chapter 3 materials and methods -...

Chapter 3 Materials and Methods

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3.1. Chemicals and microbiological media

All chemicals were of highest purity and of analytical grade. ABTS (2,2’-

Azinobis,3-ethylbenzothiazoline-6-sulfonic acid) was obtained from Sigma Chemicals

Company (St. Louis, MO, USA). NADH (Nicotinamide adenine dinucleotide reduced

disodium salt), n-propanol and Tween 80 were obtained from Sisco Research

Laboratories, India. 3,4-dimethoxy benzyl alcohol (veratryl alcohol), biochemical test kit,

Bacteriological peptone, yeast extract, glucose, sucrose, mannitol, NaCl, Potato dextrose

agar and agar powder were obtained from Hi-media laboratory, India. Ethyl acetate,

chloroform, methanol hydrogen peroxide, absolute ethanol, hydrochloric acid, sulphuric

acid, orcinol, ninhydrin, n-hexadecane and glutaraldehyde were obtained from sd-Fine

Chemicals, Mumbai, India. Trichloroacetic acid was obtained from Thomas Baker

Chemicals Limited, Mumbai, India. Urea was obtained from local market, Kolhapur,

India. Textile dye Brown 3REL was obtained from Manapasand textile processors,

Ichalkaranji, India.

3.2. Microorganisms

The cultures of Pseudomonas desmolyticum NCIM 2112, Escherichia coli NCIM

2089, Pseudomonas aeruginosa NCIM 2036, Proteus vulgaris NCIM 2027, Salmonella

typhimurium NCIM 2501, Micrococcus aureus NCIM 5021, and Bacillus subtilis NCIM

2010 (maintained on nutrient agar slants), Candida utilis NCIM 3469, Aspergillus niger

NCIM 545, and Penicillium chrysogenum NCIM 723 (maintained on potato dextrose agar

slants) were obtained from National Collection for Industrial Microorganisms (NCIM),

Pune, India. Bacillus sp. VUS NCIM 5342 was isolated from textile dye contaminated

soil in our laboratory and deposited in NCIM, Pune, India (Dawkar et. al., 2008). Pure

cultures were maintained on their respective medium at 4oC. Bacillus cereus strain MUJ

and Enterobacter sp. MS16 (maintained on nutrient agar slants) isolated and used in the

present study were deposited at NCIM with the deposition number NCIM 5391 and 5392

respectively.


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3.3. Isolation of biosurfactant producing microorganisms

The biosurfactant producing microorganisms were isolated by enrichment culture

technique. Soil samples were collected from local petrol pump. Briefly, 1 g soil sample

was inoculated into 100 ml mineral salt medium (MSM) with composition (per liter

distilled water) 1 g KH2PO4, 1 g K2HPO4, 0.2 g MgSO4.7H2O, 0.2 g CaCl2

.2H2O, 0.05 g

FeCl3.6H2O, 1 g NH4NO3. Diesel (2%, v/v) was used as carbon source and incubated at

30oC on a rotary shaker (120 rpm) for 4 days. After 4 days, 1 ml of the culture was

transferred to fresh MSM medium containing diesel (2%, v/v) and re-incubated for

another 4 days. Following five cycles of such enrichment, 1 ml of the culture was diluted

and plated on MSM agar plates containing diesel as sole carbon source. The bacterial

colonies obtained were further purified by streaking on Nutrient agar plates. Two isolates

capable of producing biosurfactant with lower surface tension were selected for further

studies. Selected isolates were deposited with National Center for Industrial

Microorganisms (NCIM), Pune, India. The isolates were maintained on nutrient agar

slants at 4oC.

3.4. Identification of microorganisms

The isolates were identified by morphological and biochemical tests and 16S

rRNA sequence analysis. 16S rRNA gene sequencing of the isolated microorganisms

were carried out at Chromous Biotech Pvt. Ltd., Bangalore, India. All the biochemical

tests were performed according to the methods described in Bergey’s Manual of

Systematic Bacteriology.

3.5. Phylogenic analysis

The partial 16S rRNA nucleotide sequence was studied for homology, on NCBI

server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using BLASTn tool. The phylogenetic tree

was linearized assuming equal evolutionary rates in all lineages (Takezaki et. al., 2004).

The clock calibration to convert distance to time was 0.2 (time per node height). The tree

was drawn to scale, with branch lengths in the same units as those of the evolutionary

distances used to infer the phylogenetic tree. The evolutionary distances were computed


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using the Maximum Composite Likelihood method (Tamura et. al., 2004) and were in the

units of the number of base substitutions per site. Codon positions included were

1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated

from the dataset (Complete deletion option). The evolutionary history was inferred using

the Neighbor-Joining method (Saitou and Nei, 1987). The percentage of replicate trees in

which the associated taxa clustered together in the bootstrap test (500 replicates) was

shown next to the branches (Felsenstein, 1985).

