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CHAPTER III

MATERIALS AND METHODS

3.1 Microalgae-Source and Culture conditions

Chlorella salina and Nannochloropsis oculata were obtained from Central Marine

and Fisheries Research Institute (CMFRI), Tuticorin, Tamilnadu (India).

A). 1 L of Culture B). 10 L of Culture

C). 22 L of Culture D). 200 L of Culture

Fig. 3 Various stages of inoculum of marine microalgae developed in

Bioelectrochemical Laboratory, Annamalai University

Chlorella salina and Nannochloropsis oculata were grown in sterile Walne’s

medium. The filtered sterilized sea water was enriched with required quantity of Walne’s

medium composition containing (gL-1): NaNO3, 100; NaH2PO4·2H2O, 20.0; Na2EDTA,

4.0; H3BO3, 33.6; MnCl2·4H2O, 0.36; FeCl3·6H2O, 13.0; vitamin B12, 0.001 and vitamin

B1, 0.02. The trace metal solution contained (gL-1

): ZnSO4·7H2O, 4.4; CoCl2·6H2O, 2.0;

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(NH4)6Mo7O24·H2O, 0.9; and CuSO4·5H2O, 2.0. The medium was adjusted to pH 8 and

autoclaved at 121ºC for 20 min. The filter sterilized vitamins were added after cooling.

The contents were later introduced into a 250-ml Erlenmeyer flask and finally transferred

to 25L photobioreactor (PBR). Mixing was provided by sparging air from the bottom of

the PBR; lighting was supplied by four cool-white fluorescent tubes with an intensity of

5000 lux under 12/12 light/dark cycle for 15 days. The culture medium was shaken every

2 hours to avoid sticking of microalgae to the surface of the photobioreactor.

3.2 Microscopic study of Marine Microalgae

Contamination of microalgae was frequently checked by microscopy. A volume

of 2 ml of microalgae culture were taken from Photobioreactor (PBR) and were

centrifuged at 3000 rpm for 5 minutes. After centrifugation was done, the supernatant

was discarded and the pellet containing cells were made into a thin smear by spreading

over a clean slide. The slide was air dried and observed under Light Microscope (Model-

Olympus CH20i BIMF, Olympus India Pvt., India) at 100X magnification by using oil

immersion.

3.3 Study of Microalgal Growth Parameters

3.3.1 Effect of Illumination Time on Microalgal Growth

The effect of illumination time was investigated in the ranges of 10:14, 12:12 and

14:10 light and dark cycle for microalgae growth. The light intensity (lux) was measured

using optometer.

3.3.2 Effect of pH on Microalgal Growth

For the effect of pH on microalgal growth, the culture medium was adjusted to

three different pH levels such as 7, 8 and 9 using 1M HCl and 1M NaOH. The pH

adjustment should be carried before autoclaving culture medium.

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3.3.3 Effect of Nitrogen on Microalgal Growth

For the effect of nitrogen on microalgal growth one reactor was filled with

nitrogen sufficient Walne’s medium and another medium supplied with nitrogen for first

4 days and after that add the nutrients medium containing medium without nitrogen for

scale up to 200 L. The culture conditions were pH 8, 12:12 h light and dark cycle for 15

days.

3.4 Analysis of Intracellular Molecules under Nitrogen Repleted and Depleted

Growth

3.4.1 Estimation of Chlorophyll a

The chlorophyll a content (mg/L) was estimated according to Su et al. (2007).

Two milliliter of culture broth was taken in centrifuge tube, ultrasonicated for 10 min in

ice bath with two milliliter of 90% methanol over night. Then the homogenate was

centrifuged at 3000 rpm for 5 min. The supernatant was separated and absorbance was

read at 665nm and the amount of chlorophyll was calculated using following formula;

Chlorophyll a (mg L-1) = 13.43 x OD665

3.4.2 Estimation of Protein by Lowry’s method

Materials required

Stock : Bovine Serum Albumin (BSA) (1mg/ml)

Working std : 10 ml stock / 100ml distilled water.

A : 2 % Sodium Carbonate in 0.1 N NaOH

B : 0.5 % CuSO4. 5H2O in 1 % Sodium potassium tartarate

C : Alkaline Copper Solution: 50 ml A: 1ml B; freshly prepared-

prior to use

D : Folin – Ciocalteau reagent

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Principle

The phenolic group of tyrosine and trytophan residues (amino acid) in a protein

will produce a blue purple color complex, with maximum absorption in the region of 660

nm wavelength, with Folin-Ciocalteau reagent which consists of sodium tungstate

molybdate and phosphate. Thus the intensity of color depends on the amount of these

aromatic amino acids present and will thus vary for different proteins. Most proteins

estimation techniques use Bovine Serum Albumin (BSA) universally as a standard

protein, because of its low cost, high purity and ready availability. The method is

sensitive down to about10 �g/ml and is probably the most widely used protein assay

despite being only a relative method subjected to interference from Tris buffer, EDTA,

nonionic and cationic detergents, carbohydrate, lipids and some salts.

Procedure

Aliquots of 0.2, 0.4, 0.6, 0.8, and 1ml of working standard solutions were added

into each test tube. An aliquot of 0.2 ml of test solution was added into another tube. The

value was made up to 1ml using distilled water. A tube with 1ml of distilled water served

as blank. To each test tube, 5ml of reagent C was added. The solution was allowed to

stand for 10 min. Then 0.5 ml of reagent D was added to tubes and incubated at dark in

room temperature for 30 min. The absorbance was read at 660 nm using a

spectrophotometer.

