Application of Cellulases in Biofuels Industries: An Overview

Abstract

Cellulases have wide applications in the biofuel industry. The three main components, namely endoglucanase, exoglucanase, and β-glucosidase effectively convert lignocellulosic biomass into fermentable sugar. The commercial production of cellulase is done using the submerged fermentation; however, it is costly and less economic for biofuels production. Moreover, microbial cellulase production process suffers from various bottlenecks. Because of the low cost, production of cellulase using solid-state fermentation by fungi is preferable. Therefore, the present review provides an overview on cellulase, main aspects of cellulase production and present scenario of cellulase production in the biofuels industry.

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Journal of Biofuels and Bioenergy (June 2015) 1(1): 55-63
DOI: 10.5958/2454-8618.2015.00007.3
Application of Cellulases in Biofuels Industries: An
Overview
Neha Srivastava1*, Manish Srivastava2, P.K. Mishra3, Pardeep Singh4,
P.W. Ramteke5
1,3Department of Chemical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi
221005, Uttar Pradesh, India
2Department of Physics and Astrophysics, University of Delhi, Delhi 110007, India
4Department of Chemistry, Indian Institute of Technology (BHU), Varanasi 221005, Uttar Pradesh, India
5Department of Biological Sciences, Sam Higginbottom Institute of Agriculture Technology and
Sciences (FormerlyAllahabad Agricultural Institute) Deemed to be University,Allahabad 221007, Uttar
Pradesh, India
Abstract
Cellulases have wide applications in the biofuel industry. The three main
components, namely endoglucanase, exoglucanase, and

-glucosidase
effectively convert lignocellulosic biomass into fermentable sugar. The
commercial production of cellulase is done using the submerged fermentation;
however, it is costly and less economic for biofuels production. Moreover,
microbial cellulase production process suffers from various bottlenecks.
Because of the low cost, production of cellulase using solid-state fermentation
by fungi is preferable. Therefore, the present review provides an overview on
cellulase, main aspects of cellulase production and present scenario of
cellulase production in the biofuels industry.
INTRODUCTION
Cellulase is the enzyme of industrial interest and plays a crucial role in hydrolysis of cellulose,
a prime component of plant cell wall (Chandra et al., 2010). Cellulase covers a broad area in the
global market of industrially important enzymes and it is considered as the third largest industrial
enzyme globally, in the industrially important enzyme market (Yoon et al., 2014). Additionally,
cellulase contributes ~20% of the total enzyme market in all over the world, because of its
massive demand in various industries such as in biofuels production, pulp, paper, textile, food,
beverages, as well as in detergent industries (Srivastava et al., 2015a).Among these, the demand
of cellulase will be strongly picked by the commercial production of biofuels in future and this
will further enhance the demand of cellulose from the biofuel industry (Yoon et al., 2014). For
the production of biofuels glucose is the most desirable product which is obtained from the
hydrolysis of cellulosic substrate via cellulase (Srivastava et al., 2015a). Further, a complete
*Corresponding Author E-mail id: sri.neha10may@gmail.com
Keywords: Biofuels
production, cellulase,
commercial cellulase,
fungal cellulase, solid-state
fermentation, submerged
fermentation
Review Article

