IBP 1812 _12 Implementing better microbial control solutions in industrial systems via microbiological & molecular biology

monitoring and the development of new microbial control solutions Geert M. van der Kraan1, Diana Jocksch2, Monica CanalizoHernandez1, Bei Yin1, Terry Williams1 & Philip A. Keene3
Copyright 2012, Brazilian Petroleum, Gas and Biofuels Institute - IBP
This Technical Paper was prepared for presentation at the Rio Oil & Gas Expo and Conference 2012, held between September, 1720, 2012, in Rio de Janeiro. This Technical Paper was selected for presentation by the Technical Committee of the event according to the information contained in the final paper submitted by the author(s). The organizers are not supposed to translate or corr ect the submitted papers. The material as it is presented, does not necessarily represent Brazilian Petroleum, Gas and Biofuels Institute opinion, or that of its Members or Representatives. Authors consent to the publication of this Technical Paper in the Rio Oil & Gas Expo and Conference 2012 Proceedings.

Abstract
New microbial control solutions as a part of industrial water cycle management in the petroleum industry are proposed via 2 case studies. Each case study addresses the different phases of the development of better microbial control solutions within this water management. Addressed in the first case study is the application of traditional microbiological methods and molecular biology techniques (viz. qPCR and 16S rDNA pyrosequencing). A survey of an oil production site and oil-water separation plant has been performed and subsequently a detailed map is produced showing the microbiological issues detected in the system. It was shown that the top site facility is severely contaminated with sulfate reducers covering at various tested locations up to 60% of the total population. Locations of high concern were identified, holding e.g. cell numbers 1000 higher than the produced water at the wells. Based on these analyses an optimized dosage regime for biocides was proposed. The second case study addresses the development of a tailored biocidal blend specifically designed for shale gas plays in the US. First organisms were enriched from the specific play. New THPS blends were formulated and optimized based on their efficacy against these isolated strains. The blends were also optimized to ensure good thermal stability and good compatibility with other chemistries used by the account. The new blends showed better efficacy at lower concentrations active ingredients, thereby making this tailored solution more sustainable from an environmental perspective.

1. Introduction
Our current society, by its very essence, is heavily dependent on fossil fuels. As an example, 40% of the global energy demand is derived from oil alone and 90% of all chemicals produced are derived from fossil resources (Bakas & Creemers, 2007). As it becomes more and more difficult to retrieve the fossil resources from the subsurface, the petroleum industry is applying secondary and tertiary oil recovery techniques, predominantly (sea)water flooding to push out more of the oil in the reservoir (on average 10 -15 % extra oil). With the introduction of water, substantial issues with unwanted growth of microorganisms are introduced (Pedersen, 2000). Frequently encountered are the sulfate reducing microorganisms and their detrimental effects like reservoir souring and microbial influenced corrosion (MIC) (Pope et al, 1991) caused by their H2S production as the end product of their metabolism (Muzyer & Stams, 2008) (Nilsen et al, 1996). But adverse effects like the plugging of filters and reservoir clogging can be caused by the unwanted accumulation of microorganisms growing on surfaces (biofilms) (Steward & Fogler, 2002) (Khazipov et al., 1993). All these processes are addressed under the term biogenic fouling. Waterflooded oilfields and the associated engineered ecosystems provide suitable environments for sulfate reducers to thrive; they are reducing (oxygen depleted) environments with a lot of sulfate introduced via seawater, and an abundant source of organic carbon in the form of hydrocarbons. The issues with unwanted growth in water are not limited to oilfields, but also occur in the recovery of shale gas where vast amounts of water are applied, especially in the actual fracturing of a well (Kerr, 2010). Issues with the growth of microorganisms in these systems are also frequently reported. With the shale gas boom happening almost overnight in the US, the increasing application of water flooding for secondary oil recovery purposes and the stricter regulations on use and disposal of industrial water, there is an increasing need within the petroleum industry for

______________________________ 1 Sr. R&D Specialist, - The Dow Chemical Company 2 R&D Technician, - The Dow Chemical Company 3 Customer Application Leader, - The Dow Chemical Company

