Inhibition of Gas Hydrate Nucleation and Growth: Efficacy of an

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Inhibition of Gas Hydrate Nucleation and Growth: Efficacy of an Antifreeze Protein from the Longhorn Beetle Rhagium mordax Christine Malmos Perfeldt,† Pei Cheng Chua,‡ Nagu Daraboina,† Dennis Friis,§ Erlend Kristiansen,§ Hans Ramløv,§ John M. Woodley,# Malcolm A. Kelland,‡ and Nicolas von Solms*,† †

Center for Energy Resources Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltoft Plads, Building 229, DK-2800 Kgs., Lyngby, Denmark # Center for Process Engineering and Technology (PROCESS), Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltoft Plads, Building 229, DK-2800 Kgs., Lyngby, Denmark ‡ Department of Mathematics and Natural Science, Faculty of Science and Technology, University of Stavanger, N-4036 Stavanger, Norway § Department of Science, Systems and Models, Roskilde University, Universitetsvej 1, P.O. Box 260, DK-4000 Roskilde, Denmark S Supporting Information *

ABSTRACT: Antifreeze proteins (AFPs) are characterized by their ability to protect organisms from subfreezing temperatures by preventing tiny ice crystals in solution from growing as the solution is cooled below its freezing temperature. This inhibition of ice growth is called antifreeze activity, and in particular, certain insect AFPs show very high antifreeze activity. Recent studies have shown AFPs to be promising candidates as green and environmentally benign inhibitors for gas hydrate formation. Here we show that an insect antifreeze protein from the longhorn beetle, Rhagium mordax (RmAFP1), the most potent protein yet found for freezing inhibition, can inhibit methane hydrates as effectively as the synthetic polymeric inhibitor polyvinylpyrrolidone (PVP). In high pressure rocking cell experiments, onset hydrate nucleation temperatures and growth profiles showed repeatable results. RmAFP1 clearly showed inhibition of hydrates compared to amino acids (L-valine and L-threonine) and the protein bovine serum albumin (BSA). This indicates that proteins or amino acids do not generally inhibit hydrate formation. The promising performance of RmAFP1 as a new green kinetic hydrate inhibitor could further the development and increased production of green hydrate inhibitors.

1. INTRODUCTION Gas hydrates are crystalline, icelike solid compounds of small gas molecules and water which form at low temperature and high pressure. In oil and gas production, formation of gas hydrates can cause pipeline blockage with subsequent loss of production and possible safety concerns.1,2 To prevent hydrate formation and ensure the flow of hydrocarbons (flow assurance), the energy industry generally uses so-called thermodynamic inhibitors (THIs), such as methanol and glycols, in high dosage concentrations (20−50 wt %).2 By adding THIs the hydrate phase boundary is shifted toward lower temperatures, meaning lower temperatures will be required to form hydrates for a given operating pressure. Due to the amounts of THIs needed, operational issues (e.g., storage, transportation, and handling) and environmental issues connected to toxicity and flammability lead to high operating (OPEX) and capital expenditure (CAPEX).1,3 An alternative class of chemicals, kinetic hydrate inhibitors (KHIs), have been developed which can be applied in much lower concentrations (0.1−1.0 wt %).1,4 Often KHIs are watersoluble polymeric compounds that prevent or delay hydrate formation. Despite the success of these compounds at very low concentrations, their poor biodegradability means that they are not certified for use in all regions (for example the North Sea) which has prompted a search for new environmentally friendly hydrate inhibitors.5,6 AFPs are a group of proteins that protect © 2014 American Chemical Society

