biocide cocktail consisting of glutaraldehyde, ethylene ...132.235.17.4/paper-gu/2012 biocide...

CORROSION SCIENCE SECTION

994 CORROSION—NOVEMBER 2012

Submitted for publication: December 8, 2011. Revised and accepted: March 30, 2012. Preprint available online: July 11, 2012, http://dx.doi.org/10.5006/0605.

‡ Corresponding author. E-mail: [email protected]. * Department of Chemical and Biomolecular Engineering, Institute

for Corrosion and Multiphase Technology, Ohio University, Athens, OH 45701.

** Department of Infectious Diseases, Infection Control, and Employee Health, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77230.

Biocide Cocktail Consisting of Glutaraldehyde, Ethylene Diamine Disuccinate (EDDS), and Methanol for the Mitigation of Souring and Biocorrosion

D. Xu,* J. Wen,* T. Gu,‡,* and I. Raad**

ABSTRACT

Souring and microbiologically influenced corrosion (MIC) caused by sulfate-reducing bacteria (SRB) are major prob-lems in the oil and gas industry as well as other industries such as water utilities. SRB biofilms are notoriously difficult to treat. More effective dosing of biocides is desired to deal with increasing environmental restrictions and costs. This work presents an effective green biocide system consisting of glu-taraldehyde (C5H8O2), ethylene diamine disuccinate (EDDS [C10H16N2O8]), and methanol (CH4O) against souring and MIC caused by SRB. Desulfovibrio vulgaris (ATCC 7757) was used in this work in the full strength ATCC 1249 medium and a modified ATCC 1249 medium with only four ingredients (MgSO4, lactate [C3H5O3

–], yeast extract, and Fe2+) at concentra-tions 1/4 of those in the full medium. Tests were carried out in 125 mL anaerobic vials. It was found that the triple combina-tion of 30 ppm (w/w) glutaraldehyde, 1,000 ppm EDDS, and 10% (v/v) methanol was considerably more effective than glu-taraldehyde alone and the combination of glutaraldehyde and EDDS in the inhibition of souring, SRB biofilm establishment, and MIC pitting of C1018 carbon steel (UNS G10180) in the presence of 2% (w/w) sand particles in batch tests.

KEY WORDS: biocide, biofilm, microbiologically influenced corrosion, pitting, souring, sulfate-reducing bacteria

INTRODUCTION

Souring caused by the formation of hydrogen sulfide (H2S) often occurs in oil reservoirs during secondary oil recovery. Microbial sulfate reduction is usually the source of H2S in reservoirs.1-2 Sulfate-reducing bacte-ria (SRB) often cause souring in the oil and gas indus-try.3 SRB use sulfate as a terminal electron acceptor and various carbon sources such as volatile fatty acids or even hydrogen gas as electron donors.4 Some SRB can even use saturated and unsaturated hydro-carbons that are readily available in oil pipelines as electron donors.3,5 As an example, the following reac-tion shows the overall anaerobic respiration reac-tion combining SRB oxidation of acetate with sulfate reduction:4

SO CH COO H CO H S H O42

3 2CO3 2COO H3 2O H CO3 2CO 2 2H S2 2H S H O2 2H O3 2O H3 2O H3 23 23 2O H3 2O H3 2O H3 2O H 22 222 2– –CH– –CH CO– –COO H– –O H+ +CH+ +CH CO+ +COO H+ +O H3 2+ +3 2CO3 2CO+ +CO3 2COO H3 2O H+ +O H3 2O H → +CO→ +CO3 2→ +3 2CO3 2CO→ +CO3 2CO3 2→ +3 23 23 23 2→ +3 23 23 2 +2 2+2 2

+3 2+3 2 (1)

Souring decreases the quality and value of oil and gas, and increases production costs consider-ably.6 H2S is highly toxic and very corrosive against carbon steel in certain concentration ranges.7-8 It also causes stressed corrosion cracking even against stain-less steels.7 Because of the diminishing oil reserves and increasing oil prices, enhanced oil recovery is practiced more and more often. Flooding, also known as water injection (often using seawater or produced water), is the most common method to increase well pressures. SRB can be introduced into a reservoir during flooding or they already may be present in the reservoir since geological times.9 Reservoir souring as

