Electrochemical Testing of Biocide Enhancement ... - ACS Publications

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Electrochemical Testing of Biocide Enhancement by a Mixture of D‑Amino Acids for the Prevention of a Corrosive Biofilm Consortium on Carbon Steel Ru Jia,† Dongqing Yang,† Hussain H. Al-Mahamedh,‡ and Tingyue Gu*,† †

Department of Chemical and Biomolecular Engineering, Institute for Corrosion and Multiphase Technology, Ohio University, Athens, Ohio 45701, United States ‡ Saudi Basic Industries Corporation, Jubail Industrial City 31961, Saudi Arabia ABSTRACT: Problematic biofilms cause microbiologically influenced corrosion (MIC) and biofouling in many industries such as the oil and gas industry, water utilities, and the power generation industry. Equipment failures cause not only economic losses but also environmental damages. A high biocide concentration is required to treat mixed-culture biofilms due to their various defense mechanisms. A biocide enhancer can reduce the dosage or make a biocide more effective. In this work, an equimolar mixture with 100 ppm (w/w) of four D-amino acids (D-tyrosine, D-methionine, D-leucine, and D-tryptophan) labeled as D-mix enhanced 60 ppm alkyldimethylbenzylammonium chloride (ADBAC) against a field biofilm consortium on C1018 carbon steel coupons by achieving at least an extra 2-log reduction of sessile cell counts in the biofilm prevention test. Scanning electron microscopy images, confocal laser scanning microscopy images, weight loss, and pitting data all corroborated the electrochemical tests.

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INTRODUCTION In natural environments, microbes often live in biofilm communities.1 Biofilms protect sessile cells with extracellular polymeric substance (EPS). In drinking water distribution systems, biofilm formation causes microbial contamination and corrosion of pipes.2 This corrosion is known as microbiologically influenced corrosion (MIC) or biocorrosion, which was first discovered more than a century ago.3 The corrosion is initiated and accelerated by microbes.4 It is more prevalent nowadays because of the aging equipment and increased awareness.5 More than 20% of annual global metal corrosion costs is caused by MIC.6 MIC is a major problem in the oil and gas industry.7 The 2006 Trans-Alaska Pipeline leak was likely due to MIC. It caused major economic losses and raised concerns over environmental damages.8 Even the U.S. Navy faces MIC problems caused by marine biofilms.9,10 In anaerobic MIC, electrons released by iron oxidation can be absorbed by an electron acceptor (oxidant) such as sulfate, nitrate, and protons.11 However, biocatalysis is required for the reduction of some of these oxidants.11 Sulfate-reducing bacteria (SRB) are commonly studied corrosive microbes.12−14 It was found that an SRB biofilm on carbon steel was more corrosive under carbon starvation because SRB could use iron as an electron donor for energy production.15 In this SRB corrosion, a biofilm carries out cross-cell-wall electron transfer to shuttle extracellular electrons released by iron oxidation for sulfate reduction in the cytoplasm.12 Planktonic cells do not perform cross-cell-wall electron transfer because electrons cannot freely © XXXX American Chemical Society

move in water; thus, they do not directly contribute to this type of MIC.15 Another important type of MIC is caused by secreted corrosive metabolites such as organic acids.15 Underneath a biofilm, the local pH can be much lower than that in the bulk fluid. Protons are reduced on the metal surface without biocatalysis. Although a biofilm is not needed for biocatalysis of proton reduction, it is required to achieve locally high acidity underneath the biofilm. It is clear that biofilms are the culprits in both types of MIC.16 Thus, MIC mitigation must treat biofilms.17 Biofilms defend inner sessile cells against harmful attacks using the following mechanisms. First, a biofilm is a diffusion barrier for antimicrobial agents.18 Second, biofilms can slow down the metabolic rate to minimize the intake of antimicrobial agents.19 Third, biofilms can form persister cells that can survive much better than other sessile cells in the community. They quickly rebound when the antimicrobial stress is removed.20 Fourth, biofilms can upregulate resistant genes to counter antimicrobials.21 Finally, sessile cells can utilize efflux pumps to pump out toxic chemicals in the cells.20 These defense mechanisms make it far more difficult to mitigate biofilm consortia than planktonic cells, requiring much higher biocide concentrations to treat sessile cells than planktonic cells.22 Received: Revised: Accepted: Published: A