organisms at subfreezing temperatures by preventing ice crystals from growing upon cooling below the freezing point. This prevention of ice growth is called antifreeze activity and is defined as the difference between the melting temperature and the freezing temperature of a solution. This thermal hysteresis can readily be measured using a nanoliter osmometer. The adsorption inhibition mechanism of antifreeze proteins is a noncolligative phenomenon where AFPs are irreversibly adsorbed on the ice crystal.7 However, the exact mechanism is not well understood, and different explanations are discussed in the literature.8,9 Ice and gas hydrates have different although related structures, and it appears that the inhibition mechanism toward ice and hydrate formation is different. In ice, AFPs allow the formation of nuclei, to which they bind, after which growth is inhibited. In hydrates the nucleation itself is inhibited.10 AFPs are expressed in a variety of organisms, such as bacteria, fungi, plants, fish and insects, where the reported activities vary from about 0.3 °C in bacteria to 5−7 °C in insects.11,12 Recent studies have shown that antifreeze proteins (AFPs) may have potential as kinetic hydrate inhibitors while at the same time being environmentally benign.10,13−21 Zeng et al.17 found that Pleuronectes americanus (winter flounder) antifreeze protein Received: February 7, 2014 Revised: April 9, 2014 Published: May 7, 2014 3666

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Thermal stability of proteins is the equilibrium between the native form and an ensemble of the nonnative form also called the unfolded form.29 For AFPs the thermal stability has been observed to vary for different organisms. Chao et al.30 reported for the type I antifreeze protein (AFP9) from winter flounder a melting temperature estimated to be 18 °C. Li et al.31 found for the beetle, Dendroides canadensis, that the thermal stability was approximately 84 °C. The melting temperature of RmAFP1 from R. mordax was recently determined by Friis et al.29 to be 28.5 °C. However, thermal stability can be affected by the composition of the buffer.32 The thermal stability of RmAFP1 might affect its performance as hydrate inhibitor. In order to avoid the influence of RmAFP1 unfolding during hydrate inhibition experiments the temperature range in this study was chosen to be well below the melting point of RmAFP1. Various techniques have been applied to investigate hydrate nucleation in the presence of inhibitors. These include highpressure differential scanning calorimetry,10,13,33−35 stirred tank,6 NMR,21,22 Raman14 and PXRD.36 In order to simulate more realistic hydrate nucleation scenarios a so-called highpressure rocking cell is increasingly being used by the oil industry and gas hydrate research laboratories.37,38 The rocking of the cell and movement of the ball inside the cell creates shear forces and turbulence which better simulate conditions in the pipelines. In addition, hydrate nucleation is a stochastic process which leads to scattered results making evaluation of the strength of these inhibitors difficult. To improve repeatability, a significant number of experiments under the same experimental conditions are preferred. Rocking cells with multiple units can more rapidly provide statistically meaningful results for inhibition activity of hydrate formation37,38 and are thus increasingly being adopted in the research community for evaluating kinetic inhibition activity. We used rocking cells with constant cooling experiments, and these cells allowed us to reliably measure hydrate nucleation and growth data in the presence of inhibitors. In this study we report repeatable results for both the hydrate nucleation and growth showing that the AFP from R. mordax performs as effectively as PVP to inhibit methane hydrate formation under realistic conditions. The goal of this study was to investigate how effective an AFP from R. mordax is as a green KHI compared to a commercial synthetic inhibitor. Furthermore, the amino acids essential to inhibit the ice growth and a nonantifreeze protein were studied in order to examine if the amino acids themselves perform as inhibitors or if the structure of AFPs is unique for this application.

