CHAPTER 3 MATERIALS AND METHODS -...

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CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

3.1.1. Chemicals

Synthetic microbial growth media such as Luria Bertani broth, nutrient broth, nutrient agar,

potato dextrose broth, Pikovskaya’s broth, noble agar powder, starch agar, DNase test agar,

nitrate broth, media components such as dextrose, glycerol, peptone, proteose peptone,

beef extract, pancreatic digest of casein, skim milk powder, tryptone, tween 80, yeast

extract, agar powder; aminoacid L-tryptophan; other salts and chemicals such as silver

nitrate, chloroauric acid, ammonium chloride, ammonium molybdate, dipotassium

hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate,

stannous chloride, zinc sulphate, manganese sulphate, ethylenediamine tetraaceticacid

(EDTA), ferric chloride, ferrous sulphate, sodium chloride, tris base, sodium dodecyl

sulfate (SDS), piperazine-, N, N’- bis (2-ethane sulphonic acid) (PIPES), indole-3-acetic

acid, boric acid, picric acid, sulphanilic acid, α-napthylamine; pH indicators such as

bromophenol blue and congo red; fluorescent dye rhodamine B; oxidase discs and hicarbo

kits were purchased from Hi-Media, India. Chrome azurol S, aminocyclopropane-1-

carboxylate (ACC), hexadecyltrimethylammonium bromide (HDTMA) and organic acid

standards viz., cis-aconitic acid, ascorbic acid, quinic acid, succinic acid, lactic acid, formic

acid, fumaric acid were obtained from Sigma-Aldrich, USA. Solvents such as chloroform,

isoamyl alcohol and sulphuric acid, hydrochloric acid, nitric acid, perchloric acid and

hydrofluoric acid were obtained from Merck, Mumbai, India. Granite, limestone and

marble were purchased from G.C.M Industries, Salem. GeneluteTM bacterial genomic DNA

miniprep kit, Gen EluteTM gel extraction kit and PCR reagents such as Taq DNA

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polymerase, magnesium chloride, 10X reaction buffer and dNTPs and chemicals such as

gultaraldehyde and cacodylate buffer were purchased from Sigma-Aldrich, USA. HPLC

column (KC-811) 8x300 mm was purchased from Waters Corp. MA, USA.

3.1.2 Microorganisms

Fungi, Alternaria sp., Sclerotium rolfsii, Sarocladium oryzae, Magnaporthe grisea,

Bipolaris oryzae, Macrophomina phaseolina, Fusarium oxysporum f. sp. cubense,

Pestalotia theae, Colletotrichum gleosporoides and human pathogenic bacteria, Bacillus

subtilis, Escherichia coli, Vibrio cholera, Pseudomonas aeruginosa, Staphylococcous

aureus, Salmonella typhi and yeast, Candida albicans were obtained from the Microbial

Culture Collection, Department of Biotechnology, Pondicherry University, Puducherry.

Microbial cultures used in this study are maintained at the Department of Biotechnology,

Pondicherry University, Puducherry.

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3.1.3 Media

Nutrient broth (NB) (Atlas 1993)

Peptone 5 g

NaCl 5 g

Beef extract 1.5 g

Yeast extract 1.5 g

pH was adjusted to 7.4 and the final volume was made to 1000 ml using distilled water.

Nutrient agar (NA)

1.5 % of agar was added to NB before autoclaving.

Potato dextrose agar (PDA) (Atlas 1993)

Potato infusion 200 g

Glucose 20 g

Agar 15 g


King’s B (KB) agar (King et al. 1954)

Proteose peptone 20 g

Glycerol 15 ml

K2HPO4 1.5 g

MgSO4.7H2O 1.5 g

Agar 15 g


Luria Bertani broth (LB) (Bertani 1951)

Tryptone 10 g

Yeast extract 5 g

NaCl 10 g


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Luria Bertani agar (LBA)

1.5 % of agar was added to LB before autoclaving.

