first phase potential CMCase producing bacterial...
Transcript of first phase potential CMCase producing bacterial...
3.0. Materials and Methods
Crop residues are the most promising non-conventional source for energy generation. With
the increase in production, the amount of crop-residues generated each year has also
increased. These residues are generally burnt in the field as a means of disposal, but these
organics are rich source of lignocelluloses. Generally they are used as animal feed, but they
can also be used as alternate source for fuel generation. In the present study rice straw was
used as agro-residue and investigations were carried out to obtain maximum fermentable
sugars and its further conversion to ethanol by fermentation process. The whole study was
carried out in different phases. In the first phase potential CMCase producing bacterial and
fungal strains were isolated from soil samples collected from different geographical areas.
Screening of cultures for qualitative and quantitative estimation was carried out to find out
potential CMCase producing isolates and their molecular identification was done.
In the second phase, various physical and chemical pretreatment methods were
optimized for the maximum delignification. Physico-chemical properties of enzyme such as
stability and activity at different pH and temperature were studied in due period of time.
Optimization of various cultural conditions were studied under stationary, submerged and
solid state fermentation for maximum production of CMCase by using different conditions of
pH, temperature, moistening agent, carbon source and nitrogen source. Optimization was also
done by response surface methodology. Response surface experiments identify the response
of a system as a function of explanatory variables. The interaction among the possible
influencing parameters can be evaluated with limited number of experiments.
In the third phase, effect of UV mutagenesis was studied on the isolates with aim to
increase the cellulolytic enzyme production.
In the final phase, fermentation of the saccharified sugars obtained from pretreated
rice straw was carried out using SHF (Separate Hydrolysis and Fermentation) and SiSF
(Simultaneous Saccharification and Fermentation) methods.
3.1 Materials
3.1.1 Maintenance of cultures
The cellulolytic microorganisms were isolated from soil and waste samples collected from
different habitats such as the sugarcane field, rice field, wheat field, cattle shed, cattle dung,
rotten fruits and vegetables. The isolated bacterial strains were maintained on nutrient agar
slants supplemented with 0.1% CMC (w/v) as a sole carbon source and fungal strains were
maintained on PDA slants at 40C respectively and sub-cultured periodically.
The microbial cultures used for fermentation i.e. Kluyveromyces marxianus var.
marxianus MTCC 4062 (Growth temperature 250C, pH 6.2) and Zymomonas mobilis subsp.
mobilis MTCC 2427 (Growth temperature 300C, pH 6.0) were procured from IMTECH,
Chandigarh and Saccharomyces cerevisiae NCIM 3280 (Growth temperature 280C, pH 6.4)
was procured from NCIM, Pune. The yeast cultures were maintained on MGYP (Malt Extract
Glucose Yeast Extract Peptone) medium and Zymomonas mobilis was maintained on the
selective medium.
3.1.2 Commercial cellulase enzyme
Commercial preparation of cellulase was obtained from Jagdamba Chemicals, Faridabad,
India. The enzyme is preparation from Aspergillus niger, having pH (4.5-5.5) and growth
temperature (30-400C). Enzyme activity was measured according to DNS method (Miller,
1959).
3.1.3 Chemicals and Reagents
All the chemicals and reagents used in the study were of analytical grade and bought from
standard manufactures i.e. SRL, Pvt. Ltd., Mumbai, Galaxo India Ltd., Mumbai, Sigma
Aldrich, USA, Himedia Laboratories Ltd., Mumbai, Merck India Ltd., Mumbai, and Genei,
Bangalore, etc.
3.1.4 Glass wares and Plastic wares
Glass wares and Plastic wares used in the present study were supplied by Borosil, Tarson,
Axygen, Scott, Duran, etc.
3.1.5 Instruments used
Table 3.1 Instruments used in the present study.
S. No. Instrument Manufacturer
1. Autoclave NSW
2. Incubator shaker NSW
3. Incubator shaker REMI
4. Centrifuge REMI
5. Spectrophotometer PG instrument
6. Electronic balance Afcoset
7. Laminar air flow system NSW
8. Microscope Magnus (Olympus)
9. Microwave oven ONIDA
10. pH meter Eutech
11. Refrigerator LG
12. Water bath NSW
13. Water distillation unit Rions
14. Mixer grinder Sujata
15. Deep freezer Blue Star
16. UV cabinet Popular traders
17. Microfuge Plastocraft
18. Magnetic stirrer Genei
19. Hot air oven Popular traders
20. Vortex shaker IKA
21. Distillation Unit Duran
3.2 Collection of samples for isolation of cellulolytic enzyme producers
Various samples of soil and wastes were collected from different regions such as sugarcane
field, rice field, wheat field, rhizospheric soil, cattle shed, cattle dung, rotten fruits and
vegetables. Different places like Ambala Cantt, Ambala city, NDRI (Karnal), Shahabad,
Yamunanagar, Gohana (Haryana), Kulu, Manali, Manikaran (Himachal Pradesh), Chennai
(Tamil Nadu) and adjoining areas were visited for sampling.
3.2.1 Raw material used
Rice straw from basmati/non-basmati rice was collected from local fields of Kurukshetra and
Karnal districts, dried at 600C and powdered in grinder mixer and sieved to obtained rice
straw of different mesh size. Rice straw of 0.5mm was selected, pretreated and used as source
of carbon for screening and production of cellulases under stationary fermentation,
submerged fermentation (SmF) and solid state fermentation (SSF) conditions.
3.3 Methods
3.3.1 Media and Glassware sterilization
Various media and distilled water used in present study were sterilized in the autoclave by
moist heating at a pressure of 15lbs/square inch and temperature of 1210C for 15 min. All the
glassware were sterilized in oven by dry heating at 1800C for 2 h. Forceps, loop and spreader
were flame sterilized prior to use.
3.3.2 Media
The composition of various media used in the present study is as follows:
3.3.2.1 Carboxymethyl Cellulose (CMC) agar medium (pH 8.0)
The medium was used for the isolation and primary screening of CMCase producing bacterial
strains and has the following chemical composition (Table 3.2).