increases the induction time of THF hydrate formation more than PVP (available as a commercial inhibitor). Zeng et al.22 also found that Choristoneura fumiferana (an insect AFP) is more effective than PVP in inhibiting THF hydrate formation. Gordienko et al.20 have observed that antifreeze proteins modify THF hydrate structure by adhering to the hydrate surface a way similar to PVP. Jensen et al.15 studied the influence of type III HPLC12 (originally identified in ocean pout) and ISP type III found in meal worm (Tenebrio molitor) on methane hydrate formation and reported that low concentrations of these proteins are more effective in reducing hydrate growth than PVP. Daraboina et al.6,13,14,21 used a multiscale approach to investigate the influence of two fish AFPs: type I AFP (AFP-I; average molecular weight, 3.3−4.5 kDa) and type III AFP (AFP-III; Swiss Protein Database accession number P19414; average molecular weight, 7 kDa) on methane/ethane/propane gas hydrate formation and decomposition. They reported that these proteins show significant inhibition activity with less structural and compositional complexity during the dissociation process. Jensen et al.,16 also showed that type III AFP showed good hydrate inhibition capability compared to PVCap. From all these results it was evident that AFPs from different species had the ability to inhibit gas hydrate formation. Furthermore, many studies were only indicative and qualitative or comparative and were not performed at conditions close to those encountered in natural gas production (such as studies on THF hydrate). The search for active antifreeze proteins continues in order to develop strong environmentally benign alternatives to commercial kinetic inhibitors. Recently, the longhorn beetle Rhagium mordax was shown to exhibit high antifreeze activity which during the cold season may be in excess of 8 °C.23 In the AFPs of R. mordax (RmAFP1), several repeats of a motif rich in the amino acid threonine (TxTxTxT motifs) is suspected to make up the ice-binding site.24 Antifreeze activity measured for mutations of the four threonine residues in fish AFP-I replaced with serine showed no antifreeze activity. However, replacement of the threonine hydroxyl-groups with methyl-groups (threonine to valine) reduced the antifreeze activity to 30−50% of the AFP-I.25−27 This indicates that the loss of the methyl group is more detrimental to the antifreeze activity than the loss of the hydroxyl group. The ice crystal growth of valine mutations are observed to cause slow crystal growth27 or bipyrimidal growth, whereas for serine mutations the crystals were growing freely.26 This indicates that the hydrophobic methyl group could be involved in water exclusion at the adsorption site of the protein. This means that the hydrophobic methyl group in threonine could be important in the antifreeze inhibition mechanism of ice.9,25 However, the slow growth of valine mutations suggests that the hydroxyl groups of threonine residues in fish AFPs (AFP-I) could be essential to make the antifreeze protein−ice interaction permanent.9 However, no data were available as to how effective this hyperactive protein from longhorn beetle R. mordax is on the gas hydrate formation. Hydrophobic amino acids such as KHIs have also been recently studied28 for CO2 gas hydrate inhibition. Glycine showed KHI performance comparable to that of PVP in fresh water, and the increasing length of the amino acid alkyl side chain showed decreasing inhibition. However, amino acids as KHIs for methane gas hydrate inhibition have not yet been investigated.

2. EXPERIMENTAL SECTION 2.1. Materials. In all the experiments methane gas and 50 mM NaCl in Milli-Q water as aqueous phase was used, which will form structure I hydrates. The methane gas was supplied from AGA (purity 99.5%). The synthetic kinetic hydrate inhibitors were two polyvinylpyrrolidones (PVP; MW 10,000 and MW 40,000) supplied from Sigma-Aldrich. The recombinant antifreeze protein (RmAFP1) originates from the bark beetle R. mordax. The genetic engineering of the gene and the expression of the protein are described in Kristiansen et al.24 Supernatant from cell lysate corresponding to 2 L original cell culture (around 30 mL), was applied to a HisTrap FF column with a column volume (CV) of 5 mL (GE Healthcare). The column was washed with 4 CV resuspension buffer (50 mM NaCl, 10 mM imidazole, 25 mM Tris-HCl, pH 8), before eluting by applying a gradient of imidazole 10−200 mM in 40 min at a flow rate of 2 mL/ min. The fraction containing the RmAFP1 was dialyzed against 500 volumes of deionized water for at least 24 h with change of water once 3667