April 12, 2017 June 1, 2017 June 13, 2017 June 13, 2017 DOI: 10.1021/acs.iecr.7b01534 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

Stericup (Millipore, Bedford, MA, USA). Liquid solutions were sparged with filtered nitrogen gas for 1 h to remove dissolved oxygen. Biofilm Prevention Test. C1018 carbon steel (UNS G10180) coupons with a top exposed surface area of 1 cm2 were used to grow biofilms. The composition of carbon steel was (wt %): C 0.14−0.20, Mn 0.60−0.90, P 0.04, S 0.05, Si 0.15−0.30, and Fe balance. Only the top surface was exposed to the culture medium. The other surfaces were coated with inert Teflon. Coupons were abraded with 180, 400, and 600 grit abrasive papers, sequentially. Then, they were cleaned with 100% isopropanol and dried under UV light. An anaerobic chamber was sparged with filtered nitrogen gas for 1 h to remove dissolved oxygen before use. In the biofilm prevention test, five coupons, 100 mL of ATCC 1249 culture medium, and 1 mL of biofilm consortium seed culture were placed into each 125 mL anaerobic vial with and without treatment chemicals in the nitrogen-filled anaerobic chamber. The initial planktonic cell concentration right after inoculation was 106 cells/mL. The vials were sealed and incubated without shaking in a 37 °C incubator. After 7 days, coupons were taken out for biofilm and corrosion analyses. The test conditions are listed in Table 1.

High concentrations of biocides are not favored because of discharge problems in view of tightening environmental regulations. In addition to MIC, biofilms also cause biofouling in many industries.23−25 In cooling systems, biofouling leads to the loss of efficiency in heat exchanger tubes.26 In water utilities, membrane biofouling leads to high energy consumption and high operating costs.27 In the field, pigging and biocides are main tools to mitigate biofilms.28 The two are sometimes combined. However, not all pipelines are piggable because of elbows and bends.29 Quaternary ammonium compounds (QAC) are commonly used as biocides in many industries to mitigate MIC30 and biofouling.31 Alkyldimethylbenzylammonium chloride (ADBAC) is a cationic membrane-active QAC, which is active against a variety of microbes.32 Repeated uses of the same biocide may lead to resistant microbes over time. Thus, more effective biocide treatments are desired to combat problematic field biofilms. Since only a small number of biocides are suitable for largescale applications and new biocides are not easy to come by, it is desirable to enhance the existing biocides. D-Amino acids are naturally occurring chemicals that have been found to disperse bacterial biofilms. They probably work by incorporating in the peptidoglycan molecules in the cell walls, thus inhibiting peptidoglycan biosynthesis and interfering with the remodeling of the cell wall to affect the biofilm formation.33,34 D-Tyrosine (D-tyr), D-methionine (D-met), D-leucine (D-leu), and D-tryptophan (D-trp) were found to disperse Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa biofilms.35 Xu et al.36,37 found that 1 ppm (w/w) D-tyr and 100 ppm D-met individually enhanced the efficacy of 50 ppm tetrakis hydroxymethyl phosphonium sulfate (THPS) against the Desulfovibrio vulgaris biofilm (an SRB biofilm) on carbon steel coupons and achieved better efficacies than 100 ppm THPS alone treatment. Jia et al.38 showed that 1 ppm D-tyr and 50 ppm D-met individually enhanced 10 ppm ADBAC against the D. vulgaris biofilm on carbon steel, achieving better efficacies than the 30 ppm ADBAC alone treatment. It was also found that individual D-amino acids had limited effect in enhancing THPS against field biofilm consortia. A mixture of D-amino acids was required probably because different bacteria in a biofilm consortium responded to different D-amino acids.29 In this work, a mixture of four D-amino acids containing equimolar amounts of D-tyr, D-met, D-trp, and D-leu (labeled as D-mix) was evaluated as a biocide enhancer for ADBAC against a field biofilm consortium on C1018 carbon steel.