3.6. Production and characterization of rhamnolipid biosurfactant produced by

Pseudomonas desmolyticum NCIM 2112

3.6.1. CTAB-methylene blue agar plate assay

Pseudomonas desmolyticum NCIM 2112 (Pd 2112) was initially assayed for

rhamnolipid production using mineral salt cetyltrimethylammoniumbromide (CTAB)-

methylene blue agar plate method (CTAB 0.2 mg/ml and methylene blue 5 µg/ml)

(Siegmund and Wagner, 1991). Pd 2112 was grown for 24 h (OD660 0.1) in MSM under

appropriate growth conditions. Shallow wells were cut into the surface of the indicator

plates. Ten micro liters of the appropriate culture was placed into each well. The plates

were then incubated at 30oC and checked periodically over a 24 h to 48 h time period.

The production of rhamnolipid was confirmed by the formation of dark blue halos around

the colonies.

3.6.2. Cultivation conditions for rhamnolipid production

Pd 2112 was grown in the nutrient broth medium for 24 h at 30oC. For

biosurfactant production, 2% inoculum of Pd 2112 was added in 100 ml MSM containing

2% (v/v) hexadecane. Cultivations were performed in 250 ml Erlenmeyer flasks and

incubated at 30oC in a shaking incubator at 120 rpm for 168 h to obtain the highest

microbial growth and rhamnolipid concentrations.


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3.6.3. Medium optimization for rhamnolipid production

Rhamnolipid production was studied using different carbon sources supplied in

MSM medium. Glucose, sucrose, mannitol, and glycerol were used individually (2 g

each) and with hexadecane at 2:1.54 (w/w) ratio. Effects of different vegetable oils (2%)

on rhamnolipid production were studied using groundnut oil, sunflower oil, and corn oil

in MSM medium. In addition, the effect of inorganic nitrogen sources NaNO3 and

(NH4)2SO4, and organic source urea were studied at 1 g/l concentration in the glucose-

hexadecane-MSM medium. The effect of carbon/nitrogen (C/N, w/w) ratio on the

rhamnolipid production was studied using glucose-hexadecane and NaNO3 as optimized

carbon and nitrogen sources respectively. The C/N ratio of 10 to 50 per litre was studied

by keeping nitrogen concentration constant. Culture flasks were incubated for 8 days and

cell growth, rhamnolipid production, and surface tension reduction were monitored

during the course of batch fermentation.

3.6.4. Screening of unconventional substrates for rhamnolipid production

Pd 2112 was grown in the nutrient broth medium for 24 h at 30oC. For

biosurfactant production, 2% inoculum of Pd 2112 was added in MSM containing natural

wastes as carbon source at the concentration of 1% (w/v). The wastes utilized include

molasses, fried oil, wheat bran and rice husks. Wheat bran and rice husk were dried and

powdered before use. Cultivations were performed in 250 ml Erlenmeyer flasks

containing 100 ml sterile MSM and incubated at 30oC in a shaking incubator at 120 rpm

for 168 h. Culture flasks were monitored for cell growth, rhamnolipid production, and

surface tension reduction during the course of batch fermentation. Cell concentration was

determined by measuring optical density at 660 nm using a UV visible spectrophotometer

(Hitachi U-2800). The dry cell weight was calculated using a predetermined correlation

between OD at 660 nm and dry cell weight.

3.6.5. Extraction and quantification of rhamnolipid

Pd 2112 cells were separated from rhamnolipid production medium by

centrifugation at 8500 rpm at 4oC for 20 min. The clear supernatant was further treated by


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acidification to pH 2.0 using 6.0 M HCl and incubated at 4oC for ~12 h to precipitate

biosurfactants. After centrifugation at 10,000 rpm for 20 min, the precipitate was

dissolved in 0.1 M NaHCO3, followed by rhamnolipid extraction using

chloroform:methanol (2:1 v/v) at room temperature. The organic phase was removed

using a rotary evaporator yielding a viscous honey-colored rhamnolipid product.

The concentration of rhamnose produced was determined using orcinol method

(Chandrasekaran and Bemiller, 1980). In brief, 333 µl culture supernatant was evaporated

to dryness and 0.5 ml of distilled water was added to it. In 100 µl samples 900 µl of

0.19% orcinol, prepared in 53% H2SO4 (v/v) was added. After heating at 80oC for 30 min,

all samples were cooled at room temperature and OD421 was measured

spectrophotometrically. Rhamnolipid concentration was calculated using L-rhamnose as

standard and expressed as rhamnose equivalents (RE).

3.6.6. Determination of surface tension and emulsification index

Surface tension of cell-free culture broth was measured according to the Du Nouy

ring method using a surface tensiometer (Jencon Company, India). The tensiometer was

calibrated before each measurement using distilled water. The ring was cleaned with

benzene at low heating for each measurement. Samples of the culture media were

centrifuged at 8000 rpm for 20 min. The ring was introduced in 50 ml cell-free culture

broth applying an ascending force until the ring was pulled out from the culture broth,

and the surface tension was recorded from the graduated dial.