Dworkin and Foster (DF) salt medium (Dworkin and Foster 1958)

(NH4)2SO4 2 g

KH2PO4 4 g

Na2HPO4 6 g

MgSO4.7H2O 0.2 g

FeSO4.7H2O 1 mg

H3BO3 10 µg

MnSO4 10 µg

ZnSO4 70 µg

CuSO4 50 µg

MoO3 10 µg


Nitrate broth (Dye 1962)

Potassium nitrate 2 g

Peptone 10 g

NaCl 5 g


Skim milk agar (Smibert and Krieg 1994)

Pancreatic digest of casein 5 g

Yeast extract 2.5 g

Glucose 1 g

Skim milk solution (7%) 100 ml

Agar 15 g


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Pikovskaya’s agar medium (Pikovskaya 1948)

Yeast extract 0.5 g

Dextrose 10 g

Ca3(PO4)2 5 g

(NH4)2SO4 0.5 g

KCl 0.2 g

MgCl2 0.1 g

MnSO4 0.0001 g

FeSO4 0.0001 g

Agar 15 g


Carboxymethyl cellulose (CMC)-Congo red agar medium (Wood 1980)

MgSO4.7H2O 0.005 g

K2HPO4 0.005 g

Carboxymethyl cellulose 0.02 g

Congo red 0.002 g

Agar 15 g

pH was adjusted to 7.0 and the final volume was made to 1000 ml using distilled water

Henderson’s medium (Henderson and Duff 1963)

KH2PO4 0.4 g

(NH4)SO4 0.5 g

FeCl3.6H2O 0.0166 g

Peptone 1.0 g

Yeast extract 1.0 g

Glucose 10 g

Pulverized rock powder 2.5 g

Noble agar 32 g

pH was adjusted to 7.0 and the final volume was made to 1000 ml using distilled water

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Lipase agar (Smibert and Krieg 1994)

NH4NO3 4 g

KH2PO4 4.7 g

Na2HPO4 0.1 g

CaCl2 0.01g

FeSO4 0.05 g

MgSO4 1 g

MnSO4 0.01 g

Yeast extract 0.1 g

Tween 80 10 ml

Rhodamine B 10 mg

Agar 20 g


Xylanase agar (An et al. 2005)

Tryptone 10 g

Yeast extract 5 g

NaCl 10 g

Xylan from oat spelts 10 g

Congo red 20 mg

Agar 15 g


Starch Agar (Jacob and Gerstein 1960)

Peptic digest of animal tissue 5 g

Yeast extract 1.5 g

Beef extract 1.5 g

Starch, soluble 2 g

NaCl 5 g

Agar 15 g


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DNase Test Agar (Schreier 1969)

Tryptose 20 g

Deoxy ribonucleic acid 2 g

Sodium chloride 5 g

Toluidine blue 0.1 g

Agar 15 g


Chrome Azurol S (CAS) agar (Milagres et al. 1999)

CAS 60.5 mg

Iron (III) solution 10 ml

(1mM FeCl3 .6H2 O, in 10 mM HCl)

HDTMA 72.9 mg

PIPES 30.24 g

NaOH 12 g

Agar 15g

3.1.4. Buffers and solutions (Sambrook et al. 1989)

Tris-EDTA (TE) buffer

Tris 10 mM

EDTA 1 mM

pH 8.0

DNA loading dye (6X)

Glycerol 50%

EDTA, pH 8.0 0.2 M

Bromophenol blue 0.05%

Xylene cyanol 0.05%

Ethidium bromide

10 mg of ethidium bromide was dissolved in 1 ml of water.

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Tris acetate-EDTA (TAE) buffer 50X

Tris base 242 g

Glacial acetic acid 57.1 ml

0.5 M EDTA (pH 8.0) 100 ml


Sulphanilic acid

0.8 g of sulphanilic acid was dissolved in 100 ml of 5N acetic acid.

α-napthylamine solution

0.5 g of α-napthylamine was dissolved in 100 ml of 5N acetic acid.