Table 3.2 Chemical composition of CMC agar medium (pH 8.0).
S. No. Component Amount (g/l)
1. NaNO3 2.0
2. K2HPO4 1.0
3. MgSO4. 7H2O 0.5
4. KCl 0.5
5. CMC 5.0
6. Agar-Agar 20.0
7. Distilled water 1L
3.3.2.2 Carboxymethyl cellulose (CMC) agar medium (pH 5.0)
The medium was used for the isolation and primary screening of CMCase producing fungal
strains and has the following chemical composition (Table 3.3).
Table 3.3 Chemical composition of CMC agar medium (pH 5.0).
S. No. Component Amount (g/l)
1. NaNO3 2.0
2. K2HPO4 1.0
3. MgSO4. 7H2O 0.5
4. KCl 0.5
5. CMC 5.0
6. Agar-Agar 20.0
7. Distilled water 1L
3.3.2.3 Nutrient Agar medium (NA)
This medium was used for general culturing and storage of isolated bacterial strains and has
the following chemical composition (Table 3.4).
Table 3.4 Chemical composition of Nutrient Agar medium.
S. No. Component Amount (g/l)
1. Peptone 5.0
2. Beef extract 3.0
3. NaCl 5.0
4. CMC 1.0
5. Agar-Agar 20.0
6. pH 6.8
7. Distilled water 1L
Nutrient broth was prepared by omitting agar-agar from the above medium.
3.3.2.4 Potato Dextrose Agar medium (PDA)
This medium was used for general culturing and storage of isolated fungal strains and has the
following chemical composition (Table 3.5).
Table 3.5 Chemical composition of Potato Dextrose Agar medium.
S. No. Component Amount (g/l)
1. Potato 200.0
2. Dextrose 20.0
3. Yeast extract 0.1
4. Agar-Agar 20.0
5. pH 5.0
6. Distilled water 1L
200 g of peeled potatoes were cut into small pieces and suspended in 1000 ml distilled water
and steamed for 30 min. The extract was obtained by filtering through the muslin cloth and
final volume was made up to 1000 ml with distilled water.
3.3.2.5 Malt Extract Glucose Yeast Extract Peptone (MGYP) medium
This medium was used for the growth of yeast cultures and has the following chemical
composition (Table 3.6).
Table 3.6 Chemical composition of MGYP medium.
S. No. Component Amount (g/l)
1. Yeast extract 3.0
2. Malt extract 3.0
3. Peptone 5.0
4. Glucose 10.0
5. Agar-Agar 20.0
6. pH 6.0-6.4
7. Distilled water 1L
3.3.2.6 Growth medium for Zymomonas mobilis
This medium was used for the growth of ethanologenic bacterial culture (Zymomonas
mobilis) and has the following chemical composition (Table 3.7).
Table 3.7 Chemical composition of growth medium for Zymomonas mobilis.
S. No. Component Amount (g/l)
1. Yeast extract 10.0
2. KH2PO4 2.0
3. Glucose 20.0
4. Agar-Agar 20.0
5. pH 6.0
6. Distilled water 1L
3.3.2.7 Screening medium (pH 8.0)
This medium was used for the quantitative screening of CMCase producing bacterial strains
and has the following chemical composition (Table 3.8).
Table 3.8 Chemical composition of screening medium (pH 8.0)
. S. No. Component Amount (g/l)
1. Yeast extract 2.5
2. K2HPO4 5.0
3. NaCl 1.0
4. MgSO4 .7H2O 0.2
5. Peptone 2.0
6. CMC 5.0
7. Distilled water 1L
3.3.2.8 Screening medium (pH 5.0)
This medium was used for the quantitative screening of CMCase producing fungal strains
and has the following chemical composition (Table 3.9).
Table 3.9 Chemical composition of screening medium (pH 5.0)
.
S. No. Component Amount (g/l)
1. Yeast extract 2.5
2. K2HPO4 5.0
3. NaCl 1.0
4. MgSO4 .7H2O 0.2
5. Peptone 2.0
6. CMC 5.0
7. Distilled water 1L
3.3.2.9 Production medium (pH 8.0)
This medium was used as production medium under stationary fermentation and SmF by
Bacillus sp. 313SI and has the following chemical composition (Table 3.10).
Table 3.10 Chemical composition of production medium (pH 8.0)
S. No. Component Amount (g/l)
1. Yeast extract 2.5
2. K2HPO4 5.0
3. NaCl 1.0
4. MgSO4 .7H2O 0.2
5. Peptone 2.0
6. Pretreated rice straw 5.0
7. Distilled water 1L
3.3.2.10 Production medium (pH 5.0)
This medium was used as production medium under stationary fermentation and SmF by
Aspergillus niger BK01 and has the following chemical composition (Table 3.11).
Table 3.11 Chemical composition of production medium (pH 5.0).
S. No. Component Amount (g/l)
1. Yeast extract 2.5
2. K2HPO4 5.0
3. NaCl 1.0
4. MgSO4 .7H2O 0.2
5. Peptone 2.0
6. Pretreated rice straw 5.0
7. Distilled water 1L
3.3.2.11 Mineral Salt medium I
This medium was used as moistening agent in parametric optimization of cultural conditions
for maximum titre of cellulolytic enzymes under SSF by Bacillus sp. 313SI and has the
following chemical composition (Table 3.12).
Table 3.12 Chemical composition of Mineral Salt medium I.
S. No. Component Amount (g/l)
1. (NH4)2SO4 10.0
2. KH2PO4 4.0
3. CaCl2 0.5
4. MgSO4.7H2O 0.5
5. pH 8.0
6. Distilled water 1L
3.3.2.12 Mineral Salt medium II
This medium was used as moistening agent in parametric optimization of cultural conditions
for maximum titre of cellulolytic enzyme under SSF by Bacillus sp. 313SI and has the
following chemical composition (Table 3.13).