Emulsification index (E24) was determined by the addition of 4 ml hydrophobic

substrate to equal volume of cell free culture broth, mixed with a vortex for 2 min and left

to stand for 24 h. Emulsification activity E24 (%) was determined using following

equation:

Eଶସ (%) =The height of emulsion layer

The height of total solution× 100

The hydrophobic substrates like hexadecane, kerosene, diesel, toluene, groundnut

oil, sunflower oil, and corn oil were tested for emulsification assay.


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3.6.7. Crystalline appearance of rhamnolipid

The crystalline appearance of the extracted rhamnolipid was determined by

examining the precipitated crystalline rhamnolipid, recovered from an acidified,

overnight incubated rhamnolipid solution, under a light microscope at a magnification of

×40, and the films were photographed.

3.6.8. Structural characterization of rhamnolipid

3.6.8.1. High performance thin layer chromatography (HPTLC)

High performance thin layer chromatography (HPTLC) was carried out using a

CAMAG thin layer chromatography system composed of an automatic TLC sampler

(CAMAG Linomat 5), automatic development chamber (CAMAG ADC2), detector

(CAMAG TLC Scanner 3), and an electronic integrator (winCATS software). An aliquot

(15 µl) of the crude rhamnolipid sample was band applied (mm) on to an HPTLC

precoated silica gel 60F254 plate (10 × 10 cm). The sample was loaded at a dosage speed

of 50 nl/s under nitrogen stream. The sample was developed (ascending) using 10 ml of

the mobile phase of CHCl3/CH3OH/H2O (65:25:4, v/v/v), in plates preconditioned for 3

min, to a migration distance of 85 mm. The plate was dried, sprayed with orcinol reagent

(0.19% orcinol in 53% H2SO4), and then put in a hot-air oven at 120oC for 15 min. The

developed chromatogram was scanned in remission type, absorbance mode at 550 nm.

The signals recovered from the scanner were integrated into absorbance chromatograms

from which peak area was automatically calculated using the winCATS software. Based

on the Rf value, the band of rhmanolipid was scratched off from the other HPTLC plate

after developing and was used for further characterizations.

3.6.8.2. Nuclear magnetic resonance (NMR)

1H and 13C-Nuclear magnetic resonance (NMR) spectra were obtained using an

OXFORD NMR400 spectrometer. The HPTLC purified rhamnolipids were deuterium-

exchanged by repeated evaporation in methanol–D2O (1:1, v/v). The NMR spectra were


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determined in deuterated methanol and DMSO at 30oC using tetramethylsilane (TMS) as

an internal standard.

3.6.8.3. Fourier transform infrared spectroscopy (FTIR)

The Fourier transform infrared spectroscopy (FTIR; Perkin-Elmer, Spectrum one)

analysis of HPTLC purified rhamnolipid was done in the mid IR region of 400-4000 cm-1

with 20 scan speed. The samples were mixed with spectroscopically pure KBr in the ratio

of 5:95. The pellets were fixed in sample holder for an analysis.

3.6.8.4. Gas chromatography-mass spectrometry (GC-MS)

The Gas chromatography-mass spectrometry (GC-MS) analyses were performed

using Varian 4000 mass spectrometer equipped with an integrated chromatograph with a

DB-5 column. Helium was used as carrier gas at a flow rate of 1 ml/min. The injector

temperature was maintained at 280oC with oven conditions as: 80oC kept constant for 2

min; increased up to 200oC with 10oC/min; rose up to 280oC with 20oC/min rate. The

negative ion mode was used throughout and scans were initiated over the 50-1000 m/z

range.

3.6.9. Application of biosurfactant in dye degradation

3.6.9.1. Decolorization of Brown 3REL by Bacillus sp. VUS NCIM 5342 in the

presence of rhamnolipid

Bacillus sp. VUS strain was grown in 250 ml Erlenmeyer flask containing 100 ml

nutrient broth at static condition for 24 h at 40oC. Before addition of dye, cells were

permeabilized with Pd 2112 produced mono-rhamnolipid (Pd mono-rhamnolipid, 1

mg/ml) for 30 min as described by Galabova et. al. (1993). A synthetic surfactant, Tween

80 (1 mg/ml, v/v) was used to compare its effect with that of mono-rhamnolipid on

decolorization process. Further, Brown 3REL (procured from Manpasand textile industry,

Ichalkaranji, India) (50 mg/l) was added in the culture medium and incubated at same

conditions. Three milliliter aliquot of the culture media was withdrawn at different time


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intervals, centrifuged at 6000 rpm for 20 min, and decolorization was determined by

measuring the change in absorbance of culture supernatants at OD440 (Hitachi U-2800).

The percent decolorization was determined as follows:

Decolorization (%) =(Initial absorbance)– (observed absorbance)

(Initial absorbance)× 100

All decolorization experiments were performed in three sets. Abiotic controls

were always included.

