CHAPTER 3: MATERIALS ETHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29696/9/09_chapter3.pdf · APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS

ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR

APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS

MATERIALS AND METHODS | 60

CHAPTER 3: MATERIALS & METHODS

This chapter contains all the methods in detail used for the study. All chemicals were of AR grade if not otherwise mentioned. Methods are divided into five main sections: isolation of microbes, screening of microbes of interest, identification of microbes, methods used in biodiesel production and methods used in methanogenesis, building of a mobile and economic bioreactor




LIST OF MATERIALS USED IN THE STUDY

Chemical/Reagent Manufacturer Batch No.

Acetic acid SRL 012885

Acetone SRL 102365

Agar SRL T8351288

Ammonium sulfate SRL P929280

Bismuth ammonium citrate Otto B1256

Calcium chloride Merck MK8M573206

Carboxymethyl Cellulose CDH 01098

Chloroform Merck IL9I590414

DEAE cellulose CDH 23014

Dextrose SRL T8351378

Diethyl ether SRL TT534319

Dipotassium hydrogen phosphate Merck ME7M563235

Ethanol SRL 135216

Ethyl acetate Nice 802266

Fatty acid methyl ester standard Sigma LB51413

Ferric Chloride SRL T3251662

Ferrous chloride SRL T8351622

Glycerol tributyrate (Tributyrin) CDH A41007

Glycine SRL T8331337

Hexane Merck SG8S580489

Magnesium sulphate Merck MH3K12352

Methanol SRL 132977

Nutrient broth Himedia 0000098170

Peptone Merck MJ7M572373

pera-nitro phenyl palmitate Sigma 109K5200

Phenophthelin Merck MD9M583592

Phosphomolybdic acid Himedia 0000022692




Potassium chloride SRL T8331126

Potassium dihydrogen phosphate SRL T8331221

Sodium chloride Merck MG7M571532

Sodium deoxycholate SRL T1033363

Sodium EDTA Sigma 085K00291

Sodium hydroxide Merck MH8D580206

Sodium nitrate Merck C532154

Sodium Sulfite Merck MC4M540332

Sulfuric acid Merck CL0L600566

TLC silica gel G 60 Merck HX011552

Yeast Extract Otto Y1215

CDH: Central Drug House Pvt. Ltd, New Delhi, INDIA

Himedia: Himedia laboratories Pvt. Ltd., Mumbai, INDIA

Merck: Merck India Pvt. Ltd, Mumbai, INDIA

Otto: Otto chemie Pvt. Ltd, Mumbai, INDIA

Sigma: Sigma Aldrich Pvt. Ltd., USA

SRL: Sisco research laboratories Pvt. Ltd., Mumbai, INDIA




3.1 SAMPLE COLLECTION AND ISOLATION OF HALOPHILES AND

THERMOPHILES

Halophiles and thermophiles could be easily isolated from sea water and hot water spring

respectively. Two different sites located in the state of Gujarat were selected for sample

collection. These sites are “The Bay of Khambhat” for halophilic microorganisms and

“Lasundra”, a hot water spring for thermophiles. (Figure. 3.1 & 3.2) Samples were collected in

autoclaved glass bottles. Bottles were rinsed thrice with sample water before collection. Samples

were immediately transferred to the laboratory under cold condition and immediately proceeded

for experiment within 12hrs of collection. Isolation of microorganisms was carried out by spread

plate method after serial dilution.

3.1.1 SPREAD PLATE METHOD

Bacteria usually grow together in populations containing a number of species. In order to

adequately study and characterize an individual bacterial species, one needs a pure culture. The

spread plate technique is one of the most widely accepted methods to do this. In this technique, a

small volume of dilute bacterial mixture containing 100 to 200 cells or less is transferred to the

center of solid media containing plate and spreaded evenly over the surface with a sterile glass

rod known as spreader. After incubation at proper temperature for desired time, some of the

dispersed cells developed into isolated colonies. A colony is a large number of bacterial cells on

solid medium, which is visible to the naked eye as a discrete entity. In this procedure, one

assumes that a colony is derived from one cell and therefore represents a clone of a pure culture.

After incubation, the general form of the colony and the shape of the edge or margin can be

determined by looking down at the top of the colony. The nature of the colony elevation is

apparent when viewed from the side as the plate is held at eye level. After a well-isolated colony

has been identified, it can then be picked up and streaked onto a fresh medium to obtain a pure

culture (Prescott, 2002).




PROCEDURE

1. The entire procedure was carried out in a laminar air flow hood, wiped with 70% alcohol and

exposed to UV for 30minutes.

2. Serial dilutions of samples were prepared in sterile distilled water. Ratios that were obtained

as a result of serial dilutions were 1(Undiluted), 1:101, 1:102, 1:103, 1: 104, 1:105, 1:106,

1:107, 1:108, 1:109 and 1: 1010

3. 0.1 ml of each diluted samples were pipetted onto the centre of different media containing

petri plates i.e. nutrient agar for bacteria and GYE (glucose yeast extract) for yeast. These

plates are prepared by mixing agar to different media. Media containing agar was sterilized

and poured in sterile petri dishes and allowed to solidify. Plates were kept at 25ºC/45oC for

24 hrs to check the presence of any contamination.