Rio Oil & Gas Expo and Conference 2012 improved water cycle management and microbial control. Biogenic fouling of industrial water is a key element that needs to be addressed in water cycle management. It is common practice in the petroleum industry to perform routine tests on various sources of water like produced water, injection water, water derived from storage tanks, etc. This often occurs via traditional culture based methods (CBM), indicating that a minute amount of water is added to a nutrient rich broth specific for the group of microorganisms of interest. The observed growth is an indication of the presence of microorganisms. During these traditional tests, also called incubations, often dilution series and plate counts are made providing some level of quantification on the number of microorganisms present. In many cases, this is also the way microbial control solutions are tested. There are two main protocols that are recognized and applied internationally (NACE Standard TM01942004-21224). The efficacy of biocidal treatments is evaluated at different concentration ranges of the actives and via serial dilutions and plate counts. Usually treatments are tested on a predefined standardized combination of organisms, often lab strains. Culture based methods, although providing information on how heavily an engineered ecosystem is bio-fouled holds limitations. Only a fraction of microorganisms (0.1-1%) can be cultured in laboratory environments (Staley & Konopka, 1985) and often the applied enrichment broth for the cultures select which microorganisms develop so the laboratory culture-based test results may not accurately predict the performance of the treatments in the field ecosystem. With the rise of molecular biology tools in the petroleum industry (techniques for characterizing the genetic content of cells viz. DNA & RNA) a new toolbox is at our disposal to be able to better determine which microorganisms are growing in industrial systems and to better evaluate the detrimental effects of these microbes (Singh et al, 2009). This deeper understanding also helps in the development of new microbial control treatments since they can be tested on relevant species in the ecosystem or against more relevant laboratory equivalents. These methods also allow for a field evaluation of novel microbial control solutions, when they are applied in real industrial systems. Having the monitoring of biological processes (biomonitoring) included in the traditional survey of an industrial system as a first step, allows a better understanding of the microorganisms thriving in the system and identifies the areas in the system of high priority for microbial control treatments. This subsequently allows the application of a better treatment and dosage regime. The development of the best microbial control solution tailored to site conditions and end user needs is the second step. This includes the testing and development of various biocidal treatments, including combinations of biocides, relevant to the ecosystem. Usually this requires extended biocide screening programs. This paper discusses via 2 case studies methods aimed at improving the applied microbial control solutions. Case study 1 discusses a full study of all the different water streams belonging to an on-shore oilfield and its associated oil water separation facility. It included a geochemical analysis and the application of traditional and molecular biology tools. Case study 2 discusses the development of new patent pending THPS blends for a shale gas play in the US. Unwanted growth of microbes was observed. A full screening was performed to prepare a tailored solution. Thereto, microorganisms from the various water streams were isolated. Efficacy of the tailored blends was then tested on these isolated wild type strains. A similar diversity survey as in case study 1 was done, but is not discussed in this paper.

2. Case study 1 - Materials & Methods

Production sites-S1 Injection wells-S6 Incoming lines-S2 Oil water separator-S4 Oil storage tank

Backfeeding pipeline-S3 Water storage tank

Basin-S5

Figure 1: Artist impression - Overview of the site in case study 1.

