Biocatalysis and Biotransformation

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Immobilization of co-culture of Saccharomyces

cerevisiae and Scheffersomyces stipitis in sodium
alginate for bioethanol production using
hydrolysate of apple pomace under separate
hydrolysis and fermentation

Shruti Pathania, Nivedita Sharma & Shweta Handa

To cite this article: Shruti Pathania, Nivedita Sharma & Shweta Handa (2017) Immobilization
of co-culture of Saccharomyces�cerevisiae and Scheffersomyces�stipitis�in�sodium
alginate for bioethanol production using hydrolysate of apple pomace under separate
hydrolysis and fermentation, Biocatalysis and Biotransformation, 35:6, 450-459, DOI:
10.1080/10242422.2017.1368497

To link to this article: https://doi.org/10.1080/10242422.2017.1368497

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BIOCATALYSIS AND BIOTRANSFORMATION, 2017
VOL. 35, NO. 6, 450–459
https://doi.org/10.1080/10242422.2017.1368497

RESEARCH ARTICLE

Immobilization of co-culture of Saccharomyces cerevisiae and

Scheffersomyces stipitis in sodium alginate for bioethanol production using
hydrolysate of apple pomace under separate hydrolysis and fermentation
Shruti Pathania , Nivedita Sharma and Shweta Handa
Division of Microbiology, Department of Basic Sciences, Dr Y S Parmar University of Horticulture and Forestry, Solan, HP, India

ABSTRACT ARTICLE HISTORY

Apple pomace as a substrate for bioethanol production is interesting due to its abundance and Received 15 February 2017
sustainable availability in varied states like Himachal Pradesh (H.P.), Jammu and Kashmir, Revised 21 July 2017
Uttarakhand and Arunachal Pradesh, India. In the current study, apple pomace which is the main Accepted 9 August 2017
fruit industrial waste of H.P. was evaluated as feedstock for bioethanol production by the process
of enzymatic saccharification using multiple carbohydrases. Microwave pretreatment of the apple KEYWORDS
pomace resulted in the efficient removal of lignin and crystalline structure of cellulose fibre. The Apple pomace;
enzymatic saccharification of the pretreated biomass was done by optimizing parameters for saccharification;
maximal saccharification leads to production of 27.50 mg/g of reduce, ng sugar. An enhanced S. cerevisiae; S. stipitis;
ethanol yield of 44.46 g/l and fermentation efficiency of 58% by immobilized co-culture of SHF; scanning electron
Saccharomyces cerevisiae MTCC 3089 and Scheffersomyces stipitis NCIM 3498 under SHF as com- microscope
pared to fermentation performed with free yeast cells, i.e. 34.46 g/l of ethanol and 45% of
fermentation efficiency.

1. Introduction Since, apple pomace main by-product of food process-

Energy security and climate change imperatives
require large scale substitution of petroleum-based
fuels. The depletion of petroleum-based fuels and
environmental problems has stimulated the develop-
ment of inexpensive production of biofuels (Yadav
et al. 2011). The production of ethanol from compara-
tively cheaper source of raw materials using efficient
fermentative microorganism is the only possible way
to meet the great demand for ethanol in the present
situation of energy crisis (Pramanik and Rao 2005).
Biofuel production from agri-food wastes/horticultural
waste can also contribute to make waste management
more socially acceptable, sustainable and cost effective
(Kim 2004). Agricultural activities and food industry
generate considerable quantities of wastes which are
rich in organic matter and could constitute new mate-
rials for value added products.
In India around 2896.59 ‘000MT of apple is pro-
duced every year, the top apple producing states of
India are Jammu & Kashmir, Himachal Pradesh,
Uttarakhand & Arunachal Pradesh with their respective
shares of 2003.07, 753.35, 106.14 ‘000 MT, respectively,
in year 2015–16 (Horticulture Statistics Division, GOI).
ing industry of Himachal Pradesh extracted from press-
ing apples for juice or cider and it accounts for
25–35% of the dry mass of apple, since it is not hard-
wood or softwood material it does not require any
harsh pretreatment method for extraction of ferment-
able sugars. Being a waste product, its disposal poses
an environmental hazard. However, owing to its high
fermentable sugars contents, it has a great potential
to produce bioethanol, single cell protein, enzymes
mild alcoholic drinks and pigments. This presents great
opportunity for developing an appropriate technology
that can effectively utilize apple pomace, thereby
reducing the environmental pollution it causes. Prior
to ethanol fermentation by ethanologenic microorgan-
sims, the feedstock needs to be processed by sacchari-
fication technology in order to release fermentable
sugars. Microwave irradiation is reported to change
the ultra-structure of cellulose, degrade lignin and
hemicellulose and increase enzymatic susceptibility
(Agu et al. 2017). Microwave pretreatments create an
electromagnetic fields that heats up the substrate
causing the weakening of its interlinkages. Therefore,
heating is volumetric and rapid when microwave is