4. Spreading was done using a spreader, which is a glass rod with a triangle end. The spreader

was dipped in 70% alcohol and sterilized by passing through a blue flame before spreading.

5. Spreading was done in a clockwise manner at least for 5 minutes with regular rotating of the

plate in one direction.

6. The spreader was sterilized each and every time before use on another plate.

7. The plates were incubated at different temperature (25oC for yeast and 45oC for bacteria) in

inverted position until visible colonies appeared on the plate.

8. The total number colonies present in the sample were counted keeping the dilution rate in

mind.




Figure 3.1 The bay of Khambhat – sample collection sites for halophilic microbes




Figure 3.2 Hot water well of Lasundra – sample collection sites for thermophilic microbes




3.2 SCREENING TECHNIQUES FOR SEPARATION OF MICROBES OF INTEREST

It is very difficult to study each and every isolated microorganism for its particular

characteristics. Screening methods which can differentiate between microbes of interest are

available. These mainly involve certain physical and chemical factors. Temperature, requirement

of oxygen and pH are most commonly used physical parameters, while chemical factors includes

media constituents (Ginalska et al., 2007; Sissons, Sharrock, Daniel, & Morgan, 1987).

For this study we were mainly interested in halophilic yeast and extracellular lipase and cellulase

producing thermophiles. Based on our interest three different media were selected. GYE (glucose

yeast extract) for screening of yeast, media containing only tributyrin as sole carbon source for

screening of extracellular lipase producing bacteria and media containing carboxymethyl

cellulase(CMC) as sole carbon source for screening of extracellular cellulase producing

microbes. CMC and tributyrin act as inducers for production of specific enzymes. Many previous

studies have used the similar procedure to cultivate as well as to screen the microbes from mixed

population (Ginalska et al., 2007; Joseph, Ramteke, & Kumar, 2006; Mingardon, Bagert,

Maisonnier, Trudeau, & Arnold, 2011; Sissons et al., 1987). For yeast isolation pH of medium

was kept on the slightly acidic side and the incubation temperature was maintained at 25oC while

for cellulase and lipase producing microbes the temperature was kept at 45oC.

Here is the composition of media of all the three media used for screening of microbes.

1. Glucose Yeast Extract (per liter) for yeast

Glucose : 20 gms

Yeast Extract : 10 gms

Peptone : 10 gms

pH : 6.0

Incubation temperature : 25oC




2. Media for screening of extracellular lipase producing microbes (per liter)

Tributyrin : 10ml

Magnesium sulphate : 0.2 gms

Calcium chloride : 0.02 gms

Monopotassium phosphate : 1.0 gms

Dipotassium phosphate : 1.0 gms

Ammonium nitrate : 1.0 gms

Ferric chloride : 0.05 gms

pH : 7.0


3. Media for screening of extracellular Cellulase producing microbes (per liter)

Carboxymethyl cellulose : 10 gms

Magnesium sulphate : 0.2 gms

Calcium chloride : 0.02 gms

Monopotassium phosphate : 1.0 gms

Dipotassium phosphate : 1.0 gms

Ammonium nitrate : 1.0 gms

Ferric chloride : 0.05 gms

pH : 7.0


All the media were autoclaved at 121oC at 15 lbs pressure for 15 minutes before use.

PROCEDURE

Similar procedure was also followed here for selective screening of microbes as mentioned in

isolation of microbes by spread plate method. But here the difference is use of selective

media rather than generalize media for cultivation of selected microbes.




Once the colonies of selected microbe are grown on the plates they were further spreaded on

new plates containing the same media to obtain pure culture. Once pure cultures were

obtained they were preserved on slants of the same media in a refrigerator at 4ºC for further

applications.




3.3 IDENTIFICATION OF MICROBES

It is a property of certain microbes when they are grown in media containing specific

constituents, they will react with the constituents and give some visible changes in colony

morphology or medium morphology. Based on these changes, it is possible to identify microbes

at primary level. The best example of this is the metallic sheen appearance of E.coli on

MacConky’s agar media (Prescott, 2002).

A similar kind of differentiating feature was observed in yeast when grown on media like

molybdate agar and BIGGY’s (Bismuth Sulfite Glucose Glycine Yeast Extract Agar) media.

Molybdate and bismuth present in the media were reduced by yeast in differentiating way and

resulted into differentia colony pigmentation. Based on these pigmentation yeast could be

identified (Atlas, 1993; Bump & Kunz, 1968; Rale and Vakil, 1984). Tthe composition of both

the media are given below.

Molybdate agar (per 101.5)

Base : 100.0mL

Phophomolybdic acid solution : 1.5mL

Agar : 2.5%

pH - 5.3±0.2 at 25oC

Composition of base (1000mL)

Sucrose : 40.0 gms

Agar : 15.0 gms

Meat peptone : 10.0 gms

pH was adjusted to 7.6 and autoclaved for 15 minutes at 15 psi at 1210C and cool it at 45-

50oC.

Composition of phosphomolybdic acid solution

P2O5.2OMoO3 : 12.5 gms




PMA was dissolved in sterile distilled water. Mixed thoroughly and without adjusting the

pH.

