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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
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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
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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
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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).
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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.
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Figure 3.1 The bay of Khambhat – sample collection sites for halophilic microbes
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Figure 3.2 Hot water well of Lasundra – sample collection sites for thermophilic microbes
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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
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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
Incubation temperature : 45oC
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
Incubation temperature : 45oC
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.
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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.
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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
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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.
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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).
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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.
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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.
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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
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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.
5. After 12 hrs of incubation precipitates were collected by centrifugation at 10,000 rpm for 15
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.
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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.
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 again chilled ethanol was added upto 80% (v/v) and allowed to
precipitate for 12 hrs.
5. After 12 hrs of incubation precipitates were collected by centrifugation at 10,000 rpm for 15
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.
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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.
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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.
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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)
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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.
Reagents/requirements
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.
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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.
Reagents/requirements
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.
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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).
Reagents/requirements
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.
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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]
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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.
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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.
Reagents/requirements
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.
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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.
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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
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‘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.
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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
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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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