medium preparation for isolation of bacterial...

TERI University, PhD Thesis 2010

MATERIALS AND METHODS

Medium Preparation for Isolation of Bacterial

Strains

Various nutrient media containing different constituents were tried for culture of

thermophilic anaerobic sulphate reducing bacteria (SRB) bacterial strains collected

from different oil well samples. The different nutrient media used in this study are

mentioned in Annexure I (media S1-S15). All the components of a medium except

the reducing agents were added to the medium. The medium in the flask were

boiled and cooled under N2; CO2 (80:20) gas mixture and the pH was adjusted to

7.5 (Jackson and McInerney, 2001). The medium was purged with anaerobic gas

mixture comprising of N2: CO2: H2 in a ratio of 90: 5: 5 and the reducing agents were

added. Then medium was dispensed into specialized anaerobic bottles using 50 ml

disposable syringe. The bottles were then properly plugged and sealed with rubber

stopper and aluminum crimps. The bottles containing media were then autoclaved

at 121 °C and 15 psi pressure for 20 min, membrane sterilized vitamin solution was

added to the autoclaved medium. Different media were inoculated on the spot at oil

field with aliquote of formation fluid. The overhead space of the anaerobic bottles

was filled with the anaerobic gas mixture of N2; CO2; H2 in a ratio of 90: 5: 5. pH of

the medium was adjusted to 7.5 before the medium was dispensed in anaerobic

bottles under nitrogen. The boiling medium (5 ml) was dispensed into 10 ml bottles

while sparging oxygen free nitrogen gas continuously. Before dispensing the

medium into anaerobic bottles, medium was reduced by adding cysteine HCl and

sodium sulfide. The anaerobic bottles containing different nutrient mixture were

capped with butyl rubber stopper carefully preventing entry of air into the bottle.

The bottles were sealed with aluminium caps using crimper. The medium was

autoclaved at 121ºC for 15 minutes and cooled in anaerobic chamber.

3

Materials and methods


49

Collection of Formation Fluid Samples from Oil

Collecting Tank

Formation fluid samples were collected from oil wells of India (Table 3.1). The

samples were collected from oil wells of Oil India, Assam in the month of December

04, February 05 and Aug 05. This was done to check the reproducibility of SRB

population in oil reservoir.

The samples from the oil wells were inoculated into the different media set

(Annexure 1) on site, after the addition of 0.1% cystine HCl. The samples were

collected from K-7 (Kathloni oil wells no. 7), K-8 (Kathloni oil well no. 8), D-13

(Deccan oil well no. 13), H– 497 (Hathiali oil well no. 497) and B -500 (Bekulajaan

oil well no. 500) oil wells mainly and also from few other oil wells.

Formation fluid from the oil wells was collected through sampler nozzle. Anaerobic

sample bottles were filled with formation fluid and bottles were capped immediately

with butyl rubber stoppers and crippled with aluminium caps.

The formation fluids collected in anaerobic bottles were used as inoculum for

cultivation of thermophilic anaerobic SRB bacterial strains. The sets of anaerobic

SRB bacterial strains from the different oil wells containing different media were

inoculated with formation water and inoculated bottles were transported to TERI,

New Delhi and incubated at different temperatures (37 oC, 55ºC, 75ºC, and 90ºC).

The bacterial strains after first enrichment cycle were purified by spread plate

technique in an anaerobic chamber. The isolates were purified by Hungate roll tube

method (Hungate, 1969). The purity of anaerobic isolates was checked

microscopically.

Table 3.1 The geographical location of the sampling sites

Sampling sites in

India

Geographical location Latitude and longitude

OIL, Assam Northeastern India 27o5′38′′ N to 27o42′30′′

N

ONGC, Ahemdabad Western India 22o46′100′′ N to

72o18′56′′



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Analysis of the Collected Formation Water

The formation water thus obtained was then analyzed for the electrical conductivity,

pH, salinity, cations and anions, total dissolved solid and volatile fatty acids were

also analyzed (Scott, 1925).

Qualitative and Quantitative Estimation of the

Different Fractions of Total Petroleum

Hydrocarbon in Crude Oil

The quantitative and qualitative estimation of degradation of the aliphatic and

aromatic fraction of total petroleum hydrocarbon (TPH) was performed as per the

protocol mentioned. The protocol is as follows:

Extraction of residual TPH

The TPH was extracted by evaporation of the crude oil with hexane and

choloroform respectively. The extracts were dried solvent was removed at 30 oC in a

fume hood. After drying the residual oil was quantified by „weight reduction

technique‟.

Fractionation of residual TPH

The residual TPH was fractionated by silica gel column chromatography (Walker et

al, 1975) into aliphatic and aromatic fractions. Residual TPH was first dissolved in

n-pentane and separated into soluble and insoluble fractions (asphaltenes). The

soluble fraction was further separated into aliphatic and aromatic fraction on silica

gel column. The column (50x 20 cms) was cleaned with water, dried and rinsed with

ethanol (to remove traces of moisture). The column was finally rinsed with hexane.

The slurry of the silica gel, which was activated overnight at 100 oC was prepared in

hexane and was packed in the glass column. A quantity of 500 mg of the crude oil

(residual TPH) was loaded to the silica gel column. The fractionation of the TPH



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was accomplished by successive elution with hexane and toluene. The aliphatic

fractions were eluted with 100 ml of hexane and aromatic fractions were eluted with

100 ml toluene.