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56 Journal of Biofuels and Bioenergy (June 2015)
hydrolysis of cellulose into glucose requires a complete cellulose system; generally it consists
of endoglucanase, exoglucanase, and -glucosidase.
Currently, cellulase production is carried out via biological route using the number of bacteria
and fungi. Among variety of known microorganisms, fungi are capable to produce a complete
cellulase system than bacteria, due to the better penetration ability (Srivastava et al., 2015a).
Though, fungi efficiently produce a complete cellulase system, meanwhile these fungi may
have the deficiency of a specific cellulase component. For example, Aspergillus niger is known
to produce afairly goodamount of -glucosidase, but it may have deficiency of cellobiohydrolase
enzyme whereas Trichoderma reesei is known for low -glucosidase production (Singhania et
al., 2010). Moreover, these fungal cellulases are produced via submerged fermentation at
industrial scale which increases the production cost. Beside cost, submerged fermentation also
suffers from number of drawbacks such as complicated operation, use of expensive materials
and low concentration of end products (Singhania et al., 2007). Low concentration of end
products further requires purification steps, and the additional cost of downstream processes
makes the overall process cost consuming and less economic. In this context, efforts are being
made to improve the cellulase production and reduce the production cost. However, separation
and purification of thermostable enzymes are easy when they over express in a suitable
heterologous host by using affinity tags, like His-Tags (Bai et al., 2010), which make their
production economical using the agriculture waste from the industrial point of you. Keeping
this in view, the present article is focused on numerous approaches for cellulase production and
its application in biofuels industries. In addition, we also summarize obstacles in the process of
cellulase production as well as the possible ways to overcome them.
CELLULASE: CURRENT PRODUCTION STATUSAND CHALLENGES
Biofuels’ production has been reached up to 105 billion liters by the year of 2010 which is
raised ~17% from 2009. Moreover, biofuel production contributed nearly 2.7% of world’s fuel
road transportation, among which bioethanol as well as biodiesel are the most preferable sources
(Srivastava et al., 2015a). For the production of fuels like bioethanol and biohydrogen, conversion
of cellulose into sugar is essential. Further, bioconversion of cellulose into fermentable sugar
requires combined action of three enzymes in cellulase system, which include -1,4-
endoglucanase, -1,4-exoglucanase, and -D-glucosidase. Endoglucanase (EG) breaks down
the crystalline structure of cellulose microfibrils to liberate individual polysaccharide chains.
On the other hand, exoglucanase [cellobiohydrolases (CBH)], gradually convert long-chain
cellulose into cellodextrins; whereas cellobiase [-glucosidase (BG)] converts cellodextrins
into the individual glucose molecules (Tuncer et al., 2004; Rawat et al., 2014). Therefore, it is
essential to haveall the three enzyme components in the cellulolytic enzyme mixture for effective
hydrolysis of cellulosic biomass.
Several fungal species have been reported for the efficient production of cellulase such as
Aspergillus niger, Aspergillus fumigatus, Trichoderma spp., etc. These fungi are further classified
as wood rotting fungi (Yoon et al., 2014). Next, wood rotting fungi can be differentiated into
three categories, namely white-rot fungi (WRF), brown-rot fungi (BRF), and soft rot fungi
(SRF). Among these, fungi belonging to the SRF are known as potential cellulase producers
(e.g. T. reesei and A. niger). Besides, number of the WRF (e.g Phanerochaete chrysosporium)

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57Srivastava et al. (Application of Cellulases in Biofuels Industries: An Overview)
and BRF (e.g. Gloeophyllum trabeum) are also known for proficient cellulase production
(Shrestha et al., 2008; Rasmussen et al., 2010; Yoon et al., 2014).Although, the aforementioned
fungi are potential for effective production of cellulase, the hydrolysis of cellulosic biomasses
using these cellulases is incomplete due to the deficiency of a particular cellulase component.
Therefore, further researches targeting to improve the efficiency of cellulase and its production
is required.
Cellulase Market Scenario
The wide applications of cellulase gradually increase its demand in industries. However, only
for bioethanol production, the available literatures have reported considerably different cost,
including $0.10/gal (Aden and Foust, 2009), $0.30/gal (Lynd et al., 2008), $0.32/gal (Dutta et
al., 2010), $0.35/gal (Klein et al., 2010), and $0.40/gal (Kazi et al., 2010). Because of the
contradictions in the cost of enzymes, specifically for biofuel applications, there are several
difficulties for techno-economic analysis and commercial production processes of biofuels. For
the economical production of cellulase, many factors such as high substrate loading, low enzyme
loading, and a short hydrolysis period are crucial. According to Klein et al. (2012), these factors
can significantly reduce the production cost of enzymes at commercial platform. At commercial
scale, there are number of industries involve in production of cellulase, whereas at the global
stage, two main companies, namely Genencor and Novozymes are known for industrial
production of cellulase. These companies have significantly contributed to bring down the cost
of cellulose by several folds. Genencor has introduced a cellulase complex named
Accelerase®1500, specifically for lignocellulosic biomass processing industries (Singhania et
al., 2010) which is further considered to be more cost effective than previously available
predecessor—Accelerase®1000 for bioethanol production industries. Moreover, the
Accelerase®1500 contains higher levels of -glucosidase enzyme activity over the other
commercial cellulases and thus it seems to be efficient for almost conversion of cellobiose into
glucose (Penttila et al., 1998; Singhania et al., 2010). There are many potential cellulases which
can hydrolyze biomass along with -glucosidase. Further, Novozymes also represent various
ranges of cellulase preparations depending on their application. In addition, Novozymes also
have cellulase preparation for hydrolysis of biomass. Besides, Amano Enzyme Inc., Japan and
MAP’s India are the other enzyme industry which has actively participated in the production of
cellulase. Although, the number of cellulase producing companies around the world have
participated in production and its marketing, but only few of them have developed cellulase for
conversion of biomass.
Cellulases for Biofuels Production
Depletion of fossil fuels and the increasing demand of alternate sources for renewable energy
have developed a huge interest in cellulase production (Pandey et al., 2012). Cellulases have
potential application in biofuels production. Bioconversion of lignocellulosic substrate using
cellulases and other enzymes are the thirst area for the commercialization of biofuels. The
cellulase preparation in biomass conversion processes is based on number of its properties such
as stability, product inhibition, synergism, and composition of lignocellulosic biomass etc.
(Srivastava et al., 2015b). Commercial cellulases are found in the market by different names

