Energy Conversion and Management: X 26 (2025) 100933

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Energy Conversion and Management: X

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Bioethanol as an alternative fuels: A review on production strategies and

technique for analysis
Mona Fatin Syazwanee Mohamed Ghazali a ,∗, Muskhazli Mustafa b
a
Program ASASIpintar, Pusat PERMATA@Pintar Negara, Universiti Kebangsaan Malaysia, UKM Bangi, 43600, Selangor, Malaysia
b
Department of Biology, Faculty of Science, Universiti Putra Malaysia, Serdang, 43400, Selangor, Malaysia

ARTICLE INFO ABSTRACT

Keywords: Bioethanol production represents an alternative source of energy that also helps minimize greenhouse gas
Analytical techniques effects. Currently, the focus of advanced technologies for bioethanol production is on the conversion of
Bioethanol lignocellulosic biomass into renewable energy for transportation as it offers a low cost of investment and non-
Bioconversion
pollution bioprocesses. However, the utilization of lignocellulosic biomass has several challenges, including
Lignocellulosic Biomass
the high cost of pretreatment, the recalcitrant nature of the biomass and the requirement for robust microbes
Gas chromatography
to ferment various types of sugars. Informations on the subject were achieved through a literature search
using various electronic databases such as Google Scholar, ScienceDirect, Scopus, and others. From literature
findings, few strategies such as separate hydrolysis and fermentation (SHF), simultaneous saccharification and
fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF), and consolidated bioprocessing
(CBP); were found established to overcome these challenges, ultimately increasing the effectiveness of the
bioconversion process and minimizing the overall cost of production. CBP was found to be the most promising
strategy as direct production of ethanol from pretreated corn cob yielded 11.1 g/L ethanol without the addition
of external hydrolytic catalyst. Various analytical techniques are commonly used to quantify bioethanol in
a sample, and these methods were theoretically analyzed in relation to established theories. Currently, gas
chromatography is known to be the most effective approach with limits of detection typically around 0.099
mg/mL, demonstrating excellent linearity and recovery rates between 91% and 109%. This paper aims to
highlight the efficiency of every strategy involved in the bioconversion process and provide insights into
every suitable analytical technique that can be employed to ensure the sustainability of biofuel by allowing
researchers to improve the productivity and quality of bioethanol, thus promoting its role as a feasible
alternative fuel.

1. Introduction considered suitable for producing bioethanol, as they are largely avail-
able in terms of quantities at very low prices, which is advantageous
With the depletion of oil reserves due to the high consumption of over the use of food-based feedstocks, as they reduce competition with
fuels by transportation and industry, the demand for alternative fuels the food supply [5]. The biomass components are influenced by sev-
from renewable resources has increased over the years [1]. One of
eral factors and are reasonably stable: 35%–50% cellulose, 20%–40%
the suggested alternative fuels for transportation is bioethanol, as this
hemicellulose and 15%–30% lignin [6]. The most common approach in
biofuel can reduce greenhouse gas emissions by up to 75% compared
with fossil fuels [2]. Bioethanol can be derived from various sources, conversion technologies is biochemical conversion, which is performed
such as wheat, corn, lignocellulose agro waste, genetically modified in sequential phases: pretreatment, enzymatic hydrolysis and fermenta-
crops or even microalgae [3]. tion [7]. Pretreatment is the most crucial step to improve the efficiency
According to the 7th Sustainable Development Goal of the United of enzymatic saccharification process and minimize the inhibitory effect
Nations (SDG: Affordable and Clean Energy), to maintain food prices of lignin by removing parts of lignin content to increase the accessibility
and supply, the transition toward second-, third- and fourth-generation of substrate surface area to the hydrolytic enzymes such as cellulase [8].
biofuels is the best strategy, as this transition helps reduce adverse The next stage is enzymatic hydrolysis, in which fermentable monosac-
impacts on the environment, mitigate climate change and provide af-
charides are released from the lignocellulosic structure of cellulose
fordable and clean energy for all [4]. Lignocellulose based resources are

∗ Corresponding author.
E-mail address: monafatin@ukm.edu.my (M.F.S.M. Ghazali).

https://doi.org/10.1016/j.ecmx.2025.100933
Received 6 November 2024; Received in revised form 7 February 2025; Accepted 15 February 2025
Available online 24 February 2025
2590-1745/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

and hemicellulose [9]. After enzymatic hydrolysis, the fermentable 3. Type of feedstock for bioethanol production
sugars are ready to be converted into various fermented products,
such as bioethanol. Both conversion processes (enzymatic hydroly- Bioethanol production can be classified into three generations on
sis and fermentation) can be performed via several strategies and at the basis of the types of feedstocks used during the bioconversion
different levels of integration. The main strategies involved are simul- process: (i) first-generation bioethanol, developed from food-based
taneous saccharification and fermentation (SSF), separate hydrolysis crops; (ii) second-generation bioethanol, developed from lignocellu-
and fermentation (SHF), simultaneous saccharification and cofermen- losic biomass or municipal solid wastes; and (iii) third-generation
tation (SSCF) and consolidated bioprocessing (CBP). These conversion bioethanol, which utilizes CO2 as feedstock.
strategies can be accomplished under different modes of operation,
such as continuous mode, batch mode and fed batch mode [10]. 3.1. First generation of bioethanol
The accuracy and precision of ethanol measurement are particularly
necessary in a wide range of biotechnological processes and industries, According to Renewable Fuel Association, leading countries such
such as pharmaceutical, cosmetics, food and alcohol beverages. Several as United States and Brazil are known to be the main contributor for
analytical techniques can be applied to quantify the alcohol content bioethanol production with an estimation of 13.9 billion gallons (53%)
to ensure that the highest standard of quality is based on physical and 8.08 billion gallons (31%), respectively, but the total number of
(hydrometry, densitometry, pycnometry and refractometry), chemical productions has been decreased to around 2.9 billion gallons due to
(dichromate assay), spectrometric and chromatographic analyses. The the COVID-19 crisis [15]. The first generation of bioethanol (1G) has
official method for the quantification of ethanol in alcoholic beverages increased the value of biofuel in terms of cost and energy efficiency
is pycnometry, which requires a well-balanced and controlled temper- as well as reducing greenhouse gas (GHG) emissions [16,17]. Typi-
ature, and this method was designed by the Association of Official cally, this generation of bioethanol originates from the fermentation
Analytical Chemists (AOAC) [11]. The long and slow process of ethanol of biomass with high content of sugar (i.e. sugar beet and sugar
fermentation, especially on industrial scale normally takes 48–72 h to cane) and/or starch (i.e. grain sorghum, barley, corn, wheat and rhi-
complete, hence, requires an instant quantitative method which is suit- zome) [18]. However, the production of 1G bioethanol has influenced
able to monitor the quality of ethanol produced and rapidly verifying the socioeconomic aspects of biorefinery and is considered unethical
the on-going process of fermentation [12]. Existing instruments avail- as it competes with the food supply, arable lands and natural resource
able for qualitative and quantitative measurement for ethanol analysis input; creating a food versus fuel scenario [19].
not only have high accuracy and precision, but also complex, expensive Fig. 1 shows there are over 200 corn-based bioethanol plants in the
and require trained personnel to operate the instruments [13]. Hence, United States that produces 15 billion gallons of bioethanol compared
this review aims to deliver comprehensive information on the types of to 10 cellulose-based bioethanol plants, which can only produce ap-
feedstocks used for every bioethanol generations, deeper understanding proximately a half-billion gallons per year [21]. To meet half of the
on every strategy available for bioconversion process to improve the transport fuel necessity, the US biofuel industry highly depends on the
production of bioethanol, and offering critical insights on the properties utilization of energy crops which demand large areas, estimated around
of analytical technique to ensure the quality of bioethanol, thus con- 24% of the total cropland for agricultural activities [22,23]. Growing
tributing towards the advancements in bioethanol technology and also concern over food security due to the increasing use of arable land for
promoting the sustainability of biofuels in the global energy landscape. cultivation of energy crops as feedstock has led to the development of
bioethanol from lignocellulosic biomass known as second-generation
2. Methodology bioethanol (2G).