Table 3.13. Chemical composition of Mineral Salt medium II.
S. No. Component Amount (g/l)
1. Na2HPO4 1.10
2. NaH2PO4.2H20 0.61
3. KCl 0.30
4. MgSO4.7H2O 0.01
5. pH 8.00
6. Distilled water 1L
3.3.2.13 Mineral Salt medium-III
This medium was used as moistening agent in parametric optimization of cultural conditions
for maximum titre of cellulolytic enzyme under SSF by Aspergillus niger BK01 and has the
following chemical composition (Table 3.14).
Table 3.14 Chemical composition of Mineral salt medium-III.
S. No. Component Amount (g/l)
1. NaNO3 3.0
2. K2HPO4 1.0
3. KCl 0.5
4. MgSO4.7H2O 0.3
5. FeSO4. 7H2O 0.01
6. pH 5.0
7. Distilled water 1L
3.3.2.14 Mandel and Sternburg’s medium (1976)
This medium was used as moistening agent in parametric optimization of cultural conditions
for maximum titre of cellulolytic enzyme under SSF by Aspergillus niger BK01 and has the
following chemical composition (Table 3.15).
Table 3.15 Chemical composition of Mandel and Sternburg’s medium.
S. No. Component Amount (g/l)
1. KH2PO4 2.0
2. Urea 0.3
3. MgSO4.7H2O 0.3
4. CaCl2 0.3
5. Peptone 0.75
6. Yeast extract 0.25
7. Trace element solution 1.0 ml
8. pH 5.0
9. Distilled water 1L
Trace elements (mg/l)
10. FeSO4.7H2O 5.00
11. MnSO4.4H2O 1.60
12. ZnSO4.7H2O 1.40
13. CoCl2.6H2O 20.0
3.3.2.15 Fermentation medium for SHF
This medium was used for the fermentation under SHF by ethanologenic strains i.e. Yeast
(Kluyveromyces marxianus var. marxianus MTCC 4062 and Saccharomyces cerevisiae
NCIM 3280) and bacteria (Zymomonas mobilis subsp. mobilis MTCC 2427) and has the
following chemical composition (Table 3.16).
Table 3.16 Chemical composition of fermentation medium under SHF.
S. No. Component Amount
1. Hydrolysate 100ml
2. KH2PO4 0.15%
3. Urea 0.3%
4. Yeast extract 0.5%
3.3.2.16 Fermentation medium for SiSF
This medium was used for the fermentation under SiSF by ethanologenic strains i.e. Yeast
(Kluyveromyces marxianus var. marxianus MTCC 4062 and Saccharomyces cerevisiae
NCIM 3280) and bacteria (Zymomonas mobilis subsp. mobilis MTCC 2427) using
commercial cellulase enzyme preparation and has the following chemical composition (Table
3.17).
Table 3.17 Chemical composition of fermentation medium used under SiSF.
S. No. Component Amount
1. Hydrolysate 100ml
2. KH2PO4 0.15%
3. Urea 0.3%
4. Yeast extract 0.5%
3.4 Congo red dye solution
This dye (0.1%) has been used for preliminary qualitative analysis for cellulolytic activity of
selected isolates.
3.5 Enzyme assay
3.5.1 Endoglucanase activity (CMCase)
Carboxymethyl cellulase (CMCase) activity was assayed by the DNS (3,5-dinitrosalicylic
acid) method (Miller, 1959). The reaction mixture contained 900 µl of substrate (CMC in 10
mM sodium phosphate buffer pH 7.0) and 100 µl of crude enzyme and was incubated at 300C
for 60 min for bacterial CMCase and 900 µl of substrate (Carboxymethyl cellulose in 0.1 M
citrate buffer pH 4.8) and 100 µl of crude enzyme and was incubated at 400C for 60 min for
fungal CMCase. An appropriate control which contained 100 µl of distilled water instead of
crude enzyme extract was also run along with the test. The reaction was terminated by adding
3 ml of 3,5- dinitrosalicylic acid reagent. The tubes were incubated for 15 min in a boiling
water bath for color development and were cooled rapidly. The activity of reaction mixture
was measured against a reagent blank at 540 nm. The concentration of glucose released by
enzyme was determined by comparing against a standard curve constructed similarly with
known concentrations of glucose.
3.5.2 Filter Paper Activity (FPA)
Filter paper activity (FPA) was estimated by the methods of (Mandels et al., 1976). To 50 mg
(1×6 cm strip) of Whatman No.1 filter paper, were added 1ml of 0.1 M citrate buffer pH 4.8
and incubated at 400C for 60 min for fungal isolate and 10mM Sodium phosphate buffer pH
7.0 and incubated at 300C for 60 min for bacterial isolate. The reaction was terminated by
adding 3ml of 3, 5- dinitrosalicylic acid reagent. The tubes were incubated for 15 min in a
boiling water bath for color development and were cooled rapidly. The activity of reaction
mixture was measured against a reagent blank at 540 nm.
3.5.3 β-glucosidase activity
β-glucosidase activity was measured according to Berghem and Petterson, (1974) with some
modification. The substrate was 5mM 4-nitrophenyl-β-D-glucopyranoside (pNPG) in Sodium
phosphate buffer pH 7.0 for bacterial isolate and 0.1 M citrate buffer pH 4.8 for fungal
isolate. One ml pre-incubated substrate was mixed with 0.1ml diluted enzyme solution and
incubated for 10 min at 40°C and 300C for fungal and bacterial isolate respectively. The
reaction was terminated by addition of 2ml of 1 M Na2CO3 solution and then diluted with
10ml distilled water. The amount of the liberated 4-nitrophenol was measured at 400 nm
against substrate blank.
One enzyme unit (IU) is defined as the amount of enzyme required to hydrolyze 1 µg
of substrate per min under the assay conditions. The amount of enzyme production under
stationary and submerged fermentation was measured as U/ml whereas it was measured as
Unit per gram dry substrate (U/gds) in case of SSF.