BiGGY’s Media (Nickerson media) (per liter)

Agar : 2.5%

Glucose : 10.0 gms

Glycine : 10.0 gms

Bismuth ammonium citrate : 5.0 gms

Na2SO3 : 3.0 gms

Yeast extract : 1.0 gms

pH – 6.8±0.2 at 25oC

PREPARATION OF MEDIA

All the compositions were added into 1 liter distilled water. It was mixed thoroughly and heated

with frequent agitation until boiling. It was then distributed in to sterile petri plates without

autoclaving.

All the isolated yeasts were streaked on both the differential media and incubated at 25oC for

48hrs. Resultant morphology was noted and compared with expected observations.

In order to confirm the identity of all the isolated microorganism 16s or 18s rRNA sequencing

was done and the sequences obtained were compare with the database available on NCBI. All the

sequences were submitted to NCBI and given universal numbers. The sequencing was carried

out at Gujarat State Biotechnology Mission (GSBTM) Laboratory, Gandhinagar, Gujarat. Slants

containing the pure microbial strains were submitted to the GSBTM for the sequencing.




3.4 TECHNIQUES/METHODS USED FOR BIODIESEL PRODUCTION

3.4.1 FORMULATION OF MEDIA FOR LIPID ACCUMULATION

It is known that yeast generally do not accumulate lipids in higher quantities under normal

conditions but they have the capacity to accumulate lipids under certain conditions. One of the

most common methods is providing metabolic stress. This stress could be provided by growing

yeast in media containing higher concentration of carbohydrates and lower concentration of

nitrogen (Gill, Hall, & Ratledge, 1977; Hall & Ratledge, 1977; C Ratledge, 2002). Under these

circumstances, yeast can only replicate for certain numbers of cycles, and after that replication is

inhibited and accumulation of glucose starts which is converted into lipids in the cell. This is

ascribed to the lack of enzyme production capability of the yeast in absence of nitrogen.

For this study, a specialized media was designed by us based on same principles of isolation

discussed above. The optimum concentration of carbon source, inorganic nitrogen source and

nitrogen source were determined to favour maximum lipid accumulation. Not only media

composition but also inoculum size and incubation times were also determined. In the

optimization experiments, different concentrations of each constituent were used and lipids were

extracted by modified Blight and Dyer method after specific incubation time.

3.4.1.1 EXPERIMENTAL SET UP FOR OPTIMIZATION OF CARBON SOURCE

Six different concentrations of glucose i.e. 30, 40, 50, 60, 70 and 80 grams/liter were added in

the media containing other components required for the growth of yeast. Media were inoculated

with 24hr old activated culture of yeast grown in GYE media. After incubation of 120hrs on a

rotary shaker at 200 rpm, biomass was collected by centrifugation at 8000 rpm for 15 minutes at

4ºC. Collected biomass was mixed with known quantity of sterile water to obtain uniform cell

suspension from which lipid was extracted by modified Blight and Dyer method (Blight & Dyer,

1959).




3.4.1.2 EXPERIMENTAL SET UP FOR OPTIMIZATION OF ORGANIC NITROGEN

SOURCE

Six different concentrations of yeast extract i.e. 40, 50, 60, 70, 80 and 90 mg/liter were added in

the media containing other components required for the growth of yeast. Media were inoculated

with 24hr old activated culture of yeast grown in GYE media. After incubation of 120hrs on a

rotary shaker at 200 rpm, biomass was collected by centrifugation at 8000 rpm for 15 minutes at

4ºC. Collected biomass was mixed with known quantity of sterile water to obtain uniform cell

suspension from which lipid was extracted by modified Blight and Dyer method.

3.4.1.3 EXPERIMENTAL SET UP FOR OPTIMIZATION OF INORGANIC NITROGEN

SOURCE

Six different concentrations of ammonium sulphate i.e. 10, 20, 30, 40, 50 and 60 mg/liter were

added in the media containing other components required for the growth of yeast. Media were

inoculated with 24hr old activated culture of yeast grown in GYE media. After incubation of

120hrs on a rotary shaker at 200 rpm, biomass was collected by centrifugation at 8000 rpm for

15 minutes at 4ºC. Collected biomass was mixed with known quantity of sterile water to obtain

uniform cell suspension from which lipid was extracted by modified Blight and Dyer method.

3.4.1.4 LIPID EXTRACTION METHOD (BLIGHT AND DYER METHOD)

PROCEDURE

Blight and Dyer method is one of the widely used methods for lipid extraction from cell

suspensions. It is a very simple and rapid method.

1. Cell suspension was prepared by suspending known quantity of biomass in known volume of

sterile distilled water.

2. To this suspension, mixture of chloroform: methanol (1:2) 3.75mL/mL of suspension was

added and the mixture was vortexed atleast for 15minutes.

3. Then 1.5mL of chloroform/mL of suspension was added and vortexed for 2 minutes.




4. In the next step, 1.5mL of distilled water/mL was added and vortexed for 2 minutes.

5. Two separate layers were obtained after centrifugation at 8000rpm for 10 minutes at 4ºC.

Lower solvent phase was collected and dried at room temperature.