3.4.3 Estimation of Total Carbohydrate (Dubois et al., 1956)

Materials required

5% phenol, 96% H2SO4, Glucose (stock: 1mg/ml; working standard: 10ml stock

in 100ml of distilled water)

Principle

In hot acidic medium, glucose is dehydrated to hydroxymethyl furfural. This

forms coloured product with phenol and has a maximum absorption at 490nm.

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Procedure

Aliquots of 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard solution were added

into a series of test tubes. Test solution of 0.2 ml volume was pipette out into another

tube. The volume of all tubes was made up to 1ml with distilled water. The blank was set

with 1 ml distilled water. A volume of phenol (5%) was added to each tube and shaken

well. The tubes were then left in a water bath (30ºC) for 10 min. The tubes were cooled

and optical density was read spectrophotometrically at 490nm.

3.4.4 In vitro Identification of intracellular lipid by Nile Red Staining

It is a specific stain to identify intracellular lipids present in biological samples. A

stock solution of Nile Red stain (9-diethylamino-5H-benzo (�) phenoxa-phenoxazine-5-

one) was prepared according to Mohammady et al. (2012). A quantity of 2.5 mg of Nile

Red was dissolved in brown bottle containing 100 ml of acetone and this was stored at

dark. Each 0.5 ml of microalgae culture broth (both nitrogen rich and nitrogen depletion)

was centrifuged at 1500 rpm for 10 minutes and the pellets were washed with sterile

distilled water (equal volume) for several times. The cell pellets were then mixed with 0.5

ml of Nile Red solution incubated for 10 min at room temperature. After washing with

distilled water, the stained cells observed under fluorescence microscopy.

3.4.5 Fourier Transform Infrared Spectroscopy Analysis

A quantity of 50mg of dried biomass was taken, mixed with 150mg of KBR

powder and ground well to fine mixture. The mixture was pressed to a disc using a

hydraulic press. The disc was subjected to FTIR spectral measurement in the frequency

range of 4000-400cm-1. The algal powder was characterized using a Fourier Transfer

Infrared Spectrophotometer (Bruker Optics, GmBH, Germany).

3.5 Harvesting of Microalgae using Chemical Flocculation

Flocculation experiments were carried out in stationary growth phase of

microalgae. All experiments were conducted in glass tubes (size 25x150, capacity 50 ml)

with 50 ml testing volume containing microalgae and flocculants. Eight different

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flocculants were used (AlCl3, Al2 (SO4)3, FeCl3, Fe2 (SO4)3, ZnSO4, ZnCl2, MgSO4 and

MgCl2), at varying dosages which ranged from 0.2 g L-1

to 1.0 g L-1

with the step of 0.2 g

L-1. The flocculation efficiency was measured for each parameter at different time intervals

like 0, 30, 60, 90, 120, 150, 180, 210, 240, 360 and 480 min respectively. All experiments

were done in triplicates each time.

3.5.1 Flocculation Efficiency

After addition of flocculants, each tube was kept in orbital shaker (Model-

Technico, Honeywell Ltd, India) and stirring speed was maintained at 250 rpm. The

initial microalgal biomass concentration in the tubes was estimated from the optical

density of 750 nm (OD750) (Papazi et al., 2010 and Salim et al., 2011), in UV-VIS

Spectrophotometer (Model- SL 159, ELICO Ltd, India). At every 30 minutes, the optical

density of the microalgal culture was measured at half the height of the clarified culture

(Vandamme et al., 2010). Culture broth containing no flocculant was used as control and

culture medium with appropriate quantity of each salt was used as blank for respective

flocculants. Flocculation efficiency was calculated by (Pan et al., 2009 and Papazi et al.,

2010):

Flocculation Efficiency (%) = 100B

A1 �

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where, A= OD750 value of sample and B=OD750 value of control

3.5.2 Effect of Temperature, Light and Darkness on Flocculation

The effective flocculants were chosen from the cell viability test to study the

effect of temperature on flocculation. Such flocculants were added to the microalgal cells

and flocculant test was carried out at different temperatures (15, 25, 35, 45ºC) in orbital

shaker incubator in light and under dark conditions separately at 250 rpm for 30 min. The

optical density of the supernatant was measured at half the height of the clarified culture.

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3.5.3 Cell Viability Test

Cell viability test was determined with Evans blue staining method (Papazi et al.,

2010). One ml of sample of each culture was centrifuged at 3000 rpm for 5 min, the

supernatant was discarded, 100µl of 1% of Evans blue solution was added to pellet, and

incubated for 10 min at room temperature. The cells were then washed twice in deionized

water. The cell pellets were examined for the viability by light microscope (Model-

Olympus CH20i BIMF, Olympus India Pvt., India) at 100x magnification. Broken cells

appeared blue as Evans blue solution diffused in the protoplasm region and stained the

cells blue.

3.6 Harvesting of Microalgae using Bioflocculation

3.6.1 Culture for Bioflocculation

The marine bacterial culture Bacillus subtilis (MTCC 10619) was used as the

bioflocculant, obtained from the Department of Marine biology, CAS in Marine biology,

Parangipettai, Annamalai University Tamilnadu, India. The bacterial culture was

cultivated for growth and bioflocculant production using Nutrient broth containing

peptone – 5g/L, beef extract –1.5g/L, yeast extract–1.5g/L and NaCl–5g/L, supplemented

with 3% NaCl subcultured periodically and stored as stocks on nutrient agar slants at 4ºC.

3.6.2 Evaluation of Bioflocculation Parameters using Response Surface Methodology

A quantity of 50 ml of Chlorella salina and Nannochloropsis oculata each were

used for optimization study. The effects of bioflocculation parameters, namely

temperature, pH, time, bioflocculant size and cationic inducer concentration, were

individually experimented by analyzing bioflocculation efficiency. For the effect of pH,

the culture was divided in different test tubes and the pH was adjusted to fixed values by

the addition of 1M HCl and 1M NaOH, ranging from approximately 6.0 to 10. Likewise,

for examining the effect of temperature the test tubes were incubated at desired

temperatures.

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After the set up of fixed parameters, each tube was kept in orbital shaker (Model-

Technico, Honeywell Ltd, India) and stirring speed was maintained at 250 rpm. The

initial microalgal biomass concentration in the tubes was estimated from the optical

density of 750 nm (OD750), using UV-VIS Spectrophotometer (Model- SL 159, ELICO

Ltd, India). At a time interval of every 30 minutes, the optical density of the supernatant

was measured at half the height of the clarified culture. Culture broth without

bioflocculant was used as control and culture medium with appropriate quantity of each

salt were used for blank to respective flocculants. Flocculation efficiency was calculated

by Papazi et al. (2010). Response Surface Methodology (RSM) is statistical tool to

construct for experiments to define medium factors by quadratic polynomial equation to

depict the interaction effect among the variables and to optimize bioprocesses like

biomass cultivation, harvesting and metabolic secretion. RSM experiments reduce time

when compared to conventional methods. Plackett Burman Designs were use to

investigate the dependence of response on a number of independent variables using 1 or -

1 as coded levels. Box Behnken Design is an independent response surface quadratic

design which requires three levels of each factors usually coded as -1, 0, +1. A Central

Composite Design is an experimental set up for constructing a second order model for

response variables.

3.6.3 Central Composite Design (CCD)

A Central Composite Design (CCD) of the experiments was formulated to

investigate five flocculation parameters. Each 50 ml culture of Chlorella salina and

Nannochloropsis oculata were added into test tubes and the parameters were set

according to the orthogonal values of Central Composite Design (CCD) (Table 3.1).

RSM is known to evaluate the interaction between the significant factors of an

experiment and optimize them (Vandamme et al., 2012). Five level factorial experiment

set up, with 7 central points, was designed using Design Expert Software version 8.0.7.1,

Stat-Ease, Minneapolis, USA and the quality of analysis model was based on analysis of

variance (ANOVA).

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The actual values of coded levels of different parameters: Temperature (X1), pH

(X2), Flocculation time (X3), Bioflocculant size (X4) and Cationic inducer concentration

(X5) is presented in Table 3.1 and its influence on harvesting of microalgae by

flocculation, represented as Y, the response variable, has been investigated. The actual

values of coded level ‘0’ were fixed based on one -factor- at - a - time method.

Table 3.1 Experimental Range and levels of independent process variables for

Bioflocculation Experiments

Code Variables -2 -1 0 +1 +2

X1 Temperature (ºC) 20 25 30 35 40

X2 pH 6 7 8 9 10

X3Flocculation

Time (hr) 2 4 6 8 10

X4Bioflocculant size

(ml) 0.1 0.2 0.3 0.4 0.5

X5

Cationic Inducer

Concentration

(mM)

0.01 0.02 0.03 0.04 0.05

3.7 Effect of different Oil Extraction methods from Microalgae

3.7.1 Soxhlet Extraction

The Soxhlet flasks were dried in the oven at 100ºC for 1 h to remove water

content. Dried microalgae biomass was ground with mortar and pestle to get fine powder.

Then 2g of powder was placed in Soxhlet apparatus and sample was covered with cotton

wool. Hexane (150 mL) was taken in a round bottom flask, fixed into a Soxhlet

condenser and kept on water bath for 12 h at 69ºC. The solvent was evaporated using

rotary evaporator at 30ºC.

3.7.2 Folch method

A 2g of dried biomass was weighed and homogenized with 40ml of

chloroform/methanol (2:1) and whole mixer was agitated for 15-20 min at room

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temperature in orbital shaker. The homogenate was filtrated (funnel with a folded filter

paper) to get liquid phase. Then the liquid phase was washed with 8 ml of 0.9% NaCl

solution, vortex for some seconds and centrifuged at 2000 rpm for 5 min and two

interfaces were obtained. The upper layer was siphoned out and rinsed with

methanol/water (1/1). Without mixing the whole preparation was centrifuged, the upper

phase was removed and the lower chloroform phase containing lipids was collected. The

solvents are evaporated using rotary evaporator.

3.7.3 Bligh and Dyer method

Dried biomass was subjected to oil extraction by Bligh and Dyer (1959) with

slight modification. In brief, the biomass suspension was mixed with 60 ml of

chloroform: methanol (1:2) ratio, vortexed for few minutes and incubated on ice for 10

minutes. Then, 30 ml of chloroform was added followed by addition of 20 ml 1M HCl

and again vortexed it for few minutes. Finally the whole suspension was centrifuged at

maximum speed for 2 minutes. Bottom layer containing lipid was transferred into fresh

previously weighed beaker. Again 60 ml of chloroform was added to reextract the lipid

from the aqueous sample.

3.8 Effect of various Pretreatment on Microalgal Cells for Oil Extraction

The quantity of 2g of dried biomass was used for all the pretreatment process and

the intracellular lipid was extracted by Bligh and Dyer (1959) with slight modification.

The amount of oil extraction was calculated according to Suganya and Renganathan

(2012) and the oil extraction yield (% w/w) was determined by following formula;

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The extracted oil from untreated algal biomass (from nitrogen rich medium and

nitrogen depletion medium) considered as control for comparing oil pretreated by

different extraction techniques.

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3.8.1 Acid pretreatment

The dried microalgal biomass was added to sterile distilled water, the pH was

reduced to 2.0 with 1M HCl and the solution was shaken for 1 h, 2 h and 3 h using orbital

shaker at 180 rpm.

3.8.2 Enzymatic treatment

The microalgal suspension was disrupted with cellulase (0.3U/mg, Sigma Aldrich,

USA). Microalgal biomass was taken in 250ml Erlenmeyer flask containing cellulase

enzyme solution prepared using 0.1M sodium citrate buffer and the enzymatic hydrolysis

was conducted at 50ºC in a water bath for 8 h, 10 h and 12 h. The concentration of

cellulase enzyme was 5mg L-1

. The pH was adjusted to 5.5 with diluted HCl before

disruption. Then cellulase was inactivated by heating at 100ºC for 10 min.

3.8.3 Ultrasonication

The pretreatment process for microalgal cell wall destruction was also performed

with Ultrasonicator (Model-Vibra Cell VX400, Sonic Ltd, USA). The algal biomass was

mixed with 15ml of sterile distilled water and sonicated at 70 amplitude for 5 min, 10

min and 15 min (2012) at 24 kHz at a temperature at 50ºC. To avoid overheating the

samples were kept in an ice bath during the ultrasonic process. All the experiments were

carried out for both microalgal biomass harvested from nitrogen rich and depleted media.

After pretreatment, the biomass slurry was subjected to drying using hot air oven for

removing excess moisture. Finally the fatty acid composition of oil extracted from both

nitrogen rich and nitrogen depleted cultures were analyzed by Gas Chromatography-Mass

Spectrometry (GC-MS-QP 2010, Shimadzu).

3.8.4 Autoclaving

For the thermal treatment the microalgal cells was mixed with sterile distilled

water and carried out using autoclave. In this experiment, the autoclave was maintained

at 121ºC, 15 lbs pressure for 10 min, 20 min and 30 min.

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3.8.5 Microwave treatment

This experiment was conducted with the microwave oven (Model-National NN-

S557WF) for 5 min, 10 min and 15min at 100ºC, 900W and 2455MHz.

3.8.6 Pretreatment with 40% NaCl solution

The algal dried biomass was treated with 40% NaCl solution by osmotic shock in

an Erlenmeyer flask and kept at 180 rpm in an orbital shaker for 24, 48 and 72 hrs.

3.9 Analysis of Microalgal cells for cell damage after pretreatment using SEM

The effective pretreated microalgal cells were subjected to morphological analysis

for cell wall damage. Small amount of sample was taken from the suspension, dried and

observed with Scanning Electron Microscope (SEM) (Model-Jeol, Japan).

3.10 Isolation of lipase producing Bacterial strain from Marine Sediment

The lipase producing bacteria was isolated from marine sediment at Parangipettai,

a coastal area of Tamilnadu, India. The samples were collected from sediment (5 cm

depth) using a sterile container and immediately transferred to laboratory. After serial

dilution, the samples were spreaded on Tween 20 agar plates followed by incubation for

24 h at 37ºC. Lipase producing bacteria produced a zone of clearance. Then the bacterial

strain was isolated and subcultured using nutrient agar with 1% olive oil, 3% NaCl and

subjected to studying morphological, characterization, spore production and biochemical

characteristics.

3.10.1 Analysis of Morphological characteristics

3.10.1.1 Gram staining

Materials required

Culture broth, Crystal violet, Gram’s iodine, Saffranin, Distilled water

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Principle

Crystal violet permeates cells. Iodine is subsequently used as mordant to form

CV-I (crystal violet-iodine) complex, so that dye cannot be easily removed. Treatment

with ethanol decolorizes by dissolving liquid layer from Gram negative cells and

remaining excessive unbound dye. The removal of liquid layer enhances the leaching of

primary stain (CV) from the cells into surrounding solvent. The solvent dehydrates the

Gram positive cells, closing the pores as the wall shrink during dehydration. As a result,

diffusion of CV-I complex out of the cell is obstructed and the cells remain stained

purple. A counterstain of saffranin is applied to the smear, to dye the decolorized G-

cells, with a pink color.

Procedure

On a clean, dry slide, a drop of distilled water placed to which a loopful of culture

was added. The mixture was spread to a very thin smear. The slide with the culture was

heat fixed. 2-3 drops of crystal violet dye was added onto the smear and left for 1 min,

after which the smear was washed with running tap water. 3 drpos of Grams iodine of

3drops was added and left for 1 min. After washing with water, few drops of ethanol was

added and left for 30 sec. Then to the washed smear, 2 drops of saffranin was added and

left for 2 min. The water- washed slide was air dried and viewed under light microscope

with 100X magnification.

3.10.1.2 Endospore stainning (Schaffer – Fulton method)

Materials required

Culture broth, malachite green, safranin, distilled water.

Principle

A primary stain, malachite green, is used to stain the endospore. Because

endospore has a keratin covering and resist staining, malachite green will be forced to

enter the endospore by heating, which acts as mordant. Water is used to remove excessive

dye, acts as the decolorizing agent, as endospores are stain resistant. Endospores are

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equally resistant to destaining, thus retains primary stain while vegetative cells lose the

stain. Cells get stained pink on counterstaining with saffranin.

Procedure

A very thin smear was prepared with a loopful of culture mixed in a drop of

distilled water, placed on a cleaned dry slide. The slide was placed carefully on stand

mounted on boiling water bath. The smear was fully covered with malachite green stain,

and was allowed to fix the smear for 10 min. Then the slide was washed with water,

completely to remove excess unbound stain. Few drops of saffranin was added to

counterstain and left for 2 min. The slide was later washed and viewed under microscope

with 100X magnification.

3.10.1.3 Negative staining

Materials required

Culture broth, India Ink, Distilled water

Principle

Capsules are mainly carbohydrate metabolites tightly bound to cell wall of

microorganisms which are also found to constitute nucleic acids and proteins. Capsules

are characterized by poor staining of standard dyes. Capsule staining can be

accomplished by using India ink. The negatively charged dye interacts with the negative

ions of the bacterial capsules and cell wall, due to which repulsion of dye occurs. Thus

under microscope, the cells with capsule appear bright with no stain while the

background appears dark.

Procedure

A drop of Indian ink was placed at one end of a clean slide. One drop of culture

broth was placed in the dye and mixed well. With the help of another slide, the mixture

was swiped throughout the slide to form a thin, transparent smear. The air dried slide was

then observed under microscope with 100 x magnification.

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3.10.1.4 Motility test (Hanging drop technique)

This technique was carried out to check whether the organism is motile or non

motile, which is due to the presence of locomotive organ, flagella. A drop of culture broth

was placed in the center of the corner slip. A cavity slide was placed in such a way that

the culture hangs into the cavity from the coverslip. The slide was viewed for motility

under microscope.

3.10.2 Biochemical characterization analysis

3.10.2.1 IMViC test

Indole production Test

Materials required

Tryptone broth, Kovac’s reagent and bacterial culture.

Principle

This test is based on the hydrolysis of tryptophan into indole and pyruvic acid,

using a hydrolytic enzyme (hydrolase) called tryptophanase. Indole produced is detected

with Kovac’s reagent. If the organism has the ability to hydrolyse, cherry red ring at the

top of the culture broth could be observed, indicating the presence of indole.

Procedure

A loopful of culture was inoculated into 5ml of sterile tryptone water broth

containing test tube and incubated at 37ºC for 48h. After incubation few drops of Kovac’s

reagent was added to interpret the result.

Methyl Red Test

Materials required

Bacterial culture, Methyl Red indicator, MR-VP broth

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Principle

Upon fermentation of glucose in MR-VP medium into mixed acids like lactic,

acetic, succinic acd and formic acids and CO2, H2 and ethanol, the pH of the medium

turns acidic. The pH indicator, methyl red, turns red, if the organism is a mixed acid

fermenter.

Procedure

A loopful of culture was inoculated into 2ml of sterile MRVP broth and incubated

at 37ºC for 24h. After incubation, 3-4 drops of methyl red indicator was added to infer

the results.

Voges Proskauer Test

Materials required

Bacterial culture, MRVP broth, Barritt’s (A) and Barritt’s (B) reagent.

Principle

This test is carried out to check whether the organism possesss the capacity to

produce a precursor, 2,3- butanediol, also called acetoin, during glycolysis, instead of

ethanol as metabolite. If glucose could be fermented by organism, medium turns bright

red upon addition of reagents.

Procedure

A volume of 5ml of MRVP broth was inoculated with a loopful of culture and

incubated at 37ºC for 24-48h. To observe the result, 15 drops of Barritt’s (A) reagent was

added to the culture broth, following which Barritt’s (B) reagent, alont 5 drops, was

added.

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Citrate Utilization Test

Materials required

Bacterial culture, Simmon’s Citrate agar

Principle

The organism is tested for its ability to utilize citrate as the sole carbon source. If

citrate is used by the organism, the medium color changes from green to prussian blue

due to alkaline pH. This is detected by pH indicator, bromo thymol blue, is the medium.

Procedure

With 5 ml of molten, sterile Simmon’s Citrate agar, a slant was prepared in a

sterile test tube on which a loopful of culture was streaked. The slant was then incubated

at 37ºC for 24 hrs.

3.10.2.2 Starch hydrolysis

Materials required

Nutrient starch agar, bacterial culture and distilled water.

Principle

The purpose is to check whether the bacterium uses starch, a complex

carbohydrate made from glucose, as a source of carbohydrate and energy for growth. Use

of starch by microbe is due to the production of hydrolyzing enzyme, �-amylase. Starch

hydrolysis can be detected as a halo surrounding the culture and upon adding iodine, if

unutilized, the medium remain blue.

Procedure

A loopful of culture was streaked on a sterile petridish containing nutrient starch

agar. The inoculated plate was incubated at 37ºC for 24 h. After incubation, iodine

solution was added onto the plate, to observe the results.

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3.10.2.3 Casein hydrolysis

Materials required

Skimmed milk, agar, bacterial culture, distilled water

Principle

The enzyme caseinase is secreted out of the cell as an exoenzyme into the

surrounding medium catalyzing the breakdown of milk protein, casein, into small

peptides and individual amino acids, which are then taken up by organism for energy or

used up as a building material. A zone of clearance was observed upon casein hydrolysis.

3.10.2.4 Gelatin hydrolysis

Ingredients

Nutrient gelatin, distilled water, bacterial culture

Principle

Gelatin gets hydrolyzed in the presence of gelatinases to poly peptides, which are

then broken down to amino acids. This results in the liquefaction of the medium.

Procedure

A loopful of culture was inoculated in 5 ml of sterile nutrient gelatin medium. The

inoculated tubes were incubated for 24 hrs at 37ºC.

3.10.2.5 Oxidase Test

Ingredients

N, N, N,l N

l – Tetramethyl p-phenylnediamine, culture broth, distilled water,

bacterial culture

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Principle

The oxidase test in a biochemical reaction assays cytochrome oxidase, an enzyme

sometimes called indophenol oxidase. In the presence of an organism that contains the

cytochrome oxidase enzyme, the reduced colourless reagent becomes an oxidized

coloured product.

Procedure

To 5 ml of culture broth, 1 ml of reagent was added and indicated for the color

change.

3.10.2.6 Catalase Test

Materials required

3 % hydrogen peroxide, bacterial culture broth.

Principle

The catalase enzyme serves to maintain the bactericidal effects of H2O2. Catalase

expedites the breakdown of hydrogen peroxide into H2O and O2. This reaction is evident

by the rapid fermentation of bubbles.

Procedure

A drop of culture broth was placed on a clean slide, to which a drop of 3% H2O2

was added and observed for results.

3.10.2.7 Nitrate Reduction Test

Materials required

Bacterial culture, nitrate broth, sulfanilic acid, �-naphthylamine

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Principle

The Nitrate reduction test is used identify the presence or absence of nitrite (after

incubation) as an indicator of nitrate reduction. Generally some of the facultative

anaerobes are capable of using nitrate as an electron acceptor at the end of the electron

transport chain and that reduce nitrate (NO3) usually produce nitrite (NO2). The nitrite

will react with indicator and produce red colour which denotes nitrate positive.

Procedure

The sterile nitrate broth was inoculated with isolated bacteria and incubated at

37ºC for 24h. After incubation period was over few drops of sulfanilic acid� -

naphthylamine was added and a colour change was compared with control (nitrate broth

with no inoculum).

3.10.2.8 Salt tolerant test

The test bacterial strain was inoculated on nutrient agar containing different

concentrations of sodium chloride such as 2%, 5% and 10%. Then the Petri plates were

incubated at 37ºC for 24 h incubation. After the incubation period the colonies were

formed based on their salt tolerant features.

3.11 Strain Identification using Gene Sequence

3.11.1 Isolation of DNA from Marine bacterial isolate

Principle

The lysis of the bacteria is initiated by suspending a bacterial pellet in a buffer

containing lysozyme and EDTA. In addition to inhibiting DNases, EDTA disrupts the

outer membrane of the bacterium by removing the Mg2+

from the lipopolysaccharide

layer. This allows the lysozyme access to the peptidoglycan. After partial disruption of

the peptidoglycan, a detergent such as SDS is added to lyse the cells. Most of the cells

will lyse after this treatment and many can even be lysed without lysozyme. Once the

cells are lysed, the solution should be treated gently to prevent leakage of DNA.

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Subsequent steps involve the separation of the DNA from other macromolecules in the

lysate. Both phenol and chloroform, with isoamyl alcohol as a defoaming agent are

commonly used to dissolve protein from nucleic acids. These reagents also remove lipids

and some polysaccharides. Proteolytic enzymes such as pronase or proteinase K are

added to further remove protein. Proteinase K is particularly a useful enzyme, it is not

denatured by SDS and works more efficiently in the presence of SDS. The nucleic acids

may then be precipitated in ice cold ethanol if the ionic strength of the solution is high.

This is followed by RNase treatment to degrade the RNA. The solution may then be

reprecipitated with ethanol, leaving purified DNA in the pellet which can then be

dissolved in an appropriate buffer.

Materials required

Culture, Luria Bertani broth, 1 M Tris HCl, pH 8 (adjust with conc.HCl), 0.5 M

EDTA, pH 8 (adjust with conc. NaOH), 5% NaCl, 20mg/ml proteinase K (working conc-

20µg/ml), 10 mg/ml lysozyme (working conc-10 µg/ml), 20% SDS, 1X RNase, Buffer

saturated phenol, 70% ethanol, Absolute ethanol, Phenol, Chloroform (1:1), Chloroform:

Isoamyl alcohol (24:1), 3 M Sodium acetate, pH 5.2 (adjust with glacial acetic acid), TE

buffer (10mM Tris HCl, pH 8, 5mM EDTA, pH 8), TES buffer (10mM Tris HCL, pH 8,

5mM EDTA pH8, 1.5% NaCl)

Procedure

A single colony of bacterial culture was inoculated in 10 ml of LB broth and it

was allowed to grow at 37ºC for 12 h at 110 rpm. The cells were harvested at 7000 rpm

for 10 min at 4�C. The media was decanted as much as possible and the pellet was tapped

gently. The pellet was resuspended in 5 ml of the ice cold TES buffer and 50 µl of

10mg/ml lysozyme was added and incubated at room temperature for 5-10 min.

