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\DOI{10.5802/crchim.268}
\datereceived{2023-04-26}
\daterevised{2023-08-29}
\datererevised{2023-10-20}
\dateaccepted{2023-10-24}
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\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Materials and Energy Valorization of Biomass and Waste : The Path for Sustainability and Circular Economy Promotion}
%%\def\cdr@specialtitle@french{Valorisation \'energ\'etique et mati\`ere de la biomasse et des d\'echets : la voie de la durabilit\'e et de la promotion de l'\'economie circulaire}
%\makeatother

\CDRsetmeta{articletype}{research-article}

\title{Bioethanol production from water hyacinth with isolated
thermophilic microbial consortium from Kenya}

\author{\firstname{Selamawit Shiferaw} \lastname{Deffar}}
\address{Department of Mechanical, Production and Energy Engineering, School of Engineering, Moi~University, P.O. Box 3900, Eldoret, Kenya}

\author{\firstname{Anil} \lastname{Kumar}\CDRorcid{0000-0002-6765-8240}}
\address{Department of Chemical and Process Engineering, School of Engineering, Moi~University, P.O. Box 3900, Eldoret, Kenya}

\author{\firstname{Anthony} \lastname{Muliwa}}
\addressSameAs{2}{Department of Chemical and Process Engineering, School of Engineering, Moi~University, P.O. Box 3900, Eldoret, Kenya}

\author{\firstname{Njira Njira} \lastname{Pili}}
\address{Department of Biological Sciences, School of Sciences and Aerospace Studies, Moi~University, P.O. Box 3900, Eldoret, Kenya}

\author{\firstname{Timothy} \lastname{Omara}\CDRorcid{0000-0002-0175-1055}\IsCorresp}
\address{Institute of Chemistry of Renewable Resources, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Stra{\ss}e 24, 3430 Tulln, Austria}
\email{timothy.omara@boku.ac.at} 
\email[T. Omara]{prof.timo2018@gmail.com}

\curraddr[T. Omara]{Department of Chemistry, School of Physical Sciences,
College of Natural Sciences, Makerere University, P.O. Box 7062,
Kampala, Uganda}

\begin{abstract}
Bioethanol production from water hyacinth pretreated thermochemically
through boiling in water with or without sodium chlorite, followed by
fermentation with a locally isolated thermophilic bacteria consortium
in a partially consolidated bioprocessing was investigated. The time
yielding the maximum total reducing sugars was 2~h using 155~mg/g
and 113~mg/g of pretreated and untreated water hyacinth samples,
respectively. The optimized factors were inoculum dose (8.1\%, v/v),
temperature (48.8~\textdegree C) and time (52.3~h) which resulted into 7.2~g/L
of predicted bioethanol concentration. In the experimental run, 7.7~g/L
of ethanol was produced, which is close to the predicted value.
\end{abstract}

\shortrunauthors

\keywords{\kwd{Bioethanol}
\kwd{Central composite design}
\kwd{\textit{Eichhornia crassipes}}
\kwd{Optimization}
\kwd{Partially consolidated bioprocessing}}

\thanks{Africa Centre of Excellence II in Phytochemicals, Textile and
Renewable Energy (ACE II PTRE), Moi University, Kenya (Credit No.
5798-KE)} 

\maketitle

\clearpage

\twocolumngrid

\end{noXML}

\section{Introduction}

The non-renewable nature of petroleum, petrochemicals and petroproducts
has shifted global attention to biofuels. This paradigm shift is
beneficial as biofuels are comparatively non-toxic, sulfur-free and
biodegradable in nature~\cite{1}. Lignocellulosic biomass-based
biofuels (e.g., bioethanol) is envisioned as one of the economically
viable alternatives to the current petroleum-based energy sources and
petrochemical products~\cite{2,3}. However, bioethanol production
occurs only when biomass are fermented with suitable microbes
\cite{4}, and this is favored by the abundance of exploitable
biomass and microorganisms~\cite{5}.

