ARTICLE

Antifreeze proteins as gas hydrate inhibitorsVirginia K. Walker, Huang Zeng, Hiroshi Ohno, Nagu Daraboina, Hassan Sharifi,S. Alireza Bagherzadeh, Saman Alavi, and Peter Englezos

Abstract: Certain organisms survive low temperatures using a range of physiological changes including the production ofantifreeze proteins (AFPs), which have evolved to adsorb to ice crystals. Several of these proteins have been purified and shownto also inhibit the crystallization of clathrate hydrates. They have been found to be effective against structure II (sII) hydratesformed from the liquid tetrahydrofuran, sI and sII gas hydrates formed from single gases, as well as sII natural gas hydrates usinga mixture of three gases, as assessed using a variety of instrumentation including stirred reactors, differential scanningcalorimetry, nuclear magnetic resonance, Raman spectroscopy, and X-ray powder diffraction. For the most part, AFPs are equalto or more effective than the commercial kinetic hydrate inhibitor (KHI) polyvinylpyrolidone, even under field conditions wheresaline and liquid hydrocarbons are present. Enclathrated gas analysis has revealed that the adsorption of AFPs to the hydratesurface is distinct from tested commercial KHIs and results in properties that should make these proteins more valuable in somefield applications. Efforts to overcome the difficulties of recombinant protein production are ongoing, but in silico models of AFPadsorption to hydrates may offer the opportunity to design commercial KHIs for hydrocarbon recovery and transport with all theattributes of these AFP ”green inhibitors”, including their benefits for human and environmental safety.

Key words: tetrahydrofuran, kinetic hydrate inhibitors, antifreeze proteins, oil and gas recovery, green inhibitors.

Résumé : Certains organismes survivent a de basses températures grâce a une variété de changements physiologiques, dont laproduction de protéines antigel, lesquelles ont évolué pour s’absorber sur les cristaux de glace. Plusieurs de ces protéines ont étépurifiées et ont montré une capacité d’inhiber la cristallisation des hydrates en clathrates. Nous avons observé qu’elles sontefficaces contre les hydrates de structure II (sII) formés a partir de tétrahydrofurane liquide, contre les hydrates de gaz sI et sIIformés a partir de gaz simples, ainsi que contre les hydrates de gaz naturel sII a partir d’un mélange de trois gaz, grâce uneévaluation réalisée a l’aide de divers instruments, dont des réacteurs agités, l’analyse calorimétrique a compensation depuissance, la résonnance magnétique nucléaire, la spectroscopie Raman et la diffraction de rayons X sur poudres. Essentielle-ment, les protéines antigel sont équivalentes ou plus efficaces que la polyvinylpyrrolidone, un inhibiteur cinétique d’hydratescommercial, et ce, même en conditions naturelles en présence d’eau saline et d’hydrocarbures liquides. L’analyse de gazenclathratés a révélé que l’adsorption des protéines antigel a la surface de l’hydrate diffère de celle des inhibiteurs cinétiquesd’hydrates commerciaux qui ont été analysés. Les propriétés qui en découlent devraient rendre ces protéines plus intéressantesdans certains champs d’application. Nous déployons actuellement des efforts pour surmonter les difficultés de productiondes protéines recombinantes, mais les modèles in silico d’adsorption des protéines antigel sur les hydrates pourraient ouvrir lavoie a la création d’inhibiteurs cinétiques d’hydrates commerciaux destinés a la récupération et au transport des hydrocarbures,et possédant tous les attributs de ces protéines antigel dites « inhibiteurs verts », y compris leurs avantages en matière de sécuritéhumaine et environnementale. [Traduit par la Rédaction]

Mots-clés : tétrahydrofurane, inhibiteurs cinétiques d’hydrates, protéines antigel, récupération de pétrole et de gaz, inhibiteursverts.

Introduction

Preamble: Honouring Dr. John RipmeesterAbout 15 years ago, John Ripmeester, already one of Canada’s

leading physical chemists, agreed to collaborate with a geneticistto determine whether some small proteins associated with over-wintering insects and fish could possibly be used for the control ofgas hydrate growth. It sounds like an unusual basis for coopera-tion. After all, antifreeze proteins have evolved over millennia to

adsorb to ice, and although gas hydrates are made of crystallineice-like cages of water that house gas molecules, the organizationof the waters in these structures are strikingly distinct from hex-agonal ice (Ih). Proteins tend to opt for their minimum energyprofiles, and it would be curious if they could equally readily”bind” to such seemingly structurally-different substrates. No oneknew this better than John Ripmeester, who with his colleaguesfrom the National Research Council of Canada (NRC) had just twoyears earlier published the novel hexagonal structure (sH) clath-

Received 29 November 2014. Accepted 6 January 2015.

V.K. Walker, H. Zeng,* and H. Ohno.† Department of Biology, 116 Barrie Street, Queen’s University, Kingston, ON K7L 3N6, Canada.N. Daraboina,‡ H. Sharifi, S.A. Bagherzadeh, S. Alavi,§ and P. Englezos. Department of Chemical and Biological Engineering, 2360 East Mall,The University of British Columbia, V6T 1Z3, Canada.Corresponding author: Virginia K. Walker (e-mail: walkervk@queensu.ca).*Present address: British Petroleum, Calgary, Canada.†Present address: Department of Civil and Environmental Engineering, Kitami Institute of Technology, Hokkaido 090-8507, Japan.‡Present address: Department of Petroleum Engineering, University of Tulsa, 800 S. Tucker Drive, OK, 74104, USA.§Concurrent address: National Research Council, NRC-SIMS, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada.This article is part of a Special Issue in honour of Dr. John Ripmeester and his outstanding contributions to science.

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1

Can. J. Chem. 93: 1–11 (2015) dx.doi.org/10.1139/cjc-2014-0538 Published at www.nrcresearchpress.com/cjc on 3 February 2015.

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rate in Nature.1 Undaunted, John agreed to co-supervise HuangZeng, a Queen’s University Biology Ph.D. student with VirginiaWalker, and thus started a long and fruitful collaboration thatcame to entangle other Queen’s University students, John’s teamand colleagues from NRC, the talents of a University of BritishColumbia (UBC) Chemical Engineer, Peter Englezos and his stu-dents, as well as Saman Alavi, a colleague of both Peter and Johnand his student.