Information and analytical data on the subject were achieved 3.2. Second generation of bioethanol
through a literature search conducted for publications using vari-
ous electronic databases such as Google Scholar, Scopus, ScienceDi- Growing concern over food security due to the increasing use of
rect, PubMed and others. The date range of the publications selected arable land for cultivation of energy crops as a feedstock and impact
for this review was from 2019 to 2024; this range was chosen to on biodiversity has led to the development of bioethanol from ligno-
make sure the information gathered is validated and updated. The cellulosic biomass (i.e. municipal, agricultural, forest and industrial
keywords ‘bioethanol production’, ‘bioconversion process’, ‘lignocel- wastes) known as second-generation bioethanol (2G). These feedstocks
lulosic biomass’ and ‘analytical techniques’ were used as primary are inexpensive, have no issue with food sustainability and require no
searches; combined using Boolean operators ‘‘OR’’ and ‘‘AND’’. The in- extra land. Globally, approximately 181.5 billion tons of lignocellulosic
clusion/exclusion criteria were used as a guideline during the selection biomass are produced in a year but only 8.2 billion tons are currently
process to make sure inclusion in this review comprises of only relevant utilized [24]. Lignocellulosic biomass (LCB), such as forest residues
studies and related to the theoretical basis of the selected keywords which mostly are woody residues, agricultural waste (paddy straw, corn
used in the search engine. While studies that do not align with the stover, sugarcane bagasse, and wheat straw) and perennial grasses, is
objectives of this review were excluded from the selection. The article suitable for producing 2G biofuels [25].
choice was made by only considering English-language publications. Agricultural wastes are rich in lignocellulose materials and con-
The information from any publication focusing on current generations tain mainly cellulose, hemicellulose, extractives and lignin as seen
of bioethanol production, strategies for bioconversion process and in Table 1. The components of LCB (refer Fig. 2) primarily consists
techniques for bioethanol analysis were gathered and then analyzed by of cellulose (40%–60%), hemicellulose (20%–40%) and lignin (10%–
summarizing the advantages, weaknesses, the main features and key as- 25%), as well as small fraction of extractive, ash and protein [36].
pects on each method of analytical processes. The methodology of this The technology applied for this generation allows the cellulose and
study emphasizes theoretical analysis, which strengthens the quality of hemicellulose fractions of a plant to be separated from lignin to al-
research by providing structure, fostering critical evaluation, guiding low the fermentation of cellulose and hemicellulose into cellulosic
methodological choices, and facilitating new insights into bioethanol ethanol [37,38]. Second-generation of bioethanol has become a favor-
production processes. Integrating a theoretical perspective enhances able fuel owing to its clean combustion properties, low toxicity and
the rigor of the review by establishing a foundation for evaluating volatility; making this generation of bioethanol suitable to address the
existing studies. This approach also enables researchers to critically environmental and social issues [39]. However, 2G bioethanol requires
assess how well previous research aligns with established theories, careful evaluation in the aspect of energy consumption, impact on
thereby identifying gaps and inconsistencies in literature [14]. the environment, maintenance involves throughout the process and is

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M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

Fig. 1. The production of 1G bioethanol by top leading countries in 2023 [20].

Fig. 2. The primary components in lignocellulosic biomass [26].

limited by the growing production costs as the substrates need to be 3.3. Third generation of bioethanol
pretreated before the fermentation process can take place [40]. Besides,
the biological limitations in the 2G bioethanol often result in more Third generation of bioethanol (3G) utilizes microorganisms as feed-
diluted ethanol products compared to 1G bioethanol. This generation stock while fourth generation of bioethanol (4G) involves genetically
struggles to obtain the minimum amount of 40 g/L or more in a modified microorganisms to attain a desirable amount of hydrogen to
standalone process while 1G bioethanol can produce up to 80–115 g/L carbon (HC) output and create artificial carbon sink to minimize or
from fermentation broth [17,41]. Hence, modifications have been made eliminate carbon emissions [55]. However, these last two generations
in the development of third-generation biofuels to compensate for 2G of bioethanol are still in early stage of development. Typically, third-
biofuels. generation biofuels (3G) are derived from algal biomass owing to

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M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

