from 170 to 314.1 mg ­g−1, 105.7–192.8 mg ­g−1,
128–299.5 mg ­g−1, and 63.2–198 mg ­g−1 biomass, respec-
tively, whereas the sugar yield released from the untreated
sample was 99.2 mg ­g−1 biomass. The difference between
the maximum and minimum yields obtained from each set
of acid pretreatment suggested the importance of pretreat-
ment optimization.
1673
The multi-regression analysis has generated the second-
order polynomial equation for acid (AA, CA, OA, HCl)
pretreated lignocellulose biomass as shown in Eqs. 2–5.
Second-order model significance was tested by using analy-
sis of variance (ANOVA) (p-value < 0.05), lack-of-fit, and
coefficient of determination (R2). The ANOVA results for the
second-order AA, CA, OA, and HCl pretreated samples are
given in Table 3 and Supplement Table 1–3. It was noticed
that the quadratic models were significant for all pretreated
samples because the p-value was lesser than 0.05. Addi-
tionally, the p-value of lack of fit tests in AA, CA, OA, and
HCl, were 0.5406, 0.0668, 0.3947, and 0.2865, respectively,
which were greater than 0.05 suggesting the confidence of
the predicted model. Furthermore, the R2 values obtained
from the models of AA, CA, OA, and HCl were 0.7917,
0.7686, 0.8139, and 0.9368, respectively. The R2 values
greater than 0.75 suggest the reliability of the predicted
model in several studies [19, 25]. Altogether, the model’s
p-value, lack of fit test, and R2 obtained from this study
advocate the confidence of the acid pretreatment models.
Yacetic = +25.67 + 2.43 ∗ A + 0.80 ∗ B − 0.13 ∗ C − 1.56 ∗ AB
(2)
+0.60 ∗ AC + 4.56 ∗ BC − 4.63A2 + 1.33 ∗ B2 − 0.72C2
Ycitric = +11.27 + 0.18 ∗ A − 0.31 ∗ B − 1.79 ∗ C + 0.36 ∗ AB
(3)
−2.80 ∗ AC + 0.14 ∗ BC + 1.88A2 + 0.31 ∗ B2 + 2.58C2
Yoxalic = +17.41 − 1.97 ∗ A − 3.18 ∗ B + 3.36 ∗ C + 3.67 ∗ AB
(4)
−2.18 ∗ AC + 0.18 ∗ BC + 1.86A2 + 0.018 ∗ B2 + 2.68C2
Yhcl = +14.21 + 2.72 ∗ A + 1.52 ∗ B + 3.20 ∗ C + 2.47 ∗ AB
(5)
+0.42 ∗ AC − 3.45 ∗ BC + 1.38A2 − 0.1.51 ∗ B2 + 2.95C2
After the analysis of model significance, the significance
of each term model representing the pretreatment parameter
was analyzed to identify the influence of individual pretreat-
ment parameters and the interacting effect of two parameters
at a time. The significant term models (p-value < 0.1) were
selected to be used to generate the contour plots that pre-
sented the influence of each pretreatment factor and interact-
ing factors on reducing sugar yields. The interaction effect of
time, temperature, and acid concentration on reducing sugar
yield for organic acid pretreated biomass is shown in Fig. 1.
Based on the three-dimensional contour plot, it was
observed that the RS yield decreased with an increase in
temperature from 100 to 140 °C, and with an increase in acid
concentration from 2 to 10% w/v for acetic acid pretreated
samples (Fig. 1a). This sugar loss during acid pretreatment
could be due to the degradation of sugar during the intense
pretreatment condition. For the citric acid pretreatment, it
was observed that the RS yield increased as the acid con-
centration and time increased from 2 to 10% w/v and 30 to
90 min, respectively (Fig. 1b). Increased acid concentration
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1674 BioEnergy Research (2023) 16:1667–1682

Fig. 1  Contour plot of reducing sugar yield (a acetic acid; b citric acid; c, d oxalic acid; e, f HCl acid against time, temperature, and acid con-
centration)

Table 4  Optimum conditions for pretreated lignocellulose biomass using different acids
Pretreatment Time (X1, min) Temperature Acidconcentra- Predicted RS yield Observed RS yield % error
(X2, °C) tion (X3,%) ­(mgg−1 biomass) (mg ­g−1 biomass)*

Acetic acid 68.32 140 10 309.7 314.1 ± 1.27a 1.40

Citric acid (CA) 89.8 106.13 2.11 211.5 220 ± 0.08b 4.26
Oxalic acid (OA) 31 101 8.52 300.9 288.2 ± 1.46c 4.16
Hydrochloric acid (HA) 89 137 6 200.2 189.2 ± 1.06d 5.82

*The small letters presented that the data of each sample was statistically different at p-value < 0.05

13
BioEnergy Research (2023) 16:1667–1682
solubilizes hemicellulose and partially changes lignin in
plant material. The removal of the hemicellulose and lignin
layers allows cellulase enzymes easier access to the cellu-
lose layer, which leads to an increase in the RS yield [29].