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CORROSION—Vol. 68, No. 11 995

a result of SRB occurs in most oil and gas fields when flooding with sulfate-containing water is practiced.10 The sulfate concentration in the injection varies depending on the source of water. Seawater, which is used often for flooding in offshore operations, typically contains 30 mmol/L sulfate.10 The Arabian seawater contains 10 mmol/L more sulfate than typical sea-water.11 Enhanced oil recovery injects large quantities of water. A large amount of H2S can be produced in a single reservoir. A maximum of 1,100 kg per day has been reported.12 The highest H2S concentration was recorded as 40,000 ppmv in the Huntington Beach oil field in California.3 Souring also occurs during hydro-testing using seawater when the system is contami-nated by SRB.

In addition to souring, SRB biofilms cause micro-biologically influenced corrosion (MIC) because the sessile SRB cells on a steel surface can couple iron oxidation with sulfate reduction to harvest energy under anaerobic conditions.13 Pitting corrosion by SRB has been blamed for pipeline failures.14 MIC was a primary suspect in the Alaskan pipeline leak in the spring of 2006.15

Nitrate injection into reservoirs often is used to promote utilization of organic carbon by nitrate-reducing bacteria to suppress sulfate reduction by SRB in soured reservoirs.10,16 Biocide treatment is another method to mitigate souring.16 It is also used in pipelines to mitigate MIC. To avoid chemical cor-rosion by biocides, non-oxidizing biocides are pre-ferred in field applications. Glutaraldehyde (C5H8O2) and tetrakis hydroxymethyl phosphonium sulfate (THPS [C8H24O12P2S]) are the two most popular bio-cides because they are broad-spectrum and readily biodegradable.17 Biocide treatment to control reser-voir souring usually requires high dosages and rather frequent periodic applications.3 During hydrotesting of pipelines, glutaraldehyde at an active concentra-tion of 50 mg/L to 75 mg/L (or 50 ppm to 75 ppm) typically is required.18 When a large amount of a bio-cide is used, its cost and environmental impact upon discharge are of serious concerns. The active biocide concentration may drop as a result of reactions with chemicals in the bulk fluid and with metal surfaces, as well as degradation.17,19-20 When the active bio-cide concentration is lower than the effective concen-tration, it cannot inhibit microbial activity because a minimum biocide concentration must be maintained. A much higher, usually 10 times higher, biocide dos-age is needed to treat sessile SRB in biofilms com-pared with that for planktonic SRB.3

It is likely that no new and more effective green biocides for large-scale oil field applications will be on the market any time soon. Therefore, it is important to develop a new biocide cocktail treatment method

for more effective use of biocides. It has been known that chelators can enhance antibiotics because of their ability to increase the permeability of the outer cell membrane of gram-negative bacteria.21 Ethylene-diaminetetraacetic acid (EDTA [C10H16N2O8]) enhanced eradication of biofilms on catheter surfaces effectively when combined with minocycline.22 EDTA is regarded to be slowly biodegradable and there are concerns about their accumulation in fresh water systems.23 Ethylene diamine disuccinate (EDDS [C10H16N2O8]) is a recommended replacement for EDTA in various appli-cations because it is readily biodegradable.24 Wen, et al., reported that 1,000 ppm EDTA or 2,000 ppm EDDS combined with 30 ppm glutaraldehyde effec-tively inhibited planktonic SRB growth in lab tests.23 It is desirable to lower the EDDS dosage and increase the biocide cocktail efficacy against SRB.

Raad, et al., found that 25% (v/v) ethanol (C2H6O) accelerated the eradication of microorganisms in bio-films on catheters when it was combined with mino-cycline (C23H27N3O7) and EDTA.25 It is reasonable to speculate that a triple biocide cocktail consisting of a biocide, a chelator, and an alcohol may be more effec-tive in the mitigation of reservoir souring and MIC caused by SRB. This work tested a triple biocide cock-tail consisting of glutaraldehyde, EDDS, and methanol (CH4O) in the mitigation of souring as a result of SRB, in the prevention of SRB biofilm establishment and in the mitigation of MIC pitting, all in the presence of sand under anaerobic conditions.

