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TERI University, PhD Thesis 2010

RESULTS

Collection of Samples

Formation fluid samples were collected from different oil wells of OIL,

Duliajaan, Assam; Oil and Natural Gas Corporation, Ahmedabad, and Bombay

High, Assets. The samples were inoculated on the spot in different media

(Annexure 1) and incubated at different temperatures, growth of sulfide

reducing bacteria was observed. The samples were collected at different time

intervals to check the seasonal variation of sulphate reducing bacterial

population in the oil reservoirs.

The details of the samples collected from different oil wells and oilfield of India

are given in Table 4.1.

Table 4.1. Details of formation fluid samples collected from oil reservoirs of India

Sample Details

Oil and Natural Gas Corporation, Gujarat (Western India)

J#104 Jhalora well # 104

ETP GGSV Emulsion treatment plant of group gathering system V

J#119 Jhallora well # 119

JETP Jhalora emulsion treatment plant

K#172 Kalol well # 172

K#489 Kalol well # 489

GGS III ETP Group gathering station III

J ETP Jhaora emulsion treatment plant

GGSV HT Group gathering station heat treatment

Chase water GGSIII

ETP

Group gathering station chase water

SS III Sour stage III

SS II Sour stage II

SS-9 Sweet Stage-9

SS-5 Sweet stage-5

NRM ETP Emulsion treatment plant

Oil India Limited, Assam (Northeastern India )

4

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KET Kathloni emulsion tank

KPD Kathloni pump delivery

K#7 Kathloni well # 7

DET Dikom emulsion tank

DPD Dikom pump delivery

D#13 Dikom well # 13

H#497 Hathali well # 497


B#500 Bekuljaan well # 500

WI#21 Water injection well 21



FW Tank Out –K Kathloni Formation water tank outlet

TPS out – D Dikom three phase outlet outlet

TPS outlet-K Kathloni three phase outlet outlet

FWT-IN Formation water inlet

Disposal water K#7 Kathloni disposal water of well no 7

Fwt-out-D Dikmom Formation water outlet



D#6 Dikom well # 6

N#155 Naharkatia well # 155




Oil and Natural Gas Corporation, Bombay High (Western India)

IA India Alpha

IB India Brabo

IR India Romeo

SPT Surj Pipe Tank 1 mixed sample

IA W#5 India Alpha well # 5

IA W#2 India Alpha well # 2

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Characteristics of the Formation Water

The pH of formation water was found to be between 7.5-8.0. The total dissolved

solids varied between 3500-4200 ppm levels (Table 4.2).

Table 4.2. The characteristics of the formation water collected from the various oil field

of India.

Location of

Sampling

Reservoir

Temperature

(oC) (Avg)

pH of

formation

water

Total dissolved

solids in

formation water

(mg/L)

Electric

conductivity of

formation water

(ms)

OIL

(Assam)

80-90 7.3-8 3200-5800 6-7.5

ONGC

(Gujrat)

100 7.0-8.5 3000-6500 6.2-7.8

ONGC

(Bombay

high)

100 7.5-8.5 4000-6100 6.2-7.5

Medium Optimisation

Amongst 15 media tried medium S-7 (Figure 4.1) gave the best results, followed

by API RP 38.

Figure 4.1. Photograph showing the presence and absence of the growth of sulfide

producing bacteria. The bottle on left shows the absence of sulfate reducing bacteria and

bottle on right shows the presence of sulfate reducing bacteria after injecting formation

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fluid in the S7 medium. The black colour (ferrous sulfide precipitate) formation is due to

the production of hydrogen sulfide thereby reacting with ferric citrate.

Isolation of Sulphate Reducing Bacteria from

Oil Reservoirs of India

The cultivable anaerobic sulphide producing bacterial strains were isolated from

various oil wells and oilfields of India. The microscopic examination of the

bacterial cells showed different morphological shapes short rods, and long rods,

slightly curved rods. The total of 96 anaerobic cultivable strains was isolated by

enrichment culture technique at 55 oC from formation water (Table 4.3).

However, there was no growth at 75 and 90 oC. There were large numbers of

mesophilic cultivable sulphide producing strains isolated which were not

undertaken because the present was focused on thermophilic anaerobic sulphide

producing bacterial strains.

Table 4.3. Total numbers of anaerobic bacterial strains were isolated at 55 oC from

various oil fields and oil reservoirs of India

Characterization of SRBs

Morphology

The cell morphology of the sulphide producing bacterial strains isolated from

the formation fluid samples were analyzed by microscopic studies. Different

types of cells were obtained from various sampling sites, viz. small rods, long

rods, spore forming rods. Morphology of some selected strains is shown in figure

4.2 and similar cell morphology of other SRB isolated from formation water

Isolation sites Total number of sulfide producing

bacterial strains growing at 55 oC

OIL, Assam 48

ONGC Gujarat 27

ONGC, Bombay

High

21

Total 96

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samples were also noticed (data not shown). All the cultivable isolates were

characterised morphologically, however, only representative strains have been

shown in the figure 4.2. The morphology of sulphide producing bacterial strains

isolated oil reservoirs of Oil India Assam (North East India) revealed that

different species of anaerobic bacteria may exist in oil reservoir of Assam.