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58 Journal of Biofuels and Bioenergy (June 2015)
for biomass hydrolysis. Nieves et al. (2009) have analyzed various commercial cellulases for
the hydrolysis of biomass. These authors performed the standard enzymes assays of Filter Paper
Activity (FPU), CM Case (EG), -glucosidase (BGL) as well asxylanase. However, no clear
relation between the enzymatic activities of cellulase on soluble and insoluble substrates could
be found. Therefore, it is unpredictable to explain the efficiency of cellulase for effective
bioconversion of cellulosic biomass. Nevertheless, cellulases having higher FPUs are desirable
for effective conversion of biomass. Figure 1 depicted the schematic diagram of biofuels
production from lignocellulosic biomass.
Figure 1: Overall view of a conventional biochemical conversion process to produce fuels and
chemicals from lignocellulosic biomass
Cellulase enzymes can be used to convert the cellulose portion of nonfood biomass, such as
agricultural waste and energy crops, into fermentable sugars for subsequent conversion to
renewable fuels and chemicals. Reprinted (adapted) with permission from (Payne et al., 2015).
Copyright (2015) American Chemical Society.
ADVANCED DEVELOPMENT IN CELLULASES PRODUCTION PROCESSES
Production of cellulase is the thirst area of research around the world for cost-effective production
of biofuels and, therefore, many researchers are consistently working in this field. Low production
and high cost have always been a major constrain which are needed to be overcome by adopting
the novel and versatile approaches. In this context, use of inexpensive raw materials as the
substrates, use of genetically modified microorganisms, use of efficient crude thermostable/
thermophilic enzyme are some of the key factors which can enhance the cellulase production,
significantly (Srivastava et al., 2015a). Srivastava et al. (2015a) have discussed the use of
thermostable enzyme using various microorganisms for partial/complete hydrolysis of cellulose.
These authors have systematically discussed the utility of thermostable cellulase over cellulase
for effective hydrolysis. Ang et al. (2013) reported thermostable cellulase production from
Aspergillus fumigatus SK1. These thermostable cellulose exhibited higher FPU activity with
effective hydrolysis for shorter time period. It has also been reported that the thermostable
enzyme can reduce the hydrolysis time (Dutta et al., 2014; Srivastava et al., 2015b).