Salkowski’s reagent

2% ferric chloride (0.5 M) in 35% perchloric acid.

Phosphate solubilization estimation

Chloromolybdic acid

(NH4)2MoO4 1.50 g

12N HCl 34.2 ml

Volume made upto 100 ml with distilled water.

Chlorostannous acid

SnCl2. H2O 2.5 g

12N HCl 10 ml

Volume made upto 100 ml with distilled water.

Lugol’s iodine

2.0 g potassium iodide and 1.0 g iodine dissolved in 100 ml distilled water.

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3.2 Methods

3.2.1 Maintenance of microorganisms

Bacterial cultures were maintained in NA slants and stored at 4°C for routine use and

maintained as glycerol stocks (50% v/v) at –86°C for long term storage. The fungal

cultures were maintained in PDA slants and stored at 4°C for long term storage and as

water cultures for routine use.

3.2.2 Sterilization

All the reagents, buffers and media were sterilized at 15 lbs/inch2 for 20 min unless

otherwise specified. Heat labile chemicals were filter sterilized using 0.22 µm filters

(Millipore, Molsheim, France.)

3.2.3 Collection and identification of plants and preparation of rock samples

Plants growing within the rocks lacking visible soil from Papanasam (8°42’N; 77°22’E) of

Western Ghats, India, were collected. The rock surrounding the plant was broken manually

with a surface-sterilized chisel and hammer and the roots were collected in sterile plastic

bags. Also, rock particles surrounding the roots were collected aseptically, stored at 4°C

and transferred to laboratory and processed within 24 h after collection.

Rock samples were collected from three different locations namely, Mahabalipuram

(12°37’N; 80°11’E), Western Ghats (Thoranamalai, 8°55’N; 77°20’E) and Gingee

(12°15’N; 79°23’E), India. The rock samples collected from the field were cleaned and

chipped to remove the weathered and altered fractions. The rock samples were submerged

in 1N HCl overnight at room temperature to remove the organic matter and then washed

thoroughly with distilled water, dried in oven at 160°C for 2 h. The fresh chips were

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ground to nearly less than 125 µm using steel and agate mortar and stored for further

studies.

3.2.4 Physio-chemical analysis of rock sample

3.2.4.1 Microscopic analysis of thin section of rocks

Rock chips were polished on one side, stuck individually on a glass slide using epoxy and

allowed to dry overnight. Then, the exposed rock surfaces were polished using petrothin

(Buehler, USA) to a thickness of 0.035 mm. These thin sections of rocks were observed

under petrology light microscope (Olympus, USA) with plane and cross polarized light.

3.2.4.2 X-ray diffraction (XRD) analysis of rock

The mineralogy of the rock samples was deciphered by XRD analysis. XRD analysis was

carried out using PanAlytical XRD (Model Xpert pro) Powder system-Philips. The

powdered rock samples were tightly packed into the well of XRD sample holder and

spectrum were taken from 10° to 74.99° 2θ, at a scan speed of 0.02° 2θ per 0.5 s and

wavelength of 1.5406 Å. The spectra obtained were matched with the database with the

help of Xpret High score PANalytical software (Ver. 2.0.1) for mineral identification.

3.2.4.3 X-ray fluorescence spectroscopy (XRF) and pH analysis of rock

Rock powder (2 g) was added to 0.5 g of boric acid, mixed well in agate mortar and was

placed in a 34 mm die. It was pressed into a pellet at a pressure of about 10 t for 20 s, in a

hydraulic press (Insmart systems, India). The chemical composition of rocks was analyzed

by XRF (Bruker (AXS) S4 Pioneer, Germany). The pH of all the rock samples were

determined using Eutech pH meter (Thermo Scientific, USA) after mixing 1 g of powdered

rock sample with 10 ml of KCl and allowing it to stand for 30 min (Warscheid et al. 1990).

