12_chapter 4.pdf - shodhganga

TERI University-PhD. Thesis, 2009

Materials and Methods

Standard solutions and buffers Aqueous stock solutions and buffers were prepared in Milli-Q water. The pH of buffers whenever necessary, were determined with a pH meter (Orion research, USA). The pH meter was standardised routinely before use with standard buffer solutions of pH 7.0, 4.0 and 9.0. The solutions of antibiotics of desired concentration were prepared in Milli-Q water as per instruction in the manual by Ausubel et al. (1989). The antibiotic solutions were stored at 4°C.

Chemicals and reagents General chemicals like sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium sulfate, magnesium chloride, calcium chloride, ammonium molybdate, ferric nitrate, zinc acetate, manganese chloride, cupric chloride, cobalt chloride, sodium borate, sodium hydroxide, sodium chloride were procured either from Merck (India) or Qualigens (India). Organic solvents and acids such as ethyl acetate, diethyl ether, methanol, acetone, hexane, benzene, phenol and acids: hydrochloric acid, sulphuric acid were purchased from Qualigens (India). Tween and molecular biology grade chemicals e.g. sodium dodecyl sulphate (SDS), proteinase K, lysozyme (chicken yolk), Tris salt, ethylene diamine tetra acetic acid (EDTA), agarose, vitamins and trace elements were obtained from Sigma chemical company (St. Louis, USA). The reagents for polymerase chain reaction (PCR) were obtained from Sigma chemical (St. Louis, USA). The primer sets were obtained from Sigma (USA). The commercial microbiological media like Luria Bertani (LB) agar, Luria Bertani (LB) broth and agar powder was obtained from Himedia (India).

The production water samples were procured from Oil India Limited (OIL) and Oil and Natural Corporation (ONGC). Both the companies are among the largest public sector oil and gas production industries in India. The commercial biocides were obtained from Navdeep Chemical Private Limited, India. Molecular biology kits The following DNA sequencing kits were obtained from PE Applied Biosystems (Foster City, USA): Genomic DNA isolation kit, partial and the full gene encoding 16S rRNA sequencing kit (Microseq™ 500 and Microseq™ 16S rRNA full gene bacterial sequencing kit), PCR product

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Materials and Methods 52

TERI university-Ph.D. Thesis, 2009

sequencing kit (Big dye™ terminator cycle sequencing kit). Qiagen’s (USA) RNA extraction kit was used for extraction of total RNA from induced cells. All the molecular biology kits were used as per the manufacturer’s instructions.

PCR primers The following are the different sets of oligonucleotide primers used in the present study for DGGE analysis

16S rRNA gene (El Fantroussi et al. 1999) 63F*: 5’- 'Cag gCC TAA CAC Atg CAA gTc-3’ 518R: 5’- ATT ACC gCg gCT gCT gg-3’

DSR gene (Geets et al., 2006) Dsr p2060F*: 5’-CAA CAT CgT (C/T)CA (C/T)AC CCA ggg-3’ Dsr 4R : 5’-GTg TAg CAg TTA CCg CA-3’

*A 40-bp GC-clamp (CgC CCg CCg CgC gCg gCg ggC ggg gCg gCA Cgg ggg g) was attached to 5’ end of the original primer sequence.

The primers were obtained from Operon and Sigma .

Following primers were used for sequencing of amplicons: M13F: 5´– gTA AAA CgA Cgg CCA g– 3´ M13R: 5´– Ag gAA ACA gCT ATg AC– 3´

Primers used for quantitative PCR are as follows: EUB341F 5’- CCT ACg ggA ggC AgC Ag-3’ EUB534R 5’-ATT ACC gCg gCT gCT ggC-3’ Dsr p2060F: 5’-CAA CAT CgT (C/T)CA (C/T)AC CCA ggg-3’ Dsr 4R : 5’-GTg TAg CAg TTA CCg CA-3’ I, Inosine; R, A or G; M, A or C; Y, C or T; K, G or T; N, A or C or G or T.

Softwares For identification of the sequences NCBI’s BLASTn search was performed.

Primers were designed using alignment tool Clustal W and software Gene Fisher and Gene Runner.

Nucleotide sequences were translated using Expasy translation tool.

Purity of nucleotide sequences were checked with Chromaspro and Bioedit softwares.

Theoretical Melting Gradient for DGGE was determined by Melt Ingeny, Ingeny, The Neatherlands



DGGE Gels analysis and Shannnon DIversity Index were calculated by Bionumerics (Applied Mathematics, Belgium)

The phylogenetic and evolutionary analyses were done using the software MEGA version3.1 (Kumar et al., 2004).

Gene quantification and melt-curve analysis was done by Rotor-gene 6 (Corbett international, Australia)

References were managed using Mendeley reference manager

Composition of media used Iron Lyngby Medium (Lorentzen et al., 2003)

Component Grams/liter Peptone 20 g Yeast ext 3 g Ferric citrate 0.3 g Sodium thio sulphate 0.3 g NaCl 5 g The pH was adjusted to 7.5 by 1 molar Sodium Hydroxide Solution.

Baar’s Medium (Tanner, 1989) Component I Quantity MgSO4 2 g Sodium Citrate 5 g CaSO4 1 g NH4Cl 1 g Milli Q 400 ml Component II K2HPO4 0.5 g Milli Q 200 ml

Component III Sodium Lactate 3.5 g Yeast Extract 1 g Milli Q 400 ml

All the components were mixed and adjust the pH to 7.5 by 1 molar Sodium Hydroxide Solution.

