27

3 Materials and Methods

Chemicals and reagents

General chemicals like potassium dihydrogen phosphate, ammonium sulfate,
magnesium chloride, calcium chloride, sodium hydroxide, calcium carbonate,
galacturonic acid, sugars like glucose, fructose, xylose, lactose and sucrose were
procured either from Merck (India) or Qualigens (India). Acids like hydrochloric
acid and sulphuric acid were purchased from Qualigens (India). Ethanol (99%)
was procured from Changshu Yangyuan Chemicals, China. Sugar standards were
procured from Sigma chemical company (St. Louis, USA). Enzymes like
amylase, pectinase, cellulase and xylanase were purchased from Biocon Limited,
India. Calcium ethylene diamine tetra acetic acid (Ca EDTA) was used for HPLC
analysis. The commercial media components like agar powder, yeast extract
were obtained from HiMedia (India).

Microorganisms explored in the study

Fruits, such as apple, sapota, grapes etc. were collected from the fruit market
and were allowed to ripen for 8–10 days at 30 ºC and 37 ºC temperatures. After
the incubation period, 2.5 g of ripened fruits were added to 45 ml of M21
medium prepared in 100 ml serum bottles sealed with rubber septum. The
composition of the M21 medium is described in the Appendix.
In addition to ripened fruits ethanol producing microbial strains were also
enriched and isolated from raw honey, molasses, fermenting sugarcane juice.
The inoculated bottles were incubated on a rotary shaker (150 rpm) at three
different temperatures 23 °C, 30 °C and 37 °C temperatures for 5 days. After
three cycles of such enrichment, 1 ml of aliquot was diluted up to 108 fold, and
100 μl of all the dilutions were plated on M21 agar plates. The bacterial colonies
obtained were further purified on M21. Six different strains were enriched and
isolated from above mentioned substrates. They were compared on the basis of
their ethanol production ability.
A known ethanol producing bacterial strain Zymomonas mobilis MTCC 92 was
procured from Microbial Type Culture Collection, Institute of Microbial
 Materials and Methods 28

Technology (IMT), Chandigarh, India. The other bacterial strain Z. mobilis DSM
473 was procured from German Microbial Culture Collection centre.

Preservation and maintenance of the strains

Each of the purified bacterial isolates was given a code for cataloguing purpose.
Frozen stock cultures of the bacterial strains were stored in 25 % glycerol at -70
°C. Cultures were sub-cultured once, every 15 days and the purity of the bacterial
strains was checked periodically. The glycerol stocks of all the bacterial strains
were thawed and streaked over M21 medium. The plates were incubated at
respective temperatures (30 °C and 37 °C) used for isolation and enrichment of
bacterial strains in earlier experiments.
Liquid cultures of bacterial strains required for ethanol production studies were
also grown at the respective temperatures (30 °C and 37 °C), maintained during
the isolation and enrichment procedures. For biochemical characterization and
colony morphology studies, the bacterial strains were grown on M21 agar plates.

Collection of Agro-industrial waste material

Fruit and vegetable waste

In the present study, thirteen different fruits and vegetables that are found
almost through out the year were tested for initiating the ethanol fermentation.
Before the fermentation experiments, characterization of all the thirteen fruit
and vegetable waste was carried out. The waste material was shredded and
smashed to extract juice, which was used as a substrate to study the ethanol
production ability of strains, Z. mobilis MTCC 92, DSM 473 and an indigenous
strain TERI SH 110. Generic process scheme for bioethanol production from
agricultural waste followed in this work can be explained as Figure 3.1.

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 Materials and Methods 29

Seasonal production pattern analysis

Collection of fruit and vegetable

Analysis and characterization of individual components and mixture of

fruit and vegetable residues

Extraction of liquid Solid waste

Estimation of reducing sugars in Dilute acid hydrolysis

liquid extract (2 step hydrolysis)

Alkali hydrolysis

Fermentation or storage of liquid Enzymatic hydrolysis

extract at 4°C

Neutralization of the hydrolysate

Estimation of reducing sugars in

hydrolysate

Fermentation or storage of

hydrolysate at 4°C

Figure 3.1 Flow Chart of general strategy of fruit and

vegetable residues processing

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 Materials and Methods 30

Thippi
Starchy waste material, thippi is a waste by-product of the tapioca (Manihot
esculenta) processing industry. It was obtained from Sri Laxmi industries,
Tamilnadu, Southern India. Thippi was in dry pellet form and stored at room
temperature. It was first ground to powder form before further processing and
then was characterized. Before starting the experiment thippi was subjected to
heat treatment at different temperatures. Initially, 10 g of thippi powder was
taken in each of the flasks and incubated at three different temperatures of 30
°C, 55 °C and 121 °C. The heat treatment at 121 °C was given for 15 min and 30
min only while the incubation of flasks at 30 °C and 55 °C was extended up to 1
h, 3 h, and 6 h.

