meterial methods

8/13/2019 Meterial Methods

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3 1 Bacteriaotob cter sp.was isolated from garden soil. The soil sample was

collected from Thazhathangady, Kottayam District. g soil was addedto 90 ml of sterile distilled water and shaken by rotation of the flask. Samplewas streaked on to Jensons agar plates. Growth was observed after 48 h. Thebacteria was identified based on Bergy s manual of systematic bacteriol0gy.~4

Composition of Jensens agar medium:5sucrose 20.0 ePotassium monohydrogen phosphate 1.0 gMamesium sulphate 0.1 sCalcium carbonate 2.0 gSodium chloride 0.5 s

pH 7.0Deionised gl ss distilled water was used to ensure that the medium is devoid of ny metal

Sodium molybdateAgarDGDW

0.005 g15.0 g1000 ml


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3 I Metal treatment of bacterial culture and measurement of Nitrogenase activityJensens' broth culture was prepared by the inoculation of bacteria from

Jensens' agar plates into the Jensens' broth medium. Broth cultures wereshaken for uniform growth. After 48 h, i.e., at logphase, the culture wassubjected to metal treatments.

Sterile metal salt solutions were added to 2 ml of 48 h old bacterialculture in 100 ml glucose bottles, so that concentration of metal in the culturewas 0.5, 1 5, 10 and 25 ppm. Chlorides of nickel, cadmium, copper andmercury (NiClz, CuC13 CdQ2 and HgC12) were used.

10 of air in the bottle was replaced by acetylene using a sterile gastight glass syringe. After incubation period 0.5 m of gas mixture was takenfrom the glass bottle using microsyringe through rubber stopper and injectedinto the gas chromatograph at intervals. Nitrogenase activity was measuredafter 24 48 and 72 h of metal treatments

GC operational parameter for RDetectorCarrier gasDetector gasInjection port temperatureOven temperatureDetector temperatureColumn usedCompany

FID (Flame Ionisation detector)N 5ml/minH2 and air100C75C120CPoro pack (stainless steel)Shimadzu


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The amount of ethylene formed was calculated by the equation andexpressed as nanomoles CzH4 produced per m g protein per min.

Area of sample ethylene Gv-VCF x 105Area of standard ethylene 22.4 (Ti To)h

Gv = Gas volume of the containerVCF = Vacuum correction factorTI TO = Difference in sampling interval22.4 L = Volume of 1mol of gas at standard temperature and pressure.

3 7 2 reparation of extracts f r enzyme assaysBacterial culture was treated with .metal chloride solutions as

described above. The bacterial culture was centrifuged at 1500rpm for 20 minat 4C and cell paste was washed with double distilled water and later with0.01 M imidazole HCI buffer (pH 7.0) (containing 2 mercaptoethanol). Thecells were disrupted in 10 ml of the buffer by sonication at 4OC. The clearsupernatant fluid, after centrifugation was used as the enzyme source.

nzyme assaysa) Glutattrine synthet se G S P

Assay mixture contained:Irnidazole HCl buffer (0.2 M) 0.2mGlutamine (0.1 M) 0.4 mManganese chloride (0.1 M 0.6mAdenosine diphosphate (0.01 M) 0.2 mlPotassium arsenate 1M 0.4mDGDW 2.2 ml

Standard: Prepared a range of standards containing 100-500 py-glutamyl hydroxamate (Sigma Co.) in the buffer.


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0.5 ml of assay mixture was added to 0.21 ml of the extract Added0.2 ml distilled water and 0.1 ml hydroxylamine solution (2 M NHz0H.HCIand 2 N NaOH mixed in equal volume). Incubated at 30C for 30 min.Reactions were terminated and colour was developed after the addition of2 mlof stopping reagent (10 ea .4 trichloroacetic acid, 6 N HCI and h:,and 0.1 ml glutamine. . , .,(0.1 M were added to the above solution and absorbance was measured.Enzyme activity was expressed as niy~omolesof NADPH oxidised per mgprotein per min.c) Glutamate dehydrogenase GDHP7

Reagents-

---~._. -___NADPHAmmonium chloride


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Reaction mixture containing 0.3 m 2-0x0-glutarate, 0.12 ml NADPHand 0.5 ml N Cl was prepared in 2.7 ml final volume of tris-HC1 bufferpH 7.6. 0.3rnl of the extract was added to it.