6. The lipids extracted was weighed and suspended in a known volume of chloroform:

methanol (2:1) which was stored at 4 -8 ˚C for further analysis.

7. Lipid accumulation by yeast was determined on the basis of lipids extracted from a known

quantity of dry biomass.

3.4.2 DIRECT TRANSESTERIFICATION FOR BIODIESEL PRODUCTION

There are several methods available for transesterification which are mediated either by acids or

bases or enzymes (M. S. Antczak, Kubiak, Antczak, & Bielecki, 2009; Fukuda, Kondo, & Noda,

2001; Peter, Ganswindt, Neuner, & Weidner, 2002; Schuchardt, Sercheli, & Matheus, 1998).

Here, yeast biomass was directly used for transesterification; hence the method is also called

direct transesterification. In the process, yeast biomass produced in the nitrogen limiting media

as well as in normal media was collected by centrifugation and dried at 70oC until constant

weight was obtained. Then it was used for biodiesel production.

Several parameters were standardized for transesterification reactions. Major among them are

ratio of alcohol (Biomass : methanol - 1:5, 1:10, 1:15, 1:20, 1:25 and 1:30), concentration of acid

(Sulfuric acid – 0.1, 0.2, 0.3 and 0.4 mol/liter), incubation temperature (40oC, 50oC, 60oC, 70oC

and 80oC) and reaction time (6hrs, 12 hrs, 18 hrs, 24 hrs and 30 hrs). As a result of

standardization, a method was set up which gives optimum production of FAMEs.

PROCEDURE

1. In this process, dried yeast biomass and methanol was mixed in a ratio of 1:20 (w:v)

containing concentrated sulfuric acid to a final concentration of 0.02 moles/L.

2. The reaction was carried out in a tightly closed glass bottle at 70˚ C with constant stirring for

24 hrs. The glass bottle was rinsed once with methanol before using for the reaction.




3. At the end of the reaction, suspension was cooled and filtered with whatman no 1 filter paper

to remove all solid impurities in the mixture.

4. To the filtrate, 1/3 volume of hexane was added and mixed vigorously to recover the

FAMEs.

5. The upper hexane layer containing FAMEs was collected in separate tubes and used for

various analyses. When not used immediately it was stored at 4oC - 8oC in a close vessel.

3.4.3 EXTRACELLULAR LIPASE PRODUCTION AND PURIFICATION

Microorganisms isolated on tributyrin agar were used for extracellular lipase production. The

initial rate of production was determined by plate assay in which activity of lipase was

determined by zone of clearance of tributyrin on agar plates (Prescott, 2002; Singh, Gupta,

Goswami, & Gupta, 2006). Based on this activity a few strains giving higher lipase activity were

selected for the experiments. Tributyrin agar plates were prepared by mixing 1% tributyrin in

50ml of Bushnell Haas media containing agar powder 2.5%.

PROCEDURE

1. Selected strains were grown in tributyrin broth (Appendix A) at 55oC on a rotary shaker at

120 rpm.

2. Aliquots of the media were taken at regular intervals for determination of lipase activity.

Lipase activity was measured by pNPP assay.

3. At a particular time when the activity of the enzyme was maximum, the broth was collected

and processed for enzyme extraction and purification using different methods.

3.4.3.1 LIPASE EXTRACTION

Salting out is one of the most common and widely accepted methods for precipitation of proteins

(Kashmiri, Adnan, & Butt, 2006; Saxena, Davidson, Sheoran, & Giri, 2003; Saxena, Sheoran,

Giri, & Davidson, 2003; Sinchaikul, Sookkheo, Phutrakul, & Pan, 2001; Zheng-yu, Jiang-ke, &

Yun-jun, 2007). Enzymes being protein in nature could be easily precipitated out from the broth




by using different salts. In this experiment ammonium sulphate and alcohol were used to

precipitate proteins.

PROCEDURE

3.4.3.1.1 AMMONIUM SALT PRECIPITATION

1. At a particular time when pNPP activity of the medium was maximum; all the cells were

removed from the broth by centrifugation. Centrifugation was done at 8000 rpm for 15 min at

4oC.

2. After centrifugation, broth was collected and benzimidine(2mM) and sodium azide (0.02%)

was added to enhance the enzyme stability. After that ammonium sulphate was added upto

40% (w/v) and allowed to precipitate for at least 4 hrs at 4oC.

3. After 4 hrs of incubation precipitates were collected by centrifugation at 10,000 rpm for 15

minutes at 4oC.

4. To the remaining supernatant ammonium sulphate was added to 80% (w/v) and allowed to

precipitate for 12 hrs.


minutes at 4oC.

6. Precipitates were dissolved in phosphate buffer pH: 7.2 (100mM) containing

benzimidine(2mM), EDTA(2mM) and sodium azide (0.02%).

7. Salt particles were removed by filtering through molecular weight cutoff filters (MWCO) at

4ºC. Filter that was used for filtration was of 3kD.

8. As a result of filtration, all the salt particles were removed from the buffer and activity of

lipase was determined by pNPP.