API-RP 38 medium (Tanner, 1989)

Component Grams/liter Sodium lactate 4 g/L



Yeast ext 1.0 g Ascorbic acid 0.1 g MgSO4 0.2 g K2HPO4 0.01 g KH2PO4 0.1 g Fe(SO4)2(NH4) 0.2 g NaCl 10 g

The pH was adjusted to 7.5 by 1 molar Sodium Hydroxide Solution

Study Area and Sampling Location The western and north eastern oil fields of India were selected for the present study. It is believed that this region of the Northeastern India have been formed out of ‘Tectonic Evolution of the Indian Continent,’ is literally floating on a vast oil field. The Major part of oil operation in north-eastern field is done by Oil India Limited, Assam. The western oil fields are located in the state of Gujarat and Mumbai High. The major oil and gas fields of Gujarat are Mehsana, Kalol, Ahmadabad, Khambat, Ankleshwar and Ghandhar. Mumbai High is an offshore oilfield 160 km off the coast of Mumbai. The first offshore well was sunk in 1974 and oil operations are run by India's Oil and Natural Gas Corporation (ONGC). Number of oil wells was drilled in these fields to produce thousands of barrels of crude oil, which is transferred to oil collecting station (OCS). The oil collecting station (OCS) process thousands of barrels of crude oil to separate water by means of oil-water separators. This separated water was known as production water. Production water is then injected into a disposal well through a pump delivery system. Production water samples from five different oil-water separation tanks installed in various oil fields of India were selected for this study. The northeastern sites were located 20 km east of Dibrugarh city in the state of Assam, India. These specific sites were known as Kathloni (K1) and Dikom (D2) oil fields. The sites located in western part of India were known as Kalol (K2) and Ghandhar (G2) oil fields, located in the state of Gujarat. The Bombay High offshore oil field was situated in the Arabian Sea in the south-western part of India. The production water process unit of Bombay high offshore oil fields is situated in Uran Process plant (D2), in the state of Maharashtra, India.

Sampling protocol Samples were collected anaerobically from different oil-water separation tanks. Oil water separators have sampling nozzles which were used to collect the samples. Production water samples were collected anaerobically from oil-water separators in sterilized 500 ml serum bottles and 10-liter carboys which were pre-flushed with nitrogen to maintain anaerobic



Figure 4.1: Geographical locations from which the production water samples were collected

Kalol, Gujarat

Ghandhar, Gujarat

Uran, Maharastra

Dikom, Assam

Kathloni,Assam

INDIA



conditions. The 500 ml serum bottles were used for onsite inoculation of the production water samples was done in various selective media for isolation and the three-tube most probable number (MPN) procedure. 3 ml of production water sample was inoculated in the 50 ml serum bottles having 30 ml of medium. The inoculated serum bottles and 10-liter carboys were transported back to the laboratory and stored at 4 °C until further use. Following precautions were taken while collecting the samples:

Bottles and carboys were pre labeled to avoid confusion

(a) Samples were filled upto the rim of the bottles to avoid oxygen

(b) Stagnant Liquid was carefully removed from the nozzle of the separator before sampling

(c) Latex gloves were worn throughout the sampling

Physio-chemical characterization of Production water samples Physico-chemical characterization of these production water samples was carried out using standard methods (APHA, 1995). Temperature and pH of the production water was measured on-site. Electrical conductivity was measured by Control dynamics conductivity meter (Scientific Systems, New Delhi, India).

Production water samples were also analyzed for total Dissolved solid (TDS), suspended solid, dissolved oxygen (DO), dissolved sulfide, sulfate and oil content.

Dissolved sulfide and sulfate was measured by sulfide and sulfate detection kit (Merck KGaA, 64271 Darmstadt, Germany) according to the manufacturer’s instructions.

Temperature Temperature was measured by Thomson scientific thermometer.

pH For most practical purposes, the pH of aqueous solutions can be taken as negative logarithm of hydrogen ion activity. pH values from 0 to 7 are diminishingly acidic, 7 to 14 increasingly alkaline and 7 is neutral. The pH of production water usually lies in the range of 5.5 to 8.5. The effect of pH on the chemical and biological properties of liquids makes its determination very important. It is used in several calculations in analytical work and its adjustment is necessary for some analytical procedures. pH was determined by digital pH meter.

Electrical Conductivity (EC) Conductivity is a capacity of water to carry an electrical current and varies both with the number and types of ions the solution contains, which in turn is related to the concentration of ionized



substances in the water. Most dissolved inorganic substances in water are in the ionized form and hence contribute to conductance. Rough estimation of dissolved ionic contents of water sample can be done by multiplying specific conductance (mS cm-1) by an empirical factor that may vary from 0.55 to 0.9 depending on the soluble components of water and on the temperature of measurement. Conductivity measurement gives rapid and practical estimate of the variations in the dissolved mineral contents of the production water. The EC of the effluent was measured using a digital EC meter (Control Dynamics, Bangalore).

Total Dissolved Solids The dissolved (filterable) solids can be determined from the difference between the residue on evaporation and total suspended solids, but if the dissolved solids content is low and the suspended solids high, a direct determination is better. It is preferable to adopt the centrifugal method of separating suspended matter in order that a sufficiently large volume of separated liquid is available for the determination. A known volume of filtered sample is evaporated and dried in a weighed dish at 105 °C to constant weight the increase in weight over the empty dish represents the dissolved solids.

To estimated the TDS in the production water, known volume of sample (50 ml) was filtered and transferred into the previously weight evaporating dishes (W1). The sample was evaporate to dryness on a steam bath and further dry at 105 °C for one or two hours in an oven. Subsequently sample was cool in the desiccator and weight (W2). Sample was again kept for heating for 15 minutes and cooling until successive results do not differ by more than about 0.4 mg. Total Dissolved solids were estimated using the following formula:

Where

W2 = Weight of residue and dish

W1 = Weight of empty and dry dish

V = Weight of sample

Dissolved Oxygen (DO) Analysis of DO is a key test in production water treatment practices. Estimation of DO is necessary levels to assess quality of raw water and to keep a check on stream pollution. The amount of DO is an important factor to determine the biological changes are fought out by aerobic or anaerobic organisms. Oxygen is an important factor in corrosion. DO test is used to control amount of oxygen in boiler feed waters either by chemical or physical methods.