Similar morphology of SRB was also noticed from other oil reservoirs.

Figure 4.2. Electron micrograph of the sulphide producing bacterial strains isolated

from oil reservoirs of India (Northeastern India). Small Rod shaped bacterial strains

isolated from the formation water Kathloni oil reservoir, Assam (A). Long slender rod

shaped bacteria isolated from formation fluid the Ahmedabad Assest (B). Spore forming

rod shaped bacteria isolated from Kathloni oil reservoir (C). Rod shaped bacteria isolated

from formation fluid of the formation water of oil reservoirs of Gujarat (D). Rod shaped

bacteria isolated from oil reservoirs of Assam (E). Slender, long rod shaped bacterium

isolated from formation water of oil reservoirs of Assam (F).

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Volatile Fatty Acid Analysis

All the isolates were estimated for volatile fatty acids (VFA) production and all

the isolates were found to produce volatile fatty acids. The volatile fatty acid like

acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and

isovaleric acid, were reported to be produced in the medium due to the growth of

sulfide producing strains. These volatile fatty acids were checked for their

presence in the bottles where bacterial strains was grown. The peaks obtained in

the test samples were compared with the control and standards previously

injected in the GC under same conditions. The isolates were shown to produce

acetic acid, propionoic acid, isobutyric acid, butyric acid, and isovaleric acid. The

data of the representative strains is shown in figure 4.3, however, the similar

pattern of VFA produced by other anaerobic bacterial isolates from various oil

reservoir were also noticed.

Figure 4.3. The gas chromatogram showing the production of volatile fatty acids by

sulfide producing bacterial isolates. (AA- acetic acid, PA- propionic acid, IBA- isobutyric

acid, BA- butyric acid, IVA- isovaleric acid, VA- valeric acid); A – Standard, B – TERI SRB

– 1001, C - TERI SRB – 1010.

A

B

C

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Evaluation of Sulfide Producing Bacterial

Diversity in Oil reservoirs of Oil India

Limited, Assam

The oil India limited is situated in the North eastern parts of India (27.33N,

95.40E). The oil field is 20 km away from the Dibrugarh (city in Assam state of

India). The ambient temperature of Assam oil field ranged from 5- 40 oC. The

company owns various oil collecting stations viz. Kathloni, Naharkatia, and

Dikom oil reservoirs.

Phylogenetic affiliation of the isolated bacterial

strains

A total of 48 sulfate reducing and sulfide producing bacterial strains were

isolated from the formation water procured from Oil India Limited, Assam. The

bacterial strains isolated from Assam were designated as TERI SRB 1001 to

TERI SRB 1048.

Of the total bacterial strains isolated, strains TERI SRB 1012, TERI SRB1013,

TERI SRB 1015, TERI SRB 1018, TERI SRB 1020, TERI SRB 1021, TERI SRB

1025, TERI SRB 1029, TERI SRB 1032, TERI SRB 1034 - TERI SRB 1048 were

phylogenetically affiliated to the genus Clostridium sporogens. Most of the

strains were affiliated to the genus Clostridium sp.

The strains TERI SRB 1010, TERI SRB 1011, TERI SRB 1014, TERI SRB 1016,


1028, and TERI SRB 1033 showed 16S rDNA sequence match to Garciella

nitratireducens. Three strains were phylogenetically affiliated to the genus

Anaerobaculum mobile. Two strains (TERI SRB 1003, and TERI SRB 1006)

showed sequence homology with Coprothermobacter sp. TERI SRB 1008, and

TERI SRB 1009 were identified as Thermosediminibacter sp. One strain TERI

SRB 1007 and TERI SRB 1004, showed affiliation to Thermodesulfotobacterium

sp. and Thermodesulfotobacter yellowstonii (Table 4.4). The strains were also

producing volatile fatty acid viz. acetic acid, propionoic acid, butyric acid,

isobutyric acid, and valeric acid.

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Table 4.4. The phylogenetic affiliation of the bacterial strains growing at 55 oC isolated

from the formation water of the Oil India Limited, Assam.

Strain number Isolation site Identification

TERI SRB 1001 KET Anaerobaculum mobile

TERI SRB 1002 KPD Anaerobaculum mobile

TERI SRB 1003 KPD Coprothermobacter sp.

TERI SRB 1004 K # 7 Thermodesulfovibrio yellowstonii

TERI SRB 1005 K # 516 Anaerobaculum mobile

TERI SRB 1006 KPD Coprothermobacter sp.

TERI SRB 1007 KET Thermodesulfotobacteirum sp.

TERI SRB 1008 KPD Thermosediminibacter sp.

TERI SRB 1009 KPD Thermosediminibacter sp.