Table 1 direct evolution, to increase productivity and output in the production

The composition of lignocellulosic biomass for the production of 2G biofuels. of biofuels [64]. The most promising option is the use of bioengineered
LCB Materials Cellulose (%) Hemicellulose (%) Lignin (%) References plants with few modifications, such as destruction of lignin enzymes,
Banana stem 30.08 27.8 6.1 [27] increasing the level of polysaccharides or the growth of plant biomass
Banana leaf 39.2 22.4 16.5 [27] and removing polysaccharides from the plant cell wall [65]. The de-
Corn stover 40.7 31.1 11.7 [28]
velopment of bioengineered crops as feedstocks with modifications to
Empty fruit bunch 46.5 33.8 32.5 [29]
Hazelnut shell 30.0 35.0 32.0 [30] the structure of the plant cell wall can potentially reduce the cost of
Sugarcane molasses 38.4 24.3 26.0 [31] pretreatment and hydrolysis processes [66]. In addition, altering the
Sweet sorghum 37.7 28.1 21.5 [32] lignin profile via targeted lignin biosynthetic genes using the use of
Switchgrass 39.4 20.3 21.2 [33] new technology can accelerate the overall biomass process [67]. In
Rice straw 24.0 27.8 13.5 [34]
Wheat straw 45.4 36.5 21.6 [35]
addition, metabolic engineering and genome editing techniques also
help in the development of specific designed crops that can produce
high-quality biocrude that is rich in alkenes and ketones to produce
biofuels [63]. This type of feedstock offers significant potential in the
their properties, such as high starch and lipid contents, rapid growth future development of biofuels by improving sugar yields, enhancing
rates, ease of cultivation, high carbon dioxide absorption and low biomass characteristics and enabling cultivation under stress condi-
land usage [56]. Red algae, green algae, brown algae, cyanobacteria tions. However, overcoming the public perception of bioengineered
(blue–green algae) and diatoms are recognized as the major groups plants as energy crops, especially concerning food security and impacts
in microalgae for bioethanol feedstocks. Among these, cyanobacteria on the environment, is important before considering the potential of 5G
are the most efficient group for manufacturing 3G bioethanol [57]. biofuels.
Fast-growing algae are usually known to have low oil content while
high-lipid algae species have slower growth rate, hence, it is important 4. Strategies for bioconversion
to select the correct species with high biomass and lipid concentrations
to ensure the efficiency of 3G bioethanol production [58]. Most impor- The recent fluctuation of fossil fuel prices and complete shift to-
tantly, productive cultivation of algae requires a direct supply of CO2 wards developing green energies have contributed to a new approach in
which can be obtained from atmospheric carbon capture or industrial biomass conversion techniques [68]. Significant studies have classified
emitters for photosynthesis and eventually for bioethanol production 4 important process configurations for a more effective bioconver-
with negative carbon footprint [23]. An example of microalgae species sion process for bioethanol production such as separate hydrolysis
such as Chlorella sp. is considered as possible microalgal feedstock for and fermentation (SHF), simultaneous saccharification and fermenta-
bioethanol production, where this species was capable to generate up tion (SSF), simultaneous saccharification and cofermentation (SSCF),
to 11 g/L of bioethanol using hydrolytic enzymes (i.e. 𝛼-amylase and and consolidated bioprocessing (CBP) [69]. Normally, the process of
glucoamylase) [51]. Besides, studies also reveal that macroalgae has substrate saccharification and ethanol fermentation are achieved sep-
better potential to produce high amounts of bioethanol by utilizing only arately but can also be accomplished efficiently by performing both
0.43 g/g substrate compared to land feedstock and consume less time processes in the same reactor, thus eliminating the possibility of in-
of production [59]. A comparison between different feedstock utilized hibitory sugars accumulation, avoid inhibition of the end-product and
for the development of every generation of bioethanol can be observed reduce the risk of contamination in the medium [70].
in Table 2.
4.1. Separate hydrolysis and fermentation (SHF)
3.4. Fourth generation of bioethanol
This process configuration involves a traditional strategy in which
the substrate is subjected to saccharification process and followed
Fourth-generation biofuels (4G) are produced from bioengineered
by fermentation of glucose to ethanol in separate reactors [71]. The
algae (i.e. Bacillariophyceae (diatoms), Eustigmatophyte, Chlorophyceae,
strategy of separating the saccharification process from fermentation is
and Chrysophyceae) with the ability to capture carbon in their leaves
to allow the enzymes to operate at high temperature while operating
and branches as a feedstock for biofuel production [60]. The ge-
the fermentation of ethanol at a moderate temperature for optimum
netic modification in microalgae intended to enhance certain traits
performance and increase the productivity of ethanol [72]. This process
in microalgae and optimized features to improve the production of is typically the most preferable method because it allows flexibility in
bioethanol such as improvised photosynthesis and carbon fixation, choosing the hydrolysis process and provides optimized conditions for
increase the synthesis of lipid and ability to utilize diverse sugar enzymes, catalysts or even fermenting microorganisms in both saccha-
especially pentoses and hexoses [61]. This generation of bioethanol rification and fermentation processes [73,74]. However, this approach
includes an emerging field in which the direct conversion of solar en- results in low efficiency of ethanol production due to the time delay
ergy into biofuel via designed microorganisms based on new synthetic between sugar release and fermentation as high sugar concentrations
techniques [62]. However, the development of 4G bioethanol has a can inhibit the performance of the enzymes [75]. Besides, there are sev-
negative impact on the environment because the release of transgenic eral major drawbacks for SHF including high-cost of operations caused
agents can disrupt the ecological balance of a habitat [40]. by having two separate reactors, enzyme inhibition by end-products,
possible microbial contamination owing to long hydrolysis period and
3.5. The future of bioethanol generation sterilizing them in a large-scale process remains challenging [76,77].
A study by [78], shows the process of saccharification involving 1.0%
Currently, four generations of biofuels have been developed, but NaOH-pretreated rice straw using 200 FPU/mL extracted crude enzyme
their availability in the world’s market relies on the support of the from Aspergillus fumigatus releasing 22.15 g/L reducing sugars in 20 h
government to provide tax credit incentives and energy subsidies [63]. and resulted in 9.45 g/L ethanol during fermentation process with
Every generation of biofuels have their advantages and disadvantages Saccharomyces tanninophilus. Besides, there is also a comparison study
from many aspects (Fig. 3). The future is expecting the development between SHF and SSF approaches using S. cerevisiae; which indicated
of the next generation of biofuels to compensate for the weaknesses SHF as a better method with a higher ethanol concentration (48.72 g/L)
of the previous generations, which constitute the fifth generation of as compared to SSF method which was able to produce 29.59 g/L after
biofuels (5G). This generation is expected to utilize knowledge of 48 h, but SHF is still unfavorable due to having longer total time for
synthetic biology, such as genome editing, genetic engineering and bioethanol production [79].

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Table 2
Comparative analysis between feedstocks used for different generations of biofuels production.
Genera- Feedstocks Pretreatment Fermentation conditions Maximum ethanol References
tion of concentrations (g/L)
biofuels
1G Barley Gelatinized with boiled distilled water (36%, w/v) 37 ◦ C, 72 h 78.48 [18]
Sugarcane Autoclaved at 115 ◦ C for 20 min 30 ◦ C, 33 h 81.59 [42]
Sweet sorghum Milled using extractor and heated to increase solid 30 ◦ C, 60 h 113.3 [43]
content
Wheat Gelatinized with boiled distilled water (36%, w/v) at 37 ◦ C, 168 h 72.1 [44]
70 ◦ C for 10 min
2G Corn stover Steam + sodium carbonate (Na2 CO3 ) 37 ◦ C, 72 h 76.8 [45]
Onion waste Mechanical + autoclaving 86 h, 30 ◦ C, pH 5 30.56 [46]
Palm wood 3% sulfuric acid (H2 SO4 ), 3% nitric acid and 3% 45 ◦ C, 84 h, pH 5 22.90 [47]
phosphoric acid separately with solid to liquid ratio of
1:10 (w/v) + 2% sodium hydroxide (NaOH)
Rice straw N-methylmorpholine N-oxide-(NMMO) and phosphoric 45 ◦ C, 72 h 63.4 [48]
acid (H3 PO4 ) pretreatment
Sugarcane molasses Pretreated with 99.8% H2 SO4 30 ◦ C, 72 h, pH 5.5 56 [49]
Wheat straw NaOH/hydrogen peroxide (H2 O2 ) for pretreatment 37 ◦ C, 96 h 31.1 [50]
3G Microalgae (Chlorella sp.) Pretreated with 1.5% H2 SO4 combined with 𝛼-amylase 117 ◦ C, 20 min 11 [51]
and glucoamylase
Dried seaweed (Gracilaria sp.) 0.1M H2 SO4 45 ◦ C, 18 h, pH 4.5 3.84 [52]
Spirulina (Arthrospira platensis) 2% NaOH for 3 days of pretreatment time 35 ◦ C, 96 h 35.52 [53]
Bloom-forming cyanobacteria 0.5N H2 SO4 for 4 h at temperature 120 ◦ C 30 ◦ C, 43.6 h 18.57 [54]
(Microcystis sp.)

Fig. 3. The advantages and disadvantages of every generation of bioethanol production.

4.2. Simultaneous saccharification and fermentation (SSF) process encountered during the process such as difficulty to find compromised
optimal temperature range between saccharification (50 ◦ C) and fer-
Simultaneous saccharification and fermentation (SSF) is the first mentation (28–37 ◦ C), and simultaneously monitor several parameters
bioprocess that integrated both hydrolysis and fermentation into a sin- of bioprocess [82]. Moreover, a slower rate of hydrolysis may result
gle vessel and has overcome the inhibitory compounds from enzymatic in lower sugar concentrations compared to SHF configuration [83].
hydrolysis, thus increased cost efficiency and minimizes the residence Another drawback of this bioprocess is that most fermentative mi-
time during bioethanol production [80]. Owing to these advantages, croorganisms, such as S. cerevisiae, are not capable to ferment xylose
SSF has been broadly studied for the production of butanol and ethanol efficiently since glucose have a stronger affinity compared to xylose
from lignocellulosic waste materials [81]. Alternatively, SSF is con- in the microbial transport system, thus glucose concentration must be
sidered as a promising microbial technology, but several problems lowered to allow more absorption of xylose [84].