Analysis of aliphatic and aromatic fractions of TPH

by gas chromatography

The aliphatic fraction was analysed by gas chromatography (GC) (Hewlett and

Packard 6890N) fitted with flame ionisation detector (GC-FID, (Hewlett Packard

USA) using a 30 m long wide bore DB 5 column (0.53 mm by 1 µm [film thickness]).

The aromatic fraction was analysed by GC-FID using a 30 m long column DB5.625

(0.25 mm inside diameter, 0.25 µm film thicknesses). During the analysis, the

injector and the detector temperature of GC were maintained at 300 oC and the

oven temperature was programmed at rise from 80-240 oC at the rate of 5 oC per

minute increments and to hold at 240 oC for 30 mins.

Preparation of standards for aliphatic and aromatic

compounds

The alkane compounds form C11 to C30 were weighed (5 mg) in a clean and dry glass

vial and were solubilised in 5 ml of hexane. The mixture was diluted (5 times) and

injected (0.2 μl) into GC (FID). The aromatic standard compounds were weighed

individually in a similar manner and dissolved in acetone.

Individual aliphatic and aromatic compounds were determined by comparing the

retention times with the aliphatic and aromatic standard compounds.

Scanning Electron Microscopy

The selected strains amongst the isolated purified strains were chosen for further

studies. The exponentially growing cultures were then subjected to electron

microscopic studies at IIT, Delhi and Department of Transmission Electron

Microscopy in All India Institute of Medical Studies, New Delhi.



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Iron Nail Test / API RP 38 Test Method

Sulphate reducing bacteria has a basic tendency to reduce sulphate ion to sulfide

ion. This phenomenon was used to detect the presence of SRB in any medium like

water etc. The American Petroleum Institute (API) has developed one standard

medium to check the presence of SRB. The medium contains ferrous ammonium

sulphate as one of its ingredients. Iron nails are also added to the medium as an

external iron source. This iron nail reacts with the sulfide ions, which is produced

due the reduction of sulphate by SRB, and gives black colored precipitate in the

medium. This black precipitate is due to the formation of FeS (Iron sulfide). Hence

the presence of SRB is detected just by the indication of the black color in the

medium.

Sulfide Detection Test

Sulfide detection is one of the most suitable methods for testing the growth of SRB.

SRB reduces sulphate to sulfide ions. In liquid solution, these sulfide ions are

present in the form of H2S. Hydrogen sulfide in the medium reacts with N, N‟-

dimethyl-1,4-phenylene diamine dichloride (reagent HS2A) to form a colorless

lucomethyl blue. This is then oxidized by ferric sulfate (HS3A) to methylene blue

and the same was determined by spectrophotometer (UV 2450, Shimadzu) at

wavelength 665 nm.

The presence of sulfide ions can be qualitative measured by using sulfide detecting

kit (Merck, Germany). The culture was centrifuged at 10, 000 x g for 10 mins, 250 µl

of the supernant was taken in test tube and 450 µl of the water was added. The

reagents HS1, HS2, HS3 were added in the ratio of 1: 5: 5 and incubated for 30 mins

at room temperature. The appearance of blue color was then measured

spectrophotometrically at 665 nm.



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Volatile Fatty Acid Analysis

The volatile fatty acid like acetic acid, propionic acid, butyric acid, isobutyric acid,

valeric acid and isovaleric acid etc. were reported to be produced in the medium due

to the growth of SRB. These volatile fatty acids were checked for their presence in

the bottles where SRB was grown. The eppendroff tubes were acidified by adding

100 µl of concentrated hydrochloric acid and incubated for 24 h. The 500 µl of

culture was added to these acidified tubes. These were centrifuged for 12, 000 x g

for 15 mins at room temperature. The resultant supernant was transferred to GC

vials. The analysis of volatile fatty acids was done by acidifying the samples and

then these were injected in the Agilent (6890N) gas chromatograph fitted with DB

waxter column (30 m x 0.53 mm x 1.0 µm); Helium was used as a carrier gas. The

oven/detector/ injector temperatures of GC were maintained at 1600C, 2300C and

2300C respectively. The signals of the test sample, control and standards were

compared.

Protein Estimation

The total cell protein was estimated to detect the presence of SRB in the medium.

Centrifuge 1 ml of SRB grown culture at 10, 000 x g for 10 mins. After

centrifugation, the pellet was resuspended in 100 µl of milli Q water. Add 1 ml of

Lowry C solution and incubate for 30 mins in dark at room temperature. The 100 of

Folin Ciocalteu reagent was added. The tubes were again incubated in dark for 30

mins in room temperature. The OD was taken at 595 nm by UV-Visible

Spectrophotometer (UV 2450, Spectrophotometer, Shimadzu). The total protein

concentration was estimated by Folin Lowry standard method of protein estimation

(Lowry, 1951).

The reagents involved in this method are: Lowry A - 2 % sodium carbonate in 0.1 N

NaOH, Lowry B – 1% CuSO4 in 1 % Sodium potassium tartarate, Lowry C- Mix 50

ml of Lowry A and 1 ml of Lowry B (freshly prepared).



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Study of growth characterization of selected

SRB isolated from Indian Oil reservoirs

The isolates TERI SRB 1001 and TERI SRB 1010 were chosen to study the growth

characteristics.

Inoculum development

The pure colony of TERI SRB 1001 from Hungate roll tube method was picked and

transferred into the 10 ml of the serum bottle containing 9 ml of the S-7 medium

and incubated for 7-14 days at 55 oC. After the growth the colony was culture was

then transferred into the 50 ml of the serum bottle containing 50 ml of the S-7

medium, and incubated for 7-14 days at 55 oC. The same procedure was followed for

preparing the inoculum TERI SRB 1010.

Effect of carbon sources on the growth of TERI SRB

1001 and TERI SRB 1010

Various carbon sources were tried for growth of TERI SRB 1001 and TERI SRB 1010

that included glucose, ramnose, ribose, cellobiose, raffinose, galactose, arabinose,

mannose, trehalose, erythritol, sodium pyruvate, xylose, sodium benzoate, sodium

succinate, sodium gluconate, sodium glutamate, sodium butyrate, fructose, sodium

propionate, sorbitol, sodium thioglycolate, sodium citrate, and sodium acetate.

Each of the 50 ml serum bottles containing 50 ml of the minimal medium

supplemented with 2% (w/v) of the carbon source, inoculated with 10% of the TERI

SRB 1001 and TERI SRB 1010 respectively and incubated at 55 oC for 28 days. The

growth was monitored spectrophometrically at 600 nm, bacterial protein was

estimated by Lowry method (Lowry et al, 1951).

Effect of nitrogen sources on the growth of TERI SRB


Different nitrogen sources were tested to test their effect on growth of each of TERI

SRB 1001 and 1010 respectively. The different nitrogen sources supplemented were



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ferric nitrate, hydrogen ammonium chloride, lithium nitrate, nickel nitrate,

magnesium nitrate, potassium nitrate, sodium nitrite, sodium nitrate, and urea at

20 mM concentration each. Each of the serum bottles were inoculated with 10 % of

the inoculums of each of TERI SRB 1001 and 1010, incubated at 55 oC for 28 days.

The growth was monitored spectrophotometrically at 600 nm, bacterial protein was

estimated by Folin Lowry method (Lowry et al, 1951).

Effect of temperature on growth of TERI SRB 1001 and

TERI SRB 1010

To determine the optimal growth temperature, growth of TERI SRB 1001 and TERI

SRB 1010 were grown at 4 different temperatures 45 oC, 50 oC, 55 oC, and 60 oC.

Each of the bottles were inoculated with 10 % inoculums of each TERI SRB 1001

and TERI SRB 1010. The inoculated bottles were incubated at respective

temperatures for 28 days. The growth was monitored spectrophoitometrically at

600 nm; bacterial protein was estimated by Folin Lowry method (Lowry et al, 1951).

Effect of various pH on growth of TERI SRB 1001 and

TERI SRB 1010

To determine optimal pH for growth conditions of TERISRB 1001 and TERISRB

1010, each of the 50 ml of serum bottles containing 50 ml of the media were set at

various pH viz.; 4, 5, 6, 7, 8, 9, 10 and inoculated with 10% of inoculum and

incubated at 55 oC for 28 days.

Effect of various electron acceptors on growth of

TERI SRB 1001 and TERI SRB 1010

The effect of various electron acceptors was determined by incubating the TERISRB

1001 and TERISRB 1010 in presence of various electron acceptors. The electron

acceptors used were sodium thio glycolate, sodium sulfite, sodium dithionite,

sodium sulfate, ferrous sulfate, ferric sulfate, magnesium sulfate, nickel sulfate,

potassium sulfate, potassium aluminium sulfate, ferric aluminium sulfate, sodium

thio sulfate, sodium di sulfite, potassium meta bisulfate, potassium bis sulfate,



56

potassium per oxo di sulfate, zinc sulfate. Each of electron acceptors were tested at

20 mM concentration, and incubated at 55 oC. The growth was monitored

spectrophotometrically at 600 nm and protein was estimated by Lowry method

(Lowry et al, 1951).

Effect of NaCl concentration on growth of TERI SRB


The effect of salinity on growth of TERI SRB 1001 and 1010 was determined by

incubating both the cultures under various sodium chloride concentrations

respectively viz.; 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%. Each of the serum bottles were

inoculated with 10% of the each inoculum and incubated at 55 oC for 28 days

respectively. The growth was monitored spectrophotometrically and protein

concentration was estimated by Lowry method (Lowry et al, 1951).

Standard Solutions and Buffers Used For

Molecular Biology Work

The aqueous solutions and buffers were prepared in milliQ waster at room

temperature. The stock solutions of 1M sodium phosphate, tris HCl, Tris-acetate

EDTA and other solutions were prepared, steam sterilized and stored at 4 oC. The

pH of buffers was determined with pH meter. The solutions of antibiotics of desired

concentrations were prepared in Milli Q water as per instruction in manual book

(Ausubel, 1989). The antibiotic solutions were stored 4 oC.

BIOCHEMICAL AND REAGENTS

The 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 from

Merck (India) or Qualigens (India). The molecular biology grade chemicals like

dithiothreitol, sodium dodecyl sulfate (SDS), proteinase K, lysozyme, ethylene

diamine tetraacetic acid (EDTA), agarose, and nuclease free NaCl were obtained



57

from Sigma chemical company (St louis USA). The reagents for PCR (polymerase

chain reaction) were obtained from sigma chemicals. The restriction enzymes were

purchased from MBI fermentas USA. Organic solvents and acids like ethyl acetate,

diethyl ether, methanol, acetone, hexane, benzene and acids hydrochloric acid,

sulfuric acid were purchased from Qualigenes (India).

Identification of Bacterial Isolates Using 16S

rDNA Sequence Analysis

Confirmatory identification of bacterial strains was done by partial sequencing (500

base pair sequence) of 16S rDNA using ABI Prism 310 automatic genetic analyzer

equipment (PE Applied Biosystem, USA). Identification of bacteria using

comparative 16S rDNA sequence analysis provides unprecedented accuracy and

reproducibility of results, especially for biochemically inert species. Unlike other

bacterial identification systems, no gram stain results, biochemical information or

species growth conditions were required for identification. DNA extraction was

performed using the PE Biosystems Prepman reagent, which can be used for all

bacteria.

The Micro Seq 500 16S rDNA bacterial sequencing kit was used for amplification

and sequencing of the first 500 base pairs of the 16S ribosomal DNA gene.

Isolation of bacterial Genomic DNA

Single isolated bacterial colony was suspended in 1 ml sterile MilliQ water.

Bacterial suspension was spinned at 8500 x g for 3 minutes. Supernatant was

decanted and bacterial cell pallets were resuspended in 200 µl of Prepman solution

(PE Biosystem, USA). Incubate this mixture at 56 °C for 30 minutes. After

incubation at 56 °C, vortex the mixture for 10 seconds and then mixture was heated

at 100 °C for 8 minutes. After heating this mixture was cooled and spinned for 2

minutes at 8500 x g. Supernatant was transferred to a new microcentrifuge tube

and supernatant was diluted at 1:10 sterile MilliQ water.



58

DNA quantification

The DNA preparations were quantified spectrophotometrically by estimating its OD

at 260 nm. The DNA samples to be quantified were diluted in 1X TE buffer. The

lamda DNA - OD 260 nm of diluted DNA sample was taken against blank. The

blank was 1X TE buffer. The DNA concentration was calculated using the formula:

-OD 260 nm X dilution factor X 50 = g/ml of DNA

The value of 1 OD was considered equivalent to 50 g/ml of ds DNA

The value of 1 OD was considered equivalent to 40 g/ml of ss DNA

The purity of the DNA was estimated spectrophotometrically by estimating its OD

at 260 nm and 280 nm. A value of 1.8 for OD 260 nm/ OD 280 nm was

considered pure DNA (Ausubel, 1989).

Agarose gel electrophoresis

The genomic DNA and the plasmid DNA were resolved on 0.8 % agarose gel. The

agarose gel was prepared in 1X TAE buffer (ref appendix I). The PCR amplified

DNA and restricted DNA fragments were separated on 2% sieving agarose gel

prepared in 1x TAE buffer. The electrophoresis was performed in 1x TAE buffer for

5-6 hrs at a constant voltage of 140 V at 25 oC. The DNA samples were visualized by

staining with 0.6 g/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.). All of the thermocycling reactions were processed in Geneamp 2400 PCR

system (Perkin Elmer, USA).

Sequencing of the Genes Encoding 16S rRNA

The MicroseqTM kit is a complete system for identification of the cultured bacterial

isolates. The kit includes reagents for broad range amplification of 16S rDNA

sequencing and subsequent identification of the bacteria by data analysis of 527

nucleotide portion and another for analysis of the complete 16S rRNA gene i.e. 1540

bp sequence. The partial and the full gene encoding 16S rRNA were sequenced with



59

MicroseqTM 500 and MicroseqTM 16S rRNA full gene bacterial sequencing kit (PE

Applied Biosystems, USA).

Partial 16S rDNA sequencing

PCR amplification of 500 bp of 16S rDNA

PCR amplification of 16S rDNA was carried out using “Microseq® 500 16S rDNA”

PCR kit. The specific 16 S primers in the kit by were provided PE Applied

Biosystems, USA. The PCR master mix contained all reagents including primers,

dNTP and Taq polymerase. PCR was carried out in 50 µl volume. In this walled

PCR tubes (0.2 ml, 25 µl) of PCR master mixture and 25 µl of DNA (1 ng/µl) was

taken. A negative (25 µl PCR master mix + 25 µl sterile MilliQ water) and positive

control (25 µl PCR master mix + 25 µl of 1 ng/µl of positive control DNA) was also

kept for PCR amplification. The PCR cycling times and temperature were as follows

(Figure 3.1).

Figure 3.1. Cycling conditions for the amplification of 16S rDNA primer set.

Purification of PCR product

PCR product was purified using Microcon 100 column (Millipore). Microcon

column was hydrated by adding 500 µl sterile MilliQ water to the column. Column

was spinned at 500xg in a fixed angle microcentrifuge for 6 minutes. After

hydration of column, 400 µl sterile MilliQ water was added to the column and then

entire PCR product (45 µl) was added to the column. The column was spinned at

500 x g in a fixed angle microcentrifuge for 15 minutes. Collection tube was

95 C

10 min

95 C

60 C

72 C 72 C

30 sec

30 sec

45 sec 10 min

4 C

30 cycles



60

removed and discarded. Now column was inverted and attached to a new collection

tube to the column. Sterile MilliQ water (25 µl) was added to the inverted column

and spinned the inverted column at 100 x g for 3 minutes to collect the purified

DNA in the collection vial.

Cycle sequencing of purified PCR product

Forward and reserve cycle sequencing of each purified PCR product was carried out.

Cycle sequencing reaction volume (20 µl) contained 3 µl of purified PCR product, 4

µl of sterile MilliQ water and 13 µl of forward or reverse sequencing mixture

procured from PE Applied Biosystem (USA). Thermal cycling times and

temperatures were as follows (Figure 3.2):

Figure 3.2. Cycling conditions for forward and reverse sequencing mixture 16Sr

DNA primers set.

Purification of cycle sequencing products

Excess fluorescence dye terminators were removed from each cycle sequencing

product by ethanol precipitation method. Transferred the entire cycle sequencing

product (20 µl) into a 1.5 ml microcentrifuge tube. Sterile MilliQ water (80 µl) was

added to make final volume of 100 µl. Now, 10 µl of 3 M sodium acetate (pH 4.6)

and 250 µl or 95% ethanol was added. Entire reaction volume was mixed

thoroughly, spinned and incubated in ice for 20 minutes. Entire content was

spinned at 14000 x g for 30 minutes. Supernatant was decanted gently. 250 µl of

95 C

10 min

95 C

50 C

60 C 72 C

10 sec

30 sec

4 min 10 min

4 C

25 cycles



61

70% ethanol was added and spinned at 14000 x g for 5 minutes. Supernatant was

decanted. Again 250 µl of 70% ethanol was added into the mirocentrifuge and

spinned at 14000 x g for 5 minutes. Supernatant was aspirated with a micropipette

without disturbing DNA pellet. Now DNA pellet was resuspended in 25 µl of

template suppression reagent (TSR supplied by PE Applied Biosystem, USA) and

heat the mixture at 95°C for 2 minutes to denature and then chilled on ice. The

samples were spinned and placed in autosampler of ABI Prism 310 automatic gene

sequencer (Applied Biosystems, USA). Sequencing of 16S rDNA was done by

automatic gene sequencer. The sequence of the unknown bacterium was compared

base-by-base with the sequence of its closest match. By using the concise alignment

option in software gave only the sequences, which differed between the unknown

bacterium and its closest match. Three based tools in software gave phylogenetic

analysis and similarly the comparisons.

Identification of the unknown bacterial isolates

To identify unknown bacterial isolates, the 16S rDNA sequences obtained were

subjected to BLAST search with microseq identification and analysis software

“MicroseqTM Analysis software v. 1.40, Microseq TM 16S rDNA Sequence

Databases v. 1.01” (PE Applied Biosystems) and the nucleotide-nucleotide blastn

search from NCBI database.

The 16S sequences were aligned using CULSTALW. The phylogenetic tree was

constructed using program PHYLIP version 3.0 software (Felsenstein, 2001).

Full gene encoding 16S rRNA sequencing

Amplification of the 1.5 Kb fragment of 16S rDNA, the full gene sequence of 16S

rRNA was amplified with “Microseq® full gene 16S rDNA” PCR module, kit

procured from PE Applied Biosystems, USA. The primer set produces an

amplification product of approximately 1540 bp in size. A volume of 1 µl of the

genomic DNA was diluted in 49 µl of nuclease free sterile water. The 100 µl of the

final reaction mixture consisted of 50 µl of diluted DNA (1ng/µl) and 50 µl of PCR

master mix.

Cycling conditions for the amplification reaction were as follows:

Initial denaturation 95 oC for 10 min

This was followed by 30 cycles of



62

Step I 95 oC for 30 sec

Step II 60 oC for 30 sec

Step III 72 oC for 45 sec

(a rapid thermal ramp of 1 oC/ sec was maintained between steps)

Final extension 72 oC for 10 mins

Final Temperature 4 oC soak

Purification of the amplified 16S rDNA

The PCR product was purified using Microcon 100 column (Millipore). Microcon

column was hydrated by adding 400 µl TE buffer to the column. Then the 100 µl of

the PCR product was added to the purification column procured from Biorad and

the column was spinned at 6500 x g for 15 mins. Collection tube was removed and

discarded. The column was now inversted and attached to a new collection tube. A

volume of 50 µl of TE buffer was added to the inverted column and spinned at

12500 x g for 15 mins to collect the purified DNA in the collection vial.

Cycle sequencing of the amplified 16S rDNA

The amplified 16S rDNA was subjected to cycle sequencing with MicrseqTM full gene

16S rDNA sequencing module. The sequencing module consisted of AmpliTaq FS

DNA polymerase and dRhodamine dye terminators. Twelve primers are used for

full length sequencing to provide a strand overlap and for complete assembly of the

16S rDNA sequence.

The six forward sequencing reaction primers used are:

5F, 338F, 515F, 776F, 1087F, 1174F

The forward sequencing reaction reagents along with the primers were arranged

sequentially (5F to 1174F) in green colour microamplifier reaction tubes.

The six reverse sequencing reaction primers used are:

357R, 531R, 810R, 1104R, 1193R, 1540R

The reverse sequencing reagents along with the primers were arranged sequentially

(357 to 1540 R) in the blue colour microamplifier reaction tubes.

The primers used for sequencing are numbered from the 5‟ end of the primer on the

forward strand of E. coli (J01859). The purified PCR product was diluted with 60 µl



63

nuclease free water. The 7 µl diluted PCR product was added to each of the 12 strip

tubes containing sequencing reaction mix.

The cycling conditions have been mentioned in table 11.

The cycling conditions for sequencing of full gene length of 16S rRNA

Cycle hold number of cycles

96 oC 10 sec 65 oC 1min - 6

96 oC 10 sec 64 oC 1 min - 6

96 oC 10 sec 63 oC 1 min - 6

96 oC 10 sec 62 oC 1 min - 6

96 oC 10 sec 61 oC 1 min - 6

96 oC 10 sec 60 oC 1 min - 6

96 oC 10 sec 59 oC 1 min - 6

96 oC 10 sec 58 oC 1 min - 6

96 oC 10 sec 57 oC 1 min - 6

96 oC 10 sec 56 oC 1 min - 6

96 oC 10 sec 55 oC 1 min - 6

Precipitation of cycle sequenced DNA

The cycle sequenced DNA of all the sequencing reactions were precipitated with

95% ethanol and 3M sod acetate (pH 4.6). The cycle sequenced product was

transferred into 1.5 ml eppendrof tube and 80 µl of sterile MQ water was added to

make the final volume to 100µl. Next 10µl of 3M sod acetate (pH 4.6) and 250 µl of

95% ethanol was added. This was incubated at 4 oC for 10 mins. The sample was

centrifuged at 15000 x g at room temperature for 30 mins. The supernatant was

decanted. The DNA pellet was washed with 250 µl of 70% ethanol by centrifuging at

15000 x g for 5 mins. The supernatant was decanted. The 70% ethanol wash step of

the DNA pellet was repeated. The supernatant was carefully aspirated. The DNA

pellets were air dried at room temperature and resuspended in 20µl of Hi-

diformamide.



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Analysis of the DNA sequences

The cycle sequenced DNA was resolved by ABI PRISMTM 310 genetic analyser (PE

Applied Biosystems). The DNA samples were sequenced with the short capillaries

(5-47 cm x 50 m) and long capillaries (5-61 x 50 m). The electrophoresis was

performed with 1x electrophoresis buffer with EDTA and performance optimised

polymer (POP 6).

The parameters set for the electrophoresis in ABI PRISMTM 310 genetic analyser

are as follows:

Temperature 50 oC

Current 4 A

Voltage 12 KV

Argon ion laser power 9.7 MW

Identification and phylogenetic analysis

The identification and phylogenetic relatedness of the isolates was assessed based

on the partial and complete 16S rRNA gene sequences. To identify unknown

bacterial isolates, the 16S rDNA sequences obtained were subjected to basic local

alignment search tool (BLAST) search. This search was performed with Microseq

identification and analysis software „MicroseqTM Analysis software v. 1.4, microseqTM

16S rDNA sequence database v. 1.01‟ (PE Apllied biosystems, USA). The sequences

were alos analysed with the BLAST (N) search against the non-redundant

Genbanmk+EMBL+DDBJ+PDB database using NCBI web services:

www.ncbi.nlm.nih.gov/blast.

http://www.ncbi.nlm.nih.gov/blast



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Interspecies diversity estimated by PCR Based

Genomic Fingerprinting

The PCR based genomic fingerpriting of the different bacterial strains were

obtained with the primer sets: enterobacterial repetitive intergenomic consensus,

repetitive extragenic palindromic and BOX element. These primer sets were based

on the short repetitive DNA sequences derived from conserved on the short

repetitive DNA sequences derived from conserved palindromic inverted repeat

regions dispersed throughout the bacterial genome.

Enterobacterial repititive intergenic consensus

(ERIC)

The primer set for ERIC PCR genomic fingerprinting were as follows:

ERIC-1R 5‟ ATG TAA GCT CCT GGG GCT TCA C 3‟

ERIC-2 5‟ AAG TAA GTG ACT GGG GTG AGC C 3‟

The 15 µl reaction mixture was prepared as follows:

Tris-HCl (ph – 8.3) 10 mM

KCl 50 mM

Gelatin (w/v) 0.01%

dNTPs 0.2 mM

ERIC-1R 1 μM

ERIC-2 1 μM

Taq polymerase 0.45 U

Tamplate DNA 2 μl (25 ng/μl)

The amplification cycling conditions were:




Step II 50 oC for 1 min 20 sec

Step III 68 oC for 3 min 20 sec

The final extension 68 oC for 8 min

Final 4 oC soak



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Repetitive Extragenic palindromes (REP)

The primer set obtaining REP-PCR genomic fingerprinting is as follows:

REP 1R-I 5‟- III ICG ICG ICA TCI GGC-3‟

REP 2-I 5‟-ICG ICT TAT CIG GGC TAC-3‟

The 15 μl reaction mixture was prepared as follows:

Tris-HCl (pH-8.3) 10 mM

KCl 50 mM

MgCl2 2.5 mM

Gelatin (w/v) 0.01 %

dNTPs 0.2 mM

REP 1R-I 1 μM

REP 2-I 1 μM


Tamplate DNA 2 μl





Step II 38 oC for 1 min 20 sec

Step III 68 oC for 3 min 20 sec


Final 4 oC soak

BOX element based fingerprinting (BOXAIR)

The primer used for BOXAIR-PCR genomic fingerprinting was

BOX-AIR 5‟-CTA CGG CAA GGC GAC GCT GAC G-3‟

The 15 μl recation mixture was prepared as follows:

Tris-HCl (pH-8.3) 10 mM

KCl 50 mM



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MgCl2 2.5 mM


dNTP 0.2 mM

BOX-AIR 1 μM


Template DNA 2 μl (25 ng/ μl)


Initial denaturation 95 oC for 2 min, 94 oC for 3 sec



Step II 50 oC for 1 min

Step III 65 oC for 8 min


Final 4 oC soak

All the rep-PCR genomic fingerprinting reactions were terminated using 1 μl of gel

loading solution (contaminating 15 % Ficoll, bromophenol blue and 0.25%

bromophenol blue and 0. 25 % xylene cyanol). The amplified products were stored

at –20 oC.

Amplified ribosomal DNA restriction analysis (ARDRA)

The complete 16S rRNA gene sequences were obtained from the strains by

amplification with universal primers set (Wang and Wang, 1997).