2

Rio Oil & Gas Expo and Conference 2012 2.1 Description of the production sites and the oil-water separation & injection facility. Oil is produced at different sites at various distances from a separation site. Production and injection wells are placed throughout the sites. No souring has been seen from the produced wells. The produced oil-water emulsion has a high water cut (90%) and is transported to an oil-water separation site via pipelines. Here the largest part of the oil phase is separated in a separator and the oil is transported to an oil storage tank. The water phase is pumped to a water storage tank which from there provides the water for the injector pumps. A feedback loop exists between the water storage tank and a water basin. From the oil storage tank some remaining water is pumped back into the separator. This amount of water is low and this pipeline is in operation for 2 of hours per day. Details can be found in Figure 1. 2.2 Sampling procedure & preparation (description of sample points). From various sites the produced water was first collected in a 10 liter jerry-can. For traditional culture based analysis 1 ml amounts of water were added to three different media types (9 ml). Two media types selected to enrich for Sulfate Reducing Bacteria, one media type selected for Acid Producing Bacteria (APB). For molecular biology analysis water was filtered on sterile filters catching the cells from the different environments. After water filtration the filters holding the cells were placed in tubes and were transported at 0 C to the laboratory where they were subjected to DNA extraction and further analysis. 2.3 Geochemical analysis The temperatures of the waters were determined via a digital thermometer. The pH was determined via standard test strips. Standard test kits were used to determine sulfide levels and free ferrous irons (Fe2+) in the different waters according to the manufacturers instructions. 2.4 Culture based methods (CBM). The inoculated anaerobic bottles with the different media were shipped on ice to the laboratory. The bottles were incubated anaerobically for 5 days at 25 C. Turbidity was observed overtime (as a presence/absence check). 2.5 DNA extraction & quality check of the DNA extracted from the cells. The DNA of the cells was obtained by applying the PowerWater Pro DNA isolation kit (MO BIO Laboratories) according to the manufacturers instructions. (Filtration section 2.2). To check for the presence and quantity of the genomic DNA, 2 methods were applied:. agarose gel electrophoresis and quantification using absorption spectroscopy (via a NanoDrop spectrometer). 5 l of the solutions that contain the purified genomic DNA were placed on a 1.5% w/v agarose gel and were run for 30 minutes at 90 Volts. The gel was placed in a 1X TAE buffer during the run and subsequently stained with DNA dye. 1 l of the genomic DNA was placed in a Nanodrop spectrophotometer. The absorption was checked at 230, 260, and 280 nm. The ratios of the wavelengths indicate the DNA quality and quantity. 2.6 Relative-qPCR program & 16S rDNA pyrotag sequencing To check if the DNA could be used for 16S rDNA pyrotag sequencing and to get a rough estimation of relative cell amounts in the different waters, relative-qPCR assays were performed. The primer pair used for this amplification was the 341f/907rM and targeted the 16S rDNA gene of Bacteria. The amplification was done on a Biorad Icycler IQ5 rtPCR instrument. The mixture contained 10.0 l IQ Sybrgreen supermix (BIO RAD), 9.1 l DNA-RNA free water (Qiagen), 0.16 l BSA, 0.15 l of each primer (Bac341F, 50 M / Bac907rM(rA+rC) (Schfer & Muyzer, 2001), 50 M stock concentration) and 0.4 l template. After this check the genomic DNA was shipped to the Research & Testing Laboratories, (Texas, USA) for 16S rDNA Pyrotag sequencing. The 16S rDNA gene is widely used in the microbial ecology to characterize community structures and dynamics. Per environment 3000 sequences have been analyzed applying the PyroTagger software package (Kunin & Hugenholtz, 2010). All obtained sequencing results have been trimmed to a usable high quality length of 250 base pairs. This screening did not include Archaea at this time.

3 Case Study 2 Materials & methods

3.1 Sampling & culture based methods Waters from diverse sources were taken from a North American shale gas play using sterile 1 L bottles. For traditional culture based analysis 1 ml amounts of water were added to two different media types (9 ml) to enrich for Sulfate Reducing Bacteria and Acid Producing Bacteria (APB). The pond water sample was taken just below the surface about half a meter from the edge. The drilling mud was sampled by taking samples in similar bottles from side stream ports. Details are found in table 1. An evaluation was done on all water samples for microbiological content. These incubations were applied to estimate the cell numbers. In addition an ATP analysis of all samples was performed. Commercial 12-ml incubation bottles (holding 9 ml of media) were purchased from standard local suppliers. After inoculation with 1 ml of water, the bottles were incubated at 30 C. Log10 ml-1 cell counts were recorded for SRBs and APBs after 28 days of incubation. This long period of 28 days was due to logistic issues of sample transport. 3

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Table 1: Sample locations and the respective temperatures of the water.

