International Journal of Food Microbiology 378 (2022) 109814

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Microbial cellulase production using fruit wastes and its applications in

biofuels production
Mohammed Y. Areeshi
Medical Laboratory Technology Department, College of Applied Medical Sciences, Jazan University, Jazan 45142, Saudi Arabia
Research and Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan University, Jazan 45142, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Keywords: The present review explores fruit wastes as potential and low-cost substrates for economical production of
Fungal cellulase cellulase enzymes. Being renewable, vast availability and having rich organic nutrient, these fruit wastes can be
Fruit waste exploited to produce cellulase enzyme for various industrial applications. This review aimed to explore recent
Solid state fermentation
insight in sustainable production of microbial cellulolytic enzymes following solid state fermentation (SSF)
Cellulose
Biofuels
wherein different types of fruit wastes as a potentially viable and alternative form of substrates have been uti­
lized. In addition, detailed about the characteristics, mechanisms and market scenario of cellulase enzymes
produced through a range of microbial species have been discussed. Further, impacts of different physico­
chemical parameters on solid-state fermentation based enzyme production and scale up issues have also
analyzed. Moreover, applications of cellulases to produce different types of biofuels have been evaluated while
emphases are made on existing hindrances and the possible strategies to improve the enzyme production process
using fruit wastes.

1. Introduction fermentative sugars which can be potentially converted into biofuels

Cellulases are highly demanded industrial enzymes in the global
commercial market owing to their broad utility in different industrial
sectors (Ejaz et al., 2021; Rehman et al., 2014). Biomass based biofuels
production, detergent manufacturing, paper & pulp and juice & bever­
ages are some of the well-known industries wherein cellulase enzymes
are being utilized at large scale (Fig. 1). Biotransformation of organic
biomass via cellulase enzyme is gaining tremendous interests nowadays
because of the number of advantages like clean and green fuel produc­
tion technology, waste management & valorisation as well as low-cost
commercial energy generation for practical applications (Gurung
et al., 2013; Mahato et al., 2021). The demand of organic wastes based
green fuel is very high, and this is due to the limited life-span, high cost
and pollution intensive nature of the existing fossil fuels, and therefore
global reformations in the biowastes to biofuels production policies are
needed to made, urgently. Additionally, organic wastes are broadly
available in the environment, and these wastes are renewable and
inexpensive resources which can be exploited to produce biofuels at
relatively very low-cost in eco-friendly manner (Adrio and Demain,
2014; Angulo-Mosquera et al., 2021; Li et al., 2020; Escuder-Rodríguez
et al., 2018). Moreover, these organic wastes are rich source of
through diverse bio-processing. Nevertheless, complexities of the
compositional content of these biomasses are the main hindrance to be
resolved immediately.
In contrast, fruit wastes are the other attractive organic wastes in
context to produce both enzymes and biofuels when employed as sub­
strate. The cost of the substrate plays a major role to impact the cost of
enzymes as well as the overall biofuels production processing and un­
timely to establish the costs of biofuels (Kuhad et al., 2011; Saini et al.,
2017). Presently, cellulase enzymes are being produced using the com­
mercial cellulosic substrate called as “avicel” and this makes the overall
production of enzymes ~40 % more costly which exerts high impact in
raising the cost of biofuels production, and thus endorses its non-
suitability for the practical implementation. Therefore, search of the
most suitable, potential and low-cost substrate is one of the main thrust
areas of the research in this field. In context to organic wastes, e.g. fruits,
vegetables and cereals wastes are widely produced in huge quantity in
our everyday life, and these wastes are very rich sources of simple,
degrading organic compounds which can be easily break down by mi­
croorganisms to produce cellulase enzymes (Menendez et al., 2015; Tlais
et al., 2020). Additionally, if not utilized properly, these wastes are
being disposed directly by dumping in the environment which causes

E-mail address: marishi@jazanu.edu.sa.

https://doi.org/10.1016/j.ijfoodmicro.2022.109814
Received 30 April 2022; Received in revised form 6 June 2022; Accepted 14 June 2022
Available online 16 June 2022
0168-1605/© 2022 Elsevier B.V. All rights reserved.
M.Y. Areeshi
Fig. 1. Industrial applications of cellulases.
(Adapted with permission from Trivedi et al., 2016.).
severe environmental pollution. Additionally, direct dumping of these
wastes in the surrounding may cause issues related to the greenhouse
gases emission and the global warming along with discharging of the
toxic compounds, for an example generation of dioxin from fruit wastes
can cause severe health issues (Miao et al., 2015; Yurdugül and Naj­
malddin, 2021). In contrast, these fruit & vegetable wastes are highly
rich in nutrient like carbohydrates and minerals, and may be explored as
substrate for cellulase enzyme production.
Therefore, based on the above analysis, the present review has been
targeted to evaluate suitability of fruit wastes as the cost-effective,
Fig. 2. Action of various cellulases enzymes on surface layer of cellulose. Figure illustrates the action of enzymes from three main classes on cellulose. Glucose
molecules in red color represents crystalline region and glucose molecules in black color are in amorphous region.
(Adapted from Kumar and Murthy, 2013 (Open access CCBY 2.0).)
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International Journal of Food Microbiology 378 (2022) 109814
environmental friendly as well as renewable substrate for cellulase en­
zymes production and their implementation for biomass to biofuels
based production technology for the environmental sustainability.
Further, the availability, role and significance of fruit wastes for cellu­
lase enzymes production has been analyzed in details with existing
hindrance. Based, of the current road blocks, possible future suggestions
have also been recommended in this review.
2. Cellulases
Cellulases make the major contribution as the bio-catalyst to degrade
and convert cellulose into simple sugar (Juturu and Wu, 2014; Sharma
et al., 2016; Yang et al., 2021). These enzymes are the combination of
the sub-component enzymes called as endoglucanase, exoglucanase and
β-glucosidases which combinedly act on cellulosic biomass for the pro­
duction of sugars through the process of enzymatic hydrolysis (Bhatta­
charya et al., 2015; Kuhad et al., 2016). Moreover, active research on
cellulases production technologies are going on since past several de­
cades owing to their vast industrial utility, and also to make them more
efficient for the hydrolysis of cellulosic substrates for more economical
production of biofuels. (Haghighi Mood et al., 2013; Menon and Rao,
2012; Sarkar and Aikat, 2012) (Fig. 2). The biotechnological advance­
ment of cellulases was begun in the early 1980s, but during the last three
decades their applications in various industries have been considerably
increased (Singh et al., 2016). Henceforth, the detail and furthermore
exposure of these enzyme systems may be helpful to develop its indus­
trial production processing more economically (Sharma et al., 2016).
