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
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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
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 61
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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APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 62
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
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 63
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).
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 64
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.
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 65
Figure 3.1 The bay of Khambhat – sample collection sites for halophilic microbes
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 66
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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APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 68
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.
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
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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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APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 70
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
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 71
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.
ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR
APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS
MATERIALS AND METHODS | 74
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,