3.4.3.1.2 ALCOHOL PRECIPITATION

This process is almost similar to the salt precipitation method. Here instead of salt, ice cold

ethanol was used for precipitating proteins.




1. At a particular time where pNPP activity of the medium was maximum; all the cells were

removed from the broth by centrifugation. Centrifugation was done at 8000 rpm for 15 min at

4oC.

2. After centrifugation, broth was collected and benzimidine(2mM) and sodium azide (0.02%)

was added. To the broth, chilled ethanol was added upto 40% (v/v) and allowed to precipitate

for at least 4 hrs at 4oC.


minutes at 4oC.

4. To the remaining supernatant again chilled ethanol was added upto 80% (v/v) and allowed to

precipitate for 12 hrs.


minutes at 4oC.

6. Precipitates were dried and stored as crude enzymes particles or dissolved in phosphate

buffer pH: 7.2 (100mM) containing benzimidine(2mM), EDTA(2mM) and sodium azide

(0.02%) for determination of lipase activity.

This method enabled storage of the crude lipase in precipitated form for longer period of time at

low temperature.

3.4.3.2 LIPASE PURIFICATION

Extracellular lipases produced by microorganisms were purified using different techniques.

Broth was considered as crude source of lipase from which lipase was extracted and purified.

Ammonium salt precipitation and alcohol precipitation was used for primary extraction and

purification as mentioned above. Affinity chromatography was further performed as done is

other studies to increase the purity of enzyme (Ferrer, Plou, Nuero, Reyes, & Ballesteros, 2000;

Kambourova, Kirilova, Mandeva, & Derekova, 2003; Saxena, Sheoran, et al., 2003; Snellman,

Sullivan, & Colwell, 2002; Zheng-yu et al., 2007). The process used here is discussed below.




3.4.3.2.1 AFFINITY CHROMATOGRAPHY

Separation of molecules in affinity chromatography depends on the affinity of molecule towards

the stationary phase used. Agarose, silica gel and DEAE sepharose are most commonly used

stationary phase. Affinity of molecules could be changed by changing salt concentration, pH, pI

and ionic strength (R Gupta, Gupta, & Rathi, 2004; Kordel, Hofmann, Schomburg, & Schmid,

1991; Saxena, Davidson, et al., 2003; Saxena, Sheoran, et al., 2003).

PROCEDURE

1. For affinity chromatography DEAE cellulose was used as a stationary material. The column

was prepared in a fiber glass column available in the market.

2. DEAE cellulose was first soaked in phosphate buffer [pH: 7.2 (100mM) containing

benzimidine(2mM), EDTA(2mM) and sodium azide (0.02%)] for 2 hrs to prepare slurry for

filling in the column.

3. The Column was filled slowly with DEAE cellulose slurry with regular taping of the column

from the outside to remove air bubbles produced during pouring of the column. It is very

essential to remove air bubbles as it can affect the separation process.

4. After packing of the column, it was washed with the same buffer for atleast 1 hr at a flow rate

of 10ml/min.

5. Crude samples precipitated by ammonium salt were loaded on the top of the column after

removal of salt and elution was carried out by salt gradient prepared by mixing different

concentration of NaCl in phosphate buffer (0-0.5M).

6. Similarly samples precipitated by alcohol were loaded on the top of the column and elution

was carried out by salt gradient prepared by mixing different concentration of NaCl in

phosphate buffer (0-0.5M).

7. Fractions of 5 ml were collected in different tubes and lipase activity was determined for

each tube. Tubes which gave activity were pooled.

8. Samples were preserved at low temperature for further analysis or applications.




3.4.4 IMMOBILIZATION LIPASE ENZYME

Enzymes could be used in free as well as in immobilized form for particular types of reactions.

When free enzymes were used in a reaction, it was very difficult to recover the enzymes from the

reaction mixture resulting in loss of the enzymes. This affects economically feasibility for certain

enzymes which are expensive or available in lesser quantities. Immobilization is a technique in

which enzymes are bound on an inert surface. There are several types of immobilization i.e.

adsorption, covalent immobilization, entrapment and microencapsulation (Knežević, Šiler-

marinković, & Mojović, 2004; Lee, Park, Yeo, & Kim, 2006; Minovska, Winkelhausen, &

Kuzmanova, 2005). Each of these methods has certain advantages and disadvantages. For this

experiment, entrapment method was selected in which enzyme purified by column

chromatography as well as microorganisms themselves were immobilized on calcium alginate

beads. Method used by Devanesan et al was used here which is explained as below (Devanesan,

Viruthagiri, & Sugumar, 2007).

IMMOBILIZATION OF ENZYME BY ALGINATE

PROCEDURE

1. The sodium alginate entrapment of cells was performed according to the standard method.

Alginate solution with a concentration range of 2.0% was used for the cell immobilization

and was prepared by dissolving sodium alginate in warm water

2. The solution was sterilized by using an autoclave at 121ºC for 15 min at 15 lbs pressure.

3. Either a cell suspension or preparation of specific quantity of enzyme was mixed with

alginate slurry and stirred for 10 min to get a uniform mixture of the alginate; cell

mixture/enzyme combination.