3.6 Screening and selection of CMCase producing fungi/bacteria
All the isolates of bacteria and fungi were screened qualitatively and quantitatively for their
ability to produce CMCase.
3.6.1 Qualitative estimation (Primary screening)
Samples for the screening of cellulase producers were collected from different environment
as mentioned earlier in this chapter. Enrichment was done by adding 1g of soil sample in 25
ml of sterile deionized water having CMC as carbon source and peptone as nitrogen source at
pH 8.0 and 5.0 and incubated under stationary conditions of growth at 370C and 28
0C for
bacterial and fungal growth respectively. Isolation was done by dilution plate method.
Primary screening was done on CMC agar medium. The plates were then incubated at 280C
for fungal cultures and 370C for bacterial cultures. Further screening for cellulolytic potential
was followed by visualizing the hydrolytic zone, when the CMC agar plates were flooded
with an aqueous solution of 0.1% Congo red for 15 min and washed with 1 M NaCl (Apun et
al., 2000). The isolated colonies on these plates were maintained on their respective agar
slants at 40C for further analysis.
3.6.2 Quantitative estimation (Secondary screening)
Each of the isolates were grown in 50 ml of screening medium containing rice straw as agro-
residue in a 250 ml flask and incubated at 370C for 48 h for bacterial culture and 28
0C for 3-5
days for fungal cultures on a rotary shaker (NSW-256) at 180 rpm. Crude enzyme was
harvested by centrifugation at 10,000 x g for 20 min at 40C and the clear supernatant was
used as the source of cellulolytic enzymes. CMCase, FPase and β-glucosidase activity was
estimated. Carboxymethyl cellulose was used as substrate for assaying the activity of
CMCase.
3.7 Identification and molecular characterization of high CMCase producing isolates
The selected isolates on the basis of CMCase activity (one each from bacteria and fungi)
were identified on the basis of their morphological and other cultural characteristics. Cultures
were routinely revived and maintained on nutrient agar medium (Bacteria) and on potato
dextrose agar medium (Fungi) and stored at 40C. The morphologically identified isolates
were subjected to genetic characterization. The isolates were genetically characterized using
the commercial service provided by Xcelris Labs Ltd, Ahmedabad, India.
3.7.1 Molecular characterization of bacterial isolates using 16S rDNA based molecular
technique
Methodology:-
• DNA was isolated from the culture and its quality was evaluated on 1.2% agarose gel,
a single band of high-molecular weight DNA has been observed.
• Fragment of 16S rDNA gene was amplified by PCR from the above isolated DNA. A
single discrete PCR amplicon band of 1500 bp was observed when resolved on
agarose gel (Gel Image-1).
• The PCR amplicon was purified to remove contaminants.
• Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with
8F and 1492R primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic
Analyzer.
• Consensus sequence of 1403 bp 16S rDNA gene was generated from forward and
reverse sequence data using aligner software.
• The 16S rDNA gene sequence was used to carry out BLAST with the database of
NCBI gene bank database. Based on maximum identity score first ten sequences were
selected and aligned using multiple alignment software program, Clustal W. Distance
matrix was generated using RDP database and the phylogenetic tree was constructed
using MEGA 4.
3.7.2 Molecular characterization of fungal isolates using D1/D2 region of LSU (Large
Sub Unit: 28S) rDNA based molecular technique
Methodology:-
DNA was isolated from the culture and its quality was evaluated on 1.2% agarose gel,
a single band of high-molecular weight DNA has been observed.
Fragment of D1/D2 region of LSU (Large subunit 28S rDNA) gene was amplified by
PCR from the above isolated plasmid DNA. A single discrete PCR amplicon band of
650 bp was observed when resolved on agarose gel (Gel Image-1).
The PCR amplicon was purified to remove contaminants.
Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with
DF and DR primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic
Analyzer.
Consensus sequence of 566 bp of D2 region of 28S rDNA gene was generated from
forward and reverse sequence data using aligner software.
The D1/D2 region of LSU (Large subunit 28S rDNA) gene sequence was used to
carry out BLAST with the nrdatabase of NCBI gene bank database. Based on
maximum identity score first ten sequences were selected the phylogenetic tree was
constructed using MEGA 4.
3.8 Preprocessing of the substrate for pretreatment
The substrate i.e. rice straw was dried at 600C in a hot air oven till constant weight and
powdered in a grinder mixer (dry milling) and sieved to obtain fine particle size i.e. 0.5 mm.
3.8.1 Pretreatment
Pretreatment is required to alter the structure of cellulosic biomass to make cellulose
accessible to the enzymes that convert carbohydrate polymers into fermentable sugars. Rice
straw was pretreated with alkali and acid at different time intervals. Rice straw was pretreated
with different concentrations of alkali such as KOH (0.1-0.5M) and NaOH (0.1-0.5M) for 8 h
respectively and acid such as H2SO4 (0.1-0.5N) and HCl (0.1-0.5N) for 4 h respectively. The
combined effect of alkali and acid on pretreatment of rice straw was also observed. The pre-
treated substrates so prepared were further used for estimation of cellulose, hemicellulose and
lignin present in sample by following NDF (neutral detergent fibre) and ADF (acid detergent
fibre) method as described by Goering and van Soest, (1975).
3.9 Detoxification
The pretreated samples from alkali, acid and alkali+acid pretreatment were mixed with wood
activated charcoal (20:1 w/w sample:charcoal) and then agitate for 2 days on magnetic stirrer
at room temperature. After charcoal treatment the sample was filtered using whatman filter
paper to remove charcoal.
3.10 Extraction of crude cellulase enzyme
The bacterial and fungal isolates were grown in 100 g of pretreated rice straw in moistening
agents i.e. moistening agent II (pH 8.0) and Mandel and Sternburg‟s medium (pH 5.0)
respectively under optimized solid state fermentation conditions. The culture broth was
centrifuged at 10,000 rpm for 20 min and clear supernatant was collected and used as the
source of enzyme, which was stored at 40C till use.