After oxalic acid pretreatment, the RS yield decreased when
the reaction temperature was increased from 100 to 140 °C.
However, the RS yield increased as an increased the acid
concentration from 2 to 10% w/v (Fig. 1c, d). In the HCl-
pretreated sample (Fig. 1e, f), the RS yield increased with
an increase in temperature from 100 to 120 °C. This is due
to an increase in reaction temperature improves the solubi-
lizes of hemicellulose and partially changes lignin in plant
material. On the other hand, the RS yield decreased with an
increase in reaction time from 30 to 60 min, but the RS yield
increased when further increased the reaction time from
60 to 90 min. Furthermore, it was found that the RS yield
increased with an increase in reaction temperature from 100
to 140 °C and with an increase in acid concentration. At
lower temperatures and acid concentrations, the RS yield
was minimum due to the low efficiency of pretreatment. A
point prediction approach was employed to predict the pre-
treatment optimum conditions to obtain maximum RS yield.
Based on the generated RSM models, the predicted
yields of reducing sugars obtained from pretreated samples
by AA, CA, OA, and HCl were 309.7, 211.5, 300.9, and
200.2 mg ­g−1 biomass, respectively (Table 4). The predicted
optimum conditions were validated by performing the pre-
treatment experiments for lignocellulose biomass using dif-
ferent acids and the sugar yields obtained from the experi-
ment were measured and compared with the predicted yields.
The experimental results showed that RS yields obtained
from pretreated samples by AA, CA, OA, and HCl were
314.1, 220, 288.2, and 189.2 mg ­g−1 biomass, respectively
(Table 4). The differences in sugar yields obtained from the
Table 5  Compositional analysis of mixed vegetable biomass after
organic acid pretreatment
Sample Biomass composition (%wt)*
Cellulose Hemicellulose Lignin
Untreated biomass 20.67 ± 0.94b 16.97 ± 2.54a 31.70 ± 4.88a
AA-pretreated 22.12 ± 2.16b N.A 25.60 ± 2.03b
biomass
CA-pretreated 18.58 ± 0.16c 6.46 ± 0.49b 17.14 ± 2.38d
biomass
OA-pretreated 28.06 ± 1.25a N.A 12.98 ± 0.84e
biomass
HCl-pretreated 20.666 ± 2.96b N.A 22.71 ± 1.79c
biomass
*The small letters presented that the data of each sample was statisti-
cally different within the same column at p-value < 0.05
N.A. not available
1675
model prediction and experiments were 1.40–5.39%, sug-
gesting the high validity of the model.
The reducing sugar yields of mixed vegetable samples,
which were pretreated with CA, OA, AA, and HA, changed
significantly during the saccharification when compared to
the untreated sample. Among all acid pretreated samples,
AA-pretreated samples released the highest sugar yields at
314.1 mg ­g−1 biomass, which was higher than the untreated
sample (at 99.2 mg ­g−1 biomass) by 3.16 fold. After the
AA-pretreated sample, OA and CA pretreatments were the
second and third acids to release the highest sugar yields,
respectively. Likewise, the HCl-pretreated sample released
a higher reducing sugar yield than the untreated sample
for 1.91 fold. In this study, it was observed that the sugar
yields obtained from organic acid pretreated samples were
higher than from inorganic acid pretreated samples (HCl).