Incubated at 37OC for 30min The change in absorbance was measuredat 340 nm in a spectrophotometer. Enzyme activity was expressed asnanomoles of NADPH oxidised per mg protein per min.

3 2 Lyngbya sp and ostoc spSoil samples were collected from Amballoor in Ernakulam District,

Manampur in Thiruvananthapuram District and Kumaranalloor in KottayarnDistrict and Lyngbya sp and Nostoc sp. were isolated using Fogg's medium.

Medium for isolation nd cultures8Potassium dihydrogen phosphate 0.2 g/LMagnesium sulphate 0 2 g/LCalcium chloride 0.1 g/LFe.EDTA solution ml/L

solution m l LFeEDTA solution-26.1 g EDTA was taken in 268 m of N potassium

hydroxide solution and to it was added 24.9 g of FeS04.7H20. The contentswere dissolved by stirring. Aerated the solution overnight till a dark strawcolour was developed, and stored in amber coloured bottles.

Preparation of Sstock solutionManganese chlorideSodium molybdateBoric acidZinc sulphateCopper sulphate


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The media prepared was autoclaved at 121C for 20 min at 5 lbs.Potassium dihydrogen phosphate was autoclaved separately. This sterilisedmedia was used for culturing the algae. The organism containing soil flakeswere washed thoroughly in distilled water and used for primary culture.

The algae obtained from primary culture was centrifuged using sterileFogg s medium 3-4 times. The pellets obtained was washed in antibioticsolution containing streptomycin and ampicillin, 6000 and 3000 wg/Lrespectively to remove surface contamination. This pellet was homogenisedto obtain short filaments. These filaments were subsequently used forculturing on Fogg s agar medium containing antibiotics. Fogg s mediumcontaining antibiotics was prepared as follows.

Fogg s medium 100mAgar 2 gAmpicillin 400 i.rgStreptomycin 200 WgThe algae were inoculated onto the solidified media in petridishes

using a wire loop. After 3-4 days, they were transferred to an antibiotic-lessmedium. The purity of culture was tested by streaking them on nutrientagar. The algae isolated were identified using standard procedure.SR

Algal culture at its log phase was washed several times with distilledwater and was transferred to freshly prepared Fogg s medium. 20 ml of theculture was dispensed into the glucose bottles so that each bottle contains100 mg fresh weight of alga. Then added metal stock solutions so that thefinal concentration of the cultures were 0.5,1,5,10 and 25 ppm.

Cultures were subjected to nitrogenase assay after 15, 30 and 50 daysof metal treatment Nitrogenase activity was measured by acetylene


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reduction assay (ARA) as described in the case of Awtobacter sp. Values areexpressed as nanomoles GI&produced per mg protein per min.

3.2.2 preparation of exiracis for enzyme assaysMetal treated cultures were prepared as in the case of Ntrogenase.

Extract used for assays of GS, GOGAT and GDH was prepared as follows.20m metal treated cultures were centrifuged and suspended the algal

pellet in about 10 m of tris buffer (50 mM 2 P-mercaptoethanol) pH 7.8.Sonicabon was done in 6 cycles of 20 se at 4OCfentrifuged for removlng thecell debris at 1500 rpm for 10min at 4'C. The resultant supernatant was usedfor enzyme assays.

(a) Glutamine synthetase (GS) #9GS was estimated using transferase assay. Assay mixture was

prepared as follows.isImidazole buffer 0.2 M - 0.2 ml qL

Glutamine 0.2 M - 4ml f

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