3.0 Material and Methods: 3.1 Collection of Raw Material

MATERIAL AND METHODS Studies on Production of Bioethanol from Cotton Stalk
3.0 MATERIAL AND METHODS
Experimental material used and methods adopted to conduct the laboratory studies
are presented in this chapter under appropriate heads.
3.1 Collection of raw material
The cotton stalk used in this research work was harvested material from the
farmer's field of Marathwada region. The obtained cotton stalks belongs to
Gossypium hirsutum NHH44, sown in kharif season during the month of June and
July, and harvesting was done from January to March of every year.
The stalks consist of leaves, cotton seed and unwanted residues, which were
delivered to Microbiology research laboratory, Government Institute of Science in
Aurangabad of Maharashtra state, for further processing.
3.2 Physical pretreatment
Physical pretreatment includes mechanical comminution which resulted in
significant changes in the physical characteristic of biomass, including smaller size as
well as lesser degree of both crystallinity and polymerization.
The cotton stalk which consist of different unwanted residues were removed
mechanically by shredding with knife, sundried, debarked, bailed and ground to 1mm
particle size with laboratory blender and stored in tightly sealed plastic bags at room
temperature for further studies.
3.3 Compositional analysis
The main characteristics of cotton stalk namely carbohydrates; lignin, moisture,
and ash were measured in triplicate.
Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 63

3.3.1 Moisture contents
Moisture content of sample was determined by Laboratory Analytical Procedure
number 001 (LAP # 001) from National Renewable Energy Laboratory (NREL)
Protocol (Ehrman, 1994).
It was measured by weighing 5g of sample placed in oven dried and pre-weighed
empty dish and was placed in oven until constant weight was achieved at 105oC and
reweight after cooling in desiccator.
The percentage of moisture was calculated by using following formulae:
W1-W2
% Moisture = x 100
W1-W
Where
W = weight of dish with lid
W1= weight of dish + sample before drying
W2= weight of dish + sample after drying.
3.3.2 Ash content
Ash content of sample was determined by Laboratory Analytical Procedure
number 005 (LAB # 005) from National Renewable Energy Laboratory (NREL)
protocol (Ehrman, 1994).
It was measured by weighing 5g of sample placed in oven dried and empty silica
dish (crucible). Dish was placed on burner under fume hood with slow increase in the
temperature until smoking ceased and sample becomes thoroughly charred. Then dish
was transferred to muffle furnace and ramping the temperature from ambient to 575oC
as per the protocol and hold it for 4 to 5 hours until white ash was obtained.
The furnace was allowed to cool to100oC and the sample was removed from
muffle furnace to desiccator till complete cooling and reweights it till constant weight
was obtained.

Ash percentage was calculated by using following formulae:
W2-W1
% Ash = x 100
W1-W
Where:
W = weight of empty silica dish with lid
W1 = weight of dish + initial sample weight
W2 = weight of dish + weight after ash formed
3.3.3 Holocellulose
Holocelluloses content was determined by method of Wise et al., (1946) in which
2.5g of sample was repeatedly (4-5 times) treated with 2g of NaClO2 and 0.5 mL
Glacial Acetic Acid (GAA) in boiling water bath till the reaction mixture was turned
white. The delignified product, holocelluloses was filtered, washed with distilled
water and dried in an oven till constant weight was achieved (TAPPI, 1992; Teramoto
et al., 2008).
3.3.4 Alpha Cellulose
Alpha Cellulose is part of holocelluloses which mainly comprises of glucose
subunits. It was determined by TAAPI Method-203 (1992), which is based on the
principle that, “α-cellulose contents was determined as amount of residue of
holocelluloses insoluble in 17.5% NaOH aqueous solution”. In this determination 25
mL of 17.5% NaOH aqueous solution was added to flask containing 1g of sample
which was obtained as holocelluloses as mention in previous procedure (3.3.3). The
mixture was stirred at 20oC for 40 minutes and 25 mL of 10% glacial acetic acid was
added to residue, and was filtered again and washed with distilled water till pH

became neutral. The final residue was then dried till constant weight and kept as α-
cellulose (Teramoto et al., 2008).
3.3.5 Hemicellulose
The hemicellulose content was determined by subtracting α-cellulose from
holocelluloses (Teramoto et al., 2008).
3.3.6 Carbohydrate analysis by High Performance Liquid Chromatography
(HPLC)
Holocelluloses are primarily composed of glucose, xylose and arabinose subunits.
These sub units were quantified by HPLC (Zodiac. Ltd), equipped with sugar
monosaccharide column with RI detector (25cm × 4.6mm) using degassed Milli Q
water as mobile phase in CFRD (Central Facilities for Research and Development)
Laboratory of Osmania University, Hyderabad, India.
3.3.6.1 Preparation of sample
Sample for HPLC analysis was prepared by using Laboratory Analytical
Proceure-002 (LAP # 002) of NREL (National Renewable Energy Laboratory)
protocol.
In this preparation, 0.3g of biomass was dissolved in 10 mL of 64% (v/v)
sulphuric acid aqueous solution in a stoppered conical flask and left to hydrolysis at
30oC in shaking water bath until complete dissolution had occurred. Distilled water
(90 mL) was added and kept at 121oC for one hour in laboratory autoclave and was
filtered through 0.2µ filters for HPLC analysis. A sugar verification standard for
glucose, xylose and arabinose were also analyzed by the same procedure to find out
the loss in sugar after the treatment (Ruiz and Ehrman, 1996).