This observation is in agreement with the previous study on
organic acid pretreatment of rice straw that the saccharifica-
tion efficiency of the OA-pretreated sample was higher than
the HCl-pretreated sample [21]. However, among three types
of organic acids in previous works, OA was the best acid for
improving enzymatic saccharification in rice straw [21] and
oil palm trunk [30]. Altogether, this pretreatment experiment
and previous works revealed the importance of optimization
work for each type of biomass to maximize the efficiency of
the biorefining process.
Influence of Acid Pretreatments on Biomass
Composition and Functional Chemical Group
Rearrangements
Acid pretreatment has previously been reported to enhance
hemicellulose solubilization leading to increased glucose
yield. The composition of the organic acid pretreated bio-
mass is represented in Table 5. It is observed that the organic
acid pretreatment helped in hemicellulose removal from the
biomass when compared to the untreated sample. Addition-
ally, the lignin content of the pretreated sample has also
reduced in comparison with the untreated sample. On the
other hand, the cellulose contents in the acid pretreated bio-
mass are enriched, except for the biomass pretreated with
citric acid and hydrochloric acid. The presence of cellulose
content in CA-pretreated and HCl-pretreated samples can be
correlated with reduced sugar yields (Table 4). Chen et al.
2020 selected 4 types of organic acids for the pretreatment
of eucalyptus biomass for Kraft pulp productions and found
that all acid pretreatments showed high efficiency in the pro-
motion of reducing sugars yields and hemicellulose removal.
However, lignin was dissolved by acetic acid pretreatment,
while other acid pretreatments unexpectedly cause severe
cellulose degradation and reduction in sugar yields [31]. The
pretreatment could have disrupted the hydrogen bonds in
the cellulose layer in addition to hemicellulose and lignin
13
1676
Fig. 2  FTIR analysis for
untreated and treated lignocel-
lulose samples
removal, resulting in cellulose solubilization and reduced
sugar yield [32]. However, the increased sugar yield
obtained from the AA-pretreated and OA-pretreated sam-
ples were the results of the lignin and hemicellulose removal
through the cleavage of acetyl groups in the hemicellulose,
thereby exposing cellulose to enzymatic hydrolysis. Gener-
ally, the solubilized sugars and the degradation products of
cellulose, hemicellulose, and lignin, which formed during
the pretreatment, are mixed and solubilized in the pretreat-
ment hydrolysate. Therefore, an additional step of detoxifi-
cation of hydrolysate is recommended to remove the inhibi-
tory compounds, such as furfural, 5-hydroxymethylfurfural,
and phenolic chemicals, which may impair or inhibit the
enzymatic saccharification and microbial activity leading
to reduced sugar and bioethanol yield [32]. Similar find-
ings about the mechanism of organic acid pretreatment were
revealed by Luo et al. 2021 [33]. When eucalyptus biomass
was pretreated by glycolic acid at the optimal condition (acid
concentration 5.40%, temperature 140 °C, time 3.0 h), hemi-
cellulose was hydrolyzed to xylose at the yield of about 56%,
whereas the repolymerization of lignin was inhibited and
cellulose content was preserved and recovered.
To understand the mechanism of acid pretreatment for
improvement of enzymatic saccharification, FTIR analysis
for treated and untreated samples was conducted to observe
the alteration and rearrangement of chemical bonding within
lignocellulose biomass (Fig. 2). The primary peaks were
observed for lignocellulose samples from the FTIR analy-
sis, such as 1049 ­cm−1, 1319 ­cm−1, 1638 ­cm−1, 1725 ­cm−1,
13
BioEnergy Research (2023) 16:1667–1682
2918 ­cm−1, and 3422 ­cm−1. In pretreated samples, there was
a difference in the transmittance strength of these peaks as
compared to untreated samples. The transmittance intensity
of peak 1049 ­cm−1 decreased in acid pretreated samples,
especially CA, OA, and HCl when compared to untreated
samples. The peak of 1049 ­cm−1 in hemicellulose was attrib-
uted to the mid-strong stretching of C–O, C–C bonds, and
the mid-strong bending of C–OH bonds [34]. This observa-
tion suggested that both organic acids and inorganic acids
remove hemicellulose or cause hemicellulose hydrolysis
during the pretreatment process [33]. In comparison to the