4. This mixture was extruded drop by drop into a cold sterile 0.2 M CaCl2 solution through a

sterile 5 ml syringe from height of 5 cms and kept for curing at 4ºC for 1 h.

5. The beads were hardened by resuspending into a fresh 0.2 M CaCl2 solution for 24 h at 4ºC

with gentle agitation.




6. Finally these beads were washed with distilled water to separate excess calcium ions,

unbound enzymes and unentraped cells.

7. When the beads were not being used, they were preserved in 0.9% sodium chloride solutions

in the refrigerator.

3.4.5 ENZYME ACTIVITY DETERMINATION BY DIFFERENT METHODS

The activity of lipase enzyme can be determined by various methods. Each method follows a

different principle. Some of them are highly sensitive methods while some are helpful in

preliminary determinations of enzyme activity. Lipase activity can be determined by titrametric

assay, turbidometric assay, spectrophotometric assay (pNPP assay), gas chromatography and

zymography (Bishop & Shihabi, 1971; Dharmsthiti & Luchai, 1999; Goujard et al., 2009;

Kulkarni & Gadre, 1998). Among all these techniques, pNPP assay and titrimetric assay were

used in this study to determine the activity of lipase.

3.4.5.1 TITRIMETRIC ASSAY

This assay is a kind of acid base titration assay. Degradation of tributyrin releases butyric acid

which reduces the pH of the medium. This could be neutralized by titrating with 0.05N NaOH.

Phenophthelin is used as an indicator. Enzyme activity was determined by the NaOH utilized for

neutralization, as one molecule of butyric acid will be neutralized by one molecule of NaOH.

Reagents/requirements

Tributyrin

Phosphate buffer- 0.1M, pH 7.2

0.05N NaOH

Acetone:ethanol mixture (1:1)

Enzyme solutions (standard- 10mg/ml and crude enzymes)

Phenolphthalein (0.2% w/v in ethanol)




PROCEDURE

1. The assay mixture was prepared by mixing 2.5 ml of tributyrin, 3.5 ml of phosphate buffer

(0.1 M; pH 7.0) and incubated at 55oC for 10 minutes.

2. 200µl of enzyme was added in the mixture and incubated for 30 min at 55oC with constant

stirring at 200 rpm.

3. The reaction was terminated by adding 10 ml of acetone:ethanol (1:1).

4. Amount of liberated fatty acids was tittered with 0.05 N NaOH the in the presence of 20µl of

phenophthalein (0.2% w/v in ethanol) as indicator. Pink colour appearance indicates

completion of titration.

5. One unit of enzyme activity was defined as mmoles of free fatty acids released per ml per

min under the assay conditions.

3.4.5.2 SPECTROPHOTOMETRIC ASSAY (pNPP ASSAY)

pNPP (p-nitrophenyl palmitate) assay is one of the most common and most widely used method

for determination of lipase activity . In this method lipase degrades p-nitrophenyl palmitate to p-

nitrophenyl, which gives yellow colour to the solution which could be measure at 405nm. The

intensity of the colour produced is directly proportional to the enzyme activity.


p-nitro phenyl palmitate

Gum arabic

Sodium deoxycholate

Phosphate Buffer : 0.1M, pH 8.8

Substrate preparation: Phosphate buffer (90ml) + gum arabic (100mg) + sodium deoxycholate

(207 mg) along with 30mg of pNPP (dissolved in 10ml of isopropanol)

PROCEDURE

1. 3 ml of substrate solution was pipetted into a cuvette and warmed at 55oC for 10 minutes.




2. 10µl of enzymes were added in the cuvette and mixed by inverting.

3. Absorbance was read at 405nm at regular intervals of 1 minute.

4. Activity of enzyme was calculated by comparison with standard lipase which was define as

the amount of enzyme releasing 1 µmol pNP per minute per mL under assay condition.

3.4.6 ENZYMATIC PRODUCTION OF BIODIESEL

In another process for biodiesel production, enzymes were used for transesterification. It is

known that enzymes in free as well as immobilized form could be used for biodiesel production

and the reaction condition used for enzyme are very mild compare to other processes. Here,

purified lipase was used in free as well as immobilized form for transesterification. In the

experiment, ethyl acetate was used as acyl acceptor instead of alcohol as it was found that

enzyme activity could be inhibited by certain alcohols (M. S. Antczak et al., 2009; Nielsen,

Brask, & Fjerbaek, 2008; Vieira, Silva, & Langone, 2006). Several other parameters like

optimum ratio of ethyl acetate : oil, optimum reaction time, optimum enzyme concentration were

also standardize.


Peanut oil

Free/immobilized lipase

Ethyl acetate

PROCEDURE

1. Mixture of ethyl acetate : oil (11:1) was taken in a dry glass bottle and 10% (w/v) enzyme

was added to the bottle.

2. The mixture was incubated at 55oC with constant stirring at 200 rpm for 12 hrs.

3. In case of immobilized lipase the mixture was filtered to collect the beads. Beads were

washed with distilled water and stored in buffer at low temperature for reuse.