3.10.1 Characterization of the crude cellulase
3.10.1.1 Effect of pH on CMCase activity and stability
The effect of pH on CMCase activity and stability of the enzyme was examined at different
pH values by incubating the enzyme in buffers of different pH values ranging from 3.0 to
10.0. The pH of the reaction mixture was varied using different buffers: 0.1 M citrate buffer
(pH 3.0 to 6.0), 10mM sodium phosphate buffer (pH 6.0 to 8.0), 0.05 M Tris-HCl (pH 8.0 to
9.0) and 0.05 M glycine-NaOH (pH 9.0 to 10.0). Then 300 µl of 0.5% CMC was added to
100 µl of enzyme. The mixture was incubated at 300C and 40
0C for bacterial and fungal for 1
h respectively. The pH stability was determined by incubating crude enzyme mixture in
above-mentioned buffers at room temperature for 1h.
3.10.1.2 Effect of temperature on CMCase activity and stability
To determine the effect of temperature on CMCase activity the enzymatic reaction was
carried out at different temperatures ranging from 20 to 600C. Crude enzyme (100 µl) and
300 µl substrate (0.5% CMC in 10 mM sodium phosphate buffer and 0.1 M citrate buffer for
bacterial and fungal isolate respectively) were pre-incubated for 10 min at various reaction
temperatures ranging from 20 to 600C before starting the experiment and the enzyme assay
was performed as described earlier to determine the optimal incubation temperature. The
temperature stability was determined by incubating crude enzyme mixture in above-
mentioned buffers at room temperature for 1 h.
3.10.1.3 Effect of incubation period on CMCase activity
The optimum incubation period of reaction mixture at which the enzyme activity was
maximum was determined by incubating at different time intervals (10-90 min) and the
enzyme assay was performed to determine the optimal incubation period.
3.10.1.4 Effect of substrate concentration on CMCase activity
Effect of substrate concentration (CMC) on enzyme activity was determined using 0.1ml
enzyme incubated with different concentrations of the substrate (0.1-2.0%) under optimal
conditions. Crude enzyme and substrate was incubated and enzyme assay was performed to
determine the substrate concentration at which CMCase activity was maximum.
3.11 Mode of Fermentation
The production of CMCase by two selected isolates was studied under stationary
fermentation, SmF and SSF using pretreated rice straw as sole source of carbon. Pretreated
rice straw and respective production medium were taken in 250 ml Erlenmeyer flask and
sterilized at 121C at 15 lbs/square inch for 20 min. After cooling, the medium was
inoculated with cell suspension under aseptic conditions. These flasks were then incubated at
different cultural conditions under stationary, SmF and SSF fermentation conditions for
respective time intervals. For obtaining extracellular enzyme, the cells were removed by
centrifugation at 10,000 x g for 15 min and the supernatant was assayed for CMCase activity.
3.12 Optimization of process parameters for CMCase production by isolates under
stationary and submerged fermentation
Different cultural conditions were optimized for maximum production of CMCase by
Bacillus sp. 313SI and Aspergillus niger BK01 under stationary and SmF considering one
factor at a time approach.
3.12.1 Inoculum development
Pure culture of bacterial isolate was inoculated in screening medium (pH 8.0) for 24 h at
370C. After 24h of fermentation period these vegetative cells were used as inoculum source.
For preparation of inoculum of fungal culture, 10 ml of sterilized distilled water
supplemented with 0.1% Tween-80 was added to 3 day old fully sporulated agar slant culture.
3.12.2 Effect of substrate concentration, time, temperature and pH on CMCase
production
The effect of substrate (pretreated rice straw) concentration (0.25-2.0%) on enzyme
production was studied. Each 250 ml Erlenmeyer flask containing 50 ml of the production
medium (pH 8.0) containing different concentrations of substrate was inoculated with
Bacillus sp. 313SI under shaking conditions (180 rpm) at 30C for 48 h and at 350C for 60h
under non-shaking conditions for stationary fermentation.
Besides, the effect of inoculum concentration (0.25-1.5%) was also studied. Same
procedure was done for Aspergillus niger BK01culture and flasks were incubated at 280C for
108 h under stationary conditions and 280C for 72 h under submerged fermentation
conditions.
Evaluation of incubation period for CMCase production was assessed by incubating
the bacterial culture for varying time intervals. For this, the flask containing 50 ml of the
production medium was inoculated with Bacillus sp. 313SI under aseptic conditions. These
flasks were then incubated at 300C for 12-84 h under shaking (180 rpm) conditions and 35
0C
for 12-84 h under stationary conditions. The cell-free culture broth was assayed for the
CMCase activity. Same procedure was done for Aspergillus niger BK01 culture for varying
time intervals and flasks were incubated at 280C under stationary and submerged
fermentation conditions.
The flasks containing pretreated rice straw, inoculated with Bacillus sp. 313SI culture
were incubated under shaking (180 rpm) as well as under stationary conditions at
temperatures ranging from 20-500C for 48h and 60h respectively. Same procedure was done
for Aspergillus niger BK01 culture and flasks were incubated for 108 h and 72 h under
stationary as well as under submerged fermentation conditions respectively.
Erlenmeyer flasks containing 50 ml of selective medium with pH ranging from 5.0-
9.0 was inoculated with Bacillus sp. 313SI. The cell free extracts were assayed for enzyme
activity to determine the optimal pH for CMCase production. Same procedure was done for
Aspergillus niger BK01 culture with pH ranging from 4.0-7.0 under stationary as well as
under submerged fermentation conditions.
3.12.3 Effect of the carbon and nitrogen sources
Pretreated rice straw was supplemented with different carbon sources (0.1% w/v) (viz.
Galactose, maltose, starch, mannitol, lactose, CMC and cellulose powder) and nitrogen
sources (0.1% w/v) (viz. ammonium chloride, ammonium nitrate, ammonium sulphate,
potassium nitrate, beef, Tryptone and urea). The flasks were inoculated with Bacillus sp.