2.1. Mechanism of cellulases
Cellulase hydrolyzes cellulose by breaking down the linkage made up
of β-1,4-D-glucan which produces soluble fermentable sugars like
glucose, oligo-sugar such as cellobiose and cello oligosaccharides as the
primary products by its diverse mechanisms (Sarkar and Aikat, 2012;
Srivastava et al., 2014). Cellulases belong to the Glycoside hydrolase
(GH) family of CAZy (Carbohydrate Active Enzyme) which is a classi­
fication based on the comparison of their 3D structures, amino acids
sequences and catalytic mechanisms. Moreover, many GH enzymes have
M.Y. Areeshi
carbohydrate binding modules (CBMs) which are non-catalytic and
involved in substrate targeting so that the catalytic domain spends less
time away from the substrate (da Costa-Latgé et al., 2021; Gupta et al.,
2016; Patel et al., 2017). Endoglucanases are the members of GH fam­
ilies [5–8, 12, 16, 44, 45, 48, 51, 64, 71, 74, 81, 87, 124 & 128] (Yang
et al., 2017), whereas exoglucanases lies in the GH family [5–7, 48] and
BGLs lies in the GH family [1, 3, 4, 17, 30 and 116] (Juturu and Wu,
2014).
2.2. Endoglucanase
An artificial substrate namely carboxy-methyl-cellulose (CMC) is
used for the determination of endoglucanase (EG), henceforth endo­
glucanases are often called as carboxy-methyl-cellulases (CMCase). The
functional actions of these enzymes are mediated via endo-acting mode
which attack randomly on to the multiple sites together in amorphous
regions of cellulose which give rise to newly constructed chain ends with
both the poles named as reducing and non-reducing end for the smooth
of exoglucanases (Romo Sánchez et al., 2015; Sharma et al., 2016).
Structural analysis have explored that EG possesses cleft shaped open
active site groove (Payne et al., 2015). This results in fast degradation of
long chain of polymer along with continuous production of monomeric
sugars (Mohanram et al., 2013). Additionally, EG does not require CBMs
for the hydrolysis of soluble forms of cellulose; like CMCase and amor­
phous cellulose (Sharma et al., 2016).
2.3. Exoglucanases
These enzymes are often called as cellobiohydrolase (CBH) and
constitute about 40–70 % of the fungal cellulase proteins. Further, these
are divided into the classification called as CBH-I and CBH-II, in which
CBH-I covers huge amount and its secretion is reported by several fungal
species (Harris et al., 2014). These are the exo-acting enzymes and
possessively attack the chain ends, reducing (GH7 CBHs) and non-
reducing (GH6 CBHs) of the both amorphous and crystalline regions
to liberate soluble cellobiose for the subsequent hydrolysis by β-gluco­
sidase (Behera and Ray, 2016). The actions of CBH on substrate leads to
rapid enhancement in concentration of monomeric sugars along with
minor changes in the length of cellulose chain (Mohanram et al., 2013).
Further, structural analysis have explored that CBHs contains a tunnel
like structure (around the active sites), which helps the enzyme system
to slide along the cellulose chain to the next cleaving site after the
product is released but without leaving the substrate (Gupta et al.,
2016).
2.4. β-glucosidases
These industrially important enzymes are also known as cellobiase
which can efficiently convert cellobiose oligosaccharides into the simple
fermentable sugar. Further, insufficient BGL secretion may result
amassing of oligosaccharides which can hinder the actions and mecha­
nisms of CMCase and CBH. Therefore, adequate amount of BGL is
necessary for the optimum production of fermentable sugars (Bhatta­
charya et al., 2015). Further, characterizations of BGLs from family GH1
and GH3 are done using the pocket-containing topology (Gupta et al.,
2016).
There should be a greater degree of coordination among these three
enzymes such as endo-exo, exo-exo and endo/exo-BGL for efficient hy­
drolysis of cellulosic biomass to convert them in to the simple sugars
(Olsen et al., 2017). Further, synergism depends upon a number of pa­
rameters like the ratio of the individual enzymes in a cellulase enzyme
system, physiochemical properties of the substrate etc. (Vuong and
Wilson, 2009). Moreover, cellulase activity, type of substrates and the
reaction conditions (pH, temperature, & other parameters) are the main
parameters that affect the bioconversion reaction (Ang et al., 2013;
Gama et al., 2017). In addition, kinetics of the enzymatic hydrolysis of
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International Journal of Food Microbiology 378 (2022) 109814
cellulosic materials is a complex process, and several classified model
based approached have been proposed to explore the reaction mecha­
nism of enzymes, yet failed to establish a clear mechanistic model for the
hydrolysis of biomass (Gupta et al., 2016). During the enzymes action,
the rate of the reaction decreases due to the various factors involved
therein, like substrate crystallinity, product inhibition, protein degra­
dations and also due to the other related factors (Donohoe and Resch,
2015; Gupta et al., 2016).
The approaches such as random mutagenesis and rational protein
design have been used to study the hydrolysis of cellulose to further
improve the activities of catalytic domain, CBMs and also to thermo-
stabilize cellulases (Schuerg et al., 2017). Rational protein design is
possible with better information about the cellulase structures and with
the advancements in modelling software (Vuong and Wilson, 2009).
Hydrolysis of cellulosic components in biomass is very crucial for their
bioconversion to produce fermentable sugars, and the implementation
of an appropriate microorganism as per the substrate features also plays
a vital role to attain the required rate of reaction.
In this context, studies based on cellulase production have been
investigated using different types of fruit wastes. Delabona et al., studied
the growth of Aspergillus fumigatus P40M2 on Orange peel by employing
SSF (Delabona et al., 2013). These authors have reported that the endo-
glucanase activity was increased from 18.42 IUg− 1 to 32.92 IUg− 1 when
substrate was changed from orange peel to the mixture of orange peel
and wheat bran (1:1 ratio). Kitcha and Cheirsilp, recorded cellulases and
xylanases from Aspergillus tubingensis using palm empty fruit bunches by
employing SSF (Kitcha and Cheirsilp, 2014). The value of cellulases and
xylanases enzymes was found to be increased up to 26.1 ± 0.1 U/gds
and 59.3 ± 0.3 U/gds, respectively, and this was due to the various
factors which involved alkali pretreatment, addition of palm kernel cake
in form of nitrogen source via optimization following the method called
as response surface methodology (RSM). Zhu et al., investigated cellu­
lase enzyme producing capability of Trichoderma reesei RUT-C30 using
oil palm empty fruit bunch fiber through the SSF (Zhu et al., 2014).