Table 1. Test Conditions for the Biofilm Prevention Test parameter biofilm culture medium treatment method concentration temperature incubation duration coupon

value/condition Consortium II ATCC 1249 medium with and without treatment chemicals ADBAC, D-mix, ADBAC + D-mix 60 ppm ADBAC, 100 ppm D-mix 37 °C 7 days C1018 carbon steel

Sessile Cell Count and Biofilm Observation. The modified Postgate’s B (MPB) for SRB, standard bacterial nutrient broth for general heterotrophic bacteria (GHB), and phenol red dextrose (PRD) for acid-producing bacteria (APB) were used to enumerate sessile cells by the most probable number (MPN) method. The MPN liquid culture media were purchased from Biotechnology Solutions (Houston, TX, USA). After 7 days of incubation, coupons with biofilms were taken out from the anaerobic vials and put in a 10 mL of pH 7.4 PBS solution. The biofilm on the coupon surface was scraped off using a small brush applicator. The coupon, the applicator, and the 10 mL of PBS solution were put in a 50 mL test tube and vortexed for 30 s to distribute sessile cells evenly in the solution. Then, the solution was serially diluted in the assay vials and incubated at 37 °C. This MPN cell counting process was repeated twice. Coupons were examined by scanning electron microscopy (SEM) using a Model JSM-6390 SEM (JEOL, Tokyo, Japan). The detailed procedures of sample preparation for SEM were described elsewhere.38 Confocal laser scanning microscopy (CLSM) with a Model LSM 510 microscope (Carl Zeiss, Jena, Germany) was used to detect live and dead cells in biofilms. The information on dyes and the staining process were mentioned in a previous work.38 Weight Loss and Pit Observation. Each weight loss data point was obtained from at least 4 coupons. Corrosion products and biofilms on coupon surfaces were removed and cleaned using the Clark’s solution according to ASTM G1−03.39 After weight loss measurements, coupons were used to observe pit morphology under the SEM. Maximum pit depth for each

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MATERIALS AND METHODS Bacteria and Chemicals. A corrosive field biofilm consortium from an oil and gas field labeled as Consortium II was used in this work. Its metagenomics data showed that the biofilm consortium contained SRB, biodegradation microbes, and fermentative microbes.29 The biofilm consortium was cultured in ATCC 1249 medium.29 Then, 100 ppm L-cysteine was added to the culture medium as an oxygen scavenger. Microbes were grown in 125 mL anaerobic vials (Wheaton Industries Inc., Millville, NJ, USA). ADBAC was purchased from MP Biomedicals (Aurora, OH, USA). D-Amino acids were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). The culture medium, pipet tips, 125 mL anaerobic vials, vial septa and caps, and tweezers were sterilized in an autoclave at 121 °C for 20 min. Solutions of ADBAC and D-amino acids were sterilized through a 0.22 μm B

DOI: 10.1021/acs.iecr.7b01534 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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incubated at 37 °C. Linear polarization resistance (LPR) was scanned at a rate of 0.1667 mV/s in the range of −10 to +10 mV versus the open circuit potential (OCP). Electrochemical impedance spectroscopy (EIS) data were measured at stable OCP with a 10 mV sinusoidal voltage signal in the 10−2−105 Hz frequency range. ZSimDemo version 3.30d software (EChem Software, Ann Arbor, MI, USA) was used to analyze the EIS data. Potentiodynamic polarization curves were obtained in the −250 to +250 mV (vs the OCP) voltage range. Corrosion current densities (icorr), corrosion potentials (Ecorr), and anodic and cathodic Tafel slopes (βα and βc) were calculated from Tafel analyses of the polarization curves.

coupon was observed using an infinite focus microscope (IFM) (Model ALC13, Alicona Imaging GmbH, Graz, Austria). Electrochemical Measurements. Electrochemical tests were performed in a 450 mL glass cell using a potentiostat VersaSTAT 3 (Princeton Applied Research, Oak Ridge, TN, USA) with a saturated calomel electrode (SCE) as a reference electrode and a platinum plate (10 mm × 10 mm × 1 mm) as a counter electrode. The glass cell was filled with 350 mL of the deoxygenated ATCC 1249 culture medium. It was sealed with a 2.44 in. diameter rubber stopper in the anaerobic chamber and