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4.3. Simultaneous saccharification and co-fermentation (SSCF) 4.5. Ideal bioconversion strategy

Another bioconversion process that can produce bioethanol is si- The ideal bioconversion strategy involves various saccharification
multaneous saccharification and cofermentation (SSCF), which is also and fermentation processes used in cellulose-based ethanol production,
similar to the SSF approach. In this method, the saccharification process such as SHF, SSF, SSCF and CBP (Fig. 4). The SSF approach has been
of the feedstock and the fermentation of both pentose and hexose shown to produce more bioethanol than the SHF process does, but this
sugars from hemicellulose and cellulose, respectively, are simultane- strategy is limited by its inability to provide compromised conditions
ously performed in a single step [85]. SSCF offers several advantages for both saccharification and fermentation processes [112,113]. In
including the combination of saccharification and fermentation for addition, this bioconversion process produces low amounts of ethanol,
various feedstocks which influences the overall efficiency of cost, time as SSF fails to fully utilize the pentose sugars [114]. To overcome this
and resources. Besides, the ability of SSCF to co-ferment sugars as they problem, SSCF has been established in which the cofermentation of
produced during saccharification has increased the efficiency of the hexoses and pentoses can be carried out simultaneously. While both
overall conversion process [86]. A great ratio of xylose to glucose can SSF and SSCF demonstrated the same ability to produce high yields of
be maintained throughout the process as glucose is utilized rapidly, bioethanol, SSCF tends to be more favorable because of its potential
which reduces the competitive inhibition from glucose and enhances to ferment various types of sugar simultaneously. All configuration of
the utilization of xylose [87]. Furthermore, the lower enzyme load, bioprocesses can be clearly compared in Table 3.
shorter process duration, utilization of hexose and pentose sugars make Although the biorefinery industry has been operating for long peri-
this process have advantages over SHF and SSF process. Still, there are ods of time, finding a suitable and robust microorganism for an efficient
few concerns similar to SSF such as finding appropriate optimize con- bioconversion process is still active study field [115]. The ideal strain
ditions to accommodate saccharification and fermentation in a single should be able to utilize various types of sugars (i.e. pentose and
vessel, inhibition cause by ethanol and increased water insoluble solids hexose) released from lignocellulosic feedstock and survive inhibitory
(WIS) [88]. Hence, it is important to achieve a compromise temperature compounds that are produced during pretreatment of biomass [116].
by separating SSCF into two different stages of prehydrolysis: (1) high According to most of the literature, the ideal bioconversion approach
temperatures for cellulolytic enzymes and (2) microbial processes at is known as consolidated bioprocessing (CBP), where this strategy
low temperatures [89]. can reduce the production cost of cellulase, eliminate the requirement
of separate hydrolysis enzymes and requires less utility than SSF or
SHF [111,117]. CBP technologies developed using biosynthetic or cel-
4.4. Consolidated bioprocessing (CBP)
lulolytic microorganisms (i.e., microbial cocultures) exhibit significant
potential as alternative pathways for bioethanol production. There
Consolidated bioprocessing (CBP) provides another sustainable
are several positive reports on the direct conversion of biomass into
strategy by integrating all steps simultaneously (i.e. enzyme produc-
bioethanol by a single microorganism. Owing to the prominent nature
tion, saccharification and fermentation) in a single reactor by utilizing
of S. cerevisiae in the fermentation of ethanol, this specific yeast has
either single culture of genetically modified microorganism or a con-
been genetically manipulated to meet the industrial demands of CBP
sortium of microorganisms [90,91]. An ideal and suitable organism
for ethanol production [118]. Recently, the bioconversion of starch-
for CBP in the production of bioethanol from lignocellulosic materials based plant biomass in a CBP system (incubated at 30 ◦ C for 192 h)
must have essential features such as the ability to secrete lignocellulose- using recombinant S. cerevisiae ER T12 and M2n T1 strains yielded
degrading enzymes, able to perform hydrolysis on biomass and fer- 89.35 g/L and 98.13 g/L ethanol with high carbon conversion rates
mentation of ethanol [92]. However, to date, no natural isolates have of 87% and 94%, respectively [119,120]. A study by [121], shows
been discovered to perform CBP in conversion process [93]. Filamen- the process of CBP using recombinant strainS. cerevisiae AC14, in
tous fungi such as basidiomycetes and zygomycetes have potential medium containing solid fraction of pretreated sugarcane bagasse with
to be a good candidate for CBP due to their ability to naturally good ethanol productivity (4.46 g/L/h). Genetic modifications on S.
grow on solid biomass, penetrates deeper into the substrate with their cerevisiae allow direct production of ethanol from hemicellulosic liquor
hyphae and convert the substrate into ethanol in a single step bio- (non-detoxified) derived from pretreated corn cob, to achieved 11.1
process [94]. There are few fungal species that have been deliberated g/L ethanol concentration, without the addition of external hydrolytic
among researchers as possible CBP candidates include Trichoderma catalyst [122]. These results represent the ability of CBP to achieve
reesei, Aspergillus spp. and Fusarium spp., but these strains are not a significant ethanol yield, but the specific yield can vary according
naturally ethanologenic (able to produce ethanol), and all research to the microorganism used or the type of feedstock chosen for the
is still in an early stage. Since most cellulosic fungi are an aerobic bioconversion process. While CBP offers a promising platform to over-
microorganism, they struggle to survive in anaerobic condition during come the high cost of bioethanol production, this process is still under
fermentation [95]. development for application in the biorefinery industry.
A study by [96] indicates the potential of T. asperellum B1581 as
a single culture for bioethanol production in CBP with the volume of 5. Techniques for bioethanol analysis
ethanol produced, 0.94 g/L. Genetically modified ethanologenic strains
like S. cerevisiae are required to secrete enzymes for saccharification Owing to the large scale of production, the determination of the
of materials and fermentation of resulting sugars to ethanol, simul- ethanol (CH3 CH2 OH) content in the alcoholic beverage industry has
taneously [97]. According to [98], recombinant S. cerevisiae ER T12 become a key parameter, as one failure will cause a very large loss of
was used as a host to improve the productivity of ethanol during expenses. Hence, a fast and reliable quantitative method is needed to
fermentation process and variant ER T12.7 displayed significant im- monitor the progress of ethanol production with the flexibility to adjust
provements with 36% higher ethanol yield than the parental strain. the current and real-time fermentation methods up to the optimum
Another modification in CBP is the integration of biodelignification operating schemes. An ideal method to address this purpose should
features, which offers better opportunities for ethanol production. Re- be simple, rapid, cost-effective, highly accurate, highly repeatable and
ports found only basidiomycetes such as Trametes versicolor, T. hirsuta highly sensitive. Moreover, methods that require inexpensive equip-
and Phlebia MG-60 were able to directly produce ethanol from non- ment/instruments are generally preferred [12]. The typical method
pretreated lignocellulosic materials [99]. These consolidation strategies used to determine the volume of alcohol is the measurement based on
have the potential to enhance the efficiency of production and reduce the density of the sample using pycnometer, hydrostatic balance, fre-
the overall cost compared to methods by SHF [100]. quency oscillator and referencing it to the International Alcoholometric

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M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

Fig. 4. Summary of the process involved in every bioconversion strategy for the production of bioethanol.