General scheme of process for thippi is presented in Figures 3.2 and 3.3.

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 Materials and Methods 31

Collection of thippi

Characterization of thippi

Pretreatment (steam 121 ºC, 20 minutes)

Acid hydrolysis Alkaline hydrolysis

(0.75% H2SO4, 30 °C/ 55 °C/ (0.75% H2SO4, 30 °C/ 55 °C/

121 °C, 3 h) 121 °C, 3 h)

Solid liquid separation

Reducing sugar analysis in

Storage of hydrolysate at 4 ºC or fermentation

Figure 3.2 Experimental layout of acid and alkaline

hydrolysis and fermentation of thippi.

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 Materials and Methods 32

Collection of thippi

Characterization of

thippi

Pretreatment (steam 121 ºC, 20 minutes)

Amylase enzyme Pectinase enzyme Cellulase enzyme

(5 U g-1 substrate, (5 U g-1 substrate, (5 U g-1 substrate,
55 ºC, 3 h, pH3 55 ºC, 3 h, pH3 55 ºC, 3 h, pH3

Solid liquid separation

Reducing sugar analysis in

hydrolysate

Storage of hydrolysate at 4 °C or
fermentation

Figure 3.3 Experimental layout of enzymatic hydrolysis

and fermentation of thippi.

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 Materials and Methods 33

Analysis of constituents of individual fruit and vegetable waste

Fruit and vegetable waste material was collected from local markets. A list of
fruits and vegetables is presented in Table 3.1. Market survey on availability
(whole year) of fruit and vegetable waste was done.

Table 3.1 List of fruits and vegetables selected for the

study
Fruits Vegetables
Apple Carrot
Mango Tomato
Pineapple Potato
Banana Other seasonal
vegetable
Orange
Grapes

Characterization of waste material

The selected substrates were characterized for total solids, moisture content,
total sugar, total reducing sugar and pH. Total sugars were estimated using the
Anthrone method (Dreywood, 1946). The total reducing sugars were estimated
with the help of Dinitrosalicylic acid method (DNS Method, Miller, 1959). The
amount of total reducing sugars was the key aspect for the selection of the
substrates. Total solids, volatile solid, ash content and percentage moisture were
estimated by the method described in Microbiological Aspects of Anaerobic
Digestion, as given below,

Total solids and Moisture

A clean and empty silica crucible was heated at 103 °C to 105 °C for an hour in a
hot air oven and allowed to cool down to room temperature in desiccators and
weighed. Substrate (10 g) was weighed accurately in the silica crucible and dried
at 103 °C to 105 °C to a constant weight. Moisture content and total solids were
calculated. The dried samples were retained for further analysis. The dried
samples then were incinerated at 600 ºC for 6 h and the ash content was
determined.

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Materials and Methods 34

Total sugars
Numerous methods have been developed to quantitatively determine the
carbohydrate content of various foods, feeds and other biomass related samples.
In the present study total sugars were estimated using the Anthrone method as
given below.
Anthrone solution was prepared by dissolving 0.1 g of Anthrone (9, 10 – dihydro
– 9-Ozoanthracene) in 76% sulphuric acid to a volume of 100 ml. Glucose stock
standard solution was prepared in double distilled water by dissolving 10 mg of
glucose in 1 ml and making a working stock of 1 mg ml-1. The standard stock
solution was taken in aliquots of 0.5, 1.0, 1.5, 2, 2.5, corresponding to 50, 100,
150, 200 and 250 µg of glucose respectively in a set of five large test tubes. To
these 4.5, 4, 3.5, 3, and 2.5 ml of distilled water was added to make up the
volume to 5 ml. A reagent blank was also run along with the standards. All the
tubes were transferred to an ice bath and 10 ml ice-cold anthrone reagent was
added to each of the tubes gradually to avoid spurting. Afterwards the tubes
were kept in boiling water bath for 16 min. During the heating, test tube mouths
were closed either by keeping the test tubes caps or large marbles. The tubes
were then transferred to an ice bath and allowed to stand for 10 minutes at room
temperature. The blue green colour developed was read for absorbance at 620
nm of wavelength using a Spectrophotometer (U-2000, Hitachi, Japan). The
samples were also treated in the same way and the total sugars were estimated
using the K factor derived out of the standard curve.

pH
The pH of waste extract was checked using Orion pH meter after calibrating the
instrument with standard pH buffers.