MATERIALS AND METHODS

Bacterium, Culture Media, and ChemicalsA laboratory SRB strain Desulfovibrio vulgaris

(ATCC† 7757†)(1) was used in this work. The full ATCC† 1249† medium and a modified ATCC† 1249† medium with only four ingredients (MgSO4, lactate, yeast extract, and Fe2+) at concentrations one-fourth of those in the full ATCC† 1249† medium were used for SRB growth. EDDS (a trisodium salt of EDDS) was obtained from Octel Performance Chemicals (now Innospec in Ellesmere Port, Cheshire, U.K.). Metha-nol and glutaraldehyde were obtained from Fisher Scientific (Pittsburgh, Pennsylvania). SRB culture media were autoclaved and then deoxygenated using N2 sparging. Sand was washed and autoclaved before use.

Substratum for Biofilm GrowthTests were carried out in 125 mL anaerobic vials.

Two percent of sand (i.e., 2 g sand in 98 mL medium) with a nominal particle diameter of 2.5 mm was placed at the bottom of each vial. Sand was added to provide surface areas for sessile SRB growth, which are available in rock formations in a reservoir. In MIC tests, coupons were buried under roughly 4 mm of sand, simulating an underdeposit condition. Each vial

† Trade name. (1) American Type Culture Collection (ATCC), 10801 University Blvd.,

Manassas, VA 20110.



contained 98 mL culture medium. A glove box filled with N2 provided an anaerobic environment for manip-ulation of vials. One hundred ppm cysteine was added as an oxygen scavenger to prevent accidental oxygen leaks. Disk-shaped C1018 (UNS G10180)(2) carbon steel coupons with a top disk surface area of 1.12 cm2 were polished with 200, 400, and 600 grit abrasive papers sequentially. Only the top coupon surface was exposed to the culture medium. The rest were painted with inert polytetrafluorethylene. The coupons were rinsed in isopropanol (C3H8O) and then sterilized under ultraviolet (UV) light for 15 min before use.

Measurement of Sulfate-Reducing Bacteria Cell Concentration

Viable D. vulgaris cells were easily identified under a microscope because they were motile. The viable cells were counted using a hemocytometer at 400X magnification. For each cell count data point, the planktonic cell suspension from the same vial was sampled three times and the three readings were aver-aged. This direct cell counting method has a detection limit of 5 × 104 cells/mL. Below this cell concen-tration, a most probable number (MPN) enumera-tion method is needed. In this work, the Sani-Check† Product #100 SRB test kit from Biosan Laboratories (Warren, Michigan) was used. The time for the solid culture medium in the test vial to turn black (because of iron[II] sulfide [FeS] produced by SRB) corresponds to a certain SRB cell concentration based on vendor’s calibration data.

Measurement of Sulfate ConcentrationSRB require a small-scale reduction of sulfate for

the synthesis of cellular materials such as some sul-fur-containing amino acids. This is known as assimi-latory sulfate reduction.26 The large-scale reduction of sulfate to sulfide is termed dissimilatory sul-fate reduction that is for energy production only.27 By measuring residual sulfate concentration in the medium, the percentage of reduction in sulfide pro-duction as a result of biocide treatment were calcu-lated indirectly by measuring the amount of sulfate consumed, assuming that the ratio of assimilatory sulfate reduction to dissimilatory reduction remained relatively constant for treated and untreated SRB cul-tures in this work. This method was more practical than measuring the total sulfide production. In a typi-cal SRB culture, sulfide is present as H2S gas in the headspace, as dissolved H2S and FeS. Because of its limited solubility, some FeS in the culture medium precipitates as black particles, giving a SRB culture its characteristic black color. It would be very diffi-cult to measure total sulfide production accurately by measuring sulfides in different forms.

Sulfate precipitation with barium is a common method used to measure sulfate concentration.28 Typi-cally, 1 mL medium sample was withdrawn from a 125 mL vial at a certain time. It was centrifuged and for each assay 0.1 mL sample was diluted with 0.9 mL distilled water, and then mixed with 60 mg barium chloride and 1 mL of testing reagent. Each liter of the testing reagent contained 150 g sodium chloride (NaCl), 100 mL glycerol (C3H8O3), 60 mL concentrated hydrochloric acid (HCl), 200 mL 95% ethanol (C2H6O), and balance distilled water. A spectrophotometer was used to measure the optical density of the reaction mixture at 420 nm wavelength.

