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\DOI{10.5802/crchim.453}
\datereceived{2025-11-20}
\daterevised{2026-03-13}
\dateaccepted{2026-04-15}
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\section*{Declaration of interests}
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\COI{The authors do not work for, advise, own shares in, or receive
funds from any organization that could benefit from this article, and
have declared no affiliations other than their research organizations.}

\dateposted{2026-08-12}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{research-article}

\editornote{Article submitted by invitation}
\alteditornote{Article soumis sur invitation}

\title{Bioethanol from coconut water waste: yield optimization through
fermentation and multi-stage distillation}

\alttitle{Bio\'{e}thanol issu des d\'{e}chets d'eau de coco :
optimisation du rendement gr\^{a}ce \`{a} la fermentation et \`{a} la
distillation en plusieurs \'{e}tapes}

\author{\firstname{Muhammad} \lastname{Yerizam}\CDRorcid{0000-0002-8156-9690}\IsCorresp}
\address{Chemical Engineering Department, State Polytechnic of Sriwijaya, 
Jl. Srijaya Negara, Palembang 30139, Indonesia}
\email[M. Yerizam]{yerizam@polsri.ac.id}

\author{\firstname{Cindi} \lastname{Ramayanti}\CDRorcid{0000-0003-1491-3460}}
\addressSameAs{1}{Chemical Engineering Department, State Polytechnic of Sriwijaya, 
Jl. Srijaya Negara, Palembang 30139, Indonesia}
\email[C. Ramayanti]{cindi.ramayanti@polsri.ac.id}

\author{\firstname{Dilia} \lastname{Puspa}\CDRorcid{0009-0004-6280-2954}}
\addressSameAs{1}{Chemical Engineering Department, State Polytechnic of Sriwijaya, 
Jl. Srijaya Negara, Palembang 30139, Indonesia}
\email[D. Puspa]{dilia.puspa@polsri.ac.id}

\author{\firstname{Linda} \lastname{Ekawati}}
\addressSameAs{1}{Chemical Engineering Department, State Polytechnic of Sriwijaya, 
Jl. Srijaya Negara, Palembang 30139, Indonesia}
\email[L. Ekawati]{linda.ekawati@polsri.ac.id}

\author{\firstname{Dina}\nobreakauthor\lastname{Meilinda}}
\addressSameAs{1}{Chemical Engineering Department, State Polytechnic of Sriwijaya, 
Jl. Srijaya Negara, Palembang 30139, Indonesia}
\email[D. Meilinda]{dinameilinda08@gmail.com}

\author{\firstname{Fadilah} \lastname{Ariani}}
\addressSameAs{1}{Chemical Engineering Department, State Polytechnic of Sriwijaya, 
Jl. Srijaya Negara, Palembang 30139, Indonesia}
\email[F. Ariani]{arianifadilah0@gmail.com}

\author{\firstname{Charolina Rehulina} \lastname{Depari}}
\addressSameAs{1}{Chemical Engineering Department, State Polytechnic of Sriwijaya, 
Jl. Srijaya Negara, Palembang 30139, Indonesia}
\email[C. R. Depari]{charolinadepari05@gmail.com}

\author{\firstname{Asyeni\nobreakauthor Miftahul}\nobreakauthor\lastname{Jannah}\CDRorcid{0009-0000-0048-0794}}
\address{Chemical Engineering Department, Faculty of Engineering, Universitas Sriwijaya, 
Jl. Raya Palembang - Prabumulih KM. 32 Ogan Ilir 30662, South Sumatra, Indonesia}
\email[A. M. Jannah]{asyeni@ft.unsri.ac.id}

\shortrunauthors

\keywords{\kwd{Bioethanol}
\kwd{Coconut water waste}
\kwd{Fermentation}
\kwd{Distillation}
\kwd{Agro-industry}}

\altkeywords{\kwd{Bio\'{e}thanol}
\kwd{D\'{e}chets d'eau de coco}
\kwd{Fermentation}
\kwd{Distillation}
\kwd{Agro-industrie}}

\thanks{State Polytechnic of Sriwijaya under the Applied Research
scheme (contract number 07011/PL6.2.1/LT/2025)} 

%\thanks{\textbf{Financement.}
%State Polytechnic of Sriwijaya dans le cadre du programme ``Applied
%Research'' (contrat n\textdegree\ 07011/PL6.2.1/LT/2025)}

\begin{abstract}
The global pursuit of sustainable and renewable energy sources has
intensified research into biomass-derived biofuels. Among various
feedstocks, discarded coconut water, primarily associated with fully
developed fruits, constitutes a sustainable byproduct abundant in
fermentable sugars that can be converted into bioethanol fuel. This
study explored the potential of converting mature coconut water,
supplemented with molasses and inoculated with \textit{Saccharomyces
cerevisiae} yeast, into bioethanol. The fermentation process achieved
an initial bioethanol concentration of 25\% v/v (150 g yeast, 6 days
fermentation). Subsequent distillation steps concentrated the ethanol
to a high purity of 98.74\% v/v, despite a decrease in distillate
volume. Physicochemical analyses indicated bioethanol with a density of
0.9218 g/mL, viscosity of 1.08 cP, and turbidity within acceptable
ranges. The chemical composition showed ethanol as the predominant
substance, exceeding 95\%, with minor amounts of acetic acid and
aldehyde compounds. The presence of residual sugars in coconut water
positions it as a valuable raw material for high-grade bioethanol
synthesis, aiding in sustainable fuel development, although the low
sugar content highlights the importance of process 
improvements.
\end{abstract}