TERI SRB 1010 KET Garciella nitratireducens

TERI SRB 1011 DPD Garciella nitratireducens

TERI SRB 1012 D # 6 Clostridium sporogens

TERI SRB 1013 N # 155 Clostridium sporogens

TERI SRB 1014 Disposal water K # 7 Garciella nitratireducens

TERI SRB 1015 K # 14 Clostridium sporogens

TERI SRB 1016 Disposal water K # 7 Garciella nitratireducens

TERI SRB 1017 Dikom TPS outlet Garciella nitratireducens


TERI SRB 1019 DPD Anaerobaculum mobile



TERI SRB 1022 Kathloni formation water

tank out

Coprothermobacter sp.

TERI SRB 1023 K # 23 Garciella nitratireducens



TERI SRB 1026 D # 6 Coprothermobacter sp.


TERI SRB 1028 D # 6 Garciella nitratireducens


TERI SRB 1030 K # 23 Coprothermobacter sp.

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TERI SRB 1031 D # 6 Thermosediminibacter


















The strain numbers TERI SRB 1012, 1013, 1015, 1018, 1020, 1021, 1025, 1029,

1032, 1034 – 46, and 1048 were found to be as Clostridium sporogens. All the

isolated strains are potent sulfide producer.

Sulfide production

The isolated strains from Assam were found to produce sulfide in the range of

150-200 ppm levels (Figure 4.4.).

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Figure 4.4. The sulfide production by bacterial strains isolated from the Oil India

Limited, Assam. A: from strains TERI SRB 1001-1025. B: from strains TERI SRB 1026-

1048.

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Crude oil characteristics

The crude oil collected from the oil reservoirs of Oil India Limited, Assam

showed aliphatic and aromatic hydrocarbon. The aliphatic fractions were in the

range of 65-75% of the TPH (Figure 4.5.).

Figure 4.5. The gas chromatogram showing the aliphatic fraction in the crude oil

collected from (A) Kathloni oil reservoir, OIL, Assam; and (B) Dikom oil reservoir, OIL,

Assam.

A

B

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Isolation of Sulfate Reducing Bacteria from

oil reservoirs of Oil and Natural Gas

Corporation Oil Fields, Gujarat

Phylogenetic affiliation of the isolated strains

Total of twenty-seven isolates were isolated (growing at 55 oC) from formation

water samples procured from the oil fields of Gujarat, India.

Total 27 sulfide producing bacterial strains were isolated from formation water

samples and were belonging to firmicutes. More than 80% of the bacterial

strains were phylogenetically affiliated to the genus Clostridium sporogens. the

strains which showed the closest 16S rDNA sequence homology to Clostridium

sporogens were as follows: TERI SRB 2001, TERI SRB 2003, TERI SRB 2004,


2009, TERI SRB 2010, TERI SRB 2012, TERI SRB 2013, TERI SRB 2014, TERI

SRB 2015, TERI SRB 2017, TERI SRB 2018, TERI SRB 2021, TERI SRB 2022,

TERI SRB 2023, TERI SRB 2024, TERI SRB 2025, TERI SRB 2026, and TERI

SRB 2027 (Table 4.5). Of the total isolated strains, 11% amongst them showed a

closest 16S rDNA sequence homology to the genus Garciella nitratireducens.

These strains were TERI SRB 2002, TERI SRB 2011, and TERI SRB 2019. The

strains TERI SRB 2016, and TERI SRB 2020 were identified as

Coprothermobacter sp.

Table 4.5. Phylogenetic affiliation of the bacterial strains growing at 55 oC isolated from

the formation water of the Gujarat oil field (Western India).



TERI SRB 2002 J#119 Garciella nitratireducens


TERI SRB 2004 GGSV HT Clostridium sporogens




TERI SRB 2008 GGS III Clostridium sporogens

TERI SRB 2009 SS II Clostridium sporogens

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TERI SRB 2010 ETP G Clostridium sporogens

TERI SRB 2011 J#104 Garciella nitratireducens


TERI SRB 2013 J # 104 Clostridium sporogens

TERI SRB 2014 SS III Clostridium sporogens


TERI SRB 2016 ETP G Coprothermobacter sp.



TERI SRB 2019 ETP G Garciella nitratireducens

TERI SRB 2020 ETP G Coprothermobacter sp.





TERI SRB 2025 GGS V Clostridium sporogens

TERI SRB 2026 N ETP Clostridium sporogens

TERI SRB 2027 J ETP Clostridium sporogens

The strain numbers TERI SRB 2001, 2003 – 2010, 2012-2015, 2017- 18, 2021-

27 were found to be as Clostridium sporogens.

Sulfide production

The bacterial strains isolated from oil and natural gas were producing sulfide in

the range of 80- 120 mg/l (Figure 4.6).

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Figure 4.6. The graph showing the sulfide production of the strains isolated from the

Oil and Natural Gas Corp., Gujarat. A: from strain number 2001-2013. B: from strain

number 2014-2027.

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Crude oil of Gujarat

The profile of the crude oil collected from the oil field of Gujarat, had

comparatively low percentage of aliphatic fraction. The aliphatics in crude oil of

Gujarat were in the range of 30-40 %. Some of the representative aliphatic

compounds present in crude oil of Gujarat is shown in figure 4.7.

Figure 4.7. The aliphatic fraction of crude oil obtained from Gujarat oil field western

India (A) Jhalora well #104, (B) Emulsion treatment plant of group gathering station.

A

B

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Isolation of SRB from Formation Water

Collected From Offshore Oil Wells of ONGC

Phylogenetic affiliation of the isolated strains

A total of 21 sulfate / thiosulfate reducing bacterial strains were isolated from

the samples procured from formation water of the oil reservoir of Oil and

Natural Gas Corporation (Table 4.6).

Table 4.6. Phylogenetic affiliation of the bacterial strains growing at 55 oC isolated from

the formation water of the ONGC (offshore).


TERI SRB 3001 Pipe tank Clostridium sporogens

TERI SRB 3002 IA w # 5 Clostridium sporogens



TERI SRB 3005 SPT Garciella nitratireducens



TERI SRB 3008 IA w # 5 Garciella nitratireducens








TERI SRB 3016 IA Clostridium sporogens




TERI SRB 3020 IR Clostridium sporogens

TERI SRB 3021 IB Clostridium sporogens

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The strain numbers TERI SRB 3001 - 4, 3006 – 7, 3009 - 12, 3014 – 3016, 3018

– 3021 were found to be as Clostridium sporogens.

Of the total bacterial strains (TERI SRB 3001 to TERI SRB 3021), all the strains

belonged to the division of firmicutes. The strains which were 16S rDNA

sequence homology to Clostridium sporogens were as follows: TERI SRB 3001,


3007, TERI SRB 3009, TERI SRB 3010, TERI SRB 3011, TERI SRB 3012, TERI

SRB 3014, TERI SRB 3015, TERI SRB 3016, TERI SRB 3018, TERI SRB 3019,

TERI SRB 3020, TERI SRB 3021. The strains TERI SRB 3005, TERI SRB 3008,

TERI SRB 3013, TERI SRB 3017 were Garciella nitratireducens. All the

bacterial isolates (TERI SRB 3001 to TERI SRB 3021) belonged to firmicutes,

80% of the bacterial isolates belonged to Clostridium sporogens and 20% to

Garciella nitratireducens.

Sulfide production

All the isolates produced sulfide in the range of 80-120 mg/l (Figure 4.8).

Figure 4.8. Sulfide production by strains isolated from formation water samples

collected from offshore oil field Bombay High of western India.

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Crude oil of Bombay High (offshore oil field)

The aliphatic fractions of the crude oil collected from the Bombay High. The

aliphatic fraction in crude oil of Bombay High constituted more than 50 %

(Figure 4.9.).

Figure 4.9. The Gas chromatogram of aliphatic fraction of crude oil of Bombay High

offshore oil field.

Growth Characteristics of selected novel

sulphide producing bacterial strains

Clostridium sporogens was the most dominant sulphide producing bacterial

strain present in most of oil reservoirs selected for present study. However,

recently identified novel species of Garciella nitratireducens and Anaerobaculum

mobile were isolated from oil reservoirs of northeast and western part of India.

There are no reports on presence of these two new species in oil reservoirs of India.

Therefore, these two species were selected for present study.

Anaerobaculum mobile

Anaerobaculum mobile was selected for further studies since it is recently

identified species (Menes and Muxi, 2002) and there are no reports of the

existence of this species from oil reservoirs of India.

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Morphology of Anaerobaculum mobile

The isolate TERI SRB 1001 was isolated from oil reservoirs of Oil India limited,

Assam. The isolate TERI SRB 1001 was gram-negative shaped rods (Figure

4.10). Colonies were white, with entire margins. These colonies were obtained on

S7 medium.

Figure 4.10. The scanning electron micrograph of Anaerobaculum mobile growing at

55 oC isolated from formation water collected from the oil wells Oil India Limited, Assam

(Northeast India).

Phylogenetic analysis of Anaerobaculum mobile

The microorganisms that are phylogenetically most closely related to this strain

were Anaerobaculum thermoterrenum and A. mobile. The sequence analysis of

the strain was compared and it was found to have the closest match with the

Anaerobaculum mobile. The phylogenetic tree was then constructed with the

help of Phylip 3.0 and the dendrogram thus obtained is shown in Figure 4.11.

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Figure 4.11. The dendrogram showing the phylogenetic position of


Utilization of carbon sources by Anaerobaculum

mobile

The A. mobile optimally utilised various carbon sources like glucose, sodium

gluconate, sodium thioglycolate. Although other carbon sources like rahmnose,

ribose, arabinose, sodium propionate, sodium acetate and sodium citrate were

also utilised by the organism. However, slight growth was observed in the other

carbon sources like, cellobiose, raffinose, galactose, trehalose (Figure 4.12).

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Figure 4.12. Growth of A. mobile on various carbon sources at 55 oC and pH 7.5

Utilization of nitrogen sources by Anaerobaculum

mobile

The effect of various nitrogen sources was studied on the growth of A. mobile

and it was found that nitrogen sources like sodium nitrate increased the growth.

These microorganisms did not optimally grow in the presence of nitrogen

sources like magnesium nitrate, nickel nitrate, lithium nitrate, ferric nitrate,

hydroxy ammonium nitrate (Figure 4.13).

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Figure 4.13. The growth patterns of A. mobile at 55 oC on various nitrogen sources.

The experimental set without nitrogen source was kept as control.

Various electron acceptors utilized by


The consequences of various electron acceptors on growth of Anaerobaculum

mobile were determined. The electron acceptors used were Sodium

Thioglycolate, Sodium Sulfite, Sodium dithionite, Sodium Sulfate, Magnesium

Sulfate, Potassium Sulfate, Sodium thio Sulfate, Sodium hydrogen sulfate,

Sodium di sulfite, ferrous sulfate, ferric sulfate, nickel sulfate, potassium meta

bisulfate, potassium aluminium sulfate, potassium bis sulfate, potassium per

oxo di sulfate, zinc sulfate, ferric aluminium sulfate. Sodium thio sulfate and

Sodium thioglycolate was utilised by A. mobile. However, the sulfate sources like

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Sodium di sulfite, potassium meta bisulfate, potassium bis sulfate, potassium

per oxo di sulfate, and zinc sulphate were not utilised (Figure 4.14 and figure

4.15).

Figure 4.14. The growth patterns of Anaerobaculum mobile at 55 oC after the utilization

of various electron acceptors.

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Figure 4.15. The sulfide production was one of the end products by Anaerobaculum

mobile under the influence of various electron acceptors at 55 oC and pH 7.5. There was

no sulphide produced by A. mobile on other electron acceptors in the same experimental

set.

Growth of Anaerobaculum mobile on different salt

concentrations

The various concentrations of sodium chloride were used to determine salinity

tolerance on the growth of A. mobile. The A. mobile could tolerate the salinity

concentrations up to 4% NaCl (Figure 4.16).

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Figure 4.16. Influence of NaCl concentrations on the growth of A. mobile at 55 oC, pH

7.5.

Growth of Anaerobaculum mobile on various pH

Concentrations

The microorganism A. mobile could optimally grow at pH between 7 and 8 (ie at

7.5), though it could grow at the pH range from 7-10 (Figure 4.17). This strain

was not found to grow in acidic conditions, however the growth was found to

increase as the pH increased upto 8; hence the optimum pH for growth of this

strain was 8.0.

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Figure 4.17. The influence of pH on the growth of Anaerobaculum mobile incubated at

55 oC.

Garciella nitratireducens

Garciella nitratireducens was chosen for detailed study since this genus was

recently identified in 2003 (Tello et al, 2003). The isolate TERI SRB 1010 was

isolated from the oil fields of Oil India Limited, Assam. This strain was further

characterised for physiological and biochemical characteristics.

Morphology of Garciella nitratireducens

The microorganism G. nitratireducens was isolated from the oil fields of Oil

India Limited, Assam. The isolate was gram positive, spore forming rods (Figure

4.18).

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Figure 4.18. The scanning electron micrograph of the Garciella nitratireducens growing

at 55 oC and pH 7.5

Phylogenetic analysis of Garciella nitratireducens

The 16S rDNA sequence of this strain was compared with all sequences available

in GeneBank database. The phylogenetic analysis revealed that the closest

relative of the strain was Garciella nitratireducens. The phylogenetic tree was

then constructed with the help of Phylip 3.0 and the dendrogram thus obtained

is shown in Figure 4.19.

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Figure 4.19 The phylogram based on 16S rDNA sequences indicating the position of

the strain Garciella nitratireducens amongst members of clusters XII of Clostridiales.

Bootstrap values from 100 replications are shown; only values above 95 were considered

significant and are reported. Bar, 5 substitutions per 100 nucleotides.

Growth of Garciella nitratireducens on various

carbon sources

The Garciella nitratireducens utilised carbon sources like glucose, galactose,

sodium pyruvate, sodium gluconate, optimally where as, sodium formate,

sodium butyrate, sodium propionate, sodium acetate and sodium lactate were

also utilised (Figure 4.20).

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Figure 4.20. The influence of various carbon sources on the growth of G.

nitratireducens at 55 oC and pH 7.5.


nitrogen sources

The effect of various nitrogen sources was studied on the growth of G.

nitratireducens and it was found that nitrogen sources like sod nitrate increased

the growth of the organisms. The nitrogen sources like urea affected the growth

of G. nitratireducens. These microorganisms did not optimally grow in the

presence of nitrogen sources like magnesium nitrate, nickel nitrate, lithium

nitrate, ferric nitrate, hydroxy ammonium nitrate (Figure 4.21).