Location 1) Pond water 2) Pit water 3) Blended water (contains pond water/pit water & produced water) 4) Treated blended water* 5) Flowback water (return after initial injection) 6) Produced water 7) Drilling mud 8) Oil-Based drilling mud * This water was treated during lab testing and thus analyzed as such. Temperature (T < 40 C) (T = 40 C) (T = 40 C) (T = 40 C) (T = 70 C) (T = 70 C) (T = 60 C) -

ATP-analysis of all water streams An ATP analysis was performed on the listed waters applying the method and kit used from LuminUltra Technologies, Ltd. using the so-called Quench-Gone Aqueous kits according to the manufacturers instructions. In short, 10-ml samples were passed through a filter and the trapped organisms were lysed to release cellular ATP. The levels of ATP present were measured using a luciferin-luciferase assay with a Kikkoman C-110 Lumitester luminometer. ATP levels were determined from an internally calibrated dose-response curve with a fresh ATP standard. Values were reported as picograms of ATP per ml of sample. (The amount of ATP present can be used to compare relative environments) Relative endospore check A malachite green spore staining protocol was applied in combination with microscope counts to check for the presence of Endospores in the waters. The Schaeffer and Fulton Spore Stain Kit (Fluka) was used for spore staining following the manufacturer's instructions. An Olympus BX 51 microscope with a fluorescence capability was used for visual observation of the spores and a relative assessment of spore presence. Efficacy screening program of biocides Enrichments of environmental samples were made in order to test different microbial control programs. Reduction in cell counts to below 102 cells ml-1 was set as the criteria for good microbial control. Biocide efficacy was tested on both groups. A total active biocide concentration of 150 p.p.m. w/v was applied in all cases. Biocides were tested alone and in synergistic combinations. In the stand-alone biocide tests, 200 p.p.m. w/v of active was applied and a softer pass criterion was used indicating that higher cell levels were acceptable. The biocides tested included glutaraldehyde, quaternary ammonium compounds, dazomet, and THPS (tetrakis hydroxymethyl phosphonium sulfate).

4. Case study 1 - Results

4.1 Chemical composition of the different waters The water produced differs per well in salinity but overall its qualified as brackish. The average temperature of the water down hole varies between 65 and 85 C. At the various production sites the sulfide and iron levels in the waters as well as the pHs of the waters were measured. The water arriving at the oil-water separation site displays differences over the produced water. Both lines have different temperatures (hot and cold) of 41 C and 10 C. This difference is caused by the varying distance from the separation site. The incoming cold line passes through a heat exchanger to boost the temperature to slightly above 40 C. The pH was still neutral but there was a distinct signature of sulfide in the water, around 3 mg L-1. The water in the oil-water separator is around 43 C but slightly more acidic than the incoming water holding a pH of around 6 to 6.5, no sulfide was detected. Unlike the produced and incoming water some free Fe2+ was detected. Water from the bottom of the oil storage tank and the backfeeding pipeline has another signature holding a temperature of 37 C and a free iron level of about 10 mg L-1, the pH returned to neutral. The aerobic water in the basin held a temperature of 32 C and a slightly acidic pH of 5.8. No sulfide and free iron were detected. The injection water held a temperature of 24.8 C and a pH of 6.5, a sulfide level of over 10 g L-1 was detected. For a detailed overview see Table 2. 4.2 Results of traditional culture based methods SRB & APB (CBM) incubations have been made from the different water sources in the system. It is shown that the water from many of the locations showed growth of microorganisms (turbidity) indicating that the cell numbers in the water are high (Table 2). It is clearly shown that in all of the tested environments microorganisms are thriving, some differences between the results indicate how sensitive these methods are with respect to media composition and the detection of microorganisms. 4

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Table 2: Results of the culture based methods and geochemical analysis performed
Location description Produced water (5Yr) Incoming line Oil storage tank O/W Separator tank Basin water Injection water nr^. S1 S2 S3 S4 S5 S6 SRB (i) SRB (comm.) Growth detected: Yes Yes Yes Yes Yes Yes APB (comm.) Yes Yes Yes Yes T (C) 44 43.6 37.5 43 32.3 24.8 H2S 0 2 0 0 0 10 Fe2+ 3 0 10 3 0 0 pH 7 7 7 6.5 5.8 6.5-7

