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International Biodeterioration & Biodegradation 63 (2009) 151–155

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ib iod

Control of sulfidogenic bacteria in produced water from the Kathlonioilfield in northeast India

Guneet Kaur a, A.K. Mandal b, M.C. Nihlani c, Banwari Lal b,*

a TERI University, Darbari Seth Block, Indian Habitat Center, Lodhi Road, New Delhi 110003, Indiab The Energy and Resources Institute, Darbari Seth Block, Indian Habitat Center, Lodhi Road, New Delhi 110003, Indiac Oil India Limited, Duliajaan, Assam 786602, India

a r t i c l e i n f o

Article history:Received 18 November 2007Received in revised form 12 July 2008Accepted 14 July 2008Available online 16 October 2008

Keywords:Sulfate-reducing bacteria (SRB)OilfieldProduced waterMicrobiocidesBiofouling controlSulfidogenicSulfide-producing bacteria

* Corresponding author. Tel.: þ91 11 2468 2100; faE-mail address: banwaril@teri.res.in (B. Lal).

0964-8305/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.ibiod.2008.07.008

a b s t r a c t

A full-scale study was conducted to evaluate microbiocide efficacy to control the indigenous sulfide-producing bacterial population in produced water at Kathloni, Oil India Limited (OIL), Assam (northeastIndia). The sulfide-producing culturable bacterial strains present in produced water of Kathloni oilfieldwere identified as Anaerobaculum mobile, Garciella nitratireducens, Clostridium sporogenes, Thermosedi-minibacter oceani, Coprothermobacter sp., Thermodesulfovibrio sp., Thermodesulfobacterium sp., Ther-modesulfotobacterium sp., and Caldanaerobacter sp. These strains could produce sulfide in the range of50–200 mg l�1 and volatile fatty acids such as acetic acid, propionic acid, isobutyric acid, butyric acid, andisovaleric acid. Out of 10 microbiocides screened, three (sodium hypochlorite, benzyl trimethyl ammo-nium chloride, and 2-bromo-2-nitropropane-1,3-diol) were selected to control the growth and sulfideproduction by the aforementioned mixed bacterial species. A strategy was designed in which these threemicrobiocides were sequentially applied in an oilfield for 132 days. There was no significant difference inthe chemical composition of the produced water after the treatment.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Produced water is the water phase generated during the processof lifting oil from water bearing oil formations. Oil productioncompanies unavoidably generate enormous amount of producedwater, especially in the case of aging oilfields where waterproduction constitutes as much as 95% of the total oil/watermixture produced. Storage of such an enormous volume ofproduced water is difficult. It is stored in large tanks, and duringstorage the sulfide-producing bacterial population increases.Hydrogen sulfide gas produced by the sulfide-producing bacteriaand sulfate-reducing bacteria (SRB) reacts with iron when it isdisposed in wells, generating a precipitate that plugs the wells.Thus, disposal of produced water causes plugging in the wells,leading to reduction in the rate of disposal.

The disposal of large volumes of produced water into the oceanhas been banned by government agencies and nongovernmentalorganizations. Other means of produced water disposal includeevaporation or percolation in pits. During evaporation, the watersin these pits become laden with very high concentrations of total

x: þ91 11 2468 2144.

All rights reserved.

dissolved solids. Many of the formation water pits are withoutsynthetic liners and pose a severe threat to the groundwater (Tellezet al., 1995).

The major problem faced by the oil-producing companies is howto control hydrogen sulfide gas generated due to metabolism ofsulfate by anaerobic microorganisms. Hydrogen sulfide is a toxicand corrosive gas responsible for a variety of environmental andeconomic losses including reservoir souring, contamination ofnatural gas and oil, corrosion of metal surfaces, and the plugging ofreservoirs and consequent low production of oil in place (Davidovaet al., 2001; Eckford and Fedorak, 2002). The rate of pitting corro-sion has been attributed to sulfate and thiosulfate reducing bacteria(Marchal et al., 2001; Crolet, 2005).