3.3.7 Klason lignin
Klason (acid insoluble) lignin of biomass was determined from the method
adopted by Browning (1967), as the amount of residue insoluble in 72% sulphuric
acid aqueous solution. In this determination 1g of sample was mixed with 15 mL of
72% sulphuric acid solution. The mixture was stirred at 20oC for 4 hours.
Subsequently 560 mL distilled water was added to it and system was refluxed for 4
hours. Thereafter, the solution was filtered and residue was washed with boiling water
and then cold water followed by drying in an oven till constant weighed was achieved
(TAPPI Method-T222, 1992; Teramoto et al., 2008).
3.4 Acid hydrolysis
Acid hydrolysis of physically pre-treated cotton stalk was carried out in two stages
including concentrated acid decrystallization followed by dilute acid hydrolysis with
steam explosion and heat treatment (Liao et al., 2006).
In this procedure, sets of different concentration of sulphuric acid (65%, 70%,
75% and 80%) were prepared from concentrated sulphuric acid solution (Merk Sp. Gr
1.84). In order to obtain the optimal condition for decrystallization, biomass was
mixed with these four concentration of acid separately to obtain homogeneous paste at
fixed sample-acid ratio of 1:2 (by weight), till colour of the paste turned dark brown
without resulting into severe oxidation by acid (Iranmahboob et al., 2002). Distilled
water was added to the paste to obtain the different normality (1N, 2N, 3N) in
different tube in each set of experiment for hydrolysis. Sets were then treated with
high pressurize steam at 121oC for 30 minutes (Somkid and Wuttichai, 2009) and
finally this steam treated hydrolyzates were heated at 90oC for four hours in water
bath. During overall process aliquots were taken at regular interval of time for
analysis of total fermentable sugars and glucose content separately.

3.4.1 Sugars estimation by 3, 5, Di nitro salicylic acid (DNSA) method
The DNSA method has been used to quantify the amount of reducing sugars
present in the sample (Miller, 1959).
3.4.1.1 Principle
DNSA (3, 5, Di nitro salicylic acid) reagent is an yellow colour compound and
under hot alkaline condition gets reduce to 3-amino 5-nitro salicylic acid in presence
of reducing sugars such as glucose, xylose etc. which is deep orange coloured
complex and can be estimated spectrophotometrically at an extinction coefficient of
540 nm.
3.4.1.2 Preparation of reagent
The DNSA reagent was prepared by dissolving 10g of DNSA powder, 2g of
crystalline phenol and 0.5g of sodium sulphite in one litre of 1% NaOH solution by
stirring, 40% of Rochelle salt (sodium-potassium tartrate ) was added to it, and after
dissolution stored in amber or dark colour bottle (Thimmaiah, 2006).
3.4.1.3 Preparation of standard graph
Standard graph was prepared by using 1mL DNSA reagent in 1mL standard
sample of glucose, from stock solution (2 mg/mL). Standard samples were prepared
by taking increasing concentration of glucose from 0.2 mg/mL to 2.0 mg/mL from
stock solution. Blank was prepared using distilled water instead of sugar solution.
All the tubes were boiled for 5 minutes in vigorously boiling water bath and after
5 minutes; the tubes were cooled in cold water bath and made up the volume up to 10
mL by adding distilled water and finally intensity of deep orange colour was
estimated spectrophotometrically at 540 nm against blank.

3.4.1.4 Estimation of sugar concentration in unknown sample
During hydrolysis and fermentation, aliquots were drawn at regular interval of
time and proceed for sugar estimation by adding 1 mL DNSA reagent in 1 mL of
respective sample, followed by boiling and dilution. Absorbance was read at 540 nm
against blank and the concentration of sugar was estimated using standard graph.
3.4.2 Glucose estimation by GOD-POD method
Glucose oxidase method is an enzymatic assay for determination of glucose
present in solution. The analysis was performed by following guideline given by
Bergmeyer et al., (1974).
3.4.2.1 Principle
Glucose is determined after enzymatic oxidation in presence of glucose oxidase
(GOD) which results in the formation of hydrogen peroxide (H2O2) reacts under
catalysis of peroxidase (POD) with phenol and 4-amino antiypyrine to form red
quanoneimine complex. The intensity of the colour is proportional to the glucose
concentration present in sample.
3.4.2.2 Equation representation
GOD
Glucose + O2 + 2H2O Gluconate + 2H2O2
POD
2H2O2 + Phenol + 4-aminoartipyrine red quanoneimine complex + 4H2O
3.4.2.3 Enzymatic reagent
GOD-POD assay kit was purchase from AMBICA diagnostics Pvt. Ltd. This
contains enzyme reagent and calibrated standard (100 mg/dL). Analysis was
performed according to given instruction on the manual available with kit.