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experiments conducted resulted in an equilibrium temperature of 12.9 ± 0.10 °C. We used the WinRC software (PSL Systemtechnik) to program the experiments and to log the temperature and pressure for each cell and the temperature for the cooling bath throughout the experiment. Two cycles were set up for each experiment in order to perform fresh and memory experiments. A typical pressure−temperature−time recording for one cell is shown in Figure 2. The cells were pressurized with

or twice during this time. The dialyzed product was lyophilized and stored at −18 °C until use. Upon use, the lyophilized RmAFP1 was dissolved in 50 mM NaCl in Milli-Q water. Concentration of the RmAFP1 was determined by amino acid analysis (AAA) (Department of Systems Biology, DTU, Denmark) to 2770 ppm. The RmAFP1 has a molecular weight of 13.9 kDa. The two amino acids, L-threonine (Thr, purity ≥98%) and L-valine (Val, purity ≥98%) supplied from Sigma-Aldrich and a nonantifreeze protein bovine serum albumin (BSA; MW 68 kDa; purity ≥98%) supplied from Sigma-Aldrich, were used as controls. 2.2. Apparatus. The hydrate nucleation experiments were conducted in a Rocking Cell produced by PSL Systemtechnik (see Figure 1). The Rocking Cell has five test cells placed on a cell drive in

Figure 1. Rocking cell (PSL Systemtechnik. (A) Rocking cell setup consisting of the cooling bath with the five cells and the high pressure panel. (B) One of the test cells.

Figure 2. Temperature and pressure recorded during the constant cooling test of PVP10 in 50 mM NaCl-Milli-Q. Two cycles are recorded where the first one is the fresh solution test and the second is the memory solution test. The red solid line indicates the pressure decrease as the temperature decreases. The measured pressure will deviate from the red solid line as hydrates nucleate. This is indicated by a black arrow pointing at the onset temperature (To).

the cooling bath. Each cell has a working pressure up to 200 bar. The temperature is adjusted by the cooling bath in the range −20 °C to +60 °C. The detailed experimental setup and procedure is explained elsewhere.37 Briefly, the apparatus consists of stainless steel test cells with a volume of 40 mL and has a stainless steel ball that freely rolls over the entire length of the cell when it is tilted. The cells can rock from an angle of −45° to +45°. When the cell is rocked, the ball inside the cell rolls along the length of the cell thereby mixing the contents. The movement of the ball creates shear forces and turbulence in the cell, better reproducing conditions in the pipeline. 2.3. Procedures. Measurements of the onset of hydrate nucleation temperatures by constant cooling experiments were studied using a rocking cell apparatus. The aim was to obtain the onset temperature difference in delay of hydrate nucleation for synthetic and biological KHIs compared to a noninhibited system in order to investigate effectiveness of AFP as an inhibitor. The sample from the first formation is then used for the second hydrate formation (memory solution) in order to obtain more repeatable nucleation temperatures. The inhibitors were tested at a concentration of 2770 ppm and at 1385 ppm. First the cell was loaded with 10 mL of test liquid. To eliminate air in the cells, they were evacuated using a vacuum pump, pressurizing with 2−3 bar of methane gas, rocking the cells, and finally using the vacuum pump again. The system without inhibitor was pressurized to 95 bar at the initial experimental temperature of 20.5 °C, and the constant cooling/heating cycle was from 20.5 to 2 °C to 20.5 °C. The baseline experiments without inhibitor were only performed with fresh solution. The KHI experiments were pressurized to 95 bar at the initial experimental temperature of 13 °C and the constant cooling/heating cycle was from 13 to 2 °C to 14 °C in order to maintain the stability of the protein while fully melting the hydrates in the cells. The equilibrium temperature for the water−methane system at 95 bar was calculated using CSMGem software to 12.5 °C. The equilibrium temperature for the methane−water system at 95 bar was determined by first forming hydrates and then heating very slowly (0.25 °C/h) from a temperature of 2 °C to a temperature about 2 °C higher than the expected equilibrium temperature. The intersection of the two curves obtained from the cooling and heating steps gives the dissociation point. This is a standard laboratory technique.39 The

methane to 95 bar and set to rock at a rate of 20 rocks/min at an angle of 40° and to cool at a constant rate of 0.1 °C/min from 13 to 2 °C. During cooling the pressure decreases at a constant rate as the temperature decreases until hydrate nucleation, where the pressure drops faster due to hydrate formation. The temperature at which hydrate nucleates is called the onset temperature, To (see Figure 2). The cells were then heated from 2 to 14 °C at a rate of 0.5 °C/min in order to quickly dissociate the hydrates. By maintaining the temperature at 14 °C for 30 min we could ensure full dissociation of the hydrates before repeating the memory cycle. Full dissociation of hydrates was established, since the pressure reached the initial set pressure (before hydrate formation) and stabilized at a constant temperature. Each inhibitor was tested at least 4 times.