From this audacious beginning involving these physical chem-ists, geneticists and engineers, was forged a new field of inquiry,all motivated by the best of reasons. It was our hope to discover”green inhibitors” for gas hydrate formation to ensure both envi-ronmental and human safety and to provide increased security tothe energy sector. Our hypothesis was that macromolecules thathad evolved in certain overwintering organisms to adsorb andrestrict ice crystal growth and thereby protect them from cellulardamage might also be useful for hydrocarbon discovery and re-covery. Such inhibitors would by their very nature be environ-mentally friendly, in that AFPs are not known to be toxic to livingorganisms and would be readily hydrolysed even if accidentlyreleased.

Gas hydrates and the problem with gas hydrate formationClathrate hydrates are inclusion compounds with a host-lattice

consisting of hydrogen-bonded water molecules that entrap guestmolecules.2,3 When water and an appropriate guest are broughttogether under suitable temperature and pressure conditions, asolid phase (hydrate crystal) is formed. For gas hydrates, the guestmolecules are gases such as methane, ethane, propane, carbondioxide, or hydrogen sulfide (4–8 Å range). Gas hydrates areknown to occur in the lab in at least half a dozen structures, butnaturally they have only been reported in three distinct struc-tures: the cubic forms, structure I (sI), structure II (sII), and thehexagonal form (sH). These consist of small water cages (12-facedpentagonal dodecahedrons, 512) as well as larger ones.4 Generally,smaller gas molecules such as methane, ethane, and CO2 promotesI formation (unit crystal = 2 × 512; 6 × 51262), with larger gasmolecules, including propane and isobutene, promoting sII for-mation (unit crystal = 16 × 512; 8 × 51264). The sH hydrates canaccommodate still larger gases, but they are rarer and are notknown to be a concern for pipeline flow assurance.

Hydrates of natural gas are found in and under permafrost andbelow the sea floor along the continental margins, constituting ahuge potential energy source estimated to be more than twicethat of all known coal, oil, and gas deposits.5,6 Such deposits pres-ent an opportunity for energy security for those nations withcontinental shelf reserves or with permafrost deposits, as well asfor other jurisdictions to offset demands on more conventionalenergy deposits. Nonetheless, mining of gas hydrate deposits, aswell as the generation of gas hydrates during formation fractureprocesses, or in pipelines carrying crude oil or natural gases,7 canbe a serious safety concern. When pressure and temperature con-ditions are favorable, gas hydrate plugs can form in pipes, andthese can lead to shut down and catastrophic financial losses.They have the potential to move at velocities up to 300 km/h,which creates more hazards.4,8 The numbers of operating prob-lems due to hydrate blockage are considered “countless” by indus-try,9 and even at the time that we began our research, the controlof hydrates in pipelines was estimated at $1 billion in annualproduction costs.10 The usual technologies for hydrate inhibitioninclude dehydration, heating, pressure reduction, and the addi-tion of substantial amounts of chemicals such as methanol orethylene glycol (15% to 50% by weight of the free water phase) asthermodynamic controls.

Initially, it was the high cost of thermodynamic inhibitors thatstimulated the search for low-dose hydrate inhibitors. These in-clude kinetic hydrate inhibitors (KHIs), which delay the onsetof nucleation and (or) control hydrate growth, as well as anti-

agglomerates that prevent hydrate particles from adhering toeach other.11–13 Either inhibitor type would reduce the probabilityof blockage during the residence time of the liquid phase in thepipeline.10–15 However, economic loses are not the greatest prob-lem. Gas hydrate plugs and the use of thermodynamic inhibitorssuch as methanol and glycol in offshore developments and on-shore processing facilities lead to safety and environmental con-cerns. There are hazards in the storage and handling of suchinhibitors to employees, and there is the potential for substantiallosses to the environment if pipelines burst. Indeed, the environ-mental impact of methanol use is of such concern that restric-tions have been imposed in some European sectors16,17 as well asin the Arctic. In the Gulf of Mexico, another alcohol, ethyleneglycol, has been used,9 and in laboratory studies ethylene glycoldeicers have been shown to be hazardous to aquatic organisms.18

Both alcohols act by lowering the crystallization point of the hy-drate, and as mentioned, large quantities of these chemicals arerequired. In practical terms, this can be as high as one barrel ofmethanol or glycol for every barrel of water in the productionstream.

Gas hydrates represent a significant accumulation of hydrocarbonsin a given volume (1 m3 methane hydrate releases �60 m3 of gas),and thus care must be taken in pipeline and fixed installations.The explosion of the Piper Alpha oil platform in the North Sea in1988 in which 167 lives were lost is an example of a disaster asso-ciated with the uncontrolled release of hydrocarbons.19 The envi-ronmental impact of several methane hydrate accidents in the Seaof Azov, Ukraine, was reported to have resulted in mass fish mor-tality, even in fish collected distant to the accident site.20 Under-sea wellheads and land pipelines are also potential hazards. Theseextend for thousands of kilometers and gas hydrates can causeblowouts or be formed subsequent to the development of cracks.We have only to remember the Deep Water Horizon disaster inthe Gulf of Mexico. After an attempt to cap the wellhead failed dueto hydrate formation, British Petroleum’s chief operation officer,Doug Suttles, said, “We did anticipate hydrates being a problem,but not this significant”.21

Antifreeze proteinsThese proteins have been variously named to follow the con-

vention associated with a particular host organism or as appropri-ate for a particular application. They are known as thermalhysteresis proteins after the first observation of this phenomenonin beetles, ice recrystallization inhibitor proteins most often inplants where this activity is protective, more recently as ice-binding proteins in an attempt to include proteins that nucleateice in the same group, ice-structuring proteins as they are calledby processed food corporations, or more generally and popularlyas antifreeze proteins (AFPs). They are produced by certain fish,insects, plants, and microorganisms that survive at temperaturesbelow 0 °C. AFPs adsorb to microscopic ice crystals and preventtheir growth, with the result that host organisms can live unfro-zen at subzero temperatures, such as fish, some insects, and var-ious microorganisms, or alternatively, survive after freezing, suchas certain perennial plants. As indicated, AFP activity was firstnoted in insects, but these proteins and the antifreeze glycopro-teins were originally characterized and cloned from cold-waterfish, such as the Type I AFP from winter flounder or Type III AFPfrom ocean or eel pouts. Later they were cloned from a broadrange of cold-adapted organisms including invertebrates, plants,fungi, and microorganisms, including bacteria (reviewed 22). AFPsinhibit freezing in a noncolligative manner by adsorbing to iceand delaying ice crystal growth.23,24 AFPs also modestly contrib-ute to ice crystal superheating during melting,25 so that the actionbetween AFPs and the ice surface results in a separation of thefreezing point and the melting point, a phenomenon termedthermal hysteresis (TH). The inhibitory activity is thought to de-rive from local ice surface curvature effects induced by the