CONTACT Shruti Pathania shrutipathania89@gmail.com Division of Microbiology, Department of Basic Sciences, Dr Y S Parmar University of
Horticulture and Forestry, Solan, HP, India
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

used to treat the lignocelluloics. It is hypothesized that
this unique heating feature causes an “explosion
effect” among particles, and improves disruption of
recalcitrant structure of lignocelluloses (Hendriks and
Zeeman 2009).
An increase at the initial hydrolysate sugar concen-
tration provides an increased ethanol concentration
which has a major effect on the energy demand
(Dehkhoda et al. 2008). Traditional microorganisms
(e.g. Saccharomyces cerevisiae and Zymomonas mobilis)
used for ethanol fermentation do not metabolize
pentoses (Hang and Woodams 2008). Further the
discovery of xylose-fermenting yeasts has attracted
widespread interest, as the economy of production of
liquid fuels from lignocellulosic materials is much
improved by the efficient fermentation of both hexose
and pentose sugars (Hinman et al. 1989). Some
yeast such as Scheffersomyces stipitis can ferment
xylose and other important hexoses with relatively
high yields and rate of fermentation. The development
of technologies that can improve the performance of
ethanol production has gained considerable import-
ance over the past 35 years. In this scenario, cell
immobilization has been used for ethanol production
in bioreactors in order to decrease the inhibition
caused by high concentrations of substrate and prod-
uct, thus increasing ethanol production and reducing
its costs (Duarte et al. 2013). There are several studies
reporting the use of immobilized cells in various sup-
ports for a more economical production of ethanol.
These systems are attractive and promising, since
production is higher than that observed for free cells
(Bai et al. 2008). Sodium alginate beads are one of
the most commonly used supports for the immobiliza-
tion of cells. They offer several advantages as a sup-
port, such as good biocompatibility, low cost, easy
availability, and ease of preparation. However, there
are some disadvantages associated with their use,
such as gel degradation, severe mass transfer limita-
tions, low mechanical strength (causing cells to be
released from the support), and large pore size (Zhou
et al. 2007).
Considering the above facts, the present work was
aimed to explore the potential of microbial hydrolytic
enzymes for lignocellulosic biomass degradation and
subsequent bioethanol production. Potentially multiple
carbohydrase from fungus Rhizopus delemar F2 isolated
from decayed wood, were used and evaluated for the
effective hydrolysis of pretreated apple pomace. The
study provides insights, and their potential for utilizing
the respective microbial hydrolytic enzymes for maxi-
mizing the yield of sugars from complex biomass and
subsequent fermentation to ethanol under separate
BIOCATALYSIS AND BIOTRANSFORMATION 451
hydrolysis and fermentation (SHF). Therefore, for effi-
cient conversion of all sugars to ethanol, co-fermenta-
tion of hexoses and pentoses to ethanol is a prime
research target.
2. Materials and methods
2.1 Raw material and processing
Apple pomace residue was procured from H.P.
Horticulture Produce Marketing and Processing
Corporation (HPMC), Parwanoo, H.P., India. The residue
was sun dried in a sun drier available in Energy park
of Dr Y S Parmar University of Horticulture and
Forestry, Nauni, Solan, HP for 2 weeks, followed by its
storage in air tight container. Apple pomace was then
grounded using a grindig mixer, sieve to particle size
of 2 mm and was stored in airtight containers for fur-
ther use.
2.2 Fungal culture and production of multiple
carbohydrase
The brown rot fungal culture, i.e. Rhizopus delemar F2
was obtained from decaying wood and grown on
Cooke Rose Bengal medium (Cooke 1954) for 5 days
and provided with accession number KX5123312.
Multiple carbohydrase was produced by solid state fer-
mentation using biomass pretreated at 450 watt of
microwave dose with moisture agent, i.e. basal salt
medium (Maniatis et al. 1982), in 1:4.5 at 35  C for 6
days. The multiple carbohydrase was partially purified
by using acetone purification in ratio of acetone:super-
natant (6:4). After 7 days of incubation, 50 mL of phos-
phate buffer (0.1 M, pH 6.9) was added to the flasks
and kept under shaking for 1 h. The flask contents
were filtered using muslin cloth and the process was
repeated twice. The filtrate was centrifuged at
12,000 rpm for 15 min at 4  C and the supernatant was
collected for further studies Bollag and Edelstein
1991).