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RESULTS Sessile Cell Count. Figure 1 shows the sessile cell counts on the coupons after the 7-day biofilm prevention test with different treatments. The sessile cell counts on the no treatment control coupon were 2.7 × 107 cells/cm2 for SRB, 7.6 × 106 cells/cm2 for APB, and 7.6 × 106 cells/cm2 for GHB in the 7-day biofilm prevention test. Treatment with 100 ppm D-mix alone did not show any log reduction of sessile cell counts compared with the no treatment control. Treatment with 60 ppm ADBAC alone achieved 1-log reduction in SRB, APB, and GHB sessile cell counts compared with that of the no treatment control. The cocktail of 60 ppm ADBAC + 100 ppm D-mix achieved an extra 2-log reduction of SRB sessile cell count and extra 3-log reductions of APB and GHB sessile cell count compared with those of 60 ppm ADBAC alone treatment. Biofilm Observation. The SEM image in Figure 2A shows different shapes of cells, confirming that it was a mixed

Figure 1. Sessile cell counts for the biofilm prevention test after the 7 days of incubation. (Error bars represent standard deviations.).

Figure 2. SEM images of biofilm Consortium II on C1018 after 7 days of incubation in the biofilm prevention test with (A) no treatment, (B) 100 ppm D-mix, (C) 60 ppm ADBAC, and (D) 60 ppm ADBAC + 100 ppm D-mix. (Scale bars in the small inserted images are 50 μm.) C


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Figure 3. CLSM images of biofilm Consortium II after 7 days of incubation in the biofilm prevention test: (A) no treatment, (B) 100 ppm D-mix, (C) 60 ppm ADBAC, and (D) 60 ppm ADBAC + 100 ppm D-mix.

culture biofilm. The sessile cells on the no treatment control coupon (Figure 2A) were abundant after the 7-day biofilm prevention test. When 100 ppm D-mix alone was used (Figure 2B), the sessile cells were as abundant as on the control coupon. This means that the D-mix treatment alone did not prevent the attachment of sessile cells. Sessile cells were also easily found on the coupon treated with 60 ppm ADBAC alone (Figure 2C). However, with the cocktail of 60 ppm ADBAC + 100 ppm D-mix (Figure 2D), the number of sessile cells were much less compared with the other conditions. The results were consistent with the sessile cell enumeration in Figure 1. The data in this work clearly indicate a synergy between the biocide and D-amino acids. It confirms that for a recalcitrant biofilm consortium a biocide stress is needed for D-amino acids to work.29 CLSM images in Figure 3 corroborate the SEM images in Figure 2 very well. Figure 3A indicates that live cells (green dots) were abundant on the untreated coupon surface after the 7-day biofilm prevention test. With the treatment of D-mix alone, live cells were fewer but still plentiful as shown in Figure 3B. Figure 3C also shows a lot of cells when 60 ppm ADBAC alone was used, but some of them were dead cells (red dots). With the cocktail treatment of 60 ppm ADBAC + 100 ppm D-mix (Figure 3D), dead cells were abundant while live cells were not. The CLSM results were generally consistent with the sessile cell counts. Both SEM and CLSM are valuable

Figure 4. Weight losses of coupons (bars) and pH values (circles) of the culture media at the end of the 7-day biofilm prevention test.

for biofilm observations because SEM tells cell morphology while CLSM shows live and dead cells. Weight Loss. Figure 4 shows the specific weight loss data after the 7-day biofilm prevention test. The average weight loss after 7 days of incubation for the abiotic control, the no treatment control, the 100 ppm D-mix alone treatment, the 60 ppm ADBAC alone treatment, and the treatment using 60 ppm ADBAC + 100 ppm D-mix were 0.3, 11.0, 11.4, 2.8, and 1.9 mg/cm2, D


