Chapter 3

METHODOLOGY
3.1

Isolation, screening, selection and identification of ethanol producing yeast

3.1.1

Sample Collection

The biomass such as orange fruit waste from juice shops, flower leaf, sugarcane straw, fruit
waste were collected from nearby area, Delhi University South campus, New Delhi. Samples
were kept in zip tight polyethylene bags and were stored at 4C until used further.
3.1.2

Sample Preparation

About 1g of sample was taken and was serially diluted using serial dilution method.
Thereafter 100l of inoculum from each dilution were spread onto the Sabouraud medium
plate containing antibiotic (ciprofloxin) (pH-60.5) and were incubated at 30C for 48 h.
3.2.1.2 Purification of cultures
The isolated colonies appeared on the incubated plates, were then picked up and maintained
on Sabouraud Agar medium containing antibiotic (ciprofloxin) by Streaking on different
plates. Plates of yeast(SA) were incubated at 30C for 48 h. The isolates thus developed were
then screened for their alcohol fermentation ability.
The composition of medium used for the isolation are shown in Table 3.1.
Table 3.1: Composition of Sabouraud agar (SA)
Medium component

Quantity(g/l)

Xylose

40.00

Casein enzyme

10.00

hydrolysate
Agar

15.00

pH

60.5

Antibiotic (Ciprofloxin)

3.3 Identification of yeast isolate:

The identification of selected alcohol producing isolate was carried out on the basis of their
cultural, morphological, metabolic characters.
3.3.1

Microscopic identification

For the identification of culture microscopically, 20l of culture grown in MXYP medium
(grown for 12 h) was mixed with equal amount of normal saline on the clean glass slide. A
drop of methylene blue stain was mixed to the sample and after 1 min of staining, a clean
cover slip was placed on the drop. The slide was observed under light microscope in oil
immersion at 1000X and the morphological features such as cell shape, size and arrangement
were observed.
3.3.2

Maintenance of Isolated Yeast Cultures

Four Yeast cultures were procured from the culture collection. The Yeast cultures were grown
in the 25 ml sterilised MXYP Broth ( pH-60.5) in 250 ml Erlenmeyer flasks at 30C and 150
rpm in an incubator shaker. 1ml culture broth of each yeast culture was mixed separately with
1.0 ml of 60% sterile glycerol and stored at -80C.
3.2

Selection of alcohol producing yeast

For the screening of alcohol producing yeast, all the isolates were grown in 50ml culture
tubes, each containing 10ml MXYP (Table 3.2) at 30C and 150 rpm for 5-6 days. Samples
were taken out at regular intervals of 24 h and analysed for residual sugars, ethanol content
and biomass produced.
Table 3.2: Composition of MXYP
Medium component

Quantity (g/l)

Xylose

20.00

Malt extract

3.00

Yeast extract

3.00

Peptone

5.00

pH

6.0

3.3

Pretreatment of lignocellulosic biomass (Prosopis Julifera)

3.3.1

Preparation of acid prehydrolysate (Acid hydrolysis)

Prosopis Julifera was size reduced, sieved (1-2 mm, ASTM standard sieves), washed and
dried overnight to remove moisture. About 200g of biomass was taken in 2 L Erlenmeyer
flask and then 3% H2SO4 was added. The samples were treated in an autoclave at 121C and
15psi for 30 min. After the treatment, the liquid and solid content was separated using
whatman No.1 filter paper and then kept at 4C until use. The liquid content was analyzed for
reducing sugar, phenolics and furan concentration.
3.3.2

To quantify sugar, phenolics and furfural assays were performed as follows:

3.3.2.1 Sugar Analysis:

Reducing sugar was measured using DNS method. About 20l of sample (Prosopis filterate)
was taken in assay tube with 980l of distilled water. Then 1ml of DNS is added to it.
Incubated at 100C for 10 min in waterbath. Absorbance was measured at 540nm.
3.3.2.2 Phenolics Analysis:
About 50l of sample (Prosopis filterate) was taken in assay tube with 3ml of distilled water.
250l of Folin Ciacalteu reagent(1N) was added to it. Incubated for 1 min. Then 750l of
10% prepared Na2CO3 (Sodium carbonate) is added to the assay tube. Incubated the assay
tubes in dark for 1 h. Absorbance was measured at 760nm.
3.3.3

Pre-treatment of acid-prehydrolysate (Detoxification)

The acid prehydrolysate (filterate) was then subjected to detoxification using 2% charcoal.
The filterate was kept on stirrer for about 30 min. After the treatment, the charcoal was
separated using whatman filter paper No.1 and then it was subjected to fermentation. The
filtered content was again analyzed for reducing sugar, phenolics and furan concentration.
The prehydrolysate was neutralized to pH 6 by adding sodium hydroxide (NaOH). The
resulting precipitate and insoluble residues were removed by centrifugation and the clear
supernatant fluid was stored at 4C until use.

3.3.4

Acid prehydrolysate medium

Pre-treated acid prehydrolysate was supplemented with (g/l): xylose, 10; malt extract, 3; yeast
extract, 0.3; peptone, 0.5; the pH adjusted to 60.5 and then it was subjected to fermentation.