untreated, AA, CA, and HCl pretreated biomass, the peak
intensity of 1319 ­cm−1 attributed to the C-O, C–C, and ­CH2
stretching vibrations in cellulose in the OA-pretreated sam-
ple was increased in OA pretreated biomass. The high cel-
lulose ratio left in the OA-pretreated sample obtained from
the composition analysis (Table 5) supported the results of
the FTIR analysis. The phenolic chemicals found in lignin
are responsible for the peak of 1638 ­cm−1, and the inten-
sity of this peak was reduced in pretreated samples with CA
and HCl when compared to untreated samples suggesting
the removal of lignin during acid pretreatment [21]. On the
other hand, oxalic acid showed a drop in peak intensity of
1725 ­cm−1 due to the C = O bond in hemicellulose followed
by AA, CA, HCl, and untreated samples implying the loss
in hemicellulose during acid pretreatment. The peak inten-
sity of 2918 ­cm−1 corresponding to CH stretching vibration
in the aromatic methoxyl, methyl, and methylene groups in
lignin was reduced in the acid pretreated samples, especially
BioEnergy Research (2023) 16:1667–1682
Fig. 3  Ethanol yield for treated
and untreated lignocellulose
samples. Different small alpha-
bet and capital alphabet repre-
sent the statistical difference at
p-value < 0.05
CA and AA pretreatment [5], which were well correlated
with the composition analysis (Table 5). Also, the broad
absorption peak of 3422 ­cm−1 representing the OH bond
stretching vibration in cellulose [35] was increased in AA
pretreated samples when compared to untreated samples
suggesting the enrichment of cellulose and it was subse-
quently hydrolyzed to high yields of reducing sugars.
Bioethanol Production from Mixed Vegetable
Lignocellulose Biomass
Figure 3 depicts the bioethanol production from mixed
vegetable biomass using the fermentation process with S.
cerevisiae TISTR5606 and S. cerevisiae TISTR5596. Using
various acid pretreatment, the ethanol yield produced from
each pretreated sample was enhanced when compared to
untreated biomass. Using S. cerevisiae TISTR5606, the
highest ethanol yield at 7.60 g ­L−1 was obtained from the
OA-pretreated sample, which was equivalent to 86.36% of
theoretical yields, followed by CA, AA, HCl, and untreated
samples, respectively. In this study, pretreatment of mixed
vegetable waste with OA improved ethanol yield by 2.75-
fold time compared to the untreated sample. It was noted
that AA pretreatment provided the highest reducing sugar
yield, while OA pretreatment was the best option for eth-
anol production. This finding was different from the pre-
vious study [30] that reported the effect of AA, OA, and
1677
CA pretreatment on the oil palm trunk. The highest sugar
yield was obtained from OA-pretreated palm trunk, while
the highest ethanol yield was obtained from CA-pretreated
sample. This anomaly observation, in which the OA-pre-
treated sample was not the method promoting the highest
sugar yields from enzymatic saccharification (Table 4) but
producing the highest ethanol yields from fermentation,
could be hypothesized to be the results of the consump-
tion of remaining oxalic acid in pretreated biomass by yeast
for ethanol production via TCA cycle. Zhang et al. applied
oxalic acid pretreatment on wheat straw and corn stover to
improve ethanol production up to 9.9% and 9.5%, v/v. This
study proposed the explanation that oxalic acid functions as
the catalyst for lignocellulose hydrolysis and simultaneously
degrade the inhibitory byproducts and finally oxalic acid was
consumed by microbes at low pH condition [36].
Similar trends in the effect of each acid on ethanol pro-
duction from S. cerevisiae TISTR5606 and S. cerevisiae
TISTR5596 were observed, although the ethanol yield
obtained from S. cerevisiae TISTR5596 was less than from
S. cerevisiae TISTR5606. The efficiency of ethanol pro-
duction of different yeast strains could be due to the char-
acteristics of yeast and biomass. Although S. cerevisiae
TISTR5606 and S. cerevisiae TISTR5596 were obtained
from alcohol factories using sugarcane molasses as sub-
strate, they were further applied for ethanol production in
the fermentation processes of fruit waste [37] and sorghum
13
1678
straw [38], respectively. Therefore, the results reported in
our work could be used as a guideline to select the yeast
strain to improve ethanol production and suggested that
both strains have the potential for 2nd generation ethanol
production.