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adsorption of AFPs at the ice/solution interface (the adsorption-inhibition hypothesis26). Due to this action, small quantities ofAFPs can have large effects on ice crystal growth.27

AFPs have emerged at various times during evolution with theappearance of some North American fish antifreeze glycoproteinsand AFPs coinciding with the freezing of the North Atlantic in theCenozoic period, 2–3 million years ago.28,29 Similarly, a NorthAmerican insect AFP appeared at approximately the same time.30

As would be expected due to their independent origins, except forclosely related organisms and other unusual metazoan excep-tions,31 their structures are distinct (Fig. 1). For example, the win-ter flounder fish Type I (referred to as AFP I here) folds into a3–4 kDa �-helix, whereas Type III AFP from the ocean pout (AFP III)is a 7 kDa globular protein.22 The spruce budworm (Choristoneurafumiferana) insect AFP (CfAFP) folds into a 9 kDa left-handed �-helixwith a triangular cross-section, and the beetle (Tenebrio molitor)insect AFP (TmAFP) takes on a 8.4 kDa right handed �-helical fold.The structure of the 13.5 kDa recombinant version of the peren-nial rye grass (Lolium perenne) protein (LpAFP) is often described asleft-handed �-solenoid, but other plant AFPs appear to have dis-tinctly different conformations. As well as different structures,these diverse AFPs have different activities. Characterized insectAFPs32,33 are hyperactive with TH activity that is 30–100 timeshigher than that reported for most fish AFPs. In contrast, plantAFPs, exemplified by LpAFP, have activities that are lower than themoderately-active fish AFPs.34 However, the low TH active plantAFPs often show superior inhibition of ice recrystallization (thegrowth of ice crystals at temperatures close to melting), which iswhy they are often referred to as ice recrystallization inhibitorproteins.35

The differences in activity of the various AFPs have been ex-plained by the abilities of the dissimilar AFPs to adsorb to differ-ent ice planes. The moderately active AFP I and III adsorb to theprism planes of an embryonic ice crystal, but hyperactive insectAFPs adsorb to the basal plane as well as prism planes and confergreater TH activity.22 Somewhat curiously, the low activity LpAFPalso shows binding to the same planes as the hyperactive AFPs. Anattempt to explain this conundrum focused on the observed mul-tiple substitutions in the ”ideal” TXT repeats (where X can be anyamino acid) that make up the LpAFP ice-binding face. It was sug-gested that these less than ideal substituted amino acids reducethe probability of adsorption to any or all of the ice crystal planes.36

In other words, the plant AFP is just an ”inefficient” hyperactiveAFP. Nevertheless, the affinity of LpAFP to adsorb to ice is stillsufficient to allow purification of the recombinant protein byice-affinity, which is hard to reconcile with its inefficient binding.Monte Carlo simulations of ice crystals containing tens of millionsof water molecules predicted that AFP activities would be re-

flected by their planes of binding on each ice face rather than thestrength of their bonds.37 Thus we speculate that although LpAFPadsorbs to multiple planes, the orientation of the protein mole-cules on the basal surface, for example, could be suboptimal (i.e.,away from the perpendicular of this ice plane), resulting in theaddition of water molecules and thus growth and therefore ac-counting for the lower observed activity and the success of ice-affinity purification. This prediction would be most easily testedusing in silico experiments.

Experimental section, results, and discussion

The use of a model sII hydrate-THF hydrateThere are challenges to be surmounted when studying the in-

hibition of gas hydrates in the laboratory. First, the formation ofgas hydrates requires elevated pressure, which is not always wel-comed in teaching laboratories. As well, the gas hydrate layerformed at the liquid–gas junction reduces the mass transfer atthis interface, and therefore by itself retards further hydrate for-mation. As a result, it is convenient to use the liquid tetrahydrofu-ran (THF), which allows the formation of hydrates not only at thewater surface but throughout the solution. THF molecules stabi-lize the large water cages of sII hydrates at atmospheric pressuresand at temperatures just a few degrees above 0 °C. These advan-tages prompted early investigations on the impact of AFPs onhydrates to use THF model hydrates. Like other crystallizationprocesses, THF hydrate formation includes two major steps: nu-cleation and growth, with the former separated into homoge-neous and heterogeneous nucleation. In practice, heterogeneousnucleation is much more important because of the ubiquity ofnucleating surfaces such as those found inside pipelines, for ex-ample. The time to nucleate in stirred test tubes was measured asinduction time, and AFPs from both a fish (AFP I; 0.25 mmol/L) andan insect (CfAFP; 0.05 mmol/L) demonstrated better inhibitioneffect on THF hydrate formation than the polymeric low dosagecommercial hydrate inhibitor, polyvinylpyrrolidone (PVP).38–40

Normally when hydrates are formed in the presence of inhibi-tors, melted, and subsequently allowed to reform again within arelatively short period of time, they recrystallize in less time orwith a lower driving force. This phenomenon is termed the ”mem-ory effect”. Although some have argued that there is no suchthing,41 chemical engineers and pipeline operators report thatthis effect is a real threat to flow assurance. Strikingly, as well asinhibiting the initial formation of the THF hydrate, both AFPsseemed to eliminate the memory effect, whereas the commercialinhibitor, PVP, was unable to do so.39,40 Both impurities andresidual hydrate structures likely contribute to the memory

Fig. 1. AFPs show diverse modeled structures as exemplified by (A) AFP I or type I AFP from the fish, winter flounder (Pleuronectes americanus),(B) AFP III or type III AFP from ocean pout (Zoarces americanus), (C) HnAFP from the snow flea (Hypogastrura nivicola), (D) TmAFP from themealworm beetle (Tenebrio molitor), and (E) CfAFP from the spruce budworm (Choristoneura fumiferana). The ice-binding surfaces of all theseproteins are relatively hydrophobic and flat. Color scheme: H, white; C, cyan; N, blue; O, red; S, yellow. (See online version for colour.)

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effect,40,42 and the impact of AFPs on this phenomenon neededmore investigation.