Several methods have been investigated for mitigation of sour-ing of produced water. Elimination of sulfate from water is onepossibility for control of sulfides; it is, however, expensive. Theaddition of nitrate to produced water could abate H2S production;however, it requires repeated treatments that result in highchemical costs (Reinsel et al., 1996). Addition of nitrate might alsoincrease the biomass of microbes in produced waters, which maycause plugging in the disposal well (Eckford and Fedorak, 2002).The control of H2S gas and sulfide production is usually attemptedthrough the use of broad-spectrum biocides. Many biocides capableof controlling microbes are commercially available. However,

G. Kaur et al. / International Biodeterioration & Biodegradation 63 (2009) 151–155152

microbes develop resistance to biocides; hence a single biocide haslimited capacity to kill all the indigenous bacteria present in theoilfield (Reinsel et al., 1996).

The aim of the present study was to examine the sulfide-producing microbial population in produced water, and evaluatea treatment based on sequential use of selected microbiocides toreduce microbial growth and sulfide production in produced waterbefore disposal in disposal wells.

2. Materials and methods

2.1. Sample collection

Kathloni oilfield of Oil India Limited (OIL), which is situated 20 km fromDibrugarh city in India’s Assam state (northeast India), was selected for this study.The Kathloni oil collection station (OCS) processes approximately 1500 kl d�1 offormation fluid (oil and water), of which 1000 kl d�1 constitutes produced waterthat is disposed of into disposal well through a pump delivery system (PD) (Fig. 1).The water samples were collected from manifold, storage tank, and pump delivery toassess the population of sulfide-producing microbes. Nine different types ofanaerobic media were inoculated with 5 ml produced water on site by using a sterilesyringe for each medium.

2.2. Characterization of produced water

The physical and chemical properties of produced water samples collected fromthe pump delivery (PD) line (just before the disposal of produced water into thewells) were characterized. Turbidity was measured by using a turbidity meter (SQ118, Merck). Sodium, chloride, calcium, magnesium, iron, and sulfide were measuredas described by Glude et al. (2003). Carbonate, bicarbonate, silica, oil, and greasewere also measured by standard methods (Scott, 1925).

2.3. Enrichment, isolation, and identification of SRB from formation water

Nine different types of basal media amended with various carbon and energysources were used in this study: Postgate B medium (Postgate, 1984); API RP 38medium (American Petroleum Institute, 1975); YE medium (Takahata et al., 2001);HSTYG medium (Magot et al., 1997); Iron Lyngby medium (Lorentzen et al., 2003);sodium acetate containing medium (Lien and Beeder, 1997; Lien et al., 1998); sodiumlactate containing medium (Nilsen et al., 1996); and thioglycolate medium (Reeset al., 1997).

All the ingredients of a medium except the reducing agents were mixed in Milli-Q water. The medium in the flask was boiled and cooled under a N2:CO2 (80:20) gasmixture and the pH was adjusted to 7.3. The medium was purged with an anaerobicgas mixture consisting of N2:CO2 in a ratio of 80:20, and the reducing agents wereadded. Then medium was dispensed into 100-ml anaerobic bottles using a 50-mldisposable syringe. The bottles were then plugged and sealed with rubber stoppersand aluminum crimps. The bottles containing media were autoclaved at 121 �C and15 psi for 20 min; 0.25 mm membrane filtered vitamin solution (0.1%) was added tothe medium after autoclaving. Three sets of different media were inoculated withaliquots of produced water and incubated under anaerobic conditions at 50, 60, and70 �C.

Total viable cells were enumerated by the standard three-tube most probablenumber (MPN) assays (Greene et al., 2006). After the first series of enrichment, the

Fig. 1. Schematic illustration of the treatment of produced water at oil collection station Kamanifold, storage tank, and pump delivery (PD) systems, and sulfide-producing microorgan

strains were isolated according to the Hungate roll-tube technique. The genomicDNA was isolated from each purified strain and the identity of each strain wasdetermined based on the sequence analysis of its 16S rDNA gene.

2.4. Selection of microbiocide against H2S producing microbes

Ten microbiocides were screened: glutaraldehyde (Qualigens), tetrakishydroxymethyl 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-producingmicroorganisms. For each microbiocide 1000 mg l�1 stock solution was freshlyprepared 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 Lyngbymedia.