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3.2.4.4 Inductively coupled plasma emission-mass spectrometer (ICP-MS)

The elements in the rocks were analyzed by ICP-MS. Powdered rock samples were

digested using a mixture of hydrofluoric acid, nitric acid and hydrochloric acid in the ratio

of 7: 3: 1 within Parr® pressure digestion vessel. The samples were then repeatedly dried

with HNO3 and analyzed by ICP-MS (Thermo scientific, USA) calibrated with the United

States geological survey (USGS) rock standards.

3.2.5 Scanning electron microscopy (SEM) analysis of plant root sample

Roots of Barleria acuminata, Ficus nervosa and F. mollis were cut into 1 cm long pieces

and fixed separately with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and

stored at 4°C, immediately after sample collection and processed for SEM analysis with a

slight modification of method described by Puente et al. (2004). Dried root samples were

mounted on aluminium stub containing double-adhesive tape and analyzed in SEM

(Hitachi Model: S-3400N, Tokyo, Japan) at a resolution of 10 nm at 5 kV HV mode.

3.2.6 Isolation of rhizoplane bacteria

The rhizoplane bacteria were isolated as described by Puente et al. (2004). Briefly, the

roots of B. acuminata, F. nervosa and F. mollis were cut aseptically into 1 cm long bits and

5 g of each sample was placed separately along with few glass beads in 50 ml of sterile

distilled water in a conical flask and incubated at 30°C on a rotary shaker at 200 rpm for

15 min. The root homogenate was then serially diluted with phosphate buffered saline

(PBS) at pH 7.0 and plated onto Henderson medium. The culture plates were incubated at

30°C for 48 h. The bacterial colonies obtained were further purified and the stock cultures

were maintained in nutrient broth containing 50% (v/v) glycerol at −86°C.

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3.2.7 Biochemical characterization

3.2.7.1 Oxidase

Bacterial culture were grown overnight in NA supplemented with 1% glucose (Schaad

1980). A loopful of cells were rubbed onto oxidase discs (Himedia Laboratories, Mumbai,

India) and observed for colour change to deep purple within 10 s which indicates the

production of oxidase by the bacteria. As traces of iron can catalyze the phenylenediamine

compound, sterile toothpicks were used for inoculation.

3.2.7.2 Catalase

Overnight grown bacterial cells were treated with few drops of 30% H2O2. Appearance of

brisk effervescence indicated the production of catalase by the bacteria (MacFaddin 1980).

3.2.7.3 Nitrate reductase

To test the production of nitrate reductase, the bacterial cells were inoculated in 5 ml of

nitrate broth and allowed to grow in a rotary shaker at 28°C for 5 d. An aliquot of 1 ml

culture was taken at 24 h interval and few drops of sulphanilic acid and α-naphthylamine

reagent were added. Colour change to distinct pink or red indicates the presence of nitrite

(Dye 1962).

3.2.7.4 Carbohydrate utilization profile

To determine the carbohydrate utilization profiles, HiCarbohydrateTM (Himedia

Laboratories, Mumbai, India) were used. Cells were grown in NB until the turbidity

reaches 0.5 O.D. at 620 nm. Cell suspension (50 µl) was inoculated to the wells of

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HiCarbohydrateTM kit, incubated at 35°C for 24 h and the results were registered according

to the manufacturer’s instructions.

3.2.7.5 Tolerance to temperature and salinity

In order to test the bacteria for salt tolerance, the NA plates were amended with increasing

concentrations of NaCl from 0.5 to 3%. Assay plates were incubated at 28°C for 2 d.

Temperature tolerance was determined by growing bacteria in NA plates and incubating at

different temperatures ranging from 25°C to 50°C for 2 days (Puente et al. 2004). Bacterial

growth was considered as positive for tolerance to salinity and temperature.

All the phenotypic traits were converted into the binary code and cluster analysis was

performed using unweighted pair group method with average (UPGMA) algorithm of

NTSYSpc2 (Version 2.02a, Exeter software, USA) as described (Sneath and Sokal 1973).