Experiment was design according to following protocol.
Blank Standard Test

Enzyme reagent 1.0 ml 1.0 ml 1.0 ml
Standard 0.01 ml
Test solution 0.01 ml
All tubes were mixed properly, incubated at 37oC for 10 minutes and absorbance
was read at 540 nm. Concentration of glucose was determined by using following
formulae.
Absorbance of test - Absorbance of blank

Glucose concentration = x 100
Absorbance of std. – Absorbance of blank
3.4.3 FTIR analysis
Fourier Transform Infrared (FT-IR) spectroscopic analysis of untreated biomass
and lignified residues (after acid hydrolysis) was carried out to detect changes in
functional group by Shimadzu spectrophotometer. Sample was prepared by mixing it
with KBr with ratio of 1:100 (sample : KBr) this mixture was turn to a disc form by
processing through agate mortar and finally absorbance was recorded between 4000
and 450 cm-1 resolution and 25 scan per sample (Derkacheva et al., 2008).
3.5 Neutralization and detoxification
The harsh condition of hydrolysis causes formation of byproduct which does not
only decrease the sugar yield; it also hampers the fermentation, since several
byproducts are inhibitory to fermenting organisms. These inhibitors (i.e. furans and
phenolics) were removed from hydrolyzate with neutralization and detoxification by
over liming and activated charcoal treatment respectively (Srilekha Yadav et al.,
2011).

3.5.1 Over liming
The optimization of over liming was carried out by increasing pH up to 12.0 using
dried lime (calcium oxide) in separate flask for each pH and keeping it with constant
stirring for an hour by stirrer. After over liming the hydrolyzate was filtrated and pH
of hydrolyzate was brought back to pH 6 using concentrated H2SO4 (Chandel et al.,
2007b).
3.5.2 Activated charcoal treatment
In order to obtain the optimization of charcoal treatment, increasing concentration
of activated charcoal was applied on over limed hydrolyzate from 1% to 5% (w/v)
separately along with stirring at room temperature for half an hour followed by
filtration through vacuum filter (Martinez et al., 2000).
3.5.3 Determination of phenolics compounds
Total content of phenolic compound in hydrolyzate was determined by Folin
Ciocalteus (FC) method (Singleton and Rossi, 1965).
In this determination each sample is neutralized and properly diluted with distilled
water. From diluted sample, 0.1 mL of sample was taken and mixed with 0.5 mL of
FC reagent in presence of 8.4 mL of distilled water and incubated for 5 minutes,
Followed by addition of 1mL of freshly prepared 20% Na2CO3 and incubated in dark
at room temperature for an hour and absorbance was read at 750 nm.
3.5.3.1 Standardization
Gallic acid was used as standard to produce calibration curve and concentration
used in stock solution was 200 µg/mL. From stock solution, different diluted sample
have been prepared by using increasing concentration from 20 µg/mL to 200 µg/mL.
Blank was prepared by mixing 0.5 mL of FC reagent with 1 mL of 20% Na2CO3
in presence of 8.5 mL of distilled water (without test sample).

3.5.4 Furans estimation
Furans (furfural and hydroxy methyl furfural) are the by-products of sugars
formed during hydrolysis and were estimated with spectrophotometric method
described by Martinez et al., (2000).
In this determination acid hydrolyzate were neutralized and properly diluted 20µL
of sample was mixed with 1980 µL distilled water so as to make up the volume to 2
mL and absorbance was read at 280 nm (UV range). Distilled water used as blank and
furfural was taken as standard to produce calibration curve with concentration of 10
mg/L as stock solution. After detoxification, neutralized hydrolyzate is ready to be
fermented.
3.6 Microorganism and their maintenance
Fermentation of detoxified hydrolyzate of cotton stalk was carried out by using
Saccharomyces cerevisiae MTCC 36 and Pachysolen tannophilus MTCC 1077
purchased from Microbial Type Culture Collection, IMTECH, Chandigarh, India.
Lyophilized culture of Saccharomyces cerevisiae and Pachysolen tannophilus
were activated separately on yeast and malt extract (YM) medium. The medium was
prepared by adding 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1%
glucose in distilled water. pH of the medium was adjusted to 6.5 (Chandel et al.,
2009a). The yeast cells were allowed to grow aerobically at 30oC on rotary shaker
incubator with 120 rpm for 48 hours or till the culture partially covered the bottom of
flask. Completely activated yeast cells were actively transferred by Nicrome wire loop
to YM agar plates which contain 0.3% yeast extract, 0.3% malt extrat,0.5% peptone,
1% glucose and 2.5% agar and pH of medium was adjusted to 6.5 and allowed to
grow at 30oC for 48 hours and purity was checked microscopically from isolated
colonies.