3.6.9.2. Effect of mono-rhamnolipid and dye concentrations on decolorization

The various concentrations of mono-rhamnolipid (0.5-2 mg/ml) and Brown 3REL

(50-250 mg/l) were added in nutrient broth in order to evaluate their effect on

decolorization ability of Bacillus sp. VUS. Percent decolorization and dry cell weight

were measured at different time intervals. The correlation between the specific

decolorization rate and dye concentration was described by Michaelis Menten kinetics

(vdye = vdye,max [Dye] / Km + [Dye]); where vdye max and Km denoted maximum

decolorization rate and Michaelis Menten constant respectively and [Dye] represents the

concentration of Brown 3REL (mg/l).

3.6.9.3. Enzyme status during dye decolorization

3.6.9.3.1. Preparation of cell free extract

Bacillus sp. VUS was grown in nutrient broth for 24 h at 40oC, harvested by

centrifugation (6000 rpm, 20 min) and suspended in 50 mM potassium phosphate buffer

(pH 7.4) and sonicated (30 seconds, 60 amplitude, 10 strokes) at 4oC. This extract was

used as enzyme source without centrifugation.

3.6.9.3.2. Enzyme assays

All enzyme activities were assayed in cell free extract as well as culture

supernatant at room temperature.


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a. Lignin peroxidase (LiP)

Lip activity was assayed by the procedure of Shanmugam et al. (1999). It was

determined by monitoring the formation of propanaldehyde at 310 nm in a reaction

mixture (2.5 ml) containing 100 mM n-propanol, 250 mM tartaric acid and 10 mM H2O2.

One unit of enzyme activity corresponds to change in 0.1 U of absorbance per minute.

Enzyme activity was expressed as units of enzyme/min/mg of protein.

b. Laccase

Laccase activity was determined by monitoring the formation of oxidized ABTS

at 420 nm in 2.0 ml reaction mixture containing 10% ABTS in 100 mM acetate buffer

(pH 4.8) (Hatvani and Mecs, 2001). Enzyme activity was expressed as units of

enzyme/min/mg of protein.

c. Tyrosinase

Tyrosinase activity was determined by the method of Zhang and Flurkey (1997).

It was determined by monitoring the formation of catechol quinone at 495 nm in a

reaction mixture (2.0 ml) containing 0.01% catechol in 100 mM potassium phosphate

buffer (pH 6.8).

d. Veratryl alcohol oxidase

Veratryl alcohol oxidase (VAO) was determined in a reaction mixture (2 ml)

containing 4 mM veratryl alcohol in 0.05 M citrate phosphate buffer (pH 3) by

monitoring the formation of veratraldehyde at OD310 (Jadhav et. al., 2009).

e. NADH-DCIP reductase

NADH-DCIP reductase assay mixture contained 25 µM DCIP, 50 µM NADH in

50 mM potassium phosphate buffer (pH 7.4) and 0.1 ml of enzyme solution in a total

volume of 5.0 ml. the decrease in dolor intensity of DCIP was observed at 595 nm. The

DCIP reduction was calculated using the extinction coefficient (ε) of 19 mM-1 cm-1


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(Salokhe and Govindwar, 1999). Enzyme activity was defined in terms of units. One unit

of enzyme activity was defined as amount of enzyme required to reduce 1 µM of

DCIP/min.

f. Riboflavin reductase [NAD(P)H:Flavin oxidoreductase]

Riboflavin reductase NAD (P)H:Flavin oxidoreductase was measured by

monitoring the decrease in absorbance at 340 nm. Cell free extract was added to a

solution (final volume, 1 ml) containing 100 M of Tris-HCl (pH 7.5), 25 M of

NADPH and 0.003 U l-1 of riboflavin. Reaction rates were calculated by using a molar

extinction coefficient of 6.3 mM l-1cm-1.

All enzyme assays were carried out at 30oC where reference blanks contained all

components except the enzyme. All enzyme activities were run in triplicate and average

rates calculated.

3.6.9.4. Extraction and analysis of dye degradation products

3.6.9.4.1. Extraction of metabolites for analysis

Culture broth was centrifuged at 10,000 rpm for 20 min after complete

decolorization of dye. Equal volume of ethyl acetate was used to extract metabolites from

clear supernatant. The extracts were dried over anhydrous Na2SO4 and evaporated to

dryness in rotary evaporator.

3.6.9.4.2. UV-Vis spectrophotometric analysis

UV-Vis spectrophotometric analysis was carried out at initial time (zero time)

when dye was added to the medium and after complete degradation of dye. Scans of clear

supernatant broth were taken in the visible region of 400-800 nm.


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3.6.9.4.3. High performance liquid chromatography (HPLC)

The biodegraded product was characterized by HPLC analysis which was carried

out (Waters model no. 2690) on C18 column (symmetry, 4.6 × 250 mm) with methanol as

mobile phase at flow rate of 0.75 ml/min and the UV detector at 316 nm for Brown

3REL.

