Article pubs.acs.org/EF
Bacterial Inhibition of Methane Clathrate Hydrates Formed in a Stirred Autoclave Iwan Townson,† Virginia K. Walker,‡ John A. Ripmeester,§ and Peter Englezos*,† †
Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Department of Biology, Queen’s University, Kingston, Ontario K7L 3N6, Canada § Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, Ontario K1A 0R6, Canada ‡
ABSTRACT: The potential for hydrate inhibition in a stirred autoclave at a subcooling of 2.3 K has been tested on two bacterial isolates. Chryseobacterium sp. C14 survives multiple freeze−thaw events and inhibits ice recrystallization, and Escherichia coli has neither of these properties but is a biofilm producer. Both strains showed methane hydrate inhibition, with a significant reduction in total hydrate formed. Chryseobacterium delayed hydrate nucleating time, to a similar level found for the commercial kinetic inhibitor, polyvinylpyrrolidone (PVP; 0.2 wt %). However, in the presence of E. coli, in comparison to PVP, the time to nucleation was almost tripled and the hydrate growth rate was reduced by half. Because of the variation inherent with the microbial samples, likely a result of the complexities associated with varying cell numbers and metabolic activity, numerous experiments were required. Analysis of the distinct hydrate growth profiles shown by the two bacterial strains indicated that different molecular sources were likely responsible for the observed inhibition. As a working hypothesis, it is suggested that the inhibition observed by Chryseobacterium cultures was partially due to ice recrystallization inhibition properties, while that of E. coli may be due to secreted macromolecules and the effect of biofilm.
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INTRODUCTION Clathrate hydrates are crystals composed of water and a guest molecular species. Many hydrocarbons (e.g. methane, ethane, propane, and butane) that are commonly found in oil and gas pipelines/processes can form hydrates if the minimum water cut, temperature, and pressure conditions are met. Hydrate prevention and control is a serious economic burden to oil and gas producers.1 The scale of the problem is growing as pipelines increasingly are used in deeper waters and colder regions, where hydrate formation is more favorable.2 One of the most common techniques for hydrate prevention is to shift conditions in pipelines out of the hydrate formation region by the injection of colligative additives, such as methanol or glycol. However, this method requires up to 60 vol % of inhibitor, thus introducing expensive, downstream treatments as well as furthering environmental concerns, because of the toxicity associated with the inhibitors. Over the last 20 years, development of low-dosage hydrate inhibitors (LDHIs) that require less than 2 wt % to be effective have gained popularity.3 LDHIs do not significantly shift the hydrate phase equilibrium but rather interfere with hydrate growth by delaying the onset of hydrate nucleation (the induction time) or reducing the tendency for hydrate particles to agglomerate, thus affecting the growth rate and/or total hydrate conversion. Although different LDHIs may be used, there is a gap in the market for new inhibitors that are both environmentally benign and inexpensive.3,4 A subclass of LDHIs, kinetic hydrate inhibitors (KHIs), delay the onset of hydrate formation, but the means by which they achieve this is not yet fully understood. There is debate as to whether KHIs adsorb to form hydrate crystals, mask nucleation sites, or even if binding is responsible for their inhibition mechanism at all.5 © 2012 American Chemical Society
The KHI polyvinylpyrrolidone (PVP) has been routinely used as a model and a benchmark for screening new inhibitors.6−8 Although the understanding of KHI-mediated inhibition is unclear, historically, it has been thought to be similar to the antifreeze protein (AFP) inhibition of ice crystals.9,10 Perhaps ironically, however, the inhibition of ice by AFPs that are found naturally in some species of cold-hardy organisms is also under debate. Nevertheless, a “flat” face on the AFP appears to complement an ice crystal plane, resulting in a growing ice front that is curved and, therefore, thermodynamically unfavorable, as described by the Kelvin effect.11,12 Although ice and gas hydrates have distinct structures, some AFPs have been reported to show stronger hydrate inhibition than PVP.6−8,10,13 AFP binding and incorporation into growing tetrahydrofuran (THF) hydrates has been shown by tagging AFPs with green fluorescent protein (GFP).4 Interestingly, GFP, a protein that does not bind ice and was not incorporated into THF hydrate crystals, nevertheless showed low inhibition of natural gas hydrates.6 Mutant fish and plant AFPs with no activity toward ice showed THF hydrate inhibition.13,14 Furthermore, melting points and hydrate heterogeneity in the presence of commercial KHIs and AFPs have been shown to be clearly distinct.7,15,16 Taken together, these observations suggest that the mechanism for hydrate inhibition is distinct from ice inhibition and that the mechanism differs between classes of inhibitors. Although AFPs have shown strong hydrate inhibition, the associated cost of protein purification prohibits the application of AFPs as KHIs in industry. One option is to screen ice-active Received: June 26, 2012 Revised: November 12, 2012 Published: November 12, 2012 7170