3. RESULTS AND DISSCUSION 3.1. Hydrate Nucleation. Previous studies showed that AFPs do inhibit structure I hydrate nucleation and structure II hydrate nucleation.6,10,13,14,16,21,22 The efficacy of AFPs in delaying hydrate nucleation has been shown to vary according to type, and no correlation between protein type and inhibition efficacy has yet been found. Previous studies conducted at high pressure with methane gas are with AFPs sourced from plants and fish. Hence, the performance of hyperactive insect AFPs on methane hydrate nucleation at relevant pressures could lead to new potential hydrate inhibitor candidates. The measured onset hydrate nucleation temperatures are summarized in Table 1 and 2 for the fresh and memory tests. The relative standard deviation in the onset of hydrate temperature values were for fresh solution tests between 0.4− 6.9% (SD 0.05−0.63 °C) and for the memory solution tests between 1.0−5.7% (SD 0.11−0.53 °C) indicating good repeatability of the tests. When using fresh solutions (Table 1) of the amino acids L-valine and L-threonine, hydrate 3668

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Table 1. Experimental Conditions and Average Results for Fresh Solutions and Constant Cooling Tests for Methane Hydrate number of tests

solution Milli-Q 50 mM NaCl− Milli-Q L-valine L-threonine BSA RmAFP1 PVP10 PVP40 a

concentration (ppm)

4 4

− −

5 5 5 1 14 8 6

2770 2770 2770 2770 1385 2770 1385

avg. onset temp. (°C)

SD (°C)

RSD (%)

11.8 11.5

0.19 0.05

1.6 0.4

11.5 11.3 10.3 8.6a 8.8 9.0 9.3

0.05 0.31 0.08 − 0.42 0.63 0.31

0.5 2.3 0.8 − 4.8 6.9 3.4

Only one test was conducted due to limited amount of AFP.

Figure 3. Onset hydrate nucleation temperatures for the fresh solutions and memory solutions at a concentration of 2770 ppm (error bars represent standard deviations (SD)).

Table 2. Experimental Conditions and Average Results for Memory Solutions and Constant Cooling Tests for Methane Hydrate solution L-valine L-threonine

BSA RmAFP1 PVP10 PVP40

number of tests

concentration (ppm)

avg. onset temp. (°C)

SD (°C)

RSD (%)