U5‟ – 5‟ TTA CCT GAT AGC GGC CGC AGA GTT TGA TCC TGG CTC AG 3‟

(nucleotide 8 to 27 of E. coli 16S rRNA gene)

U3‟ – 5‟ TAC AGG ATC CGC GGC CGC GGC CGC TAC GG{C/T} TAC CTT GTT ACG

ACT T 3‟ (nucleotide sequences of 1492-1513 of E. coli 16S rRNA gene).

The 15 μl of the reaction mixture was prepared as follows:

Ttris-HCl (pH-8.3) 10 mM

KCl 50 mM

MgCl2 2.5 mM



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Deoxynucleotide phosphate 0.2 mM

(dATP, dCTP, dTTP, dGTP)

U5‟ 1 μM

U3‟ 1 μm


Tempalate DNA 2 μl (25 ng/ μl)

The amplification conditions were as follows:

Initial denaturation 95 oC for 30 sec




Step III 72 oC for 45 sec

Final extension 72 oC for 10 mins

Final 4 oC soak

The 5 μl of the amplified fragment was checked on 2% agarose gel

Restriction analysis of the amplified 16S rRNA gene

sequences

The amplified 16S rDNA were purified with Microcon PCR centrifugal filter devices

as per the protocol mentioned above. In order to perform the digestion of the

amplified 16S rDNA with a single restriction enzyme, below given steps were

followed:

All the reagent tubes were assembled on ice bath. The contents of all the reagent

tubes were thawed and mixed so as to have a homogenous suspension (except for

the restriction enzymes, which was not to be thawed or vortexed mixed). The

purified fragments were digested with single restriction enzyme HaeIII, EcoRI,

HindIII, and SauIIIA. The restriction enzyme was obtained, as concentration of 10X

and the working concentration of the buffer was 1x.



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The 20 μl of the reaction mixture was prepared, which consisted of:

Nuclease free sterile MQ water 9 μl

PCR product 10 μl

Restriction enzyme 10 U/ μl

Total volume 20 μl

The reaction content was mixed by pipetting and the reaction mixture was

incubated at 37 oC for 2 h. the reaction was terminated by inactivating the enzyme

by heating the reaction mixture at 65 oC for 10 min. the restricted DNA was checked

on a 2% agarose gel.

PCR based ribotyping

The template DNA was prepared by the protocol mentioned above. The primers

were selected from the conserved sequences in 16S and 23S region within 40 bases

of intervening ITS 1 (Jensen et al, 1995).

GIRRN 5‟ GAA GTC GTA ACA AGG 3‟

(30 to 40 nucleotide upstream from rrs-rrl ITS)

LIRRN 5‟ CAA GGC ATC CAC CGT 3‟

(20 bases downstream from rrs-rrl ITS)

A 15 μl reaction mixture was prepared as follows:

Tris-HCl (pH – 8.3) 10 mM

KCl 50 mM

MgCl2 2.5 mM

Gelatin (w/v) 0.01%

Deoxynucleotide phosphate 0.02 mM

(dATP, dTTP, dCTP, dGTP)

GIRRN 1 μM

LIRRN 1 μM


Template DNA 2 μl (25 ng/μl)


Initial denaturation 95 oC for 2 mins



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This was followed by 35 cycles



Step III 72 oC for 6 min

The final extension at 72 oC for 8 min

Final 4 oC soak

PCR based restriction fragment length polymorphism of 16S-23S rDNA internally

transcribed spacer region

Amplification of the 16S-23S rDNA ITS

The amplification of the 16S-23S rDNA ITS region was performed as per the

protocol mentioned above. The primer set for the amplification of 16S-23S rDNA

ITS was GIRRN and LIRRN (Jensen et al, 1995).

RFLP analysis of amplified 16S-23S rDNA ITS

The amplified 16S-23S rDNA ITS were purified with Microcon PCR centrifugal filter

devices as per the protocol mentioned above. In order to perform the digestion of

the amplified 16S-23S rDNA ITS with a single restriction enzyme, following steps

are given below:

All the reagents tubes of were assembled on ice bath. The contents of all the reagent

tubes thawed and mixed so as to have homogenous suspension (except for the

restriction enzyme, which was not to be thawed or vortexed mixed). The purified

fragments were digested with single restriction enzyme HaeIII, EcoRI, HindIII, and

SauIIIA. The 10X restriction enzyme buffer was diluted to a working concentration

of 1x.

The 20 μl of the reaction mixture was prepared, which consisted of:

Nuclease free sterile MQ water 9 μl

PCR product 10 μl

Restrcition enzyme 10 U / μl

Total volume 20 μl



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The reaction content was mixed by pippetting and the reaction mixture was

incubated at 37 oC for 2 h. The reaction was terminated by inactivating the enzyme

by heating the reaction mixture at 65 oC for 10 min. the restricted DBA was checked

on 2% agarose gel.

Data analysis (rep-PCR, ARDRA, ITS I-RFLP)

The computer assisted data analysis was performed for the PCR based genomic

fingerprints. The gel profiles were visualized (photographed) with UVI gel

documentation (UVItec) and analysed with UVI photo version 99 and UVI

band/map version 99 software (UVItec). Whenever a distinct PCR profile was

observed in terms of the number and position of a clearly visible band, the

corresponding strains were given a unique number or letter designation.