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Table 1. Average Induction Time and Hydrate Growth Characteristics total hydrates formed, NT
number of runs label water TSB Chryseobacterium + TSB E. coli + TSB PVP
system
fresh
memory
induction time, ti (h)
growth rate, RG (mol min−1)
(mol)
(%)
2 3 1
4 3 5
0.4 7.1 30.7
0.00027 0.00040 0.00072
0.119 0.286 0.274
9 22 21
1 4
3 2
118.5 38.5
0.00007 0.00016
0.284 0.407
22 32
distilled water 0.5 wt % TSB aqueous Chryseobacterium in 0.5 wt % TSB aqueous, A625 nm = 0.83 E. coli in 0.5 wt % TSB aqueous, A625 nm = 0.31 0.2 wt % PVP aqueous
bacteria for hydrate inhibition, to find a cheaper alternative to protein purification.17 The cell wall material of microbial communities indigenous to seabed methane deposits have been shown to inhibit hydrate formation.18 Chryseobacterium sp. C14 was recovered as the most freeze−thaw-resistant isolate from a high-altitude soil community.19 This isolate showed ice recrystallization inhibition (IRI), a property also shared by AFPs. Although the putative protein has not been purified, this isolate showed some inhibition of THF hydrate crystals.20 IRI has been associated with biofilms and polymeric substances,21 and thus, Escherichia coli, which produces biofilm, was also tested. The objective of the work presented herein was to test selected bacteria, Chryseobacterium and E. coli, as hydrate inhibitors. Questions about the suitability of THF hydrate to reliably assess KHIs have been previously raised.22 THF is toxic to bacteria, and one Pseudomonas bacterial strain that appeared to inhibit THF hydrate showed no activity when tested with natural gas hydrate.23 Thus, an important criteria was that the strains would be tested using a traditional industry-standard inhibition assay with an autoclave containing methane as the hydrocarbon guest.
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curve, 20 min after the induction time. Hydrate conversion, NT, was taken as the total amount of moles of gas consumed after the hydrate growth curve plateaued. For the hydrate growth profiles, time zero represents the induction time of the trial. Repeats were performed on both fresh solutions and solutions that were recrystallized (memory runs). Prior to experimentation, the CR was cleaned (with ethanol and distilled water) and purged 3 times with methane (at 2−3 MPa) and the temperature was reduced to 273.8 K. Subsequently, the CR was charged to 2.6 MPa with methane and allowed to settle for 15 min before again being charged to the operating conditions of 3.5 MPa. Stirring was initiated 15 min later, marking the start of the experiment. After hydrates had formed and the gas consumption had reached a plateau, the experiment was stopped and the system was slowly vented to dissociate the hydrates. As indicated, recrystallization experiments were only started 4 h after complete dissociation and venting. Hydrate dissociation experiments were performed by recording the pressure at which the last crystal could be seen in the autoclave. For the water (no additive system), we recorded this pressure to be 2700 kPa and estimated an error (including the pressure gauge, thermocouples, and human error of judging the point when the last crystal dissociates to be ±100 kPa). This corresponds with the CSMGem software27 prediction of 2770 kPa. All experiment sets that included additives were also checked for THI properties but yielded the same dissociation pressure of 2700 (±100) kPa. Bacterial Preparations. Chryseobacterium sp. strain C14 and E. coli TG-2 were cultured on 0.5 wt % tryptic soy broth (TSB; Becton, Dickinson) agar plates. Both of these microorganisms are classified as risk group one and are, therefore, not human pathogens. A single colony was used to inoculate 0.5 wt % TSB medium, which was incubated at 300 K for 5−7 days. Subsequently, the cultures were transferred to 277 K for 5−7 days,19 prior to estimating the cell concentration using optical density (OD; 625 nm) and placing in the CR. It should be noted that both of these strains can be kept for several months at 300 K without the addition of fresh medium and remain viable. For experiments without the TSB growth media, Chryseobacterium and E. coli were grown on TSB plates, as above, but bacterial colonies were picked and added to water (10 mL) until OD625 was approximately 1. The bacterial solutions were added to 110 mL of sterile distilled water in the CR. All equipment and solutions (prior to bacterial inoculation) were sterilized at 394 K and 0.1 MPa for 20 min.