3.6.9.4.4. Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR analysis was done to characterize the biodegraded products of Brown

3REL formed by mono-rhamnolipid treated cells and compared with control dye.

Analysis was carried out in the mid IR region of 400-4000 cm-1 with 16 scan speed. The

samples were mixed with spectroscopically pure KBr in the ratio of 5:95, pellets were

fixed in sample holder, and the analyses were carried out.

3.6.9.5. Scanning electron microscopy of biosurfactant treated cells

Permeabilized cells and control cells were fixed in 2% (w/v) glutaraldehyde for 2

h at 4oC, washed with saline solution, and dehydrated for 5 min in increasing ethanol

concentrations (30, 50, 70, and 90% v/v) and for 15 minutes in absolute ethanol. The

samples were air dried then coated with gold in argon atmosphere to an approximate

thickness of 50 nm with the help of sputtering. The Scanning electron microscopy (SEM)

observations were carried out using a scanning device JEOL JSM-6360.

3.6.9.6. Effect of rhamnolipid on the dye decolorization activity of purified laccase of

Pseudomonas desmolyticum NCIM 2112

Pd 2112 was grown in nutrient broth for 12 h (absorbance at 660 nm: 0.7) and

then inoculated in 3 l nutrient medium (10% inoculum) and incubated for further 12 h at

30oC. Cells were collected by centrifugation at 8000 rpm for 15 min and suspended in 50

mM sodium phosphate buffer (pH 7.0) (buffer A) (150 mg cells/ml) containing lysozyme

at 5 mg/ml concentration. Cells were further incubated at 37oC for 90 min in water bath

and then disrupted by sonication by keeping sonifier output at 40 amplitude maintaining


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temperature below 4oC and giving 8-10 strokes each of 5 s with 2 min interval. This cell

free extract was solubilized in cholic acid (0.33 mg/mg protein) on magnetic stirrer at 5oC

for 30 min. The cell lysate obtained was centrifuged twice at 15000 rpm for 30 min at

4oC. The clear supernatant was collected and used as crude source of laccase. The

supernatant containing laccase activity 0.04 U/mg protein/min was heated at 60◦C for 10

min and centrifuged at 8000 rpm for 20min. The clear supernatant obtained after

centrifugation was loaded on a DEAE cellulose fast flow column (15mm×120mm),

equilibrated with buffer A. The column was washed with the same buffer by two times of

the column volume and the enzyme was eluted with a linear gradient of 0–1.0 M NaCl.

Fractions containing laccase activity were pooled and dialyzed against 1 mM sodium

phosphate buffer (pH 6.0). The dialyzed sample was loaded on Biogel P100 column

(10mm×500mm) equilibrated with 50 mM sodium phosphate buffer (pH 6.0, buffer B).

The protein elution was carried with the same buffer at 6 ml/h flow rate. Fractions

containing laccase activity were used for further studies (Kalme et. al., 2009).

To study the effect of rhamnolipid on the dye decolorization ability of purified

laccase, decolorization studies were carried out in absence and presence of rhamnolipid.

The decolorization reaction was carried out at 30◦C for 24 h in 2 ml reactions mixture

containing 100 mg/l dye prepared in 50 mM acetate buffer (pH 4.8) and 0.5 U/ml purified

laccase. To study the effect of rhamnolipid on the enzyme 0.5 ml of 1mg/ml rhamnolipid

was added in the above reaction mixture. Control containing heat-denatured enzyme was

used to measure decolorization of dye at different time interval. The decolorization was

monitored by scanning the UV–vis spectrum between 200–800 nm using Hitachi (U-

2800) double beam spectrophotometer.

3.7. Production and characterization of lipopeptide biosurfactant cerefactin

produced by Bacillus cereus NCIM 5391

3.7.1. Biosurfactant production using various carbon sources

Bacillus cereus NCIM 5391 was grown in nutrient broth medium for 24 h at 30oC.

For biosurfactant production, 2% inoculum of B. cereus was added in MSM containing

natural wastes (1%, w/v) as carbon sources. The carbon sources used were groundnut oil


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cake, sunflower oil cake, molasses and fried oil. Cultivations were performed in 250 ml

Erlenmeyer flasks containing 100 ml sterile MSM and carbon source and incubated at

30oC in a shaking incubator at 120 rpm for 168 h to obtain the highest microbial and

biosurfactant concentrations. To determine dry cell weight, 10 ml culture broth was

centrifuged at 8000 rpm for 20 min and cell pellets were washed twice with distilled

water and dried by heating at 50oC until constant weight attained.