3.6.1 Microscopic examination and preservation of yeast cells
Microscopic examination is rapid screening method for detection of yeast and
bacteria. The yeast cells were checked for their purity by staining with methylene blue
solution (0.2 g dissolved in 100 mL distilled water and diluted 10 times with sterile
distilled water) under compound microscope.
Checked culture was aseptically transferred to freshly prepared YM agar slants
and incubated at 25oC for 48 hour and stored at 4oC for further studies. Stored slants
were regularly sub cultured on monthly basis.
3.7 Growth curve of test organism in cotton stalk hydrolyzate
The different phases of growth curve of Saccharomyces cerevisiae and
Pachysolen tannophilus have been studied to evaluate its potential for utilization of
cotton stalk hydrolyzate as growth medium and specific time period for inoculation of
inoculum. Experiment with both the organisms was carried out separately under
similar set of condition. Each medium has fix sugar concentration (11%) obtained
from cotton stalk hydrolyzate and were supplemented with 0.5% yeast extract, and
1% peptone with 5.5 pH sterilized at 110oC for 40 minutes and 5% inoculum of each
test organism was inoculated separately in each flasks. Blank was prepared by same
supplementations but without any organisms. Flasks were allowed to grow aerobically
at 30oC. At every 4 hours 1 mL of medium was pipette out under strict aseptic
condition from each flask and transferred to spectrophotometric glass cuvette and
absorbance was read at 600 nm. A graph was plotted to study the growth curves of
organism, exhibiting various phases of growth, with time (in hours) on X-axis and
optical density value proportionate to growth on Y-axis.

3.8 Determination of ethanol tolerance of test organisms
The ethanol stress is probably one of the most interesting conditions to be
analyzed due to high amount of this substance during alcohol fermentation. Maximum
ethanol concentration is the stage above which the yeast ceases to function. The test
were carried out by transferring colonies from YM agar slants to cotton stalk
hydrolyzate supplemented with 0.5% yeast extract and 1% peptone. Absolute alcohol
was added in increasing amount in each tube so that for each organism the only
variable was alcohol concentration. Control, without alcohol was included in each set.
Two sets were prepared including Saccharomyces cerevisiae in one set and
Pachysolen tannophilus in another set. Immediately after adding yeast, the initial
sugar content was determined and tubes were incubated at 30oC for 4 days (Gaber,
2010) following which all the samples were analyzed for residual sugars by DNSA
method.
3.9 Inoculum development
3.9.1 Growth of cell mass
Biomass require for batch fermentation was obtained by growth of yeast cell on
YM medium (refer section 3.6) in Erlenmeyer flask and was sterilized at 110oC for 40
minutes (above this temperature charring of sugar takes place). The flasks were
cooled and cells from slants were aseptically transferred into the flask with the help of
wire loop and were allowed to grow aerobically on rotary shaker incubator with 120
rpm at 30oC for 48 hours. After incubation, completely activated yeast cells were
harvested by centrifugation at 4000 rpm at 4oC for 10 minutes and repeatedly washed
with distilled water and used as cell mass for inoculum development.

3.9.2 Inoculum preparation
Inoculum was prepared in detoxified hydrolyzate solution of cotton stalk
supplemented with 0.5% yeast extract, 1% peptone and pH was 5.5%. The yeast cells
which were harvested by centrifugation were added in inoculum and incubated on
rotary shaker incubator with 150 rpm at 30oC for 24 hours (Srilekha Yadav et al.,
2011) and grown aerobically to promote healthy growth of yeast cells in hydrolyzate
and used as inoculum for fermentation. The volume of inoculum again set to 10% to
the total volume used for fermentation.
For quantifying the cell mass, One millilitre aliquot from each suspension was
taken to performed serial dilution up to 105 and 100 µL of diluted culture was spread-
plated on to YM agar plates by adding 0.3% yeast extract, 0.3% malt extract, 0.5%
peptone, 1% glucose and 2.5% agar and were incubated at 30oC for 48 hours and
yeast colonies were counted to ensure that each time the inoculation stayed at
approximately 6.0 × 107 cfu/mL corresponding to 10g dry weight/Litre.
3.10 Fermentation
The detoxified hydrolyzate was employed as sugar solution for fermentation by
transferring separately developed inoculum of Saccharomyces cerevisiae MTCC 36
and Pachysolen tannophilus MTCC 1077.
3.10.1 Fermentation medium
The sugar solution of cotton stalk was supplemented with 0.1% (w/v) yeast
extract, peptone, NH4Cl, KH2PO4 and 0.05% (w/v) of MgSO4.7H2O, MnSO4,
CaCl2.2H2O, FeCl3.2H2O and ZnSO4 in 250 mL Erlenmeyer flasks, adjusting the pH
5.5 and autoclaved at 110oC for 20 minutes (Pasha et al., 2007).

3.10.2 Inoculation of inoculum and processing for batch fermentation
Fermentation was initiated by adding inoculum to respective fermentation broth
having detoxified hydrolyzate of cotton stalk as sole carbon source. In case of mono
culture fermentation the volume of inoculum must be 10% of total volume which was
used for fermentation while in case of co-culture fermentation volume of inoculum
used for growth of Saccharomyces cerevisiae was 6% and for Pachysolen tannophilus
was 4% (Sharma et al., 2007). Flasks were sealed with aluminium file for
development of anaerobic condition required for ethanol production during
fermentation by yeast and incubated at 30oC on rotary shaker incubator with 120 rpm
for first 24 hours for utilization of oxygen in flask and then in static mode for 72
hours.(Srilekha Yadav et al., 2011). Parallel flasks were employed under similar set of
experimental methods to evaluate more than one parameter in same experiment.
3.10.3 Determination of ethanol, residual sugars and cell growth
Sample obtained during fermentation was transferred to pre weighted centrifuged
tube and were centrifuged at 10000 rpm for 10 minutes at 4oC. The supernatant was
collected and analyzed for concentration of ethanol and residual sugars in broth while
pellet was repeatedly washed with distilled water and dry in hot air oven at 60oC till
constant weight. The difference between initial and final weight was recorded as cell
biomass and express in g/L (Oberoi et al., 2010).
3.10.4 Ethanol estimation by potassium dichromate colorimetric method
Potassium dichromate spectrophotometric method is well known method of
ethanol estimation. This method is popular because of its convenience, easy
availability of reagent and solution is indefinitely stable in air.