4. To the reaction mixture hexane was added and two phases were allowed to separate.

5. Upper hexane phase containing biodiesel was collected for further analysis.




3.4.7 HPTLC ANALYSIS

HPTLC was preformed to confirm the presence of fatty acid methyl ester. It works on the

principle of adsorption and partition. Many previous studies have shown that non-polar

compounds could be easily separated out by using non-polar solvent systems (Freeman & West,

1966; Morris, 1966). So, only non polar solvent systems were used for separation of the non

polar compounds. High performance thin layer chromatography (HPTLC) was done on CAMAG

V (Germany).


Silica gel G 254 plate

Hexane

Diethyl ether

Phopshomolybdic acid (5% in ethanol)

Ethanol

PROCEDURE

1. In the very first step the chamber was saturated with solvent system. For this 40mL of

solvent mixture (Hexane:diethyl ether – 9:1) was added to the chamber and allowed to

saturate atleast for 45 minutes.

2. On a clean dry silica gel G254 plate produced biodiesel as well as lipid extracted from yeast

were loaded using the CAMAG V applicator.

3. Plates were dried with drier before transfer to the chamber for development.

4. Plates were kept in the chamber and allowed to move with the solvent to develop the plate up

to a certain height in ascending chromatographic mode.

5. After development plates were dried again and spread with phosphomolybdic acid solution.

6. Plates were allowed to develop by keeping at 105oC.

7. Once the spots were visualized plates were scanned by CAMAG scanner at 546nm.




3.4.8 INFRARED SPECTROPHOTOMETRIC ANALYSIS

Infrared spectrophotometry analysis helps in interpretation of functional groups present in the

compound based on the different type of movement which occurs upon exposure of laser light.

Biodiesel has mainly two functional groups i.e. ester (C=O) and alkyl (C-H). IR analysis was

carried out using an IR affinity-I instrument (FTIR), Shimadzu, Japan. An additional component

DRS 8000A was used for determination of IR. This component gives better results and need not

require sample preparation.

PROCEDURE

1. IR instrument was put on and checked for the humidity.

2. Background scanning was done to avoid any background peak while scanning for samples.

3. IR of the samples and standard were taken using DRS 8000A.

4. The results obtained were compared with FAMEs standard and purity was determined.

3.4.9 GAS CHROMATOGRAPHIC ANALYSIS

Presence of fatty acid methyl/ethyl ester was confirmed by gas chromatographic analysis. It was

also used to determine the composition and concentration of fatty acid esters in the produced

biodiesel. Gas chromatographic analysis was also done for comparative analysis of biodiesel

composition produced by yeast grown in nitrogen limiting media and in normal media. Here a

non-polar capillary column was used which gives separation on basis of boiling point as well as

degree of unsaturation. Unsaturated fatty acid esters elutes first as compare to their respective

saturated fatty acids. Gas chromatographic analysis was carried out using a GC-2014 instrument,

Shimadzu, Japan.

PROCEDURE

1. GC instrument were put on and all the parameters were set for the analysis. [Restek 1

capillary column 15 meters, Column gradient 150ºC - 270oC FID Detector, detection temp.

300˚C, injection volume-1 µL with spilt ratio 1:10]




2. The instrument was allowed to stabilize for 30 minutes before injection of samples/standards.

3. 1µl of sample was injected for the analysis and instrument was allowed to analyze the

sample.

4. The obtained data were compared with external standards (FAMEs standards) for

determination of composition and concentration of produced biodiesel.




3.5 TECHNIQUES/METHODS USED FOR METHANOGENESIS

3.5.1 ADAPTATION OF MICROBES FOR CELLULASE PRODUCTION

Microorganisms isolated on CMC agar were used for extracellular cellulase production. Rate of

production was determined by release of glucose monomer which was measure by DNS assay.

Based on this activity a few strains giving higher cellulase activity was selected for the

experiment.

PRODUCTION OF CELLULASE

Production media was the same as the screening media.

PROCEDURE

1. Selected strains were grown in CMC broth (Appendix A) at 55oC on a rotary shaker at 120

rpm.

2. Aliquots of the media were taken at regular interval for determination of cellulase activity.

3. DNS assay was performed to determine the concentration of librated glucose in the media.

3.5.2 DINITOSALICYLIC ASSAY FOR DETERMINATION OF CELLULASE

ACTIVITY

Reducing sugars have the property to reduce many of the reagents. One such reagent is 3,5-

dinitrosalicylic acid (DNS). 3,5-DNS in alkaline solution is reduced to 3 amino 5 nitro salicylic

acid.


Reagent 1: Sodium potassium tartrate: 45 gms of sodium potassium tartrate was dissolve in 75

mL of H2O.

Reagent 2: 3,5-DNS solution: 1.5 gm of DNS reagent was dissolve in 30 mL of 2 M/liter NaOH.




2 molar NaOH: 80 gms of NaOH was dissolved in 1 liter of water.

DNS reagent was prepared fresh by mixing the reagents (1) and (2) to make up the volume to

150 mL with water.

Standard sugar solution:

(i) Stock standard sugar sodium: 250 mg of glucose in water to make up the volume to 100 mL.

(ii) Working standard sodium: 10 mL from the stock solution was taken and volume was made

upto 100 mL.