The efficacy of each microbiocide against a mixed culture of sulfate-reducingbacteria was examined based on logarithmic reductions in viable count of the testorganisms at different concentrations of microbiocides. Assays were performedunder anaerobic conditions in 150-ml serum bottles containing 50 ml of producedwater. The produced water was inoculated with 0.5 ml of a log-phase culture adjustedto108 cfu ml�1 of the mixed population comprised of strains of Anaerobaculummobile, Garciella nitratireducens, Clostridium sporogenes, Thermosediminibacter oceani,Thermodesulfovibrio sp., Coprothermobacter sp., Thermodesulfobacterium sp., Ther-modesulfotobacterium sp., and Caldanaerobacter sp. A single biocide at variousconcentrations (25, 50, 75, 100, 150, and 200 mg l�1) was added to each bottle. Thetest suspensions were incubated under anaerobic conditions at 55 �C for variouscontact times (0, 0.5, 1, 2, 4, and 6 h). The MPN assays were performed in Iron Lyngbymedium at 55 �C to determine the viable count. Growth was evaluated from visualblackening of the media. The log reductions were estimated according to Selvarajuet al. (2005). The initial log value was obtained from the mean values of the untreatedproduced water used as control.

2.5. Full-scale microbiocide treatment of produced water with selectedmicrobiocides

The Kathloni Oil Collection Station situated in Assam was selected for treatmentof produced water by microbiocides and disposal of produced water into disposalwells. Microbiocides selected for the treatment were SHC, BTMAC, and BNPD; theywere added sequentially in the system following the two-cycle schedule describedbelow. Each microbiocide used in the treatment was added gradually upstream ofthe emulsion tank (ET) to reach a final concentration of 50 mg l�1. This was achievedby adding a 200-l volume of appropriate concentration of the microbiocide at therate of 40 l h�1. It therefore took 5 h to reach the final concentration of 50 mg l�1 ofthe desired microbiocide in produced water. The two-stage 66-day treatmentapplied to the test field was as follows: During the first 3 days SHC was added oncea day to a final concentration of 50 mg l�1 of produced water. Then, for the next15 days SHC was replaced by BTMAC, which was added once a day to reach 50 mg l�1

and for last 15 days BTMAC was replaced by BNPD, which was added once a day to50 mg l�1 of produced water. A second 33-day period was then initiated in whichBNPD addition was stopped and SHC was added once a day to reach 50 mg l�1 for3 days. Then SHC was replaced for the next 15 days by BTMAC, which was addedonce every second day to reach 50 mg l�1. Finally, for the last 15 days BTMAC wasreplaced by BNPD, 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

thloni situated in northeastern India. The produced water samples were collected fromisms were identified.

Table 1Relative abundance of the different taxa recovered from different sample sources

Genera Relative abundance of microorganisms (%)

Manifold Storage tank Pump delivery

Anaerobaculum mobile 9 0.2 NDGarciella nitratireducens 15 0.5 NDCoprothermobacter sp. 12 0.4 NDThermodesulfobacterium sp. 18 0.8 NDThermosediminibacter oceani 6 ND NDCaldanaerobacter sp. 4.4 ND NDClostridium sporogenes 14.6 98 100Thermodesulfovibrio sp 12 ND NDThermodesulfotobacterium sp. 9 ND ND

ND, not detected.

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at the 132nd day. During the experiment, samples were collected from the storagetank and pump delivery every day and the rate of disposal of produced water to thedisposal well was monitored.

3. Results and discussion

3.1. Characterization and identification of thermophilic SRB inwater samples

A total of nine culture media were used for enumeration ofmicrobes that could produce sulfide. Gram-positive and Gram-negative bacteria as well as fermentative sulfidogens were isolatedfrom the produced water collected at the Kathloni oil collectionstation. However, it was observed that the most commonly culturedmicroorganisms belonged to Firmicutes.