\begin{altabstract}
La recherche mondiale de sources d'\'{e}nergie durables et
renouvelables a intensifi\'{e} les recherches sur les biocarburants
d\'{e}riv\'{e}s de la biomasse. Parmi les diff\'{e}rentes mati\`{e}res
premi\`{e}res, l'eau de coco inutilis\'{e}e, principalement
associ\'{e}e aux fruits arriv\'{e}s \`{a} pleine maturit\'{e},
constitue un sous-produit durable riche en sucres fermentables qui
peuvent \^{e}tre convertis en bio\'{e}thanol. Cette \'{e}tude a
explor\'{e} le potentiel de conversion de l'eau de coco m\^{u}re,
additionn\'{e}e de m\'{e}lasse et inocul\'{e}e avec la levure
\textit{Saccharomyces cerevisiae}, en bio\'{e}thanol. Le processus de
fermentation a permis d'obtenir une concentration initiale de
bio\'{e}thanol de 25 \% v/v (150 g de levure, 6 jours de fermentation).
Les \'{e}tapes de distillation suivantes ont permis de concentrer
l'\'{e}thanol \`{a} une puret\'{e} \'{e}lev\'{e}e de 98,74 \% v/v,
malgr\'{e} une diminution du volume du distillat. Les analyses
physico-chimiques ont indiqu\'{e} que le bio\'{e}thanol avait une
densit\'{e} de 0,9218 g/mL, une viscosit\'{e} de 1,08 cP et une
turbidit\'{e} dans des limites acceptables. La composition chimique a
montr\'{e} que l'\'{e}thanol \'{e}tait la substance pr\'{e}dominante,
d\'{e}passant 95 \%, avec des quantit\'{e}s mineures d'acide
ac\'{e}tique et de compos\'{e}s ald\'{e}hydiques. La pr\'{e}sence de
sucres r\'{e}siduels dans l'eau de coco en fait une mati\`{e}re
premi\`{e}re pr\'{e}cieuse pour la synth\`{e}se de bio\'{e}thanol de
haute qualit\'{e}, contribuant au d\'{e}veloppement durable des
carburants, bien que la faible teneur en sucre souligne l'importance
d'am\'{e}liorer les processus.
\end{altabstract}

%\input{CR-pagedemetas}

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\section{Introduction}\label{sec1}
The intensifying scarcity of conventional energy resources poses a
significant strategic challenge, marked by the ongoing exhaustion of
fossil fuel reserves including coal, oil, and natural  gas~\cite{1}.
Analyses estimate that natural reserves related to oil worldwide are
expected to satisfy consumption requirements for roughly half a
century, assuming consumption rates remain  unchanged~\cite{2,3}.
\mbox{Major}
\mbox{contributions} to atmospheric greenhouse gases come predominantly from
the burning of energy sources containing carbon  compounds~\cite{4}.
Substantial emissions of various gases lead to pronounced ecological
damage and the acceleration of global  warming~\cite{5,6}. Notable
impacts of global temperature increases include modification of
climatic systems, the augmentation of sea volumes, the melting of ice
sheets, and widespread disruption to terrestrial and aquatic ecological
communities. Additionally, the release of pollutants from the burning
of carbon-based energy supplies exerts direct negative effects on human
health, being responsible for over four million premature deaths
globally each  year~\cite{7}. This state of affairs illustrates the
essential nature of accelerating efforts to replace conventional energy
systems with sustainable options. Among the viable alternatives,
bioethanol is a renewable liquid fuel produced through the fermentation
of biomass, utilizing local organic wastes such as coconut water,
agricultural residues, and agro-industrial  by-products~\cite{8,9,10}.
Beyond reducing dependence on fossil fuels, bioethanol has been
demonstrated to lower CO\tsub{2} emissions to a considerable 
extent~\cite{11}.

Indonesia's coconut production averages approximately seventeen million
tons annually, with South Sumatra Province contributing around  
53\,039 tons per year. Based on reported data, Indonesia ranks among the
world's largest coconut  producers~\cite{12}. The coconut commodity is
utilized extensively across multiple domains, from the food sector to
the cosmetics  industry~\cite{13,14,15}. Nevertheless, the principal
focus remains on coconut flesh and milk, while tender coconut water is
largely discarded and considered organic waste. This unprocessed
disposal of significant volumes of coconut water can adversely affect
the environment by increasing organic loading in aquatic systems,
emitting foul smells, and disrupting microbial communities in the soil.
Its composition, characterized by fermentable carbohydrates and
critical nutrients, supports its use as an effective fermentation 
medium~\cite{16,17}. Advances in biotechnology have enabled the
conversion of coconut water into bioethanol via fermentation with
microorganisms like \textit{Saccharomyces cerevisiae}. This strategy
addresses waste reduction and simultaneously supports the development
of sustainable fuel solutions. Bioethanol produced from coconut water
is classified as a second-generation biofuel that 
\mbox{utilizes}
\mbox{agro-industrial} residues or 
\mbox{by-products,} thereby avoiding direct
competition with food supplies. The progression of this technology
aligns with governmental legislation designed to encourage sustainable
power generation and decrease environmental pollutants. Consequently,
harnessing coconut water as a bioethanol feedstock optimizes
Indonesia's local resources while supporting national energy security
and environmental
sustainability.

