MATERIALS AND METHODS - Materials and Methods MATERIALS AND METHODS 3.1 Pesticides and chemicals...

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  • Materials and Methods

    MATERIALS AND METHODS

    3.1 Pesticides and chemicals

    Lindane/γ-Hexachlorocyclohexane (γ-HCH, 99% pure) was purchased from

    Sigma Chemical Co., USA. HCH-muck was a generous gift from Kanoria Chemicals and

    Industries Ltd. (U.P., India). Alufolein kiesel gel 60 F254 plates were purchased from

    Merck (Darmstadt, Germany). Acetone and ethyl acetate (purity >99.0%) were purchased

    from Thomas Baker, Mumbai, India. The media components were purchased from

    HiMedia Lab., India. All other solvents and reagents used were of highest available

    purity. The oligonucleotide primers for polymerase chain reaction (PCR) were supplied

    by Sigma-Genosys (USA) and PCR reaction-mix was purchased from Genei, Bangalore,

    India.

    3.2 Media used

    3.2.1 Medium used for growth and storage of isolates:

    The cultures were grown in nutrient broth and were stored on nutrient agar slants

    at 4°C. The stored microbial strains were sub-cultured every month. The cells were also

    maintained as 20% (v/v) glycerol stock in nutrient broth at -80°C. The composition of the

    nutrient agar medium used is as given below:

    Components Quantity (g l-1)

    Peptone - 5.00

    Beef extract - 3.00

    NaCl - 5.00

    Agar-agar - 20.0

    Distilled water to make - 1000 ml

    pH - 7.0

    3.2.2 Biosurfactant production medium:

    Mineral salt medium (MSM) of the following composition was used for

    biosurfactant production studies, unless specified otherwise.

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  • Materials and Methods

    Components Quantity (g l-1)

    Na2HPO4 (anhydrous) - 3.60

    (NH4)2SO4 - 1.00

    KH2PO4 - 1.00

    MgSO4.7H2O - 1.00

    Fe(NH4)Citrate - 0.01

    CaCl2.2H2O - 0.10

    Yeast extract - as per requirement

    Glucose - as per requirement

    Trace element solution - 10.0 ml l-1

    Distilled water to make - 1000 ml

    pH - 7.0

    Composition of trace element solution:

    Components Quantity (mg l-1)

    ZnSO4.7H2O - 10.0

    MnCl2.4H2O - 3.00

    CoCl2.6H2O - 1.00

    NiCl2.6H2O - 2.00

    Na2MoO4.2H2O - 3.00

    H3BO3 - 30.0

    CuCl2.2H2O - 1.00

    Distilled water to make - 1000 ml

    The stock solutions of yeast extract (25.0% w/v) and glucose (50.0% w/v) were

    sterilized separately and added to MSM as per the requirement. Similarly, stock solutions

    of other sugars (fructose, mannitol and sucrose) and organic extracts/supplements (beef

    extract, malt extract, peptone, soy peptone and tryptone) used in optimization studies

    were prepared and used as per the requirement of a particular experiment.

    3.2.3 Chloride-free medium:

    Chloride-free mineral salt medium (CFMSM) of the following composition was

    used to assay the release of free chloride-ion during HCH-biodegradation.

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  • Materials and Methods

    Components Quantity (g l-1)

    Na2HPO4 (anhydrous) - 3.60

    (NH4)2SO4 - 1.00

    KH2PO4 - 1.00

    MgSO4.7H2O - 1.00

    Fe(NH4)Citrate - 0.01

    Ca(NO3)2 - 0.10

    Distilled water to make - 1000 ml

    pH - 7.0

    3.3 Isolation of microbial strains

    3.3.1 Isolation of biosurfactant-producing microorganisms:

    The microbial populations in soil samples collected from sites contaminated with

    petrochemicals (petrol stations, motor workshops, etc.) were isolated. The soil samples

    were collected in plastic bags from just below the surface, appropriately labeled and

    stored at 4°C. To a 9 ml water blank, 1 g of soil sample was added and contents of the

    test tube were vortexed and the suspension was used as inoculum (10% v/v) to inoculate

    50 ml MSM in 250 ml Erlenmeyer flask supplemented with 0.1% (w/v) yeast extract.