2.3 Enzyme activity
The activities of cellulase, i.e. filter paper activity was
determined using standard methods of FPase (Grazek
1987), xylanase, amylase and pectinase by Miller
(1959). One international unit (IU) of enzyme activity
represents lmoles of xylose/glucose/p-nitrophenol
released/min/ml of enzyme.
2.4 Optimization of enzymatic hydrolysis
Optimization of the enzymatic saccharification of pre-
treated apple pomace was carried following one factor
452 S. PATHANIA ET AL.
at a time response (OFAT). The hydrolysis was carried
out in conical flask with working volume of 50 ml
using multiple carbohydrase from Rhizopus delemar F2.
The parameters were- biomass loading (1–6 ml/g), tem-
perature (30–55  C) and time period (12–72 h). The
hydrolysis experiment was performed in 50 mM of
phosphate buffer at 45  C in a shaking incubator. The
numerical optimization of functional parameters was
done using one factor at a time (OFAT).
2.5 Structural characterization of native and
pretreated biomass
In order to study the surface properties of native, pre-
treated and enzymatically hydrolyzed apple pomace.
Scanning electron microscopy (SEM) images were
taken using Hitachi S-3400 N field emission SEM
(Hitachi High-Tech, Japan). The samples were first
coated with gold in a sputter coater system Model
Q150 RS (Quorum Technologies, UK) and the sample
was observed under a voltage of 25 kV.
2.6 Estimation of sugars from microwave and
multiple carbohydrases treated apple pomace
Glucose and xylose were determined by HPLC method
with the Ultra C18 (Restek Corp.), 250 mm  4.6 mm,
5 mm under the following conditions: sample volume
30 ll; mobile phase 90:10 water:methanol, 10 mM
ammonium formate; flow rate 0.5 ml/min; column tem-
perature 50  C The yield of hydrolysis was defined as
the ratio of the quantity of the liberated simple sub-
stances from the fibrous fraction of the raw material
to the amount of these substances theoretically pos-
sible to obtain determined on the basis of the dry
matter content of the apple pomace (Białas et al.
2012).
2.7 Preparation of yeast inoculum
2.7.1 Hexose yeast
Saccharomyces cerevisiae MTCC 3089 was maintained
on YEPD (yeast extract, peptone, and dextrose)
medium consisted of yeast extract. 10 g/L; peptone,
20 g/L; glucose, 20 g/L; and agar, 25 g/L, pH: 5.0.
2.7.2 Pentose yeast
Yeasts P. stipitis (NCIM 3498) were procured from
NCIM culture collection centre. It was maintained on
MGYP (malt extract, glucose, yeast extract, peptone,
and xylose) medium consisted of malt extract, 5 g/l;
yeast extract. 5 g/l; peptone, 20 g/l; glucose, 5 g/l;
xylose 30 g/l and agar, 25 g/l, pH: 5.0.
2.8 Inoculum development from co-culture
fermentation and their immobilization
The contents of the flask obtained were centrifuged at
10,000 rpm for 10 min and the pellet thus obtained was
given washing with sterile water so as to wash out any
nutrient components of the broth. The washed yeast
cells were then prepared as a suspension in sterile
water containing viable cell count of 1.3  108 cells/ml
and used as inoculum for carrying out fermentation.
The immobilization in calcium alginate beads was car-
ried out as follows: the yeast cells slurry and the
sodium alginates were mixed with sterile water to final
volume 300 mL, with 2.0% (w/v) sodium alginate and
2% (w/v) yeast cells slurry (1:3 ratio). The mixture
obtained was extruded dropwise with sterile syringes
into gently stirred 3% (w/v) CaCl2 and kept for 4 h for
stabilization. The beads obtained containing cells were
stored at 4  C and used for further studies.
2.9 Ethanol fermentation by co-culture
SHF reaction mixture contained fermentation medium
of enzymatically hydrolyzed sugary syrup of apple
pomace inoculated with 20% of yeast inoculum (10%
of S. cerevisiae and 10% of S. stipitis) and the pH of the
resulting mixture was adjusted to 5.6 using NaOH.
2.10 Analytical techniques
Fermentation efficiency was calculated as: Practical
yield/Theoretical yield 100.
Practical yield is the ethanol produced (g/l) and the
theoretical yield (%) is 0.511 per gram of sugar
consumed.
Reducing sugars: The total reducing sugars, released
after acid hydrolysis were estimated by DNS method
(Miller 1959).
Ethanol estimation: The ethanol estimation was
done by using Caputi and Wright (1969).
3. Result and discussion
3.1 Biochemical composition of biomass and
pretreatment of substrate
The various chemical components in the biomass var-
ied depending upon the growing conditions, maturity
of plant and environmental conditions. Quantification
of the structural component yielded 36.6% cellulose,
 BIOCATALYSIS AND BIOTRANSFORMATION 453