Triple Biocide Cocktail Treatment to Inhibit Planktonic Sulfate-Reducing Bacteria Growth and Souring

One mL of a 3 day old SRB seed culture was used to inoculate each 125 mL vial. The initial SRB cell concentration in each vial right after inoculation was around 106 cells/mL. After adding treatment chem-icals, the vials were sealed and then incubated at 37°C. After each day, 1 mL culture medium was taken out of each vial using a syringe. Planktonic SRB con-centrations were measured. Sulfate concentration was also assayed each day. No coupons were needed for this set of tests.

Triple Biocide Cocktail Treatment to Prevent Biofilm Establishment on Coupon Surfaces

The planktonic SRB inhibition test procedure was reused. This time, two disk coupons were added into each vial. Coupons were harvested after 7 days of incubation. The preparation of coupons for scanning electron microscopy (SEM, JEOL† model JSM-6390) observation of the biofilms on the coupon surfaces fol-lowed the procedure described elsewhere.29 Because sessile cells were not evenly distributed on a coupon surface, especially for those coupons with weak bio-films, an SEM image was taken focusing on the spot with the most sessile cells. To view the pits on a cou-pon surface, the same coupon for biofilm observation under SEM was reused by removing the biofilm and Au coating using the Clark’s solution (ASTM G1-90 solution for corrosion specimen preparation).30-31 A SEM image was taken focusing on the spot with the largest pits. This spot was not necessarily the same as the spot with the most sessile cells.

RESULTS

Inhibition of Planktonic Sulfate-Reducing Bacteria Growth

Figure 1 shows that 10% methanol (CH4O) alone, 1,000 ppm EDDS + 10% methanol, and 30 ppm glu-taraldehyde + 10% methanol did not prevent plank-tonic SRB cell counts from taking off, while 25% methanol reduced the planktonic SRB cell count by

(2) UNS numbers are listed in Metals and Alloys in the Unified Num-bering System, published by the Society of Automotive Engineers (SAE International) and cosponsored by ASTM International.



test kit had to be used to enumerate the planktonic cell count because the hemocytometer method could not detect a concentration below 5 × 104 cells/mL.

FIGURE 1. Effects of methanol alone, 1,000 ppm EDDS + 10% methanol, and 30 ppm glutaraldehyde + 10% methanol on planktonic SRB growth in ATCC† 1249† medium.

FIGURE 2. Inhibition of planktonic SRB growth in (a) ATCC† 1249† medium with 2% sand, and in (b) modified (1/4 strength) ATCC† 1249† medium with 2% sand.

(a) (b)

0.4 log10 at the end of the test. Figure 2(a) shows that 30 ppm glutaraldehyde showed very little inhibition against planktonic SRB growth in the full ATCC† 1249† medium. However, when 30 ppm glutaralde-hyde was combined with 1,000 ppm EDDS, the inhi-bition effect was significant, because the planktonic SRB count after 7 days was about 2 log10 lower than that without the treatment. The corresponding P value for statistical significance was found to be 0.0016, suggesting that the inhibition effect exhibited by the binary combination after 7 days was very significant. Furthermore, Figure 2(a) shows that the triple bio -cide cocktail consisting of 30 ppm glutaraldehyde, 1,000 ppm EDDS, and 10% methanol achieved kill effect compared with the initial cell count after 7 days. It indicated that 10% methanol enhanced the binary biocide combination consisting of 30 ppm glutaralde-hyde and 1,000 ppm EDDS in the 7 day test (P < 0.0016). After 7 days, the planktonic SRB cell count was 1.3 log10 lower than its initial value, but the SRB cell count stabilized. In the 1/4 strength culture medium, the kill effect of the triple combination was more rapid and the trend continued when the test ended as shown in Figure 2(b). Figure 2 proves that 10% methanol enhanced the mitigation of planktonic SRB growth by 30 ppm glutaraldehyde combined with 1,000 ppm EDDS considerably. The data shown in Figures 1 and 2 were hemocytometer cell counts of motile SRB cells.

