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
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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
1001 and TERI SRB 1010
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,
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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
1001 and TERI SRB 1010
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
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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.
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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
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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
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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
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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
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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
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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.
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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:
Initial denaturation 95 oC for 2 min
This was followed by 53 cycles of
Step I 95 oC for 30 sec
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
Taq polymerase 0.45 U
Tamplate DNA 2 μl
The amplification cycling conditions were:
Initial denaturation 95 oC for 2 min
This was followed by 53 cycles of
Step I 92 oC for 30 sec
Step II 38 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
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
Gelatin (w/v) 0.01 %
dNTP 0.2 mM
BOX-AIR 1 μM
Taq polymerase 0.45 U
Template DNA 2 μl (25 ng/ μl)
The amplification cycling conditions were:
Initial denaturation 95 oC for 2 min, 94 oC for 3 sec
This was followed by 53 cycles of
Step I 92 oC for 30 sec
Step II 50 oC for 1 min
Step III 65 oC for 8 min
The final extension 68 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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Gelatin (w/v) 0.01 %
Deoxynucleotide phosphate 0.2 mM
(dATP, dCTP, dTTP, dGTP)
U5‟ 1 μM
U3‟ 1 μm
Taq polymerase 0.45 U
Tempalate DNA 2 μl (25 ng/ μl)
The amplification conditions were as follows:
Initial denaturation 95 oC for 30 sec
This was followed by 30 cycles of
Step I 95 oC for 30 sec
Step II 60 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
Taq polymerase 0.45 U
Template DNA 2 μl (25 ng/μl)
The amplification cycling conditions were:
Initial denaturation 95 oC for 2 mins
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This was followed by 35 cycles
Step I 94 oC for 30 sec
Step II 50 oC for 30 sec
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
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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
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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.
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