PROCEDURE

1. Standard sugar solution was pipetted out in the range of 0 to 3 mL in different test tubes

and volume was made to 3mL in all test tubes with distilled water to obtain different

concentrations of standard ranging from 0 to 750 mg. 1 mL of the aliquots of digested

cellulose by cellulase enzyme was taken as sample.

2. 1 mL DNS reagent was added in to all the test tubes and mixed. Marble were kept to

avoid evaporation and test tubes were kept in a boiling water bath for 5 minute.

3. Tubes were cooled down and the optical density was measured at 540 mm against the

blank.

4. Standard curve was prepared and used to estimation the concentration of the unknowns.

5. Activity of enzyme was calculated based on glucose liberated by the digestion of

cellulose.

6. One unit of enzyme activity was defined as mmoles of glucose released per ml per min

under the assay conditions.




3.5.3 A SPECIAL APPARATUS FOR METHANOGENESIS DESIGNED IN THE

LABORATORY

A special apparatus was designed by us in our laboratory to study the rate of methanogenesis

as well as to determine the effect of different parameters on the rate of methanogenesis. Design

of the apparatus is shown in the figure below.

B

B

C D A

Figure 3.3 Apparatus designed in our laboratory for determination of biogas production




‘A’ is methanogenesis vessel (fermentor chamber), filled with slurry,

‘B’ is inlet of apparatus for addition of components if required later,

‘C’ is the gas collection vessel, filled with coloured fluid whose displacement indicates

the amount of gas produced (in mL)

‘D’ is the effluent tube for removal of excess fluid.

All the parts of the apparatus were of international standard quality. This apparatus works on a

very simple principle of displacement of fluid. Biogas which is produced in the fermentor

chamber by methanogenesis will go to the collecting vessel where it creates pressure on the fluid

kept in the collecting vessel. Because of this pressure, fluid from the collecting vessel will be

displaced from the vessel and this displacement is directly proportional to the biogas produced in

mL during methanogenesis at normal atmospheric pressure. Before using the apparatus for any

experiment, all the junctions were checked properly to prevent leakage of gas produced during

methanogenesis.

Advantages of apparatus

1) It works on a very simple phenomenon

2) It is very economic

3) Construction and application is very easy

4) It gives highly accurate results under standard conditions.




3.5.4 EFFECT OF DIFFERENT ENVIRONMENTAL FACTORS (ABIOTIC AND

BIOTIC) ON METHANOGENESIS RATE

During the study several other parameters were also studied to determine the effect on rate of

methanogenesis. These factors include certain biotic and abiotic factors like addition of

microorganisms, effect of partial digestion and undigested cellulose, effect of temperature.

Effects of individual parameter and different combinations of parameters were also studied to

determine the effect on production rate.

PROCEDURE

3.5.4.1 TO DETERMINE THE EFFECT OF UNDIGESTED AND PARTIALLY

DIGESTED CELLULOSE

Partial digestion of cellulose was carried out using thermophilic cellulase producting

bacteria by incubating them with plant extract prepared in sterile water (10%) at 55oC at

120 rpm for 72 hrs. Extract prepared in sterile water was taken as undigested feedstock.

In two different vessel designed for methanogenesis, mixture of sterile water, cow dung

and either partially digested or undigested cellulose was fed and incubated at 55ºC for 30

days. Production rate was determined by the displacement of water in the collecting

vessel.

3.5.4.2 TO DETERMINE THE EFFECT OF EFFECT OF TEMPERATURE

Experiments were carried out at three different temperatures of 37ºC, 45ºC and 55ºC.

Vessels containing cowdung and partially digested cellulose was maintained at

mentioned temperatures and incubated for 30 days. Production rate was determined by

the displacement of water in the collecting vessel.

3.5.4.3 TO DETERMINE THE EFFECT OF EFFECT OF MICROORGANISMS

Five different extracellular cellulase producing strains were used to study the effect of

microorganisms on rate of methanogenesis. In these experiments, vessels containing




cowdung and partially digested cellulose were fed with culture of different

microorganisms and incubated for 30 days at 55ºC. Production rate was determined by

the displacement of water in the collecting vessel.

3.5.5 GAS CHROMATOGRAPHIC ANALYSIS

Varieties of columns and detectors can be used for determination of composition of biogas.

WLOT and PLOT are amongst the top priority in the columns while TCD and FID are most

preferable detectors. Detector should be very sensitive as many of the produced gases have lower

concentration in ppm or less than that. Here, for gas chromatography of biogas HP-PLOT Q

column was used. Helium was used as carried gas. TCD detector was used to detect the presence

of different gases.

PROCEDURE

1. GC instrument were put on and all the parameters were set for the analysis. [HP PLOT Q

column, Column gradient 60ºC - 240oC, TCD Detector, detection temp. 250˚C, injection

volume-0.25cc with spilt ratio 1:20]

2. Instrument was allowed to stabilize for 30 minutes before injection of samples/standards.

3. 0.25cc of sample was injected for the analysis and instrument was allowed to analyze the

sample.

4. Obtained data were compared with standard.




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CHAPTER 3: MATERIALS ETHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29696/9/09_chapter3.pdf · APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS