2. materials and methodsshodhganga.inflibnet.ac.in/bitstream/10603/4573/11/11_chapter 2.pdf · 2....

24 G. SIVARANJANI, Ph.D THESIS, 2011 MATERIALS AND METHODS

2. MATERIALS AND METHODS

2.1. Glass ware:

All the glass ware used in the present experiments including test

tubes, measuring cylinders, culturing flasks, petriplates were of

borocil or Duran or Anumbra brand.

2.2. Cleaning:

The glass ware used in the experiments were initially soaked in dilute

H2SO4 (20% v/v) for 24 h and cleaned with tap water and tepol, a

detergent. After removing all the traces of the detergent, the glass

ware was rinsed with deionized water and kept in oven for drying at

100 ºC.

2.3. Deionized water:

Deionized water obtained from deionizer plant (Bhanu Aqua DM600,

Bhanu Scientific Instruments Company) was routinely used for final

rinsing of glass ware after washing and for media preparation.

2.4. Distilled water

Double glass quartz-distilled water stored in white polypropylene (pp)

Carboys was used for preparation of stock solutions.

2.5. Chemicals

All the chemicals used in the present study were of analytical grade

from sigma, S.D. fine chemicals, Hi-Media, Qualigens, E-Merck or

LOBA.

2.6. Sterilization

Sterilization of the culture media and glass ware was done by

autoclaving at 15 lbs for 15min. Heat labile compounds were sterilized


by filtration of their aqueous solutions through a 0.45µ cellulose

acetate membrane (Millipore, Bangalore).

2.7. Organism

Strain JC1, a bluish purple colored organism was isolated from

demineralized water sample of sri Krishna pharmaceuticals limited.

Culture was purified and characterized.

2.8. Purification of the strain JC1: The isolated strain JC1 was

purified by repeatedly streaking on nutrient agar [Difco Manual, 1998]

plates (g/L peptone- 5, yeast extract-1.5, beef extract 1.5, NaCl-5 and

agar-20) and incubating aerobically in an incubator at 35 ºC.

Contamination from other chemotrophic bacteria was checked by

monitoring the cultural characters like color of the culture, colony

morphology and by few morphological characters like cell shape etc.

2.9. Morphological and cultural characterization

Direct microscopic observation: morphological properties such as cell

shape, cell size were visualized through phase contrast microscopy

(Olympus-13201).

2.10. Staining methods:

2.10.1. Gram staining: A loopful of argon flushed anaerobically

grown culture was taken on a neat clean slide, smear was prepared

and was fixed by heat. The slide was then flooded with crystal violet

(10 g of crystal violet is dissolved in 100 ml of absolute ethanol, filter

sterilized and made the volume to 1000 ml with double distilled

water). And stained for a minute then washed under tap water. In the

next step Gram iodine solution (Iodine 1g, potassium iodide 2 g,


dissolved in 300 ml double distilled water) a mordant was added to

the slide for a minute. After washing with water decolorization of the

dye was done with acetone. In the final step safranin is applied as a

counter stain. The slide was observed under light microscope at 100 X

magnification after the final rinsing and air drying.

2.10.2. Spore staining:

The heat fixed smear was flooded with malachite green (5% w/v in

double distilled water) and the slide heated on steam bath till fumes

come out as color less water droplets, the smear was counter stained

with safranin for one minute washed and air dried slide was observed

under light microscope in oil immersion.

2.11. Pigment analysis:

2.11.1. Whole cell absorption spectrum:

Absorption spectrum of whole cells was measured by the sucrose

method of Troper and Pfennig (1981). To 3.5 ml of the liquid culture, 5

g of sucrose was added and mixed thoroughly on a vortex spinner. The

absorption spectrum from 300-1100 nm was measured on a

spectronic Genesys 2 spectrophotometer using sucrose in the medium

as a blank.

2.12. Physiological/Biochemical characterization:

2.12.1. Utilization of organic compounds as carbon sources:

For both growth and pigment production a 10 % inoculum of culture

was inoculated into Biebl and Pfennig's basal salt medium with NH4Cl

(0.12 % w/v) and yeast extract as source of nitrogen and growth

factors, respectively with the test organic compound serving as the


carbon source. Growth was monitored turbidometrically (O.D at

720nm) in 15×125 mm test tubes after incubation in an incubator at

35 ºC for 7 days. Various organic carbon sources such as sugar, TCA

intermediates and aromatic compounds were tested for their

utilization as carbon source. Sugars and TCA cycle intermediates were

used at a concentration of 10mM. Aromatic compounds at 1mM

concentration were used after filtrate sterilization. In addition, 0.1 %

(w/v) of filter sterilized bicarbonate was supplemented in the medium

in case of aromatic compounds.

2.12.2. Utilization of various nitrogen sources:

Biebl and pfennig's (1981) medium with pyruvate (0.3% w/v each) as

carbon source in presence of various inorganic (NH4Cl, NaNO3,

NaNO2, KNO3, urea, (NH4)2SO4, NH4H2PO4) serving as sources of

nitrogen at 10mM concentration. Media with out any nitrogen source

served as the control. Growth of the inoculated media was monitored

turbidometrically in test tubes after incubation in an incubator at 35

ºC.

2.12.3. Vitamin requirements: A 10% (v/v) inoculum of the culture

was inoculated in Biebl and pfennig's (1981) medium with pyruvate

(0.3% w/v) and NH4Cl (0.12%w/v) as carbon and nitrogen source,

respectively devoid of yeast extract and replaced with the test vitamin

(Biotin [15µ/L], Thiamine [500µg/l], Nicotinic acid [500µg/L], Para-

Aminobenzoic acid [300 µg/L], Pyridoxal phosphate [15µg/l], B12

[15µg/L] and yeast extract [0.6 g/L])filter sterilized on a 0.45 µm


cellulose acetate membrane) growth was monitored in 15×125 mm

test tubes under aerobic conditions at 35 ºC.

2.12.4. Tolerance of sodium chloride:

Nutrient broth containing a total of 0.1% to 5% (w/v) of sodium

chloride were inoculated with 10% of young broth culture and

incubated at 35 ºC for 7 days. Media with out NaCl served as control.

2.12.5. Growth temperature: Growth of the strain at different

temperatures of 5-45 ºC was monitored by incubating the inoculated

cultures in the growth media under aerobic conditions.

2.12.6. Growth at different pH:

Growth of the strain JC1 at different pH range (3-9) was monitored in

the nutrient broth by inoculating with the 10 % young culture and

incubated 35 ºC under aerobic conditions in an incubator.

2.12.7. Growth mode- aerobic/anaerobic

Gelatin hydrolysis: gelatin hydrolyzing ability of the strain is tested by

streaking on gelatin agar [Difco manual, 1998] plates incubated at 35

ºC. After 72 h of assay, grown culture was directly tested for gelatin

hydrolysis by adding 5ml of mercuric chloride solution (15g of HgCl2

in 20 ml of HCl (conc.) in 80 ml of distilled water. Appearance of clear

zone indicates starch hydrolysis.

2.12.8. The catalase effect: Catalase activity was determined by the

addition of a few drops of 30% H2O2 on 18h fresh culture. Appearance

of effervescence indicates catalase positive.


2.12.9. Casein hydrolysis: Casein hydrolyzing ability of the strain is

tested by streaking on casein agar plates [Difco manual, 1998] and

incubated at 35 ºC.

2.12.10. Starch hydrolysis: Starch hydrolyzing capacity of the strain

is tested by streaking on starch agar plates [Difco manual, 1998] and

incubated at 35 ºC up to 72 h

2.12.11. Test for urease: Ammonia formation from urea was studied

by inoculating logarithmically growing culture in the Bacto-urea broth

(HiMedia). Tubes were incubated at 35 ºC for 48 h. observed for color

change. Appearance of red from yellow color indicates urease positive.

2.12.12. Indole production from L-Tryptophan: Indole formation

from L-Tryptophan was studied for the purple bacterial strain by

inoculating a loopful of logarithmically growing culture in tryptophan

medium (Difco manual, 1998) and incubated at 35 ºC for 48 h. 0.5 ml

of Kovac's reagent (5g of p-dimethylaminobenzaldehyde in 75 ml of

amyl alcohol and adding 25 ml of concentrated hydrochloric acid)

were added to 5 ml of culture. Appearance of a dark red color in the

surface layer constitutes a positive test.

2.12.13. Methyl red test: The strain was inoculated into a tube

(16×150mm) containing methylred voges proskauer (MRVP) medium

incubated at 35 ºC for 24 h. To 5 ml of culture 5 drops of methyl red

solution [0.1g Bacto-Methyl Red in 300 ml of 95% alcohol and diluting

to 500 ml with distilled water] was added. Appearance of red color

indicates positive test.


2.12.14. Test for voges-proskauer (VP) reaction: Inoculated the

strain to a tube (16×150 mm) containing methyl red-voges proskauer

(MRVP) medium and incubated at 35 ºC for 24 h. To 1ml of the

culture 0.6 ml of 5% (w/v) alpha Naphthol solution in absolute ethyl

alcohol and 0.2 ml of 40% (w/v) potassium hydroxide solution were

added. The tube was mixed well and allowed to stand exposed to the

air, and observed at intervals of 2, 12 and 24 h for the appearance of a

pink color, which indicates a positive reaction.

2.12.15. Citrate utilization test: Citrate utilization ability of the test

strain was determined by streaking on Simmon's citrate agar (Difco

1892) tubes (16×150 mm) and incubated at 35 ºC. After 48h observed

for color change from green to blue which indicates positive reaction.

2.12.16. Lecithinase and lipase activity: Both lecithinase and lipase

activities were determined by streaking the strain on egg yolk

emulsion agar (Difco 1892) and incubated at 35 ºC for 48h.

Lecithinase activity appear as colonies surrounded by an insoluble

opaque precipitate. Lipase activity appears as colonies with a sheen or

pearly surface. Appearance of a clear zone indicates lecithinase

activity.

2.13. GENETIC CHARACTERIZATION

2.13.1. DNA mol% determination:

2.13.1.1. DNA extraction and purification The culture was

harvested by centrifugation (10,000 rpm for 15 minutes) and their

genomic DNA was isolated by the method of Marmur (1961) modified

as illustrated below. Except the solvents used, glassware, buffers,

solutions were all sterilized by autoclaving.


Protocol for DNA extraction and purification

2 to 3 g wet packed cells

Suspended in 50 ml Saline – EDTA (0.15 M NaCl + 0.1 M EDTA pH 8)

Centrifuged (10,000 rpm, 10 minutes)

Suspended the cells in a total volume of 25 ml of Saline – EDTA

*Lysed the cells

[*2 ml of 25% (w/v) Sodium Dodecyl Sulfate (SDS) was added to the

cell suspension and the mixture was placed at 60 oC in a water bath

for 10 minutes and then cooled to room temperature.

OR

Ten milligrams of lysozyme was added to the cells suspended in

25 ml Saline - EDTA. The mixture was incubated at 37 oC with

occasional shaking till increase in viscosity was observed which

indicated the lysis of the cells (30–60 minutes). When lysozyme was

used, SDS was also added after the cells had lysed followed by the 60

oC heating and cooling].

Sodium Perchlorate (5M) was added to a final concentration of

1 M to the viscous, lysed suspension

Equal volume of chloroform – Isoamyl alcohol (24:1 v/v) mixture was

added to the lysed cell suspension and shaken for 30 minutes

Centrifuged (10,000 rpm for 15 minutes)


Formation of three layers

Pipetted out upper aqueous layer which contained nucleic acids

Added 95% (v/v) ethyl alcohol to precipitate nucleic acids

Removed the precipitated nucleic acids by stirring with the help of a

glass rod (removed excess alcohol by pressing the glass rod against

the container)

Transferred and dissolved the precipitate into 10 – 15 ml

of dilute saline citrate (0.015 M NaCl +0.015 M Tri sodium citrate; pH

7)

Adjusted the above solution approximately to standard saline citrate

concentration by adding concentrated saline citrate solution (1.5 M

NaCl + 0.15 M Tri sodium citrate pH 7)

Shaken well with an equal volume of chloroform – Isoamyl alcohol

(24:1 v/v) for 15 minutes

Centrifuged and removed the supernatant

(Repeated three times to remove all the proteins)

Added 95% (v/v) ethyl alcohol to the supernatant; dispersed the

precipitate in ½ to ¾ of the supernatant volume


Added ribonuclease [(50 g ml-1) ribonuclease was dissolved in 0.15 M

sodium chloride pH 5 and heated at 80oC for 10 minutes to inactivate

any DNAase present]

Incubated for 30 minutes at 37 C

Added chloroform – Isoamyl alcohol (24:1 v/v) and centrifuged (10,000

rpm for 10 minutes) (Repeat three times to remove all the proteins)

To the supernatant 95% (v/v) ethyl alcohol was added to precipitate

the nucleic acids

Dissolved the precipitate in 9 ml dilute saline citrate

Added 1.0 ml acetate – EDTA solution (3 M sodium acetate + 0.001 M

EDTA pH7)

While stirring the solution rapidly with glass rod isopropyl alcohol was

added drop wise into the vortex

DNA as fibrous material was collected on glass rod

Washed the DNA isolated with ethyl alcohol

DNA was dissolved in dilute saline citrate (0.015 M NaCl + 0.0015 M

Tri Sodium citrate) and then stored in refrigerator

Twenty five microlitres of the DNA stock in concentrated buffer

solution was diluted to standard saline buffer concentration with

diluted saline citrate buffer (0.015M NaCl + 0.0015 M trisodium

citrate) and the absorption at 25C was noted by spectrophotometry.


2.13.1.2. Spectrophotometry

By using quartz absorption cells, absorbency was measured at 260

nm in a thermostated Spectronic Genesys 2 spectrophotometer. For

DNA solution the absorbency was determined at different

temperatures. A graph of ‘Absorbency of the DNA’ versus the

‘temperature’ was plotted and the melting temperature (Tm) of the DNA

was determined from the graph. The mol% G+C was calculated using

the formula given:

mol% G+C = (Tm - 69.3) x 2.44

2.13.2. 16S rRNA gene sequence analysis

2.13.2.1 DNA extraction from colony Well grown isolated colony

was picked up from the slant or plate, suspended in 20 to 50 l of

sterile milli-Q water. The bacterial suspension was lysed in a thermal

cycler (MJ Mini Personal Thermal Cycler – BIO-RAD) at 96 C for 10

minutes.

2.13.2.2. Amplification of 16S rRNA gene Amplification is routinely

performed on 50 l volumes in 0.2 ml microfuge tubes using a DNA

thermal cycler (MJ Mini Personal Thermal Cycler – BIO-RAD). All

plastic ware were autoclaved and ultraviolet irradiated. The primers

used for the amplification of the 16S rRNA gene are Eub27F

(5GAGTTTGATCCTGGCTCAG-3) and Univ1492R (5-

GGTTACCTTGTTACGACT T-3). The concentration and volume of the

reaction mixture are as follows.


1. Primers: 2 l of each primer (10 pmol l-1),

2. Template: 2 l of DNA template (25ng l-1),

3. Water: 19 l and

4. Master mix: 25 l (Obtained from Bangalore GENEI [Cat.

No.105908]).

The cycling parameters were given as follows.

No. of

cycles

Denaturation Annealing Elongation

Temp (C) Time Temp

(C)

Time Temp (C) Time

1 96 2 min

32 96 40 s 50 40 s 72 1

min

1 42 1

min

72 5

min

Specific PCR for chemotrophic bacteria was performed by using 599

(5-ACTTGGCGCAAGGGTGA-3) and 31387 (5-

CGGGCGGTGTGTACAAGG-3) primers using touch-down PCR


2.13.2.3. Agarose gel electrophoresis 5 l of amplified DNA, 5 l of 1

Kb DNA marker were electrophoresed in 2% (w/v) horizontal agarose

gel as described earlier, in TAE buffer at 15 V cm-1, stained in 0.5

g/ml ethidium bromide and visualized on UV transilluminator.

2.13.2.4.PCR amplicon purification: The amplified product was

purified by using the QIAquick PCR Purification Kit (Cat. No.28104)

and the quality and concentration of the purified product was checked

by agarose gel electrophoresis as described in method 15.B.ii.

2.13.2.5. 16S rRNA gene sequencing and assembling of the 4

partial sequences The complete length of the 16S rRNA gene

sequence was obtained by sequencing with 4 primers Eub27F (5-

GAGTTTGATCCTGGCTCAG-3), 5-372F (5-TACGGGAGGCAGCAG-3),

5-790F(5-GATACCCTGGTAGTCC-3) and Univ1492R (5-

GGTTACCTTGTTACGACT T-3). The 16S rRNA gene amplicon was

sequenced at MWG, Bangalore, India. The four sequences obtained as

*.scf format were assembled using software SeqMan in the DNA STAR

Lasergene 6 package.

2.13.2.6. BLAST search The single contig of sequence of length

approximately 1350 to 1450 bp was submitted to the NCBI-BLAST

search in order to know the nearest phylogenetic relative. EzTaxon

server (web based database of 16S rRNA gene sequences – Easiest way

to the accurate identification of prokaryotes) is more useful for

comparison of 16S rRNA gene sequences with type strain sequences.


2.13.2.7. Collection of 16S rRNA gene sequences of the type

strains Based on the blast search results, type strain sequences of

the closely related members and an out group sequence were obtained

in fasta format from National Center for Biotechnology Information

(NCBI) - Nucleotide search or from Ribosomal Database Project-II

(RDP-II) Release 9.56. The type strain numbers were either obtained

from Bergey’s Manual of Systematic Bacteriology (2005) or from List of

prokaryotic names with standing in nomenclature (LPSN –

http://www.bacterio.cict.fr/index.html).

2.13.2.8. 16S rRNA gene Sequence Alignment Sequences (all the

closely related type strain sequences along with an out group

sequence and the sequence to be analysed) were aligned using the

CLUSTAL X program (Thompson et al., 1997). The alignment file was

opened with BioEdit software and the alignment was corrected

manually, and the file was saved with “.phy” extension which is an

input file for the programs used for phylogenetic analysis.

2.13.3. Methods for phylogenetic tree construction Distance and

character based methods were used for phylogenetic analysis.

2.13.3.1. Distance based methods Two distance based methods

were used for phylogenetic tree construction, one is neighbor joining

method and the other is Fitch method.

2.13.3.2. Neighbor joining method The evolutionary distance matrix

was calculated using the distance model of Jukes and Cantor (1969).

The evolutionary tree was constructed using the neighbor-joining

method (Saitou and Nei, 1987) and the resultant tree topologies were

http://www.bacterio.cict.fr/index.html


evaluated by bootstrap analysis based on 100-1000 resamplings,

using the SEQBOOT and CONSENSE programs in the PHYLIP

package.

2.13.3.3. Fitch method: The evolutionary distance matrix was

calculated using the distance model of Jukes and Cantor (1969). The

evolutionary tree was constructed using the Fitch-Margoliash Program

(Fitch and Margoliash, 1967) and the resultant tree topologies were

evaluated by bootstrap analysis based on 100 resamplings, using the

SEQBOOT and CONSENSE programs in the PHYLIP package.

2.13.3.4. Character based methods Maximum likelihood method in

both phylip package and PhyMl were used for phylogenetic tree

construction.

2.13.3.4.1. Maximum likelihood method in Phylip package The

CLUSTAL X alignment file with “.phy” extension was used as the input

file to the program SEQBOOT in the PHYLIP package (Felsenstein,

1993) and the output file of SEQBOOT was used as the input file for

maximum-likelihood [in the PHYLIP package (Felsenstein, 1993)]

analysis with 100 datasets and five times jumbling. One single tree

was produced using 100 trees generated during maximum-likelihood

analysis using the program CONSENSE. A final dendrogram with

evolutionary distances was constructed by taking the alignment file

with “.phy” extension as the in file and the consensus tree as the in

tree in the maximum-likelihood program of the PHYLIP package.

2.13.3.4.2. Maximum likelihood method in PhyML program The

dendrogram was constructed using PhyML (Guindon and Gascuel,

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Guindon%20S%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Gascuel%20O%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus


2003) program using 100 replicates of non parametric bootstrap

analysis, GTR model of nucleotide substitution and 4 substitution rate

categories.

2.14. Effect of N-heterocyclic aromatic compounds on the growth

and violacein production of the strain JC1 in mineral salts media:

Herbicides used: Atrazine [2-chloro-4-ethylamino-6-isopropylamine-5-

triazine] commercially marketed atrataf, 50% w/w purity, Butachlor

tech [N-(butonymethyl)-2-chloro-N-(2,6-diethylphenyl) acetamide; 92%

v/v purity], 2,4D [2,4 Dichlorophenoxy acetic acid; 98% w/w purity],

and pendimethalin tech [N-(1-ethylpropyl)-3,4-dimethyl-2,6-

dinitrobenzennamine;95.5% w/w purity] were the herbicides used for

this study. Pendimethalin tech was procured from Rallis India limited.

Atrazine and 2,4-D from sigma, USA and Butachlor tech from

Hindustan insecticides limited.

The herbicides were added (100µm, unless otherwise mentioned) from

their stock solutions in ethanol (99%v/v) to the autoclaved media or to

the assay cultures with the alcohol concentration not exceeding

50µl/5ml. No significant change in the pH of assay was observed at

the concentrations of herbicides used. 10% of inoculum was used to

inoculate the test tubes (15× 125mm) containing 5ml of media and

incubated at 35 ºC in an incubator. The results were compared with

the control tubes containing only alcohol without herbicide.


2.15. Effect of aromatic compounds on the growth and violacein

production of the strain JC1:

Aromatic compounds used: Benzoate, cresol, dichloroaceticacid,

indole, indole-3-acetic acid, malonic acid, pyrazines (2-Pyrazine-

carboxylic acid), pyrrole, 2-nitroaniline, NN-Methylene Bis-

Acrylamide, 3(3,4 Dichlorophenyl)-1,1 Dimethyl urea, imidazole and

furfurol were the N-heterocyclic aromatic compounds used. The

compounds were added (1.6 µl/ml) to the assay from their stock

solutions (neutralized with 0.5N NaOH in case of compounds that

bring about a change in pH) in ethyl alcohol (99% v/v) to a final

concentration of 0.1mM (unless otherwise mentioned).

2.16. Effect of antibiotics on violacein production by the strain

JC1.

Antibiotics of different concentrations (µg/disc) were procured from

Hi-Media. The antibiotics used were ampicillin (10), Augmentin (20),

cefoperazone (75), cefotaxine (30), cefuroxime (30), penicillin (10)

amikacin (30), azithromycin (15), clarithromycin (15), erythromycin

(15), gentamycin (10), netilmicin (30), roxythromycin (15),

streptomycin (10), tetracycline (30) chloramphenicol (30),

ciprofloxacin(5), sparfloxacin (5), nalidixic acid (30), norfloxacin (10)

and cotrimoxazole (25).

2.17. Antibiotic susceptibility test:

Antibiotic susceptibility of the strain JC1 was determined using the

procedure of Bauer et al (1966). An activily growing 18 h culture (108

cells/ml) was inoculated on the surface of the agar plate to make a


lawn. Using alcohol steriled forcep each antibiotic disc was placed on

the dried agar plates. All the plates were incubated at 35 °C for 48 h.

The zone of inhibition arround tthe antibiotic disc was measured in

mm and compared to that of standardized chart.

2.18. Effect of streptomycin and chloramphenicol at different

concentrations: Growth and violacein production by strain JC1 was

studied in test tubes (15 × 125 mm) containing 5 ml Muller and

Hinton broth supplemented with streptomycin and chloramphenicol

at 10 µg and 30 µg concentrations respectively. 2 fold dilutions were

done to attain different concentrations. 10% of inoculum was added to

the media. Growth and violacein production was determined after 48

h incubation at 35 °C.

2.19. Violacein production through solid state fermentation:

2.19.1 Raw materials:

Different milled agricultural wastes like Bengal gram husk, black

gram husk, coconut cake, cotton seed cake, deoiled rice bran, ground

nut cake, red gram husk (different varieties), saw dust, maize bran

and wheat bran were obtained from local market are used as

substrates for SSF. The moisture content in each bran was

determined by drying it to constant weight at 100 ˚C in an oven.

2.19.2. Media employed:

Biebl and pfennig's mineral (1981) salt medium without carbon and

nitrogen source adjusted to neutral pH using 1N NaOH/1N H2SO4 was

used to moisten the solid substrate.


2.19.3. Inoculum development: culture was developed at 35 ºC in

Biebl and pfennigs mineral salt medium with bran (0.3 % w/v) as sole

carbon and nitrogen sources. An actively growing 18 h age culture

was used to inoculate into solid state wheat bran.

2.19.4. Screening for suitable substrate for violacein production

in SSF:

Violacein production in SSF by the strain JC1 was studied using

various milled agricultural wastes. Different substrates 3g were taken

in 4 inches petriplates. The production of violacein was studied by

varying the ratio of substrate to moistening agent with nutrient

concentration maintained constant. Care was taken to maintain the

final moisture content by taking into account the volume of the

inoculum and the moisture content present in bran. pH of the

moistening agent was adjusted to 7 before adding to wheat bran. An

actively growing 18 h old culture of 1.5 ml containing 108 cells / ml

was inoculated mixed well and incubated at 35 ˚C for 48 h. Violacein

produced after fermentation was extracted and estimated from

fermented bran

2.19.5. Growth curve and yield studies of the strain JC1 on

selected substrate.

Growth curve and violacein production of the strain JC1 was studied

with wheat bran and deoiled rice bran. To 10g of deoiled rice bran and

wheat bran 70% and 75% of moisture content respectively was

provided. The initial pH of the substrate was adjusted to 7 by

adjusting the pH of moistening agent. An actively growing 18 h old


culture of 1.5 ml containing 108 cells/ml was inoculated and

incubated at 35 ºC for 2d. Samples were with drawn at every 3 hour

intervels regularly from the time of inoculation. The samples collected

were analyzed for total bacterial count (c.f.u/g dry substrate) and

violacein yields (mg crude violacein /g dry substrate).

2.20. Optimization of fermentation conditions

2.20.1. Physical parameters

2.20.1.1. Effect of incubation time on violacein production: Time

course study was carried out in wheat bran with 75% initial moisture

content at 7pH and inoculated with 1.5 ml of an actively growing 18 h

culture containing 108 cells/ml. The plates were incubated for 5d.

Samples were drawn for every 24h for the analysis of biomass and

violacein.

2.20.1.2. Effect of pH: Effect of pH on violacein production by strain

JC1 was determined in the range of 3 to 10 after sterilization. pH was

adjusted by adjusting the pH of the moistening agent before adding to

3g of bran. 1.5 ml of an actively growing 18 h culture was used to

inoculate and incubated for 3d at 35 ºC with 75% moisture.

2.20.1.3. Effect of inoculum size: An 18h old actively growing

culture was centrifuged. The pellet was suspended in double distilled

water was used in the range of 0.75 to 4.5ml as an inoculum. Care

was taken to maintain constant final moisture content of 75% and

nutrient content in all the plates. The pH was adjusted to 6.5 and

incubated the plates at 35 ºC for 72h.


2.20.1.4. Effect of temperature: Experiments were conducted in

4inches petriplates containing 3g of wheat bran with 75 % moisture

content at 6.5 pH. Inoculated with 2 ml of actively grown 18 h culture

and were incubated for 3 d at various temperatures ranging between

25 ºC to 40 ºC at 5 ºC intervels.

2.20.1.5. Effect of bed thickness, mm: The effect of bed thickness

was studied in the range of 2 to 10 mm. Even though the quantity of

bran differed in each petridish the initial moisture content and

inoculum size were kept constant to 75 % and 2 ml respectively.

Plates were incubated at a pH of 6.5 at 35 ºC for 3 d.

2.20.1.6. Effect of different moistening agents: Different

moistening agents such as tap water, deionized waer, mineral salts

media and industrial effluents (corn steep liquor and molasses diluted

to 1:10) were studied. Experiments were conducted in 4inches

petriplates containing wheat bran of 2mm bed thickness with 75 %

moisture content at a pH of 6.5. 2 ml of actively grown 18 h culture

was used as inoculum and plates were incubated for 3 d at 35 ºC.

2.20.1.7. Effect of buffers: Two types of buffers such as phosphate

buffer and sodium phosphate buffer (pH 6 to 8) were used at 0.1M

concentration.

Potassium phosphate buffer (the pH range of 6-7):0.1M KH2PO4 +

0.1M K2HPO4

Sodium phosphate buffer (pH range 6 to 8):1M Na2HPO4 (57.7ml) and

1M NaH2PO4 (42.3 ml) in 900 ml distilled water. Experiments were

conducted in 4inches petriplates containing wheat bran of 2mm bed


thickness with 75 % moisture content at 6.5 pH. 2 ml of actively

grown 18 h culture was used as an inoculum and plates were

incubated for 3 d at 35 ºC.