EXPERIMENTAL SECTION
Apparatus and Materials. All experiments were performed in the semi-batch apparatus described elsewhere,24,25 using ultra-high-puritygrade methane (Praxair Technology) as the hydrate guest. Hydrates were formed in aqueous solution (140 mL) in a 323 mL SS316 stainless-steel crystallizer (CR), fitted with two Plexiglas windows. The CR was stirred using a magnetic stirring bar coupled with an external stirrer shaft. A baffle inside the vessel reduced vortex formation to keep the gas/liquid interfacial surface more consistent while stirring.25 Distilled and deionized water was sterilized prior to use in all microbiological trials. PVP (average molecular weight of ∼10 kDa; Sigma-Aldrich) was used at 0.2 and 0.5 wt %. Because there were problems with stirring when using the more viscous solution, the lower PVP concentration was routinely used. Induction times and hydrate growth characteristics for both PVP concentrations were similar.26 The pressure in the crystallizer was kept constant through a PID control valve at a pressure of 5−7 MPa. A pressure drop in the supply vessel indicated gas consumption in the CR because of hydrate formation. Pressure measurements were converted to moles using equations described elsewhere.27 Procedure. All experiments were performed at 273.8 (±0.2) K and 3500 (±50) kPa (subcooling of 2.3 K) and agitated at a constant stirrer speed of 330 (±25) rpm. A low driving force was chosen to observe weak inhibition as well as to give more consistent induction times and well-understood hydrate growth characteristics of the pure water solutions (not shown). Once the solution to be tested was placed in the CR, gas consumption was recorded to determine hydrate nucleation, growth rate, and total crystal conversion. The induction time, ti, was taken as the time from starting the stirrer to the time of nucleation as judged by the exotherm and a sudden increase in gas consumption.24,25 The initial hydrate growth rate, RG, was taken to be the average of the gradient over a 1 h range of the gas consumption
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RESULTS AND DISCUSSION The potential for the bacteria to inhibit methane hydrates was initially tested with cultures and compared to PVP, 0.5 wt % TSB medium, and water controls. The medium was chosen for the cultures because it is suitable for high-density growth of both bacterial species. In addition, although it contains potential thermodynamic hydrate inhibitors in the form of peptides, salts, and carbohydrates, at a dilution of 0.5 wt %, the effect was calculated as almost negligible (e.g., 0.5 wt % NaCl shifts the methane hydrate phase boundary by less than 50 kPa; using phase equilibrium software CSMHYD27). In the course of determining the appropriate experimental conditions, another 7171
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As indicated by the standard deviations, induction times in the presence of both bacterial cultures were not routinely predictable. Although changes to the CR and water impurities could impact induction times, these would have been seen with the PVP addition. Consequently, we attribute the observed bacterial sample variation to the complexities inherent with the use of living systems, for example, changes in medium composition parallel bacterial growth, which, in turn, results in changes in cell number and metabolic products, such as polypeptides, polysaccharides, and lipids, some of which could impact hydrate nucleation. As well, it would be important for future experiments to optimize bacterial concentrations. Hydrate Growth. As well as induction times, hydrate growth characteristics and the total amount of hydrate formed were also significantly altered for all of the samples when compared to the water controls (Figures 2 and 4). Remarkably,
27 experiments were performed (not shown) in addition to the 28 reported here. Hydrate Induction. As expected, water solutions gave consistently short induction times (Table 1), averaging 0.4 h (standard deviation; s = 0.05 h). Hydrate formation was delayed an average of 7.1 h (s = 4.2 h) in the presence of culture medium (0.5 wt % TSB). PVP and Chryseobacterium cultures showed modest inhibition with average induction times of 38.3 h (s = 8.8 h) and 30.7 h (s = 29.1 h), respectively. Even though the cell density was higher in the Chryseobacterium cultures than those of E. coli (Table 1), of all tested samples, E. coli cultures showed the longest delay (118.5 h; s = 59.4 h) for hydrate formation (Figure 1 and Table 1).
Figure 1. Average induction times (bars) of the individual runs (dots) within a particular experimental solution set. The sample size (n) of each set is shown, and error bars are shown as ±1 standard deviation from the mean.
Figure 2. Average initial hydrate growth (bars) of individual runs (dots) within an experimental solution set. The sample size (n) of each set is shown, and error bars are given as ±1 standard deviation from the mean.