Proteinase K was added to a final concentration of 40µg/ml and was incubated at 55ºC

for 10 min. SDS was added to a final concentration of 1% and incubated in a water bath

at 55ºC for 45 min. An equal volume of buffer saturated phenol was added, vortexed well

and centrifuged at 12000 rpm for 10 min. The aqueous layer was transferred to a fresh

tube and 2 volumes of ice cold 70% ethanol was added and was incubated on ice for 5-10

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min. The precipitated DNA was spooled to a fresh tube. It was resuspended in 5 ml of TE

buffer completely. A concentration of 100 µg/ml of RNase was added and was incubated

at 45ºC in a water bath for 15 min followed by the addition of an equal volume of phenol:

chloroform (1:1), vortexed well and centrifuged at 12000 rpm for 10 min at room

temperature. The tap aqueous layer was collected and extracted once with chloroform:

isoamyl alcohol (24:1). The top aqueous layer transferred to a sterile tube and 1/10th

volume of 3M sodium acetate was added and 2 volumes of ice cold 70% ethanol was

added to precipitate the DNA. It was centrifuged at 10000 rpm to pellet the DNA. The

DNA pellet was air dried and was redissolved in 200µl of TE buffer. A volume of 5 µl of

DNA sample was checked using 0.8% agarose gel electrophoresis.

3.11.2 Molecular identification using 16S rRNA Sequencing

The molecular identification of the characterized culture was done by analyzing

the genomic DNA. PCR analysis was performed with 16S rRNA primers: 27F (5’-AGA

GTT TGA TCC TGG CTC AG-3’) and 1492R (5’- TAC GGT TAC CTT GTT ACG

ACT T-3’). A volume of 25µl reaction mixture for PCR was carried out using 10ng of

genomic DNA, 1X reaction buffer (10mM Tris HCl pH 8.8, 1.5mM MgCl2, 50mM KCl

and 0.1% Triton X 100), 0.4 mM dNTPs each, 0.5U DNA polymerase and 1mM reverse

and forward primers each. The reaction was performed in 35 amplification cycles at 94ºC

for 45 sec, 55ºC for 60 sec, 72ºC for 60 sec and an extension step at 720C for 10min. The

sequencing of 16S amplicon was performed according to manufacturer instructions of

Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing

products were resolved on an Applied Biosystems model 3730XL automated DNA

sequencing system (Applied BioSystems, USA). The 16S rRNA gene sequence obtained

from the organism was compared with other Bacillus strains for pairwise identification

using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and multiple sequence

alignments of the sequences were performed using Clustal Omega version of EBI

(www.ebi.ac.uk/Tools/msa/clustalo). Phylogenetic tree was constructed by Clustal

Omega of EBI (www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny) using neighbor

joining method.

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3.12 Enzymatic Biodiesel production using Methyl acetate in solvent free system

3.12.1 Fermentation of Lipase Production using Isolated Strain

The lipase production was carried out in 250ml Erlenmeyer flask using 100 ml

basal medium containing 1% olive oil, 0.2% CaCl2·2H2O, 0.01% MgSO4·7H20, 0.04%

FeCl3·6H2O and 3% NaCl. The contents were incubated for 48 h at 37ºC at 200 rpm and

neutral pH was maintained. After incubation, the culture was centrifuged at 10,000 rpm

for 10 min at 4ºC. The supernatant of crude lipase was quantified using lipase assay and

used for immobilization.

3.12.2 Lipase Assay and Protein Estimation

Lipase activity was determined for free and immobilized enzymes according to

Burkert et al (2004) and Padihla et al (2012). The olive oil emulsion was prepared by

mixing 25ml of olive oil and 75ml of 7% Arabic gum solution in a homogenizer for 5

min at 500rpm. The reaction mixture containing 5ml of emulsion, 2ml of 10mM

phosphate buffer (pH 7.0) and 1ml of the culture supernatant was incubated at 37ºC for

30 min in orbital shaker. The reaction was stopped by addition of 15ml of acetone-

ethanol (1:1v/v), and the liberated fatty acids were titrated with 0.05 N NaOH. The lipase

activity was calculated using Equation (3.5). One unit of lipase activity was defined as

the amount of enzyme, which liberated 1 µmol of fatty acid per minute. The protein

content in the crude enzyme was determined by Lowry et al. (1951) with BSA as a

standard.

Lipase activity (U/ml)

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3.12.3 Immobilisation of crude lipase

Crude lipase (6ml) was mixed with 14ml of sodium alginate solution (2%). The

mixer was dripped into cold sterile 0.2 M CaCl2 using sterile syringe from a constant

distance and was cured at 4ºC for 1 h. The beads were hardened by suspending it again

in a fresh CaCl2 solution for 24 h at 4ºC with gentle agitation. After immobilization, the

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beads were separated by filtration and washed with 25 mM phosphate buffer (pH 6.0) to

remove excess calcium chloride and enzyme. Then the beads were preserved using 0.9 %

NaCl solution for future use (Vimalarasan et al 2011; Kavardi et al 2012).

3.12.4 Determination of Molecular Weight of Microalgae oil

The molecular weight of the oil was calculated as (Xu et al 2006):

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where, M is the molecular weight of the oil, SV the saponification value and AV

is the acid value. The saponification value and acid value was determined according to

Sathasivam and Manickan (1996).

3.12.4.1 Estimation of Free Fatty Acid

Principle

Generally oils contain small quantity of free fatty acids along with the

triglycerides and the free fatty acid content is known as acid number or acid value. It

increases during storage. The free fatty acid in oil is estimated by titrating it against KOH

in the presence of phenolphthalein indicator. The acid number is defined as the mg KOH

required neutralizing the free fatty acids present in 1g of sample.

Materials required

1 % phenolphthalein, 0.1 N KOH, Neutral solvent: Mix 25ml ether, 25ml 95%

alcohol and 1ml of 1% phenolphthalein were mixed and neutralized with N/10 alkali.