5 5 21 8 7 28 20 8

2770 2770 2770 1385 2770 1385 2770 1385

11.5 11.4 10.4 10.5 9.6 9.2 9.2 9.5

0.11 0.36 0.13 0.23 0.24 0.26 0.53 0.30

1.0 3.2 1.2 2.2 2.5 2.8 5.7 3.1

basis of these results BSA, PVP, and RmAFP1 perform better than the noninhibited system in delaying hydrate formation. The statistical differences between the KHIs are determined by comparing the onset hydrate nucleation values using independent sample t tests with equal variances assumed. For ranking the performance of KHIs it is assumed that p values lower than 0.05 confirm a statistically significant difference between two sets of onset hydrate nucleation values. On the basis of these results, we find the ranking of the inhibitors to be the same, regardless of the concentration (2770 or 1385 ppm) or the history of the water (memory or fresh). This indicates that both methodologies result in the same ranking. At the high concentration (2770 ppm): RmAFP1 = PVP10 > BSA > Lthreonine = L-valine = 50 mM NaCl in Milli-Q water > Milli-Q water. It should be noted that only one RmAFP1 in fresh solution was performed at 2770 ppm due to limited amount of RmAFP1. At the low concentration (1385 ppm) (Supporting Information, Figure S1): PVP10 > PVP40 > RmAFP1 > 50 mM NaCl in Milli-Q water > Milli-Q water. For the synthetic inhibitors and the RmAFP1 a shift to higher values in the recorded onset hydrate nucleation temperatures is observed when using memory rather than fresh solutions. Using memory solutions clearly results in higher temperatures which are expected as the memory effect makes it easier to form hydrates. Similar observations have been reported by others for AFPs:6,10 Daraboina et al.6,13,14,21 observed similar results for PVP and fish AFPs I and III for inhibition of natural gas hydrates. By using an isothermal nucleation procedure to obtain the nucleation time (time delay of hydrate formation) it was shown that PVP and AFP obtained very similar nucleation times using both a stirred tank, highpressure microdifferential scanning calorimeter (HP μDSC), and magnetic resonance imaging (MRI). The RmAFP1 was also compared with two PVPs of different average molecular weights at a lower concentration of 1385 ppm (Tables 1 and 2). As expected, the lower-molecular weight PVP10 performs better than its higher-molecular weight counterpart, PVP40. O’Reilly et al.40 explain the better nucleation inhibition of the lower-molecular weight polymer PVP by reasoning that this KHI has greater surface to volume ratio. Hence, the polymer perturbs the water structure more than higher-molecular weight polymers which might in addition be curled up or entangled through intramolecular hydrogen-

nucleation temperatures were obtained at 11.5 and 11.3 °C, respectively. Comparison of these nucleation temperatures to that of the noninhibited system (11.5 °C) shows that the amino acids do not exhibit any KHI effect by themselves as they do not delay hydrate nucleation. Furthermore, addition of 2770 ppm RmAFP1 in a fresh solution decreased the hydrate nucleation temperature from 11.5 to 8.6 °C compared to the noninhibited system by a depression in temperature of approximately 3 °C. This was more effective than the performance of BSA (10.3 °C) and comparable to PVP10 (9.0 °C) under similar experimental conditions. When repeating the experiment using memory solution (Table 2), the average hydrate nucleation temperature of 2770 ppm RmAFP1 (9.6 °C) again showed better inhibition than BSA (10.4 °C). Experiments conducted at a lower concentration of 1385 ppm RmAFP1 (10.5 °C) showed poorer inhibition than PVP10 (9.2 °C) and PVP40 (9.5 °C). We suspect that a protein concentration of 1385 ppm is too low to inhibit hydrates as efficiently as PVP at the same concentrations. The main reason for the discrepancy in the number of tests performed for the different substances was the limited amount of antifreeze protein at our disposal. Nevertheless, we believe the number of tests done for AFP with memory water at 2770 ppm (8) were enough to conclude that it was comparable to PVP10 and better than the other substances tested. Similarly, the smaller number of amino acid tests performed were nevertheless enough to see that they were not good inhibitors. Since synthetic inhibitors such as PVP10 are the industry benchmark, a larger number of tests were performed here. In Figure 3 the onset hydrate nucleation temperatures for fresh and memory cycles are illustrated at 2770 ppm. On the 3669