313SI or Aspergillus niger BK01 and are incubated under their optimized cultural conditions
under shaking as well as stationary conditions respectively. Furthermore, different
concentrations of the selected carbon and nitrogen source were also tried for having optimum
response with respect to the production of CMCase.
3.13 Optimization of process parameters for CMCase production under solid state
fermentation (SSF).
Various physico-chemical parameters were optimized under SSF considering one factor at a
time approach.
3.13.1 Effect of substrate concentration, time, temperature and pH on CMCase
production
The effect of substrate (pretreated rice straw) concentration (1.0-5.0%) on enzyme production
was studied. Each 250 ml Erlenmeyer flask containing 50 ml of the production medium (pH
8.0) containing different concentrations of substrate was inoculated with Bacillus sp. 313SI
under SSF. Same procedure was done for Aspergillus niger BK01 culture with varying
substrate concentration (4.0-9.0%).
The effect of inoculum size on enzyme production was analyzed under SSF. Each 250
ml Erlenmeyer flask containing 50 ml of the production medium was inoculated with
different inoculum concentrations (1-4%) in case of Bacillus sp. 313SI and (6-9%) in case of
Aspergillus niger BK01. The enzyme was extracted and CMCase activity was determined.
The SSF was carried out at different incubation temperature (20-500C) for Bacillus sp.
313SI to evaluate effect of incubation temperature on enzyme production. The enzyme was
extracted and the activity of crude enzyme was assayed. Same procedure was followed for
Aspergillus niger BK01 culture and effect of incubation temperature on enzyme production
was evaluated at different incubation temperature (20-400C).
The bacterial culture was inoculated in the autoclaved SSF flasks containing
moistening agent maintained at different pH ranging from 5.0 to 9.0. The contents were
mixed thoroughly and then the flasks were incubated at 350C in case of Bacillus sp. 313SI.
Same procedure was followed for Aspergillus niger BK01 culture and flasks were moistened
with different moistening agents maintained at different pH ranging from 4.0 to 7.0 and
incubated at 280C.
3.13.2 Effect of moistening agents and moisture level
Different types of moistening agents such as mineral salt medium-I, II, tap water (Cl- 0.08%;
Ca++
0.5%, Mg++
0.5%, HCO-3 0.4%) and distilled water were examined for the enzyme
production. These contents were mixed properly, autoclaved, inoculated with Bacillus sp.
313SI. The enzyme was extracted and activity was determined. Same procedure was followed
for Aspergillus niger BK01 when culture was moistened with Mandel and Sternburg‟s
medium, mineral salt medium III, tap water and distilled water.
The selected moistening agent was examined for its moisture level. The solid
substrate was moistened by using mineral salt medium-II for Bacillus sp. 313SI. For the
bacterial strain, different ratios (w/v) of substrate: moistening agent ranging from 1:1, 1:2,
1:3, 1:4 and 1:5 was used to determine the best ratio for enzyme production. The enzyme was
extracted and assayed for cellulolytic enzymes production. Same procedure was followed for
Aspergillus niger BK01 culture but the solid substrate was moistened by Mandel and
Sternburg‟s medium at different ratios.
3.13.3 Effect of the carbon and nitrogen sources
The effect of different carbon sources (0.1% w/w) (viz. Galactose, maltose, starch, mannitol,
lactose, CMC and cellulose powder) and nitrogen sources (0.1% w/w) (viz. ammonium
chloride, ammonium nitrate, ammonium sulphate, potassium nitrate, beef, tryptone and urea)
were studied in case of both bacterial and fungal isolates. Each flask containing pretreated
rice straw in selective moistening agent was supplemented with different carbon and nitrogen
sources. After autoclaving, the flasks were inoculated with Bacillus sp. 313SI and Aspergillus
niger BK01 and incubated under optimized cultural conditions. Different concentrations of
the selected carbon and nitrogen sources were also tried for having optimum response with
respect to the production of CMCase.
3.14 Statistical Model
Response Surface Methodology (RSM) using the Central Composite Design (CCD) of
experiments was used to develop a mathematical correlation between four independent
variables on production of CMCase by the bacterial strain of Bacillus sp. 313SI.
3.14.1 Response Surface Methodology (RSM) and Central Composite Design (CCD) of
experiments
Based on one factor at a time approach experiments, four independent variables were chosen
for further optimization by Response Surface Methodology (RSM) using the Central
Composite Design (CCD) of experiments. This was used to develop a mathematical
correlation between four independent variables on production of CMCase by the bacterial
strain of Bacillus sp. 313SI. The four independent variables, pretreated rice straw
concentration (A), ammonium sulphate concentration (B), temperature (C) and pH (D) were
chosen to study their effect on CMCase production by Bacillus sp. 313SI. The four
independent variables were studied at five different levels (-α, -1, 0, +1, +α). All variables
were taken at a central coded value of zero. The minimum and maximum ranges of variables
investigated are listed in Table 3.18 and a set of 30 experiments were carried out (Table
3.19). The statistical software package „Design Expert 8.0.7.1‟ was used to analyze the
experimental data.
Upon completion of experiments, the average maximum CMCase biosynthesis yields
were taken as the responses (R1). A multiple regression analysis of the data was carried out
for obtaining an empirical model that relates the response measured to the independent
variables. A second order polynomial equation for a four factor system is:
R1 = β0 + β1A+ β2 B + β3C + β4D + β11 A2 + β22 B
2 + β33C
2 + β44 D
2 + β12 AB + β23
BC + β34 CD + β13 AC + β14 AD + β24 BD
Where R1 is the predicted response, β0 intercept, β1, β2 , β3, β4 linear coefficients,
β11, β22, β33, β44 squared coefficients, β12, β23, β34, β13, β14, β24 interaction coefficients
and A, B, C, A2, B
2, C
2, D
2, AB, BC, CD, AC, AD, BD are levels of the independent
variables. A total of 30 experiments were necessary to study the coefficients of model. The
response surface curves were obtained from „Design Expert 8.0.7.1‟ software for determining
the optimum levels of the variables for maximum production of CMCase and to generate
response surface contours graphs.