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Figure 4.21. The influence of various nitrogen concentrations on the growth of G.

nitratireducens growing at 55 oC and pH 7.5.


electron acceptors

The strain G. nitratireducens reduced thiosulfate to H2S. The electron acceptors

utilized by this strain were Thioglycolate, Sodium Sulfite, Sodium dithionite,

Sodium Sulfate, Ferrous sulfate, Magnesium sulphate, Potassium sulfate,

Potassium aluminium sulphate and Sodium thio sulfate. Sodium thio sulfate and

sodium thioglycolate was utilised by G. nitratireducens. However, the sulfate

sources like sodium di sulfite, potassium meta bisulfate, potassium bis sulfate,

potassium per oxo di sulfate, and zinc sulphate were not utilised by either of

them (Figure 4.22). The strain reduced thiosulfate to H2S and nitrate to

ammonium. Growth was enhanced in the presence of thiosulfate or nitrate.

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Figure 4.22. The growth of G. nitratireducens at 55 oC and pH 7.5 under the influence

of various electron acceptors.


salt concentrations

The various concentrations of sodium chloride were used to determine the effect

of salinity on the growth of G. nitratireducens. G. nitratireducens could tolerate

sodium chloride concentrations up to 0-7%, with the optimum NaCl

concentration of 1.5 % (Figure 4.23). The ability of growing in high salinity

conditions enhances the chances of the microorganism to be found in extreme

environmental conditions.

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Figure 4.23. The growth patterns of Garciella nitratireducens on various NaCl

concentrations at 55 oC and pH 7.5.

Growth of Garciella nitratireducens on different

pH

The G. nitratireducens was not found to grow in lower pH values however it

could multiply at pH values from 6-8, however, the optimum pH for its growth

was 7.5 (Figure 4.24).

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Figure 4.24. The influence of pH on the growth of G. nitratireducens at 55 oC.

Control of sulphide production by A.mobile

and G. nitratireducens

The sodium hypochlorite (SHC), Benzyl trimethyl ammonium chloride

(BTMAC) and Bronopol (BNPD) were the most efficient microbiocides to control

sulfide producing bacteria. For each of them, at concentration of each 25 mg l-1 it

took only 2 h of contact time to kill 100% of the mixed sulfide producing

bacterial population used in the assay. BTMAC affects the membrane

permeability, inhibition of enzyme activity, coagulation and finally death,

whereas BNPD reacts with sulphydryl groups leading to accumulation of free

radicals and finally death. A great variety of microbiocides were screened against

indigenous population, as microbiocide activity varies greatly between different

types of strains of the same species.

Among all the strains growing in the presence of potassium nitrate, in some

cases the sulfide production was decreased and most cases it was increased.

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There was 25%, 92% reduction in sulfide production by potassium nitrate

amendment in cases of A. mobile, G. nitratireducens respectively; however the

sulfide production could not be ceased (Figure. 4.25). On the contrary, there was

24.5%, 2.2%, 4.14%, 3.41% and 0.659% increase in sulfide production in the

presence of potassium nitrate in case of Coprothermobacter sp., C. sporogens,

T. oceani, Thermosulfobacterium sp. and Thermodesulfotobacterium sp.

respectively.

Figure 4.25. Influence of potassium nitrate on the sulfide production by selected

strains. The control shown in figure is the sulfide production by respective strain in

absence of nitrate.

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Intraspecies Diversity Among Different

Strains Of G. nitratireducens

Total of 18 strains of Garciella nitratireducens were isolated from Indian oil

reservoirs. The aim of the study was to determine intraspecies diversity among

18 strains of G. nitratireducens.

The genetic diversity in different strains of Garciella nitratireducens isolated

from different oil reservoirs and oil fields of India was selected to evaluate the

intraspecies diversity. Of the 18 strains of G. nitratireducens selected for present

evaluation, eleven were isolated from the oil reservoirs of Oil India Limited,

Assam. The evaluation of genetic diversity was performed by PCR based

ribotyping strategies such as rep-PCR genomic fingerprinting, PCR based

ribotyping, Amplified Ribosomal DNA Restriction Analysis (ARDRA), 16S-23S

rDNA, ITS-RFLP analysis. The strain number of the G. nitratireducens in the

present study has been listed in the Table 4.7.

Repetitive-PCR genomic fingerprinting

The rep-PCR methodology for the 18 different strains of G. nitratireducens

(Table 4.7) yielded complex genomic fingerprinting consisting of 8-12 amplified

bands of varying intensity. The amplified banding profiles were clearly

distinguishable with the sizes ranging from 100 bp to 9000 bp. Visual inspection

followed by analysis of the DNA fingerprints enabled the identification of novel

genotypes. Of the 18 strains of G. nitratireducens, REP-PCR produced 11

patterns, and BOX-PCR produced 5 patterns. Among the primers sets studied

REP-PCR produced the most complex amplified banding patterns which

reflected a high degree of intraspecies diversity among the G. nitratireducens

strains isolated from the oil reservoirs of India.