There is available literature on studies investigating the
destructuring of cell wall polysaccharides to fermentable (total
reducing) sugars, which is a critical step for lignocellulose
conversion into bioethanol. For example, the utilization of thermal
liquefaction~\cite{6} as well as acid and alkaline hydrolysis
\cite{7} have been explored. Nevertheless, the extent to which this
deconstruction is effected largely depends on the type of pretreatment
and biomass chosen~\cite{8}. This, on the other hand, is dictated by
the overall cost, environmental footprint and energy efficiency of the
method~\cite{9}. Among the different pretreatment methods, very few
approaches (e.g., hot water pretreatment) provide high total reducing
sugars (TRS) with little investment~\cite{10}. Hot water pretreatment
requires no or little additional chemicals, shorter residence times,
and is comparatively more energy efficient. At high pressures and
temperatures, water becomes acidic, and it induces autohydrolysis of
glycosidic bonds, thereby resulting in the solubilization of
hemicellulose and partial delignification of the biomass~\cite{11}.
Similarly, acetic acid/sodium chlorite solution has been used for
delignification of biomass~\cite{12}. In a recent investigation,
addition of sodium chlorite (NaClO\tsub{2}) directly to
fermenting water hyacinth (WAHT) without prior pretreatment was neither
harmful to the microbes nor expensive (as it eliminated the cost of
acetic acid)~\cite{13}. This particularity prompted us to
investigate the use of NaClO\tsub{2} with thermal treatment in
WAHT fermentation. 

On the other hand, consolidated bioprocessing is a unit operation where
more than one microorganism hydrolyses and ferments biomass in a single
step. For this reason, thermophilic microbes are more useful because
they can utilize both pentose and hexose sugars, and are provenly
resistant to fermentation inhibitors~\cite{14}. When used as a
consortium, thermophilic microbes tend to be more stable, productive
and functional at optimal conditions~\cite{15}. Thus, establishing
the best (optimal) parameters for fermentation using microbial
consortium and enzymes is crucial to improve substrate utilization and
increase the yield of bioethanol \cite{16}. This step is usually
achieved using Response Surface Methodology, a multivariate statistical
technique that allows the determination of multivariate equations for
the experimental data and realizing an optimized experimental design
\cite{17}. Therefore, this study focuses on the optimization of
bioethanol production from WAHT (\textit{Eichhornia crassipes}), a
pleustophytic ecological plague and noxious weed found in frost-free
water bodies globally. In the lentic waters of Lake Victoria (East
Africa), WAHT has presented various challenges. For example, it has
been cited to constrain navigation, water treatment, electricity
generation and irrigation operations as well as enhancing the spread of
waterborne diseases \cite{18}. This study proposes to pretreat WAHT
using boiling water with NaClO\tsub{2}, followed by fermentation with a
thermophilic microbial consortium isolated from some selected
environmental matrices of Kenya, East Africa. To the best of our
knowledge, no previous study has used this pretreatment combination for
optimized bioethanol production via partially consolidated
bioprocessing approach.


\section{Materials and methods}
\subsection{Feedstock collection}

The WAHT feedstock for this research was manually harvested from Ahero
irrigation scheme in the propinquity of River Nyando 
($0\text{\textdegree}11^{\prime}~0\text{\textdegree}19^{\prime}\mathrm{S}$, 
$34\text{\textdegree}47^{\prime}~34\text{\textdegree}57^{\prime}\mathrm{E}$)
on the Kenyan portion of Lake Victoria, East Africa. The plants were
collected in bags and transported to the laboratory at Moi University,
Kenya. They were cleaned thrice with tap water, and the roots were cut
off. The feedstock was left to dry at room temperature under shade for
a fortnight, and then oven-dried for 3 days at 105~\textdegree C
to constant weight~\cite{19}. Thereafter, the feedstock was ground
to a final particle size of 0.2~mm using a 
NutriBullet$^{\text{\textregistered}}$
600 Series electric grinder (Capbran Holdings, LLC Los Angeles, USA).


\subsection{Feedstock pretreatment and charcterization}


Accordingly, WAHT was subjected to thermochemical pretreatment with
boiling water only or with NaClO\tsub{2} solution. Precisely
weighed 3.0~g of WAHT powder was dissolved in 100~mL of a 1~mg/L
NaClO\tsub{2} solution and the mixture kept in a boiling water
bath for 4~h. Measured 2~mL of the hydrolysate was drawn every one
hour to quantify the TRS. After 4~h, the entire sample was
centrifuged at 6000~rpm for 30~min, and the supernatant was used to
quantify the TRS. 

To characterize the feedstock, both untreated and pretreated WAHT were
subjected to Fourier-transform infrared (FTIR) spectral analysis using
an IRAffinity-1S FTIR spectrophotometer (Shimadzu Corporation, Japan)
in the attenuated total reflectance mode ($4~\mathrm{cm}^{-1}$
spectral resolution scanning between 4000 and $400~\mathrm{cm}^{-1}$). 