Several biomass byproducts were identified and charac-
terized as inhibitors to hydrolysis enzymes and microbial
activities. The effects of remaining inhibitory byproducts
generated during acid pretreatment on bioethanol produc-
tion were demonstrated previously [39]. The study reported
that unwashed samples obtained the minimum reducing
sugar yield and ethanol after the saccharification and fer-
mentation process. This is due to the inhibition caused
by furfural and 5-hydroxymethylfurfural bound to the
biomass. Also, the furfural and 5- hydroxymethylfurfural
exhibited a stronger inhibitory effect on enzymes than
formic acid, acetic acid, and phenolic acid compounds
produced during the pretreatment. Furthermore, CA-pre-
treatment on rice straw was demonstrated to generate a
low concentration of furan and phenolic compounds, which
subsequently improved ethanol production when compared
to untreated rice straw [40]. The difference between acid
pretreatment on sugar yield and ethanol yield of various
types of biomass highlight the significance of optimization
and tailor-made process for each feedstock to establish a
highly efficient process. A similar finding was observed
by Marsden et al. [41] that CA-pretreated biomass had
Fig. 4  Cumulative biogas
production from treated and
untreated lignocellulose sam-
ples. Different small alphabets
represent the statistical differ-
ence at p-value < 0.05
13
BioEnergy Research (2023) 16:1667–1682
higher ethanol yields than AA-pretreated biomass. For
ethanol production, wild yeast species, e.g., S. cerevisiae,
depend on C6 carbohydrates, because native yeast species
are incapable of converting xylose to ethanol during the
fermentation process. However, the commercialization of
genetically modified yeast capable of fermenting C5 car-
bohydrates to ethanol has yet to occur [42]. Alternatively,
co-culturing of yeast strains, S. cerevisiae and Pichia stip-
ites, which can ferment C6 and C5 sugars, respectively, is
proposed to be the solution to fully utilize lignocellulose
biomass for the production of ethanol. Similarly, organic
acid pretreatment of cotton gin by using lactic acid, oxalic
acid, citric acid, and maleic acid is optimized to maximize
ethanol production from the co-culturing of S. cerevisiae
and Pichia stipites. It was demonstrated that lignin in
maleic acid-pretreated biomass was removed up to 88%,
686.13 g/g saccharification yield was obtained, and the
highest ethanol yield at 18.74 g/l was achieved when using
an optimum pretreatment condition [43]. Comparatively,
based on the results of our work (using mixed vegetable
waste) and other works (using a single type of biomass) on
organic acid pretreatment [21, 30, 36, 40, 41, 43], it was
demonstrated that the optimization experiment of pretreat-
ment parameters, including type of organic acid, pretreat-
ment time, pretreatment temperature, and acid concentra-
tion was necessary to select the suitable method with high
efficiency for each type of biomass. Also, a single acid for
BioEnergy Research (2023) 16:1667–1682
pretreatment is not recommended for all types of biomass
before preliminary experiments or optimization experi-
ments are conducted. Furthermore, when using one single
type of biomass, it was demonstrated that different types
of organic acids were specifically suitable for improvement
of enzymatic saccharification, ethanol fermentation, and
biogas production suggesting the crucial role of optimiza-
tion experiment of each type of biomass.
Biogas Production from Mixed Vegetable
Lignocellulose Biomass
The influence of organic acid pretreatment on biogas produc-
tion from mixed vegetable biomass using an anaerobic pro-
cess was investigated. The anaerobic process was carried out
for the pretreated samples, and the pH value of the samples
was adjusted to 7.0 using 1 N NaOH. The pH condition plays
an important role in biogas production and can cause the
system to fail during the reactor startup stage. As a result,
pH neutralization to 7.0 is required for biogas production
during anaerobic digestion of mixed vegetable lignocellulose
biomass. The cumulative biogas yield for untreated, acetic,
citric, oxalic, and hydrochloric acid pretreated solids varied
as 276.5 mL, 634 mL, 1394 mL, 1098 mL, and 469.5 mL,
respectively (Fig. 4). After 35 days of the AD process, the
biogas yield of acetic, citric, oxalic, and hydrochloric acid
pretreated biomass increased by 2.29, 5.04, 3.97, and 1.69
times, respectively, as compared to the untreated sample.