11% hemicellulose, 16.6% pectin, 8% starch, 19% lig- Crude Acetone purificaon

nin of the total dry biomass of apple pomace. 18

16
Currently, the interest in the search of novel fungal

Enzyme activity (IU)

14
species capable of producing a mixture of depolyme- 12
rizing enzymes is increasing, so that an enzyme mix- 10
8
ture can be obtained for the complete hydrolysis of
6
different polysaccharides of microbial origin leading to 4
the production of fermentable sugars which can be 2
converted to value added products such as bioethanol. 0
Cellulase Xylanase Pecnase Amylase
Rhizopus delemar F2 producing multiple carbohydrase Acetone purification
is one of the common brown rot fungi associated with Figure 1. Partial purification of multiple carbohydrases from
the decay of substrate capable of producing multiple R. delemar F2.
carbohydrases, i.e. cellulase, xylanase, pectinase and
amylase for enzymatic saccharification of biomass.
Before enzymatic saccharification, a pretreatment is 20
18
applied on the substrate to simplify the structure of
16

Reducing sugar (mg/g)

substrate and to provide the site for multiple carbohy- 14
drases to act and release the sugars. The latest pre- 12
treatment method applied to biomass for production 10

of enzymes and fuels was the microwave treatment. 8

Microwave technology was supposed to simplify three
structural polymers along with extraneous compo-
nents. This physical pretreatment increases the
amorphous region of hemicellulose and thus has a
dramatic effect on decomposition because of lignocel-
lulose softening. The electromagnetic field used in the
microwave might cause non-thermal effects that also
quicken the destruction of crystal line structures
(Verma et al. 2011). After pretreatment, monomeric
sugars were formed from the enzymatic hydrolysis of
cellulose and hemicelluloses, which were further fer-
mented into ethanol using ethanologenic cultures of
yeast. When enzymatic hydrolysis and fermentation
are performed sequentially, it is referred to as separate
hydrolysis and fermentation (SHF). The efficacy of the
pretreatment strategies largely depends upon the
removal of lignin and breakage of complex structure
of lignin to remove hemicelluloses and cellulose. Using
microwave irradiation, it is often possible to speed up
the rate of reactions and hence reduce the reaction
time and the energy consumption.
3.2 Enzymatic hydrolysis
After identifying the optimum pretreatment condi-
tions, optimization of the enzymatic saccharification
was carried out on the pretreated apple pomace. The
efficacy of multiple carbohydrase from R. delemar F2,
in hydrolyzing apple pomace residues was evaluated.
The multiple carbohydrates, i.e. cellulase, xylanase,
pectinase and amylase were partially purified from
R. delemar F2 using cold acetone precipitation. The pre-
cipitation was attained at 6:4 (acetone: supernatant),
6
4
2
0
5 10 15 20 25 30
Enzymatic dose (ml/5g)
Figure 2. Effect of enzymatic dose (ml/5gm) on enzymatic
hydrolysis of apple pomace.
resulting in 2.34 IU/ml of cellulase, 16.30 IU/ml of xyla-
nase, 9.05 IU/ml of pectinase and 2.7 IU/ml of amylase.
The final recovery of partially purified multiple carbo-
hydrase resulted in 64.46% of enhanced purification
fold (Figure 1).
3.2.1 Effect of enzymatic loading
Statistical analysis of Figure 2 showed significant vari-
ation among different enzymatic dose (ml/5gm) used
for sugar production ranging from 5, 10, 15 … to
30 ml from multiple carbohydrases of R. delemar F2.
Optimum level of enzymatic dose was found to be
20 ml/5gm, showing significantly higher reducing
sugar of 18.28 mg/g whereas least reducing sugar yield
of 16.72 mg/g was recorded in 10ml/5gm of enzymatic
dose. Increase in enzymatic dose other than optimum
showed a consistent decrease in sugar production.
In this trail, applying an extremely high enzyme
dosage of 4 ml/g, the saccharification occurred to a
relatively high extent giving 18.28 mg/g of reducing
sugar and 25.76 mg/g of total sugar after 72 h of incu-
bation, allowing reasonable enzymatic hydrolysis activ-
ities, whereas a lower yield of reducing sugar of
12.70 mg/g obtained at lower enzymatic dose