The rep PCR gel images were digitized with a charge couple device camera (CCD-

UVI) and the images were stored as TIFF images. The images were analysed with

the standard molecular weight markers using UVI bands Maps. The similarity

matrices were calculated by Jaccard‟s coefficient and the cluster analysis of

similarity matrices was performed by “unweighted pair group method‟ (UPGMA).

The band-matching algorith (UPGMA) was calculated with mean Jaccard‟s

coefficient (band matching tolerance). The dendrograms were constructed for the

combination of rep-PCR genomic fingerprints. Likewise the ARDRA and the ITS I-

RFLP profiles were analysed by constructing dendrograms.

Pilot Scale Microbiocide Treatment Of Produced

Water to Control Of H2S Gas and Sulfide

Producing Microbes in Produced Water at

Kathloni Oilfield

Characterization of produced water

The physical and chemical properties of produced water sample collected from the

pump delivery (PD) line (just before the disposal of produced water into the wells)

were characterized. Turbidity was measured by using a turbidity meter (SQ 118



72

Merck). Sodium, chloride, calcium, magnesium, iron, sulfide were measured as

previously described by Glude (Glude et al., 2003). Carbonate, bicarbonate, silica,

oil and grease were also measured by standard methods (Scott, 1925).

Selection of microbiocide against H2S producing

microbes

Ten microbiocides were screened against SRB obtained from formation water of

Kathloni Oil field India (Northeast India) , including glutaraldehyde (Qualigens),

tetrakis hydroxymethyl phosphonium sulfonate (THPS) (Navdeep Chemicals Pvt

Ltd, Pune), benzyl trimethyl ammonium chloride (BTMAC) (Navdeep Chemicals

Pvt Ltd, Pune), formaldehyde (Qualigens), 2-bromo-2-nitropropane-1,3-diol (BNPD)

(Qualigens), Benzenol (Qualigens), 2-methyl-4-isothiazolin-3-one (Qualigens),

ethylene oxide (Qualigens), propiolactone (Sigma), and sodium hypochlorite (SHC)

(Qualigens). These microbiocides were screened against mixed cultures of sulfide

producing microorganisms. For each microbiocide 1000 mg l-1 stock solution was

freshly prepared in autoclaved oxygen free water. A final concentration of 25, 50, 75,

100, 150, and 200 mg l-1 of each microbiocide was prepared in API RP 38 and Iron

Lyngby media. The efficacy of each microbiocide against a mixed culture of sulfate

reducing bacteria was determined based on logarithmic reductions in viable count

of the test organisms at different concentrations of microbiocides. Assays were

performed under anaerobic conditions in 150 ml serum bottles containing 50 ml of

produced water. The produced water was inoculated with 0.5 ml of a log phase

culture adjusted to 108 cfu ml-1 of the mixed population obtained from formation

water of Kathloni oil field (northeast India) comprised of strains of Anaerobaculum

mobile, Garciella nitratireducens, Clostridium sporogenes, Thermosediminibacter

oceani, Thermodesulfovibrio sp., Coprothermobacter sp.,

Thermodesulfobacterium sp., Thermodesulfotobacterium sp. and

Caldanaerobacter sp. A single biocide at various concentrations (25, 50, 75, 100,

150 and 200 mg l-1) was added in each bottle. The test suspensions were incubated

under anaerobic conditions at 55oC for various contact time (0, 0.5, 1, 2, 4 and 6 h).

The MPN assays were performed in Iron Lyngby medium at 55 oC to determine the

viable count. Growth was evaluated from visual blackening of the media. The log



73

reductions were estimated (Kaur et al, 2009). The initial log value was obtained

from the mean values of the untreated produced water used as control.

Full scale microbiocide treatment of produced

water with selected microbiocides

The Kathloni Oil Collection Station situated in Assam North East India, was

selected for treatment of produced water by microbiocides and disposal of produced

water into disposal wells. Sodium hypochlorite, Benzyl tri methyl ammonium

chloride and Bronopol (2-bromo-2-nitropropane-1,3- diol) were selected as

microbiocides for the treatment and added sequentially in the system following the

two-cycle schedule described below. Each microbiocide used in the treatment was

added gradually upstream of the emulsion tank (ET) to reach a final concentration

of 50 mg l-1. This was achieved by adding a 200 l volume of appropriate

concentration of the microbiocide at the rate of 40 l h-1. It therefore took 5 h to

reach the final concentration of 50 mg l-1 of the desired microbiocide in produced

water. The two-stage 66 days treatment applied to the test field was as follow.

During the first 3 days sodium hypochlorite was added once a day to a final

concentration of 50 mg l-1 of produced water. Then, for the next 15 days sodium

hypochlorite was replaced by benzyl trimethyl ammonium chloride which was

added once a day to reach 50 mg l-1 and for last 15 days benzyl trimethyl ammonium

chloride was replaced by Bronopol which was added once a day to 50 mg l-1 of

produced water. A second 33 days period was then initiated where bronopol

addition was stopped and sodium hypochlorite was added once a day to reach 50

mg l-1 for 3 days. Sodium hypochlorite was then replaced for the next 15 days by

benzyl tri methyl ammonium chloride which was added once every second day to

reach 50 mg l-1. Finally, for the last 15 days benzyl trimethyl ammonium chloride

was replaced by bronopol which was added once a day to reach 50 mg l-1. The same

two-cycle treatment was repeated for another 66 days before the end of the

experiment at the 132nd day. During the experiment, samples were collected from

the storage tank and pump delivery everyday and the rate of disposal of produced

water to the disposal well was monitored.

medium preparation for isolation of bacterial...

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