\subsection{Sampling and isolation of thermophilic microbes}

Samples with thermophilic bacteria were obtained in sterilized plastic
containers from two different sites. The first sample was taken from a
new and an old biodigester at Moi University, Kenya 
$(0\text{\textdegree}17^{\prime}3.2^{\prime\prime}\mathrm{N}
~35\text{\textdegree}17^{\prime}31.2^{\prime\prime}\mathrm{E})$. 
The temperature and pH of the digesters were 28~\textdegree C and 5.6,
respectively. The second site was a hot spring in the Chemurkeu area on
the shores of Lake Bogoria 
$(0\text{\textdegree}13^{\prime}33^{\prime\prime}\mathrm{N}$ and 
$36\text{\textdegree}05^{\prime}41^{\prime\prime}\mathrm{E}$), an East
African soda lake. On April 17th 2021, water samples were drawn using
sterilized thermal flasks from a hot spring (at 92~\textdegree C and pH
8). Similarly, sediments and microbial mats (at 56.7~\textdegree C and pH
8.4) were sampled. 

To isolate thermophilic bacteria, serial dilution followed by plating
on nutrient agar was done on the same day the samples were collected.
Measured $1~\upmu\mathrm{L}$ of the inocula were taken from all the
dilutions, transferred onto prepared media and then incubated at
50~\textdegree C for 48~h. Thereafter, they were subcultured to
obtain pure cultures. 


\subsection{Microbial profiling of the isolates}

Morphological characterization of the microbial isolates was achieved
through microscopic observation of the colonies, texture as well as
pigmentation~\cite{20}. Gram stains of each isolate was examined
under a microscope, and this was done in duplicate~\cite{21}. Gram
staining result was repeated with potassium hydroxide test.
Physiological tests were based on the growth of the bacteria at various
sodium chlorite concentrations (2--10\% w/v), pH ranges
(5.5--8.5) and incubation temperatures
(22--60~\textdegree C). 

Further, the isolates were subjected to different biochemical
tests---namely, glucose fermentation~\cite{19}, Triple Sugar Iron,
starch hydrolysis~\cite{22}, catalase test, MacConkey
agar~\cite{23}, eosin methylene blue agar~\cite{24}, oxidase
test~\cite{25}, methyl red test~\cite{26}, mannitol salt
agar~\cite{27}, Simmon's Citrate agar~\cite{28} and Kligler
Iron Agar test~\cite{29}.  

\subsection{Feedstock utilization spectrum and microbial consortium
development}

A total of eight pure bacterial isolates were obtained through serial
dilution technique, and were separately tested under the same
conditions for their ability to utilize different substrates. The
fermentation broth medium was prepared according to previous authors
\cite{30} with slight modifications. It contained (per liter) 1~g of
NH\tsub{4}SO\tsub{4}, 1~g of peptone, 2~g of
MgSO\tsub{4}${\cdot}$7H\tsub{2}O, 0.1~g FeSO\tsub{4} and 0.1~g
CaCl\tsub{2}, 2~g of trisodium citrate, 1 g of K\tsub{2}HPO\tsub{4},
5~g of yeast extract and 0.5~g of KH\tsub{2}PO\tsub{4}. The experiment
was conducted at pH 7 and 50~\textdegree C. The pH was adjusted by
addition of either 1M hydrochloric acid or 1M sodium hydroxide
solution. Isolates were grown separately on a medium containing: 10~g/L
of glucose, 20~g/L of starch, Whatman filter paper (cellulose) and WAHT
separately as the sole carbon (energy) source. The Whatman paper
consisted of 99\% cellulose \cite{31}. Growth of bacteria in the
soluble substrate was monitored by optical density of the cultures
(OD600) using ultraviolet visible spectrometry~\cite{14,32}
whereas the OD600 for insoluble substrate (filter paper and WAHT) were
determined from the supernatant, after centrifugation at 6000~rpm for
20~min. Further, the TRS was also quantified from clear supernatant
to establish if there were any degradation of polysaccharides.
\looseness=-1

For the development of microbial consortium, three out of the eight
isolates (coded OL, BO1 and BO2) which possessed cellulolytic
activities were selected for consortium development. Further, the three
bacterial isolates were separately grown at 50~\textdegree C in
broth media containing 10~g/L of untreated WAHT powder. The microbial
consortium was developed using two isolates from the three isolates. To
obtain a stable consortium, they were repeatedly cultured in the broth
medium with WAHT five times with the same condition as that of the
individual strains.


\subsection{Optimization of bioethanol production from WAHT feedstock}


Bioethanol was produced in a 250~mL conical flask with a working
volume of 100~mL. The fermentation was carried out using sugar
hydrolysate obtained from WAHT and the broth medium. The medium was
then sterilized and its pH adjusted to 7. The fermentation experiment
was based on partially consolidated bioprocessing using the
thermophilic microbial consortium, rather than a single microbe,
because it increases the yield of the finished product and more
efficiently consumes the substrate~\cite{33}.