To determine if the protein face that is important for ice-binding was also important for hydrate inhibition, an ice-inactiveAFP I mutant protein bearing an amino acid substitution at the17th residue (A17L) was used in analogous experiments. The mu-tant showed somewhat higher inhibition activity for THF hydratethan the wild-type protein, although it had no capability to elim-inate the memory effect.39,40 An absence of a memory effectsuggested to us that ice-active properties may be involved in ”poi-soning” nucleation sites. Some AFPs can indeed lower bacterial icenucleating protein-mediated freezing points by up to 2 °C43, and itis possible that an analogous mechanism is applicable here. Mostimportantly, however, these early experiments demonstrated thepotential of AFPs to inhibit hydrate clathrate induction. In addi-tion, they showed that with respect to protein adsorption, the twoice-like structures appeared to be functionally distinct, furthersuggesting that ice activity might not be an exacting predictor ofhydrate activity.

To visualize AFPs on THF crystals, DNA sequences correspond-ing to the coding regions of LpAFP and AFP III were joined to agreen fluorescent protein (GFP) sequence originating from a jellyfish and then transferred to the bacterial host, Escherichia coli. Theresulting recombinant AFPs were then visualized on the THF hy-drate crystals. Polycrystalline hydrates grown in the presence ofeither of the labelled AFPs were uniformly fluorescent green withno evidence of appreciable binding by control proteins.44 It mustbe noted that the presence of the GFP tag appeared to decrease theaffinity toward THF hydrate, but nevertheless these experimentswere compelling visible evidence of hydrate adsorption. Curi-ously perhaps, the plant LpAFP-GFP with low TH activity towardsice but with a higher ice recrystallization inhibition activity thansome other AFPs, showed high affinity to polycrystalline THF hy-drate, just as did the moderately ice-active AFP III-GFP. Polycrys-talline THF hydrate ”popsicles” have also been used to show thatthe amino acid residues important for ice-binding are not alwaysimportant for hydrate, at least for LpAFP,45 substantiating theresults of the mutant AFP I experiments. Labeled AFPs, as well asproteins without fluorescent tags, were also incorporated intosingle THF hydrate crystals. Although these are tricky to form,when successfully grown, both fluorescently marked LpAFP andType III AFPs bound to the large single crystals, showing no pref-erence for any of the 8 identical {111} hydrate faces44 (Fig. 2). Athigher driving forces (4.4 and 5.9 °C subcooling) these faces were

the slowest growing regions of the hydrate crystals, suggestingthat the heat of crystallization was more easily dissipated on thecrystal edges. AFPs were effective at low concentrations, support-ing their designation as low-dosage hydrate inhibitors; single THFcrystals grown in AFP III without a fluorescent tag showed pro-nounced morphological changes even at concentrations as low as15 �g mL−1 (2.2 �mol/L). The images in Fig. 2 show hydrate growthinhibition where the AFPs adsorb to the crystal below the equilib-rium melting point, changing the morphology to ”hopper-like”crystals and providing strong visual evidence that AFPs can in-hibit hydrate growth via an ”adsorption-inhibition” mechanism.

Carbon dioxide and methane hydrate experimentsAfter the encouraging results from a model sII THF hydrate

system, it was of interest to examine their efficacy with a distinctgas hydrate structure. CO2 forms sI hydrates and others showedthat these were inhibited in the presence of AFP III, as determinedby morphological changes of droplets formed in a pressurizedcrystallizer.46 AFP clearly acted as a kinetic inhibitor since even atlow protein concentrations (0.01 mg mL−1), CO2 hydrate formationtook three times longer than in control droplets. Like CO2, meth-ane also preferentially forms a sI hydrate. To examine theseclathrates, the power of nuclear magnetic resonance (NMR) wasexploited, a technique that had previously proved to be so success-ful in John Ripmeester’s hands. This time a 1H NMR-based micro-imaging technique was facilitated by a custom-designed inset forthe high-pressure imaging tube; this allowed the rate of methanehydrate formation in small water droplets (�1 mm) to be moni-tored using the reduction of the 1H signal.47 The rate was reduced�3-fold in the presence of AFP I. Thus when combined with theTHF hydrate data, these results effectively demonstrated the inhi-bition activity of these proteins against the two most commonhydrate structures (Table 1). Subsequently, these observations onmethane hydrate were augmented by results from the lab ofPhilip Servio (trained with Peter Englezos), who not only notedthe inhibition activity but was able to directly visualize the dis-tinct crystal morphology produced in the presence of AFP I(eg.48,49). Subsequently other groups have also verified and ex-tended these observations.50

More recently, the investigation of AFP-mediated inhibition ofmethane hydrate formation has employed a ”rocking cell”, con-sisting of stainless steel tubes, each containing a steel ball thatis made to rock back and forth as the apparatus is tipped.51

The authors, who include another trainee from our labs, Nagu

Fig. 2. Typical single THF hydrate crystals grown without additives (A) or with 200 �g mL−1 recombinant green fluorescent protein (GFP) (B)show no major changes in morphology. Crystals grown in solutions containing 200 �g mL−1 (30 �mol/L) AFP III-GFP (C) or 15 �g mL−1

(2.2 �mol/L) LpAFP-GFP (D) show skeletal growth, as do those grown in 200 �g mL−1 (20 �mol/L) PVP (E). Crystals with adsorbed AFP III-GFP(100 �g mL−1; 3.1 �mol/L) (F) and LpAFP-GFP (100 �g mL−1; 2.4 �mol/L) (G) show skeletal or hopper-like growth and fluoresce green under UVlight. The THF hydrate crystals were grown in triplicate at 3 °C to a diameter of �4.5–5 cm (images modified from reference 44). (See onlineversion for colour.)

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Daraboina, showed that an AFP from a longhorn beetle acted as aneffective inhibitor of methane hydrate reformation and growth inpreviously crystallized solutions. TmAFP and AFP III also inhibitedmethane hydrate growth,52 strengthening the evidence that AFPsshow efficacy against both hydrate types. To our knowledge, no-body has tested these proteins with sH, which would be veryinteresting given that the common structure of ice is also hexag-onal, but perhaps of not much practical utility to industry.