(Figure 1). Monosaccharide was the main product
454 S. PATHANIA ET AL.
obtained after enzymatic hydrolysis of the pretreated
apple pomace. High enzymatic loading may counteract
the saccharification by increasing the rate of transgly-
cosylation reactions along with hydrodynamic instabil-
ity, improper mixing, and suspension of slurry
(Alrumman 2016). The observed increase in the yield
of sugars could be due to the higher availability of
simplified sugars from cellulose, hemicelluloses, pectin
and starch after enzymatic hydrolysis. Enzyme loading
above the saturation level beyond the level resulted in
the reduced efficiency due to nonspecific binding of
the enzyme as well as lesser availability of substrate
(Ioelovich and Morag 2012). The saccharification of
POME and OPFF using Novozyme/Celluclast (N/C ratio)
at a dosage of 1 ml/g and 2 ml/g substrate produced
30.26 g/l reducing sugar and 16.73 g/l glucose as
revealed by Khaw and Ariff (2009). The release of
reducing sugar doubled, i.e. 30.26 g/l with the increase
in enzyme dosage 0.2 ml CellicVHTec2/g WIS, which
could achieve a glucan conversion of 80% in 45 h
(Pengilly et al. 2016).
3.2.2 Effect of temperature
The effect of temperature was studied to identify the
optimum temperature for maximum hydrolysis of
pretreated apple pomace into monomeric sugars.
Different fixed temperatures, i.e. 30  C, 35  C, 40  C,
45  C, 50  C and 55  C were used for assessing the
enzymatic hydrolysis of apple pomace for releasing
reducing sugars. Data pertaining to enzymatic hydroly-
sis are presented in Figure 3. The maximum yield of
reducing sugar 20.87 mg/g was observed at 50  C, fol-
lowed by 45  C with 18.28 mg/g of reducing sugar,
whereas the minimum reducing sugar of 12.85 mg/g
was observed at 30  C using enzymatic dose of
20 ml/5 gm. The rate of hydrolysis was slow at 30  C,
which would be due to insufficient energy provided
by the temperature for the carbohydrases to react
with the substrates. In the present study, the rate of
25
20
Reducing sugar (mg/g)
15
10
5
0 0
30 35 40
Temperature (degree celsius)
Figure 3. Effect of temperature on enzymatic hydrolysis of
apple pomace.
hydrolysis increases at 50  C, due to the exposure of
peptide bonds which leads to the increase in the
degree of hydrolysis, indicating the thermostable
nature of multiple carbohydrases. Thermostable
enzymes would expectedly also allow hydrolysis at
higher consistency due to lower viscosity at elevated
temperatures, more flexibility in the process configura-
tions and also lead to hemicelluloses degredation and
lignin transformation.
The decreased effect on saccharification efficiency
with an increase in the temperature above 55  C
resulted in the loss of enzyme activity due to thermal
denaturation resulting in a decrease in the hydrolysis
rate, which could also be due to other factors such as
decrease in the concentration of peptide bonds avail-
able for hydrolysis, enzyme inhibition and enzyme
deactivation.
A combination of enzymes from A. oryzae P6B2
R
(WM) and A. oryzae P27C3A (EE) were used for enzym-
atic hydrolysis of sugarcane baggase which released
the maximum reducing sugars of 34.5% after 72 h at
50  C under SHF (Pirota et al. 2014). Similarly the high-
est degree of saccharification (DoS) was also achieved
by Trichoderma sp. which was 45.71% at 50  C under
separate hydrolysis and fermentation using agrowaste
(Mahamud and Gomes 2012).
3.2.3 Effect of time period
Optimization of time for enzymatic hydrolysis of micro-
wave pretreated apple pomace for satisfactory yield of
monomeric sugars was done under SHF. Figure 4
shows a decreased yield of reducing sugar in the ini-
tial incubation time period of 12 h, i.e. 14.45 mg/g
reducing sugar which afterward showed an increase
up to the time period of 46 h with 26.56 mg/g of
reducing sugar, as a longer incubation time period
allowed the enzyme to act more extensively on the
structure of substrate (Figure 4), a further increase in
time period reduces the saccharification process.
30
25
Reducing sugar (mg/g)
20
15
10
5
45 50 55 1 2 3 4 5 6
Time period (h)
Figure 4. Effect of time period on enzymatic hydrolysis of
apple pomace.