Process parameters for the partially consolidated bioprocessing
approach were optimized using central composite design of response
surface methodology. The model was built with three factors and three
levels to explore their effects and interactions on bioethanol yield.
These factors, namely, fermentation temperature (X\tsub{1}),
fermentation time (X\tsub{2}) and inoculum dosage (X\tsub{3}), were
optimized. Experimental designs were performed using Design--Expert
statistical software for Windows (version 13.0.5.0, Stat-Ease Inc.,
USA). The parameters included temperature (40--60~\textdegree C), time
(48--96~h) and inoculum ratio (8--12\%~v/v). A total of 20
experimental runs were conducted. Experimental significance of the
obtained model was checked by $F$-test (calculated $P$-value) while
goodness of fit was assessed using multiple correlations and
determination of $R^{2}$ coefficients. Analysis of Variance (ANOVA)
was used to estimate the statistical parameters for maximum bioethanol
yield. Statistical significance was considered at $P< 0.05$. 

Growth on the different substrates was determined based on the OD600
using DU 720 General Purpose UV/Vis spectrophotometer. The optimal pH
was measured using a digital pH meter. The TRS were determined at 
540~nm using 3,5-dinitrocyclic acid (DNS) method with glucose as the
standard~\cite{34}.

Ethanol concentration was determined by back titration with acidified
potassium dichromate. When an alcohol vapor makes contact with the
orange dichromate, the color changes from orange to green. The degree
of colour change is directly related to the level of alcohol in the
samples. The ethanol concentration was analyzed via the spectrometric
method by dissolving 7~g of K\tsub{2}CrO\tsub{7} in 5M sulphuric acid
at a working volume of 250~mL as described by previous authors
\cite{35}. To prepare the dichromate--alcohol reaction,
$300~\upmu\mathrm{L}$ of alcoholic samples were mixed with 3~mL of
dichromate solution and then incubated at room temperature for 
30~min. The absorbance was measured at 590~nm, and the ethanol
content was calculated using an ethanol standard curve.


\section{Results and discussion}
\subsection{Pretreatment and FTIR spectra of WAHT feedstock} 

The effect of pretreating WAHT in boiling water with and without
NaClO\tsub{2} was investigated. Results (Figure~\ref{fig1}) show that
the amount of TRS increases from the start of the experiment up to 
2~h, and does not show any appreciable increase after 3~h, i.e.,
the maximum TRS yield of 155~mg/g of WAHT is realized at 2~h for
the pretreated WAHT biomass. This result is expected since the use of a
delignifying agent reduces the amount of cellulase enzyme adsorbed on
lignin while simultaneously improving cellulose digestibility
\cite{36}. The obtained yield was higher than 88 and 97~mg/g of WAHT
feedstock pretreated with formic and acetic acids~\cite{37} as well
as 22.41 and 99.12~mg/g of WAHT after alkaline and microwave/alkaline
combined pretreatments reported by previous authors~\cite{19}.
Compared to the other pretreatment methods, lower-cost chemicals were
used and there was no loss of polysaccharides. In addition, the
obtained TRS in the hydrolysate was directly fermented without washing
and no inhibitory effects were observed possibly due to very low
concentration (10~mg/L) of the acid used during the pretreatment. Thus,
a combination of boiling water and low concentration of acidified
sodium chlorite solution as a pretreatment condition herein is a
promising approach. The mechanism of WAHT treatment involving
NaClO\tsub{2} occurs via its reaction with acetic acid produced during
fermentation process, resulting into the production of chlorine dioxide
(ClO\tsub{2}) which is a strong delignifying agent \cite{38}. In
addition, the sodium ions that are released into the \mbox{fermentation}
medium enhance the catalytic activity of hydrogen-producing bacteria
\cite{39}.

\begin{figure}
\includegraphics{fig01}
\caption{Yield of total reducing sugars from untreated and pretreated
WAHT.\label{fig1}}
\end{figure}

FTIR spectroscopy was harnessed to confirm if any changes occurred in
the chemical composition and structure of WAHT before and after
pretreatment (Figure~\ref{fig2}). The spectra obtained suggest that
structural changes in WAHT occurred upon pretreatment. For instance,
the peak at $895~\mathrm{cm}^{-1}$ for C--H deformation is associated
with $\upbeta$-glycosidic linkages and monosaccharide units in
saccharides and cellulose. This confirms that the cleavage of
$\upbeta$-glucoside bond (considered a rate-limiting step in
lignocellulosic biomass destructuring) occurred following
pretreatment~\cite{40}, giving rise to the peak that was not present
in the untreated WAHT. 


\begin{figure}
\includegraphics{fig02}
\caption{FTIR spectra of untreated and pretreated WAHT feedstock.\label{fig2}}
\end{figure}