With the triple biocide cocktail, the planktonic SRB cell count continued to decline after 7 days. The SRB



SRB test kit data in Table 1 indicate that 30 ppm glu-taraldehyde and the binary combination of 30 ppm glutaraldehyde plus 1,000 ppm EDDS showed no inhibition effect against planktonic SRB cells after 10 days. Yet, the triple biocide cocktail consisting of 30 ppm glutaraldehyde, 1,000 ppm EDDS, and 10% methanol achieved 3 log10 kill effect in the ATCC† 1249† medium (P < 0.020) and 4 log10 kill effect in the modified ATCC† 1249† medium (P < 0.019) after 10 days.

Mitigation of SouringFigure 3(a) shows that 30 ppm glutaraldehyde

alone showed almost no effect in reducing sulfate con-sumption (P < 0.12). Wen, et al., showed that EDDS acting alone had almost no effect against D. vulgaris culture.23 When 1,000 ppm EDDS was added to 30 ppm glutaraldehyde, sulfate consumption was reduced only slightly. However, with the triple biocide cocktail consisting of 30 ppm glutaraldehyde, 1,000 ppm EDDS, and 10% methanol, sulfate con-sumption was cut by nearly half when the test ended on day 7 (P value < 0.0022). Based on the stoichio-metric molar ratio of 1:1 for sulfate consumption vs. sulfide production, this is equivalent to nearly 50% reduction in sulfide production. Figure 3(b) (with cou-pons) shows almost identical results as Figure 3(a) (without coupons). This means that the coupons in the vials did not impact sulfate consumption signifi-cantly. The maximum coupon weight loss for one cou-

pon was found to be 5 mg. Stoichiometrically, based on iron oxidation coupled with sulfate reduction, it corresponded to 0.044 mmol/L of sulfate consump-tion or a 0.44 mmol/L of sulfate consumption in a vial with two coupons. Therefore, the impact of the coupon on sulfate consumption was negligible. This means that Figure 3(b) may be considered a duplicate of Figure 3(a). Table 2 shows the cumulative sulfide production data (based on sulfate reduction measure-ments) from the 7 day data corresponding to Figure 3.

Tests in Figure 3 were conducted in the full SRB medium that was able to grow very robust SRB bio-

FIGURE 3. Residual sulfate concentrations in ATCC† 1249† medium containing 2% sand (a) without coupons and (b) two coupons in each vial.

(a) (b)

taBlE 1Planktonic Sulfate-Reducing Bacteria Cell Count Measured

Using Sulfate-Reducing Bacteria Test Kit After 10 Days’ Treatment

Cell Count Cell Count (cells/ml) (cells/ml) atCC† 1249† Modified atCC† treatment Medium 1249† Medium

No treatment ≥106 ≥106 30 ppm glutaraldehyde ≥106 ≥105 30 ppm glutaraldehyde + ≥106 ≥105 1,000 ppm EDDS 30 ppm glutaraldehyde + ≥103 ≥102 1,000 ppm EDDS + 10% methanol



films. In reality, the fluid is often less nutritious than full-strength laboratory culture media designed for optimal growth. Therefore, the triple biocide cocktail tests were repeated with the modified (1/4 strength) ATCC† 1249† medium. Only final sulfate concentra-tions after 7 days were measured because the overall sulfate reduction was much less than that in the full medium. Table 3 shows that the triple biocide cocktail treatment achieved a 69% reduction in sulfide pro-duction without coupons (or 74% with coupons). It was much more effective than the dual combination without methanol because 30 ppm glutaraldehyde combined with 1,000 ppm achieved only 51% reduc-tion without coupons (or 55% with coupons).

Prevention of Biofilm Establishment on a Coupon Surface

Figure 4 showed SEM images of coupons from 7 day tests in ATCC† 1249† medium with no treat-ment, with 30 ppm glutaraldehyde plus 1,000 ppm EDDS, and with the triple biocide cocktail consist-ing of 30 ppm glutaraldehyde, 1,000 ppm EDDS, and 10% methanol. Without treatment, numerous SRB cells were present (Figure 4[a]). Figure 4(b) shows that there were still some sessile SRB cells present, while in Figure 4(c), sessile SRB were absent, indicat-ing successful prevention of SRB biofilm establish-ment on the coupon surface with the triple biocide cocktail treatment. Similar effects were observed for tests using the modified (1/4 strength) ATCC† 1249† medium as shown in Figure 5.