With the conditions chosen and with a 4 h gap between hydrate dissociation and recrystallization, only a small memory effect (manifested as a shorter induction time) was observed in the case of water, where the induction time was 0.4 and 0.3 h, respectively. In the presence of inhibitors, we observed the following. The average induction time for four fresh solutions was 37.8 h (26.9−43.7 h) and 33 h (30.8 and 35.2 h) for two memory PVP solutions. These observations are consistent with previous reports in the literature that, in pure water systems and solutions with chemical inhibitors, the nucleation or induction time observed for the solution with memory of hydrate formation is shorter. The same trend was observed with the TSB media. The average induction time for three fresh solutions was 8.9 h, and that for three recrystallizations was 5.2 h. On the other hand, the observations with the Chryseobacterium and E. coli with TSB were not so conclusive. The induction time for one fresh Chryseobacterium + TSB system was found to be 19.9 h. The five memory solutions had induction times ranging from 1.2 to 69.8 h, with an average of 32.8 h. The induction time for one fresh E. coli system was 164.9 h, and the average from three recrystallization runs was 103 h (33.4, 122.7, and 152.8 h). Investigation of the memory effect was not an objective of the present work, but clearly, our understanding is not complete.
the highest initial growth rate after induction (7.2 × 10−4 mol min−1) was shown by Chryseobacterium cultures, a rate more than double that shown by the water controls (2.7 × 10−4 mol min−1; Figure 2). In contrast, E. coli cultures showed promise as hydrate inhibitors, with a growth rate that was half of that of PVP (0.7 × 10−5 mol min−1 versus 1.6 × 10−4 mol min−1; Table 1). Significantly, although the rate of hydrate growth was slow in the presence of PVP, the total amount of hydrate formed was 1.45-fold more (0.407 mol of gas consumed) than in the presence of the culture medium and the two bacterial cultures (at about 0.280 mol; Figure 3). Previously, PVP has been reported to delay hydrate nucleation while promoting overall conversion to hydrate.28 In fact, PVP solutions appeared to form a thick hydrate slurry in the CR (not shown). In contrast, all other samples, including the bacterial cultures, agglomerated, forming a hydrate cap between the gas−liquid interface. This cap is believed to reduce the mass transfer of the guest gas into the water, thus, reducing hydrate growth.28 The hydrate cap in the presence of the TSB culture medium appeared to have trapped bubbles throughout the hydrate layer. We speculate that these bubbles may have made the hydrate cap more porous, allowing for increased hydrate conversion and a correspondingly greater observed gas consumption, albeit still less than that seen for the PVP samples (Table 1). 7172
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Figure 3. Average total hydrate accumulation (bars) of individual runs (dots) within a particular experimental solution set. The sample size (n) of each set is shown, and error bars are given as ±1 standard deviation from the mean. Figure 5. (Top) Hydrate growth profiles of Chryseobacterium cultures from the first fresh run (F) to the last memory run (M5) and (below) corresponding solution temperatures.
The overall hydrate growth profile for water, medium, and the two bacterial strains are shown in Figure 4. As indicated,
hydrate nucleators or inducers under different circumstances.29 It should be noted that purified AFPs demonstrated effective yet complex behavior when tested as inhibitors of natural gas hydrates.16 For the E. coli cultures, nucleation and the appearance of hydrate crystals was significantly delayed in comparison to all of the other samples. The crystals were observed to first grow above the interface and later throughout the solution to form a cap. Unlike Chryseobacterium, E. coli does not inhibit ice recrystallization. However, E. coli produces extracellular polymeric substances (EPS), and this accumulates to form biofilm,21,30 a protective polymer matrix containing water channels. The sigmoidal curves characteristic of E. coli growth profiles were similar to those seen when Xe hydrates were grown on ice surfaces, prompting the suggestion that, although some nucleating preclusters occurred quickly, a subsequent dense layer impeded guest access and, thus, delayed growth.31 Although the effect of biofilm on hydrate formation has yet to be explored, such a partial barrier at the gas−liquid interface could explain the initial reduction in the hydrate growth rate as the hydrates cross from above the interface to the bulk solution. The inhibition strength of the E. coli solution may also be a result of the biofilm covering heterogeneous nuclei, which has been suggested as a possible biological hydrate inhibition mechanism.13 In contrast, Chryseobacterium cultures did not show a S-shape growth profile and do not appear to form biofilm.21 It should be noted too that some biological inhibitors can alter the hydrate growth to give an initial low growth rate region followed by higher growth,8 which would result in a Sshaped growth profile. Thus, it is always possible that, apart from the biofilm, other unidentified biological hydrate inhibitors could be responsible for the significant inhibition seen here. Hydrate Inhibition Independent of Culture Medium. Because the presence of the TSB culture medium showed some impact on hydrate growth and accumulation, bacteria were recovered directly from agar plates and placed in water. These preparations were referred to as extracted bacteria. The inhibition of nucleation seen with the bacterial extracts was more modest than the corresponding bacterial cultures (Figure 6 versus Figure 1). However, the concentrations of the
Figure 4. Typical hydrate growth profiles showing gas uptake over time in water, culture medium, and the two bacterial cultures (Chryseobacterium and E. coli).