3.2.8 Molecular characterization

3.2.8.1 Isolation and quantification of genomic DNA

Overnight grown bacterial cells were used for the isolation of genomic DNA using

GeneluteTM bacterial genomic DNA miniprep kit and the genomic DNA was extracted

according to the manufacturer’s instructions (Sigma–Aldrich, USA). The quality and

quantity of DNA were verified by electrophoresis through 0.8% agarose gel and the purity

of the DNA was analyzed spectrophotometrically by checking the O.D. at 260 nm. An

O.D. value of one corresponds to approximately 50 µg/µl of double stranded DNA

(Sambrook et al. 1989). Based on the O.D. values, DNA samples were quantified.

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3.2.8.2 Amplification and sequencing of 16S rRNA

Approximately 10 ng of purified genomic DNA from each bacteria was used as template

for 16S rRNA gene amplification, using the universal primers, fD1

(5’-GAGTTTGATCCTGGCTCA-3’) and rP2 (5’-ACGGCTACCTTGTTACGACTT-3’)

as described by Weisburg et al. (1991). The PCR products were purified using

Gen EluteTM Gel Extraction Kit (Sigma–Aldrich) and were sequenced with automated

DNA sequencer using the facility at Macrogen Inc. (Seoul, Korea). The nucleotide

sequences of 16S rRNA were deposited in Genbank.

3.2.8.3 Phylogenetic tree analysis

In order to perform molecular phylogenetic tree analysis MEGA 4.0 software was used

(Tamura et al. 2007). The test sequences were compared with the reference sequences

available in European Molecular Biology Laboratory (EMBL) database

(http://www.ncbi.nlm.nih.gov/Genbank). CLUSTAL V was used for multiple sequence

alignment of the 16S rRNA sequences (Higgins et al. 1992). The sequences were checked

for gaps manually, arranged in a block of 600 bp in each row and saved in molecular

evolutionary genetics analysis (MEGA) format. The pairwise evolutionary distances were

computed with the help of Kimura 2-parameter (Kimura 1980) and the original data set

was resampled 1000 times using bootstrap analysis method to obtain confidence values.

The bootstrapped data set was used for phylogenetic tree construction using MEGA 4.0 by

neighbour-joining (NJ) method (Saitou and Nei 1987).

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3.2.9 Functional characterization

3.2.9.1 Rock-weathering potential

3.2.9.1.1 Qualitative assay

Pikovskaya’s agar medium (pH 7.0) supplemented separately with each rock powder (<125

µm) of igneous (granite), sedimentary (limestone) and metamorphic (marble) rocks and

was spot-inoculated with rhizoplane bacteria. The assay plates were incubated at 28°C for

3 d. Halo formation around the bacterial colony was considered as positive for weathering

of rocks (Chang and Li 1998).

3.2.9.1.2 Quantitative assays

3.2.9.1.2.1 Production of organic acid

Rhizoplane bacteria were checked for the production of organic acids by HPLC using

slight modification of the method described by Moreau and Savage (2009). Briefly,

fermentation culture medium was centrifuged at 5000 g for 5 min and the supernatant was

collected. The supernatant was filtered through nylon syringe filters, 0.22 µm (Whatmann,

USA) and loaded in HPLC vials. Sample filtrate (1 µl) was injected onto the column (KC-

811 column, 8x300 mm, Waters Corp. MA, USA). The mobile phase was of 0.0125 M

H2SO4 in water with a flow rate of 0.5 ml/min and 30 min run time. The organic acids were

detected at 210 nm and the spectrum was recorded between 190-320 nm using a diode

array detector (DAD). Before the injection of sample the instrument was calibrated with

the mixture of organic acids with a concentration range from 0.11 to 0.55 ng. The

estimation of organic acids in the sample filtrate was carried out with respect to that of the

calibration curve obtained by known concentration of standard organic acids.