Oxygen present in sample oxidizes the divalent manganeous to its higher valency, which precipitates as a brown hydrates oxide after addition of NaOH and KI. Upon acidification,



manganese reverts content of the sample. The liberated iodine is titrated against standard (N/80) solution of sodium thiosulphate using starch as an indicator. The reagents required to estimate DO are:

a. Manganous sulfate solution: 480 g MnSO4.4H2O, 400 g MnSO4.2H2O or 364 g MnSO4.H2O was dissolved in distilled water, filter, and dilute to 1 L. The MnSO4 solution should not give a color with starch when added an acidified potassium iodide (KI) solution.

b. Alkali-iodide-azide reagent: 500 g NaOH (or 700 g KOH) and 135 g Nal (or 150 g KI) was dissolved in distilled water and dilute to 1 L. Add 10 g NaN3 dissolved in 40 mL distilled water. Potassium and sodium salts may be used interchangeably. This reagent should not give a color with starch solution when diluted and acidified.

c. Sulphuric acid, H2SO4 conc.: one millilitre is equivalent to about 3 mL alkaliiodide-azide reagent.

d. Starch: To prepare an aqueous solution, 2 g laboratory-grade soluble starch was dissolved and 0.2 g salicyclic acid, as a preservative, in 100 mL hot distilled water.

e. Standard sodium thiosulfate titrant 0.025 N: 6.205 g Na2S2O3 .5H2O was dissolved in distilled water. Add 1.5 mL 6N NaOH or 0.4 g solid NaOH and dilute to 1000 mL. The solution was standardizing with potassium dichromate.

f. Standard Potassium Dichromate solution 0.025 N: Approximately 2 g K2Cr2O7 is dried in oven at 105 °C for 1 hour and put in desiccator for 1 hour. Accurate weight 1.2258 g is dissolved in distilled water and volume make up 1 L.

g. Standardization of Sodium thiosulphate Solution: 25 mL K2Cr2O7 of 0.025 N + 10 mL conc. HCl + 1 g Kl solid was taken. Dark yellow colour develops followed by titration with Na2S2O3. 5H2O. if light yellow colour develops; starch indicator (0.5 mL) is added and titrated to end point green.

2 ml MnSO4 solution was added to the production water sample collected in a 250 to 300 ml bottle, followed by addition of 2 ml alkali-iodide-azide reagent. The solution was mixed carefully and when precipitates has settled sufficiently (to approximately half the bottle volume); to clear supernatant above the manganese hydroxide precipitate, 2.0 ml concentrated H2SO4 was added. Solution was mixed again using stopper and by inverting several times until dissolution is complete. Titration was done of a volume corresponding to 200 mL original sample after correction for sample loss by displacement with reagent. Thus, for a total of 4 mL (2 mL each) of MnSO4 and alkali-iodide-azide reagents in a 300-mL bottle, titrate 200 x 300/ (300-4) = 203mL, titrate it against Sodium Thiosulphate solution. It becomes light yellow, add 2 or 3



drops starch blue, titrate till colorless end point. For titration of 200 mL sample, 1 mL 0.025 M Na2S203 = 1 mg DO L-1

Where:

N=Normality of Na2S2O3.5H2O

V=Volume consume in titration of Na2S2O3.5H2O

S=Sample taken

Total Petroleum Hydrocarbon (TPH) To extract the TPH, 100 ml of production water sample was taken in a flask and 50 ml of hexane was added to it. The flask was then put on a shaker for 3 hours and then left overnight for the suspended particles to settle down. Solvent from the top was collected in a flask and fresh hexane (50 ml) was added and the procedure was repeated. Consecutive extractions were done with hexane, toluene, methylene chloride and chloroform. All the three extracts were pooled and dried at room temperature by evaporation of the solvents under a gentle stream of nitrogen in a fume hood. After evaporation, the amount of residual TPH recovered was determined gravimetrically. For gravimetric analysis, the collecting dish was weighted prior to the extraction and also after the fraction was collected and dried. The difference between the two would give the amount of that fraction. Fractionation of TPH

Following, gravimetric quantification, the TPH was fractionated into alkane, aromatic, asphaltene and NSO fraction on a silica gel column. The TPH (500 mg) was dissolved in n-pentane and separated into soluble and insoluble fractions (asphaltene). The soluble fraction was then loaded onto silica gel columns and eluted with different solvents. The alkane fraction was eluted with 100 ml of hexane followed by the aromatic fraction, which was eluted using 100 ml of toluene. Finally, the NSO fraction was eluted with methanol and chloroform (100 ml each). The alkane fraction was analyzed by gas chromatography with flame ionization detector (GC-FID Hewlett Packard, 5890 Series II) using DB 2887 column (specifications) while the aromatic fraction was analyzed by GC-FID using a 30 m long DB 5.625 column (0.25 mm I.D, 0.25 μm film thickness). During analysis, the injector and detector were maintained at 300°C and the oven temperature was programmed to rise from 80°C to 270°C in 5°C/min increments and to hold at this temperature for 30 min. Individual compounds present in the alkane and aromatic fractions were determined by matching the retention time with authentic standards (Sigma chemicals, USA).

Dissolved Sulfide Determination



Dissolved sulfide in the production water was determined by sulfide detection kit (Merck KGaA, 64271 Darmstadt, Germany) and a spectrophotometer, according to the manufacturer instruction. The principle of this kit is based on the reaction of hydrogen sulfide with N, N’- dimethyl-1-4-phenylene-diamine dihydrochloride and its oxidation with Iron (III) to methylene blue. A standard curve was made with sodium sulfide ranging from 0 to 1.5 mg/l. The measurement of absorbance was done against a blank at 665 nm. For sulfide measurement by isolated cultures, media were prepared without the addition of iron salts. The sulfide production by isolated cultures was measured on the 0, 7, 14 and 28th day.

Acridine Orange Direct Count (AODC) and most probable number (MPN) estimation in production water sample The acridine orange direct count (AODC) procedure was followed to determine the total number of bacteria present in the production water samples (Hobbie et al., 1977). For enumeration of total viable cells, the three-tube MPN procedure was performed in modified API RP 38, Baar’s and Lynbay medium. Anaerobic media were prepared by dispensing 9 ml medium in glass bottles under oxygen-free nitrogen and quickly sealed with rubber stoppers and aluminum caps. 1 ml of anaerobically collected production water was inoculated in 9 ml of medium in triplicate. Medium was shaken well and 1 ml was transferred to a set of new bottles. This process was repeated to produce a series of eight ten-fold dilutions. The anaerobic bottles were incubated at 37 °C for 28 days. Culture growth was indicated by black FeS precipitate of FeS. Statistical analysis of the MPN was done by a computer program (Hurley and Roscoe, 1983). This was used to calculate MPN values and the standard errors for the MPN estimates.

Medium preparation, enrichment and isolation of hydrogen sulfide producing bacterial strains Medium preparation Three basal media were used to cultivate hydrogen sulfide producing bacteria. To mimic the conditions of an oil-water separator, all media were prepared in filtered (0.22 micron) and autoclaved production water. Sterilized production water was again filtered to remove precipitation. To cultivate SRB, samples were inoculated in Baar’s medium and modified API RP38 medium as described by Tanner [19]. To cultivate TRB, samples were inoculated in S7 (supplemented by thiosulfate) medium [20]. After adding all the components, pH was adjusted to 7.5 with 1 M NaOH and medium was boiled under a stream of O2-free nitrogen gas. The media was then dispensed in 50 ml serum bottles under oxygen free nitrogen gas and sterilized at 121°C and 15 psi pressure for 20 min.



Enrichment Strategy The bacterial strains were isolated by enrichment culture technique from production water samples collected from oil fields located in various part of India. For enrichment, 3 ml produced water samples were inoculated into 30 mL of anoxic selective media and incubated at respective temperatures as noted during sample collection for 28 days. The composition of various media is detailed elsewhere. The enrichment temperatures were maintained similar to oil-water separation tanks to enrich the same microbial flora which proliferate in these tanks. It was observed that maximum hydrogen sulfide production takes place when the crude oil is pumped out of oil reservoirs where temperature is favorable for these microbes to proliferate. Three series of enrichments were performed before isolation.

Isolation of bacterial strains with Hungate Roll Tube method Strains were isolated by repeated use of the Hungate roll tube method [21], with 1.8% solidified agar medium. All the medium components were added and pH was adjusted to 7.5 with 1 M NaOH and medium was boiled under a stream of O2-free nitrogen gas. Ten ml of it was dispensed anoxically in roll bottles (50ml) and sterilized at 121 °C and 15 psi pressure for 20 min. These bottles were inoculated with 100 μl of dilutions from 10-1 to 10-8 enriched production water samples using disposable syringes. Now these bottles were rolled continuously on ice to form a uniform layer on the side of walls. Bottles so prepared were incubated at respective temperatures. In order to check the purity of the cultures, the serially diluted roll tube process was performed three fold. Isolated pure cultures were differentiated on the basis of colony and cell morphology and gram staining.

Characterization of the bacterial strains isolated from production water samples Gram’s staining and morphology study of the selected strain

Isolated pure bacterial colonies were suspended in 100 μl of 0.85 % NaCl and smeared and spread evenly on a clean glass slide. The smear was dried; heat fixed and allowed to cool. The smear was then stained with crystal violet for 30 sec. The stain was drained off and washed with water. The smear was covered with fresh Lugol's iodine solution for 60 sec and then washed off with distilled water. Ethanol (95 %) was used to remove excess crystal violet. The smear was then counter stained with saffranine for 60 sec. The slide was washed with water, dried and observed under a compound microscope (Olympus BH-2, USA) at 100× magnification. The



images of the bacterial cells were photographed with the CCD (charge couple device) camera (Image Pro plus version 4.10). The composition of the stains is described in the Annexure.

Electron microscopy study of the selected bacterial strain The morphology was studied using Scanning Electron Microscopy (SEM) for those isolates which showed very distinguished morphology in gram staining. For SEM analysis, the selected bacterial strain was grown overnight at 55˚C on a Luria Agar plate. The cells picked picked up using a sterile loop and fixed in 2.5% glutaraldehyde (Annexure 1) for 6 hours. Further, the fixed sample was mounted on a gold-coated grid and stained by 0.5% (w/v) uranyl acetate solution. The grid was air-dried and then observed using scanning electron microscope (LEO 435 VP, 30 kV voltage). The Scanning electron microscopy analyses were done at the Indian Institute of Science (IIT), New Delhi, India. Thermo-tolerance of the isolated bacterial strain

Temperature has important effect on the growth, activity and H2S production by isolated bacteria, so optimal growth temperature and temperature tolerance was determined. The growth temperature of the isolates was tested on the basis of blackening of media at different temperatures. The strain was grown at incubation temperatures of 30°C, 35°C, 37 °C, 40°C, 45°C, 50°C, 55°C, 60°C and 70°C. Experiment was done in batches of 10 to 15 isolates per batch and uninoculated media were used as control. The appearance of blackening in the media was indicative of growth. The growth profile at these temperatures was also tested by growing the bacterial strain in respective media and measuring the dissolved sulfide after 72 hours. The thermo tolerance was measured because these bacteria survive at high temperature in oil reservoirs but have higher activity in external production facilities, near well heads and near to injection well head.

Hydrogen sulfide determination Hydrogen sulfide production by isolated cultures was also measured on the 0, 3, 7, 14 and 28th day with a sulfide detection kit (Merck KGaA, 64271 Darmstadt, Germany) as earlier mentioned. This sulfide estimation was also carried out during the optimum temperature determination experiment.

Identification and phylogenetic analysis Genomic DNA extraction Genomic DNA of purified cultures was extracted according to the protocol from Hendrickx et al., (2006) with some modifications. Two ml of bacterial cell culture was suspended in 4 ml of Tris-



glycerol buffer (10mM Tris +15% glycerol, pH 7). To this suspension 160µl of Lysozyme solution (50mg/ml) was added and incubated at 37 °C for 30min. After incubation 120µl of 20% sodium dodocyle sulfate and 32 µl of proteinaseK (20mg/ml) was added, mixed gently and incubated at 50°C for 30 min. To this 2 ml of NaKPO4 buffer (5.3397g Na2HPO4 and 4.0827g K2HPO4 in 500ml H2O, pH 8) was added. Vials were kept on ice for 30 sec and centrifuged for 10 mins at 7000rpm.Supernatant was carefully transferred in to fresh vials and was extracted with phenol:chloroform:isoamyl (25:24:1). DNA was precipitated with 2 volumes of absolute ethanol and 3M Sodium acetate pH 5.2 and was kept at -20°C overnight. DNA was washed with 70% ethanol and pallet was resuspended in 50µl of TE –buffer. Quality of DNA was analyzed by 0.7% agarose gel stained with etidium bromide and DNA concentration was measured spectrophotometrically by Nanodrop. Agarose gel electrophoresis

Genomic DNA was resolved on 0.8 % agarose gel. The agarose gel (0.8 %) was prepared in 1× TAE buffer (Annexure 1). The electrophoresis was performed in 1× TAE buffer for 5 to 6 hrs at a constant voltage of 140 V at 25 C in Bio-Rad gel casting apparatus (Bio-Rad, USA). The DNA samples were visualized by staining with 0.6 μg/ml of ethidium bromide. The agarose gel DNA profiles were observed and photographed in UVI gel documentation (UVItec, Cambridge, UK). The data analysis was done with UVI photo V.99 and UVI band / map V.99 software (UVItec.). Identification by sequencing of 16S rRNA gene

The identification of the bacterial strains was done by sequencing and analysis of the 16S rRNA gene as per the protocols described below.

Amplification of the partial 16S rDNA sequences

The 500 bp sequences of 16S rDNA were amplified with Microseq™ 500 16S rDNA-PCR module (PE Applied Biosystems, USA). A volume of 1 μl of the genomic DNA was diluted in 24 μl of nuclease free sterile de ionized water.

Figure 4.2: Cycling conditions for amplification of partial 16S rRNA gene

96 °C 94°C

60 °C

5 min 1 min

30 sec 4°C∞

35 cycles

1 min 5 min72 °C 72°C



The various reagents of PCR: primers, dNTPs, AmpliTaq Gold DNA polymerase, MgCl2 and buffer are pre-mixed into a single tube as the PCR master mix. A 50 μl of the reaction mixture was prepared which consisted of 25 μl of the diluted genomic DNA (1 ng/μl) and 25 μl of the PCR master mix. The cycling conditions for the amplification reaction included an initial denaturation step at 96°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 30 sec and elongation at 72°C for 1 min. There was a final extension step of 5 min at 72°C (Figure 4.2). A rapid thermal ramp of 1 C/sec was maintained between the above steps. A 5 μl of the amplified 16S rDNA was confirmed on a 2% agarose gel as mentioned earlier

Purification of the amplified 16S rDNA

The PCR products were purified with Microcon 100 PCR centrifugal filter device (Millipore, USA). The Microcon column was hydrated by adding 500 µl of sterile MilliQ water to the column. The column was centrifuged at 500 × g in a fixed angle microcentrifuge for 6 min. After hydration of column, 400 µl sterile MilliQ water was added to the column and then 45 µl of the PCR product was loaded on to the column. The column was spined at 500 × g in a fixed angle microcentrifuge for 15 min. Collection tube was removed and discarded. The column was now inverted and attached to a new collection tube. A volume of 25 µl sterile MilliQ water was added to the inverted column and spinned the inverted column at 10000 × g for 3 min to collect the purified DNA in the collection vial. The purified DNA was recovered in 25 μl of de-ionized water.

Cycle sequencing of the amplified 16S rDNA

The amplified 16S rDNA was subjected to cycle sequencing with Microseq™ 500 16S rDNA sequencing module. The forward and reverse sequencing reactions were assembled in separate reactions. The 20 μl of the reaction mixture consisted of 3 μl Purified PCR product and 13 μl of sequencing reaction mix. The final volume of 20 μl was made up by 4 μl of de-ionised water.

Figure 4.3: . Cycling conditions for cycle sequencing of the amplified partial16S rRNA gene

96 °C

50 °C

60 °C10 sec

5 sec

4 min

4°C∞

25 cycles



The cycling conditions for the amplification reaction included 25 cycles of denaturation at 96°C for 10 sec, annealing at 50°C for 5 sec and elongation at 60°C for 4 min (Figure 4.3). There was no final extension step. A rapid thermal ramp of 1 C/sec was maintained between the above mentioned steps.

Precipitation of cycle sequenced DNA

The cycle sequenced DNA of all the sequencing reactions (partial and full length) were precipitated with 95 % ethanol and 3M sodium acetate (pH 4.6). The cycle sequenced product was transferred into a 1.5 ml eppendorf tube and 80 μl of sterile MilliQ water was added water to make the final volume to 100 μl. Next 10 μl of 3M sodium acetate (pH 4.6) and 250 μl of 95 % ethanol is added. This was incubated at 4°C for 10 min. The sample was centrifuged at 15000-rpm at room temperature for 30 min. The supernatant was decanted. The DNA pellet was washed with 250 μl of 70% ethanol by centrifuging at 15000-rpm for 5 min. The supernatant was decanted. The 70 % ethanol wash step of the DNA pellet was repeated. The supernatant was carefully aspirated. The DNA pellet was air dried at room temperature and re-suspended in 20 μl Hi-Di Formamide (PE Applied Biosystems, USA).

The cycle sequenced product was transferred to sequencing vials to proceed with sequence analysis (ABI prism 310 genetic analyzer, PE Applied Biosystems). Analysis of the DNA sequences

All the cycle sequenced DNA for the present study was resolved by ABI PRISMTM 310 genetic analyzer (PE Applied Biosystems, USA). The DNA samples were sequenced with either the short capillaries (5-47 cm × 50 μm) and long capillaries (5-61 cm × 50 μm). The electrophoresis was performed with 1× electrophoresis buffer with EDTA and performance optimized polymer (POP6). The parameters set for the electrophoresis in ABI PRISMTM 310 genetic analyzer is as follows:

Temperature 50 °C

Current 4 μA

Voltage 12 kV

Argon ion Laser power 9.8 MW

The purity of the 16S rRNA sequences was checked with Microseq software and Chromaspro version 1.41 (http://www.technelysium.com.au/ChromasPro.html). A chimera check was done with RDP 9 (http://rdp.cme.msu.edu/). To identify the isolates, sequences were subjected to a BLAST search with the NCBI database (Altschul et al., 1997). Multiple sequence alignments of approximately 500-bp sequences were performed using CLUSTAL W, version 1.8 (Thompson et al., 1994). A phylogenetic tree was constructed with the evolutionary distances using the



neighbor-joining method (Saitou and Nei, 1987). Tree topologies were evaluated by performing bootstrap analysis of 1000 data sets with the MEGA 4 package (Tamura et al., 2007). The 16S rRNA sequences of representative isolates from this study have been submitted to the NCBI Genbank Database with accession numbers as EU664980 to EU664988.

The PCR based 16S rDNA fingerprinting of bacterial isolates using Denaturating Gradient Gel Electrophoresis The pcr based genomic fingerprints of the different bacterial strains were obtained with the 16S rDNA primer sets: 63F and 518R, followed by DGGE profiling. The heterogeneity of 16S rDNA sequence makes it a sensitive method to assess species diversity. The DGGE based profiling method has been earlier used to assess inter and intraspecies diversity of culturable bacterial strains (A.de Souza F et al., 2004).

Polymerase chain reaction

Polymerase chain reaction on the extracted DNA was performed in a volume of 50 μl. A 495 bp eubacterial 16S rRNA gene fragment was amplified using the primer set GC-63F/518R, described by Marchesi et al. (1998). 1 μl of 1:10 or 1:50 dilution of template DNA was added to 49 μl of PCR mix consisting of 5 μl of 10x exTaq reaction buffer (20 mM MgCl2), 0.25 μl exTaq Polymerase (5 U.μl-1), 4 μl dNTP (deoxynucleoside triphosphate; 2.5 mM each), 0.25 μl of both primers and 39.25 μl sterile nuclease free water. The exTaq Polymerase, dNTPs and PCR reaction buffer were purchased from TaKaRa (TaKaRa Shuzo Co., Biomedical Group, Japan).

Figure 4.4: Cycling conditions for amplification of 16S rRNA gene

The PCR profile consisted of an initial denaturation of 5 min at 94 °C, followed by 35 further denaturation cycles of 1 min at 94 °C, annealing of 30 sec at 55 °C, an delongation for 1 min at 65 °C. The last step included an extension for 5 min at 65 °C (figure 4.4). PCR was performed on a Biometra thermocycler (Biometra, Göttingen, Germany). 10 μl of the PCR products were analysed by agarose gel electrophoresis to evaluate their size and quality, (1.5 % agarose (Invitrogen, Paisley, Scotland, UK), 1 x EY running buffer (10 x EY-buffer: 0.4 M Tris, 0.02 M

5 min

94°C

55°C

65°C 65°C94°C

1 min

30 sec

1 min 5 min

4°C ∞

35 cycles



EDTA, on pH = 7.9 with acetate in H2O), 1 hour at 85 V). DNA bands were visualized by ethidium bromide staining (1 mg.L-1).

Purification of the amplified 16S rDNA The PCR products were purified with Microcon 100 PCR centrifugal filter device (Millipore, USA). The Microcon column was hydrated by adding 500 µl of sterile MilliQ water to the column. The column was centrifuged at 500 × g in a fixed angle micro centrifuge for 6 min. After hydration of column, 400 µl sterile MilliQ water was added to the column and then 45 µl of the PCR product was loaded on to the column. The column was spined at 500 × g in a fixed angle microcentrifuge for 15 min. Collection tube was removed and discarded. The column was now inverted and attached to a new collection tube. A volume of 25 µl sterile MilliQ water was added to the inverted column and spinned the inverted column at 10000g for 3 min to collect the purified DNA in the collection vials. The purified DNA was recovered in 25 μl of de-ionized water.

Denaturing gradient gel electrophoresis Bacterial diversity was examined by denaturing gradient gel electrophoresis. In DGGE analysis DNA fragments of the same length but with different base-pair sequences can be separated, thus obtaining a band pattern in a denaturing polyacrylamide gel in which each band theoretically corresponds to one type of bacterium. Eubacterial 16S rRNA gene PCR products obtained with the primer set GC-63F/518R were analysed in 8% polyacrylamide gels with a denaturing gradient of 35% to 65% urea-formamide (100% denaturant gels contain 7 M urea and 40% formamide). PCR products obtained with the GC-P2060F/DSR4R primer set were analysed in 8% polyacrylamide gels with a denaturing gradient (40% to 70%). In both cases, DGGE was performed at a constant voltage of 120 V for 15 h in 1 x TAE (Tris-acetate-EDTA) running buffer at 60 °C. The electrophoresis was performed on an INGENY phorU-2 DGGE apparatus (INGENY International BV, The Netherlands). After electrophoresis, the gels were stained in a 1 x TAE buffer containing 1 x SYBR Gold nucleic acid stain (molecular Probes Europe BV, Leiden, The Netherlands) and photographed under UV light with a Pharmacia digital camera system with Liscap Image Capture 1.0, Pharmacia Biotech, UK). Photo files were processed and analysed with Bionumerics software (version 2.5, Applied Maths, Kortrijk, Belgium).

Total diversity studies using dsrB gene as a functional marker DNA extraction from production water samples



DNA extraction from production water samples was done according to the modified protocol of Hendrickx et al. (2006), as described earlier. Prior to DNA extraction 500 ml of production water samples were centrifuged at 12000 rpm for 15 min and the resultant pellet was pooled in 2 ml Tris-EDTA buffer, which was then used for DNA extraction. This was done to settle down the microbial flora exist in the production water samples. The supernatant was discarded carefully, so not to disturb the pellet.

Several pure strains of SRB were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), GmbH, Braunschweig, Germany to prepare a standard dsrB gene based DGGE Ladder. Genomic DNA extraction of these strains was also done by according to the protocol of Hendrickx et al. (2006), as described earlier.

PCR amplification and DGGE analysis of dsrB gene fragment The schematic representation of strategy followed for PCR-DGGE is given in figure 4.5. The extracted DNA of the pure SRB strains and the production water samples, were used as template for PCR amplification using the primers DSRp2060F and DSR4R (Geets et al., 2006). The PCR amplification of dsrB gene fragment was done in a 50 µl reaction mixture containing 1X PCR buffer containing 15 mM MgCl2, 800µM of dNTP mix, 50 nM of each primer and 5 units of TAKARA Taq DNA polymerase. Thermo-cycling conditions were: initial denaturation at 95°C for 10 min, followed with 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 1 min followed by 72°C for 10 min. These primes amplified approximately 350bp of the dsrB gene fragment. Residual dNTPs and primers were removed using PCR Microcon columns (Millipore, USA) according to the manufacturer’s protocols. Finally the PCR amplified product was resuspended in 25 µl nuclease free water (MoBio Inc., Solana, CA, USA). The PCR product (5 µl) was diluted to 1:1000 and used as template for semi-nested PCR with the primers gcDSRp2060F and DSR4R (Forward primer consisted of a 40 bases long GC clamp). While using these primers, the reaction mixture and amplification conditions were kept identical.

Community pattern based on dsrB gene was analyzed by dsrB gene based DGGE analysis. Denaturation conditions for DGGE analysis were determined by Melt95 software according to melting temperature of the amplicon (Melt analysis software 1.0.1, Ingeny International BV, The Netherlands). Denaturation gradient was made from 40% to 70% (w/v) by urea and formamide for 8% polyacrylamine gel (1 mm thick). DGGE was performed with the INGENYphorU-2 system. 1X TAE was used as running buffer. Electrophoresis was carried out at 60 °C initially at 200 V for 10 min followed by 120 V for 15 h. Following electrophoresis DGGE gels were stained with 1X SYBR Gold nucleic acid stain (Molecular probes, Europe BV, The Netherlands)



in 100 ml 1X TAE for 15 min. Stained gels were visualized under UV light using Pharmacia digital camera system with Liscap capture software version 1.0 (Pharmacia Biotech, England). The DsrB-DGGE ladder was used for normalization of banding pattern in DGGE gels.

Figure 4.5: Strategy for DGGE, cloning and sequencing

Cloning and Sequencing of dsrB gene fragment

Cloning of purified dsrB gene fragment was done in pCR 2.1-TOPO plasmid vector and was transformed in E. coli TOP10 cells according to manufacturer’s instructions (Invitrogen, CA). Clones were screened for the presence of positive insert by PCR using the gcDSRp2060F/ DSR4R primer set. PCR amplified dsrB gene product of positive clones were compared with dsrB-DGGE pattern of original oil field samples. A mixture of PCR amplified dsrB gene products of positive clones of pure SRB strains was used as dsrB gene based DGGE ladder.

The dsrAB gene from Desulfomicrobium norvegicum ( DSMZ) and 16S rDNA from Hydrogenophaga species were amplified by using DSR1F-DSR4R and 27F-1492R primer sets respectively in order to construct the plasmids to be used for standard curve in qRT-PCR, (Lane, 1991; Wagner et al., 1998). Amplified PCR product was cloned in pCR 2.1-TOPO plasmid vector

Production waters

Nucleic Acid Extraction

Pure SRB Strains

gc-dsrB gene amplification

dsrB gene amplification

1:1000 dilution Cloning

gc-dsrB gene amplification

Positive clone selectionby gc-dsrB PCR

DGGEstandardization

Ladder for DGGE

DGGETo be used with dsrB-DGGE

of production waters

Sequencing of selected dsrB clones



and was transformed in E.coli TOP10 cells according to manufacturer’s instructions (Invitrogen, CA).

Positive clone inserts representing different DGGE fragments were selected for sequencing of the expected 350bp dsrB fragment (UZA, Antwerp, Belgium). Sequences so obtained were run through BLAST search tool of NCBI for similarity analysis using nr database (Altschul et al., 1997). The dsrB gene sequence of the clone obtained in this study was deposited at the NCBI GenBank database (accession numbers: FJ648427 to FJ648451).