The highest biogas yield (equivalent to 110.72 mL/g-
VS) was found in the following order: citric acid > oxalic
acid > acetic acid > HCl acid > untreated sample (equivalent
to 21.96 mL/g-VS). This is because of the maximum sugar
content remaining in the biomass during pretreatment when
compared to other pretreated samples. Further, the degree of
sugar solubilization is dependent on the pretreatment operat-
ing parameters.
In previous reports, the CA pretreatment on rice straw and
palm oil trunk was demonstrated to be a more effective method
to enhance biogas production than other acid pretreatments
(AA, OA, and HCl) [21, 30]. In comparison to the other treated
and untreated samples, CA pretreatment caused chemical and
compositional changes in the lignin, hemicelluloses, and cel-
lulose of lignocellulose to be more susceptible to enzymatic
hydrolysis [14]. Unlike ethanol production, when using wild
yeast strain for fermentation, only C6 sugars are consumed and
converted to ethanol; the AD process overcomes the disadvan-
tages of fermentation by utilizing C5, C6 sugars, and VFA as
substrates by the functions of microbial consortium in AD
reactor. The efficiency of acid pretreatment is governed by the
severity of pretreatment conditions, which are depending on
the type of chemical and type of biomass. The pretreatments
of winery waste, cotton gin waste, olive pomace, and juice
industry waste by using eight different types of chemicals were
1679
optimized to improve methane production. It was demonstrated
that each pretreatment chemical needs optimization to obtain
appropriate pretreatment severity to improve methane produc-
tion because each chemical has a specific mechanism for pre-
treatment. For example, the moderate severity of citric acid and
­H2O2 pretreatment conditions was used; the methane yield from
winery waste was maximized up to 163.7 and 190.2 mL/g-VS,
respectively. On the other hand, using ethanol pretreatment pre-
ferred a more severe condition to be a highly effective pretreat-
ment [44]. Although in this study, the digestate of the biogas
reactor was not further analyzed for its qualitative values, it has
been proposed in several works that the digestate obtained after
pretreatment and anaerobic digestion were applied as feedstocks
for microalgae cultivation [45] and as fertilizers of plants [46].
The sustainable processes to utilize the anaerobic digestate of
lignocellulose biomass for the production of algal biomass for
further biorefining process of other value-added products were
reviewed recently by Javed et al. 2022 [47]. The main bottle-
neck of the utilization of lignocellulose digestate was described
to be the lacking of an efficient pretreatment method to improve
the conversion of lignocellulose biomass [47].
ANN Results in Comparison with Experimental
Values
In this study, the ANN models were additionally used to predict
the reducing sugar yield of pretreated lignocellulose biomass
with time, temperature, and acid concentration as input vari-
ables. The tansigmoid transfer function was adopted for both
hidden and output layer neurons in the selected feed-forward
network [1]. The transfer function gives neural network non-
linearity, which makes it more efficient than the linear trans-
formations and the performance function to be optimized by
mean square error. The numbers of neurons in the hidden layer
were increased from two to twenty-nine for each optimal itera-
tion. The MSE was used as a criterion throughout the topology
research. The neural network was built and trained by using the
data given in Table 2. The ANN is built using 29 data points in
total. The network is trained with 70% of the total experimental
data, while the rest of the data was used for model testing and
validation. Prior to the training session, the input and target val-
ues are normalized. The goal of the normalization is to prevent
overflow situations. The neural network training is forced to
stop when the network error (MSE) decreases to a low number
(MSE ≤ Eo). In the present study, the training came to end after
9 iterations (epochs) [20].
The optimal architecture for reducing sugar yield with acetic
acid pretreated biomass was found to be three neurons in the
input layer, sixteen neurons in the hidden layer, and two neu-
rons in the output layer (3–16-1), while the optimal architecture
for RS yield pretreated with citric acid was found to be three
neurons in the input layer, eighteen neurons in the hidden layer,
and two neurons in the output layer (3–18-1). For oxalic acid
13
1680
pretreated biomass, the optimum architecture for RS was three
neurons in the input layer, fourteen in the hidden layer, and one
in the output layer (3–17-1), whereas the optimum architecture
for RS yield pretreated with HCl was found to be three neurons
in the input layers, nineteen neurons in the hidden layer, and one
neuron in the output layer (3–19-1). The optimum architecture
for the performance model was found to be (4–16-3), whereas
the optimal architecture for the emission model was found to
be (4–14-6) [1].