Table 3.18 Ranges of the four independent variables used in RSM.
Factors Name Levels
-α -1 0 +1 +α
A Pretreated rice straw (% w/v) -0.75 0.5 1.75 3 4.25
B Ammonium sulphate (% w/v) -0.1 0.1 0.3 0.5 0.7
C Temperature (0C) 15 30 45 60 75
D pH 3.5 5 6.5 8 9.5
Table 3.19 Experimental plan for optimization of CMCase production using RSM.
Runs
Pretreated
Rice straw
concentration
(%w/v)
Ammonoium
sulphate
concentration
(%w/v) Temperature (0C) pH
1 1.75 0.3 45 6.5
2 1.75 0.3 75 6.5
3 1.75 0.7 45 6.5
4 0.50 0.1 60 5.0
5 -0.75 0.3 45 6.5
6 0.50 0.5 30 8.0
7 0.50 0.1 30 8.0
8 0.50 0.5 60 8.0
9 0.50 0.1 30 5.0
10 4.25 0.3 45 6.5
11 0.50 0.5 60 5.0
12 1.75 0.3 45 9.5
13 0.50 0.5 30 5.0
14 1.75 0.3 45 6.5
15 3.00 0.5 30 8.0
16 1.75 0.3 45 6..5
17 1.75 0.3 45 6.5
18 0.50 0.1 60 8.0
19 1.75 -0.1 45 6.5
20 3.00 0.1 30 5.0
21 3.00 0.1 30 8.0
22 1.75 0.3 45 6.5
23 1.75 0.3 45 3.5
24 3.00 0.1 60 8.0
25 3.00 0.5 60 8.0
26 3.00 0.5 30 5.0
27 1.75 0.3 45 6.5
28 3.00 0.1 60 5.0
29 1.75 0.3 15 6.5
30 3.00 0.5 60 5.0
3.15 Effect of UV mutagenesis on CMCase production
Improvement in enzyme production by mutagenesis was sought to isolate hyper-producer
mutant derivatives of Bacillus sp. 313SI and Aspergillus niger BK01 under shaking
conditions of growth. For this, the selected fungal and bacterial strain was treated with
mutagenic agent ultraviolet (UV) irradiation. Various experiments were designed to see the
effect of UV mutagenesis at different intervals of time in UV chamber. Effect of different
other parameters of UV mutagenesis were also analyzed on CMCase activity i.e. effect of UV
exposure with petri-plate lid and without petri-plate lid at different intervals of time. Different
strength of UV rays were also analyzed on CMCase activity in UV chamber i.e. exposure of
short UV wavelength, long UV wavelength and intermittent UV wavelength.
3.16 Optimization of ethanol production using SHF (Separate Hydrolysis and
Fermentation) and SiSF (Simultaneous Saccharification and Fermentation) methods.
Fermentation was carried out using SHF (Separate Hydrolysis and Fermentation) and SiSF
(Simultaneous Saccharification and Fermentation) methods.
Under SHF, hydrolysis of pretreated rice straw was carried out and compared using
bacterial crude cellulase, fungal crude cellulase and commercial cellulase enzyme
preparations. The hydrolysate was centrifuged at 5000 rpm for 15 min and the effect of
different concentration of respective enzyme preparation and incubation time on the amount
of reducing sugars released and consequently on percent saccharification was investigated.
The saccharification value was calculated as:
Reducing sugars produced × 0.9 × dilutions
Cellulose produced from substrate
The hydrolysate having maximum reducing sugar was further subjected for maximum
bioethanol production. The fermentation was carried out in 250 ml conical flasks containing
100 ml hydrolysate supplemented with urea (0.3%), potassium-di-hydrogen phosphate
(0.15%) and yeast extract (0.5%) The ethanologenic strains (yeast or bacteria) were used for
the maximum bioethanol production at different temperatures and after different intervals of
time.
Under SiSF, fermentation was carried out by suspending pretreated rice straw in
distilled water (1:10) with commercial cellulase supplemented with yeast nutrients. The
fermentation flasks were inoculated with different ethanologenic strains and incubated at
different temperatures and fermentation profile with respect to bioethanol production was
checked over a range of period. Ethanol estimation was done by spectrophotometric method
(Caputi et al., 1968). Fermentation efficiency, ethanol yield and volumetric ethanol
productivity was calculated by the following equation
Fermentation efficiency (%) = Actual ethanol produced × 100
Theoretical ethanol produced
Where Theoretical ethanol produced = Reducing sugar present in fermentation solution ×
0.51
Ethanol yield (g/g) = Ethanol produced (g)
Reducing sugar (g)
Ethanol productivity (g/l/h) = Ethanol concentration (g/l)
Time (h)
3.17 Chemical Analysis
3.17.1 Estimation of Cellulose, Hemicellulose and Lignin
The cellulose, hemicellulose and lignin were estimated by a method developed by Goering
and van Soest, (1975) as mentioned below:
Neutral Detergent Fibre (NDF)
Preparation of neutral detergent solution: In a 200 ml of distilled water taken in a beaker,
18.61 g of disodium ethylenediamine tetraacetate and 6.81 g of sodium borate decahydrate
were dissolved by heating. To this about 100-200 ml of a solution containing 30 g of sodium
lauryl sulphate and 10 ml of 2-ethoxy ethanol were added. To this about 100 ml of a solution
containing 4.5 g of disodium hydrogen phosphate was added. Then the volume was made up
to one litre and pH was adjusted to 7.0.