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3.2.9.1.2.2 Biogeochemical deterioration of the rock sample

To test the biogeochemical deterioration, strains RB9, RB15, RB21 and RB24 were grown

overnight in NB at 28°C and the cells were pelleted by centrifugation at 3000 rpm for

20 min. The cell pellets were washed completely with sterile distilled water and were

resuspended in 50 ml of 0.85% saline solution. A 25 ml aliquot of each bacterial

suspension were inoculated separately in the flasks containing glucose (2.5 g) and

pulverized rock (0.375 g of three different rock powders each separately) in 250 ml of

distilled water and incubated for 28 d at 28°C in a rotary shaker at 150 rpm. Uninoculated

flask containing rock powder and glucose alone served as control (Puente et al. 2004).

After incubation, the suspended rock powder and the cells were centrifuged at 8000 rpm

for 20 min. The pellets were separated from organic matter and bacteria by continuous

treatment with hydrogen peroxide. The final pellet residue containing only the rock powder

was dried completely. About 0.2 g of all the dried rock powder was suspended in 2 ml

distilled water and the supernatant were analyzed for particle size reduction using particle

size analyzer (Malvern-NanoSeries, Zeta sizer, UK.) and SEM. The pellet minerals were

also analyzed by XRD and ICP-MS to observe for the change in mineralogy and chemical

composition.

3.2.9.2 Plant growth-promoting traits

3.2.9.2.1 Phosphate solubilization

Bacterial strains were inoculated on to Pikovskaya’s agar medium and incubated at 28°C

for 3-5 d. Halo formation around the colony was considered to positive for phosphate

solubilization (Ravindra Naik et al. 2008). Phosphate solubilizing ability of the bacterial

strains was quantified using standard method (King 1932). Briefly, strains were inoculated

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in 25 ml of Pikovskaya’s broth and incubated in a rotary shaker at 28°C at 180 rpm. An

aliquot of 2.5 ml culture was harvested at different time intervals (1, 3, 5, 7 and 10 d). 2 ml

culture filtrate was used for pH determination. To 0.2 ml of culture filtrate, 1 ml of

chloromolybdic acid was added and vortexed. Then 3.3 ml of distilled water was added

followed by 25 µl of chlorostannous acid. The volume was made up to 5 ml with distilled

water and the absorbance was measured immediately at 600 nm. Amount of phosphate

liberated was calculated by comparing with the caliberation curve obtained using KH2PO4

standards. The experiments were done in triplicates.

3.2.9.2.2 Production of siderophores

To determine the production of siderophores modified chrome azurol S (CAS) assay was

used (Milagres et al. 1999). Initially, all the glasswares were rinsed with 6M HCl to

remove iron and also with double distilled water thoroughly. Iron (III) solution (10 ml) was

added to CAS (60.5 mg) in 50 ml deionised distilled water to which, 72.9 mg

Hexadecyltrimethylammonium bromide (HDTMA) dissolved in 40 ml water was added

slowly with constant stirring. The resulting dark blue liquid was autoclaved. 30.24 g of

PIPES buffer was dissolved in 750 ml of distilled water (pH 6.8 adjusted to using 12 g

solution of 50% NaOH) and autoclaved separately after the addition of 15 g of agar. The

contents were mixed and added to media plates already half filled with NA, to nullify the

toxic effect of HDTMA on Gram-positive bacteria. Bacterial strain was inoculated on the

half containing NA and incubated for 5 d at 28°C. Change in the colour of the CAS

medium from blue to orange indicates siderophore production.

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3.2.9.2.3 Production of IAA

Bacteria were streaked onto LB plates supplemented with 5 mM L-tryptophan, 0.06 %

SDS and 1 % glycerol and the plates were overlaid with sterile Whatman no. 1 filter paper

and incubated at 28°C for 3 d after which, the filter papers were saturated with Salkowski’s

reagent containing the formulation of 2 % of 0.5 M ferric chloride in 35 % of perchloric

acid for 1 h at room temperature. Formation of a red halo on the paper surrounding the

colony confirms the production of IAA by the bacterial strains (Bric et al. 1991).