Further, the maximum FPase, CMCase and β-glucosidase activities were
found to be 20.2 IU g− 1, 132.5 IU g− 1 and 101.1 IU g− 1, respectively.
Palm empty fruit bunch was utilized to produce cellulase following SSF
wherein Trichoderma viride and Trichoderma reesei were employed as the
fungal species (Wonoputri et al., 2018). It was found that Trichoderma
viride exhibited better efficiency to produce cellulase enzyme as
compared to Trichoderma reesei and the optimum activity could be found
to be 0.79 FPU/g when solid to liquid ratio set to 1:1, whereas the
optimized temperature and pH to perform the fermentation were
recorded to be 30 ◦ C and 5.5, respectively. Similarly, chestnut seed
(Terminalia catappa Linn.) has been employed to produce cellulases using
Aspergillus niger IOC 3998 (Uchida and Silvino dos Santos, 2020). It was
noticed that the pre-treatment using NaOH 2 % was effective to produce
CMCase and FPase. Additionally, activity of CMCase and FPase were
significantly influenced by the moisture content wherein the maximum
CMCase could be recorded to be 13.2 U/g using pH 5.5 and 54 %
moisture. Besides, it was found that CMCase and FPase exhibited better
stability over the temperature range 30 ◦ C to 50 ◦ C, meanwhile CMCase
showed better resistance to temperature increase as compared to FPase.
In continuation, production of pectinase and cellulase has been reported
using orange peels by Aspergillus species NCIM -1432 (Labrath and Gai­
kar, 2020). The activity of pectinase and cellulase were found to be 155
U/g and 239 CMC/g, respectively. In a recent study combination of
Trichoderma sp., tape yeast, and tempeh yeast was used to produce
cellulase using groundnut (Arachis hypogaea) shell (Abduh et al., 2022).
The optimum cellulase activity of 0.12 FPU/ml in case of mixed culture
of Trichoderma sp. and tape yeast could be noticed.
2.5. Microbial sources for cellulase production
Cellulolytic enzymes are secreted by several microorganisms for
example; fungi, bacteria, actinomycetes and fungi are the dominating
M.Y. Areeshi
microbial species which are capable to produce cellulase enzymes (Mesa
et al., 2016). In addition, based on their environmental adaptation, these
enzymes producing groups are further categorized as aerobes & anaer­
obes followed by mesophiles, thermophiles, extremophiles and com­
plexes & non-complex microbial systems (Fig. 3) (Kuhad et al., 2016;
Rathinam et al., 2017). Though many bacterial and fungal species are
well reported for the potential cellulase production (Fariq, 2016; Mesa
et al., 2016; Sethi et al., 2016), the aerobic fungal as well as bacterial
species produce free enzymes in non-complex forms (Acharya and
Chaudhary, 2012; Gusakov, 2011). Further, the complex extracellular
cellulolytic enzyme secreted by anaerobic microorganisms are the high
molecular weighted compounds, known as cellulosomes (consists of
scaffoldins and cellulosomal enzymes) that are capable of degrading the
plant cell walls, but it is difficult to recover individual active enzyme
species (A. Ferreira et al., 2016; Behera and Ray, 2016). The large size of
cellulosomes limits their movement compared to free enzymes; and also
for the equal mass loading, the molar concentrations of cellulosomes are
lower than that of free enzymes, and this resulting in substantial loss of
activity (Bhattacharya et al., 2015). Therefore, for the commercial
purpose aerobic microbes are preferred over the anaerobic, because free
cellulases have greater activity and it is easy to recover the individual
active enzyme species.
The ability of certain aerobic, filamentous fungi (such as soft rot
fungal strains of Trichoderma and Aspergillus) to release large amount of
extracellular, non-complex protein make it suitable for the commercial
production of cellulase enzyme, as it is easy for the extraction and pu­
rification process (Sarkar and Aikat, 2012; Yoon et al., 2014). Also, the
capacity of producing sufficient enzyme titers completes the enzyme
system (having EG, CBH & BGL) for efficient saccharification of cellu­
lose, and the hyphal penetration makes filamentous fungi preferable
over the unicellular organism e.g. bacteria (A. Ferreira et al., 2016).
Further, filamentous fungal species are well known for their versatile
Fig. 3. Microbial cellulase sources, diversity, and forms.
(Adapted with permission from Leo et al., 2019.)
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International Journal of Food Microbiology 378 (2022) 109814
nature and flexibility to develop on broad substrates like fruit, cellulosic
and vegetables wastes (Julia et al., 2016). Additionally, these fungal
species are capable to break down lignin and makes cellulose free for the
enzymatic hydrolysis (Romo Sánchez et al., 2015). Currently, these
filamentous fungal species are the main source of commercial cellulase
enzymes, for examples, Trichoderma and Aspergillus (Bischof et al.,
2016; Gusakov, 2011).
The growing temperature of the fungal species which produce
commercial cellulolytic enzymes are found between 5 ◦ C to 37 ◦ C along
with the optimal temperature range of 25–30 ◦ C (Cerda et al., 2017;
Tuomela et al., 2000). Since the hydrolysis step is an endothermic re­
action, increase in temperature results in increased yield of sugar which
is the result of lesser viscosity and greater solubility of substrates at
elevated temperatures (Kaur et al., 2017). However, there is a high risk
of contamination of mesophilic enzyme at high temperatures. Therefore,
to reduce the risk of the contamination at higher temperature, thermo­
philic microorganisms can be employed because such microorganisms
possess good thermal stability and the optimum activity at elevated
temperatures (Kaur et al., 2017). Thermoascus aurantiacus, Chaetomium
thermophilum, Sporotrichum thermophile, Talaromyces emersonii, Sporo­
thrix carnis are some of the thermophilic fungi that are capable to pro­
duce thermostable cellulases having complete enzyme system
(Olajuyigbe and Ogunyewo, 2016).
On the other hand, bacteria can withstand under the harsh condi­
tions like high temperature and extreme pH values. Thus, bacteria
produce enzymes which are stable under the harsh conditions, usually
face during a range of industrial bio-processing (Gaur and Tiwari, 2015).
In a study production of cellulase enzyme using fruit wastes by Strep­
tomyces Sp has been reported (Rathnan and Mechoor, 2011). These au­
thors recorded the maximum production of cellulase enzyme at pH 5.0
and 40 ◦ C and the enzyme exhibited ~50 % activity after 5 h at 50 ◦ C.