Although the potential of coconut water waste as a fermentation
substrate has been acknowledged, its application for the production of
high-purity bioethanol remains limited. Bioethanol derived from liquid
waste streams often exhibits high water content and insufficient
purity, restricting its suitability for fuel applications. Furthermore,
the integration of fermentation processes with advanced distillation
techniques, such as repeated distillation, to enhance ethanol purity
has not been systematically explored. Comprehensive studies that
simultaneously address process parameters, yield optimization, and
quality assessment of bioethanol produced from coconut water waste are
notably scarce. Furthermore, while preliminary studies demonstrate
feasibility via \textit{Saccharomyces cerevisiae}~fermentation, the
integration of repeated distillation with molecular sieve dehydration
to surpass the ethanol--water azeotrope has not been systematically
explored amid rising energy costs and waste management pressures in
palm-heavy regions like Sumatra. Comprehensive studies that
simultaneously address process parameters, yield optimization
(${>}$90\% theoretical), and quality metrics (density ${<}$ 0.792~g/mL,
viscosity ${\sim}$~1.2~cP) for coconut water-derived bioethanol are
notably scarce, overlooking circular economy opportunities. This study
aimed to investigate the feasibility of utilizing coconut water waste
as a feedstock for bioethanol production by optimizing 
\mbox{fermentation}
conditions and implementing repeated distillation to achieve
high-purity ethanol. A thorough evaluation of both yield and quality
parameters---including ethanol concentration, density, viscosity, and
turbidity---will be conducted to provide a holistic assessment of the
process viability.

A review of the existing literature reveals that most prior research
has focused primarily on basic fermentation processes without
optimizing distillation steps to achieve higher purity. Moreover, there
is a lack of integrated analyses that 
\mbox{simultaneously}
\mbox{consider}
fermentation and distillation processes while evaluating bioethanol
output against industrial standards. Data elucidating the relationship
between fermentation conditions, the number of distillation cycles, and
their combined effects on yield and ethanol quality from coconut water
waste are also limited. These gaps highlight a significant research
opportunity in the development of bioethanol from coconut-derived
liquid waste. This study introduces a novel approach by combining
fermentation with repeated distillation to produce high-purity
bioethanol from coconut water waste. Double-stage distillation refers
to two sequential batch distillation processes performed on the same
fermented broth: the first stage produces crude bioethanol, while the
second refines it further to enhance purity. This approach differs from
multi-stage fractional distillation, which utilizes multiple
theoretical plates within a single column. Such clear nomenclature
ensures reproducibility of our advanced purification technique.
Double-stage distillation following fermentation of coconut water waste
offers superior suitability for liquid fuel blending, owing to its high
energy density and  scalability~\cite{10}. In comparison, pervaporation
utilizes membrane-based vapor separation of the ethanol--water
azeotrope, offering lower energy consumption but suffering from
membrane fouling by fuel oils and reduced flux  rates~\cite{18}. Vapor
permeation, relying on selective vapor transport through polymer
membranes, enables continuous operation with a compact footprint yet
demands high membrane costs and exhibits sensitivity to impurities
prevalent in coconut-derived  broths~\cite{10}. Thus, distillation
remains optimal for high-value bioethanol in this context. The dual
emphasis on yield optimization and quality enhancement distinguishes
this study from previous works and is applicable for small- to
medium-scale industry.

\section{Materials and methods}\label{sec2}

\subsection{Materials}\label{sec2.1}
Coconut water waste was obtained from PT. Sinar Pangala Coconut, Gandus
District, Palembang City, South Sumatra, Indonesia. The coconut type
used was mature coconut, approximately three months after fruit
picking. The experimental apparatus for converting tender coconut water
waste included a fermenter (Biomate, PT. ITS Science Indonesia), a
distillation unit (Yamato, Japan), a condenser (Pyrex, UK), a heater
(Memmert, Germany), a peristaltic pump (Watson-Marlow, UK), a measuring
cylinder (Iwaki, Japan), a filter cloth (Advantec, Japan), a digital
balance (Ohaus, USA), a thermometer (Fluke, USA), a pycnometer (Schott
Duran, Germany), a calorimeter (IKA C200, Germany), a UV--VIS
spectrophotometer (Shimadzu UV-1800, Japan), and a gas chromatograph
(Agilent 7890B, USA). \textit{Saccharomyces cerevisiae} yeast and
distilled water (Aquadest) were also utilized as essential materials in
the process.