Furthermore, Mahamud and Gomes (2012) reported
that the rate and extent of saccharification depend on
pre-treatment of the substrate, enzyme and substrate
concentration, product inhibition, and enzyme stability,
all of these make the rate of hydrolysis to decrease
rapidly with the time. The decline in reducing sugar
yield could be attributed to the adsorption of liquid
over solid biomass resulting in decline in viscosity
impeding the enzymatic hydrolysis as well as the end
product inhibition of enzyme by glucose and cello-
biose (Kuhad et al. 1999).
Leenakul and Tippayawong (2010) reported the
maximum yields of total reducing sugar, i.e. 85 mg/g
at 1.2% H2SO4 concentration (120  C and 60 min) using
bamboo. Ballesteros et al. (2006) studied steam pre-
treatment of wheat straw impregnated with H2SO4
at a higher concentration (0.9%) and obtained the
highest yield in enzymatic hydrolysis, i.e. 23 g/100 g
dry straw at a temperature of 180  C for 10 min.
Besides its positive effects on fermentation process,
the pretreatment of lignocellulosic substrate also
leads to the formation of inhibitory compounds
which may have negative effect on the growth of
the fermenting microbes and decrease ethanol yields
(Palmqvist and Hagerdal 2006). These inhibitors gen-
erate the phenolic compounds which negatively
affect the potential effects of pretreatments followed
by enzymatic hydrolysis (Ximenes et al. 2011). It was
found that among the various inhibitory compounds
monitored, 5-HMF and acetic acid were generated in
the highest concentration after sulphuric acid and
hydrogen peroxide pretreatment of apple pomace
respectively that could also inhibit the ethanol
production.
3.3 Biomass morphology study by scanning
electron microscope
Figure 5 represents the images of apple pomace
observed by scanning electron micrograph (SEM) for
verification of the structural changes caused by micro-
wave pretreatment and enzymatic hydrolysis.
Structural changes in the biomass during pretreatment
were analyzed using scanning electron microscopy
(SEM). A comparison of surface morphology of native
pretreated biomass indicated that microwave swelling
of the fibre and an increase in surface area the gap
between the fibres were increased on pretreatment
which possibly aid in better enzymatic binding and
hydrolysis. A comparison of surface morphology of
native pretreated and enzymatic hydrolysis biomass
indicated that the pretreatment resulted in a swelling
of the fibre and an increase in the surface area.
BIOCATALYSIS AND BIOTRANSFORMATION 455
The microwave treatment affects the crystalline struc-
ture of cellulose at least in several types and the
carbohydrate polymers are left undisturbed. The gap
between the fibres was increased further by enzymatic
hydrolysis, which aid in better release of sugars
required for fermentation. Previous studies have also
shown that the surface of rice straw treated with acid
assisted with steam became loose and pores were
formed (Gong et al. 2010). Sigler (2013) also proved
that acid pretreatment had improved the rice straw
digestibility by forming large pores.
3.4 Estimation of sugar by HPLC
HPLC analysis depicted that each of the chromato-
grams has a common peak with retention time of
around 6.37 with the wide peak area containing glu-
cose. The quantity of glucose present in the native
sample of apple pomace was observed to be of
6.76 mg/ml as estimated by the chromatogram of
HPLC which further increased to 16.60 mg/ml after the
microwave pretreatment damage to the crystalline
structure of apple pomace. The reducing sugar, i.e.
