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).


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


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.


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


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.


1. Potato 200.0

2. Dextrose 20.0

3. Yeast extract 0.1

4. Agar-Agar 20.0

5. pH 5.0


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.



2. Malt extract 3.0

3. Peptone 5.0

4. Glucose 10.0

5. Agar-Agar 20.0

6. pH 6.0-6.4


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.



2. KH2PO4 2.0

3. Glucose 20.0

4. Agar-Agar 20.0

5. pH 6.0


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)


2. K2HPO4 5.0

3. NaCl 1.0

4. MgSO4 .7H2O 0.2

5. Peptone 2.0

6. CMC 5.0


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)

.



2. K2HPO4 5.0

3. NaCl 1.0

4. MgSO4 .7H2O 0.2

5. Peptone 2.0

6. CMC 5.0


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)



2. K2HPO4 5.0

3. NaCl 1.0

4. MgSO4 .7H2O 0.2

5. Peptone 2.0

6. Pretreated rice straw 5.0


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).



2. K2HPO4 5.0

3. NaCl 1.0

4. MgSO4 .7H2O 0.2

5. Peptone 2.0

6. Pretreated rice straw 5.0


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


Table 3.12 Chemical composition of Mineral Salt medium I.


1. (NH4)2SO4 10.0

2. KH2PO4 4.0

3. CaCl2 0.5

4. MgSO4.7H2O 0.5

5. pH 8.0


3.3.2.12 Mineral Salt medium II


for maximum titre of cellulolytic enzyme under SSF by Bacillus sp. 313SI and has the


Table 3.13. Chemical composition of Mineral Salt medium II.


1. Na2HPO4 1.10

2. NaH2PO4.2H20 0.61

3. KCl 0.30

4. MgSO4.7H2O 0.01

5. pH 8.00


3.3.2.13 Mineral Salt medium-III


for maximum titre of cellulolytic enzyme under SSF by Aspergillus niger BK01 and has the


Table 3.14 Chemical composition of Mineral salt medium-III.


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


3.3.2.14 Mandel and Sternburg’s medium (1976)


for maximum titre of cellulolytic enzyme under SSF by Aspergillus niger BK01 and has the


Table 3.15 Chemical composition of Mandel and Sternburg’s medium.


1. KH2PO4 2.0

2. Urea 0.3

3. MgSO4.7H2O 0.3

4. CaCl2 0.3

5. Peptone 0.75


7. Trace element solution 1.0 ml

8. pH 5.0


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


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


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


% 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.

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