Starch content (Thippi)

Starch content of thippi was determined by acid hydrolysis method described
elsewhere (Microbiological Aspects of Anaerobic Fermentation, 1986). Slurry of
thippi was made in flasks by dissolving grinded thippi into 10% HCl solution.
The solution was incubated at 100 °C for 3 h with shaking. After incubation,
solid liquid separation was done by filtration and total reducing sugars were
estimated in the hydrolysate.

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 Materials and Methods 35

Characterization of potential strain

The strains were studied for morphological characteristics by staining. Different
morphological changes were studied in case of the selected indigenous strain.
The culture was grown in different physical conditions like pH, temperature etc.
Then the cultures were grown on M21 agar plates using thippi hydrolysate to
observe their colony characteristics. Glucose of the medium was replaced by the
thippi hydrolysate and salt components were added at the time of pouring the
plates. After streaking plates were incubated at different temperatures and
observed for colony morphology. Plates were prepared with different pH
medium. For biochemical characterization, cultures were grown on different
sugar media, glucose, xylose, fructose and sucrose. Sugar concentration and
incubation time was optimized using these sugars.

Growth curve
The M21 medium was prepared with thippi hydrolysate medium. Medium was
inoculated with the selected strain and samples were withdrawn at 4 h interval.
Growth of the culture was monitored by measuring OD at 600 nm and protein
concentration was estimated by Biuret method (Layne, 1957). Morphological
changes were also monitored in every 4 H sample.

Strain Selection- Ethanol production

potential of ethanologenic strains
The bacterial strains of Zymomonas mobilis were procured from German
Microbial Culture Collection Center (DSM) and Microbial Technology Culture
Collection (MTCC), Chandigarh. The two procured strains and isolated (in
laboratory) strains were compared on the basis of their ethanol production
potential. In M21 medium, the glucose was replaced by autoclaved and pH
adjusted fruit waste extract/vegetable waste extract/thippi slurry. The medium
was prepared in anaerobic bottles sealed with rubber septum. The concentration
of reducing sugars in the media was adjusted to come to a final concentration of
50 g l-1 using appropriate amount of fruit/vegetable waste extract or thippi
slurry. The other components of the media i.e., yeast extract, glucose,
magnesium chloride, ammonium sulphate and potassium dihydrogen phosphate
were autoclaved separately and added at the time of inoculation. Anaerobic
bottles containing media were inoculated with above mentioned strains and

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 Materials and Methods 36

incubated for a period of 96 h. The samples were withdrawn at every 24 h

interval and analysed for reducing sugars and ethanol production.

Optimization of hydrolysis process

Acid hydrolysis of the substrate

Fruit and vegetable waste

Fruit and vegetable were shredded and were soaked in to the different
concentrations of sulphuric acid (0.25%, 0.5%, 0.75%, 1.0%, 2.0%, 3.0% and
4.0% v/v) solution. The mixtures were then incubated at 37 °C and 55 °C for 3 h
with shaking. Release of reducing sugars in the hydrolysate was estimated.

For further optimization, National Renewable Energy Laboratory (NREL)

protocol was followed for standardizing acid hydrolysis of fruit and vegetable
waste to maximize reducing sugar content. Standardization was carried out
using a two-step hydrolysis process.

1st step hydrolysis

Fruit and vegetable waste mixture was soaked into 0.75% sulphuric acid
(optimized concentration) and kept at 55 °C for 3 h. Afterwards it was heated at
150 °C for 10 min to hydrolyse the hemicellulose. This slurry was pressed to
obtain liquid portion and neutralization was done by adding lime, which forms
gypsum precipitate. Gypsum was then removed by solid-liquid separation.

2nd step hydrolysis

Remaining solid was impregnated into 0.75% H2SO4. This acid soaked portion
was kept at 55 °C for 3 h again, then heated at 180 °C for 5-7 min, neutralized by
adding lime and stream was subjected to fermentation process.