Biocide Mitigation of Microbiologically Influenced Corrosion Pitting

Figure 6 shows a comparison of the effects of dif-ferent biocide treatment methods in the mitigation of MIC pitting of coupon surfaces. Disk coupon surfaces were examined using SEM to locate the largest pits. Figure 6 correlates with Figure 5 very well. In Figure

6(a), a relatively large, round-shaped pit with a sur-face diameter of roughly 14 μm is seen for the cou-pon without biocide treatment. With biocide treatment using 30 ppm glutaraldehyde and 1,000 ppm EDDS, the largest pit size (based on surface diameter) was about 5 μm as shown in Figure 6(b). With the triple biocide cocktail treatment, no obvious characteristic MIC pits are seen in Figure 6(c), which is consistent with the absence of sessile SRB cells in Figure 5(c).

DISCUSSION

In this work, 10% methanol showed considerable enhancement of glutaraldehyde and EDDS combina-tion treatment of SRB planktonic and sessile cells. All three components in the triple biocide cocktail were needed because glutaraldehyde alone, and glutaralde-hyde combined with EDDS, proved to be inadequate.

EDDS enhancement of glutaraldehyde could be explained by its removal of divalent cations such as

taBlE 2Stoichiometric Sulfide Production After 7 Days

Corresponding to Figure 3

Sulfide Reduction Production in Sulfide Without and Production With Coupon Without and treatment (mmol/l) With Coupon

No treatment 2.7 (2.6)(A) 0% (0%)(A) 30 ppm glutaraldehyde 2.7 (2.5) 0% (4%) 30 ppm glutaraldehyde + 2.4 (2.2) 11% (19%) 1,000 ppm EDDS 30 ppm glutaraldehyde + 1.4 (1.3) 48% (52%) 1,000 ppm EDDS + 10% methanol

(A) Number in brackets is with coupon.

taBlE 3Residual Sulfate Concentration and Cumulative Sulfate Consumption After 7 Days in Modified

(1/4 Strength) ATCC† 1249† Medium With 2% Sand Without Coupon. Initial [SO42–] = 5.9 mmol/L(A)

total Sulfate Stoichiometric Consumption/ Sulfide Reduction Residual [SO4

2–] Production in Sulfide treatment (mmol/l) (mmol/l) Production

No treatment 1.4 (1.7)(A) 0.45 (0.42)(A) 0% (0%)(A) 30 ppm glutaraldehyde 2.2 (2.3) 0.37 (0.36) 18% (14%) 30 ppm glutaraldehyde + 3.7 (4.0) 0.22 (0.19) 51% (55%) 1,000 ppm EDDS 30 ppm glutaraldehyde + 4.5 (4.8) 0.14 (0.11) 69% (74%) 1,000 ppm EDDS + 10% methanol

(A) Data in brackets were obtained for the same conditions, except with two coupons in each vial.



Mg2+ and Ca2+ from lipopolysaccharide in cell mem-branes, resulting in increased permeability for glu-taraldehyde through the outer cell membrane.21,23,29 EDDS alone showed almost no effect against SRB.23,29 Piet and Rossmoore demonstrated that monocop-per citrate (II) (MCC), also a chelator, was not able to

inhibit microorganisms, but it had a synergistic effect with antimicrobial agents against Pseudomonas aeru-ginosa.32

Chelators are already used as treatment chemi-cals in oilfield applications. To prevent the precipi-tation of solids including iron hydroxide and iron sulfide, chelators are frequently added to acidic stim-

FIGURE 4. SEM images for 7 day coupons in ATCC† 1249† medium containing 2% sand with (a) no biocide treatment, (b) with 30 ppm glutaraldehyde + 1,000 ppm EDDS, and (c) with 30 ppm glutaraldehyde + 1,000 ppm EDDS + 10% methanol. Arrows indicate SRB cells. Scale bars for the small inserted images are 500 μm.