3.4

Ethanol fermentation

3.4.1

Inoculum production

The primary inoculum was prepared by inoculating a loop full of yeast culture from a 24 h
old MXYP agar plates into the inoculation medium containing MXYP. While the secondary
inoculum was developed by inoculating 4% (v/v) of primary inoculum in the inoculation
medium.
3.4.2

Time course of ethanol fermentation using Pichia Stipitis strain

Time course of ethanol production from Pichia Stipitis was studied by growing in 50ml acid
prehydrolysate medium, supplemented with (g/l): xylose, 10.0; Malt extract, 3.0; yeast
extract, 0.3; Peptone, 0.5. For this, separate 250ml Erlenmeyer flask containing 50.0 ml acid
prehydrolysate medium (pH 6.0) were inoculated with 4.0% (v/v) of 12 h old inoculum and
incubated at 30C under shaking (150 rpm) conditions. The samples were withdrawn at
regular intervals of 4 h and centrifuged at 10,000 rpm for 5 min and assayed for the alcohol,
residual sugar and biomass.
3.4.3

Optimization of physical parameters for Ethanol production

Effects of different physical parameters were evaluated in order to maximize ethanol

production from Pichia Stipitis. Each factor examined for optimization was incorporated
further in subsequent experiments. All other experimental conditions were kept constant
unless otherwise stated.
3.4.3.1 Effect of initial pH of the medium on alcohol production
The effect of initial pH of acid prehydrolysate, supplemented with MXYP broth (3.0-7.0) on
fermentation to ethanol by Pichia Stipitis was studied. Each 250ml Erlenmeyer flask
containing 50ml of acid prehydrolysate broth (supplemented with MXYP) was inoculated
with 4% (v/v) of 12 h old inoculum and incubated at 30C and 150 rpm. The samples were
withdrawn at regular interval of 4h and centrifuged at 10,000 rpm for 5 min and assayed for
the alcohol, residual sugar and biomass.

3.4.3.2 Effect of incubation temperature on alcohol production

The effect of different incubation temperatures (20-40C) on alcohol production by Pichia
Stipitis was studied. Each 250ml Erlenmeyer flask containing 50ml of acid prehydrolysate
broth (supplemented with MXYP) was inoculated with 4% (v/v) of 12 h old inoculum and
incubated at different temperatures under shaking (150 rpm) conditions. The samples were
withdrawn at regular interval of 4h and centrifuged at 10,000 rpm for 5 min and assayed for
the alcohol, residual sugar and biomass.
3.4.3.3 Effect of agitation on alcohol production
The effect of varying levels of agitation, ranging from 0-200 rpm on alcohol production by
Pichia Stipitis was studied. Each 250ml Erlenmeyer flask containing 50ml of acid
prehydrolysate broth (supplemented with MXYP) was inoculated with 4% (v/v) of 12 h old
inoculum and incubated at 30C under shaking at different agitation rates. The samples were
withdrawn at regular interval of 4h and centrifuged at 10,000 rpm for 5 min and assayed for
the alcohol, residual sugar and biomass.
3.4.5

Optimization of nutritional parameters for ethanol production

Effect of inoculums size was studied in order to maximize the ethanol yield from Pichia
Stipitis during fermentation of acid prehydrolysate. Factor examined for optimization was
incorporated further in subsequent experiments.
3.4.5.1 Effect of inoculums size on fermentation
The effect of varying levels of inoculums sizes, ranging from 2-10% (v/v) of 12h old culture
on fermentation of acid prehydrolysate to ethanol by Pichia Stipitis was studied. Each 250ml
Erlenmeyer flask contained 50ml of acid prehydrolysate (pH 6.0) supplemented with nutrient
components ((g/l) malt extract, 3; yeast extract, 3; peptone, 5). The flasks were inoculated
with different sizes of 12h old inoculums and incubated at 30C and 150 rpm. The samples
were withdrawn at regular intervals and centrifuged at 10,000 rpm for 5 min and assayed for
the alcohol, residual sugar and biomass.
3.5

Analytical methods

Cell growth was measured turbidometrically at 600nm after the samples were diluted 1:10 in
distilled water. Total reducing sugars were estimated by the DNS method (Miller 1959). The
saccharification yield was calculated as follows:
Hydrolysis (%) = (Amount of reducing sugars released x 100)
s

Holocellulose content of pretreated substrate

Ethanol was estimated by Gas chromatography (GC) (Perkin Elmer, Clarus 500) with an
elite-wax (cross bond-polyethylene glycol) column (30.0 m x 0.25 mm) at oven temperature
of 90C, injector temperature 150C and flame ionization detector (FID) at 200C. The
ethanol standards were prepared using commercial grade ethanol (Merck, Darmstadt,
Germany). Nitrogen with a flow rate of 0.5 ml/min was used as carrier gas. While the ethanol
yield was calculated as follows:
Ethanol yield (%) =

Ethanol concentration

Holocellulose content