Propane hydrate experimentsLike THF, propane also preferentially forms sII hydrate, but it

has the advantage that it is a ”real” gas hydrate and not a modelsystem. As had been done with methane hydrate, the inductiontime and formation rate were measured in multiple samples us-ing NMR micro-imaging. In the presence of 0.25 mmol/L AFP I, theinduction time increased three-fold with an overall reduction inthe rate of hydrate formation of 5-fold, behavior that allows AFP Ito be classified as a low-dosage hydrate inhibitor (Table 1). A strongmemory effect is normally seen when propane hydrate is re-formed after melting. Remarkably, AFP I eliminated this and wasthen, to our best information, the first additive that had shownsuch a potentially valuable property as a gas hydrate inhibitor.47

Natural gas (mixed) hydratesAlthough AFPs were effective against sI and sII hydrates formed

from single gases, we were mindful that single gases did notmimic ”real” hydrates found in oil and gas fields. Thus it wasimportant to also test these proteins with a gas mixture (methane,ethane, and propane in a composition of 93/5/2 mol%), designed tomimic natural gas to help ensure the efficacy of these green in-hibitors under a wider range of practical conditions. These mixedgas hydrates were then used to compare AFPs with different ac-tivities toward ice, under conditions that more closely mimickedundersea and pipeline situations, and for an investigation of theformation and decomposition of hydrates as detailed below.

Comparing AFPs with different activities towards iceAs indicated, the experiments with single gases had demon-

strated the efficacy of both hyperactive insect AFPs (CfAFP) as wellas the moderately active fish AFPs towards gas hydrates. Never-theless, it was important to also make side by side comparisons ofthese different proteins using the same experimental system. As aresult, AFPs with a range of TH activities were employed: LpAFP,AFP III, and TmAFP. Facilitating data collection and possibly fur-ther modelling gas formations in situ, the proteins, gas mixture,and water were dispersed in the pore space of silica beads.53 Afternucleation, hydrate inhibition was monitored by calculating thewater-to-hydrate conversion ratio over time from the hydrate gasconsumption. Strikingly, all the AFPs were effective inhibitors,even more than PVP, which was again used as a benchmark chem-ical KHI (Fig. 3). There was some evidence that the GFP tag on someof the AFPs may have modestly reduced hydrate affinity. Notablyhowever, AFP III, AFP III-GFP, and TmAFP were more effective thanLpAFP-GFP as sII growth inhibitors, as might be expected if hy-drate inhibition loosely mimicked ice growth inhibition. Again,

this is consistent with the THF-hydrate single crystal studies,which showed that AFP III and AFP III-GFP were more effectivethan LpAFP-GFP.44

High pressure differential scanning calorimetry (HP-DSC) hadpreviously proved its value in the assay of hydrate phase transi-tions and nucleation temperatures, and thus it was pressed intoservice to help compare the efficacy of the different AFPs usingthe silica gel matrix. This procedure ensured nucleation in areasonable period. In temperature ramping experiments (4 MPawith temperatures dropping from 25 to −45 °C), all the AFPs(0.1 mmol/L) shifted the exothermic peaks to lower temperatures,demonstrating effective inhibition of hydrate nucleation. Asmight be predicted then, in DSC isothermal experiments (4 MPaand −12 °C), comparison of the cumulative heats of reactionshowed that all of the AFP-containing samples considerably de-layed nucleation.53 Faster nucleation occurred in samples con-taining PVP or no additives (Fig. 4). Notably, the AFPs retainedtheir nucleation inhibition effects over long periods, givinghope that they might be useful in the field even given fluid resi-dence times that can extend to days.54 In nucleation inhibitionactivity, the moderately TH active AFP III proved superior to thehyperactive TmAFP. Thus, AFP nucleation and growth inhibition

Table 1. Inhibition, shown by a plus sign (or if not tested; nt), of sI or sII hydrates (formed withtetrahydrofuran, THF, or various gases including a three-gas component natural gas) by a range ofantifreeze proteins (AFPs), classified by their thermal hysteresis (TH) activity against ice.

Low TH Moderate TH High TH

Hydrate formerHydratestructure LpAFP AFP I AFP III

CfAFP, TmAFPor other Selected Refs.

THF sII + + + + 38,40,44–45Methane/CO2 sI nt + + + 46,47–52Propane sII nt + nt nt 47Natural gas sII + + + + 53,56,59–64

Fig. 3. A comparison of the efficacies of AFPs with different thermalhysteresis activities towards ice as inhibitors of hydrate gasconsumption with mixed gas (93% methane, 5% ethane, and 2% pro-pane), water and dispersed with silica beads. Samples included0.1 mmol/L commercial KHI, PVP (black line), the beetle proteinTmAFP (green line), fish Type III AFP (AFP III) with and without agreen fluorescent protein (GFP) tag (pink and orange lines,respectively), and the plant AFP LpAFP-GFP (mauve). Afternucleation, hydrate inhibition was monitored by calculating thewater-to-hydrate conversion ratio (adapted from reference 53,copyright Wiley-VCH Verlag GmbH & Co.KGaA; reproduced withpermission). (See online version for colour.)

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activities did not appear to be tightly correlated with their ice-binding activities, implying differences in their adsorption sitesor the mechanism of inhibition.

Closer to real world models with natural gas (mixed) hydratesThe gas and oil industry contain tough critics. Although this

body of work had clearly shown the utility of AFPs as KHIs againstdifferent structures of hydrates and with mixed gases, the ques-tion remained, would they work under real world situations inoffshore pipelines? We addressed these industry concerns byagain using the gas mixture but adding saline (3.5% wt% of NaCl)and heptane, to mimic liquid hydrocarbons.

AFP I and III (0.1 mmol/L) in the presence of both saline andheptane continued to perform as inhibitors by increasing the in-duction time and controlling post nucleation hydrate growth,similar to other KIs.55–58 There was some impact of both theseamendments however. Under saline conditions, AFP III prolongedinduction time more than AFP I, but the opposite was true in purewater. Similar to observations reported for other KIs, however,AFP I was not effective in prolonging the induction time in pres-ence of heptanes.56,59,60 We speculate that this amphipathic AFPmight have reduced the interfacial tension between the liquidhydrocarbon and aqueous phases and thus decreased the masstransfer barrier for hydrate nucleation, whereas the presence ofheptane and saline did not impact the induction time of the glob-ular AFP III (Fig. 1). For future industrial application consider-ations, it is useful to note that with both AFPs, the gas hydrategrowth rate was controlled more effectively in the presence of theliquid hydrocarbon with no acceleration of gas hydrate growthdetected. Nonetheless, AFP III performed better under these con-ditions than AFP I but also appeared superior to previously re-ported chemical KHIs, which also reduced gas hydrate inductiontime in the presence of a light crude mimic.56,59,60