Thippi
Thippi pellets were grinded into small granules before processing. Standardized
quantity of water was then added to the granules and the mixture was treated
with steam at 121 ºC and 15 lb pressure for 20 min before hydrolysis process.

Thippi pellets were ground and treated with steam before hydrolysis process.
Steam pre-treated thippi were subjected to dilute acid treatment with different

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 Materials and Methods 37

strength (0.25%, 0.5%, 0.75%, 1.0%, 2.0%, 3.0% and 4.0% v/v) of sulphuric acid
in water. Different concentrations of H2SO4 solutions were added to respective
bottles of the substrate and incubated at 37 °C and 55 °C with shaking. The
release of reducing sugars was estimated. NREL protocol was followed for
further optimization of acid hydrolysis process. Two-step hydrolysis was done
with 0.75% (v/v) H2SO4. Waste material was soaked into 0.75% (v/v) H2SO4 and
incubated at 55 °C for 3 h, then, heated at 150 °C for 10 min. This slurry was
pressed to separate liquid and solid. Then remaining solid was again
impregnated into 0.75% (v/v) H2SO4 followed by incubation at 55 °C for 3 h and
heating at 180 °C for 5-7 min.. Lime was added to increase the pH of the liquid
to 6.0. There was gypsum formation at the bottom of the hydrolysate, which was
removed by solid liquid separation and liquid stream was subjected to
fermentation process after reducing sugar analysis.

Alkaline hydrolysis
Fruit and vegetable wastes were soaked into the different concentrations of
alkali solution as explained for acid hydrolysis procedure. Mixture was
incubated at 37 °C and 55 °C with shaking. The release of reducing sugars was
analysed.

Thippi pellets were steam pre-treated before hydrolysis process. Afterwards

thippi slurry was subjected to hydrolysis with different alkaline concentrations
(0 to 4% of NaOH) and release of reducing sugars was estimated.

Enzymatic hydrolysis

Fruit and vegetable waste

Fruit and vegetable waste were sorted and individual components of the waste
were subjected to enzymatic hydrolysis. The enzymatic hydrolysis was
standardised for enzyme concentration, pH, incubation temperature and time.
The hydrolysis of apple, carrot, mango, sapota and pineapple was done by
adding three different enzymes i.e., amylase, pectinase and xylanase.

Standardization Incubation time

A fifty gram pre-weighed substrate was incubated with different enzymes (1 ml
each of the three different enzymes in different flasks). The reactions were

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 Materials and Methods 38

sampled after specified intervals of time (0, 0.5, 1.0. 1.5, 2.0, 2.5 and 3.0 h). The
total reducing sugar released after a specific incubation time was estimated.

Enzymatic hydrolysis of the individual waste substrates using pectinase,

xylanase and amylase
After optimization of incubation temperature, 50 g of each substrate was taken
in 5 different flasks. Three such sets were made for the three enzymes. A positive
control was also kept for each enzyme. The experiments were conducted in the
similar manner for all the waste material: apple, pineapple, mango, carrot and
sapota. Enzyme concentration of 0, 5, 10, 15 and 20 U g-1 was added to individual
flask for each of the enzyme. The enzymes were added in citrate buffer. All the
sets were incubated at 55 °C for 3 h and the release of reducing sugars was
estimated.

Scale up of the enzymatic hydrolysis of the fruit and vegetable waste

A pre-weighed substrate of 500 g was taken in each of four different flasks and 5,
10, 15, 20 enzyme units of respective enzymes were added per gm of substrate to
all the flasks. Optimum pH was adjusted and maintained throughout the
process. A positive control (containing only substrate, without enzyme) was also
kept and all the flasks were incubated at 55 °C for 3 h on a shaker.

Thippi
The thippi was then enzymatically hydrolysed using different enzymes.
Optimization of enzyme concentration was done by using 5, 10, 15 and 20 U
enzyme g-1 substrate. Thippi was hydrolyzed by adding optimized concentrations
of different combinations of enzymes (5 U g-1 thippi). After optimization of
enzyme concentration, 50 g of each substrate was taken in 5 different flasks. Two
such sets were made for enzyme treatment and control. The flasks were
incubated at 30 °C, 37 °C 50 °C, 55 °C and 60 °C for 3 h and the release of
reducing sugars was estimated. Similar procedure was followed for all the
enzymes. Then, steam pre-treated thippi was mixed with enzymes (5 U g-1) at
pH 5, 5.5 and 6. Reaction as incubated for 3 h and a control was kept for every
pH.