On the other hand, the wavenumbers ranging from 1000 to
$500~\mathrm{cm}^{-1}$ are from cellulose and hemicelluloses~\cite{41}.
Upon pretreatment, these absorption bands were broadened, particularly
for $1024~\mathrm{cm}^{-1}$. In this range, a slight increase in the
number of peaks was also observed, and this results from an increase in
the content of cellulose and hemicellulose in the lignocellulose
\cite{41}. The FTIR peaks at $1361~\mathrm{cm}^{-1}$ and
$1317~\mathrm{cm}^{-1}$ are associated with cellulose, while
$2900~\mathrm{cm}^{-1}$ is C--H stretching vibration band of
cellulose~\cite{42}. The band around $3300~\mathrm{cm}^{-1}$
corresponds to the stretching vibration and overlapping of O--H, which
is recognized as the main infrared sensitive group in
lignocelluloses~\cite{40}. At the $3300~\mathrm{cm}^{-1}$ band, both
treated and untreated sample had almost the same wavenumber but the
pretreated sample had a broader band.


\subsection{Characteristics of isolated thermophilic bacteria}

A total of eight thermophilic bacterial strains were isolated and coded
as BO1, BO2, BOY, BOW, OL, NW, CF and YF. One of these strains (BO2)
which was a bioethanol-producing and lignocellulose-degrading
thermophilic bacteria isolated at 50~\textdegree C is shown in
Figure~\ref{fig3}.

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig03}
\vspace*{-5pt}
\caption{Bacterial isolate, BO2 (a) on nutrient agar and (b)
gram-stained smear under a light microscope.\label{fig3}}
\vspace*{-5pt}
\end{figure*}
            
Morphologically, the isolated colonies ranged from white, cream, cream
yellow to yellowish in pigmentation. Microscopy revealed that all the
isolates from Lake Bogoria were Gram positive, with three rods (BO2,
BOY and BOW) and one coccus (BO1). On the other hand, four of the
biodigester isolates were Gram negative cocci. Based on Gram staining
results, four of the isolates were \textit{Bacillus} species (one Gram
negative and three Gram positive). The remaining five isolates were
Gram negative cocci. \textit{Bacillus} species have straight rod-shaped
cells, measuring between 0.5--2.5 ${\times}$ 1.2--10.25~$\upmu\mathrm{m}$
and are often clustered~\cite{43}. 

All the isolates from Lake Bogoria resemble the isolates reported
previously \cite{44} in terms of color, staining, utilizing most of
the carbon sources and a majority of them belong to genus
\textit{Bacillus}. In consonance with the report of Duckworth et al.\ 
\cite{45}, a majority of soda lakes are mainly associated with
bacteria of the \textit{Bacillus} taxon. \textit{Bacillus} presence
could be due to the genus' tolerance to harsh environmental
circumstances \cite{46}.

It is interesting to note that all the isolates were able to grow at
30~\textdegree C to 60~\textdegree C, though the growth increased
between 35~\textdegree C and 50~\textdegree C in most of the isolates.
At room temperature (22~\textdegree C), only isolate BOY exhibited some
little growth. This indicates that the isolates are heat-loving and can
survive from mesophilic to thermophilic conditions. This could be since
thermophilic bacteria have different protein structures compared to
mesophilic bacteria so that they are able to survive at extreme
temperatures~\cite{47}.

Further, the isolates were subjected to 12 biochemical tests and the
results show that they were all amylase-producing thermophilic
bacteria. Two (eosin methylene blue and oxidase) tests provided
evidence of \textit{Enterobacter} in the samples. In eosin methylene
blue medium, \textit{Enterobacter aerogenes} grows well and pink in
colour without sheen, but \textit{Escherichia coli} grows with a green
metallic sheen~\cite{48}. Isolates BO1, BO2, BOW and OL were
oxidase-negative. Aerobes and facultative aerobes exhibit oxidase
activity whereas \textit{Enterobacteriaceae} are
oxidase-negative~\cite{20}. Mannitol Salt Agar (MSA) is a selective
differential medium for some gram-positive bacteria
(\textit{Staphylococcus} and  \textit{Enterococcus})  that tolerate
high salt concentrations. In this study, only NW was negative for MSA
test. Lactose broth was also a differential medium and all the isolates
were able to ferment lactose except BOY, whereas BO1 fermented lactose
with gas production. Isolates BO1, BOY and BOW were Triple Sugar
Iron-negative, BO1 and BO2 were MacConkey-positive, BO1 and BOY were
Kligler Iron Agar-positive. Isolates BO1, OL, and SO were
catalase-positive, BOY and SO were Simmons' citrate-positive, and BO2,
BOW, YF, and OL were all methyl red-positive. 