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Figure 5. SEM pit images on coupon surfaces after 7 days of incubation in the biofilm prevention test: (A) abiotic control, (B) no treatment, (C) 100 ppm D-mix, (D) 60 ppm ADBAC, and (E) 60 ppm ADBAC + 100 ppm D-mix. (Scale bars in the small inserted images are 50 μm.)

where Wuninh is the weight loss of the no treatment control and W represents the weight loss with treatment. The ηp values calculated from the average weight losses for the treatments of 100 ppm D-mix alone, 60 ppm ADBAC alone, and 60 ppm ADBAC + 100 ppm D-mix are 0%, 74.5%, and 82.7%, respectively. From these efficiency values, the calculated synergism parameter is 1.47 based on the formula used by Hegazy et al.40 This value is greater than unity, suggesting the synergistic effect of the cocktail treatment. Pit Observation. Figure 5 shows the images of pits on the coupon surfaces of the abiotic control, no treatment control, 100 ppm D-mix, 60 ppm ADBAC, and 60 ppm ADBAC + 100 ppm D-mix after 7 days of incubation. Figure 5A shows that the chemicals had negligible corrosion in the abiotic medium in the anaerobic condition. In contrast, large pits were found on the no treatment control coupon surface (Figure 5B). With 100 ppm D-mix alone treatment, the pit surface diameter was

respectively. The weight loss of the abiotic control shows that chemicals had a very minor effect to weight loss in an anaerobic condition. A similar weight loss was obtained with 100 ppm D-mix treated alone compared to the no treatment control. With a biocide in the medium, lower weight losses were observed compared with the no treatment control. Particularly, the combination of 60 ppm ADBAC + 100 ppm D-mix resulted in a lower weight loss compared with the 60 ppm ADBAC alone treatment. The results showed the D-amino acids enhanced ADBAC’s biofilm mitigation and resulted in less corrosion. The pH values in the media (Figure 4) for all treatments after 7 days of incubation were all above 6.5. With this kind of pH, the acid attack contribution was negligible. The corrosion inhibition efficiency or corrosion protection efficiency (ηp) was calculated from the following equation:40 ⎛W − W⎞ ηp = ⎜ uninh ⎟ × 100% ⎝ Wuninh ⎠

(1) E


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Figure 6. IFM pit depth profile of coupons after 7 days of incubation in the biofilm prevention test: (A) abiotic control, (B) no treatment, (C) 100 ppm D-mix, (D) 60 ppm ADBAC, and (E) 60 ppm ADBAC + 100 ppm D-mix.

smaller compared with that of the no treatment control. However, the former still did not have a reduced density on the coupon surface. The combination of 60 ppm ADBAC + 100 ppm D-mix (Figure 5E) led to fewer and smaller pits compared with those of 60 ppm ADBAC alone treatment (Figure 5D). The pit images were consistent with weight loss data. The maximum pit depth data after 7 days of incubation with different treatments are shown in Figure 6. Figure 6A confirms the Figure 5A SEM surface image, indicating that the abiotic control coupon did not exhibit any pitting. The no treatment control after 7 days of incubation led to a maximum pit depth of 48.3 μm. With 100 ppm D-mix alone treatment, the maximum pit depth was 23.9 μm. The maximum pit depth was reduced to 9.7 μm with the 60 ppm ADBAC alone treatment. It was further reduced to 4.7 μm with the 60 ppm ADBAC + 100 ppm

D-mix treatment. The pit depth profiles from IFM in Figure 6 are consistent with the SEM pit morphologies in Figure 5. Pit depths are usually more important than weight losses in MIC corrosion because MIC failures are typically due to pinhole leaks.8 In 7-day MIC lab tests against carbon steels under a strictly anaerobic condition, pure-strain SRB biofilms usually led to maximum pit depths of 5−10 μm in literature reports.15 In this work, biofilm Consortium II achieved 48.3 μm, suggesting that it was a much more corrosive biofilm. Electrochemical Measurements. Figure 7A shows the variation of OCP vs time of coupons in the ATCC 1249 medium under different treatments during 7 days of incubation. It can be seen that at the very beginning (t = 0), all treatments gave a similar OCP. However, after only 1.5 h of incubation, the OCP values of the no treatment control and the 100 ppm