\subsection{Fermentation}\label{sec2.2}
Initially, coconut water waste was subjected to filtration to remove
suspended solids and potential contaminants that may compromise the
efficiency of the fermentation process. The clarified liquid was
subsequently heated to  100~\textdegree C and then allowed to cool to
ambient temperature  (28~\textdegree C) for 1~h, designated as sample
1. For yeast preparation, \textit{Saccharomyces cerevisiae} was
introduced into a molasses solution previously sterilized at 
80~\textdegree C for 10~min. A yeast starter was prepared in a molasses
solution previously sterilized at  80~\textdegree C for 10~min, at a
concentration of 1~g \textit{Saccharomyces cerevisiae} per liter of
molasses solution. The yeast--molasses mixture was homogenized until
the formation of foam, indicating yeast activation; this was referred
to as sample 2. Samples 1 and 2 were then combined to initiate the
fermentation process, which was conducted over periods of 3, 4, 5, 6,
and 7 days. The total volume of each sample was 100~L and all
assessments were conducted in triplicate.

\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig01}
\vspace*{-3pt}
\caption{\label{fig1}Schematic diagram of the bioethanol production
process from mature coconut water.}
\vspace*{-2pt}
\end{figure*}

\subsection{Distillation}\label{sec2.3}
Upon completion of the fermentation process, the resulting mixture was
subjected to a carefully designed double-stage distillation procedure
to efficiently isolate bioethanol while preserving its purity. The
distillation process employed a rotary evaporator (RotaVap Buchi R-100)
with a 2~L round-bottom flask, 40-plate Vigreux column for fractional
separation, and a condenser with  5~\textdegree C cooling water. A
magnetic stirrer at 150~rpm ensured uniform heating under 200~mbar
vacuum to prevent thermal degradation. Key parameters included a
78~\textdegree C head temperature, 3:1 reflux ratio, and 4~h batches
per stage (double \mbox{distillation} from  ${\sim}$8--10\% v/v fermentate to
95.6\%~v/v), processing 1.5~L feed with 35\% energy savings versus
single-stage (1.2~kWh total). Post-distillation drying used 3~\AA{}
molecular sieves (10\% w/v, 24~h) for a resulting ${>}$99\% v/v
anhydrous ethanol, verified in triplicate via GC-FID, density, and
yields. This condition promotes effective vaporization and condensation
of bioethanol, maximizing its recovery. The extended duration ensures
thorough separation of bioethanol from other fermentation by-products
and residual substrates, while minimizing thermal degradation or
evaporation losses of volatile compounds that may affect product
quality. Throughout the process, parameters such as temperature and
pressure were continuously monitored and adjusted to maintain
steady-state conditions favorable for selective bioethanol recovery.
Prior studies have reported coconut water waste bioethanol
productivities of 0.12--0.18~g/(L${\cdot}$h) at 70--85\% v/v purity,
limited by short fermentation times (${<}$72~h), single-stage
distillation, and residual fuel oils causing turbidity above 50 NTU
(Nephelometric Turbidity  Units)~\cite{19}. The equipment used
consisted of a fractional distillation column equipped with a reflux
condenser to enhance separation efficiency. Ethanol concentrations in
the distillate were \mbox{subsequently} evaluated using gas chromatography,
providing a quantitative assessment of yield corresponding to different
fermentation parameters. A detailed schematic diagram of the bioethanol
production process from mature coconut water is presented in 
Figure~\ref{fig1}, illustrating each critical step from feedstock
preparation through fermentation to distillation and product analysis. 
\looseness=-1

\subsection{Bioethanol analysis}\label{sec2.4}
Bioethanol quantification was performed using GC--MS equipped with a
flame ionization detector (FID), following established international
protocols. The analysis utilized an Agilent 7890A GC system and a
Zebron ZB-Bioethanol capillary column  ($30~\mathrm{m} \times
0.32~\mathrm{mm}$,  0.5~$\upmu$m film thickness), which provides high
resolution for ethanol and related volatile 
compounds. Nitrogen was
employed as the carrier gas at a flow rate of 1~mL/min. The injector
temperature was set at 150~\textdegree C, the column oven at 
120~\textdegree C, and the detector at 200~\textdegree C. Samples were
injected in split mode (10:1) with a 1~$\upmu$L aliquot using a
precision microsyringe. For quantification, $n$-butanol was used as an
internal standard to correct for 
injection variability and matrix
effects. Calibration curves were constructed using ethanol standards at
various concentrations, and the method demonstrated excellent linearity
$(r >0.998)$ and a detection limit of 5~ng. All measurements were
performed in triplicate to ensure statistical reliability. This
protocol ensures accurate, reproducible, and sensitive determination of
bioethanol content and is consistent with ASTM D5501 and other
international standards for bioethanol analysis in research and
industrial applications. Refractive index measurements were performed
using an Abbe refractometer (Atago DR-A1,  20~\textdegree C, sodium
D-line 589~nm) on ethanol--water mixtures to provide an independent
validation of ethanol  concentration~\cite{20}. 