Procedure: The delignified substrate of 0.5 g was taken in a refluxing flask. 100 ml of cold
neutral detergent solution, 2 ml of decahydronaphthalene (Decalin) and 0.5 g of sodium
sulphite were added. The mixture was heated to boiling. Then the heat was reduced to avoid
foaming and refluxed for one hour. After cooling, the sample was filtered through a
previously weighed gooch crucible of G-1 grade under suction using a vacuum pump. The
residue remained in the gooch crucible was washed with hot water repeatedly. Finally the
residue was given two washings of acetone. The crucible containing residue was dried at
1000C for 8 h in a hot air oven. Then it was cooled in a desiccator and the dry weight was
recorded.
% NDF = Y - X × 100
W
Where,
Y: Weight of crucible + NDF
X: Weight of empty crucible
W: Weight of the sample
Acid Detergent Fibre (ADF)
Acid detergent solution: In one litre of 1 N sulphuric acid, 20 g of cetyl trimethyl ammonium
bromide was dissolved. 72% H2SO4: 73.5 ml of concentrated sulphuric acid (98% pure) was
added to a beaker containing distilled water of 26.5 ml.
Procedure: The sample of 0.5 g was transferred to a refluxing flask. To this 100 ml of acid
detergent solution and 2 ml of decahydronaphthalene were added. This mixture was heated to
boiling and the heat was reduced to avoid foaming and refluxed for one hour. After one hour
of refluxing, the mixture was cooled and filtered through a previously weighed gooch
crucible of G-1 grade under suction using a vacuum pump. The sample in the crucible was
washed with hot water to remove acid followed by two washings with acetone. The crucibles
were dried at 1000C for 8 h in a hot air oven. After 8 h, the crucibles were cooled in a
desiccator and dry weight was recorded.
% ADF = Y - X × 100
W
Where,
Y: Weight of crucible + ADF
X: Weight of empty crucible
W: Weight of the sample
Acid Detergent Lignin
Procedure: Crucibles containing ADF (acid detergent fibre) were placed in 50 ml beaker and
the contents of crucibles were covered with cooled 72% H2SO4. The contents were stirred
with a glass rod to break lumps of residue if any. As the acid drain away the crucibles were
filled half way with acid and frequent stirring was done. After 3 h of intermittent stirring, the
contents were filtered off under suction to retain the residue and to remove acid using hot
water. Then the crucibles with residues were dried at 1000C for 8 h. After this, the crucibles
were cooled in a dessicator and weighed (L). After weighing, the contents in crucibles were
kept inside a muffle furnace for ashing at 5000C for 2h. After the furnace temperature came
down, the crucibles were taken out, cooled partially in air, then in a desiccator and the weight
(A) of the ash was recorded.
Where,
Y: Weight of ADF + crucible
L: Weight of crucible + lignin
A: Weight of crucible + ash
W: Weight of the sample
% Hemicellulose = % NDF - % ADF
% Cellulose = Y - L × 100
W
% Lignin = L - A × 100
W
3.17.2 Estimation of reducing sugars
The amount of reducing sugars was estimated by dinitrosalicylic acid (DNS) method (Miller,
1959).
Preparation of Reagent
DNS: One gram of 3, 5-dinitrosalycylic acid (DNS), 200 mg of crystalline phenol and 50 mg
of sodium sulphite were dissolved in 100 ml of 1% NaOH and was stored at 40C. As the
reagent deteriorates due to sodium sulphite, if long storage is required, sodium sulphite may
be added at the time of use.
Rochelle salt solution (40%)
It was prepared by dissolving 40 g of potassium sodium tartrate in 100 ml distilled water.
Preparation of stock solution of glucose
Standard stock solution having the concentration of 1 mg glucose/ml was prepared by
dissolving 100 mg of D-glucose in small amount of distilled water and final volume was
made up to 100 ml with distilled water.
Preparation of working standard
About 10 ml of the stock was diluted to 100 ml with distilled water in a 100 ml volumetric
flask to obtain the glucose concentration of 100 μg glucose/ml
Procedure: About 0.5 ml of sample was drawn from every treatment into test tubes. The
volume was made up to 3 ml using distilled water. DNS reagent of 3 ml was added to each
sample, mixed well. The reagent blank containing 3 ml of distilled water and 3 ml of DNS
reagent was also prepared. Similarly, standards were also included whose glucose
concentration ranged from 10 μg to 100 μg. All tubes viz., samples, standards and blank were
kept on boiling water bath for 5 min. After this one ml of 40 % Rochelle salt solution was
added when the reaction mixture was still warm. Then the tubes were cooled. The absorbance
in terms of optical density of the standards and sample were read at 510 nm using UV
spectrophotometer. The standard graph of glucose was plotted.
3.17.3 Estimation of ethanol
The ethanol was estimated calorimetrically as described by Caputi et al., (1968).
Preparation of reagents
Potassium dichromate (K2Cr2O7) 0.23 N: About 34 g of K2Cr2O7 was dissolved in 500 ml
of distilled water. To this 325 ml of concentrated sulphuric acid was added and the volume
was made up to 1000 ml with distilled water.
Preparation of stock solution : It was prepared by mixing 12.6 ml of analytical grade
ethanol (789 mg/ml) with little amount of distilled water and making up the volume to 100 ml
using distilled water, this gives 100 mg ethanol/ml.
Procedure: 3 ml of representative sample from each treatment was transferred to 250 ml
round bottom flask connected to the condenser and was diluted with 30 ml distilled water.
The sample was distilled at 74-750C. The distillate was collected in 25 ml of 0.23 N K2Cr2O7
reagent which was kept at the receiving end. The distillate containing alcohol was collected
till total volume of 45 ml was obtained. Similarly, standard (20-100 mg ethanol) were mixed
with 25 ml of K2Cr2O7 separately. The distillate containing alcohol was collected till total
volume of 45 ml was obtained. These, samples and standards were kept in water bath at 600C
for 20 min and were cooled. The volume was made up to 50 ml with distilled water and
optical density was measured at 600 nm using spectrophotometer. The standard curve was
plotted considering the concentration of ethanol against absorbance.