3.10.4.1 Principle
Dichromate colorimetric method has been used to quantify the amount of ethanol
by oxidation reaction in presence of sulfuric acid with the formation of acetic acid.
Dichromate (Cr2O7--, Cr (IV)) is yellowish in color and reduced to chromic product
(Cr+++, Cr (III)) which is intensely green. This has absorption at 660 nm which can be
determined in colorimeter using blank.
3.10.4.2 Theoretical reaction
2Cr2O--7 + 3C2H5OH + 16H+ 4Cr+++ + 3CH3COOH + 11 H2O
3.10.4.3 Preparation of reagents
i. Ethanol standard (10 mg/mL): Prepared by dissolving absolute ethanol in
water to get 10 mg/mL concentration
ii. Potassium dichromate solution (10%): This was prepared by dissolving 10 g
of potassium dichromate (K2Cr2O7) in 100 mL distilled water.
iii. Concentrated sulphuric acid solution (36 N)
3.10.4.4 Preparation of standard graph
Standard graph was prepared by using standard ethanol solution aliquots in
separates tubes to get the concentration range of 1-10 mg per tube (0.1 -1.0 mL) from
stock solution (10 mg/mL) and make up the volume up to 5 mL in each tube with
distilled water. Blank was prepared by keeping a tube without ethanol but with water
and to each tube one mL of potassium dichromate reagent was added. All the tubes
were kept in ice bucket and after cooling, 4 mL of concentrated sulphuric acid was
added to each tube from the side wall of test tube to avoid spurting (bumping).
Finally, the intensity of green colour was read at 660 nm in spectrophotometer against
blank.

3.10.4.5 Concentration of ethanol in unknown sample (fermentation broth)
During experimental work, aliquots were taken from fermentation broth,
centrifuged it and absorbance was read at 660 nm by preparing sample as mentioned
above (section 3.7.7.4) and ethanol concentration of fermentation broth was
determined (Williams et al., 1950; Gopal Reddy et al., 2008).
3.10.5 Ethanol estimation by Gas Chromatography (GC)
After each experiment, part of supernatant was filtered by 0.22 µm cellulose
acetate filter and analyzed by Gas Chromatography (Shimadzu Japan). All analysis
was carried out according to NREL (National Renewable Energy Laboratory)
procedure LAP # 011 (David, 1994) using ZB-Wax column (30mm × 0.25mm) with
Flame Ionization Detector (FID). The temperature of column, injector and detector
was 150oC, 175oC and 250oC respectively. Program run and ethanol retention time
was about 5.5 and 2.3 minute respectively. Nitrogen (16 kPa), flow rate: 40 mL/min,
split ratio: 1/50, velocity of H2 flow: 60 mL/min and amount of sample used for
analysis was 10µL (Srilekha Yadave et al., 2011).
3.10.6 Fermentation efficiency
Fermentation efficiency was calculated as
Practical yield of ethanol

Fermentation efficiency = x 100
Theoretical yield of ethanol
Practical yield is the ethanol produced and theoretical yield is 0.511 per gram of
sugar consumed.
3.10.7 Cell density
Cell density was measured turbidometrically at 600 nm by using UV-VIS
spectrophotometer (Shrilekha Yadav et al., 2011).

3.10.8 Parametric optimization of ethanol production
The parametric optimization were carried out by varying pH, temperature,
agitation, aeration, inoculum concentration and incubation time to optimize the most
ideal condition for maximum ethanol production. Fermentations were performed in
250 mL of Erlenmeyer flask while the sugar concentration obtained from detoxified
hydrolyzate of cotton stalk becomes kept constant as 11g/L. All the experiment
related to fermentation parameters were conducted in triplicate.
3.10.8.1 Effect of pH and Temperature on ethanol production
Parametric optimization was started with optimizing temperature and pH
separately. Effect of temperature on ethanol fermentation by using co-culture of
Saccharomyces cerevisiae and Pachysolen tannophilus was carried out by varying the
temperature from 20oC to 40oC (20, 25, 30, 35 and 40oC). The temperature was
established only at the beginning of experiment and no change was made during the
process. Similarly pH of various ranges from 4.0 to 6.5 (increasing by 0.5 pH) was
also tested for optimization purpose. It was adjusted by adding drops acid (H2SO4) or
alkali (NaOH) at room temperature under agitation. The pH adjusted medium was
then fermented by co-culture of Saccharomyces cerevisiae and Pachysolen
tannophilus. pH adjustment was also made before the fermentation started and no
change was made in between. The flasks were sealed with aluminum foil and
incubated on rotary shaker incubator with 100 rpm for first 100 hours and then kept in
static mode for 72 hours. The samples were analyzed for sugar left and ethanol
production.
3.10.8.2 Effect of aeration on ethanol production
Aeration of fermentation broth was optimized by following the procedure given
by Chandel et al., (2009a). In this process 250 mL of Erlenmeyer flasks were used for