Production of cellulases and xylanases using Pineapple crown leaves
waste has been performed wherein optimum pH and temperature to
produce cellulase (0.261 U/ml) and xylanase (1.683 U/ml) were noticed
to be 6.0 and 35 ◦ C, respectively (Evelyn et al., 2020). Commercially,
thermostable enzymes secreted by bacterial species like Clostridium
thermocellum, Dictyoglomus turgidum, etc., are available. Beside this, ef­
forts have been made to find thermophilic, cellulolytic microorganisms
and to produce thermostable cellulase enzymes systems with the
implementation of nanoparticles using biomass other than fruit wastes
(Pereira et al., 2015; Rathinam et al., 2017). In a study reported by
Srivastava et al., impact of Fe3O4 NPs and Fe3O4/Alginate nano­
composite have been observed on the cellulase production, activity and
stability (Srivastava et al., 2015). The nanomaterials treated cellulases
showed 35 % and 40 % more activity than that of control enzymes.
Additionally, Fe3O4 NPs and Fe3O4/Alginate nanocomposites treated
cellulase showed 56 % more stability while control enzymes hold it only
19 %. In continuation, Azizi et al., reported new thermophilic cellulase
producing bacterium isolate, Isoptericola variabilis sp. IDAH9 that could
produce significant amount of thermostable endoglucanase using cost
effective substrates for example rice barn and commercial CMC (Azizi
et al., 2015). Zhengang et al., identified a bacterium strain from lime-
applied garbage dumps, named as SWU-27 whose crude enzyme activ­
ities (EG, CBH, BGL) could tolerate treatment of high temperatures (up
to 80 ◦ C) (Zhengang et al., 2015). Moreover, EG, BGL activities could
even tolerate high pH (> 10) conditions. These authors concluded that
lime-applied dumps are better places to isolate thermostable alkaline
cellulase producing bacterial strains (Zhengang et al., 2015). Further,
Srivastava et al., observed that ZnO NPs supported raw cellulase pro­
duced by Aspergillus fumigatus AA001 using rice straw substrate via SSF
showed thermal stability at 10 h at 65 ◦ C and pH stability of 53 % at pH
10.5 (Srivastava et al., 2016). Olajuyigbe and Ogunyewo, reported that
the crude cellulase enzyme obtained from corncob by Sporothrix carnis
was highly stable at elevated temperature (50–90 ◦ C) and had optimum
activity at 80 ◦ C (Olajuyigbe and Ogunyewo, 2016). Moreover, it was
noticed that in presence of Mn2+, Cu2+ and Mg2+, cellulase production
M.Y. Areeshi
level was increased having activities of 959.94 U/ml, 840.52 U/ml and
442.83 U/ml which were 336 %, 294 % and 155 %, respectively higher
than the enzyme yielded in absence of any supplements (Olajuyigbe and
Ogunyewo, 2016). Further, cellulolytic enzymes production can be
increased with the optimization of process parameters following the
statistical approach (Jain et al., 2017; Sharma et al., 2018). Besides, a
number of studies have reported that hot springs are the reservoirs of
useful thermophiles which could produce thermostable enzymes of
cellulase, amylase etc., and these are useful for many industrial appli­
cations (Wajdi et al., 2016; Zhao et al., 2017). It is concluded since very
limited studies about the production of thermostable enzyme using fruit
wastes have been performed and therefore more efforts should be made
to explore the possibility to produce thermostable cellulase enzyme
using the variety of fruit wastes with the implementation of
nanoparticles.
2.6. Cellulase market scenario
The total market for industrial enzymes is increasing drastically
which was estimated to be $3.3 billion in 2010, $4.2 billion in 2014,
$4.61 billion in 2016, and estimated to cover $6.30 billion by 2022
along with the compound annual growth rate of ~5.8 % from 2017 to
2022 (Adrio and Demain, 2014). These demands have been increasing
due to the diverse industrial and R&D activities. On the other hand due
to the depletion of non-renewable energy reserves, biofuels productions
from renewable resources (from starch materials like corn, sugarcane
juice & molasses etc. and lignocellulosic materials like rice husk, saw
dust, etc.) seemed to be a potential alternative in near future (Behera
and Ray, 2016). Since, starch based biofuels utilize food materials which
are imperative for the human consumption such types of food materials
are not favourable to produce biofuels, and hence cellulosic wastes
biomass based biofuels production technology are gaining global
attention (Guerriero et al., 2016). Additionally, the large amounts of
wastes are being produced around the globe which is directly disposed
in to the environment (Jasani et al., 2016). For the production of bio­
fuels from cellulosic and other organic biomasses, cellulase plays a vital
role in the hydrolysis which is the crucial step in the production of
sugars and their conversion in to biofuels (Sharma et al., 2016). Thus,
increasing demand of biofuels is boosting the demand of cellulases in the
commercial market. Moreover, cellulase has many known conventional
applications like textile, food, brewery, paper and pulp industries, etc.,
(Sharma et al., 2016). The US Department of Energy has estimated that
the cellulases annual market would be reaching about $ 9 billion by the
year 2030 (Zarafeta et al., 2016).
Further, “Genecor International Inc.” and “Novozyme” are the
leading producers of cellulase enzyme in the world, supplying cellulase
blends for a wide range of applications (Kuhad et al., 2016; Singhania
et al., 2015; Srivastava et al., 2014). Cellic technology is being used by
the large scale biomass conversion ethanol plants, globally. The latest
cellic product of novozymes “Cellic® CTec3” decreases the cost and
increases the yield of cellulosic ethanol. To make 1 ton of bioethanol,
only 50 kg of the Cellic® CTec3 enzyme is required which makes the
process highly effective. In addition for the commercial production of
these cellulase enzymes using the sub-merged fermentation (SmF),
genetically modified Trichoderma species, Aspergillus species are
generally preferred (Kuhad et al., 2016).
The low yield and high cost of cellulolytic enzyme are the impeding
factors for the economical generation of biofuels and bio products
(Cerda et al., 2017; Jasani et al., 2016). For the cost effective biotrans­
formation of lignocellulosic biomass in to biofuels and bio-based prod­
ucts, cost-effective production of cellulolytic enzymes is essential
(Jasani et al., 2016; Subhojit et al., 2016). Use of cheaper substrates like
easily available fruit wastes and cost effective fermentation process like
solid state fermentation (SSF) may perhaps decrease the production cost
of cellulases, effectively (Julia et al., 2016). Additionally, SSF resembles
the natural habitat of fungi which requires less energy, less capital and
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International Journal of Food Microbiology 378 (2022) 109814
gives more yield of enzymes as compared to SmF (Behera and Ray, 2016;
Julia et al., 2016). The enzyme yields can be increased by screening and
isolation of cellulase producing microbes from the diverse environments
to yield novel cellulases or by tailoring the existing strains (Hussain
et al., 2017; Juturu and Wu, 2014).
Pectin rich wastes like fruit wastes have low amount of lignin as
compared to lignocellulosic wastes such as agricultural wastes (rice
straw, wheat straw, wheat bran) which consist of high lignin concen­
trations about 10–30 % (Dhillon et al., 2013). Since, lignin, cellulose and
hemicellulose are strongly intermeshed in the lignocellulosic materials;
the contents of lignin significantly interferes the enzymatic hydrolysis of
cellulose and hemicellulose (Hendriks and Zeeman, 2009). Therefore,
lignocellulosic substances often require energy intensive delignification
treatments (i.e., pre-treatments) such as physical, chemical and/or
biological as compared to pectin rich (i.e., lignin poor) materials (Con­
esa et al., 2018; Mosier et al., 2005). This pre-treatment process in­
creases the overall production costs of cellulases, and may also degrade
lignin & the component of sugars into the inhibitory compounds (e.g.
furfural) (Dhillon et al., 2013; Hendriks and Zeeman, 2009). Hence, the
production of cellulases enzyme using fruit wastes as substrate is more
potential as compared to the agricultural wastes such as rice straw,
wheat straw, etc. (Dhillon et al., 2013).
3. Factors affecting the performance of SSF
3.1. Microorganisms viability in SSF
SSF closely resembles the natural habit of many micro-organisms
where they remain in close vicinity with substrate (Kuhad et al., 2016;
Pandey, 2003). Micro-organisms like fungi, bacteria, yeast, actinomy­
cetes etc., can be employed in SSF under the aerobic or un-aerobic
conditions to produce various value added products (Behera and Ray,
2016). Classical examples include use of Aspergillus oryzae for koji pro­
cess and Penicillium roqueforti for cheese production. Based on the
theoretical concept of water activity (aw), microorganisms having lower
aw [like fungi and yeast] are suitable for the SSF processes than micro­
organisms having higher aw [like bacteria] (Pandey, 2003; Thomas
et al., 2013). Fungi and yeast exhibit water activity around 0.5–0.6 aw
whereas; bacteria have higher water activity around 0.8–0.9 aw. But
choice of the microorganism based on this theoretical concept seems to
be failed, as there are well established SSF processes which are based on
bacteria (Pandey, 2003). Further, selection of the microorganism should
be based upon the kind of substrate used and also on the products
required (Thomas et al., 2013). Several filamentous fungi like Tricho­
derma sp., Aspergillus sp., Penicillium sp., Humicola insolens, etc., and
bacteria like Bacillus sp., Clostridium sp., Cellulomonas sp., etc. have been
reported for the production of cellulases via SSF (Kuhad et al., 2016; Li
et al., 2014).
3.2. Temperature suitability for SSF
Temperature is one of the main process variables of SSF, influences
the cellulase production significantly (Subhojit et al., 2016; Sun et al.,
2010). It has been observed that the enzyme production increases with
increase in incubation temperature till an optimum temperature, and
further increase in temperature leads to decrease in the production
(Kupski et al., 2013; Subhojit et al., 2016). This phenomenon has been
reported in several studies. Usually, the incubation temperature for the
optimum cellulase production falls in the range of 25-30 ◦ C and mainly
depends upon the kinetics of microorganism employed therein (Yoon
et al., 2014). In an experiment conducted by Silva et al., the effect of
temperature was studied on the cellulase activity of Lichtheimia ramosa
on fruit wastes including Bocaiuva (Acrocomia aculeata), guavira
(Campomanesia pubescens), and pequi (Caryocar brasiliense) following
SSF (Silva et al., 2013). Further, it was noticed that the β-glucosidase
activity obtained on pequi waste was higher among the activities
M.Y. Areeshi
obtained using other wastes. Besides, CMCase activity obtained at 30 ◦ C
(0.656 U/ml) after 120 h was found to be higher than the activity ob­
tained at 35 ◦ C (0.787 U/ml) after 96 h. A similar kind of effect has been
also observed in the study of Irshad et al., wherein significant impact of
temperature on the activity of cellulase enzyme of Trichoderma Viride on
orange peels employing SSF was investigated (Irshad et al., 2013).
Further, the experimental results explored that the activity follows an
increasing trend with rise in temperature up to 35 ◦ C, and further de­
creases with increase in temperature beyond 35 ◦ C. This report was also
in agreement to the observations reported by Subhojit et al., where
maximum cellulase production by Trichoderma sp. RCK65 employing
SSF was obtained at 30o C (Subhojit et al., 2016). Subsequently, further
increase in temperature decreases the yield. This phenomenon may be
attributed to decrease in fungal growth at higher temperature.
In addition, other than the heat supplied from the external source,
there may be heat generation from the inside. This type of heat is
associated with the metabolic reaction, usually released during the mi­
crobial growth under the aerobic conditions (Ray and Behera, 2017).
The removal of metabolic heat is one of the major problems for the scale
up of SSF (Nagel et al., 2001). In this context, evaporative cooling and
wall cooling are the methods which are generally used for the removal of
the generated heat (Nagel et al., 2001). In large-scale mixed bioreactors
evaporative cooling is preferred because wall cooling is not effective.
Further, evaporative cooling should be combined with the water addi­
tion to maintain the required moisture content in the reactor (Nagel
et al., 2001). Moreover, heat transfer may not be uniform in SSF and
therefore increased temperature may cause denaturation of enzymes,
and this may appears in terms of harmful effects on microorganisms and
consequently on enzyme secretion. Thus, it is difficult to control the
temperature in SSF, and at large-scale this is even more difficult. Finally,
it can be concluded that the impact of temperature on the kinetics of
growth and enzyme secretion for the processed microorganism should
be monitored for achieving efficient performance of SSF (Farinas, 2015).
3.3. Particle size
Particle size of substrate is a very crucial factor and plays an
important role during the production of cellulase via SSF. Usually, larger
surface area of substrate particle is a desirable one. Though, smaller
particles offer larger surface area but, too small particles size of substrate
may cause agglomeration, interference with microbial respiration i.e.
aeration which result in poor growth (Agrawal and Matkar, 2016). On
the other hand, larger particles size provides more void space, better
aeration but, at the same time it creates low surface area for attack of
enzymes. Therefore, there should be comprise between the both types of
particle size for the particular process and can be decided by conducting
the experiments (Agrawal and Matkar, 2016). In a study, Krishna and
Chandrasekaran, noticed that the activity was maximum for the specific
particle size (Krishna and Chandrasekaran, 1996). Ang et al., investi­
gated the effect of particle size of substrate (untreated oil palm trunk) on
cellulase production by Aspergillus Fumigatus SK 1 via SSF, and reported
that the optimum particle size of 125 μm was suitable to achieve the
maximum cellulase activities (Ang et al., 2013). In another study,
Agrawal and Matkar, reported that the optimum particle size of sub­
strate allows better nutrient absorption, gas exchange and heat transfer
thus helps to increase the cellulase production (Agrawal and Matkar,
2016).