Table 3
Strategies of the bioconversion process for bioethanol production.
Strategies Process Involved Advantages Disadvantages Maximum Ethanol Yield Refer-
(g/L) ences
Separate Hydrolysis Enzymatic SHF demands a smaller The accumulation of 44.7 [101–
and Fermentation hydrolysis and quantity of cellulolytic glucose and cellobiose 103]
(SHF) fermentation are enzymes compared to SSF. initiate partial inhibition to
performed Low risk of contamination occur and influences the
sequential order. as saccharified liquid with conversion process of
fermentable sugar can be biomass to glucose.
sterilized.
Simultaneous Enzymatic Minimizing inhibition on Since the optimal 77 [74,81,
Saccharification and hydrolysis and feedstock and cost of temperature of enzymatic 104,105]
Fermentation (SSF) fermentation are production. Instantaneous hydrolysis is higher than
performed utilization of resulting the fermentation
simultaneously in sugars, reduced time of the temperature, finding an
the same vessel. process and improved the optimum and equilibrium
productivity. Manage to point is necessary to
produce higher ethanol ensure the process works
concentration than SHF. efficiently
Simultaneous Simultaneous Low cost of production. Temperature has become 68.7 [85,106–
Saccharification and enzymatic Constantly eliminates the the limiting factor of this 108]
Co-Fermentation hydrolysis of end-product inhibition that process as the ideal
(SSCF) glucose and xylose can reduce the cellulases temperature for
with the or 𝛽–glucosidases. Short saccharification process is
co-fermentation of time of processing, high 50 ◦ C while microbial
xylose and glucose productivity as it avoids process take place is
in a same vessel. the end-product inhibition 30-37 ◦ C.
and low risk of
contamination.
Consolidated All processes such Minimizes capital Cellulolytic microorganisms 37 [109–
Bioprocessing (CBP) as production of investment, simplified total have lacked in the 111]
enzyme, reactions, improves the metabolic pathways for
saccharification and efficiency of hydrolysis and desired products.
fermentation are reduces the risk of
achieved in a single contamination.
step process by the
same
lignocellulolytic
microbes.

Tables, which provide the strength of alcohol by volume correspond- of the resulting sugars generated by saccharification processes, such
ing to the density measurement [123]. Currently, there are several as gas chromatography (GC), high-performance liquid chromatography
complex analytical techniques for the determination and quantification (HPLC), and enzymatic methods.

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M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

5.1. Colorimetric enzymatic assay analysis) [132]. After the fermentation of alcohol, the major compo-
nents left in the broth are ethanol and total water, thus, separation
Although enzymatic methods are typically applied for the deter- is necessary to recover and purify ethanol from the broth through
mination of a single sugar type, the most common method used for distillation process [133]. Unfortunately, the distillation process de-
ethanol analysis is the calorimetric enzymatic assay. The method is mands an enormous amount of energy along with high capital cost that
based on the oxidation of ethanol catalyzed by the alcohol dehydro- does not align with the principals of circular economy and SDG [134].
genase enzyme. Two important parameters in the process of ethanol Instrumental methods are divided into four main types: (a) chromato-
fermentation, (1) reducing sugar concentration and (2) total amount graphic methods, (b) spectrophotometric methods, (c) electrochemical
of alcohol, provide information on the optimization process used to methods and (d) other techniques. These approaches normally re-
increase the quality and yield of the products. Although chromato- quire instrumentation that utilizes low analyte concentrations (less than
graphic techniques have been more popular and widely used for the 0.001) and focuses on the physico-chemical properties of the analyte
determination of ethanol, owing to their high cost of operation and such as absorption or emission of electromagnetic radiation, electrical
expensive instruments, the quantification of ethanol via the titer acidic conductivity of the analyte and separation properties [135].
dichromate method has become the most convenient and applicable
method in most laboratories [124]. 5.3. Chromatographic techniques

5.1.1. Potassium dichromate method Chromatographic method integrates separation techniques in a two-
The effect of ethanol can be determined via the use of oxidizing phase system: (a) stationary phase (absorbed mixture of components
reagents such as potassium dichromate. The oxidation of ethanol by and separate in the next phase) and (b) mobile phase (migration of com-
potassium dichromate reagents has been commonly applied to quan- ponents can be observed) [136]. There are few analytical techniques
tify the amount of ethanol in alcoholic beverages, blood plasma and using chromatography such as gas chromatography (GC) and high-
even breathalyzers. Ethanol reacts with dichromate, where hexavalent performance liquid chromatography (HPLC) (refer Fig. 5). Today, GC is
chromium and Cr6+ are reduced to Cr3+ and ethanol is oxidized into practically used as a measuring instrument to determine qualitative and
acetic acid, making the orange color of the solution green [125]. quantitative compositions of volatile organic components (VOCs) using
Spectrometric analysis using dichromate as reagents for ethanol oxi- several internal/external standard calibrations that are responsible for
dation have integrated strong advances to analytical procedures, par- the excellence quality of alcoholic products [137].
ticularly in terms of analytical productivity, simplicity, practicality,
cost-effective instrumentation and reduces waste generation [126]. 5.3.1. Gas chromatography
However, this approach requires harmful and toxic reagents such as Gas chromatography (GC) is an instrument used to separate volatile
cerium (IV) and chromium (VI) along with extremely acidic conditions or semivolatile liquids and gases in relation to their affinity for a sta-
and longer reaction times [127]. In addition, this approach fails to tionary phase (liquid thin or layer of polymer on a solid). The volatile
determine the concentration of ethanol accurately, as a variety of side compound from an injection port is carried by a gaseous mobile phase
products and reducing sugars present in fermentation broth can also to a column with stationary phase and finally to an amdetector [138].
respond to potassium dichromate [128]. The operation of GC depends on sample volatilization in the heated
injector, followed by the separation of component mixtures in a pre-
5.1.2. Ethanol assay kit pared column, which allows the components to be vaporized without
Several modern approaches are available to monitor the fermenta- decomposing [139]. There are four main techniques in capillary GC
tion of ethanol and control the quality of product yield for the success of to vaporized sample and transfer it onto the analytical column: (a)
commercial products. One of the most commercially available Ethanol direct, (b) on-column, (c) split and (d) splitless injections. The common
Assay Kit is manufactured by Megazyme company (catalogue no. K- technique used are split and splitless injections [140]. Combination
ETOH) includes all essential components that are necessary for the between two or more dimensions creates a robust platform for instru-
analysis. The quantification process is based on ethanol oxidation to mental analysis by providing various types of information and offers
acetaldehyde by alcohol dehydrogenase (ADH) and continues with the great reliability in the identification and quantification process [141].
oxidation of acetaldehyde by ADH by converting NAD+ to NADH [129]. Generally, two-dimensional gas chromatography (2D-GC) is applied to
There is also another commercial enzymatic test kit, Enzytec™ Liquid identify each of the complex compounds in a sample, as it specifies
Ethanol used to determine the concentration of ethanol in alcohol- a much greater resolution of chromatography than conventional GC
free beer, fruit juices, kombucha and vegetable juices up to 0.5% methods do [142]. Mass spectrometry (MS), flame ionization (FID)
alcohol by volume (ABV) which is equivalent to 3.95 g/L. The kit and thermal conductivity are the most widely detectors used in GC.
includes all the essential reagents prepared in the form of ready-to-use The common analytical technique for alcohol detection is GC-FID as
and was approved as AOAC Official Method𝑆 𝑀 2017.07 First Action in this technique provides rapid speed of detection, excellent reliability
September 2017 [130]. The assay kit possesses high enzyme specificity and simplicity in sample preparation or extraction [143]. Although
that enables it to perform complex analysis with little interference and FID is not capable of performing the identification of compounds, this
avoid complication during sample preparation, which on contrary may detector is known for high sensitivity, good retention times, easy to
happen during chromatographic coelution. Although enzymatic assays operate and has wide linear range of detection; making FID as a great
are favorable due to short period of time for the sample analysis, the ex- detector for determination of alcoholic concentration [144]. A GC–
pensive cost for each sample compared to traditional laboratory-based MS instrument is used to identify small and trace organic compounds,
method weigh the scale in favor of the latter [131]. whereas a GC–FID instrument is commonly applied to determine ma-
jor compounds [2]. The application of MS allows specific detection
5.2. Separation technique of ions from ionized analytes, hence, results in high quantification
accuracy of target components by comparing the ion mass to library
Analytical methods can be classified into instrumental and classi- data [145]. The development of standard operating procedures can
cal methods, in which the classical approach is divided into 3 main be time-consuming and require extensive preparation. Typical GC-FID
types which are: (a) separation of analyte (i.e. extraction, distillation, techniques demand for a long period of analytical run time and long
precipitation and filtration), (b) qualitative analysis (i.e. boiling point, columns to allow excellent chromatographic separation, hence, avoid-
freezing point, color, odor, density, reactivity and refractive index) ing co-elutions to happen [146]. The analysis time of ethanol in olive
and (c) quantitative analysis (i.e. gravimetric analysis and volumetric oils using GC-FID and GC–MS requires approximately 40 min [147].