fermentation, and categorization was made as; Highly aerobic (50 mL of fermentation
medium in 250 mL of flask), Aerobic (100 mL of fermentation medium in 250 mL of
flask), Semi aerobic (150 mL of fermentation medium in 250 mL of flask), Semi
anaerobic (200 mL of fermentation medium in 250 mL of flask), Anaerobic (250 mL
of fermentation medium in 250 mL of flask). Flasks were sealed and no air was
supplied externally during process and was incubated with co culture for fermentation
process.
3.10.8.3 Effect of inoculum size on ethanol production
Inoculum concentration is highly influencing parameter on rate of ethanol
formation. Optimization of inoculum size on ethanol production by using co-culture
of Saccharomyces cerevisiae and Pachysolen tannophilus were carried out separately
by varying the concentrations of co-culture form 6% to 16% separately (6%, 8%,
10%, 12%, 14% and 16%). Proportion of Saccharomyces cerevisiae and Pachysolen
tannophilus in each inoculum was in the ratio of 60:40, as was optimized by Sharma
et al., (2007).
3.10.8.4 Effect of agitation on ethanol production
Agitation mainly affected the fermentation by forming a homogeneous system
and was carried out for complete utilization of oxygen which leads to produce
anaerobic condition and increases in activated cell mass. In order to prove the effect
of agitation on fermentation process, stirring process were applied in two ways;
including optimization of agitation rate and agitation time. The agitation rate was
optimized by stirring the flasks with various velocities including 60 rpm to 160 rpm
(60, 80, 100, 120, 140 and 160) on rotary shaker for 18 hours. Agitation time was also
optimized by stirring the flasks for various time period, increases from zero hour to 48
hours (0, 12, 24, 36 and 48 hours) with pre optimized agitation rate. During the

process all previously optimized parameters were kept constant and were fermented
with co culture for 72 hours.
3.10.8.5 Ethanol production through optimized parameters (Effect of
incubation time)
Resultant data obtained during parametric optimization of pH, temperature,
aeration, agitation, and inoculum concentration for fermentation was used to produce
ethanol from cotton stalk hydrolyzate using co-culture of Saccharomyces cerevisiae
and Pachysolen tannophilus, by varying incubation time period from 6 to 96 hours
with an interval of 12 hours, while all the other optimized parameters were maintain
constant. Separate flasks were used for each time interval which helps to maintain
ideal parametric condition during fermentation process and results were evaluated by
analysing three parameters simultaneously including ethanol production, sugar
utilization and cell mass concentration.
3.11 Alkaline pretreatment and enzyme hydrolysis of cotton stalk
The present process comprises of delignification by using alkaline pretreatment
followed by optimization of enzyme hydrolysis, which is as follows.
3.11.1 Alkaline pretreatment
Chemical pretreatment comprises mainly on delignification process which have
been carried out by using different concentration of NaOH solution treated with
biomass for different time periods.
In this regards, 0.5-2.0% (w/v) concentrations of alkaline solution has been
prepared from NaOH pellets (Qualigens. Ltd) in aqueous medium. 5 gram of
physically pre-treated cotton stalk powder at a substrate loading of 10% (w/v) was
subjected to each alkaline solution separately. The flasks were sterilized at 121oC for
30, 60 and 90 minutes to optimize time period for steam explosion. After

pretreatment, the biomass has been separated from lignified liquor by centrifugation at
10000 rpm for 10 minutes and supernatant (black liquor) was separately collected
form each sample for quantitative detection of lignin content. The delignified biomass
was repeatedly washed with distilled water till to become neutral pH and dried in hot
air oven at 60oC to constant weight and was stored at 4oC for further studies
(Silverstein et al., 2007).
3.11.2 Enzymatic hydrolysis of pre-treated biomass
Enzymatic hydrolysis of pretreated biomass was carried out using commercial
cellulases purchased from Sisco Research Laboratories Pvt. Ltd. Mumbai, India.
Initial optimization of pretreatment was performed by least amount of enzyme i.e. 40
CMC (carboxymethyl cellulose) unit of enzyme per gram and finally enzyme
concentration was also optimized by exposing different concentrations of enzymes per
gram of biomass.
Pre-treated cotton stalk was incubated with 5% solid loading in 50mM acetate
buffer (pH 4.8) with respective concentration of enzymes and was incubated at 50oC
and 150 rpm for 72 hours, as was performed by Silverstein et al., (2007). After
incubation, the sample was centrifuged in chilled condition at 5000 rpm for 10
minutes and supernatant was collected for detection of fermentable sugar.
3.11.3 Preparation of acetate buffer (pH 4.8)
Acetate buffer of pH 4.8 was prepared by mixing 410 mL of 0.2M acetic acid
(11.55 mL of acetic in 1000 mL distilled water) and 590 mL of 0.2M sodium acetate
(22.22g of sodium acetate in 1000 of distilled water) (Thimmaiah, 2006).
3.11.4 Optimization of enzyme concentration
After alkaline pretreatment of cotton stalk, optimization of enzyme concentration
was also performed using different concentration of enzyme, ranging from 20 CMC