2.20.2. Nutritional parameters

2.20.2.1. Effect of different carbon sources: Effect of carbon

sources such as glucose, glycerol, sodium pyruvate and sodium

succinate [10 mM (w/w)] was determined. Carbon sources were

supplemented through Biebl and pfennig's (1981) mineral salts

medium. To 2mm bed thickness of wheat bran 2ml of inoculum was

added and 75% moisture content was maintained. The pH of the

wheat bran was adjusted to 6 by providing Sodium phosphate buffer.

The plates were incubated at 35 ºC for 72 h. Control plate was

maintained without carbon supplementation.

2.20.2.2. Effect of different purines and pyrimidines: Adenine,

guanine and uracil were provided at 1mM (w/w) concentration

through mineral salts medium to 3g of wheat bran. The conditions

provided were 75% moisture content, pH at 6 by providing sodium

phosphate buffer, supplementation of sodium succinate at 10mM

concentration and 2ml of inoculums. The plates were incubated at

35 ºC for 3d. Control plate was maintained without purines and

pyrimidines supplementation.

2.20.2.3. Effect of different nitrogen sources on violacein

synthesis: Different inorganic (10 mM) and organic nitrogen sources

(1mM) such as NH4Cl, KNO3, (NH4)2H2PO4, (NH4)2SO4, NaNO2, NaNO3,

DL-Alanine, L-Aspartic acid, Glutamine, Glycine, Phenyl alanine, L-


serine, L-tryptophan, DL-methionine, Urea, Peptone, yeast extract and

beef extract were supplemented through Biebl and pfennig's (1981)

mineral salts medium. Control was maintained without any nitrogen

sources to compare the effect of nitrogen sources on violacein

production. The conditions provided were 75% moisture content, pH

at 6 by providing sodium phosphate buffer, supplementation of

sodium succinate at 10 mM and adenine at 1mM (w/w)

concentrations and 2ml of inoculum. The plates were incubated at 35

ºC for 3d.

2.20.2.4. Effect of different precursors on violacein synthesis: A

study was carried out with L-tryptophan, indole and pyrrole

supplemented at 1mM (wt/wt) concentration. L-tryptophan was

provided through mineral salts medium before autoclaving. Indole and

pyrrole were added to the mineral salts medium after autoclaving from

a filter sterilized concentrated stock solution in ethylacetate (99% v/v)

pH changes were neutralized with 1N NaOH and 0.5 N HCl. Results

were compared with control plates without any compound.

2.21. Small scale fermentation: After optimizing the conditions of

crude violacein production at laboratory level in petriplates. Small

scale fermentation studies of SSF were carried out at one kg level in

34×25.5cm plastic trays One kg of wheat bran was weighed and mixed

with mineral salt solution and sterilized at 121 ºC for 20 min. After

sterilization wheat bran was cooled to room temperature and

inoculated with 20% (v/w) of actively growing 18h culture of strain

JC1. After inoculation, wheat bran was mixed thoroughly by using a


sterile glass rod and was spread uniformly and the trays were

incubated in an incubator at 35 ºC for 3d.

2.22. Analytical methods:

2.22.1. Determination of growth and biomass:

2.22.1.1. Optical density: Increase in optical density (OD)

turbidometry was used to monitor the growth of purple colored

bacterial strain JC1 in broth cultures. Optical density of the bacterial

suspension was directly measured in a Systronics make (model 112)

colorimeter at 720nm (filter 8) against uninoculated medium as blank.

2.22.1.2. Determination of dry weight: Biomass was estimated in

terms of dry weight. Dry weight was determined from an OD720 versus

dry weight graph. An aliquot of the logarithmic culture was

centrifuged at 10,000 rpm for 10minutes and the pellet was washed

(twice) and suspended in distilled water. Known dilutions of the

concentrated cell suspension were made and their optical densities

determined, transferred to previously weighed aluminum boats, dried

to constant weight at 60 ºC and weighed (Dhona, 100DS). An O.D

verses dry weight graph was plotted and an emperical relationship

between the two was determined. Further measurements of dry weight

was done by taking the OD of cell suspensions at 720 nm and

calculating the dry weight from the emperical relationship derived for

the test strain JC1 (0.1 O.D = 0.26 mg dry weight/ml).


Fig. 2.1. O.D. verses dry weight geaph of strain JC1

2.22.1.3. Bacterial enumeration in SSF: Bacterial cells were

enumerated from the samples by adding 1g (wet weight) fermented

substrate to 100 ml of the distilled water in 250 ml flask and

vigorously shaken for 5 minutes in vortex mixer. Later, 1ml from the

flask was drawn for serial dilutions and 0.1 ml from the each dilution

was plated on nutrient agar media by spread plate method. The

number of colony forming units was counted and the biomass

expressed as colony forming units (cfu) gram dry substrate-1.

2.22.2. Extraction and Determination of violacein content:

The violacein extracted was estimated by the O.D versus

concentration of crude pigment. A culture of strain JC1 in 1 L

nutrient broth was centrifuged at 8000 rpm for 15 min. The pellet

collected was washed thrice with double distilled water. 50ml of

acetone was added to the dry pellet and mixed in orbital shaker for 20

min. Acetone was evaporated in rota vapour bath at 60 ºC and the


crude pigment was dried in a hot air oven at 50 ºC (Rettori and

Duran, 1998). An O.D versus dry weight graph was plotted and an

emperical relationship between the two was determined. Further

measurement of dry weight was done by taking the OD of violacein

extract at 570 nm and calculating the dry weight from the emperical

relationship. (0.1 O.D = 2.1 µg dry weight/ml).

2.22.3. Solubility of the crude violacein pigment in different

solvent systems: To 1g wet weight of the fermented wheat bran

different solvents 5ml such as acetone, Dimethyl sulphoxide, ethanol

and methanol were added and mixed for 10 min using cyclomixer

(Remi CM 101) at room temperature. The extract was centrifuged at

10,000 rpm for 15 min. and the O.D of the supernatant was recorded

to determine the maximum solubility of the pigment in different

solvent systems.

2.22.4. Moisture content estimation in various brans:

The moisture content present in each substrate was determined by

drying it to constant weight (Robert 1967). 1g of the bran was weighed

in a petriplate and the mass was noted. The plate was placed in the

oven and sample was dried at 105 ºC to 110 ºC to constant mass. The

weight was noted accurately and calculated as follows.

100(M1-M2) Moisture content, percent by mass = M1

M1= Mass in grams of the sample before drying

M2= Mass in grams of the sample on drying to constant mass.


2.22.5. Extraction and estimation of crude violacein produced in

solid state fermentation (SSF):

Crude violacein produced after fermentation was extracted from 1g

fermented wheat bran with 5ml of acetone by vigorous mixing in

cyclomixer. The extract was centrifuged at 10,000 rpm for 15 min.

Supernatant was collected and the optical density of the supernatant

was measured at 570 nm in a colorimeter and quantified from a

standard graph. The amount of violacein produced was expressed as

mg crude violacein g-1 dry substrate.

2.22.6. Separation of violacein:

2.22.6.1. Liquid – Liquid extraction Violacein produced after

fermentation was extracted from 5g of fermented bran with 30 ml of

acetone by vigorous mixing in orbital shaker 150 rpm for 20 min. The

extract was centrifuged at 10,000 rpm for 15 minutes. Supernatant

collected was evaporated in rota vapour bath at 60 ˚C and dried at 50

˚C in hot air oven. The purple pigment was re dissolved in methanol

(30 ml) and collected into separating funnel and 10 ml of ethyl acetate

was added to it. Pigment was extracted using ethyl acetate (DeMoss

and Evans, 1959). The mixture was then shaken and after releasing

its gas the funnel was placed into the holder in order to separate the

organic and aqueous phases. The organic layer which contains the

pigments was collected and subjected to evaporation at 50 ˚C in a

rotavapour bath ( ). The pigment deposit was re dissolved in methanol

(10 ml) and used for column chromatography separation.


2.22.6.2. Column Chromatography Column chromatography was

carried out using a column with the following diameter: 12 cm height

and 18 mm width. The column was then packed with neutral silica gel

(column chromatography grade) and washed twice with 70% (v/v)

methanol. The concentrated sample obtained in methanol was placed

on the top of the column and separation initiated with the addition of

solvent. Two different fractions were collected.

2.22.7. Purity determination

2.22.7.1. UV-VIS spectrophotometric analysis

UV-VIS analysis was carried out for sample extracted from methanol.

UV-VIS sample was prepared by diluting the concentrated methanol

sample to 10 fold.

2.22.7.2. Thin layer chromatography analysis (TLC): Purity of the

violacein produced was detected using thin layer chromatography

(TLC). Plates coated with silica gel at a thickness of 0.1mm from

Merck was used for the detection of violacein. Chloroform: acetone:

pyridine (5:4:1) was used as a solvent system. Methanolic extract of

the pigment was spotted on the plates and developed. Later the plates

were exposed to iodine and also sprayed with sulphuric acid followed

by heating at 110 ˚C to identify the impurities.

2.23. Stability test of pigment towards pH changes

The effect of pH on the stability of the pigment extracted from wheat

bran was carried out by adjusting the pH to extreme acidic pH (2) and

extreme alkaline pH (10) using 0.1 N HCl and 0.1 N NaOH

respectively.


2.24. Dyeing of the cheese cloth with pigment extract from SSF:

The acetone extract of crude violacein produced by the strain JC1.

through solid state fermentation in wheat bran was used for dyeing

cheese cloth by dipping it for 30 min at room temperature [uer et al

(1966)] and drying under shade at ambient room temperature.

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