Industry not only requires kinetic inhibitors to postpone gashydrate nucleation and control the growth rate, but it is alsocrucial to evaluate how easily hydrates dissociate after beingformed in the presence of inhibitors. If gas hydrates block pipe-lines, hydrocarbon transport must be shut down, and hydratesmelted before resumption of activity. In both saline and saline–heptane systems, gas hydrates crystallized in the presence of AFPs

appeared to require more time to completely dissociate in thehigh pressure stirred crystallizer, similar to that observed forchemical KHIs.55,56,59 These investigations were confirmed byDSC experiments that showed that higher temperatures were re-quired to completely melt the formed hydrate in the presence ofall the tested inhibitors.55,56 Perhaps analogously, AFPs adsorb toice and inhibit the melting of small crystals or ice recrystalliza-tion, which is normally seen at temperatures just below theequilibrium freezing point and has been experimentally seento contribute to the ”superheating” of ice crystals, as previouslynoted. The observation that hydrates formed in the presence ofAFPs required more time to fully dissociate need not cause indus-try any alarm, however, since in comparison to controls, only halfthe volume of hydrate formed in the presence of AFPs. This sug-gests to us that if AFPs were used in practical situations, it wouldbe better from a flow assurance point of view. Interestingly, thesaline and heptane experiments, which were originally designedto mimic conditions in some pipelines, had unexpected benefitsin that they have provided more insight into the understanding ofAFPs as inhibitors.

Formation and decomposition of hydratesAs mentioned, crystallization structures and melting of hy-

drates formed in the presence of KHIs formation may provideenormous insights into the nature of hydrate inhibition. HP-DSCexperiments allow melting temperatures to be very accuratelyassessed, and with this technique, the melting point of hydratesformed in the methane/ethane/propane mixture in the presenceof AFP III was 2 °C lower than hydrates formed in the presence ofchemical KHIs (all at 0.1 mmol/L61). With AFPs, the lower meltingtemperature was completely reproducible after several rounds ofmelting and recrystallization, again exhibiting no memory effect.Hydrate crystals formed in the presence of the chemical KHIs (PVPand Baker’s HIW85281) showed evidence of complex structureswith melting in multiple DSC peaks that varied with the refor-mation cycle over a surprising 6 °C range. Strikingly however,hydrates crystallized in the presence of AFP III showed decom-position as a single, presumably homogenous melting peak.Therefore, although the AFP ranked between PVP and the BakerKHI as effective inhibitors for crystallization of fresh solutions,based on the DSC assays, one could argue that the AFP would havereal utility under field conditions where reformation is likely.

Since some oil and gas companies seem to favor stirred reactorsas models for pipeline conditions, it was important to repeat theseexperiments with the natural gas mixture in this type of appara-tus. AFP III outperformed polyvinylcaprolactam (PVCap), anothercommercial KHI, but AFP I was more effective than AFP III atdelaying induction.62 Comfortingly, the delay assessed using thestirred reactor correlated well with the DSC experiments. Hydrategrowth, as judged by gas consumption, was reduced with all in-hibitors with reductions of 13% for PVP and AFP III, 30% for AFP I,and 54% for the new Baker KHI. Decomposition was achieved inthe reactors by heating and monitored by increases in pressure,duly accounting for temperature-dependent gas expansion.Again, there were striking differences between the chemical andbiological inhibitors tested. Whereas the hydrates formed in thepresence of commercial KHIs were harder to decompose and ap-peared to melt in two phases, possibly reflecting the presence ofboth sI and sII hydrates, in comparison those formed in the pres-ence of the AFPs melted more quickly and uniformly and indeedwere more similar to unamended control solutions.62 This consis-tency with the DSC results has strengthened our contention thatunlike the chemical KHIs, AFP incorporation into hydrates doesnot appear to stabilize the cage structures.

Further investigation of the crystal structures required addi-tional instrumentation including magnetic resonance imaging(MRI), X-ray powder diffraction (XRD), Raman and NMR spectros-copy, as well as the stirred autoclave and gas analysis.61–64 All of

Fig. 4. A comparison of the efficacy of different AFPs as hydrateinhibitors as monitored by differential scanning calorimetry (DSC).The cumulative heat of reaction for freshly made samples with0.1 mmol/L hydrate inhibitors, either as estimated by integrating theexothermic peaks observed during the DSC isothermal run at −12 °C.The samples tested are the same as those described in Fig. 3. Eachline represents 36 samples (adapted from reference 53, copyrightWiley-VCH Verlag GmbH & Co. KGaA; reproduced with permission).

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the investigated inhibitors showed a delay in hydrate nucleationand a reduction in hydrate growth, with the order of strengthtowards hydrate inhibition as follows: AFP III < PVP < AFPI < HIW8528 (all at 0.1 mmol/L). Gas phase analysis, Raman andNMR spectroscopy all indicated that the different classes of KHIscould have an impact on guest gas incorporation during hydrateformation. Interestingly, the methane concentration in the gasphase increased from 93% to 96% in the presence of the chemicalKHIs. As determined by 13C NMR spectroscopy, this increase in gasphase methane was concomitant with an overall increase in theheavier hydrocarbons in the hydrate phase. Enclathrated ethaneconcentration increased in the presence of the inhibitors, com-pared with controls, but especially with the chemical KHIs(�30% increase) compared with biological KHIs (�15% increase).Such compositional heterogeneity was further confirmed by lo-calized hydrate spectra (within each sample) obtained by Ramanspectroscopy. When Raman spectra were analyzed for differentlocations within the same sample that had been crystallized with-out inhibitors or in the presence of AFP III, no significant differ-ences were observed. However, the presence of the chemical KHIsresulted in substantial within-sample variation. This latter com-positional heterogeneity reflects hydrate structural changes inthe presence of the commercial KHIs. In this case, XRD patternsconfirmed the presence of sI and sII together, even though theformation of sII hydrates were expected. In contrast, any struc-tural changes in the AFP-containing hydrate crystals were so smallthat they were difficult to detect.64

Thus the complexity of the hydrates formed with commercialKHIs appears to explain their heterogeneous melting behavior,but what process leads to this change in the crystal structure? Toinvestigate this, the small and large cage occupancies of individ-ual gas guests were calculated from the 13C NMR data coupledwith a statistical thermodynamic model.65,66 Strikingly, the smallto large cage occupancy ratio of methane in the presence of thehydrate inhibitors increased from 4.1 to 5.2–5.7, compared withcontrols due to a �25% reduction in methane in the large cages(Table 2). Rather than methane, these large cages tended to hostheavier hydrocarbons, especially ethane. Overall, however, occu-pancy of the large cages was lower when hydrates were formed inthe presence of the AFPs (92%) compared with the chemical KHIs(97%–99%). This then explains the smaller increase in ethane oc-cupancy and in the hydrate composition analysis. We speculatethat this may be due to the availability of methyl groups associ-ated with the residues on the AFPs to occupy a gas position in thehydrate cage, effectively ”anchoring” the inhibitor to the crystaland thus reducing the overall number of ethane guests. The lowerlarge cage gas occupancy in the presence of the AFPs would alsoexplain the generally quicker dissociation of the formed hydratescompared with those crystallized in the presence of the chemi-cal KHIs. Furthermore, the preferential occupancy of heavierhydrocarbons in the chemical KHI-established hydrates likelyresulted in an increase in methane concentration in the gasphase.64,67 Shifts in the gas phase composition would then drivetheformationofheterogeneoushydratestructuresincludingmeth-

ane sI hydrate or methane–ethane sI hydrates, as was detectedusing XRD and NMR spectroscopy, rather than the theoreticallyexpected sII hydrate.