3.2.9.2.4 Production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase

Bacteria were tested for ACC deaminase production using DF minimal salts medium

(Dworkin and Foster, 1958). A 3 mM solution of filter sterilized ACC was spread over the

agar plates and was allowed to dry for 10 min. Then the plates were inoculated with the

bacteria and incubated at 28°C for 2 d. Bacterial growth indicates the production of ACC

deaminase (Penrose and Glick 2002).

3.2.9.2.5 Production of HCN

Bacteria were streaked onto NA plates amended with 4.4 g of glycine per liter. Filter paper

strips treated with 0.5 % picric acid and 2 % sodium carbonate were placed on the lid of

the Petri plates and sealed with parafilm. Colour change of the filter paper from yellow to

orange after incubation at 28°C for 3-5 d indicates the microbial production of HCN

(Bakker and Schippers 1987).

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3.2.9.3 Antagonistic potential

3.2.9.3.1 Antifungal activity

Strains were screened for in vitro antifungal activity against phytopathogenic fungi such as

Altenaria sp., Cylindrocladium gleosporoides, Bipolaris oryzae, Magnaporthe grisea,

Sarocladium oryzae, Fusarium oxysporum f. sp. cubense, Pestolatia theae, Sclerotium

rolfsii and Macrophomina phaseolina by co-inoculation technique described by Sakthivel

and Gnanamanickam (1987). Agar plugs from 48 h grown bacterial cultures were placed

on to the centre of PDA plates inoculated with 50 µl of fungal spore suspensions

(106 conidia ml-1). The plates were incubated at 28°C for 3-5 d and observed for fungal

growth inhibition around the bacterial plug.

3.2.9.3.2 Antibacterial activity

Strains were also tested for antibacterial activity against human pathogenic bacteria such as

Bacillus subtilis, Escherichia coli, Vibrio cholera, Pseudomonas aeruginosa,

Staphylococcous aureus, Salmonella typhi and yeast Candida albicans by modification of

agar spot test method (Flemming et al. 1975). An aliquot of 50 µl of overnight grown

human pathogenic strains (0.6 of O.D at 620 nm) were spread on to the surface of nutrient

agar plates and were allowed to dry. Then the rhizobacteria were spot-inoculated and

incubated at 28°C for 24 h. Formation of inhibition zones around the bacterial colony

indicates the antibacterial activity against the pathogens.

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3.2.9.4 Production of industrial enzymes

3.2.9.4.1 Amylase

To screen the bacteria for production of amylase, bacteria were spot-inoculated on starch

agar plates and were incubated at 32°C for 2 d after which the plates were flooded with

Lugol’s iodine. Appearance of a halo around the colony confirms production of amylase by

the bacteria (Jacob and Gerstein 1960).

3.2.9.4.2 Cellulase

To test the bacteria for cellulase production, bacteria were spot-inoculated on CMC-Congo

red agar plates and incubated at 28°C for 2 d. Appearance of a halo around the bacterial

colony indicates cellulase production by the bacteria (Wood 1980).

3.2.9.4.3 DNase

To check the bacteria for DNase production, bacteria were spot-inoculated on DNase test

agar with toluidine blue dye and incubated at 28°C for 2 d (Schreier 1969). Formation of a

halo around the colony shows the production of DNase by the bacteria.

3.2.9.4.4 Lipase

To test for lipase production, bacteria were spot-inoculated on lipase medium containing

Tween 80 as substrate and Rhodamine B as an indicator dye (Smibert and Krieg 1994).

Appearance of clear halo around the colony after 3 d of incubation at 28°C shows the

production of lipase by the bacteria.

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3.2.9.4.5 Protease

Production of protease was tested by spot-inoculating the bacteria on skim milk agar and

incubating the plates at 28°C for 2 d. Halo formation around the inoculated colony

indicates the production of protease by the bacteria (Smibert and Krieg 1994).