Procedure

1 g of microalgal oil was dissolved in 50ml neutral solvent in a 250ml conical

flask and few drops of 1% phenolphthalein was added. The content was titrated against

0.1 N KOH and shaken until a pink colour which persists for few seconds was obtained.

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3.11.4.2 Estimation of Saponification value

Principle

Saponification is the process by which the fatty acids in the glycerides of the oil

are hydrolysed by an alkali. A known quantity of oil is refluxed with an excess amount

of alcoholic KOH. After saponification, the remaining KOH is estimated by titrating it

against a standard acid.

Materials required

HCl 0.5 N, Alcoholic KOH-40g of KOH was dissolved in 1 L of distilled alcohol

keeping the temperature below 15.5ºC while the alkali was being dissolved,

Phenolphthalein-1% in 95% alcohol and air condenser.

Procedure

1g of microalgal oil was taken in dry clean flask and 50 ml of alcoholic KOH was

added using burette by allowing it to drain for a definite period of time. Blank was also

prepared only with alcoholic KOH except oil sample. The air condenser was connected to

the flask and boiled gently for 1 h. After the flask and condenser was cooled, 1 ml of

indicator was added and titrated against 0.5 N HCl until the pink colour just disappeared.

3.12.5 Optimization of Enzyme Interesterification process by solvent free system

The enzymatic interesterification reaction was carried out in 20 ml screw cap

bottle. No solvent was added in this reaction. The oil to acyl acceptor (methyl acetate)

was optimized ranging from 1:4, 1:6, 1:8, 1:10, 1:12 and 1:14. The effect of temperature

was studied at various intervals of 25, 30, 35 and 40ºC. In order to investigate the effect

of water, enzymatic transesterification was carried out by adding small amount of water

at the concentrations of 0, 2, 4, 6, 8 and 10 weight % of the total amount of reaction

mixture. Effect of reaction time was investigated in the range of 12-72 h and agitation

was conducted between 100 and 300 rpm. The initial interesterification reaction was

allowed for 48 h at constant speed of 200 rpm with 1 g of biocatalyst, oil to methyl

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acetate molar ratio of 1:4 at 30ºC. The biodiesel yield was calculated according to Umdu

et al (2009):

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3.13 Gas Chromatography Analysis of Fatty Acid Methyl Esters

Fatty acid methyl ester composition of biodiesel produced from Chlorella salina

and Nannochloropsis oculata oil was analysed by Gas Chromatography-Mass

Spectrometry (GC-MS-QP 2010, Shimadzu) equipped with VF-5 MS capillary column

(30mm length, 0.25mm diameter and 0.25µm film thickness). The column temperature of

each run was started at 70ºC for 3 min, then raised to 300ºC and maintained at 300ºC for

9 min. GC conditions were: column oven temperature-70ºC, injector temperature-240ºC,

injection mode split, split ratio-10, flow control mode-linear velocity, column flow-

1.51ml/min, carrier gas-helium (99.9995% purity) and injection volume-1µl. MS

conditions were: ion source temperature-200ºC, interface temperature-240ºC, scan range-

40-1000m/z, solvent cut time-5 min, MS start time-5 min, end time-35 min and

ionization-EI (-70eV) and scan speed-2000.

3.14 Analysis of Biodiesel Properties

3.14.1 Density

Density is an important parameter for diesel fuel injection systems. Many

performance characteristics, such as cetane number and heating value, are related to

density. The weight of a small empty bottle was determined using an electronic weighing

balance. The bottle was then filled to the brim with the oil and the weight of the bottle

and oil determined.

3.14.2 Kinematic Viscosity

In this test method, time is measured for a fixed volume of liquid to flow under

gravity through the capillary of a calibrated viscometer under a reproducible driving head

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and a closely controlled and known temperature. The kinematic viscosity is a product of

the measured flow time and the calibration constant of the viscometer.

3.14.3 Flash Point

The flash point of petroleum products in the temperature range from 40 to 360°C

by a manual Pensky-Martens closed apparatus or an automated Pensky-Martens closed

cup apparatus. Each of the blended samples was poured into the test cup of the automated

Pensky-Martens apparatus. The automated apparatus brought the sample material to a

temperature of 15 ±5˚C, as the stirrer thoroughly mixed the water provided in the water-

bath. Then the test flame was applied automatically at the lowest temperature at which

sample vapours were ignitable.

3.14.4 Fire Point

The fire point of a fuel is the temperature at which it will continue to burn for at

least 5 seconds after ignition by an open flame. At the flash point, a lower temperature, a

substance will ignite briefly, but vapor might not be produced at a rate to sustain the fire.

Pour point and fire point parameters have great importance while determining the fire

hazard temperature at which fuel will give off inflammable vapour).

3.14.5 Pour Point

Liquids have a characteristic temperature at which they turn into solids, known as

their freezing point. On the other hand, pour points define the lowest temperature at

which the fuel can still be moved, before it has gelled. The pour point and freezing point

are used to characterize the cold flow operability of fuel, because the pour point and the

freezing point of fuel affect the utility of a fuel, especially in cold climate conditions. The

specimen is cooled inside a cooling bath to allow the formation of crystals. At about 9°C

above the expected pour point, and for every subsequent 4°C, the test jar is removed and

tilted to check for surface movement. When the specimen does not flow when tilted, the

jar is held horizontally for 5 sec. If it does not flow, 4°C is added to the corresponding

temperature and the result is the pour point temperature.