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EIS was measured under the stable OCP for different treatments on days 1, 4, and 7 during 7 days of incubation. The Nyquist and Bode plots are shown in Figure 8. The Nyquist plot diameter in the abiotic control was significantly higher than those of the inoculated media. The Nyquist plot diameters of the no treatment control and the 100 ppm D-mix alone treatment were smaller than those of the other biocide treatments, suggestion less corrosion with the biocide treatments. On day 1 (Figure 8A), the Nyquist plot diameter in the treatment of 60 ppm ADBAC + 100 ppm D-mix was similar to that in the 60 ppm ADBAC alone treatment. This means that the two treatments had similar corrosion rates because the diameter corresponded to the corrosion resistance. On days 4 and 7, the impedance in the treatment of 60 ppm ADBAC + 100 ppm D-mix was higher than that in the 60 ppm ADBAC alone treatment. The impedance spectra of coupons with different treatments were analyzed using equivalent electrical circuits.45 The one time constant model and the two time constant model (Figure 9) were used to fit the impedance spectra. In the equivalent electrical circuits, Rs is the solution resistance. Qdl and Rct represent the double layer capacitance and the charge transfer resistance, respectively. Qb and Rb stand for the capacitance and the resistance of the biofilm or the corrosion product film, respectively. The EIS fitting results of Rct + Rb values with time are shown in Figure 10. The Rct + Rb value is closely related to the corrosion rate. A lower Rct + Rb value means a higher corrosion rate.46 Rct + Rb values for the no treatment control and the D-mix alone treatment were much lower than those involving ADBAC. The treatment of 60 ppm ADBAC + 100 ppm D-mix showed higher Rct + Rb values than those in the 60 ppm ADBAC alone treatment. Results here also show that the addition of D-amino acids to the ADBAC treatment increased the charge transfer resistance thus decreasing the corrosion rate. Figure 11 shows the Tafel plots of all cases at the end of 7 days of incubation. The electrochemical parameters (corrosion potential, corrosion current density, cathodic Tafel slope, and anodic Tafel slope) are listed in Table 2. The abiotic control showed a very low corrosion current density. The icorr of the no treatment control and the 100 ppm D-mix alone treatment were much higher than those of the treatments of 60 ppm ADBAC alone and 60 ppm ADBAC + 100 ppm D-mix. The icorr was smaller with the treatment of 60 ppm ADBAC + 100 ppm D-mix compared with the 60 ppm ADBAC alone treatment. The Ecorr of the treatments of 60 ppm ADBAC alone and 60 ppm ADBAC + 100 ppm D-mix underwent a positive shift compared with those of the no treatment control and the 100 ppm D-mix alone treatment. This was probably due to the inhibition effect of the anodic oxidation reaction.47 The βa values of the abiotic control and the cocktail of 60 ppm ADBAC + 100 ppm D-mix were much smaller than those of the no treatment control, the 100 ppm D-mix alone treatment, and the 60 ppm ADBAC alone treatment. The potentiodynamic polarization curve measurements here are consistent with the EIS results and weight loss data above.

Figure 7. Variations of OCP (A) and LPR (B) for different treatments vs time during 7 days of incubation in the biofilm prevention test. D-mix