3.7.2. Purification and quantification of lipopeptide biosurfactant

The fermentation medium was centrifuged at 8500 rpm, 4oC for 20 min. The clear

supernatant was acidified to pH 2.0 using 6.0 M HCl and was left overnight at 4oC for

precipitation of biosurfactant. The pellets were obtained by centrifugation at 10000 rpm

for 20 min at 4oC. The crude lipopeptide was extracted three times using

chloroform:methanol (2:1, v/v) solvent and dried over rotary evaporator. The residual

lipopeptide was recovered with methanol, placed in a vial and weighed after evaporation

of the solvent.

3.7.3. Surface tension measurement and emulsification index

Surface tension of cell-free culture broth was measured by the method described

in the section 3.6.6. Similarly, emulsification index was determined for kerosene, diesel,

hexadecane, hexane, toluene, groundnut oil and olive oil as stated in the section 3.6.6.

3.7.4. Structural characterization of lipopeptide biosurfactant

3.7.4.1. Thin layer chromatography (TLC)

Ten microliter of biosurfactant in methanol was spotted on a silica gel thin layer

chromatography (TLC) plate (Silica gel 60; Merck, Darmstadt, Germany). The

compounds were separated using a mobile phase of chloroform/methanol/water (65:25:4,

v/v/v). For the detection of peptides, the dry plates were sprayed with 0.25% ninhydrin in

acetone and kept at 105oC for 5 min. For the detection of lipids, the plates were exposed

to iodine. For further analysis, TLC purified lipopeptide biosurfactant was used.


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3.7.4.2. FTIR

The fourier transform infrared spectroscopy (FTIR; Perkin-Elmer, Spectrum one)

analysis of lipopeptide was done as mentioned in the section 3.6.8.3.

3.7.4.3. MALDI-TOF analysis

MALDI-TOF analysis of lipopeptide was carried using Synapt high definition

mass spectrometry (Waters, USA). The lipopeptide was mixed with an equal volume of

matrix solution containing 0.1% α-cyano-4-hydroxycinnamic acid in acetonitrile-water-

trifluoroacetic acid (50:50:0.01, v/v/v). The masses in the range 600–4000 Da were

measured.

3.7.4.4. Fatty acid and amino acid analysis

Fatty acid and amino acid analysis were done by acid hydrolysis of the

lipopeptide. Purified lipopeptide (100 µg) was hydrolyzed with 6.0 M HCl at 100oC for

24 h. The solution was cooled at room temperature and fatty acids (FAs) were extracted

at least three times using ether. The FAs were esterified with 3.0 M HCl in methanol at

100oC for 1 h. After cooling, the fatty acid methyl esters (FAMEs) were extracted using

hexane and concentrated by evaporation at room temperature. The FAMEs were analyzed

on a GC–MS apparatus (Shimazdu 2010 MS Engine) equipped with integrated gas

chromatograph with a HP1 column (60 m long, 0.25 mm i.d., nonpolar). Helium was

used as carrier gas at a flow rate of 1 ml min-1. The injector temperature was maintained

at 280oC with oven conditions as: 80oC kept constant for 2 min; increased up to 200oC

with 10oC min-1; rose up to 280oC with 20oC min-1 rate. The negative ion mode was used

throughout and scans were initiated over 50-1000 m/z range.

The aqueous fraction containing free amino acids was subjected to TLC analysis

with the solvent system n-butanol/acetic acid/water (4:1:1, v/v/v). For visualization of

amino acids, the dry plates were sprayed with a solution of 0.25% ninhydrin in acetone

and kept at 105oC for 5 min.


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3.7.5. Applications of cerefactin

3.7.5.1. Antimicrobial assay

The antimicrobial activity of the isolated lipopeptide against several pathogenic

microbial strains was determined by broth micro-dilution assay in 96-well flat-bottom

microtiter plates (Hi-Media, India). The bactrial and fungal strains used in the study

include Pseudomonas aeruginosa NCIM 2036, Proteus vulgaris NCIM 2027, Salmonella

typhimurium NCIM 2501, Micrococcus aureus NCIM 5021, and Bacillus subtilis NCIM

2010 (cultured in nutrient broth), Candida utilis NCIM 3469, Aspergillus niger NCIM

545, and Penicillium chrysogenum NCIM 723 (cultured in potato dextrose broth) For

each strain, appropriate medium was used as described in methods section 2.1; briefly,

125 µl of sterile double strength medium (nutrient broth and potato dextrose broth) were

placed into the first column of the 96-well microtiter plate, and 125 µl of sterile single

strength growth medium in the remaining wells. Subsequently, 125 µl of lipopeptide

solution in PBS (50 mg/ml) was added to the first column of the microtiter plate and

mixed with the medium; this results in a lipopeptide concentration of 25 mg/ml; serially,

125 µl were transferred to the subsequent wells, discarding 125 µl of the mixture in the

tenth column. This process results in two-fold serial dilutions of the lipopeptide in the

first 10 columns (25–0.048 mg/ml). Columns 11 and 12 did not contain biosurfactant and

served as negative and growth controls, respectively. All the wells (except for the 11th

column) were inoculated with 2.5 µl of a pre-culture grown overnight in nutrient broth at

37oC. Microtiter plates were covered and incubated for 48 h at 37oC. After 48 h of

incubation, the optical density at 600 nm was determined for each well. The percent

growth of each microorganism at different biosurfactant concentrations were calculated

as:

%Growthୡ ൌ ൬ODୡ

OD୭൰ൈ ͳͲͲ

Where, ODc represents the optical density of the well with a biosurfactant

concentration c and ODo is the optical density of the control well (without biosurfactant).