\subsection{Microbial consortium development and substrate utilization
property} 

The ability of the isolated strains to utilize different carbon
substrates was tested using broth media supplemented with multiple
carbon substrates (glucose, starch, filter paper and untreated and
pretreated WAHT) and all of the isolates were positive for glucose,
starch and powder WAHT though their substrate utilization rates were
different. Further, the ability of a single strain and microbial
consortium to produce bioethanol was evaluated. Screening of
microorganisms that possess the preferred characteristics is an
important prerequisite to eliminate the inclusion of non-essential
microbes that do not contribute to the desired product yield
\cite{8}. 

In comparison to the microbial consortium containing two strains, the
microbial consortium with three strains shows higher growth and sugar
yields from hydrolysis of WAHT (Figure~\ref{fig4}). Thus, three
isolates (BO1, BO2 and OL) with cellulolytic activity were chosen for
microbial consortium development for the purpose of bioethanol
production in partially consolidated bioprocessing. Further experiments
were conducted using the three bacteria as a microbial consortium and
they were cultured \mbox{multiple} times to obtain a stable consortium. The
effect of pH on consortium development was studied and it was found
that the best pH was 7, with no growth below pH~5. Thus, pH~7 condition
was used in all the experiments. 


\begin{figure}
\vspace*{3pt}
\includegraphics{fig04}
\caption{Total reducing sugars obtained from water hyacinth hydrolysed
with consortium (BO1 and BO2, as well as BO1, BO2 and OL) with direct
supplementation of 8~mg/L NaClO\tsub{2} (in blue) and 10~mg/L
NaClO\tsub{2} (in red).\label{fig4}}
\end{figure}


Our results are similar to a previous study~\cite{13} which
reported that direct supplementation of 8~mg/L NaClO\tsub{2} increased
lignin destruction by about 7.6 times. In fact, our consortium was able
to grow in broth media containing 8, 10, and 20~mg/L of NaClO\tsub{2}
but less growth was observed at 20~mg/L. Although higher concentrations
of NaClO\tsub{2} enhanced the amount of TRS produced, there appeared to
be a decrease in the number of microbes. Thus, 10~mg/L of NaClO\tsub{2}
was used in the partially consolidated bioprocessing.

\subsection{Optimization of bioethanol production}

Bioethanol was produced from thermo-chemically pretreated hydrolysate
fermented with a locally isolated thermophilic microbial consortium.
One of the main reasons for increased interest in using thermophilic
bacteria for second generation ethanol is because of their broad
substrate spectrum~\cite{49}. The production of bioethanol was
obtained using central composite design for temperature, time and
inoculum dosage. According to the experimental run, the maximum
bioethanol concentration was 7.7~g/L, which was achieved at
50~\textdegree C and 8\% (v/v) inoculum dosage after 72~h of
fermentation. Under these conditions, it was expected that it would
achieve 7.561~g/L according to the predicted value of the model. On the
other hand, according to the model numerical analysis and point
prediction, the optimal conditions for maximum ethanol production were:
inoculum dosage 8.1\% (v/v), temperature 48.8~\textdegree C, and time
52.3~h with a predicted bioethanol concentration of 7.192~g/L. The
actual yield is closer to response surface methodology yield; this
indicates the reliability of the presented model. The actual ethanol
concentration obtained and the predicted ethanol concentration for 20
runs are presented in Table~\ref{tab1}.


\begin{table*}
\caption{Experimental design matrix prepared using central composite
design with the experimental and predicted responses\label{tab1}}
\begin{tabular}{cccccc}
\thead
Run &
\parbox[t]{1.8cm}{\centering  Temperature (\textdegree C)} &
\parbox[t]{2cm}{\centering  Incubating time (h)} &
\parbox[t]{1.3cm}{\centering  Inoculum (\%, v/v)} &
\parbox[t]{2.2cm}{\centering  Actual ethanol (g/L)} &
\parbox[t]{2.6cm}{\centering  Predicted ethanol (g/L)} \\ 
\endthead
\01 & 50 & 72 & 10 & 7 & 6.98 \\ 
\02 & 40 & 96 & \08 & 6.11 & 6.03 \\ 
\03 & 60 & 48 & \08 & 5.7 & 5.85 \\ 
\04 & 50 & 72 & 10 & 6.6 & 6.98 \\ 
\05 & 66.82 & 72 & 10 & 4.3 & 4.05 \\ 
\06 & 50 & 72 & 6.64 & 7.7 & 7.56 \\ 
\07 & 50 & 72 & 10 & 7.1 & 6.98 \\ 
\08 & 50 & 72 & 10 & 6.9 & 6.98 \\ 
\09 & 50 & 72 & 13.36 & 6.33 & 6.44 \\ 
10 & 60 & 96 & 12 & 5.3 & 5.32 \\ 
11 & 60 & 48 & 12 & 5.8 & 5.89 \\ 
12 & 40 & 48 & \08 & 6.23 & 6.23 \\ 
13 & 33.18 & 72 & 10 & 3.6 & 3.82 \\ 
14 & 40 & 48 & 12 & 5.7 & 5.55 \\ 
15 & 50 & 72 & 10 & 7.3 & 6.98 \\ 
16 & 50 & 112.36 & 10 & 6 & 6.03 \\ 
17 & 60 & 96 & \08 & 5.8 & 5.97 \\ 
18 & 50 & 31.64 & 10 & 6.73 & 6.68 \\ 
19 & 50 & 72 & 10 & 6.96 & 6.98 \\ 
20 & 40 & 96 & 12 & 4.8 & 4.66  
\botline
\end{tabular}
\vspace*{-5pt}
\end{table*}