alone treatment shifted to the negative direction and then shifted to the positive direction after 5 h of incubation. At 1.5 h after incubation, the culture media in the no treatment control glass cell and the 100 ppm D-mix treated glass cell turned black, indicating SRB growth. At the same time, the culture media in the 60 ppm ADBAC and the 60 ppm ADBAC + 100 ppm D-mix treated glass cells showed only a slight tint of black color, suggesting much less growth. After 1 day of incubation, the OCP values of the no treatment control and the 100 ppm D-mix alone treatment changed little and remained steady during 7 days of incubation. With 60 ppm ADBAC and 60 ppm ADBAC + 100 ppm D-mix, the OCP values shifted to the positive direction after the 1.5 h incubation and then shifted to the negative direction during the first day of incubation. After 1 day of incubation, the OCP values slightly shifted to the positive direction during 7 days of incubation. The positive shift could be due to the growth of sessile cells and formation of corrosion products.41,42 The OCP shifted to the positive direction in the presence of biofilm Consortium II compared with the abiotic control. Similar results of the positive shift in the presence of biofilms were reported before.43,44 The polarization resistance (Rp) measured from LPR are shown in Figure 7B. A larger Rp value means less corrosion. The polarization resistances were similar between the no treatment control and the 100 ppm D-mix alone treatment. Polarization resistances involving ADBAC were higher than those of the no treatment control and the D-mix alone treatment, suggesting decreased corrosion. The treatment of 60 ppm ADBAC + 100 ppm D-mix led to a slightly higher polarization resistance than the treatment of 60 ppm ADBAC alone. Not surprisingly, the highest polarization resistance was for the abiotic control in the medium. The LPR polarization resistance data corroborated with weight loss data, SEM pit images, and IFM pit depths.

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DISCUSSION In the field, biofilms always bounce back after a biocide treatment because either the system is not completely sterilized or microbes are reintroduced by fluid flow. Therefore, repeated biocide treatment cycles are needed.48 This raises many issues including environmental concerns and increased cost. G


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Figure 8. Nyquist and Bode plots for the samples during 7 days of incubation in the biofilm prevention test: (A, A′) 1 day, (B, B′) 4 days, and (C, C′) 7 days.

Figure 9. Equivalent circuits used for simulating the impedance spectra. Figure 10. Time-dependent changes of Rct + Rb of samples during 7 days of incubation in the biofilm prevention test. (Error bars represent standard deviations.)

D-Amino

acids can work as biocide enhancers to enhance biocides against sessile cells. Here, the combination of 60 ppm ADBAC + 100 ppm D-mix achieved 2 extra log (102) SRB sessile cell reduction and 3 extra log (103) APB and GHB sessile cell reduction compared with those of the 60 ppm ADBAC

alone treatment. A larger log reduction of sessile cells after each treatment is desired because microbes will bounce back H


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against a field biofilm consortium on C1018 carbon steel. The cocktail of 60 ppm ADBAC + 100 ppm D-mix achieved at least an extra 2-log reduction of sessile cell counts compared with the 60 ppm ADBAC alone treatment. The cocktail of 60 ppm ADBAC + 100 ppm D-mix also led to lower weight loss and less severe pitting corrosion. Electrochemical measurements confirmed these data. The experimental data clearly demonstrated the synergetic effect of the cocktail against the biofilm consortium. This work demonstrated that properly conducted electrochemical tests could be used to assess biocide effects in real time.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 740-593-1499. Fax: 740-593-0837. ORCID

Figure 11. Tafel plots obtained at the end of 7 days of incubation in the biofilm prevention test.

Tingyue Gu: 0000-0002-4208-210X Notes

more slowly.38 Thus, D-amino acids can slow down biocide dosage escalation.36 Lower biocide dosage is obviously better for the environment. In this work, the combination of ADBAC and D-mix showed a synergetic effect against biofilm Consortium II. D-mix alone did not show significant efficacy because biofilm Consortium II was recalcitrant. Therefore, a biocide stress was necessary. Individual D-amino acids were not tested here because it was reported that individual D-amino acids (e.g., D-tyr and D-met) at a high concentration had limited effect in the enhancement of THPS in the prevention of field biofilm consortia.29 These four D-amino acids were chosen to make the D-mix based on the work of Kolodkin-Gal et al.35 It is possible that specific biofilms require specific D-amino acids for best efficacy due to microbial diversity. Currently, the mechanisms of why D-amino acids disperse biofilms are still not fully elucidated. It is hypothesized that D-amino acids can replace the D-alanine terminus in the peptidoglycan molecules of bacterial cell walls, which triggers biofilm disassembly.35 Xu et al. confirmed that high concentration addition of D-alanine in the medium could hinder the effect of 37 D-met from enhancing THPS against the D. vulgaris biofilm. Lam et al. found that D-amino acids could modulate the synthesis of the peptidoglycan by incorporating into it and thus influencing the composition and strength of the peptidoglycan.34 It was also suggested that D-amino acids regulated the remodeling of bacteria cell walls.33 Leiman et al. showed that D-amino acids inhibited growth and expression of biofilm matrix genes leading to inhibition of biofilm formation.49 Regardless of dispersal mechanisms by D-amino acids, the use of D-amino acids as biocide enhancers seems to be promising technology, especially in view of D-amino acids being green chemicals and that they are even present in some protein-rich food products.50