glucose released after the enzymatic hydrolysis with
multiple carbohydrase of the substrate resulted in
release of 35.93 mg/ml of glucose (Figure 6). The pres-
ence of other simple substance, including glucose and
small quantity of xylose was determined indicating
that during the microwave treatment the cellulose and
xylose fraction of the apple pomace was susceptible
to hydrolysis. The observed quantities of released glu-
cose indicated effective hydrolysis of the cellulose frac-
tion. Visser et al. (2015) analyzed the glucose obtained
after the enzymatic saccharification of sugarcane bag-
gase yielded 3.74 g glucose as estimated by HPLC.
Similarly glucose and xylose content in an enzymati-
cally hydrolyzed agricultural waste was estimated by
using HPLC (Boonmee 2012).
3.5 Ethanol production from apple pomace using
free and immobilized co-culture of yeast cell
Hydrolysate obtained from the enzymatic hydrolysis by
multiple carbohydrases form fungus under optimized
parameters was used as a fermentation medium for
ethanol production. The fermentation was carried out
using free and immobilized beads of S. cerevisiae and
S. stipitis at an inoculum size of 20% (10% of each
yeast cultures prepared separately in the flask) for
36 h. Since, the obtained sugary syrup contains both
glucose and xylose, fermentation was carried out using
co-culture of yeasts; S. cerevisiae (which can ferment
only glucose) and S. stipitis (capable of fermenting
456 S. PATHANIA ET AL.
Figure 5. SEM image showing structure modification resulting from microwave pretreatment of apple pomace (a) untreated apple
pomace showing intact structure, (b) microwave pretreated apple pomace showing increase in surface area, (c) enzymatic
pretreated apple pomace affecting the structure.
xylose) for obtaining maximum bioethanol. Figure 7
shows the comparison of ethanol yield using free and
immobilized yeast cells, the results clearly indicates
the enhanced ethanol yield of 44.56 g/l with fermenta-
tion efficiency of 58% using immobilized co-culture of
S. cerevisiae and S. stipitis over free co-culture of yeast
cells with 34.46 g/l of ethanol and 45% of fermentation
efficiency. The advantages claimed for the use of
immobilized cells for ethanol fermentation compared
to whole (free) cells fermentation process are higher
cell density per volume of reactor, easier separation
from the reaction medium, continuous operation with-
out cells being carried away downstream, reduction of
the adaptation phase (lag phase), a higher substrate
conversion, less inhibition by products, reduced reac-
tion time, and control of cell replication (biomass
growth) (Duarte et al. 2013). Saccharomyces cerevisiae
proved to be superior to S. stipitis in yielding ethanol
which could be attributed to the presence of glucose
in the medium as more of the cellulose was hydro-
lysed by the cellulase enzyme, as compared to S. stipi-
tis which yielded less ethanol, as it hydrolysate xylose
and its content was quite low, the enzyme acted was
not able to hydrolyse the xylose required for fermenta-
tion of S. stipitus. According to Jefferies et al. (2007),
maximum yield of ethanol was obtained when a mix-
ture of S. cerevisiae and S. stipitis were introduced into
medium containing both glucose and xylose.
Figure 8 shows the scanning electron microscope
(SEM) images to the sodium alginate beads containing
entrapped yeast cells. SEM images of the beads were
acquired before being used in fermentations to show
the adherence of yeast cells to the beds. The micro-
graphs of inner side of beads before and after use are
 BIOCATALYSIS AND BIOTRANSFORMATION 457