Triplicate assays were performed at all the biosurfactant concentrations for each strain.


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3.7.5.2. Anti-adhesion assays

The anti-adhesive activity of the isolated lipopeptide against several microbial

strains (the same microorganisms that were used in the antimicrobial assay) was

determined according to the procedure described by Heinemann et al. (2000). Briefly, the

wells of a sterile 96-well flat-bottomed microtiter plates (HiMedia) were filled with 200

µl of a lipopeptide solution in PBS at concentrations ranging from 3.12 to 25 mg/ml. The

plate was incubated for 18 h at 4oC and subsequently washed twice with PBS. Control

wells contained PBS buffer only. An aliquot of 200 µl of a washed bacterial suspension

in PBS was added and incubated in the wells for 4 h at 4oC. Unattached microorganisms

were removed by washing the wells three times with PBS. The adherent microorganisms

were fixed with 200 µl of 99% methanol per well and after 15 min the plates were

emptied and left to dry. Then the wells were stained for 5 min with 200 µl of 2% crystal

violet used for Gram staining. Excess stain was rinsed by placing the plate under running

distilled water. Subsequently, the plates were air dried, the dye bound to the adherent

microorganisms was resolubilized with 200 µl of 33% (v/v) glacial acetic acid per well

and the optical density for each well was determined at 595 nm. The inhibition of

microbial adhesion activity at different lipopeptide concentrations was calculated as:

% Inhibition of microbial adhesionୡ = ͳെ ൬ODୡ

OD୭൰൨× 100

Where, ODc represents the optical density of the well with a biosurfactant

concentration c and ODo is the optical density of the control well. Triplicate assays were

performed at all the biosurfactant concentrations for each strain.

3.7.5.3. Biofilm formation in catheters

Sterile vinyl urethral catheters were treated with lipopeptide overnight at 4°C. Ten

microliter of overnight grown cultures were inoculated into 500 µl of nutrient broth

medium and injected into clear sterile vinyl urethral catheters (Medivik, India). The

catheters were capped at both ends and incubated at 30°C overnight. Media and growth

conditions were as described above in section 3.5.7.1. Cultures were removed and the


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catheters were rinsed with sterile distilled water. After drying at room temperature for 15

min, 700 µl of crystal violet (CV, 1%) was added to the catheters for 20 min. The stained

biofilms were rinsed several times with distilled water and allowed to dry at room

temperature for 15 min before examination.

3.7.5.4. Scanning electron microscopy

Bacterial and fungal cells were treated with lipopeptide at their inhibitory

concentration for 12 h. Negative controls were run in the absence of lipopeptide. The

samples for SEM were prepared and observed as described in the section 3.6.9.4.

3.7.5.5. FITC uptake assay

The fluorescence reagent fluorescein isothiocynate (FITC) was used to assess the

antimicrobial action of lipopeptide. Microbial strains were grown in their respective

medium (as mentioned in section 3.5.7.1). Cells were suspended in 10 mM sodium

phosphate buffer (pH 7.4) and incubated in the presence or absence of lipopeptide for 60

min at 30oC. Then the lipopeptide treated and untreated samples were stained with 6

µg/ml FITC in 10 mM sodium phosphate buffer and incubated at 30oC for 30 min. After

washing with phosphate buffer saline, the stained samples were immediately analyzed

with fluorescence microscope (Carl Zeiss, Germany) with an excitation wavelength of

490 nm and emission at 520 nm.

3.8. Production and characterization of glycolipid biosurfactant produced by

Enterobacter sp. NCIM 5392

3.8.1. Biosurfactant production using various carbon sources

Enterobacter sp. NCIM 5392 was grown in nutrient broth medium for 24 h at

30oC. For biosurfactant production, 2% inoculum of Enterobacter was added in MSM

containing natural wastes (1%, w/v) as carbon sources. The carbon sources used were

groundnut oil cake, sunflower oil cake and molasses. Cultivations were performed in 250

ml Erlenmeyer flasks containing 100 ml sterile MSM and carbon source and incubated at


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30oC in a shaking incubator at 120 rpm for 168 h to obtain the highest microbial and

biosurfactant concentrations. To determine dry cell weight, 10 ml culture broth was

centrifuged at 8000 rpm for 20 min and cell pellets were washed twice with distilled

water and dried by heating at 50oC until constant weight attained.

3.8.2. Purification and quantification of glycolipid biosurfactant

Purification and quantification of the glycolipid biosurfactant was done as

mentioned in the section 3.2.2.2.