3.2.9.4.6 Xylanase

To screen the bacteria for xylanase production, bacteria were spot-inoculated on xylanase

agar plates with Congo red indicator dye (An et al. 2005). Formation of a clear halo around

the colony after 2 d of incubation at 28°C confirms the bacterial production of xylanase.

3.2.9.5 Nanoparticle production

3.2.9.5.1 Biosynthesis of metal nanoparticles

The bacterial strains were inoculated in NB and incubated at 28°C on a rotary shaker at

150 rpm till the culture reached the mid log phase. The bacterial cells were harvested by

centrifugation at 3000 rpm at 4°C for 10 min. The pellets were washed thoroughly with

sterile distilled water to remove the media components. The bacterial biomass were

incubated with 100 ml of 100 mM silver nitrate and 100 ml of 1 mM gold chloride solution

in deionised water at 30°C and 120 rpm. Periodic wave scans were performed in

spectrophotometer (Amersham pro 600 UV-Vis spectrophotometer) after observing for the

colour change to brown and pink for the production of silver and gold nanoparticles

respectively.

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3.2.9.5.2 Characterization of metal nanoparticles

3.2.9.5.2.1 UV-Visible spectrophotometric analysis

The colour change was observed in the metal ion solution incubated with bacterial

biomass. The bioreduction of metal ions in solution was monitored by periodic sampling of

aliquots (0.1 ml) of supernatant and measuring the UV-Vis spectra of the solution in quartz

cuvettes of 1 cm optical-path-length with UV-Vis spectrophotometer (Amerhsam pro 600)

at a resolution of 1 nm between 200 and 1100 nm.

3.2.9.5.2.2 X-ray diffraction (XRD) studies

The bioreduced gold nanoparticle solution was drop-coated onto glass substrate and X-ray

diffraction measurements were carried out on a PANalytical X’pert PRO X-ray

diffractometer (Netherlands). The pattern was recorded by Cu Kα1 radiation with λ of

1.5406 Ǻ and nickel monochromator filtering the wave at tube voltage of 40 kV and tube

current of 30 mA. The scanning was done in the region of 2θ from 30° to 80° at a speed of

0.02°/min and the time constant was 2 s. The size of the nanoparticles was calculated

through the Debye-Scherrer’s formula D = 0.94λ/β1/2 cosθ, where D is the average crystal

size, λ is the x-ray wavelength (λ = 1.5406 Å), θ is Bragg’s angle (2θ), β1/2, full width at

half-maximum (FWHM) in radians (Borchert et al. 2005).

3.2.9.5.2.3 Fourier transform infrared (FT-IR) spectroscopy

The bioreduced metal nanoparticle solution was centrifuged at 10,000 rpm for 15 min and

the pellet was washed with deionized water. The sample was dried and grinded with KBr

pellets and analyzed on a FT-IR-instrument (Thermo Nicolet model 6700) in the diffuse

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transmittance mode operating at a resolution of 4 cm-1 over 4000-500 cm-1. In order to

obtain a good signal/noise ratio, 512 scans were recorded.

3.2.9.5.2.4 Scanning electron microscopy (SEM) and energy dispersive x-ray analysis

(EDAX)

The nanoparticles were mounted onto the copper stubs and the images were analysed using

scanning electron microscope (HITACHI model S-3400N) with secondary electron

detectors at an operating voltage of 20 kV and for elemental analysis energy dispersive

x-ray analysis (EDAX) was done in Noran-System Six X-ray microanalysis system

(Thermo Electron Corporation, USA) coupled to SEM.

3.2.9.5.2.5 High resolution-transmission electron microscopy (HR-TEM)

The size and shape of nanoparticles were analysed using high resolution-transmission

electron microscopy (HR-TEM). The samples were placed on carbon-coated copper grids

and were allowed to dry in air prior to measurements on a microscope (JEOL model 3010)

operated at an accelerating voltage of 200 keV with wavelength (λ) of 0.0251 Ǻ. The size

and morphology of nanoparticles were examined.

CHAPTER 3 MATERIALS AND METHODS -...

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