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profiles are very similar. Thus, not only the onset hydrate nucleation temperature but also the hydrate growth can be repeated in the rocking cells. In order to compare the hydrate growth for the different systems, hydrate growth profiles starting from the onset hydrate nucleation temperature are normalized according to pressure. The profiles at a concentration of 2770 ppm for the fresh solutions are shown in Figure 5A and for the memory solutions in Figure 5B. The measured pressure drops are indicative of the amount of hydrate formed during growth. In fresh solution the noninhibited system and the control L-valine show a similar growth profile and pressure drop of 10%, whereas L-threonine (11%) shows a slightly greater hydrate conversion. In fresh solution, RmAFP1 (11%) shows rapid initial growth which is in contrast to the other KHIs. BSA (12%) shows slower hydrate growth rate but greater hydrate conversion. However, a larger pressure drop was observed in the presence of PVP10 (14%), whereas the noninhibited system showed the least growth (10%). This indicates that the KHIs promote hydrate growth although they significantly delay hydrate nucleation. It is reported that once hydrates are formed in the presence of inhibitor the hydrate growth can be promoted compared to a noninhibited system.37,43 O’Reilly et al.40 observed that higher-molecular weight PVP prevents THF hydrate growth better than low-molecular weight PVP. This is explained by the longer PVP chains more strongly adsorbing onto the hydrate crystal surfaces. In our study the growth of hydrates using BSA is slower than AFP. This could be due to the size of the BSA molecule. However, RmAFP1 shows slightly less growth which indicates that the structure of the protein is important for growth inhibition. In the memory solutions the growth profiles of L-valine and L-threonine appears to be similar. After 375 min of growth Lvaline shows additional rapid growth resulting in a hydrate conversion of 11% for L-valine, whereas the hydrate conversion is 10% for L-threonine. The growth profile of RmAFP1 (8%) changes significantly by showing the least growth of hydrates which means RmAFP1 changes from promoting to inhibiting the hydrate growth. Again, PVP10 (12%) shows significant growth, but BSA (13%) shows even greater growth although at a slower rate. Inhibition of methane hydrate growth was also reported16 when using similar methodology for the insect AFP,

bonding, making it correspondingly less available to perturb the free water structure. The molecular weight difference between the proteins (RmAFP1 and BSA) might be thought to explain the observed results for hydrate nucleation. BSA (having high molecular weight) shows less inhibition than the low-molecular weight RmAFP1 as BSA might be entangled in the solution. However, for antifreeze proteins at equimolar concentrations an increase in protein size decreases the solubility of the protein in the water phase. This, in turn, will lead to increased antifreeze activity.9 Most importantly though, AFPs have a unique structure consisting of a hydrophobic flat binding site that structurally matches the water molecules in ice41 which other proteins such as BSA generally will not have. 3.2. Hydrate Growth. Hydrate inhibition studies show that AFPs inhibit hydrate growth when using plant, fish, and insect AFPs.10,13,15,21,42 It is reported that AFP may act as inhibitor or promoter, depending on the temperature and pressure conditions during experiments.19 The efficacy of RmAFP1 on hydrate growth was studied and compared to that of the other KHIs in order to evaluate its performance. The recorded hydrate growth profiles in the presence of RmAFP1 are illustrated in Figure 4. The figure shows that the recorded

Figure 4. Hydrate growth recorded for RmAFP1 during memory solutions with cooling tests at a concentration of 2770 ppm. The graph shows the seven repeated experimental recordings.

Figure 5. Normalized hydrate growth profiles at a concentration of 2770 ppm for (A) fresh solutions and (B) memory solutions. The graph begins at time zero which is the onset of hydrate nucleation. 3670

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Tenebrio molitor (TmISP), fish AFP (ISP type III HPLC12), and PVP. In that work, pressure was increased or decreased to move into or out of the stable hydrate zone. Here we do so by changing temperature. Different concentrations (0.01−0.1 wt %) were studied for the ISP type III HPLC12, and by increasing concentration the amount of hydrate formed decreased. We observed that the hydrate growth for RmAFP1 at 1385 ppm initially was more rapid than at 2770 ppm. However, the pressure dropped by 8% at both concentrations, indicating a similar amount of hydrate growth (Supporting Information Figure S2). This could be due to the high concentration of RmAFP1, as Jensen et al.16 only observe a very small increase in inhibition of growth from the ISP type III HPLC12 at 0.05 to 0.1 wt %. Furthermore, they also observed that 0.1 wt % PVP performed as hydrate growth inhibitor, whereas in our study we used the same molecular weight of PVP and observed that 0.277 wt % PVP promoted the hydrate growth. This again could be due to the higher concentration of PVP. Kumar et al.44 reported that, for methane/propane hydrates in the presence of 1 wt % PVP, catastrophic hydrate growth was observed compared to low concentration (0.1−0.5 wt %) PVP. Conversely, Daraboina et al.45 observed that by increasing concentration of PVP from 0.1 wt % to 1.0 wt % increasing hydrate growth inhibition was obtained. 3.3. Stability of AFP. The onset hydrate nucleation temperatures for RmAFP1 (2770 ppm) were observed to change throughout the test period of 16 days (Figure 6). In