3.8.3. Surface tension measurement and emulsification index

Surface tension of cell-free culture broth was measured by the method described

in the section 3.6.6. Similarly, emulsification index was determined for kerosene, diesel,

hexadecane, hexane, toluene, groundnut oil and olive oil as states in the section 3.6.6.

3.8.4. Structural characterization of Glycolipid biosurfactant

3.8.4.1. Thin layer chromatography (TLC)

Ten microliter of biosurfactant in methanol was spotted on a silica gel thin layer

chromatography (TLC) plate (Silica gel 60; Merck, Darmstadt, Germany). The

compounds were separated using a mobile phase of chloroform/methanol/water (65:25:4,

v/v/v). For the detection of lipids, the plates were exposed to iodine. For further analysis,

TLC purified glycolipid biosurfactant was used.

3.8.4.2. FTIR

The fourier transform infrared spectroscopy (FTIR; Perkin-Elmer, Spectrum one)

analysis of lipopeptide was done as mentioned in the section 3.6.8.3.

3.8.4.4. Fatty acid and sugar analysis

Fatty acid and amino acid analysis were done by acid hydrolysis of the

lipopeptide. Purified glycolipid (100 µg) was hydrolyzed with 6.0 M HCl at 100oC for 24


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h. The solution was cooled at room temperature and fatty acids (FAs) were extracted at

least three times using ether. The FAs were esterified with 3.0 M HCl in methanol at

100oC for 1 h. After cooling, the fatty acid methyl esters (FAMEs) were extracted using

hexane and concentrated by evaporation at room temperature. The FAMEs were analyzed

on a GC–MS apparatus (Shimazdu 2010 MS Engine) equipped with integrated gas

chromatograph with a HP1 column (60 m long, 0.25 mm i.d., nonpolar). Helium was

used as carrier gas at a flow rate of 1 ml min-1. The injector temperature was maintained

at 280oC with oven conditions as: 80oC kept constant for 2 min; increased up to 200oC

with 10oC min-1; rose up to 280oC with 20oC min-1 rate. The negative ion mode was used

throughout and scans were initiated over 50-1000 m/z range.

The sugar composition of the biosurfactant was determined by gas

chromatography and mass spectrometry. Biosurfactant (1 mg) was hydrolyzed in a sealed

tube with 150 µl of 2 M trifluoroacetic acid (CF3COOH) at 120oC for 4 h. After

evaporation the residue was washed twice with methanol; the sample was then reduced

with 1 M aqueous sodium borohydride (NaBH4, 100 µl) and acetylated with a mixture of

potassium acetate (100 µg) and acetic anhydride (100 µl) at 100oC for 2 h. The excess

reagent was removed by evaporation and the sample washed several times with ethanol.

The alditol acetates were extracted with ethyl acetate and water (1:1, v/v) and analyzed by

GC–MS (SHIMADZU, model QP 5050 A) equipped with a HP1 column using He as

carrier gas. Column temperature was programmed to increase from 100oC (1 min) to

200oC at a rate of 4oC/min, followed by 20oC/min to 300oC, and the column was

maintained at this temperature for 5 min. These procedures were carried out for crude and

pure biosurfactant samples, without hydrolysis, to verify the presence of free

carbohydrates in these samples. Sugars were identified by comparing the relative

retention times of sample peaks with standards. The following sugar standards were used

for the identification: glucose, galactose, rhamnose, and arabinose.


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3.8.5. Applications of glycolipid

3.8.5.1. Antifungal assay

The antifungal activity of the isolated glycolipid against Aspergillus niger NCIM

545, and Penicillium chrysogenum NCIM 723 was determined as mentioned in the

section 3.7.5.1.

3.8.5.2. Activity of glycolipid on fungal spore germination

The evaluation of glycolipid activity on germination of fungal spores was

performed by addition of 0.5 ml of biosurfactant solution to 0.5 ml of fungal spore

suspension (containing 5×103/ml spores). In case of control set biosurfactant solution was

replaced by 0.5 ml of the potato dextrose broth medium and was incubate at 25oC for 18

h. The inhibition of hyphal extension or spore germination was compared with control by

microscopic observations.

3.9. Data analysis

The optical images of the CV stained biofilms in the catheters were recorded with

a Canon Digital IXUS 960 IS digital camera. The images obtained for biofilm formation

of various microorganisms were processed using ImageJ 1.41n (MacBiophotonics

ImageJ) to obtain the intensities in different regions of interest (ROI). Five ROI points

were randomly picked for each catheter. The difference in the intensities of CV stained

biofilms on lipopeptide treated and untreated catheters were reported as percent inhibition

in biofilm formation.

All values reported are the mean of three independent measurements. The

analyses were done using one-way analysis of variance (ANOVA) with Tukey-Kramer

multiple comparisons test. Brightness and contrast were adjusted in order to show images

more clearly in the figures.

chapter 3 materials and methods -...

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