3.4. Moisture level
The substrate to moisture ratio is another important factor for
determining the growth/metabolism of microorganism, designing and
operation of the SSF bioreactor (Sun et al., 2010; Zhu et al., 2014).
Further, fungi and yeast are the suitable microorganisms according to
the theoretical concept of water activity whereas; bacteria are unsuit­
able (Thomas et al., 2013). However, several researchers have reported
6
International Journal of Food Microbiology 378 (2022) 109814
that the bacterial cultures can be used for the SSF (Thomas et al., 2013).
It has been reported that the enzyme production increases with increase
in moisture content, reaches it maximum value but, any further increase
in moisture levels resulting in decreased production (Garcia et al., 2015;
Sun et al., 2010). This phenomenon can be explained on the basis of
hindrance in microbial growth in both the cases at extreme levels
(Delabona et al., 2013; Raghuwanshi et al., 2014). In addition, in case of
low moisture content high water tension, decreased solubility of nutri­
ents and less substrate swelling are usually noticed, whereas in case of
high moisture content presence of excess free water creates an addi­
tional diffusion barrier which decreases the enzyme yield (Garcia et al.,
2015; Subhojit et al., 2016). Raghuwanshi et al., investigated the effect
of moisture on the cellulase production by mutant strain T. asperellum SR
1–7 under the SSF at 30 ◦ C, pH 10.0 and concluded that the moisture
content ratio from 1:2 to 1:2.5 could enhance the cellulase production at
optimal level and beyond which the production of cellulase was
decreased (Raghuwanshi et al., 2014). The related observations were
also recorded by Garcia et al., to produce optimum β-glucosidase (274
U/gds) using fungus Lichtheimia ramose at moisture content 65 % (Garcia
et al., 2015). These results were also supported by the study of Subhojit
et al., (Subhojit et al., 2016).
From the above studies, it is evident that the appropriate information
about the moisture content in the reactor is very important, and there
are few models which have been developed for predicting the moisture
content in the reactor. Nagel et al., developed a model based on on-line
measurements for the prediction of the moisture content of wheat grains
in 1.5 L & 35 L mixed bioreactor of SSF, cultured by Aspergillus oryzae
(Nagel et al., 2001). Nevertheless, more researches should be done in
this area for the development of simple models for the commercial
production of cellulase enzymes via SSF.
3.5. pH
The pH is one of the key parameters for the microbial growth and
enzyme production (Sarkar and Aikat, 2012; Subhojit et al., 2016).
Dhillon et al., studied the variation of pH on cellulase activity of
Aspergillus niger NRRL-567 on apple pomace using SSF and reported low
enzyme activity at pH 4.0 than pH 4.8 (Dhillon et al., 2012). In a study
by Irshad et al., it is reported that the maximum endoglucanase activity
(435 U/ml), exoglucanase activity (233 U/ml) and β-glucosidase activity
(392 U/ml) of Trichoderma Viride on orange peels employing SSF was
obtained at optimum pH 5.0 (Irshad et al., 2013). Raghuwanshi et al.,
showed the production of cellulase using lignocellulosic material via SSF
by fungus T. asperellum RCK2011 as a well mutant strain of the same
tagged as SR 1–7, maintained 90 % of the optimum activity over the pH
range of 4.0–10.0 (Raghuwanshi et al., 2014). In continuation, Subhojit
et al., investigated cellulase production through SSF using Trichoderma
sp. RCK65 over the pH range of 3.5–7.5, and found that the yield was
maximum at pH 4.5 (Subhojit et al., 2016). In a study reported by Sri­
vastava et al., it has been explored that the nanoparticles can help to
enhance the enzyme stability (Srivastava et al., 2016). Further, it was
noticed that the cellulase system produced using A. fumigatus AA001
exhibited maximum stability at pH 10.5. Thus, the obtained cellulase
system can be used in the production of biofuels. From the above studies,
it can be concluded that the pH plays a significant role in changing and
improving the activity of cellulase enzyme.
3.6. Scale up issues
To design and operate a bioreactor either in pilot scale or large scale,
thorough knowledge of heat and mass transfer effects, kinetic and
modelling studies are essential (Abu Yazid et al., 2017; Soccol et al.,
2017). Development of novel bioreactors design and mathematical
model for the optimization of physicochemical parameters of SSF such
as temperature, pH, substrate concentration, type, particle size & pre-
treatment, incubation time, moisture and aeration etc. are the
M.Y. Areeshi
challenges as well as opportunities to be addressed in SSF for the com­
mercialisation purpose (Cerda et al., 2017; He and Chen, 2013).
Nevertheless, SSF is a promising technology to produce cellulases and
other value added products. Recent developments in SSF have increased
the scope of this technique in diverse fields to produce value added
products. Further, this process should be exploited using different agro
wastes like fruit wastes and with the combination of different microbes
for the commercial production of enzymes like cellulase. However,
intense researches are needed to make in the direction to optimize
numerous process parameters, development of design of bioreactor and
mathematical models.
4. Applications of cellulases in biofuels
4.1. Biohydrogen
Among the existing cellulosic biofuels, production of biohydrogen is
considered as one of the potential alternatives in view of future
renewable energy scenario. Hydrogen via biological mode is produced
from various routes such as photosynthesis [by algae], fermentative
mode [by fermentative bacteria] and photo-fermentative mode [by
photo-fermentative bacteria] (Reddy et al., 2017; Saratale et al., 2015).
Among different approaches for the biological hydrogen generation,
dark fermentative hydrogen production is preferable because of its ad­
vantages which include diverse substrate versatility, high production
rates, easy operation in simple designed reactors and does not requires
light energy and it is cost-effective as well Fig. 4 (Reddy et al., 2017).
Though, biohydrogen production through fermentative mode is a po­
tential option for biofuels production, it suffers from two major draw­
backs e.g. (i) unavailability of suitable substrate and (ii) the low
Fig. 4. Schematic representation of biological production via different modes.
(Adapted with permission from Khanna and Das, 2013.)