The generated ANN model is significant and may be
utilized to predict the best topology, as evidenced by the
strong correlation coefficient (R2 = 0.997) and adjusted cor-
relation coefficient (R2 = 0.987) is in reasonable agreement
with each other. The degree of agreement between experi-
mental and model-predicted values shows that the major-
ity of the experimental results lie on the 45-degree line,
indicating that the predicted results are extremely similar to
the experimental data. The generalization potentiality of the
developed ANN model was increased by using the follow-
ing techniques such as normalization, the ideal numbers of
neurons, and separating the target data into training, valida-
tion, and test data subsets [22]. The result reveals that the
R and RMSE values of RS yield pretreated with different
acids are in the range of 0.921–0.986 and 0.0123–0.0166,
respectively. Hence, the ANN model can be utilized as an
effective tool for modeling and predicting RS yield from
lignocellulose vegetable waste pretreated with organic and
inorganic acids. Das et al. [20] optimized the enzymatic
saccharification of water hyacinth biomass for bioethanol
production using the ANN and RSM approach. The study
revealed that the correlation coefficient values were in the
range of 0.98 to 0.99. Similarly, Neto et al. [48] observed
correlation coefficient values between 0.68 and 0.99 in their
research work. If the R value is close to 1, it seems that the
ANN model has a lower error percentage.
The overall correlation coefficient of the developed network
for RS yield pretreated with acetic acids were analyzed and
showed that the R values in training, testing, and validation were
0.97, 0.97, and 0.98, respectively, with an overall R value of
0.97. A similar finding was observed from the previous research
works. Kumari et al. [49] optimized the pretreatment process for
Leucaena leucocephala using the ANN approach. It found that
the LM neural network model has better prediction with MSE
and R values of 0.07 and 0.99 respectively. Similarly, Esfand-
yari et al. [50] used the BPFFNN network for the prediction of
Fisher-Tropsch synthesis of natural gas with carbon monoxide
using an aluminium oxide catalyst. The study revealed that (4–7-
9–3) was the optimal architecture with a correlation coefficient
and an RMSE value of 0.93 and 0.66, respectively. Another
study reported the R values in training, testing, and validation
of ANN emission were 0.99, 0.98, and 0.98 respectively, with an
overall R value of 0.99. The ANN models were in good agree-
ment with statistical error measurement.
13
BioEnergy Research (2023) 16:1667–1682
Conclusion
The effect of organic acid pretreatment on mixed vegetable
waste conversion into bioethanol and biogas was modeled and
experimentally investigated based on RSM and ANN tech-
niques. The predicted reducing sugar yields obtained from
RSM are relatively nearer to the experimental results, and
low error percentage, e.g., 1.4% error in prediction of acetic
acid pretreated sample. The Levenberg–Marquardt algorithms
along with transigmoid transfer function have accurately pre-
dicted the RS yield, which was in good agreement with experi-
mental results. In this study, the highest conversion yield of
reducing sugar at 314.1 ± 1.27 mg/g biomass was obtained
from acetic acid-pretreated sample, whereas the highest yields
of ethanol (7.60 g/L, equivalent to 86.36% of theoretical yields)
and biogas (110.72 mL/g-VS) were obtained in oxalic acid and
citric acid pretreated samples, respectively. In addition, organic
acid pretreatment has the advantage of breakdown the inhibi-
tory chemicals, which obstruct enzyme and microbial action.
Also, organic acids could be additionally consumed by micro-
bial fermentation to increase the yields of the targeted product.
Therefore, organic acid has been suggested for the pretreatment
of mixed vegetable wastes as a greener chemical for biofuel
production to reduce the dependency on using a single type of
biomass that is often used in current R&D activities.
Supplementary Information The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s12155-​022-​10517-y.
Funding This work was financially supported by King Mongkut’s
University of Technology North Bangkok (Grant Contract Nos.
KMUTNB-Post-65–09, KMUTNB-66-KNOW-03) and Srinakharin-
wirot University (Grant Contract No. 410/2565-SWU).
Declarations
Conflict of interest The authors declare no competing interests.
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