units to 140 CMC units per gram of biomass for the purpose to evaluate the most
suitable concentration for maxim hydrolysis of pre-optimized biomass.
3.12 Ethanol productions from enzymatic hydrolysate of cotton stalk
The filtrated product obtained from enzymatic hydrolysis of cotton stalk was used
as sole carbon source for fermentation and was supplemented with 0.1% (w/v) yeast
extract, peptone, NH4Cl, KH2PO4 and 0.05% (w/v) of MgSO4.7H2O, MnSO4,
CaCl2.2H2O, FeCl3.2H2O and ZnSO4 in 250 mL Erlenmeyer flasks. Fermentations
were performed in semi aerobic mode (250 mL Erlenmeyer flask containing 150 mL
of fermentation medium) having pH 5.5 and sterilized at 110oC for 20 minutes as was
given in section 3.10.1. The flasks were inoculated with 10% co-culture of
Saccharomyces cerevisiae and Pachysolen tannophilus at concentration of 6% and
4% respectively as was describe in section 3.10.2. The flasks were sealed with
aluminium foil and incubated on rotary shaker with 120 rpm for first 24 hours and
then kept in static mode at 30oC for 96 hours. Sample was removed from each flask at
one time at the interval of 12 hours and analyzed for ethanol, residual sugar and cell
biomass concentration.
3.13 Immobilization studies
Immobilization of cells for fermentation has developed to eliminate inhibition
caused by high concentration of substrate and products also to enhance the
productivity and yield of ethanol production (Baptista et al., 2006). In this regards,
immobilization studies of Saccharomyces cerevisiae and Pachysolen tannophilus was
carried out to evaluate its potential for ethanol production from cotton stalk
hydrolyzate in batch reactor. In this study, immobilization has performed using
alginate particle as carrier and to develop comparative account with free cell
fermentation.

3.13.1 Inoculum development
Inoculum of yeast cell for immobilization was developed in two steps. In the first
step, the cell mass of Saccharomyces cerevisiae and Pachysolen tannophilus were
grown on Yeast and Malt (YM) medium; and during second step, inoculum was
prepared from activate yeast culture using cotton stalk hydrolyzate as sole carbon
source (as mention in section 3.9).
3.13.1.1 Growth of cell mass for inoculum development
Cell mass of Saccharomyces cerevisiae and Pachysolen tannophilus required for
the growth of inoculum was prepared by transferring it aseptically from slants to YM
medium (0.3% malt extract, 0.5% peptone and 1% glucose in distilled water). The
cells were allowed to grow aerobically in separate flask at 30oC on rotary shaker
incubator with 120 rpm for 48 hours. After incubation, completely activated yeast cell
were harvest by centrifugation with 4000 rpm at 4oC for 10 minutes, washed with
distilled water and used as cell mass for inoculum development.
3.13.1.2 Inoculum preparation
Activate cell mass of Saccharomyces cerevisiae and Pachysolen tannophilus
grown separately on YM medium was transferred to cotton stalk hydrolyzate
supplemented with 0.5% yeast extract, 1% peptone and pH was adjusted to 5.5. The
flasks were incubated on rotary shaker incubator with 120 rpm at 30oC for 24 hours
and grown aerobically to promote healthy growth of yeast cell in hydrolyzate. After
incubation the activated cells of Saccharomyces cerevisiae and Pachysolen
tannophilus were mixed in the proportion of 6% and 4% respectively so as the total
volume became 10% of total hydrolyzate used for fermentation. Cells were quantified
to ensure the initial inoculation stayed at approximately 6 × 107 cfu/mL
corresponding to 10 gram dry weight/liter. Total suspension of yeast cell was

centrifuged at 5000 rpm for 5 minute at 4oC and cell pellets were washed with
distilled water and stored at 4oC for beads preparation.
3.13.2 Preparation of beads from co culture inoculum of yeast cells
To carry out immobilization of co culture of Saccharomyces cerevisiae and
Pachysolen tannophilus, 2% CaCl2 solution was prepared in an aqueous medium and
kept at 4oC for chilling. Subsequently sodium alginate solution (2% w/v) was
prepared in hot water by stirring on magnetic stirrer. Dry pellets (inoculum) of yeast
cell which was stored for bead preparation, mixed with sodium alginate solution
thoroughly to form homogenous mixture. This homogeneous mixture was extruded
slowly drop by drop through stainless steel needle using syringe pump into 2% chilled
CaCl2 solution which form the calcium alginate beads of yeast cell containing co-
culture of Saccharomyces cerevisiae and Pachysolen tannophilus. These beads were
aseptically stored at 4oC for further studies.
3.13.3 Ethanol production using immobilized yeast cells
Ethanol production from immobilized co-culture of yeast cell was performed in
250 ml of Erlenmeyer flask containing 150 ml of cotton stalk hydrolyzate to fulfill the
requirement of semi aerobic mode of aeration. All the fermentations were operated in
batch manner with a fixed parameter (pH 5.5 and temperature of 30oC). The
fermentation was initiated with inoculation of calcium alginate beads of
Saccharomyces cerevisiae and Pachysolen tannophilus into flasks and sealed with
aluminium foil and allowed to ferment on rotary shaker incubator with an agitation
rate of 120 rpm. Samples were collected at 12 hours interval throughout the
fermentation from individual flask at one time so as to maintain the ideal
environmental condition during fermentation (72 hours), and were subjected to