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M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

Fig. 5. Several analytical techniques with varying analysis times are used to determine the ethanol content in the sample.

Another major drawback of this technique is the reliance on a unique 5.4.1. Near-infrared (NIR) spectroscopy
retention period of analytes for the identification process, as it can lead Near-infrared spectroscopy (NIR) provides physical and chemical
to misclassification or miscalculation if the peak measured is not a pure information related to the vibrational nature of molecular bonds such
compound [148]. as N-H, C-H, and O-H bonds [154]. Over the years, the choice of
screening method has been based on infrared spectroscopy (IR) tech-
5.3.2. High-performance liquid chromatography (HPLC) nologies, which can identify up to 10 analytes, such as total sugars,
High-performance liquid chromatography (HPLC) is an analytical
ethanol, propanol and methanol, in a single assay [155]. This approach
technique with the ability to control the quality of natural products
has become one of the popular choices of analysis over other con-
and allows the identification of specific compounds in the medium.
temporary analytical techniques as it offers rapid measurement with
Normally, GC is used to conduct the quantification of acetone, ethanol,
little or no sample preparation, the non-invasive and non-destructive
butyric acids and acetic, while HPLC is frequently applied for the
quantification of sugars (i.e. glucose, arabinose and xylose) concen- properties, low cost of operations and portability of the spectrom-
trations [149]. This analytical technique can be coupled with several eters [156]. The NIR spectrum has been frequently used in several
detectors and become the most employed methods due to their wide studies to evaluate the quality of food and beverages as well as char-
range of application, cost-efficiency and robustness [150]. With the acterizing each sample corresponds to their molecular bonds from its
utilization of UV and mass detectors, HPLC can split and discover any chemical composition [157]. Moreover, NIR spectroscopy has been
hazardous compounds, active compounds or even contaminants in the successfully applied by the biorefinery industry for the assessment
products [151]. One of the common detectors used by HPLC is tandem on the properties and quality of biofuel as well as inline monitoring
mass spectrometry (HPLC-MS/MS), which enhances several features for transesterification reactions [158]. The selection of wavelengths is
such as speed, high sensitivity, selectivity and improves the efficiency important in multivariate calibration analysis because it can remove in-
for detection [152]. By operating both instruments simultaneously, terfering and uninformative variables in the spectra to achieve greater
the principal cost and time needed to complete an analysis can be performance in model prediction and enhance interpretability [159].
increased. In addition, this technique can be laborious and against the However, IRs have several limitations, such as poor sensitivity and
philosophy of green chemistry but can still be avoided by choosing overlapping signals, inability to perform direct analysis, indirect meth-
fewer toxic solvents to reduce the generation of chemical waste [153]. ods of calibration based on the model of partial squares regression,
Hence, many alternative protocols have been intensively developed to and low sensitivity for detecting analytes such as 5-(hydroxymethyl)
overcome these limitations.
furfural (HMF) and acetaldehyde, which typically occur at relatively
low concentrations [160].
5.4. Spectroscopic techniques

Analytical techniques such as MS, NIR and NMR are suitable for the 5.4.2. Nuclear magnetic resonance (NMR)
immediate detection of ethanol and provide alternative methods with Compared with gas chromatography (GC) and high-performance
various detection specificities and sensitivities, but few studies have liquid chromatography (HPLC), nuclear magnetic resonance (NMR)
shown that the quantification of ethanol via direct injection in aqueous is considered among the unpopular options for quantitative analysis,
matrices has lower sensitivity. especially in the characterization of biofuels [161]. NMR allows the

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M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

detection and quantification of intermediate reactions as well as non- 5.5.1. Electrochemical biosensors
invasive product molecules in a fixed-time manner with very high Electrochemical biosensors have a wide range of applications, such
chemical specificity [162,163]. In addition, one of the advantages of as food safety, clinical diagnostics and environmental monitoring, and
NMR is the simplicity of the method in terms of sample preparation are adapted to detect a variety of substances depending on the type
and speed and the need for a low volume of solvent; however, it also of bioreceptor applied. These biosensors are classified according to the
has several disadvantages, such as the high cost of instrumentation and type of signal obtained by transduction, such as current intensity or
maintenance costs, including the use of personal or chemical materials conductance changes and potential differences [179]. There are various
(liquid nitrogen and helium) used for the cooling of the superconductor electrochemical techniques, such as voltammetry, potentiometry and
magnet [164]. impedance spectroscopy. However, the response time varies depending
on the type of transduction and design of the mechanism applied.
5.4.3. Raman spectroscopy
With the rapid development of chemometrics, the use of molec- 5.5.2. Cyclic voltammetry
ular spectroscopy (i.e. Raman spectrometry and NIR) techniques in
Voltammetry is known to have great reliability, high detection
various industries has also increased over the years [165]. Raman
speed, good sensitivity and accuracy, making it as one of the most
spectroscopy is another non-destructive technology that eliminates the
applied electrochemical techniques in biosensors technology. Not only
need for chemical reagents and has become the most suitable candidate
does it offer an efficient way to study the reaction mechanisms of an an-
because of its flexibility in various samples and because the spectra
alyte in the electrochemical field but can also measure the parameters
are not limited to water. In addition, the interface of a sample can
of the sample using various voltametric techniques [180]. There are
be determined via an immersion probe to directly monitor the culture
many voltametric techniques which can be applied based on the type
and composition changes in the bioreactor while completing qualitative
of voltage sweep such as square-wave voltammetry, cyclic voltammetry
and quantitative analysis [166]. This analytical technique relies on
and differential pulse voltammetry [181]. Cyclic voltammetry (CV)
the inelastic scattering of incident light via molecules, which involves
is undeniably the most widely utilized electrochemical technique for
changes in the polarization of the irradiated molecule and photon
quantification analysis. The efficiency of this technique relies on the
emission as well as the delivery of information on the vibrational
simplicity of the application and its capacity to offer insights into intrin-
energy bonds of the molecule [167]. A good polarization of nonpolar
sic aspects such as kinetics of the interfacial electron, thermodynamics
bonds leads to intensive Raman bands, whereas chemical bonds with a
and chemical mechanism [182]. During analysis, a triangular voltage
high dipole moment normally result in poor polarization, which causes
wave sweeps between two limits (cyclic form), producing peaks that
a decline in Raman activity [168]. Raman spectroscopy provides useful
represent the events of redox reactions [183]. Measurement of CV are
insights into molecular structure and enables both qualitative and
normally performed with three-terminal cell arrangement made up of
quantitative measurements on the substance as well as producing better
and sharper peaks compared to NIR while maintaining minimum inter- a working electrode (WE), a counter electrode (CE) and a reference
action with water [169]. However, even with these advantages, Raman electrode (RE); which this approach is used to determine the current
spectroscopy is normally considered for qualitative analysis rather than response to a sweeping voltage potential when measuring the ana-
a quantitative technique, and the application of a standard is compul- lyte using working electrode [184]. This technique provides various
sory for quantification analysis via Raman spectroscopy [170]. Besides, ranges of potentials for the working electrode to enhance oxidation
the scattering intensity in Raman is rather moderate, thus causing or reduction of the analyte and develops a corresponding response for
difficulties in determining the presence of analytes at low concentration the current to allow quantification of the analyte [185]. However, this
in the fermentation broth [171]. The utilization of laser waves within system is restricted by its sensitivity towards redox-active substances,
visible light range results in excitation of fluorescence; leading to the but the range of application can be improved by altering the surface
interference with the Raman signal and cause sample decomposition of the working electrode with different materials to enhance their
due to the high intensity of excitation radiation [172]. selectivity and sensitivity for various compounds [66].