FIGURE 5. Same as Figure 4, except the medium was modified (1/4 strength) ATCC† 1249† medium. Scale bars for the small inserted images are 500 μm.

(a)(a)

(b) (b)

(c)(c)



ulation fluids. In addition, chelating agents are often used as scale removers. Frenier, et al., report that 3% tetrasodium EDTA was applied in acidizing formula-tions.33 Abdul-Latif, et al., used 20,000 ppm disodium EDTA to achieve a complete scale removal from tubes of heat exchangers in the cooling system of a refin-ery.34 To remove the H2S from biogas, 0.4 M Fe/EDTA was applied.35 EDDS, a readily biodegradable chelator, is a recommended replacement for EDTA in industrial applications because EDTA is accumulating in fresh water systems as a result of its relatively low biodeg-radation rate.24 It is reasonable to believe that the use of 1,000 ppm EDDS is not a prohibitively high con-centration for field applications.

Ethanol is a denaturant that has been used widely as a disinfectant because it can kill bacteria by denaturing proteins and dissolving the lipids in the cell membranes or cell walls of microorganisms.36 Ethanol also can enhance the permeability of the cells.37 Methanol likely has similar effects as ethanol. As a matter of fact, methanol, ethanol, and isopropa-nol showed almost identical effects in our lab tests (data not shown). In oilfield applications, methanol is more practical than ethanol because methanol is the most widely used and common hydrate inhibitor to inhibit gas hydrate in oil and gas industries.38-39 A significant amount of methanol was used to prevent hydrate formation when temperature and pressure conditions in the field become more severe. The concen tration of methanol applied often exceeds 60 wt%.40 Methanol already is used also as a winteriz-ing agent for oil wells.41 With the help of EDDS and methanol, glutaraldehyde achieved better inhibition and kill effects against planktonic and sessile SRB.

This work tested a triple biocide cocktail consist-ing of 30 ppm glutaraldehyde, 1,000 ppm EDDS, and 10% methanol. In actual applications, the dosages of the three components are expected to vary based on local nutritional and microbiological conditions. For heavily contaminated situations and for well-estab-lished biofilms, a higher glutaraldehyde dosage is expected. However, a large saving of glutaraldehyde is likely when using the triple combination instead of using glutaraldehyde alone.

The enhanced biocide efficacy results in this work should be viewed relative to the unenhanced biocide efficacy results rather than the desired complete kill of SRB in the field, because of the interference by organic carbons in the culture medium. Ten percent methanol may be too high for batch treatment in large-scale applications, but plug-flow treatment may be adopted if possible such as in pipelines. This method is not suitable if methanol is utilized by the local bac-teria as a carbon source and the triple combination cocktail fails to kill or suppress their growth. When methanol is already being used as a hydrate inhibitor or antifreeze in the field, the enhancement of metha-nol will come without the extra cost for methanol.

In conclusion, the experimental data in this work indicated that the triple biocide cocktail consisting of 30 ppm glutaraldehyde, 1,000 ppm EDDS, plus 10% methanol was considerably more effective than glutar-

FIGURE 6. SEM images of bare coupon surfaces. Coupons were obtained after a 7 day test in modified (1/4 strength) ATCC† 1249† medium with (a) no treatment, (b) with 30 ppm glutaraldehyde + 1,000 ppm EDDS, and (c) 30 ppm glutaraldehyde + 1,000 ppm EDDS + 10% methanol. Scale bars for the small inserted images are 100 μm.

(a)

(b)

(c)



aldehyde alone and the combination of glutaraldehyde and EDDS in the inhibition of planktonic SRB growth, souring, biofilm establishment and MIC pitting of car-bon steel. In all these cases, the methanol enhance-ment of the biocide treatment was clearly observed. The data presented here were obtained from lab test-ing against a pure strain of D. vulgaris in anaerobic vials. Further laboratory testing using mixed-culture microbial consortia isolated from oil fields is desired before field trials.

ACKNOWLEDGMENTS

This work was supported by a grant from the M.D. Anderson Cancer Center in Houston, TX.

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biocide cocktail consisting of glutaraldehyde, ethylene ...132.235.17.4/paper-gu/2012 biocide...

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