Under experimental conditions, Lyngby and API RP38 mediaappeared to be the best suited for cultivation of the microbial florafound in produced water. Nine cultivable genera were found inproduced water and their relative abundance is shown in Table 1.All these isolated strains were producing sulfide (25–225 mg l�1)and volatile fatty aciddviz. acetic acid (600–700 mg l�1), propionicacid (200–250 mg l�1), isobutyric acid (150–200 mg l�1), butyricacid (3–16 mg l�1), and isovaleric acid (150–300 mg l�1). All thespecies were able to grow at temperatures ranging from 37 to 65 �C.However, their optimum temperature was found to be 55 �C. Thetotal population of sulfide-producing microbes was increased fromthe 2.5 � 102 cfu ml�1 seen in manifolds to 107 cfu ml�1 in storagetanks where the storage retention time was 3 h. The H2S gasproduction in the produced water storage tank is due to theheterotrophic sulfidogenic bacteria (Orphan et al., 2000). It is

Fig. 2. Determination of the efficacy of microbiocides presented as the logarithmic reducticontact time at 55 �C.

important to identify the microbial communities, and to screenmicrobiocides against these microbial communities to control H2Sand sulfide production, as the microbiocides are not same for SRBand thiosulfate-reducing bacteria (Crolet, 2005).

3.2. Biocidal activity against H2S and sulfide-producing strains

Data based on the efficiency assay described in the materialsand methods section identified SHC, BTMAC, and BNPD as the mostefficient microbiocides to control sulfate-reducing bacteria. Foreach of them, at concentrations of 25 mg l�1 it took only 2 h ofcontact time to kill 100% of the mixed sulfate-reducing bacterialpopulation used in the assay (Fig. 2). BTMAC affects the membranepermeability, inhibition of enzyme activity, and coagulation, finallyresulting in death, whereas BNPD reacts with sulfhydryl groups,leading to accumulation of free radicals and finally death (Maillard,2002). A great variety of microbiocides were screened againstindigenous populations, as microbiocide activity varies greatlybetween different types of strains of the same species (Maillard,2002).

3.3. Pilot-scale microbiocide treatment to control H2S gas andsulfide-producing microbes in produced water at Kathloni oilfield

Preliminary laboratory experiments confirmed that the additionof SHC, BTMAC, and BNPD was effective in inhibiting H2S produc-tion and sulfide-producing bacteria completely (data not shown).Thus, these results were further applied to the Kathloni oilfield,situated in Assam, in northeast India.

Without any treatment, cleaning of the soured disposal wells(i.e., removal of precipitate due to H2S production by sulfidogenspresent in produced water) was required every 20 days at theKathloni oilfield. The water disposal rate averaged 711 �8 kl d�1

during the first 10 days; thereafter, it started declining to anaverage of 648 � 34 kl d�1 for the next 5 days and then to anaverage of 529 � 25 kl d�1 until the 20th day, at which time thewells got blocked due to iron sulfide solids accumulation.

Before applying the sequential treatment described in thematerials and methods section, an initial cleaning of wells wasdone to remove iron sulfide precipitates. At the onset of treatment,the population of sulfidogens was 107 cfu ml�1 in the storage tankand 108 cfu ml�1 in the pump delivery sample (Fig. 3A). However,after a few days of the treatment H2S gas production had ceasedcompletely, and no viable sulfide-producing bacterial cells weredetected any longer in the produced water. As a consequence, the

on in the cell concentration of the mixed culture of sulfate-reducing bacteria after 2 h

Fig. 3. (A) Total population of sulfide-producing microorganisms in the producedwater of storage tank (6) and in pump delivery (C) before and after the biocidetreatment. Viable counts were determined by MPN assays performed in Iron Lyngbymedium at 55 �C. (B) The rate of disposal of produced water in the wells before andafter the treatment.

G. Kaur et al. / International Biodeterioration & Biodegradation 63 (2009) 151–155154

rate of disposal of the produced water into wells was maintained at707 � 5.35 kl d�1, 722 � 7.69 kl d�1, 751 � 4.45 kl d�1,760 � 3.98 kl d�1 respectively, for each of the four sequences of thetreatment (Fig. 3B). Hence no well cleaning was required after the132 days of the treatment.

Maintenance of an effective residual concentration of microbiocidethrough the system was monitored by collecting samples from severalpoints (storage tank and pump delivery) to overcome losses by physicaladsorption and chemical reaction of microbiocide(s). There was 100%reduction in the population of sulfidogens at the emulsion tank,storagetank, and pump delivery. There was no sulfide production after the

Table 2Chemical analysis of produced water before and after the biocide treatment