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59Srivastava et al. (Application of Cellulases in Biofuels Industries: An Overview)
Among various advanced strategies for cellulase production, solid-state fermentation (SSF) is a
potential and cost-effective approach (Pandey, 1994). The SSF is done without the presence of
free water in large amount (Pandey et al., 2000) but in the presence of ample amount of moisture
which provides support for the growth of fungi on lignocellulosic substrate (Singhania et al.,
2009). Because of this, the cost of dewatering step during the downstream processing can be
significantly reduced. Although, at the commercial scale cellulase production is carried out in
the submerged fermentation, but due to the lowyield and high cost this process is not economical.
On the other hand, SSF has the efficiency to be up-scaled for greater volume production.
Additionally, SSF is advantageous for higher enzymes concentration, higher productivity as
well as low requirement of the sterility equipments (Holker et al., 2004). Besides, the crude
enzyme components obtained from the SSF can be directly used for the hydrolysis of the
lignocellulosic substrate. Further, it is expected that the production cost of SSF can be reduced
by tenfold compared to the submerged fermentation (Raghavarao et al., 2003) due to the lower
energy consumption (Holker et al., 2004) and suitable low cost lignocellulosic substrate
(Singhania et al., 2010). SSF provides a suitable condition for filamentous fungi due to easy
cultivation (Holker et al., 2004). Filamentous fungi such as T. reesei,A. niger, A. fumigates are
well-known fungi for cellulase production under the SSF. Several studies have been reported
on effective cellulase production under the solid-state fermentation (Ang et al., 2013; Srivastava
et al., 2014; Chandra et al., 2010). Table 1 summarizes different fungus with different
lignocellulosic substrate under the SSF.
Besides SSF, enhancement of cellulase production depends on selection of lignocellulosic
materials used for SSF. Lignocellulosic biomass are found in huge quantity and considered as
the potential substrates for biofules industries. These biomasses are obtained as waste products
of agricultural practices, especially from various agriculture based industries (Perez et al., 2002).
Since these biomasses are natural and renewable resources of energy, they are the focal point of
modern industries. Additionally, these lignocellulosic biomasses can effectively be converted
into different value-added products such as cellulase production, sugar generation for bio-fuels
production as well as cheap energy sources for microbial fermentation and enzyme production
(Asgher et al., 2013; Iqbal et al., 2013). Thus, addition of suitable lignocellulosic substrate
under the SSF can improve the cellulase production in greater extent.
Apart from SSF and suitable substrate, cellulase production can be further enhanced by using
the genetically modified organisms. Although, many filamentous fungi are able to produce
cellulase but due to the lower yield, feasibility in the area of commercialization is not possible.
Insertion of high cellulase producing gene into thermophilic organism can improve the cellulase
production, but very few information is available in the literatures. Therefore, a good
understanding about the genetics of the organism is needed. Improvements in specific activities
of cellulose can be possible via cellulase engineering which depend on rational design. The
concept of artificial designing of cellulase seems to be more promising for cellulases having
desired features. In addition with genetic modification, co-culture concept is advantageous for
cellulase production under the SSF. Improvement in cellulase production can be achieved via
co-culture of different fungi in the single medium (Kalyani et al., 2013). Co-culture also has
several advantages, e.g. higher productivity, adaptability and substrate utilization compared to
pure culture (Holker et al., 2004). The enzymes system obtained via co-culturing of different
fungi may interact with each other and forms a complete cellulase system. The performance of