Post settler tank line * Yes Yes 47.7 0 0 7 Produced water (35Yr) * Yes 24.3 / / / ^ =Sample number taken for molecular biology analysis; * = No water was filtered no molecular biology analysis - Indicates no turbidity in the incubations; / indicates no data available. (comm.=commercial source) 4.3 DNA quality & relative qPCR results. To assess the quality and amounts of the extracted DNA 2 different methods were applied. 1) The HMW-DNA was loaded on an agarose gel, 2) the DNA was placed in a NanoDrop spectrometer. A qPCR was performed giving the relative cell numbers. In the qPCR reactions 1/10 dilutions were used to assess if indeed there were no discrepancies between the dilutions (data not shown). From all environments a positive PCR signal was obtained indicating that all samples were fit for pyrosequencing. It was shown that between the 1 and 10 fold dilutions a difference of 3 or 4 cycles was found indicating an acceptable DNA quality and PCR conditions. From the relative-qPCR results a relative comparison of DNA amounts could be made giving a rough estimation of multitudes of cell amounts taking the lowest amount as the reference point (S1-S6). Results are found in Table 3. Differences in the filtered water amounts have been taken into account for relative comparison calculation. It can clearly be seen that the system enriches for the number of microorganisms as the water phase migrates via the pipelines. Especially the backfeeding pipeline from the waterphase of the oil storage tank back to the oil-water separator is high in cell levels (S3). The incoming water from the production wells and outgoing water arriving at the injection wells are similar in cell levels. The produced water from the young well and the basin display lower levels when compared to the oil-water separation system. The sample S1 was chosen as the comparative environment as cell numbers increase from that point, indicating that the system enriches for bacteria when compared to the produced water that comes up.

Table 3: relative qPCR results

Sample name S1 S2 S3 S4 S5 S6 Ct values (1/10) 27/20 16/20 16/19 16/20 21/25 16/20 Cycles less than S1 (produced W) 11 11 11 6 11 Factor difference More than S1 1 102 103 102 1 102 Factor difference More than S2 0.01 1 10 1 0.1 1

4.4 Community structures (16S rDNA pyrosequencing) To present all pyrosequencing results is beyond the scope of this paper, as over 17000 different clusters have been identified in the 6 studied environments. Instead a summary of the important environments is provided in Table 4 which shows the most dominant species distribution and major findings. The data is presented in percentage of the total population. A summary of the most important findings is listed below. From Table 4 interesting observations can be made. The production water that comes up from the young production well, is dominated by species that cannot be found anywhere in the rest of the system and seem to play a role of no significance (light grey column of S1). The incoming hot lines enrich for a specific Desulfocaldus (Duncan et al, 2010) species that is also enriched in the injection pipeline, in the tank this species is also present but at a lower level with respect to the total community (grey rowDesulfocaldus). The water in the basin displays Gammaproteobacteria, which is not uncommon for aerobic slightly saline waters. In the following sections, more detail will be given to the different environments. As an example on the level of detail pyrosequencing can deliver, the results of the incoming water are provided in Figure 2.

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Table 4: Most abundant sequences (% of the total sequences) present in the samples.
Name of closest relative Bacillus sp. str. LAMI 010 Desulfocaldus sp. str. Hobo Desulfovibrio desulfuricans str. G20 Gammaproteobacterium-like Desulfotomaculum solfataricum str. V21 Clostridium-like Desulfovibrio sp. str. M1 Desulfacinum subterraneum str. 101 Desulfotomaculum-like Carboxydibranchium-like Bacteroides sp. str. SA-7 Isolate str. 52651 Pseudomonas balearica st101 S1 S2 42.08 S3 2.68 S4 64.2 S5 0.53 0.01 52.86 0.05 0.09 0.15 0.07 12.88 0.07 0.01 0.09 0.05 10.7 0.17 0.07 S6 46.73 Identity 100% 100% 100% 100% 100% 100% 100% 98.23% 99.56% 100% 100% 100% 100%