Our optimum bioethanol concentration is higher than concentrations
reported for some of the wild-type strains of thermophilic cellulolytic
bacterium \textit{C. thermocellum}, an extensively researched
candidate for thermophilic consolidated bioprocessing: strain ATCC
27405 in fermenter (4~g/L of ethanol obtained)~\cite{49},
\textit{Thermoanaerobacter} J1 from hydrolysates obtained from
different lignocellulosic biomass with the highest ethanol
concentration 1.56~g/L~\cite{50}, \textit{Bacillus paranthracis}
and \textit{Bacillus nitratireducens} microbial consortia with the
highest bioethanol concentration of 0.39~g/L from 7.5~g/L of substrate
\cite{8}.

On the other hand, Sato et al.~\cite{51} reported a higher ethanol
concentration of 23.1~g/L under optimized medium after 168~h of
fermentation with \textit{Clostridium thermocellum} wild-type
strain I-1-B from cellulolytic biomass. These differences in yields
with previous studies could be attributable to the type of biomass, the
effectiveness of the microbial consortium, the type of strain used in
the consortium development, and most importantly, the fermentation
techniques used and other factors influencing fermentation.


\subsection{Mathematical modelling and significant test of bioethanol yield}

To assess the effect of different reaction parameters on bioethanol   
production, quadratic models were selected based on the suggested
model, lack-of-fit test and model summary statistics 
(Table~\ref{tab2}). The actual concentrations of bioethanol obtained
varied from 3.6 to 7.7~g/L. ANOVA indicated a model $F$-value of 42.02,
which implies that the model is significant. Values of ``Prob\ ${>}$\ $F$''
less than 0.05 indicate that the factors B, C, AC, A\tsup{2} and
B\tsup{2} are significant terms in the model that affect the production
of bioethanol (Equation~\ref{eqn1}). The Lack-of-fit $F$-value of 0.94
implies the lack of fit is not significant relative to the pure error.
There is a 52.5\% chance that a ``Lack-of-fit $F$-value'' this large
could occur due to noise. The model-predicted $R^{2}$ of 0.8856 is
close to the adjusted $R^{2}$ of 0.9511 and the model's adequate
precision of 23.1632 indicates that the model can be used to navigate
the design space. High $R^{2}$ value (0.9742) shows that the model
described 97.42\% of the variation in the response variable, leaving
only 2.58\% to the residuals. It was shown that the regression model
was highly significant ($P < 0.05$).
{\begin{eqnarray}\label{eqn1}
&&
\mathrm{Ethanol}~(\mathrm{g}/\mathrm{L}) = 6.98 + 0.0686\mathrm{A} 
- 0.1939\mathrm{B} 
\nonumber\\
&&\quad -\, 0.3328\mathrm{C} + 0.0775\mathrm{AB}
+ 0.1800\mathrm{AC}
\nonumber\\
&&\quad -\, 0.1725\mathrm{BC} - 1.07\mathrm{A}^{2} 
- 0.2214\mathrm{B}^{2} + 0.0085\mathrm{C}^{2} 
\end{eqnarray}}\unskip
In which A $=$ incubation temperature (\textdegree C), B $=$ fermentation
time (hours) and C $=$ inoculum dosage (\%).