7
International Journal of Food Microbiology 378 (2022) 109814
production yield (Zagrodnik and Laniecki, 2015). In this context use of a
suitable cellulosic substrate and efficient cellulase enzyme may be
helpful to overcome these drawbacks in near future. There are numerous
studies available on the production of fermentative hydrogen using
cellulosic biomass, but limited reports on the production of biohydrogen
via biological mode using fruit wastes used as the substrate are found
(Cieciura-Włoch et al., 2020; Reddy et al., 2017). Thus, this area is ur­
gently needed to explore where cellulose present in the fruit wastes can
be either used to produce cellulase and subsequently for the generation
of biofuels. In a study production of biohydrogen from fruit waste by
Clostridium strain BOH3 has been reported (Mahato et al., 2020). It was
found that the Clostridium strain BOH3 was capable to directly ferment
food waste (52.57 g TS/l) and thereby could produce 10,720 ml/l of H2.
Similarly, date fruit wastes were used to produce biohydrogen through
dark fermentation with the implementation of nanocomposites based on
Fe3O4/activated carbon (Rambabu et al., 2021). It was also found that
the implementation of 150 mg/L nanocomposite could increase the yield
of biohydrogen by a factor of 2.05 as compared to control.
Cellulase enzyme produced using different types of fruit wastes can
be further exploited for the enzymatic hydrolysis of a variety of biomass
to produce sugar, and then obtained sugars is utilized to generate bio­
hydrogen following fermentation. In this context, Chen et al., reported
dark fermentative H2 production via untreated rice straw using heat-
treated mixed anaerobic sludge obtained from the municipal wastes
water treatment plant (Chen et al., 2012). These authors yielded
maximum hydrogen production of 24.8 ml/g using rice straw with a
concentration of 90 g at pH 6.5 & 55 ◦ C in a batch reactor. Further, it was
suggested that the dark fermentation of lignocellulosic residues using
mixed thermophilic microbial cultures may avoids the pre-treatment
steps and thereby process can becomes relatively inexpensive (Chen
M.Y. Areeshi
et al., 2012). Bansal et al., investigated the impact of heat pre-treated
mixed inoculum on dark fermentative bio‑hydrogen production via
vegetable wastes and reported that the heat pre-treatment of microbial
inoculum may enhance the biohydrogen production (Bansal, 2013).
Moreover, heat treatment of inoculum is one of the possible ways to
suppress the methanogenic activity to decrease the methane production
and thereby helped to enrich the anaerobic microbes for H2 production
that might survive in high temperature to enhance H2 generation
(Sakthiselvan et al., 2015). Zagrodnik et al., carried out an experiment to
achieve an enhanced hydrogen production by co-culturing of dark-
fermentation using Clostridium acetobutylicum & photo-fermentation
using Rhodobacter sphaeroides (Zagrodnik and Laniecki, 2015). Based
on the experimental results it was argued that the pH plays a very
important role in hydrogen production, and at optimum pH 7.0,
maximum hydrogen yield of 6.22 mol H2/mol glucose was noticed.
In one of the studies, Saratale et al., reported cellulase and xylanase
enzymes for the enzymatic hydrolysis of pre-treated Sorghum husk (SH),
followed by dark-fermentation for H2 production by C. beijerinckii
(Saratale et al., 2015). The maximum H2 production was recorded at
optimal conditions [like temperature of 35 ◦ C, an SH hydrolysate
loading of 5.0 g Reducing Sugar/L, pH maintained at 5.5] and concluded
that the mild acid treatment (0.2 %) of Sorghum husk could enhance the
accessibility of SH to enzymes consequently, increased the production of
reducing sugars (Saratale et al., 2015). In a study, Reddy et al., con­
ducted batch dark fermentation experiments on sugarcane bagasse hy­
drolysates which was obtained at optimum condition of steam-acid
hydrolysis and examined the impact of many factors such as initial pH,
substrate to biomass (S/X) ratio and the effect of Fe2+ and magnetite
nanoparticles (Reddy et al., 2017). As the pre-treatment and acid hy­
drolysis increases the overall cost of the process, similar process can be
done using the mixed culture (fermentative and hydrolytic) and thereby,
helps to eliminate the pre-treatment steps as suggested by Chen et al.
(2012). Nevertheless, to the best of the knowledge, commercial pro­
duction of biohydrogen is not reported yet due to the high production
costs and storage problems. The production cost may be lowered by
using fruit wastes employed as substrate for the both production of
cellulase enzymes and biohydrogen thereby, advancement towards ful­
filling the dream of commercial production of biohydrogen can be made.
4.2. Bioethanol
Besides biohydrogen production from cellulosic substrate, bio­
ethanol is also a potential and feasible alternative to produce biofuels
(Braide et al., 2016; Cherian et al., 2016; Corbin et al., 2015). Bioethanol
Fig. 5. The general process for production of biofuels.
(Adapted from Tatsis and O’Connor, 2016 (open access article under the CC BY-NC-ND license (http://creative-commons.org/licenses/by-nc-nd/4.0/)).)
8
International Journal of Food Microbiology 378 (2022) 109814
can be produced from the both starchy and cellulosic materials, and the
ethanol produced from the starchy foody materials [like molasses, rice,
rice bran, corn, etc.] is called to be Ist generation bioethanol while
ethanol produced from the cellulosic non-foody materials is called as IInd
generation bioethanol (Fig. 5) (Ali Mandegari et al., 2017; Gaurav et al.,
2017). Brazil and America, the world leading producers of bioethanol
are producing Ist generation bioethanol (John et al., 2017). The pro­
duction of Ist generation bioethanol can be no longer encouraged since,
it is produced from the starchy materials which are competing with the
food resources and raises concerns to food security (Singhania et al.,
2014). On the other hand, IInd generation bioethanol is gaining global
attention as enormous amount of cellulosic waste biomasses are being
generated every year which are cheap source of substrate (John et al.,
2017). Generally, two methods are used to produce IInd generation
bioethanol which involve separate hydrolysis and fermentation (SHF) as
well as simultaneous scarification and fermentation (SSFs) (Nguyen
et al., 2017; Srivastava et al., 2015). In case of separate hydrolysis and
fermentation (SHF), hydrolysis and fermentation steps are performed
separately in sequential manner at optimal pH & temperature (John
et al., 2017; Srivastava et al., 2014). The major drawback of this process
is the activity of cellulases inhibited by the sugar accumulated in the
hydrolysis step (John et al., 2017; Singhania et al., 2014). Thus,
resulting better hydrolysis and ethanol yield in SSFs as compared to SHF.
But, the drawback of SSFs process is that, the process is carried out at
non-optimal conditions which leads to longer hydrolysis time, may
require high enzyme loadings and therefore, increases the overall cost of
the produced bioethanol (Bhalla et al., 2013; Srivastava et al., 2015).