This multiscale investigation of hydrate crystallization andmelting underscores our contention that chemical KHIs and AFPsare distinctive in their inhibition activity. The existence and util-ity of biological inhibitors for hydrate inhibition are clearly evi-dent (Table 1). Moreover, since hydrates formed in the presence ofbiological inhibitors decompose earlier than with KHIs, thisshould be a valuable attribute for practical circumstances associ-ated with hydrocarbon recovery such as long shut in periods.

Towards a greener future: What is next?

Multiscale assessment of the performance of KHIsAssessment of the efficacy of KHIs is challenging due to the

complexity of the dependence of hydrate crystal formation anddissociation on a number of factors (Fig. 5). From a practical pointof view, perhaps the most important question is how can onepredict the performance of KHIs in the field using laboratoryobservations? At the 6th International Conference on Gas Hy-drates, Ulfert Klomp stated, “One may also think that there existlaboratory experiments and procedures the results of which canunambiguously be translated into the performance of an LDHI inthe field. Here it is argued that this may not be so.”68 Nonetheless,we feel that it is well worth the effort to try to predict KHI fieldperformance by devising laboratory assessment strategies thatwill evolve into robust engineering practice. No one type ofmethod will be satisfactory to industry, but as described here, wehave developed a multi-scale KHI assessment approach that issufficiently flexible to allow laboratory experiments at differentscales and with different techniques (Fig. 6). Although we favorstirred vessels for hydrate experiments at the macro scale, andHP-DSC to probe the energetics of the associated phase changes,additional instrumentation and techniques provide additional,valuable insights. A multiscale approach can easily accommodatechanges in protocol, as evidenced by the comparison of formationand decomposition of mixed gas (methane/ethane/propane) hy-drate in the presence of AFPs or different commercial KHIs. Theobservation that complex and multiple hydrate melting peakswere observed in calorimetric assays with the chemical KHIs,whereas AFP addition resulted in a single, less-stable hydratepeak,61 suggests that field operators using Baker’s H1W85281, forexample, would benefit from its superior inhibition activity, but ifreformation is likely, the elevated stability of its formed hydrateswould likely make its use more challenging. The multiscale ap-proach using a variety of instrumentation should also continue toprovide insight into the complexity of the hydrate structuresformed in the presence of different inhibitors,67 and future re-search in this field would benefit from this approach.

Table 2. Hydrate cage occupancy of individual gases obtained with 13C MAS NMR spectra in thepresence of two commercial KHIs (PVP and Baker's KHI, H1W85281) as well as AFP III, with significantdifferences shown in bold-face numbers (adapted with permission from reference 64; copyright, 2011,American Chemical Society).

Methane Ethane Propane

InhibitorSmall cageoccupancy �S

Large cageoccupancy �L

Large cageoccupancy �L

Large cageoccupancy �L

Total large cageoccupancy

None 0.96 0.23 0.26 0.48 0.97PVP 0.95 0.18 0.35 0.46 0.99H1W85281 0.93 0.17 0.33 0.49 0.99AFP III 0.97 0.17 0.28 0.47 0.92

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Investigation of catastrophic hydrate growth and thepotential for pipeline plugging

In the future, a multiscale assessment will likely prove useful toa lesser known phenomenon, the pipeline plugging potential.KHIs are known to effectively reduce the induction time, but oncehydrates start to form, their growth can be explosive.57,58,69 Thephenomenon has been explained by a change in hydrate morphol-ogy due to KHI inclusion, which facilitates the capillary move-ment of water across the formed hydrate layer from the aqueousphase to the natural gas–aqueous interface. It is noteworthy thataccelerated hydrate growth has not been observed when gas hy-drates form from ice rather than liquid water. This perhaps indi-cates that either the inhibitors cannot bind bulk pre-formed ice,or that hydrates formed in the presence of KHIs and ice are dis-tinct. Analogously, explosive growth is also seen when the equi-librium temperature of the AFP-ice interaction is exceeded,leading to uncontrolled growth of ice at these lower tempera-tures. Since water molecules join embryonic ice crystals at sitesnot as well covered by AFPs and crystallization heat is more easilydispelled from the edges, this leads to the characteristic needle-like burst patterns of the fish AFPs or the petal-like pattern burstof the plant and insect AFPs, for example. In our labs we haveapplied a multiscale approach to quantify what we have termedthe hydrate catastrophic index or HCI, but more work needs to bedone to describe this phenomenon. As well, until the potential forhydrate plugging is better understood and can be addressed, weadvise that caution be exercised when employing KHIs in the

field. Catastrophic hydrate growth in field applications would ob-viously have unwanted safety and economic consequences.

Alternative methods for the production of AFPsTo be useful for industry, AFPs must be available in quantity.

Although AFPs can be purified from the organisms that producethem, to date this has not been practical. Instead, the DNA se-quences encoding an assortment of these proteins have beendetermined, and these have been transferred to various host or-ganisms for the production of recombinant AFPs. At differenttimes bacteria, yeast, insects, algae, and plants have all been usedas hosts in our own labs, and other organisms may well have beenused elsewhere. The difficulty is that large-scale production of theoften temperature-sensitive, highly-folded, and sometimes post-translationally modified proteins is not straightforward. The di-rect use of bacteria that produce AFPs on the surface of theirmembranes is an attractive option that we have explored initiallyby the selection of ice and hydrate-associating extremophiles.70–73

A Chryseobacterium strain with secreted AFP activity was recoveredfrom soil, based on its impressive freeze–thaw resistance. When itwas tested for its ability to inhibit THF hydrate, it appeared prom-ising; there was an increase in THF hydrate induction time as wellas the presence of crystalline slurry, rather than the larger blocksseen in the absence of these bacterial species. However, results ina stirred autoclave pressurized with methane were not impres-sive. Although the bacteria statistically delayed nucleation time asmuch as PVP addition, there were large variations between prep-arations.74 Indeed, a non-cold tolerant, biofilm-producing E. colistain also reduced methane hydrate growth by 50%, but againshowed batch to batch variation, which would be unacceptablefor field applications.