    Different supplements viz. n-hexadecane, diesel-petrol mix (1:1), mobile oil and

    vegetable oil were added at the rate of 2.0% (v/v) to the respective flasks for enrichment

    of microbial populations present in the soil samples. The flasks were incubated at 30°C

    on an orbital shaker at 100 rpm. The cell suspension was used as 10% (v/v) inoculum in

    fresh MSM medium, containing respective the carbon sources to further enrich the

    microbial populations, after every 15 days for a period of two months. At each transfer, 1

    ml of sample was withdrawn and serially diluted and 0.1 ml of the appropriately diluted

    culture broth was spread plated onto MSM agar plates supplemented with 0.1% (w/v)

    yeast extract and 2.0% (v/v) of the respective carbon sources, separately. The plates were

    incubated at 30°C and observed for growth. Morphologically distinct bacterial colonies

    were isolated and purified.

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  • Materials and Methods

    The enrichment of microbial population on molasses and whey was carried out

    using 1/100 diluted nutrient broth. Five grams of the soil sample was added to 250 ml of

    Erlenmeyer flask containing 50 ml of nutrient broth (N/100) and incubated on a rotary

    shaker at 30°C and 100 rpm for 24 h. 5 ml of culture was transferred to 50 ml MSM

    supplemented with 0.01% (w/v) yeast extract and carbon sources viz. molasses (2.0%

    w/v) and whey (1.0% v/v), respectively. Finally, 0.1 ml of culture was plated onto

    nutrient agar plates and incubated at 30°C for 24 h. After incubation, plates were

    enumerated and morphologically distinct bacteria were screened for biosurfactant

    production.

    The microbial populations present in spoiled curd and cheese samples were

    screened for their culture diversity in MSM supplemented with n-hexadecane (1.0% v/v),

    casein (1.0% w/v) and tributyrin (1.0% v/v), separately. The enriched samples were

    plated onto nutrient agar plates and morphologically distinct isolates were selected for

    further studies.

    The isolates were stored on nutrient agar slants and stored at 4°C. Regular sub-

    culturing was carried out on fresh medium at a regular interval of 15 days. The cells were

    also maintained as 20% (v/v) glycerol stock at -80°C after growing in nutrient broth.

    3.3.2 Isolation of HCH-degrading microbes:

    The microbial populations present in soil samples collected from sites polluted

    with organochlorine pesticides, in and around the city of Amritsar, were enriched in the

    presence of γ-HCH. Ten gram (10 g) of soil was used to inoculate 90 ml MSM

    supplemented with 0.1% (w/v) glucose, 0.01% (w/v) yeast extract and 0.01 g of γ-HCH.

    The flasks were incubated at 30°C and 100 rpm. The process of enrichment was

    continued for two months, using the enriched culture as inoculum (10% v/v) to fresh

    MSM supplemented with the components mentioned above. At each transfer, the samples

    were plated onto MSM plates containing glucose (0.1% w/v), yeast extract (0.01% w/v)

    and 100 ppm γ-HCH. The plates were incubated at 30°C and observed for the microbial

    growth. The morphologically distinct colonies were purified by several transfers on the

    same medium.

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  • Materials and Methods

    3.4 Screening for potential biosurfactant-producing strains

    3.4.1 Blood agar method:

    The isolates were screened for their ability to produce surface-active molecules on

    blood agar plates. The overnight-activated cells in 20 ml nutrient broth were point

    inoculated onto the nutrient agar plates supplemented with 5.0% (v/v) sheep blood and

    incubated at 30°C. The plates were observed at a regular interval of 24 h up to 7 days for

    a clear zone around the colony due to hemolysis of red blood cells.

    3.4.2 Measurement of surface tension (mN m-1):

    The microbial strains were checked for biosurfactant production in 250 ml

    Erlenmeyer flasks, under shake flask conditions. The activation of the respective isolates

    was carried out by inoculating a single colony to nutrient broth and the flasks were

    incubated overnight at 30°C and 100 rpm. The activated cells were used as inoculum

    (5.0% v/v) in 50 ml MSM supplemented with glucose