Although, IInd generation bioethanol is a promising alternative of the
petroleum based transportation fuels but, due to the high production
cost as compared to the presently used fuels in the market, only few
large-scale production units and pilot plants are being operated around
the world (Balan, 2014; Poonam and Dutt, 2016). In this context, huge
researches are going on to decrease the cost of biofuels through the
various possible approaches including optimization of pretreatment,
hydrolysis, fermentation and low-cost enzymes (Gaurav et al., 2017). In
a study, Parmar and Rupasinghe, produced 4 % (v/v) bioethanol (77 %
fermentation efficiency) and 61.4 g acetic acid/100 g DM from apple
pomace after the sequential bio-processing using Saccharomyces cer­
evisiae and Acetobacter aceti, respectively (Parmar and Rupasinghe,
2013). Sánchez - Orozco et al., carried out studies on the chemical
characterization and compositional analysis such as proximate, total
sugar, thermal, fourier transform infrared spectroscopy (FT-IR), scan­
ning electron microscope (SEM) and energy dispersive X-ray analysis
(EDAX) of fruit wastes [like orange bagasse and orange, mango and
M.Y. Areeshi
banana peels] and reported that these materials have high potential to
be used for the production of bioethanol because of their acceptable
content of cellulose, hemicelluloses, and low lignin (Sánchez - Orozco
et al., 2014). Moreover they are cheaply available, abundant and
renewable resources.
In a study, Li et al., investigated four different types of fermentation
strategies including simultaneous saccharification and fermentation
(SSFs), fed-batch SSFs, semi-SSFs, and fed-batch semi-SSFs to produce
cellulosic ethanol from NaOH soaked sugarcane bagasse, and also
investigated the effect of solid and cellulase loading on ethanol pro­
duction (Li et al., 2014). However, these authors did not notice any
significant difference between all these four processes on the final pro­
duction of ethanol (Li et al., 2014). Nevertheless, SSFs is concluded to be
best method to produce ethanol if yield is the only factor of consider­
ation. Further, it is reported that the requirement of cellulase loading
considerably high when solid loading is high to attain the maximum
hydrolysis efficiency. Singhania et al., through their experiment proved
that the capability of hydrolysis of cellulosic biomass is similar for the
both, extracted cellulase enzymes and whole fermented matter con­
taining cellulase (Singhania et al., 2015). In addition, these authors
suggested on-site preparation of cellulase enzymes and utilization of the
whole fermented matter containing cellulase for the hydrolysis of
biomass thereby this may help to decrease the overall production cost of
bioethanol by removing the step of extraction of cellulase enzymes
(Singhania et al., 2015).
Nguyen et al., reported that the mixed biomass of two/all of Coffee
husk, Coconut coir and Cassava stem was a suitable approach for the
production of bioethanol (Nguyen et al., 2017). Further, it was noticed
that popped pre-treatment of substrate could increase the production in
both the cases i.e. separate hydrolysis and ferementation (SHF) and
simultaneous saccharification and fermentation (SSFs). Besides, exper­
imental results suggested that the SSFs was superior to yield more
ethanol as compared to the SHF from popping the pre-treated individual
and mixed substrate both (Nguyen et al., 2017). Moreover, at global
stage researches are continuously growing to improve the yield and
simultaneously decreasing the production costs of ethanol by applying
various sustainable approaches like, exploring new substrates, and in
this context fruit wastes can be a potential option but need to explore
more (John et al., 2017).
4.3. Biobutanol
Among different types of biofuel, biobutanol is also a potential
alternative to the transportation fuels (Boonsombuti et al., 2015). In
fact, the physiochemical properties of biobutanol includes higher energy
density, lower volatility, lower octane number, less corrosive and less
hygroscopic than ethanol, and these properties are comparable to gas­
oline (Khedkar et al., 2016). Thus, biobutanol can be a substitute of
gasoline and also be used in gasoline engines without any modification
in technology (Bastidas-Oyanedel et al., 2015; Belletante et al., 2016).
Following the conventional method, biobutanol production is per­
formed via acetone-butanol-ethanol (ABE) fermentation using the
fermentative Clostridium sp. Biological method of ABE fermentation
involves the following steps; pretreatment, detoxification, hydrolysis,
fermentation, separation and the purification of the product (Belletante
et al., 2016; Khedkar et al., 2016). Generally, the microorganisms which
are capable to produce butanol are the bacteria belonging to the class
Clostridia species (Belletante et al., 2016). The fermentation process also
produces by-products namely, acetone and ethanol (Belletante et al.,
2016). The major drawbacks in the commercialisation of biobutanol are
the cost of the substrate and the separation of biobutanol from the
diluted broth (Maiti et al., 2016). Therefore, the use of inexpensive and
abundant agro waste materials like fruit wastes as the substrate in the
production of IInd generation biobutanol can improve the viability of the
process. The following are the few studies reporting on the production of
biobutanol. Further, Maiti et al., reported biobutanol production by
9
International Journal of Food Microbiology 378 (2022) 109814
Clostridium beijerinckii NRRL B-466 from the apple pomace ultra-
filtration sludge by anaerobic fermentation which could be increase
with 3 (w/v) % supplementation of glucose (Maiti et al., 2016).
5. Conclusion
Production cost of cellulase enzymes plays very important role in
determining the viability of biomass based biofuels production tech­
nology at practical scale. Presently, commercial cellulases are produced
using the commercial purified substrate with following extra purifica­
tion methods that make the overall cost very high to be employed for
biofuels production process. Therefore, substrate selection and its suit­
able utilization are the main goal of the bioprocessing industries to
develop economical cellulase production technology at commercial
scale for the biomass conversion into biofuels. The rich cellulose &
organic contents, renewable characteristics, low-cost and abundant na­
ture make the fruit wastes one of the potential substrates to produce low-
cost cellulase and this may play a key role to bring down the enzymes
production cost and thereby economical production of a variety of
biofuels. This review explored numerous studies about the utilization of
fruits wastes as a promising substrate to produce cellulase enzyme and
discussed about different process parameters to influence the SSF and
thereby improved production of cellulase. Additionally, applications of
cellulases have been evaluated to produce different types of biofuels
including biohydrogen, bioethanol, and biobutanol with the utilizations
of diverse biomass. It may be concluded that, though this area is highly
promising it requires much exposures to develop the low-cost technol­
ogies to produce cellulase enzymes and their implementation to produce
a number of biofuels for a sustainable and green energy source. In
addition, implementation of nanomaterials can further be explored to
advance this area of research.
CRediT authorship contribution statement
Mohammed Yahya Areeshi: Conceptualization, writing review and
editing.
Declaration of competing interest
Author of the manuscript declare there is no conflict of interests.
Acknowledgment
MYA sincerely acknowledges Jazan University, Saudi Arabia for
providing access of Saudi Digital Libarary facility for this study.
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