Chryseobacterium cultures increased the rate of hydrate growth, so that the growth profile quickly plateaued at the same time as the water controls. The growth profiles changed, however, as the samples were recrystallized (Figure 5), with the slope of the gas uptake curve increasing after the first fresh run (F) and the first recrystallization run (M2), with subsequent experiments showing consistent profiles. The crystallization mediated by the Chryseobacterium cultures also increased the temperature of the solution by over 1 K (Figure 5), consistent with the rapid rate of hydrate formation. These observations are in contrast to the results with other samples that gave consistent growth profiles for each trial (not shown), resulting in no noticeable temperature change, because of the low driving force selected for all experiments. It is not known why Chryseobacterium cultures displayed such idiosyncratic behavior. It is possible that these cells have dual properties of hydrate promotion and inhibition but that the molecules conferring hydrate inhibition, such as molecules associated with IRI, are more labile. Indeed, it is also possible that the same molecules could serve as 7173
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than that found in the presence of the commercial hydrate inhibitor. The distinctive S-shaped hydrate growth profile could be a result of the biofilm formation of E. coli at the gas−liquid interface, which could also be at least partly responsible for the observed delay in the induction time. At present, we can only speculate on the nature of the inhibiting molecules, although the observed differences between these two Gram-negative bacteria may rule out some possibilities, including the cell membrane. However, considering the abundance of species of bacteria, it is remarkable that these two species chosen for this research gave such strong inhibition. In all likelihood, stronger hydrate-inhibiting bacteria exist. Further research will hopefully validate these results in other systems and will identify sources of the microbial hydrate inhibition, so that such samples can be manipulated to improve hydrate inhibition consistency.
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AUTHOR INFORMATION
Corresponding Author
Figure 6. Induction times of individual runs (dots) and the corresponding averages (bars) of five different bacterial extracts prepared by direct recovery from plates and added to water. The first exposure to hydrates (F) is represented by the green dot, with the last memory run shown by the red dot.
*Telephone: +1-604-822-6184. Fax: +1-604-822-6003. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and the NSERC Strategic Grants Program for financial support. We also thank anonymous reviewers for their suggestions.
extracted bacteria were also significantly less than the tested bacterial cultures (not shown). For these extracted bacteria experiments, hydrate growth profiles were not dramatically different than the water controls (0.4 h),26 likely because of the result of the low concentrations used. However, in the absence of the TSB medium, there was a clearer time dependency upon inhibition strength and the solution hydrate history (from the first fresh run to the last memory run; Figure 6). Similar to the bacterial cultures, the bacterial extracts showed delayed average induction times, with the most highly concentrated E. coli extract (solution 2; Figure 6) showing the greatest delay. After a certain number of recrystallization events, however, the induction times were reduced as if the longer time in the CR in the absence of culture media was detrimental to the bacteria. The first fresh run also generally showed lower induction times than subsequent runs, likely because bacteria prepared in this way were not pretreated at low temperatures. Bacteria express specific cold-adaptive molecules, including cold-shock proteins, after being exposed to low temperatures or ice.19,32 Therefore, we speculate that the optimal delay in hydrate induction time shown by the bacterial extracts was at a midpoint because of the necessity to express low-temperature-adaptive molecules and prior to cell death.
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REFERENCES
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CONCLUSION The freeze−thaw-resistant Chryseobacterium sp. C14 and the biofilm-forming E. coli both delayed the formation of methane hydrates in a stirred tank CR for a longer period than the water (no additive) solutions. An experimental complexity was the high variation for the bacterial-mediated delay in induction times compared to the simpler single-component solutions, likely because of the nature of the living system, for both cell viability and metabolism. Because Chryseobacterium inhibits ice recrystallization, it is tempting to attribute the delay in hydrate induction time to this property because certain AFPs are known hydrate inhibitors. Of the two bacterial strains, however, E. coli shows the greatest promise as a hydrate inhibitor. E. coli cultures mediated a delay in hydrate nucleation that was 3 times that of PVP, a calculated growth rate that was half of that of PVP, and a total hydrate formation that was significantly less 7174
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