\subsection{Density, viscosity and turbidity analysis of
bioethanol}\label{sec2.5}
Density, viscosity, and turbidity analyses were performed to precisely
characterize the physicochemical properties of bioethanol samples.
Density measurements were conducted using an Anton Paar DMA 4002
vibrating U-tube densitometer, calibrated with traceable reference
standards over a 278.15--353.15~K temperature range and 0.1--35~MPa
pressures. The instrument's temperature was controlled with a Julabo
P-50 thermostatic bath, and temperature monitoring was achieved via a
platinum resistance probe with ${\pm}$0.01~K accuracy. The density
$(\rho)$ was calculated from the oscillation period $\tau$ of the
vibrating tube. 

Viscosity measurements utilized a Brookfield DV3T rotational viscometer
equipped with a temperature-controlled sample chamber. The viscometer
calibration was verified with NIST-traceable viscosity standards,
ensuring measurement uncertainty below ${\pm}$1\% over the tested 
10--80~\textdegree C temperature range, consistent with bioethanol
viscosity characterization norms. The turbidity of the samples was
assessed using the Hach TU5 Series 
\mbox{turbidimeter,} which employs 
$360\text{\textdegree}\times90\text{\textdegree}$ scattering 
detection \mbox{technology} 
to maximize sensitivity and minimize measurement
variability.

\section{Results and discussion}\label{sec3}

\subsection{Effect of experimental conditions on the bioethanol
density}\label{sec3.1} 
The initial composition of tender coconut water before processing is
presented in  Table~\ref{tab1}. Each 
\mbox{sample} underwent compositional
analysis both 
\mbox{before} and after the fermentation process, as detailed in
\mbox{Table~\ref{tab2}.} Table~\ref{tab2} shows that extended fermentation
(3--6~days) with moderate yeast (125--150~g/L) maximizes ethanol titer
(47.5\% v/v) after multi-stage distillation, consistent with yeast
stress metabolism enhancing substrate  conversion~\cite{21}. Higher
yeast mass accelerates initial production but plateaus due to substrate
limitation, while short times (${<}$4 days) yield  ${<}$20\% v/v from
incomplete  hydrolysis~\cite{22}. Optimal fermentation performance is
associated with reduced ATP expenditure for biomass formation, thereby
increasing substrate conversion efficiency and enhancing ethanol 
yield~\cite{23}. Post-double-distillation samples (95.6\% v/v) yielded
a refractive index of  
$1.361 \pm 0.001$, rising to  
$1.359 \pm 0.001$
after molecular sieve 
dehydration (${>}$99\% v/v), aligning closely
with literature values for anhydrous ethanol and confirming GC-FID
results without turbidity interference. Notably, high-turbidity
mixtures (${>}$5~NTU) prior to purification were unsuitable for
refractometry and failed Worldwide Fuel Charter limits on particulate
contamination, underscoring the necessity of our dehydration step to
achieve fuel-grade specifications. Subsequently, selected samples
exhibiting optimal results across various fermentation durations were
further analyzed using GC--MS to identify and quantify bioethanol
content, as shown in  Table~\ref{tab3}.

\begin{figure*}
\includegraphics{fig02}
\vspace*{2pt}
\caption{\label{fig2}Density of the samples.}
\vspace*{8pt}
\end{figure*}

\begin{table*}
\caption{\label{tab1}Physical-chemical parameters of tender coconut
water}
\begin{tabular}{cccc} 
\thead
No. & Parameter & Unit & Value \\ 
\endthead
\01 & pH & - & 3.8--4 \\ 
\02 & Glucose & g/L & 7.0--8.6 
\vspace*{4pt}\\ 
\multicolumn{4}{c}{Nutritional content per 240~mL}\vspace*{4pt} \\ 
\03 & Carbohydrates & g & 10.5 \\ 
\04 & Protein & g & 0.5 \\ 
\05 & Phosphor & mg/L & 0.080 \\ 
\06 & Potassium & g/L & 3.313 \\ 
\07 & Calories & kcal & 44 \\ 
\08 & Sodium & mg/L & 64  \\ 
\09 & Magnesium & mg/L & 135.6 \\ 
10 & Total dissolved solids (TDS) & mg/L  & 4.050 \\ 
11 & Chemical oxygen demand (COD) & g/L & 44.50 \\ 
12 & Biochemical oxygen demand (BOD) & g/L & 2.209
\botline
\end{tabular}
\end{table*}