Figure~\ref{fig5} shows a three-dimensional (3D) response surface
graphical diagram depicting the effect of temperature, inoculum dosage,
time and their mutual interactions on bioethanol production.
Figure~\ref{fig6} is a counter plot representing the interactive
effects of the optimal conditions. According to the obtained results,
Equation~(\ref{eqn1}) represents the effect of the different factors
and interacting factors on bioethanol concentration (in terms of the
coded factors).

\begin{figure*}
\includegraphics{fig05}
\caption{Response surface plots showing the effect of (a) temperature,
(b) inoculum dosage, and (c)~time, and their mutual interactions on
production of bioethanol in 3D.\label{fig5}}
\end{figure*}


\begin{figure*}
\includegraphics{fig06}
\caption{Response of bioethanol production to fermentation time,
inoculum dosage and fermentation temperature in 2D.\label{fig6}}
\vspace*{3pt}
\end{figure*}



\begin{table*}
\caption{Analysis of variance and the quadratic model for total
bioethanol production\label{tab2}}
\begin{tabular}{cccccc}
\thead
Source &  Sum of squares & df & Mean square  & $F$-value & $P$-value (Prob~${>}$~$F$) \\ 
\endthead
Model & 19.72\0\0 & \09 & \02.19\0\0 & \042.02\0\0 & ${<}$0.0001 \\ 
A-temp & \00.0643 & \01 & \00.0643 & \0\01.23\0\0 & \xless0.2926 \\ 
B-time & \00.5133 & \01 & \00.5133 & \0\09.85\0\0 & \xless0.0105 \\ 
C-inoculum & \01.51\0\0 & \01 & \01.51\0\0 & \029.00\0\0 & \xless0.0003 \\ 
AB & \00.0480 & \01 & \00.0480 & \0\00.9218 & \xless0.3597 \\ 
AC & \00.2592 & \01 & \00.2592 & \0\04.97\0\0 & \xless0.0499 \\ 
BC & \00.2381 & \01 & \00.2381 & \0\04.57\0\0 & \xless0.0583 \\ 
A\tsup{2} & 16.66\0\0 & \01 & 16.66\0\0 & 319.58\0\0 & ${<}$0.0001 \\ 
B\tsup{2} & \00.7062 & \01 & \00.7062 & \013.55\0\0 & \xless0.0042 \\ 
C\tsup{2} & \00.0010 & \01 & \00.0010 & \0\00.0198 & \xless0.8910 \\ 
Residual & \00.5213 & 10 & \00.0521 &  -  &  - \\ 
Lack of fit & \00.2530 & \05 & \00.0506 & \0\00.9427 & \xless0.5250 \\ 
Pure error & \00.2683 & \05 & \00.0537 & - & - \\ 
Cor total & 20.24\0\0 & 19 &  &  &  
\botline
\end{tabular}
\end{table*}

\subsection{Effect of mono-culture and co-culture fermentation} 

Both the single strain and the microbial consortia were examined
separately for ethanol production from glucose. The microbial
consortium has obtained the highest ethanol concentration of 9.01~g/L,
which is 88.3\%  of the theoretical yield. The highest
concentration from a single strain was 3.22~g/L which was obtained
during fermentation with strain BO1. Microbial consortium produced 2.8
to 4 times the amount of bioethanol produced by a single strain from
glucose (Figure~\ref{fig7}). A previous study~\cite{50} which
constructed dual \textit{Caldicellulosiruptor} and
\textit{Thermoanaerobacter} co-cultures revealed up to 8-fold increased
ethanol yields compared to the monocultures of
\textit{Caldicellulosiruptor} strains and \textit{Thermoanaerobactor}.
But the ethanol produced from WAHT was lower as compared to ethanol
from glucose. This behavior is because glucose can easily be utilized
by microbes, whereas WAHT contains a lignocellulose component which is
not easily degraded by microbes. Ethanol production by co-cultures was
strongly dependent on the composition of the substrate~\cite{50}.

\begin{figure}
\includegraphics{fig07}
\caption{Ethanol production from glucose fermentation with single
strain and microbial consortium.\label{fig7}}
\end{figure}

\section{Conclusions}

The obtained results suggest that WAHT pretreated with boiling
water/NaClO\tsub{2} enhanced the yields of TRS (155~mg/g of WAHT). This
relatively high amount of TRS is a promising outcome as it is less
costly and the hydrolysate is directly fermented to bioethanol without
washing. The resulting hydrolysate was fermented to bioethanol by a
locally isolated thermophilic microbial consortium in a partially
consolidated bioprocessing process, which reduced the cost of enzyme
synthesis as well as the risk of contamination. A maximum experimental
bioethanol yield of 7.7~g/L was obtained at 50~\textdegree C, 72~h
of fermentation and 6.64\% (v/v) inoculum dose. This fact suggests that
WAHT has a promising potential to be embraced for more enhanced
bioethanol production. 

\section*{Declaration of interests} 
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.


\section*{Funding}
This research was funded by the Africa Centre of Excellence II in
Phytochemicals, Textile and Renewable Energy (ACE II PTRE), Moi
University, Kenya (Credit No. 5798-KE). 

\CDRGrant[MU]{5798-KE}

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