After optimizing hydrolysis with individual enzymes, mixtures of enzymes were

employed to treat thippi to obtain maximum available reducing sugars. Steam

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 Materials and Methods 39

pretreted thippi was weighted in different flasks and combinations of enzymes

were added to those flasks. Combinations of thippi and enzymes were,
(i) Thippi + amylase,
(ii) Thippi + pectinase,
(iii) Thippi + cellulase,
(iv) Thippi + amylase + pectinase,
(v) Thippi + amylase + cellulase,
(vi) Thippi + pectinase+ cellulase,
(vii) Thippi + amylase + pectinase+ cellulase.

These combinations were incubated at optimized temperature, 55 °C for 3 h with

shaking. After incubation, hydrolysate was obtained by solid-liquid separation
and then reducing sugars were estimated in the hydrolysate.

Conversion efficiency of thippi into reducing sugars was standardized in

different dilutions to obtain the maximum reducing sugars and hydrolysate
volume. Thippi powder (100 g) was taken in five flasks and 200 ml, 300 ml, 400
ml 500 ml and 600 ml water was added to the respective flasks. Mixtures were
steam pretreated and hydrolysed by using optimized amylase (5 U g-1 thippi)
enzyme at 55 °C for 3 h with shaking. Process was scaled up to hydrolyse 10 kg
thippi. Reducing sugars released during hydrolysis were checked and conversion
efficiency (thippi to reducing sugars) was calculated.

Evaluation of nutrient profile of the

hydrolysate
After hydrolysis optimization studies, the hydrolysate was evaluated
qualitatively as well as quantitatively. Quantitative analysis was done by
reducing sugars analysis using DNS method. On the basis of reducing sugars
yield, conversion efficiency was also calculated and substrates were compared.
While qualitative analysis was done by High Performance Liquid
Chromatograph (HPLC). Detail procedures are discussed later in the analytical
methods section.

Optimization of agitation, temperature and

pH by Z. mobilis MTCC 92 and TERI SH 110

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 Materials and Methods 40

Optimum ethanol production conditions were determined by standardization

process, which involved M21 medium, varying pH and incubation temperatures
of 30 ºC and 37 ºC and with shaking at 100 rpm and 200 rpm.
M21 medium (prepared with thippi hydrolysate, reducing sugar was 12%) was
inoculated with 6% inoculum of overnight grown culture (OD 0.6). The culture
was incubated at 30 ºC and 37 ºC with an agitation of 100 rpm and 200 rpm.
The M21 medium was set at pH 5.4, 6.0 and 6.8.

Fermentation of agro-industrial waste

Fruit and vegetable waste

Total reducing sugars were estimated in the hydrolysate. Medium (M21) was
then prepared with the optimum concentration of reducing sugars.
Fermentation was done in 100 ml bottles (with 50 ml head space) sealed with
rubber septum and aluminum cap. Other components of the medium, MgCl2,
(NH4)2SO4 and KH2PO4 were autoclaved separately and added aseptically.
Fermentation parameters were optimized and further fermentation studies were
carried out with the optimized conditions. Overnight grown cultures
Zymomonas mobilis MTCC 92 and Candida tropicalis TERI SH 110 were
inoculated at 6% (v/v). The fermentation was carried out at 30 ºC. The samples
were collected at 12 h interval. Ethanol production and reducing sugars
utilization were estimated. Reproducibility of the process was checked in six
repeat runs with the above conditions. For optimization of scaling up process,
fermentations were done either in 100 ml anaerobic bottles, a 1 L fermentor or a
5 L fermentor (Bioflo 3000, New Brunswick Scientific).

Thippi
Total reducing sugars were estimated in the hydrolysate and M21 medium was
prepared with optimum concentration of reducing sugars. Fermentation was
performed in bottles sealed with rubber septum and aluminum cap. Other
components of the medium, KH2PO4, MgCl2 and (NH4)2SO4 were added
aseptically. Fermentation studies were carried out with the optimized
conditions. Overnight grown cultures of Zymomonas mobilis MTCC 92 (2.8 X
108 CFU ml-1) and Candida tropicalis TERI SH 110 (1.2 X 107 CFU ml-1) were
used for seeding (6% inoculum) the fermentation broth. The fermentation was
carried out at 30 °C. The samples were collected at 12 h intervals; the ethanol
production and reducing sugars were estimated. Fermentation studies were