44.56

0.14

0.04

0.06 0.1 0.01

4.7

0.22 18.6

8.86 0.14 0.08 0.05 0.66

33.99

0.12 0.74 11.59 0.01

4.5 A detailed description of the communities found in the different water sources. In the produced water (S1) from one of the wells it was found that 2 species cover almost 80% of the community, Desulfotomaculum solfataricum and an uncultured species. The first species is described in the literature as a rod shaped microorganism that can form spores that can use a wide range of carbon sources. It uses sulfate, thiosulfate and sulfite as electron acceptors (Goorissen et al, 2003). Optimum growth of the organism is 60 C with a higher limit of 65 C. PH range of this organism is 6.4-7.9. It can grow in water with a max salinity of 1.5 g L-1. This fits with the description of the ecosystem (Table 2). The incoming water (S2) from the production lines at the separation site has by then reached a temperature of 41 C and shows a turnover in the bacterial community when compared to the production water community. The water arriving at the separation site is now dominated by a Desulfocaldus member, which occupies over 40% of the community. The cell amount roughly has gone up 10 fold when compared to the water produced at the well. In addition traces of sulfide were detected indicating an actively H 2S producing population. It is clear that the Desulfocaldus species is playing a dominant role in the souring of the system. This Desulfocaldus species has also been detected in the separators of Alaskan North Slope Oil facilities (T = 50 C). The backfeeding pipeline (S3) is only in operation for 2 of hours per day and serves to pump back the remaining water from the separated oil phase back into the separator tank. It holds a temperature of 37.5 C. From the relative comparison of cell amounts on the direct environmental samples it showed the highest cell amount. Also it was found that the community from the bottom of the oil storage tank is more diverse indicating a high microbial activity. The pH is around 7 and a substantial amount of free Fe2+ was detected indicating an MIC process which potentially also explains the low H2S amount. Interestingly the Desulfocaldus species (that is detected in the incoming water) at low levels persists here. It is clear that a lot of sequences detected have closest relatives associated to oil degradation and sulfur associated waters. The water extracted from the oil-water separator tank (S4) is a combination of all the waters from the various production locations. The average temperature is 43 C. The water is similar to the water from the incoming hot line species wise. A big dominance of the same Desulfocaldus species is found, covering almost 65% of the community. Surprisingly no sulfide was detected in the water; however the presence of free Fe 2+ can explain this absence. The water in the basin (S5) is fully open to the outside air and is therefore completely aerobic and represents a different ecosystem. The temperature of the basin is lower than in the system (32 C when measured) but this can be prone to seasonal changes. The community holds species that are typical for aerobic brackish water and is of less relevance. The water arriving at the injector wells (S6) displays a lot of similarity with the water arriving from the incoming lines. From an ecosystem perspective they are indeed similar. At the end of the pipeline the temperature has dropped to 25 C and gradually drops when getting further away from the water tank. The injection water enriches again for the encountered Desulfocaldus species which covers almost 50% of the community. The levels of sulfide detected were high such as was found in the incoming cold line with values over 10 mg L-1. That the injected water contains such high levels of sulfide could be an issue for the reservoir.