A dendrogram that was calculated with Jaccard’s similarity coefficients using the

Unweighted Pair Group Method clustering (UPGMA) for the combination of

rep- PCR results segregated the 18 strains into unique genotypic groups. The

DNA fingerprinting with the primer sets were not identical.

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Table 4.7. The strain number and the isolation sites of the strains of G.

nitratireducens

S.No. Isolated code Isolation site

1 TERI SRB 1010 Kathloni emulsion tank

2 TERI SRB 1011 Dikom pump delivery

3 TERI SRB 1014 disposal water # K7

4 TERI SRB 1016 disposal water # K7

5 TERI SRB 1017 Dikom TPS outlet

6 TERI SRB 1023 Kathloni well # 23



9 TERI SRB 1028 Dikom well # 6

10 TERI SRB 1033 Dikom well # 6

11 TERISRB 1048 Dikom well # 6

12 TERI SRB 2002 Jhalora well # 104

13 TERI SRB 2011 Jhalora well # 104

14 TERI SRB 2019 Emulsion treatment plant of group gathering

system V

15 TERI SRB 3005 Surj Pipe Tank 1 mixed sample

16 TERI SRB 3008 India Alpha well # 5



PCR based ribotyping

PCR based ribotyping characterization of the 18 strains of G. nitratireducens led

to recognition of 8 distinguishable genomic patterns. The ribotype pattern of all

the strains showed intensely staining multiple ampliers of sizes 9000 bp to 1500

bp. The strains showed either one or two amplified bands in their ribotype

patterns. This indicated polymorphism in the rRNA operons among the strains

of G. nitratireducens. The ribotype patterns were not distinct for a particular

geographical location.

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110

Amplified 16S ribosomal DNA restriction analysis

of G. nitratireducens

The amplification of the 16S rRNA genes with the primer set U3’ and U5’ could

amplify nearly the full length 16S rDNA. A single amplicon of 1540 bp was

obtained for all the G. nitratireducens isolates and the corresponding ARDRA

profile was obtained for all the strains studied. These profiles were obtained

after complete digestion of the PCR amplified 16S rDNA fragment since a

prolonged incubation of 4 h of the restriction mix did not alter the banding

pattern (which was obtained after 2 h of digestion). This was also confirmed by

summed molecular weight of the restricted fragment did not exceed the original

amplicon of 1540 bp. The restriction analysis of the amplified 16S rDNA with

the enzymes Alu III, EcoRI, HaeIII, Sau III (Figure 4.26, Figure 4.27, Figure

4.28) could reveal the variation existing within the strains.

Figure 4.26. The cluster analysis of ARDRA (Alu) of 18 strains G.

nitratireducens. The UPGMA algorithm was applied to the similarity matrix using

at and above Jaccard’s coefficient. The 18 strains got delineated into 11

genotypic groups.

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111

The variation was observed between the fragments of sizes ranging from 900 bp

to 200 bp. The variation was observed between the fragments of 900 to 700 bp

sizes where as fragments of 500 bp to 300 bp sizes were consistent in all the G.

nitratireducens strains (Figure 4.26, Figure 4.27, Figure 4.28).

Figure 4.27. The cluster analysis of ARDRA (hae) of 18 strains G.


at and above Jaccard,s coefficient. The 18 strains got delineated into 9

genotypic groups.

Results


112

Figure 4.28. The cluster analysis of ARDRA (EcoR1) of 18 strains G.



genotypic groups.

Results


113

16S-23S rDNA ITS RFLP of G. nitratireducens

The restriction of amplified 16S-23S rDNA ITS with EcoRI, AluI, Hind III, Hae

were analysed (Figure 4.29, Figure 4.30, Figure 4.31, Figure 4.32).

Figure 4.29. The cluster analysis of ITS RFLP (EcoR1) of 18 strains G.



genotypic groups.

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114

Figure 4.30. The cluster analysis of ITS RFLP (HaeII) of 18 strains G.



genotypic groups.

Results


115

Figure 4.31. The cluster analysis of ITS RFLP (HindIII) of 18 strains G.



genotypic groups.

Results


116

Figure 4.32. The cluster analysis of ITS RFLP (SauIII) of 18 strains G.



genotypic groups.

Results


117

IN SITU CONTROL OF SULFIDOGENS

Feasibility study of biocide action

The biocide(s) tried against 1 liter of formation water (obtained from the

Kathloni oilfield) were found to effective against the indigenous SRB and the

contact time 2h was optimized for the trial. There was no sulfide production

after the treatment was done. The biocides were also found effective against the

thiosulfate, sulfate reducers and other sulfide producing bacteria also.