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 Materials and Methods 41

done with Z. mobilis alone, C. tropicalis alone and mixed culture of both.
Different parameters like inoculum size, agitation, (NH4)2SO4 concentration
were changed among the batches. For optimization of scaling up process,
fermentations were done in 100 ml, 250 ml anaerobic bottles, 1 L fermentor and
10 L fermentor (BIOFLO 3000, New Brunswick Scientific, Edison, N J, USA).
Time course experiment was also performed at 1 L and 10 L level. In first run,
reducing sugars in hydrolysate were 155.25 g l-1, 6% inoculum was used and
agitation was 150 rpm. Second run contained, reducing sugars 153.78 g l-1,
inoculated with 6% culture and fermentation was carried out at 150 rpm,
(NH4)2SO4 concentration was increased to 0.5% (w/v). Then, reducing sugars in
the next run were 156.51 g l-1 and medium was inoculated with 6% culture and
(NH4)2SO4 concentration was 0.5% (w/v), agitation was increased to 200 rpm.
Reproducibility of the process was checked in repeat runs with the above
conditions in 1 L and 10 L fermentor and all the conditions were kept same as
above.

Sugar tolerance studies

The selected cultures were studies for their sugar tolerance capability and it was
further increased by continuous adaptation of cultures to high sugar medium.
Initially, M21 medium was prepared with thippi hydrolysate to obtain up to 150
gl-1 reducing sugars in the media and ethanol production was observed. Then,
cultures were gradually shifted to high sugar medium. After several such cycles,
growth and ethanol production of the cultures was observed and compared. The
adapted strains were inoculated into the 180 gl-1 media and samples were
withdrawn at every 24 h and reducing sugars, ethanol and growth were
estimated.

Analytical methods

Microbial growth-optical density

Growth of cultures was monitored by measuring optical density. Broth cultures
were withdrawn from the fermentation reaction and optical density of cultures
was measured by spectrophotometer (U-2000 Hitachi, Japan) at 600 nm.

Samples were then centrifuged, cell pellet was used for protein analysis by Biuret
method and supernatant was used for ethanol and sugar analysis.

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 Materials and Methods 42

Protein estimation- Biuret method

Composition of Biuret reagent is mentioned in annexure. Initially a standard
curve was prepared using protein Bovine Serum Albumin (BSA) as standard. A
10 mg per ml stock solution of BSA was prepared. Different volumes of this stock
were taken into the test tubes. Volume of each test tube was made 2 ml by
distilled water. To each of these test tubes 1 ml Biuret reagent was added and
tubes were vortexed and incubated at room temperature for 10 minutes.

Sample analysis
Bacterial cell protein was estimated to check growth of isolates in different
media. 1 ml culture was removed and centrifuged at 10,000 rpm for 10 min.
Pellet was used to analyse total protein. It was dissolved in 0.2 ml distilled
water. An aliquot (100 µl) of dissolved pellet was taken into a test tube. 1 ml of
distilled water (1.9 ml) was added to this dissolved pellet. 1 ml of Biuret reagent
was added to this mixture. Reaction mixture was vortexed and incubated in dark
at room temperature for 15 min. Readings were taken at 540 nm using
spectrophotometer (U-2000 Hitachi, Japan).

Sugar estimation
The total reducing sugars were estimated by DNS method, modified according to
National Renewable Energy Laboratory, USA. Initially a standard curve was
prepared using standard glucose solution (10 mg ml-1). The standard curve was
prepared as per following protocol (Table 3.2).

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 Materials and Methods 43

Table 3.2 Protocol for reducing sugars estimation

followed in present study
Gluco Deioni Incuba Amount Incuba Dilut OD (
se zed tion of DNS tion ion nm)
conce at added at of
ntrat Water 50°C 100°C react
ion (ml) (min) ion
(mg) (min) sampl
e
Blank 1.5 60 3.0 5 1:10 540
0.5 1.45 60 3.0 5 1:10 540
1.0 1.40 60 3.0 5 1:10 540
1.5 1.35 60 3.0 5 1:10 540
2.0 1.30 60 3.0 5 1:10 540
2.5 1.25 60 3.0 5 1:10 540

The concentration of unknown samples were calculated by using

the formula K1x =Abs +

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 Materials and Methods 44

Qualitative sugar analysis by HPLC

Preparation of mobile phase
The mobile phase was prepared by dissolving the 50 mM calcium ethylene di-
amine tetra acetic acid (Ca EDTA) into HPLC grade water, Qualigens (India).
This was used as mobile phase for doing HPLC of sugar standards and samples.
The mobile phase was filtered through 0.22µ membrane filters (Millipore
Corporation, BedFord, MA).