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Figure 2: Pyrosequencing results of the incoming water (community structure) 5. Case study 2 Results
5.1 Microbiology analysis of the water sources (SRB/APB Incubations and observations) All waters presented significant contamination in all enrichments. Cell counts were between 104 and 107 cells ml-1 both for SRB and APB bacteria. It was also shown that the pond and pit waters contained significant levels of yeasts and fungi (data not shown). The endospore counts especially in the flowback and the produced water were elevated when compared to the other water sources. The flowback and produced water showed the highest temperature tolerance for the microorganisms in the various samples tested. This is expected since these downhole environments are subjected to higher in situ temperature ranges. Details are found in Table 5. 5.2 ATP analysis of all water sources From the ATP analysis, it was clear that the pit water displayed the highest ATP amount of 7000 pg ml-1, followed by the pond and the blended and the flow back waters (each around 2000-2500 pg ml-1). A clear observation was that the ATP content of (lab) treated water was significantly lower, for example at around 70 pg ml-1 after 6 hours of treatment. ATP analysis from the drilling muds could not be obtained due to interference of the mud with the ATP measurement. This can be seen in Table 5. 5.3 Biocide screening and combinations for optimized performance In order to provide a sustainable solution to the biofouling problem different biocide screenings and combinations have been tested on the enriched environmental strains isolated from the shale gas play. Of particular interest are the treatment evaluations of the flow back and produced water. As shown in Table 6, it can be seen that the newly proposed biocide combinations gave better control than the traditionally used biocides, over a longer period of time. In this case the pass-criterion was set at reducing viable counts to less than 102 cells ml-1. In the screening, the combined level of actives applied was 150 p.p.m. w/v. In comparison to the tested combinations, the application of single biocides, even when dosed in higher concentrations viz. 200 p.p.m. active and with a softer pass criterion (3 log reduction per ml on cell counts) did not give as good of control. Details can be found in Table 7.

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Table 5: Cell numbers and ATP analysis of the different water sources
Source Pond water Pit water Blended water Blended water (treated) Flowback water Produced water Drilling mud Oil-based drilling mud Cell counts (to power of 10) SRB APB 3 5 6.5 6.5 5.5 7 3 3.5 4 5 3.5 3 4 7 5 7 Average ATP picogram ml-1 2200 7000 2600 (70 (6 hours) 15 (24 hours) 2300 200 * * Endospores low low low low high high * *

Table 6*: Biocide combination screening performed on enrichments from flow back water isolates
Applied combinations 24 hour efficacy SRB APB 48 hour efficacy SRB APB 7 day efficacy SRB APB

Glutaraldehyde/ Quaternary ammonium salt** Dazomet/ Quaternary ammonium salt** Gultaraldehyde/New active 1 THPS/ New active 1 Glutaraldehyde/New active 2 THPS/New active 2

* A indicates: did not pass the set criteria of cell counts below 102 cells ml-1, a indicates: did pass this criterion. ** The applied quat in this case is proprietary to the play operator and can therefore not be disclosed.

Table 7*: Biocide (stand alone) screening performed on enrichments from flow back water isolates.
Applied biocides 24 hour efficacy SRB APB 48 hour efficacy SRB APB 7 day efficacy SRB APB

Glutaraldehyde Dazomet THPS

* A indicates: did not pass the set criteria of a 3 log reduction of cells ml-1, a indicates: did pass this criterion.

6. Discussion
Performing an in depth molecular biology screening in addition to a traditional culture-based screening delivered information which is key in the application of the biomonitoring concept. Information has been obtained that could not be obtained using traditional tests alone. One interesting scientific observation is that indeed species relate to the ecosystem properties retrieved by a simple geochemical analysis (salinity, T, pH, sulfide and ferrous iron levels). The production water that comes up holds species associated with the well ecosystem and it is found that they play no significant role in the rest of the system. Subsequently (for Case study 1) the species that the hot pipeline enriches for is a Desulfocaldus strain that seems to be dominant throughout the entire system. The incoming hot line and injection water pipeline are similar to that extent; especially the injection water displays high levels of sulfide indicating a high microbial activity. The greatest point of concern is the backfeeding water from the pipeline in between the oil-water separator and the oil storage tank, this water contained the highest microbial level and diversity. The molecular biological analysis showed that this area is the likely source of infection for the entire industrial system. Figure 3 summarizes the molecular biology results. Each of the tested environments was assigned a level of concern based on the analysis. Recommendations were prepared in order to improve the water cycle management. A new biocide addition point in the water tank was proposed since the injection water was a significant concern due to the high H2S levels. An alternative fate for the water from the bottom of the oil storage tank was proposed. The current situation is that on a 8