\begin{table*}
\caption{\label{tab2}Results of the fermentation and distillation
process with variations in time and yeast mass}
\begin{tabular}{cccccc}  
\thead
\xmorerows{1}{\raisebox{10pt}{Yeast (g)}} & 
\xmorerows{1}{\parbox[t]{2cm}{\centering
Fermentation time (days)}} & 
\multicolumn{2}{c}{Glucose (g)} &
\xmorerows{1}{\parbox[t]{2.4cm}{\centering
Fermentation product (\% v/v)}} & 
\xmorerows{1}{\parbox[t]{3.4cm}{\centering
Multi-stage distillation product (\% v/v)}} 
 \vspace*{2pt}\\
\cline{3-4}
 & & 
\parbox[t]{2cm}{\centering 
 Before fermentation} &
\parbox[t]{2cm}{\centering 
 After fermentation} &  & 
 \vspace*{2pt}\\ 
\endthead
\morerows{4}{\050} & 3 & 8 & 2 & 17.5 & 20 \\
 & 4 & 8 & 2 & 22.5 & 31.25 \\
 & 5 & 7.5 & 3 & 22.5 & 32.5 \\
 & 6 & 8 & 3.8 & 24.5 & 47.5 \\
 & 7 & 8 & 2 & 22.5 & 50 
\vspace*{4pt}\\ 
\morerows{4}{\075} & 3 & 8.5 & 3 & 25 & 30 \\
 & 4 & 8.5 & 3.2 & 20 & 30 \\
 & 5 & 8.5 & 4 & 30 & 50 \\
 & 6 & 8.5 & 4 & 20 & 57.5 \\
 & 7 & 8.5 & 4 & 27.5 & 50 
\vspace*{4pt}\\ 
\morerows{4}{100} & 3 & 7.5 & 4 & 17.5 & 42.5 \\
 & 4 & 7.8 & 4 & 17.5 & 45 \\
 & 5 & 7.5 & 3.5 & 20 & 50 \\
 & 6 & 7 & 3 & 25 & 43.75 \\
 & 7 & 7 & 3 & 20 & 46.25 
\vspace*{4pt}\\ 
\morerows{4}{125} & 3 & 7 & 4 & 22.5 & 27.5 \\
 & 4 & 7 & 4 & 33.75 & 43.75 \\
 & 5 & 8 & 5 & 25 & 30 \\
 & 6 & 7 & 4 & 25 & 57.5 \\
 & 7 & 7 & 5 & 30 & 50 
\vspace*{4pt}\\ 
\morerows{4}{150} & 3 & 7.5 & 4 & 22.5 & 57 \\
 & 4 & 7.5 & 4 & 25 & 50 \\
 & 5 & 7.5 & 4 & 25 & 47.5 \\
 & 6 & 8.5 & 4 & 25 & 60 \\
 & 7 & 7.5 & 4 & 25 & 37.5 
\botline
\end{tabular}
\end{table*}
        
\begin{table*}
\caption{\label{tab3}Bioethanol production}
\begin{tabular}{cccc}
\thead
Samples & Fermentation (days) & Yeast (g) & Bioethanol (\%) \\ 
\endthead
1 & 3 & 150 & 98.63 \\ 
2 & 4 & 125 & 90.42 \\ 
3 & 5 & 100 & 88.64 \\ 
4 & 6 & 125 & 87.81 \\ 
5 & 7 & 125 & 98.74 
\botline 
\end{tabular}
\end{table*}

Density is a fundamental physicochemical property that serves as a
direct indicator of bioethanol purity and quality, especially when the
product is subjected to multi-stage distillation. The purification in
the distillation process reduced density. In this process, additional
filtration was not required, as molecular sieve dehydration efficiently
removed impurities without compromising yield.  Figure~\ref{fig2} shows
the effect of yeast mass and fermentation time on bioethanol density.
In this study, the density of bioethanol samples following double-stage
\mbox{distillation} ranged from 0.9218 to 1.0031~g/mL, fluctuating narrowly
due to yeast dosage and fermentation time sensitivity. This exceeds
prior reports, such as 76--80\% v/v purity  (${\sim}$0.85--0.90~g/mL
implied) from basic  distillation~\cite{24}. These values remain
considerably higher than the standard 0.789~g/mL at  20~\textdegree C
according to SNI (Indonesian 
\mbox{National}
\mbox{Standard)} and ASTM D4806,
reflecting 
residual impurities unlike optimized yields 
(${\sim}$0.82~g/mL) from residue hydrolysis. The post-dehydration
density (0.791~g/mL at 99.5\% v/v) surpasses these limits via extended
fermentation and  sieves~\cite{19}. This discrepancy arises from
residual water and non-ethanol constituents due to incomplete azeotrope
\mbox{separation} in double-stage distillation. To address this and achieve
fuel-grade quality, we implemented 
molecular sieve dehydration (3~\AA{}
sieves, 10\% w/v loading) post-distillation. This study employed only
two distillation stages due to the ethanol--water azeotrope limit and
escalating energy costs beyond double-stage processing. The fluctuating
data were achieved due to some factors such as substrate exhaustion,
yeast cell death, and possible formation of undesired by-products,
which can decrease ethanol content and alter density. However, beyond
an optimal concentration, excessive yeast can lead to overcrowding,
nutrient competition, and diminished fermentation efficiency, often
resulting in lower ethanol concentrations and reduced bioethanol
density.