Preparation of sugar standards

Sugar standards of glucose, sucrose fructose and xylose were prepared by using
HPLC grade water, Qualigens (India). Sugars concentration used for the
standards were 500, 750, 1000, 1250, 2500 and 5000 ppm. The standards were
filtered through 0.22µ membrane filters (Millipore Corporation, BedFord, MA)
using Swinnex mini filter assembly (Millipore Corporation, BedFord, MA).

Preparation of Samples for HPLC

Then, after acid/enzyme treatment, hydrolysate was obtained by solid-liquid
separation and was centrifuged at 10,000 rpm for 10 min. pH of the acid
hydrolysate was adjusted to neutral. The clear supernatant was double filtered
by passing through 0.22µ membrane filters (Millipore Corporation, BedFord,
USA) using Swinnex mini filter assembly (Millipore Corporation, BedFord, MA).
The filtrate was dispensed in autosampler vials and mounted on the autosampler
tray.

Samples analysis
The hydrolysate was analysed by HPLC (Agilent 1100 Series, Agilent
Technologies) for the constituent reducing sugars, which were used for ethanol
production. The sugars were analysed using HPLC fitted with Sugar-PAK I
column 300 mm x 6.5 mm ID (Waters). The mobile phase constituted 50 mM Ca
EDTA. The column temperature was set as 75 oC, and flow rate was kept at 0.5
ml min-1. The sugars were detected by Refractive Index Detector (RID). The
Hydrolysate sample (20 µl) was injected into column and samples were then
analysed through settings on the software supplied by the manufacturer.

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 Materials and Methods 45

Ethanol analysis
The ethanol produced in the culture broth was analyzed with Gas
chromatography (GC) by following methods;

1 ml of the culture broth was taken in 1.5 ml eppendorf tube from the actively
growing culture medium at regular intervals of 24 h for a period of 120 h. The
culture broth was centrifuged at 10,000 rpm for 5 min. The supernatant was
again filtered through sterile 0.22 µ membrane filter. The filtrate was analysed
by using Nucon GC. One micro litre of the filtrate was injected into the GC fitted
with chromosorb-101 column at the chromatography conditions of 155 °C oven
temperature, 175 °C injector temperature and 250°C detector temperature with
nitrogen as carrier gas with a flow rate of 30 ml min-1. An internal standard of
isopropanol was used in the making of a standard curve along with 99% ethanol
for estimating the ethanol concentration in the culture broth. Then, ethanol
estimation was further standardised by following method;

Standardization of ethanol analysis was carried out with DB Wax ETR (60 mt x
0.32 mm) J & W 123-7364 USA column by Agilent Technologies 6890 N, USA
gas chromatograph. 1 ml fermentation broth was collected and centrifuged at
10,000 rpm for 10 min, filtered with 0.22µ filter. Approximately 550 µl of the
filtrate was added to each of the autosampler vial and the vials were mounted on
to the autosampler. An injection split ratio of 1:5 was given. The oven
temperature was set at 40oC for 5 minutes and a ramp of 10 oC per minute to 230
oC and 5 minute hold at 230 oC for oven. The injector temperature was 250oC
and the detector temperature was 250 oC. The flow rate of helium mobile phase
was kept at 5 ml per minute. Calibration curve was made as given above. Both
qualitative and quantitative analysis was carried out using this method.

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 Materials and Methods 46

Statistical Analysis
The data generated during the study was processed using various statistical
tests.

Data characteristics and Analysis of Variance

The data characteristics such as Mean, Standard deviation, range, etc. were
determined. Analysis of variance (ANOVA) was used to test the hypothesis that
several means are equal. This procedure is an extension of the two-sample‘t’ test
technique. In addition to determining the existence of differences among the
means, the data was further processed to specifically to check, which means
differ, following the post hoc tests (as the post hoc tests are run after the
experiment has been conducted).

Significance Level
The significance level was chosen to be 0.05 (or equivalently, 5%) by keeping in
view the consequences of such an error. That is, we want to make the
significance level as small as possible in order to protect the null hypothesis and
to prevent, as far as possible, from inadvertently arriving at false conclusions.

T E R I University- Ph.D. Thesis, 2007