estimation of ethanol and residual sugar by potassium dichromate and DNSA method
respectively.
3.13.4 Recycling of immobilized yeast cells
In order to examine the recycling potential of immobilized cells, subsequently
repeated batch fermentation was carried out for ethanol production. At the end of each
fermentation cycle, the immobilized Saccharomyces cerevisiae and Pachysolen
tannophilus were retained, washed with sterile saline and transferred to the similar
volume of fresh medium for the next cycle of cultivation. The cycles were repeated
again and again till change in productivity was observed. After each cycle of
fermentation, the fermented liquor was analyzed for ethanol production and residual
sugar.
3.14 Statistical analysis
All the experiments were conducted in Completely Randomized Design (CRD) in
triplicate, whenever required appropriate standard error (SE), critical differences (CD)
and standard deviation (SD) were worked out to compare the results. Standard
deviations were presented on figures in the form of error bar. All these statistical
analysis was carried out by software MAUSTATE developed by Department of
Statistics, Vasantrao Naik Marathwada Agricultural University Parbhani,
Maharashtra, India.

3.0 Material and Methods: 3.1 Collection of Raw Material

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3.0 Material and Methods: 3.1 Collection of Raw Material

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MATERIAL AND METHODS Studies on Production of Bioethanol from Cotton Stalk

3.0 MATERIAL AND METHODS

are presented in this chapter under appropriate heads.

3.1 Collection of raw material

farmer's field of Marathwada region. The obtained cotton stalks belongs to

delivered to Microbiology research laboratory, Government Institute of Science in

Aurangabad of Maharashtra state, for further processing.

3.2 Physical pretreatment

Physical pretreatment includes mechanical comminution which resulted in

significant changes in the physical characteristic of biomass, including smaller size as

well as lesser degree of both crystallinity and polymerization.

temperature for further studies.

3.3 Compositional analysis

The main characteristics of cotton stalk namely carbohydrates; lignin, moisture,

and ash were measured in triplicate.

Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 63

3.3.1 Moisture contents

Moisture content of sample was determined by Laboratory Analytical Procedure

Protocol (Ehrman, 1994).

It was measured by weighing 5g of sample placed in oven dried and pre-weighed

reweight after cooling in desiccator.

The percentage of moisture was calculated by using following formulae:

W = weight of dish with lid

W1= weight of dish + sample before drying

W2= weight of dish + sample after drying.

3.3.2 Ash content

Ash content of sample was determined by Laboratory Analytical Procedure

protocol (Ehrman, 1994).

Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 64

Ash percentage was calculated by using following formulae:

W = weight of empty silica dish with lid

W1 = weight of dish + initial sample weight

W2 = weight of dish + weight after ash formed

Holocelluloses content was determined by method of Wise et al., (1946) in which

3.3.4 Alpha Cellulose

Alpha Cellulose is part of holocelluloses which mainly comprises of glucose

subunits. It was determined by TAAPI Method-203 (1992), which is based on the

principle that, “α-cellulose contents was determined as amount of residue of

holocelluloses insoluble in 17.5% NaOH aqueous solution”. In this determination 25

mL of 17.5% NaOH aqueous solution was added to flask containing 1g of sample

which was obtained as holocelluloses as mention in previous procedure (3.3.3). The

Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 65

cellulose (Teramoto et al., 2008).

The hemicellulose content was determined by subtracting α-cellulose from

holocelluloses (Teramoto et al., 2008).

3.3.6 Carbohydrate analysis by High Performance Liquid Chromatography

Holocelluloses are primarily composed of glucose, xylose and arabinose subunits.

monosaccharide column with RI detector (25cm × 4.6mm) using degassed Milli Q

Laboratory of Osmania University, Hyderabad, India.

3.3.6.1 Preparation of sample

Sample for HPLC analysis was prepared by using Laboratory Analytical

Proceure-002 (LAP # 002) of NREL (National Renewable Energy Laboratory)

In this preparation, 0.3g of biomass was dissolved in 10 mL of 64% (v/v)

Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 66

3.3.7 Klason lignin

adopted by Browning (1967), as the amount of residue insoluble in 72% sulphuric

acid aqueous solution. In this determination 1g of sample was mixed with 15 mL of

(TAPPI Method-T222, 1992; Teramoto et al., 2008).

3.4 Acid hydrolysis

including concentrated acid decrystallization followed by dilute acid hydrolysis with

steam explosion and heat treatment (Liao et al., 2006).

In this procedure, sets of different concentration of sulphuric acid (65%, 70%,