5.5. Sensor technologies 5.5.3. Enzyme-based biosensors

The application of enzyme-based biosensors for the quantification
Biosensors are classified according to the type of transducer, such as of ethanol in samples offers specificity and simple step treatment.
piezoelectric biosensors, optical biosensors, electrochemical biosensors, These analytical devices utilize an enzyme as a bioreceptor or coupled
thermal biosensors or natural biological components. Currently, typical tightly to a physical transducer to deliver a continuous or discrete dig-
biosensors, such as electrochemical sensors, are popular because of ital/optical signal that is proportional to the amount of analyte in the
their high sensitivity, rapid detection and low detection limits [173]. sample [186]. Alcohol oxidase (AOx, EC 1.1.3.13) and alcohol dehydro-
This device represents a breakthrough in scientific research, as it re- genase (ADH, EC 1.1.1.1) from microorganisms such as S. cerevisiae, are
lies on the reactions of specific biochemicals involving immune sys- enzymes that are widely employed for the application of alcohol biosen-
tems, isolated enzymes, cells, tissues, or organelles during the thermal, sor [187]. Biosensors based on ADH use the enzyme as a catalyst for
optical or electrical signal detection of chemical compounds [174]. the oxidation of ethanol to acetaldehyde and results in NAD+ reduced
Some electrochemical instruments are portable, which makes it easy to NADH, therefore monitored by amperometrical [188]. While this
to monitor the environment [175]. Electrochemical detection is very enzyme provide advantages such as high selectivity towards ethanol
sensitive towards electroactive molecules and provides selective de- without relying on oxygen, it faces with other challenges including
tection as various molecules can be oxidized and reduced at different the enzyme instability and dependency on the constant recovery of
potentials [176]. Ethanol can be quantified and determined electro- NAD+ in the assay [189]. Even with a variety of studies involving
chemically as it is known as an electroactive molecule with relatively the application of alcohol biosensors using enzymes, further analysis
high theoretical energy density (approximately 8.10 kWh/kg), better of the influence of enzymes on alcohol chain structure, temperature,
efficiency in theoretical fuel cell (97%), and great number of electron strain, pH, operational conditions, membrane materials and electrodes
transfer per ethanol molecule [177,178]. of designed biosensors is still lacking [190].

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M.F.S.M. Ghazali and M. Mustafa Energy Conversion and Management: X 26 (2025) 100933

5.5.4. Capillary electrophoresis excellent precision and reliability [124]. Although regression analysis
Capillary electrophoresis (CE) is a technique employed to separate by [216] between the enzymatic assay and HS/GC yielded a linear
the molecular charges that are present in solution/medium on the relationship described by the equation y=0.966x + 2.084 with a corre-
basis of electrophoretic mobility [191]. This approach includes the lation coefficient R2 = 0.9987, indicating strong agreement between the
separation that happens inside a thin silica capillary occupied by an two methods, enzymatic assays have disadvantages such as low ethanol
electrolyte solution which allows the analyte to move through the capil- specificity and high tendency for cross-reactivity with toxic alcohols
lary and the occurrence of separation due to the differences between the like methanol and LDH interferences.
amplitude of electro-osmotic flow (EOF) and the electrophoretic mobil- The choice of analytical techniques for determining bioethanol
ity of the analytes [192]. There are many approaches in CE which have concentration depends on the specific requirements of the analysis,
led to positive results such as Micellar ElectroKinetic Chromatography including sensitivity, specificity, speed, complexity, and cost. Gas chro-
(MEKC), Capillary Zone Electrophoresis, Non-Aqueous Capillary Elec- matography and HPLC are preferred for their high sensitivity and
trophoresis and MicroEmulsion Electrokinetic Chromatography [193]. specificity, whereas biosensors and NIR offer rapid and real-time analy-
This technique can be considered a complementary technique to high- sis. Capillary electrophoresis provides a fast alternative, while NMR and
performance ion chromatography because of its promising nature, such Raman spectroscopy offer detailed structural information but at higher
as low instrumentation costs, the small volume of sample and reagent costs and complexity. A comparison study using LF-NMR indicated that
used, short period of analysis, portability, potential for miniaturization quantification based on the ethanol CH2 group signal is reliable and
and outstanding separation productivity [194]. Compared with that comparable to GC methods, offering advantages in speed and precision
of GC, the major advantages of this analysis are the simplicity of the for ethanol concentrations between 80%–100%, but the quantification
sample preparation process, and the high performance of separation based on the CH3 group signal was found to be less accurate [226]. A
compared with liquid chromatography (LC) [195]. Most importantly, research study by [227] tested reliable methods for quantifying ethanol
CE has reduced the collection of sample workload, which the sample concentrations in hand sanitizers using NIR. The study demonstrated
required (< 1mL) is significantly less than other techniques [196]. How- the effectiveness of partial least squares (PLS), achieving a prediction
ever, CE is known to have poor sensitivity in UV detection. According error of 1.83%. NIR is preferred for its rapid result performance, but the
to Beer’s law, the absorbance of an analyte is directly associated with presence of ‘‘false correlations’’ between spectral variables and proper-
the path length and concentration, which is equivalent to the inner di- ties makes it less favorable compared to the precision and reliability of
ameter of a capillary [197]. The sensitivity of the CE-UV concentration GC analysis [159].
using a typical 50 μm inner diameter capillary can be poorer than that Analysis using HPLC with refractive index detection (HPLC-RI) was
of LC paired with a UV detector compared with traditional GC via paired t-test, showing no signif-
icant statistical differences, confirming the reliability of the HPLC
5.6. Flow injection analysis method with recovery rates ranged from 98.6% to 103.2% [228].
However, GC typically provides faster analysis times than HPLC for
Flow injection analysis (FIA) is an automated method and frequently volatile compounds like ethanol as rapid separation capabilities of GC
used for chemical analysis. The sample is analyzed by injecting it columns can result in shorter run times, making it more efficient for
into a flow carrier solution before arriving to a detector [198]. This high-throughput analysis scenarios [155]. Each method has unique
method is favorable because of its practicality in coupling with vari- advantages and limitations, making it essential to select the appropriate
ous detectors, such as UV–VIS spectrometers, amperometers, Rayleigh technique on the basis of the context of the analysis. Since thousands of
light scattering or fluorescence spectrometers. Owing to its advantages, analyses are performed every day to ensure the quality of the ethanol
such as rapid analysis; simple operation; low consumption of sam- produced and its safe use, maintaining analytical techniques/methods
ples and reagents; and high accuracy, this method has been largely and developing a new technique to improve the overall analytical
applied in food tests and biochemical, agronomical, industrial and process are important. Currently, GC is known to be the most effective
environmental assays [199]. The FIA systems integrate techniques from approach for analyzing organic solvents such as ethanol [229]. This
laboratory routines to enhance the analytical output and reduce the risk technique provides great information on the amount of ethanol and is
of contamination as well as the analyte losses [200]. In comparison able to detect ethanol at very minimal levels. The HS-GC-FID method
to the voltametric technique, the main advantages of this amperomet- validated by [230] demonstrated high selectivity and precision, with
ric approach include the capacity to simultaneously analyze different an estimated expanded uncertainty of 2%, indicating its reliability for
samples, real-time monitoring and reduces the contact time between characterizing ethanol in reference materials. Moreover, this analytical
the sample and the surface of the electrode to minimize the absorption technique is considered as the gold standard for ethanol analysis due
effects [201]. to its high specificity and sensitivity. It can detect low concentrations
of ethanol with limits of detection typically around 0.099 mg/mL,
5.7. Comparison between analytical techniques demonstrating excellent linearity and recovery rates between 91% and
109% [231]. Owing to its high potential for output, high resolution,
To measure the concentration of bioethanol across different fields specificity, sensitivity, reproducibility and accuracy, this analysis gar-
and applications (i.e., fuel production, alcoholic beverage and fermen- ners major interest worldwide [232]. These advantages make it an ideal
tation processes), various analytical techniques can be employed. A analytical technique in both research and industrial applications for
detailed comparison of several prominent methods, such as gas chro- bioethanol production.
matography (GC), high-performance liquid chromatography (HPLC),
biosensors, capillary electrophoresis (CE), calorimetric assays, flow 6. Conclusion
injection analysis (FIA), near-infrared spectroscopy (NIR), nuclear mag-
netic resonance (NMR), and Raman spectroscopy, is shown in Table 4. Biofuels, especially bioethanol, have appeared to be promising alter-
Despite the various advanced analytical techniques available, such as native renewable energy sources that can reduce the emission of green-
enzymatic analysis and chromatographic techniques, owing to their house gas, burn cleanly and improve the performance of automobile
high operational costs, the titration of acidic dichromate has been engines. However, the production of second-generation (2G) bioethanol
considered a popular option, as it is inexpensive and available in most faces a few challenges that need to be overcome before it becomes a
biofuel laboratories and wineries. The self-colorimetric method demon- sustainable source of energy. The key challenges for this production in-
strated limits of detection and quantification of 0.17% and 0.56%, clude the accessibility and cost of feedstock, limitations in technology,
respectively, with a high correlation coefficient (R2 = 0.999), indicating impacts on the environment and the feasibility of the economy. Seizing