Characterization of produced water at the

treatment site

Oil, water and gas were separated in a three phase separator as shown in Figure

4.33. After separation of oil, gas and water, produced water was pale yellow and

turbid black with oil drops.

Figure 4.33. The schematic illustration of the treatment of produced water at oil

collection station at Kathloni situated in Northeastern India. The samples were

collected from Manifold, Storage tank, and Pump delivery (PD) systems.

The produced water obtained from pump delivery before treatment was pale

yellow and turbid black with oil drops. The total dissolved solids of formation

water varied from 3500-4200 mg l-1 and were slightly alkaline in pH. The sulfide

Results


118

production at the TPS (Three Phase Separator) was 40 ± 4 mg l-1 and it

increased to 52 ± 3 mg l-1 at the emulsion treatment tank, and the same

increased to 101 ± 5 mg l-1 at the storage tank and pump delivery. Physical and

chemical properties of produced water at the treatment site are shown in Table

4.8. The pH of produced water slightly declined after the treatment; however,

the water was clear and transparent as the turbidity was decreased. The

carbonate was not present during the study. There was no significant increase in

the salinity, chloride, indicating that the chlorine from the biocide did not make

much contribution to the produced water (Table 4.8.). Calcium and magnesium

did not increase significantly after the treatment indicating that the hardness of

the water was not affected. The sodium sulfate was increased after treatment

since it was not reduced sulfide and iron being the requirement of SRB was not

consumed, hence it also increased in the absence of SRB. There was an increase

in the total dissolved solids and decrease in suspended solids after the treatment

as the produced was clear and no precipitate was visible.

Table 4.8. The physiochemical characterization of produced water of Kathloni

oil collection station

Characteristics Units Before After

pH - 6.9 .2 6.42 .1

Salinity (as NaCl) mg l-1 4250 50 4350 25

Cl- mg l-1 2578 25 2639 20

CO3 mg l-1 ND ND

HCO3 mg l-1 439 30 445 22

Na mg l-1 1550 30 1541 20

Pot mg l-1 50 5 69 7

Cal mg l-1 178 20 243.5 35

Mag mg l-1 26 7 11 5

Sulfate mg l-1 70 2 90 3

Iron mg l-1 1.83 0.2 2.51 0.1

TDS mg l-1 4800 62 5080 78

SS mg l-1 250 25 300 32

Oil and grease mg l-1 80 10 62 15

Turbidity NTU 192 15 166 20

Silica mg l-1 85 5 84.2 8

ND – not detected

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119

Pilot scale treatment of formation water to

control of sulfidogens in produced water at

Kathloni oilfield

The oil producing companies are producing enormous amount of formation

water along with crude oil. After separation of oil and water, water must be

reinjected for reservoir pressure maintenance. However, due to high population

of sulfate reducing and sulfide producing bacteria in formation water, injection

of water into depleted reservoir through injector wells become difficult. To avoid

this problem a pilot scale trial on control of these sulfide producing and sulfate

reducing bacteria in formation water was undertaken. Firstly SRB present in

formation water were isolated and identified and then a consortium of SRB was

developed my mixing of all the species of SRB. Then microbiocides were selected

against consortium of SRB present in formation water.

Cleaning of the injector wells lead to the injection of on an average 649 ± 78 kilo

liters per day of produced water for 20 days. However, the rate of injection was

maximum (711 ± 8 kilo liters per day) for initial 10 days, thereafter, it started

declining as in 5 days the rate of injection was decreased to 648 ± 34 kilo liters

per day. The rate of injection for the next 5 days declined to 529 ± 25 kiloliters

per day. After 20 days the produced water could not be injected into the injector

wells, and hence the cleaning of the injector well was again required for injection

of produced formation water. At zero day of the treatment, the population of

sulfidogens was 107 cfu ml-1 in the storage tank and 108 cfu ml-1 in the pump

delivery sample (Figure. 4.34 a), and injection of untreated produced water lead

to souring and injection losses, a decline in injection rate (Figure. 4.34 b).

However, after biocide treatment there were no viable cells of SRB found in

produced water at the storage tank and at the pump delivery tank during the all

the four treatments, revealing 100% kill of the sulfidogens at the emulsion

treatment tank. However, the rate of injection of the produced water into well

was maintained up to 707 ± 5.35 kiloliters per day, 722 ± 7.69 kiloliters per day,

751 ± 4.45 kiloliters per day, 760 ± 3.98 kiloliters per day during treatment I, II,

III, and IV respectively. Hence after 132 days of the treatment there was no

requirement of cleaning the wells. The population of sulfidogens was controlled

and the frequency of cleaning the wells was also reduced. The pressure for the

reinjection of the produced water was kept constant before and after the treatment.

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Figure. 4.34 - (A) Total population of sulfidogens in the produced water of

storage tank (■) and in pump delivery (▲) before and after the biocide

treatment. (B) The rate of injection of produced water in the wells before and

after the treatment.

B

A

Onset of treatment

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