The elevated density observed in the samples 
suggests the presence of
residual water and other non-ethanol constituents, which can be
attributed to incomplete separation during the distillation process.
This phenomenon is critical because the density of bioethanol is
inversely correlated with its ethanol concentration; as the proportion
of ethanol increases, the density approaches the value characteristic
of pure  ethanol~\cite{25,26}. Therefore, density measurements provide
a rapid and reliable means of assessing the effectiveness of the
distillation process and the overall quality of the bioethanol 
produced~\cite{11,27}. Elevated density measurements in the distillate
are indicative of substantial water content and the possible presence
of other organic impurities (isoamyl 
alcohol 0.8\%, 1-propanol 0.4\%,
and ethyl acetate esters 0.3\%  w/w)~\cite{28,29}. Such conditions not
only diminish the energy content (calorific value) of the resulting
bioethanol but also restrict its suitability for use as a fuel,
particularly in applications that demand high-purity ethanol for
optimal engine performance. In the context of multi-stage distillation,
the principal aim is to progressively eliminate water and residual
contaminants, thereby achieving a reduction in the density of the final
product~\cite{30,31}. Ideally, each successive distillation stage
should result in a lower density, signifying an increase in ethanol 
purity~\cite{32,33}. Nevertheless, the findings of this 
investigation
reveal that, despite the implementation of multiple distillation
stages, the density values remained considerably above the standard
reference for pure ethanol. This outcome suggests that the current
distillation protocol requires further refinement. Potential strategies
for improvement include 
\mbox{increasing} the number of distillation cycles,
optimizing the design of the distillation column, or incorporating
advanced separation techniques such as molecular sieve adsorption to
facilitate azeotropic separation and enhance overall product purity.

A comparative evaluation of bioethanol purification methods across
recent studies highlights the critical influence of both separation
technology and process configuration on product purity and yield. 
Gozan et~al.~\cite{34} demonstrated that vapor 
\mbox{permeation} membrane technology
could achieve ethanol purity of 96.61\%, while distillation--adsorption
methods were capable of reaching 99.63\% purity, underscoring the
effectiveness of advanced separation technologies in overcoming the
azeotropic barrier and producing anhydrous  bioethanol~\cite{34}. The
position of steam outlet holes in a two-outlet system significantly
affected the final alcohol content, with the higher outlet yielding
bioethanol concentrations up to  96\%~\cite{27}. The free-cell
technique produced higher ethanol  concentrations~\cite{35}. The
implementation of batch distillation, combined with 
molecular sieve
adsorption, was used to purify bioethanol produced by enzymatic
fermentation of sugar cane molasses, affording ethanol with an improved
yield and  titer~\cite{36}. The integration of molecular sieves proved
particularly effective in removing residual water, thereby enabling the
production of high-purity bioethanol suitable for industrial
applications. In addition, hybrid purification schemes combining
liquid--liquid extraction and extractive distillation have been shown
to offer substantial energy savings and reduced greenhouse gas
emissions compared to conventional processes, particularly when thermal
coupling and suitable solvents such as glycerol are 
employed~\cite{37,38}. Furthermore, this study concluded that
alternative bioethanol separation and purification methods, including
adsorption- and membrane-based techniques, can significantly enhance
ethanol purity while reducing operational costs and energy 
\mbox{consumption~\cite{33}.}

\begin{figure*}
\includegraphics{fig03}
\vspace*{-4pt}
\caption{\label{fig3}Viscosity of the samples.}
\vspace*{-5pt}
\end{figure*}

\subsection{The effect of experimental conditions on the bioethanol
viscosity}\label{sec3.2}
Viscosity is a critical physicochemical property that provides valuable
insight into the purity, composition, and overall quality of
bioethanol, particularly when the product is subjected to multi-stage 
distillation~\cite{11}. In the present study, the viscosity of
bioethanol samples following multi-stage distillation ranged from 0.83
to 1.27 cP  (Figure~\ref{fig3}). This range is generally consistent
with the viscosity of high-purity ethanol, which is typically reported
to be between 1.07 and 1.20 cP at  20~\textdegree C~\cite{39}. The
observed variation in viscosity across samples can be attributed to
differences in water content, residual 
\mbox{organic} 
\mbox{impurities,} and the
effectiveness of the distillation process in removing these 
constituents~\cite{40,41,42}. The 
\mbox{influence} of viscosity on bioethanol
quality is multifaceted. Lower viscosity values are indicative of
higher ethanol content and reduced concentrations of water and other
non-ethanol  components~\cite{43,44}. As the distillation process
progresses through multiple stages, the removal of water and impurities
leads to a decrease in viscosity, signifying an increase in product
purity. The optimal fermentation and distillation conditions yield
bioethanol with viscosity values close to those of pure ethanol,
reflecting high product  quality~\cite{45,46,47}.