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from TOTAL and SABIC. REFERENCES

(1) Marsh, P. D. Dental plaque: biological significance of a biofilm and community life-style. J. Clin. Periodontol. 2005, 32, 7−15. (2) Liu, S.; Gunawan, C.; Barraud, N.; Rice, S. A.; Harry, E. J.; Amal, R. Understanding, monitoring, and controlling biofilm growth in drinking water distribution systems. Environ. Sci. Technol. 2016, 50, 8954−8976. (3) Gaines, H. A. Bacterial activity as a corrosion induced in the soil. J. Ind. Eng. Chem. 1910, 2, 128−130. (4) Coetser, S. E.; Cloete, T. E. Biofouling and biocorrosion in industrial water systems. Crit. Rev. Microbiol. 2005, 31, 213−232. (5) Skovhus, T. L.; Eckert, R. B. Practical aspects of MIC detection, monitoring and management in the oil and gas industry. CORROSION 2014, NACE International, San Antonio, TX, 2014; Paper No. C2014-3920. (6) AlAbbas, F. M.; Williamson, C.; Bhola, S. M.; Spear, J. R.; Olson, D. L.; Mishra, B.; Kakpovbia, A. E. Influence of sulfate reducing bacterial biofilm on corrosion behavior of low-alloy, high-strength steel (API-5L X80). Int. Biodeterior. Biodegrad. 2013, 78, 34−42. (7) Liu, H.; Gu, T.; Zhang, G.; Wang, W.; Dong, S.; Cheng, Y.; Liu, H. Corrosion inhibition of carbon steel in CO2-containing oilfield produced water in the presence of iron-oxidizing bacteria and inhibitors. Corros. Sci. 2016, 105, 149−160. (8) Jacobson, G. A. Corrosion at Prudhoe Bay: A lesson on the line. Mater. Perform. 2007, 46, 26−34. (9) Wikieł, A. J.; Datsenko, I.; Vera, M.; Sand, W. Impact of Desulfovibrio alaskensis biofilms on corrosion behaviour of carbon steel in marine environment. Bioelectrochemistry 2014, 97, 52−60. (10) Liang, R.; Aktas, D. F.; Aydin, E.; Bonifay, V.; Sunner, J.; Suflita, J. M. Anaerobic biodegradation of alternative fuels and associated biocorrosion of carbon steel in marine environments. Environ. Sci. Technol. 2016, 50, 4844−4853. (11) Gu, T. New understandings of biocorrosion mechanisms and their classifications. J. Microb. Biochem. Technol. 2012, 4, 3−6.

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CONCLUSIONS This work shows that a mixture of four D-amino acids containing equimolar D-tyr, D-met, D-trp, and D-leu enhanced ADBAC

Table 2. Electrochemical Parameters Fitted from the Potentiodynamic Polarization Curves for Different Treatments at the End of 7 Days of Incubation treatment abiotic control no treatment 100 ppm D-mix 60 ppm ADBAC 60 ppm ADBAC + 100 ppm D-mix

icorr (μA/cm2) 0.5 19.8 17.6 4.0 1.8

± ± ± ± ±

Ecorr (V vs SCE) −0.739 −0.714 −0.746 −0.616 −0.599

0.1 1.6 1.8 0.8 0.4 I

± ± ± ± ±

0.008 0.007 0.008 0.006 0.005

βa (mV/dec) 54 225 248 206 85

± ± ± ± ±

6 17 15 8 7

βc (mV/dec) −139 −63 −65 −235 −246

± ± ± ± ±

9 5 4 13 12


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