Figure 6. Ethanol production from apple pomace using free and immobilized yeast cell under Separate Hydrolysis and
Fermentation (SHF). (a) Sugars in native apple pomace; (b) sugars in microwave pretreated apple pomance; (c) sugars in pretreated
apple pomace hydrolyzed by bacterial enzyme; (d) sugars in pretreated apple hydrolyzed by fungal enzyme.

shown in the magnification of 300 and 2000. There
was no apparent leakage of cells from the beads into
the bulk of fluid and the active sites were potentially
available for ethanol production without diffusion
problems. The average sizes of beads were 4.5 mm as
was measured by the ocular microscope. The high
alginate beads (2%) were found to be very hard and
did not break after being pressed manually with
approximately 21.4  104 cfu/ml of S. cerevisiae cells
and 27.2  104 cfu/ml of S. stipitis cells viable cells
entrapped within the beads as measured using dilu-
tion method.
Chatanta et al. (2007) used the leftover apple pom-
ace, collected from HPMC, Parwanoo as a substrate
for bioethanol production. Enzymatic hydrolysis with
carbohydrases from Aspergillus foetidus MTCC 151
(pectinase) and Fusarium oxysporum MTCC 1755 (cellu-
lase) and its further fermentation with consortium of
yeast cultures, i.e. S. cerevisiae MTCC 173 for obtaining
8.44% (v/w) ethanol after 72 h of incubation at 30  C.
Similarly the saccharified biomass of household waste
was fermented by using cellulase and xylanase of ther-
mophillic fungus Myceliophthora thermophila with
ethanol production of 19.27 g/L along with the
458 S. PATHANIA ET AL.

Ethanol yield (g/l) Fermentaon efficiency (%) reducing sugars after enzymatic hydrolysis with overall
50 70
high ethanol yield of 15.6 g/l and fermentation effi-
45
60 ciency of 61.1% (Kaushal et al. 2016).

Fermentation efficiency (%)

40
35 50
Ethanol yield (g/l)

30 40
25 Conclusions
20 30

15 20 The potential use of apple pomace as a fuel source

10
10
for bioethanol production was investigated with
5
0 0
enzymatic hydrolysis and fermentation of fermentable
free immobilized sugars from pretreated apple pomace as main focus
*Ethanol (g/l)= ethanol (%)×absolute density of ethanol
of this study. The novel fungal strain R. delemar F2
was found to have potential of producing multiple
**Fermentation ethanol produced (g/g)
efficiency =
× 100 carbohydrase for effective degradation of apple pom-
theoretical yield of ethanol
ace for bioethanol production. Upon enzymatic sac-
Figure 7. Ethanol production from apple pomace using free
charification, it was found that the microwave
and immobilized co-culture of yeast cell.
pretreated substrate containing 4ml/g of enzymatic
dose, upon hydrolysis at 50  C for 48 h lead to a high
yield of reducing sugar. An enhanced ethanol yield
of 44.56 g/l with 58% of fermentation efficiency was
obtained using immobilized co-culture of yeast cells
when compared to free yeast cells under SHF, indi-
cating the importance of using immobilized yeast
cells in ethanol production. The present work on bio-
fuel production from apple pomace, can further be
scaled up in industries to make the technology were
eco-friendly and cheap.

Disclosure statement
There is no conflict of interest.

Funding
The present work was funded by the State Council for
Science, Technology and Environment, Shimla, Himachal
Pradesh, India.

ORCID
Shruti Pathania http://orcid.org/0000-0002-1455-611X

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