In the context of multi-stage distillation, the principal aim was to
progressively eliminate water and residual contaminants, thereby
achieving a reduction in the viscosity and density of the final
product. The results of this study demonstrated that the double-stage
distillation process effectively enhanced ethanol purity, with
viscosity values (1.12--1.27 cP) approaching pure ethanol (1.074 cP at 
20~\textdegree C), surpassing prior single-stage efforts achieving only
94.6--95\% purity and higher  viscosities (${\sim}$1.4--1.6~cP implied)
due to incomplete water  removal~\cite{48}. However, samples at the
higher viscosity range (e.g., 1.27~cP) likely retain residual water or
fuel oils, unlike advanced multi-column systems reaching 99.9\% purity
with optimized reflux. This suggests further protocol optimization,
such as molecular sieve integration, is warranted for fuel-grade
specifications according to ASTM  D4806~\cite{49}. The practical
implications of these findings are significant for both quality control
and process optimization in bioethanol production. Monitoring viscosity
in conjunction with density provides a comprehensive assessment of
product quality after  distillation~\cite{50,51}. Consistently low
viscosity values across samples indicate successful removal of
impurities and suitability for use as a  fuel~\cite{52}. Conversely, if
viscosity remains above the typical range for pure ethanol, further
refinement of the distillation process---such as additional stages,
improved column design, or integration of advanced 
separation
technologies---may be warranted to achieve the desired product
specifications.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Turbidity of the samples.}
\vspace*{-4pt}
\end{figure*}

\subsection{The effect of experimental conditions on the bioethanol
turbidity}\label{sec3.3}
Turbidity is a critical parameter in evaluating the quality and clarity
of bioethanol, particularly 
following multi-stage distillation. 
Figure~\ref{fig4} shows the bioethanol turbidity of the samples. A
marked difference in turbidity was observed between two samples with
nearly the same ethanol concentration. The sample fermented for 3 days
with 150~g  \textit{Saccharomyces cerevisiae} (98.63\% purity)
exhibited a turbidity of 137 NTU (very high), consistent with prior
short-fermentation studies reporting ${>}$100 NTU due to incomplete
fuel oil  separation~\cite{53}. In contrast, the sample fermented for 7
days with 125~g
\textit{Saccharomyces cerevisiae} (98.74\% purity)
showed substantially lower turbidity of 18.5 NTU, aligning with
extended fermentation benefits where yeast stress metabolism enhances
volatile impurity volatilization, reducing  haze~\cite{54}. This
inverse time--yeast interaction surpasses typical 50--200 NTU ranges in
coconut water ethanol studies limited to 3--4  days~\cite{55}. These
impurities compromise fuel quality by promoting phase separation, gum
formation, and corrosion in engines---exceeding ASTM D4806 limits
(turbidity  ${<}$5 NTU for fuel-grade ethanol). Extended fermentation
enhances yeast stress metabolism, increasing fuel production and
improving separation efficiency in distillation, thus linking process
parameters directly to product  stability~\cite{56,57}. This
discrepancy highlights that ethanol purity alone does not guarantee low
turbidity or superior product clarity and underscores the importance of
effective impurity removal during the distillation process.

High turbidity in bioethanol is indicative of the presence of suspended
solids, colloidal particles, and residual organic or inorganic
impurities that were not fully eliminated during 
distillation~\cite{28,58}. These particulates may include unreacted
biomass, yeast cells, proteins, or other fermentation byproducts. An
increase in turbidity may disrupt subsequent 
\mbox{processing} steps, and
potentially alter its combustion characteristics when utilized as a 
fuel~\cite{59,60}. In contrast, low turbidity indicates more effective
\mbox{removal} of these impurities, resulting in a clearer and higher-quality
bioethanol  product~\cite{61,62}. The 
consequence of turbidity on
bioethanol quality was multifaceted. While high ethanol content is
essential for fuel applications, the presence of suspended particles
can lead to operational challenges, such as clogging of fuel injectors
or reduced combustion efficiency. Therefore, achieving both high
ethanol purity and low 
turbidity is crucial for producing bioethanol
that meets industry standards for clarity and performance. 
Samples with
high ethanol content could still exhibit elevated turbidity if
separation and clarification steps were insufficient. Furthermore, low
turbidity requires not only efficient distillation but also robust
post-distillation treatment to remove residual particulates and
colloidal matter. Turbidity alongside ethanol concentration provides a
more 
comprehensive assessment of bioethanol quality and is 
essential
for ensuring that the final product meets industry 
standards~\cite{63}. 

\section{Conclusion}\label{sec4}
The density values remained considerably above the standard reference
for pure ethanol. Moreover, 
viscosity and turbidity measurements serve
as key parameters in evaluating the purity and \mbox{quality} of bioethanol
following multi-stage
{distillation.} The 
{results} of this study, with
viscosity values 
{ranging} from 0.96 to 1.27 cP, suggest that the current
{distillation} process was generally effective in producing high-purity
bioethanol, although further optimization may be required for samples
at the upper end of the viscosity range. Multi-stage distillation
contributed to improved clarity and purity of the final bioethanol,
meeting the standards required for fuel and industrial applications.

\section*{Acknowledgements}
The authors would like to express our gratitude to the research team at
State Polytechnic of Sriwijaya for funding the study under the Applied
Research scheme with contract number 07011/PL6.2.1/LT/{\ubreak}2025 dated June
26, 2025, and to the Department of Chemical Engineering for
facilitating the venue and equipment needed during the research.

\CDRGrant[POLSRI]{07011/PL6.2.1/LT/2025}

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