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Table 4
Analytical techniques were applied to determine the amount of bioethanol produced in the bioconversion process.
Analytical Reliability and Speed Advantages Disadvantages Refer-
Techniques accuracy ences
Biosensors Have low detection Fast analysis. Simple build up, portable Great loss of enzyme [173,202–
(Enzyme-Based) limit and high and easy to use in a wide caused by their interaction 204]
sensitivity. range of fields or with the surface of
applications. Cost-effective, electrode; reducing the
high sensitivity, and do not lifespan of biosensor to
require trained personnel only 2–4 weeks.
to operate the instrument.
Capillary Manage complicated Complete within a few Demands only small Poor reproducibility and [205–
Electrophoresis samples with great seconds/minutes. injection volumes and this the high cost of 207]
resolution but poor might be crucial if there is instruments.
sensitivity due to only limited volume of
small volume of sample. Analyzing a
detection. broad-spectrum compound
ranging from small to
large molecules and ions.
Calorimetric Assay Poor ethanol Processed swiftly by simple This technique is simple, Tedious sample [208–
specificity. step of mixing reagents. rapid response and preparation and demands 210]
affordable. for large sample volume.
Poor accuracy, low
reproducibility and
minimal stability of
enzyme–substrate.
Cyclic Voltametric Naturally, sensitive, Rapid response time and Produce very low noise; The presence of [184,211,
highly selective, able to simultaneously lead to reproducible data background noise limits 212]
good specificity and determine the analytes. with great specificity and the sensitivity as it can
low detection limits. sensitivity. mask the Faradic current.
Flow Injection High analytical Rapid response time with Simple method with low Demands for an automated [213–
Analysis frequency, accuracy great reproducibility. cost of operation, high rate diluted system to perform 215]
and precision. of sampling, able to be in the minimal linear range
automated, low risk of
contamination, utilizing
modest amounts of
samples and reagents.
Gas Great resolution, In comparison to HPLC, Have very high resolution, The requirement for [139,216,
Chromatography high sensitivity and GC respond in a shorter selectivity, good analyte to be thermally 217]
ethanol specificity amount of time. sensitivity, great accuracy stable and volatile has
(detect other and precision, as well as limits the range of
alcohol such as wide dynamic compounds that can be
isopropanol, and concentration range. Can determined by the
methanol). be coupled with detectors instrument.
such as Flame Ionization
Detector (FID) and mass
spectrometry.
High Performance Offers a very Slower than GC. Able to analyze volatile Involves sample [155,218]
Liquid accurate technique and nonvolatile compounds derivatization prior
Chromatography for identification of including carbohydrates, analysis and this process
(HPLC) specified chemical amino acids and drugs. can be time-consuming,
components in Can be coupled with hence, affecting the
prepared sample. UV/VIS, fluorescence and accuracy and reliability of
mass spectrometry. the results. Requires
expensive organic
materials, power supply
and regular maintenance.
Reliability of HPLC pump
depends on the mobile
phase, purity of the sample
and the usage of right
procedures.
Near-Infrared Restricted sensitivity Rapid analysis. Nondestructive analysis, Time-consuming and [219]
Spectroscopy (NIR) for detection of which is eco-friendly, has require expensive tools.
ethanol low cost of production and
concentration. does not require sample
derivatization. Provide an
excellent depth of
information and stability.

(continued on next page)

these challenges requires recent technological development, especially this strategy requires less preparation steps and instruments compared
to improve the bioconversion process using lignocellulosic biomass to other bioprocess configurations such as SHF, SSF and SSCF, thus
and develop suitable analytical techniques for monitoring bioethanol reducing the overall cost of production. Besides, many studies have
concentration. The ideal bioconversion approach is known as CBP as shown the capacity of CBP to reach significant amount of ethanol

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Table 4 (continued).
Analytical Reliability and Speed Advantages Disadvantages Refer-
Techniques accuracy ences
Nuclear Magnetic Low resolution of Rapid analysis Great capacity for Hard to detect metabolites [220–
Resonance (NMR) spectrum, reproducibility and able to at minimal concentration, 222]
overlapping signals, recognize all components hence requires solvent
and restricted in a mixture without any suppression method to
sensitivity. pretreatment or prior enhance the resolution for
separation. Offers improved metabolite
simplified process of recognition.
sample extraction and
automation of protocols.
Raman Spectroscopy Narrow bandwidth Simple technique and fast Requires no sample The Raman signal is weak [223–
and better spectral analysis. preparation, simple for detection of minimal 225]
resolution. Great measurement, no concentration of
sensitivity and interference from water substances. Easily
specificity to variety and CO2 , produces fast and influenced by optical
chemical nondestructive analysis and system, for example, the
components. less contact with the signal of the sample can
sample. be affected by the
fluorescence scattering and
the fluorescence impurities
may conceal the Raman
spectrum.

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