Rio Oil & Gas Expo and Conference 2012 routine (daily) basis the bottom water is transferred from the oil tank to the settlement tank. Data generated from the analyses shows that this untreated water contains both SRBs and& APBs. The oil tank can be considered a continuous source of infection to the settlement tank and system. The transferred water and/or bottom water in the tank needs to be treated to emilinate this key source of untreated contamination to the system. Alternatively it was proposed to feed the water directly to the basin water where aerobic conditions dominate so the levels of SRBs and APBs would be greatly reduced in the system. With these changes we can adjust treatment and monitor the injection water to reduce the suspected biotic H2S and SRB/APB levels. The effectiveness of these measures can be determined by routinely monitoring the system applying the same screening methodologies.

Desulfocaldus, high levels of H2S (T=24.8 C, pH 6.5, H2S, 30 mg L1) This pipeline is of high concern. Both produced water and the water in the basin are of low concern with respect to the species and cell numbers.

Diverse community & high cell levels. This pipeline is a source of infection for the system and is of high concern. (T=37.5 C, pH 7, 10 mg L-1 Fe2+)

The area around the O/W separator are of some concern (T=43 C, pH 6.5-7; 3 mg L-1 Fe2+)

Figure 23: Showing the areas of concern: green indicates low, red indicates high
The right selection of biocides for the specific play and water stream is a key step, but represents a challenge in each different environment. In the US shale gas play study, it was found that the down-hole waters were also heavily contaminated with microorganisms. The screened biocide combinations perform potentially better than the current applied microbial control program. It was found that these waters contain resistant thermophilic microorganisms including high levels of spore formers. Treatment here is of the essence to reduce and inhibit the germination of endospores. This research indicates that the current treatment is ineffective and that an effective treatment of the drilling fluids with amended microbial control products is required. In addition the low volume of the fluids minimizes the preservative needed. For the injection fluid the conditions are different. In this case a temperature stable and long lasting microbial control application should be applied since the water will stay in the formation for a prolonged period of time. From the field monitoring data, which did not include a biocide treatment program, a heavy microbial contamination was detected (SRB & APB levels significantly above the criteria of 102 cells ml-1). The lab studies provided several solutions for bringing the system under control again. These solutions included innovative sustainable biocide combinations that offer effective control at lower active concentrations. The proposed biocide combinations still can be further optimized regarding ratio and dosage regimes. A qPCR analysis will be performed to look better at the total number of cells group-wise. A field trial will be performed when the above parameters have been optimized. Biomonitoring (not shown) programs indicated which of the water sources needed the most robust treatment.

7. Conclusions
The results presented in case study 1 give a proof of concept for how the molecular biology toolbox can be applied for the development of water cycle management in the petroleum industry. It allows a deeper understanding of the community dynamics and metabolic processes in industrial systems and can help in the identification of places of high concern and changes can be made in location and type of biocide addition to improve microbial control. Also molecular biology can help in the early identification of potential issues allowing a proactive microbial control solution strategy. Usually when issues are in an early stage, less biocide is required to regain effective control allowing for even more sustainable treatment programs. In both scenarios the microbial control solutions are applied more effectively. In the second case routine monitoring of the system is required. 9

Rio Oil & Gas Expo and Conference 2012 Results presented in case study 2 show that the development of specific biocidal combinations tailored to the ecosystem is a valuable next step which requires an extensive screening protocol taking various parameters into account, having efficacy and compatibility with the water and other chemicals added to that water as the key parameters. In the second case study new biocide combinations based on THPS as the main active have been developed specifically for shale gas plays in the US area. This case study showed clearly that an integrated water lifecycle management program has to include a dedicated microbial control program based on the individual environmental parameters of the shale gas play. Microbiological screening requires a specialized lab which can mimic specific customer environmental conditions. This is essential to develop a sustainable effective microbial control solution. Both case studies demonstrate that a good monitoring program of industrial sites in combination with the development of tailored biocidal trreatments specific to site and end user conditions, can in the near future lead to the development of a more effective and sustainable microbial control program.

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8. References
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