DsrB gene sequences were translated using Expasy program (Gasteiger et al., 2003). Multiple sequence alignment of obtained protein sequences along with dsr gene protein sequences of SRB representative strains from NCBI database was performed using CLUSTAL W, version 1.8 (Thompson et al., 1994). Aligned sequences were edited manually to remove the regions of ambiguous positional homology. A phylogenetic tree was constructed with the evolutionary distances using the neighbor-joining method (Saitou and Nei, 1987). Tree topologies were evaluated by performing bootstrap analysis of 1000 data sets with the MEGA 4 package (Tamura et al., 2007).

Quantification of SRB in production water samples using quantitative real-time PCR assay and its application in evaluation of the biocidal efficiency A quantitative real-time assay using dsr gene fragment was developed with the aim for quick and reliable quantification of SRB in production water samples, This assay was checked to evaluate the efficiency of the biocidal treatments, so that this method can be used by oil industries Quantitative Real-Time PCR assay (qRT-PCR) The qRT-PCR was performed in a Rotor-Gene 3000 real-time PCR machine (Corbett Research, Sydney, Australia) in a 72- well rotor. The thermocycling program for dsrB gene qRT-PCR were as follows: Initial denaturation at 94 °C for 15 min, followed by 40 cycles of 30 sec at 94 °C, 20 sec at 60 °C, 30 sec at 72 °C. The amplification conditions for 16S rRNA gene qRT-PCR were: Initial denaturation at 95 °C for 15 min, followed by 40 cycles of 10 sec at 95 °C, 15 sec at 60 °C, 20 sec at 72 °C. Data acquisition was performed using Sybr and FAM/Sybr detection channel in the PCR extension step. For every run the standard curve was incorporated and all the samples were calculated against this standard curve. Reaction mixture of RT-PCR were prepared in 25 µl



total volume containing 12.5 µl Ready reaction mix (AB gene), 5 µl of each primer (70nM), 1.5 µl of nuclease free water (MoBio Inc., Solana, CA, USA) and 1 µl of DNA template. Samples and standards were prepared in triplicate. Melt curve analysis protocol was added after thermocycling in order to verify that the used primer pair produced only one PCR product. Melt curve analysis was performed from temperature 72 °C to 98 °C with 1 °C hold for 5 sec.

Primer ratio optimization Primer ratio optimization was done to find the optimal concentration of both forward and reverse primers for quantitative PCR reaction. Various concentrations of primers (50nM, 100nM, 200nM, 300nM, 400nM and 500nM) were used in combination with each other in the reaction mixture with known amount of plasmid. The primer set giving lowest Ct value was further evaluated for primer–dimer formation without adding template (No template control).

Standard curve preparation Concentration of purified plasmids were measured with Picogreen (molecular probes, Eugene, OR, USA) and three serial dilution series were prepared from 4.5 × 107 to 4.5 × 102 dsrB gene copies/ µl and 9 × 108 to 90 16S rRNA gene copies/ µl . Copies/µl was calculated according to the formula as mentioned by Smith et al. (2006). In order to verify that standard curves are able to determine absolute copies in the chromosomal DNA samples, a serial dilution of known number of copies of Desulfovibrio vulgaris genomic DNA was calculated by standard curve. Serial dilution of Desulfovibrio vulgaris genomic DNA was mixed with a 50 ng/µl Escherichia coli chromosomal DNA and was used for assay to mimic the effect of foreign DNA in the samples. To analyze the effect of PCR inhibitors in the environmental samples, serial dilution of Desulfovibrio vulgaris genomic DNA was mixed in the environmental sample diluted till below detection limit.

Quantification of samples Real-time PCR assay was performed in triplicate for the samples along with the standard curve. Obtained gene copies/µl DNA was changed to gene copies/ml production water. Melt-curve analysis was done to check the specificity of the amplicon.

Biocides tests with enriched production water To mimic the conditions of an oil-water separator, all media were prepared in filtered (0.22 micron) and autoclaved production water. Sterilized production water was again filtered to remove precipitation. In the present study, production water samples were inoculated in modified API RP38 medium as described by Tanner (1989). After adding all the components, pH was adjusted to 7.5 with 1 M NaOH and medium was boiled under a stream of O2-free nitrogen gas. The media was then dispensed in 50 ml serum bottles under oxygen free nitrogen



gas and sterilized at 121 °C and 15 psi pressure for 20 min. For enrichment, 3 ml formation water sample was added to 30 ml medium and incubated at 37 °C for 15 days. The Desulfovibrio vulgaris was also inoculated in the same medium to be used as positive control for the experiment.

Six commercial biocides were initially screened against the actively growing cultures. To study the effect of each biocide, the actively growing cultures were inoculated in modified API RP 38 medium containing different concentration of biocide (200 mg/litre to 25 mg/litre and control). The inoculated cultures were again incubated at 37 °C for 28 days. The most efficient biocide was further investigated for lower range lower of concentration (5 mg/l to 25 mg/l) which can inhibit SRB. For this freshly prepared modified API RP38 medium containing mentioned concentration of selected biocide was inoculated with actively growing production water mixed cultures.

Determination of lowest concentration of selected biocide to inhibit SRB using quantitative PCR Quantification of SRB in these habitats is critical for the design of efficient treatment for the corrosion causing microorganisms. 16S rDNA gene copy number and dsrB gene copy number was calculated in the enriched culture with the above mentioned concentration (5 mg/L to25 mg/L) of BNDP. Quantitative PCR assay was performed as mentioned earlier. All the samples were quantified in triplicate and corresponding average value were converted to gene copies per ml of produced water. Standard deviation in the samples was also calculated.

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