Characteristic Unit Before biocide treatment After biocide treatment

pH – 7.5 � 0.2 7.2 � 0.1Sulfide mg l�1 101 � 5 0Salinity (as NaCl) mg l�1 4250 � 50 4350 � 25Chloride mg l�1 2578 � 25 2639 � 20Carbonate mg l�1 ND NDBicarbonate mg l�1 439 � 30 445 � 22Sodium mg l�1 1550 � 30 1541 � 20Potassium mg l�1 50 � 5 69 � 7Calcium mg l�1 178 � 20 213.5 � 35Magnesium mg l�1 26 � 7 11 � 5Sulfate mg l�1 70 � 2 90 � 3Iron mg l�1 1.83 � 0.2 2.51 � 0.1TDS mg l�1 4800 � 62 5080 � 78Suspended solids mg l�1 300 � 32 250 � 25Oil and grease mg l�1 80 � 10 62 � 15Turbidity NTU 192 � 15 166 � 20Silica mg l�1 85 � 5 84.2 � 8

ND, not detected; NTU, nephelometeric turbidity unit.

treatment, indicating that non-cultured sulfidogens were either killedor did not contribute to sulfide production. The rate of disposal beforethe treatment was zero, as the wells were blocked. However, after themicrobiocide treatment, there was neither plugging nor any require-ment of periodic redevelopment to restore the capacity of disposalwells by physical scrubbing, acidification, pumping, etc., to dispose ofthe produced water.

3.4. Characterization of Kathloni oilfield produced water

The produced water obtained from pump delivery beforetreatment was pale yellow and turbid black with oil drops. Thesulfide production at the three-phase separator was 40 � 4 mg l�1

and it increased to 52 � 3 mg l�1 at the emulsion treatment tank,and increased to 101 � 5 mg l�1 at the storage tank and pumpdelivery. Physical and chemical properties of produced water at thetreatment site are shown in Table 2. The pH of produced waterdeclined after the treatment; however, the water was clear andtransparent. No carbonate was detected during the study. Therewas no significant increase in the salinity or chloride, indicatingthat the chlorine from the microbiocide did not make much ofa contribution to the produced water (Table 2). Sulfate and ironwere found to increase after the treatment.

The produced water was found to contain a black precipitateduring the first 66-day treatment cycle; this could be because of thedetachment of the biofilm attached with the oil pipes. Since themanual cleaning of oil pipes was not feasible, the increase in ironcontent after the treatment could be due to the absence of thesulfidogens, and sulfate-reducing bacteria, which have a highrequirement for Fe2þ (Marchal et al., 2001).

4. Conclusion

The aim of this investigation was to design a microbiocidetreatment for controlling the produced water sulfide-producingbacterial population that would be optimal for the Kathloni oilfield.The treatment was designed on the basis of the following twopremises: First, it is critical to identify precisely the sulfidogenicbacterial population against which the treatment needs to bedesigned, in order to find the most suitable microbiocides foroptimal effect; second, it is best to apply more than one micro-biocide, working sequentially, in order to prevent the developmentof resistant strains.

Although most of the sulfide-producing bacteria found inproduced water at the Kathloni oilfield have also been found atother oilfields (Rees et al., 1997; Orphan et al., 2000; Tello et al.,2003; Fardeau et al., 2004), Coprothermobacter sp. and Thermose-diminibacter sp. identified in this study had not been detectedpreviously at other oilfields. A preliminary laboratory investigationhas identified three microbiocidesdSHC, BTMAC, and BNPDdasmost efficient in controlling the viability and activity of the sulfi-dogenic bacteria isolated from the produced water of the Kathlonioilfield. Sequential application to produced water of these threemicrobiocides at optimal inhibitory concentrations allowedcomplete control of the sulfidogenic microbial population for the132 days of treatment. As a consequence, the rate of disposal of theproduced water into wells was maintained above 700 kl d�1 duringthe 132 days of treatment, resulting in a reduction of frequency ofwell cleaning. Therefore, this investigation shows that applying ourtwo premises for the design of a produced water microbiocidetreatment is a promising option for controlling sulfide production.

Acknowledgments

We thank the Department of Biotechnology, Government ofIndia, and Oil India Ltd for partial funding of this research, and the

G. Kaur et al. / International Biodeterioration & Biodegradation 63 (2009) 151–155 155

Council of Scientific and Industrial Research, New Delhi, forproviding a fellowship to one of the authors. We also thank Ms.Simrita Cheema for her help in writing the manuscript.

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