Article

CONCLUSION The efficacy of antifreeze protein from the longhorn beetle, Rhagium mordax (RmAFP1), for inhibiting methane hydrate nucleation and growth was investigated using high-pressure rocking cells. The performance of the antifreeze protein was compared to that of the synthetic inhibitor PVP, the protein BSA, and the amino acids L-valine and L-threonine. Constant cooling experiments showed that the amino acids (L-Val and LThr) do not exhibit kinetic hydrate inhibition. However, using fresh solutions showed that RmAFP1 (2770 ppm) performed as effectively as PVP for inhibition of hydrate nucleation. In addition the (non-antifreeze) protein BSA showed slight inhibition of hydrates. The efficacy of RmAFP1 in memory experiments was also comparable to PVP. The efficacy seems to decrease with time though, indicating possible decomposition of the protein. For hydrate growth the RmAFP1 changes from being hydrate growth promoter in the fresh solution to becoming hydrate growth inhibitor in the memory solution. In contrast, all other KHIs perform as hydrate promoters in both fresh and memory solutions. By comparing onset nucleation temperatures and growth profiles it is observed that there is no clear correlation between the efficacies of KHIs. However, RmAFP1 in memory solution performs as an effective inhibitor for hydrate formation. These results confirm that AFPs such as RmAFP1 have a unique ability to inhibit hydrate formation and growth. Hyperactive insect AFPs such as RmAFP1 may therefore represent a new class of green hydrates inhibitors.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +45 4525 2867. Fax: +45 4588 2258. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■ Figure 6. Onset hydrate nucleation temperatures for RmAFP1 and BSA in fresh and memory solutions at a concentration of 2770 ppm over time.

ACKNOWLEDGMENTS This work was supported by Grant 10-082261 from The Danish Council for Independent Research - Technology and Production Sciences, the Danish National Advanced Technology Foundation, and the Technical University of Denmark

comparison BSA showed no change in hydrate nucleation temperatures during the test time. The first data at day zero is from a constant cooling test using fresh solutions. The memory RmAFP1 solution was then kept at 5 °C for a week before further tests were conducted. The constant cooling results using the memory solution indicate an increase in the average onset temperature from 9.3 to 9.9 °C over 7.5 days. This could suggest an increased memory effect of the RmAFP1 or that the protein may be decomposing with time. The BSA solution was kept at 8 °C between the tests and no change of performance was observed.

(1) Kelland, M. A. History of the Development of Low Dosage Hydrate Inhibitors. Energy Fuels 2006, 20 (3), 825−847. (2) Koh, C. A. Towards a Fundamental Understanding of Natural Gas Hydrates. Chem. Soc. Rev. 2002, 31 (3), 157−167. (3) Frostman, L.; Thieu, V.; Crosby, D.; Downs, H. Low-Dosage Hydrate Inhibitors (LDHIs): Reducing Costs in Existing Systems and Designing for the Future. International Symposium on Oilfield Chemistry; SPE, Houston, TX, 2003 (4) Perrin, A.; Musa, O. M.; Steed, J. W. The Chemistry of Low Dosage Clathrate Hydrate Inhibitors. Chem. Soc. Rev. 2013, 42 (5), 1996−2015. (5) Del Villano, L.; Kommedal, R.; Kelland, M. A. Class of Kinetic Hydrate Inhibitors with Good Biodegradability. Energy Fuels 2008, 22 (5), 3143−3149.

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dx.doi.org/10.1021/ef500349w | Energy Fuels 2014, 28, 3666−3672