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60 Journal of Biofuels and Bioenergy (June 2015)
Table 1: Cellulase production under the SSF using different fungi and substrates
Fungus Substrate Yields of enzymes (IU/gds) Ref.
CMCase FPase -glucosidase
A. heteromorphus Rice straw 14.00 225 186 Singh and Bishnoi, 2012
A. fumigatusSK1 Oil palm trunk 54.27 3.36 4.54 Ang et al., 2013
A. fumigatusAA001 Rice straw 18.00 211 301 Srivastava et al., 2015
Neurospora crassa Wheat straw 13.30 97 5.80 Romero et al., 1999
Penicillium Sugarcane bagasse 5.50 215 23.10 Adsul et al., 2004
janthinellum
Mixed culture: RiceChaff/ 5.64 NA NA Yang et al., 2004
Trichoderma reesei, Wheat Bran
Aspergillus niger (9:1)
Thermoascus Wheat straw 4.70 987 48.80 Kalogeris et al., 2002
auranticus
Trichoderma reesei Wheat straw 3.80 NA NA Singhania et al., 2007
RUT C30
NA: not available
co-culturing fungi for cellulase production has also been reported by Hu et al. (2011). These
authors found that the lignocellulosic components were depolymerized to a greater extent when
the fungi were co-cultured on lignocellulosic substrate during the SSF and showed better
efficiency. In one of the other study by Kalyani et al. (2013), -glucosidase activitywas recovered
by co-culturing of Sistotrema brinkmannii and Agaricus arvensis.
In addition of various approaches to improve the cellulase production at the industrial scale,
number of different co-factors such as addition of metal ions can also enhance the cellulase
production, significantly (Srivastava et al., 2014). Recently, concept of nanomaterials has aroused
as a new era in the revolution of renewable energy production. Recently, Dutta et al. (2014)
reported enhanced cellulase production in the presence of hydroxyapatite nanoparticles. In this
study, an improved thermal stability of cellulose was achieved along with reducing sugars at
the hydrolysis temperature of 80°C, when rice husk/rice straw wasused as the substrates. In one
of the very recent study, by Srivastava et al. (2015b), an improved celluase production, thermal
stability as well as sugar productivity in the presence of Fe3O4/alginate nanocomposite has been
reported. In this study, a higher yield of cellulases using A. fumigatus AA001 under the SSF
was achieved in the presence of Fe3O4/alginate nanocomposite. Further, cellulase production
and its thermal stability were also improved in the presence of Fe3O4nanoparticle and Fe3O4/
alginate nanocomposite. The results reported by the authors clearly exposed that nanoparticles
can play an important role to improve the cellulase production as well as the entire bioconversion
process.In addition, an improved cellulase production and thermal stability has also been reported
in the presence of nickel cobaltite (NiCo2O4) nanoparticle under the SSF using the thermotolarent
A. fumigatus NS (Class: Eurotiomycetes) [Srivastava et al. (2014)]. Thus, nanoparticles may be
considered as one of the key factor to enhance the cellulase production in the near future. Apart
from the aforementioned studies there are other literatures which have reported an improvement
of cellulase production, thermal stability as well as its hydrolysis efficiency in the presence of

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61Srivastava et al. (Application of Cellulases in Biofuels Industries: An Overview)
nanoparticles (Ansari and Husain 2012; Shakeel and Qayyum 2012; Verma et al., 2013).Ansari
and Husain (2012) have discussed the immobilization of cellulase enzyme in the presence of
magnetic nanoparticles. These authors have proposed that the nanoparticle works as a carrier
and improves not only the thermal stability but also the pH stability and tolerance, from inhibitor
during the enzymatic hydrolysis. Verma et al. (2013) reported that the thermal stability of -
glucosidase enzyme was increased in presence of magnetic nanoparticles and exhibited half-
life of the same enzyme at 70°C. Although, nanoparticles have shown potential for improved
cellulase production, thermal stability, and hydrolysis efficiency, their mechanism is not well
understood; therefore, emphasize should be made on cellulase production using the
nanomaterials.
CONCLUDING REMARKS
This review focused on the application of cellulase in biofuels production and its industries.
Specifically, the demand of cellulase is gradually increasing in biofuel industry and therefore,
development of cost-effective methods to produce cellulase at large scale is needed. Although,
it is challenging to develop the cost-effective technology and economy in biofuels industries,
continuous efforts are being made in this field. The use of cheaper and waste material,
thermotolraent/thermophilic organisms, thermostable/thermophilic enzyme, and addition of
certain co-factor can enhance the cellulase and consequently the biofuels production. In addition,
the genetic modification of cellulase producing microbes is another potential area to explore
the higher amount of cellulase for biofuels production.
ACKNOWLEDGEMENTS
Authors N.S. and Prof. P.K. Mishra thankfully acknowledge to DST, New Delhi, India for
providing the Women scientist-B fellowship (SEED/DISHA/WOSB/047/2012/G) and
Department of Chemical Engineering and Technology, IIT (BHU), Varanasi, India. M.S.
acknowledges the Department of Science and Technology, Govt. of India for awarding
DSTINSPIRE Fellowship [IFA13-MS-02] 2014.P. Singh thankfully acknowledges Department
of Chemistry, IIT (BHU), Varanasi, India.
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