An alternative strategy that we have pursued involves display-ing better-characterized AFPs on the surface of vector E. colistrains. Recombinant bacteria are incorporated into polycrystal-line hydrates, but we have recently discovered that these strainsare not ”decorated” with AFPs over their entire outer membrane,but rather AFP display is restricted to the polar regions of thecell.75 Unfortunately, such restrictive protein display would re-duce the probability of interaction with the hydrate surface. Thus,although our labs have worked hard to produce a bacterial hy-drate inhibitor preparation suitable for testing in pipelines andwill continue to pursue this effort, it may not be a practicallyachievable goal in the short term.

Fig. 7. Monte Carlo simulated gas hydrates grown withoutinhibitors and showing an octahedral morphology (left) and whenvolumes representing AFP I molecules (right) were added to thesimulation. It must be noted that morphologies could be changeddepending upon the programmed probability of binding, and theorientation of the inhibitor molecules. Gas molecules are shown insmall light dots, water cages are dark red, and AFP I shown as whiteoblong structures (image on the right modified from reference 79).(See online version for colour.)

Fig. 5. A diagram showing some of the many potential factors thatinfluence gas hydrate formation and dissociation, all of which needto be considered.

Fig. 6. A multiscale approach for laboratory investigation ofhydrate inhibitors is recommended, and this is made more complexby the experimental incorporation of conditions mimicking thosefound in the field.

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Computer models of AFPs and hydratesThe understanding of how AFPs interact with gas hydrate nu-

cleation and growth is important to assist in the development ofnew molecules or other strategies to reduce the probability ofhydrate formation in the field. Monte Carlo (MC) or moleculardynamics (MD) models may provide important insights into AFP-mediated hydrate inhibition. Previously, MD simulations havebeen used to study the interaction of CfAFP and TmAFPs withice.76,77 These show that water molecules around the ice-bindingsurface of the AFPs are compatibly arranged so that they have a”good fit” to the grooves found on the rough ice face. In contrast,water molecules in other regions of CfAFP did not match with theice lattice coordinates.76

MD simulations are precise but constrained by computationalresources, whereas MC approaches use probabilities in exchangefor scale and thus can be used to simulate the growth of large,three-dimensional crystals. As previously noted, MC techniquessuggested that AFP activity was dependent on ice-binding orien-tation.37,78 When MC computations were applied to the interac-tion of sII gas hydrate with the commercial inhibitor, PVP, growthreduction was associated with irreversible binding of the KHI onthe crystal surface, and since these molecules were more likely tooccupy the central portions of hydrate cages, they therefore re-duced the probability of gas incorporation.79,80 The morphologiesof the simulated hydrate crystals were dominantly and appropri-ately octahedral (Fig. 7). These also showed bound proteins whenAFP I was added to the MC simulations. Indeed the in silicocrystals, which were considerably smaller than experimentallyderived crystals (even though they contained thousands ofsimulated water molecules and hundreds of AFP molecules), nev-ertheless still showed a striking similarity to single laboratory-grown THF hydrate crystals and their bound AFPs (compare Figs. 2and 5). Hydrate growth inhibition was also achieved in the bestsimulations.79

In silico models are often hampered by the unknown solutionstructures of commercial KHIs. Nevertheless, MD simulations

have utilized PVP and PVCap in contact with hydrate surfaces81–82

Models showed charge distributions on the PVCap molecules thatcoincided with water molecules on the simulated non-standard sIIhydrate surface, resulting in strong binding to the empty cages ofthe {100} surface.82 However, it is not clear if the same mechanismwould hold for the association of KHIs to the more usual sI meth-ane hydrate surface with its smaller cages. In contrast to the dif-ficulties associated with commercial KHIs, structural informationis available for many AFPs (Fig. 1). When these are used in MDsimulations, growth inhibition can be more easily studied. Forexample, a typical configuration of such simulation is afforded byTmAFP in contact with sI methane hydrate (Fig. 8). MD simulationsof AFP I and the sI methane hydrate surface showed that similar tothe protein’s interaction with ice, adsorption is driven by hy-drophobic interactions between water and the protein, with hy-drogen bonding providing added stability. The combination ofamino acid groups containing methyl groups on the protein chaincomplements the hydrate surface structure and facilitates surfacebinding (Fig. 9). The spacing between these hydrate-binding resi-dues is appropriate for the hydrate edge, in the range of 5.4 to7.2 Å.

We believe that such investigations of the interactions of AFPswith the hydrate surface should allow the determination of thestructural/functional relationship for inhibitory action and there-fore facilitate the rational design of a ”super inhibitors”. Suchmolecules would have the benefits of AFPs but could also outper-form the current commercial KHIs.

ConclusionsOrganisms that survive low temperatures can produce AFPs,

which we have shown have efficacy against sI and sII gas hydratesas well as ice. These represent green hydrate inhibitors, which aswell as being non-toxic, slow hydrate induction time, limit thehydrate mass formed, reduce or eliminate memory effect, and inmixed gas systems melt as a single hydrate type. As such they have

Fig. 8. A typical configuration for the investigation of the mechanism of hydrate growth inhibition in the presence of AFP molecules. In thiscase, a 7 × 7 × 4 hydrate block is shown on the left with contact to the solvated mealworm beetle AFP (TmAFP) via hydrogen bonds.

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several attributes that should be enormously beneficial to indus-try. Considering that the journey to investigate AFPs as potentialhydrate inhibitors started only 15 years ago, we are pleased withthe progress. The 8th International Conference on Gas Hydratesrecognized John Ripmeester with a Lifetime Achievement award.The citation read in part, “…three achievements stand out: dis-covery of structure H hydrate, discovery of numerous new hydrateguest molecules, and pioneering NMR hydrate methods, that pro-vide our foundation for basic hydrate understanding”. We urgethat a fourth legacy be added to this already impressive list, that ofhis pioneering work with green inhibitors. John’s role in marry-ing the rigor of experimental and theoretical chemistry withthe inherent complexity of biology and applying this to an impor-tant industrial problem has contributed enormously to the suc-cess of this emerging area of inquiry. Of course, there are manychallenges to come, not the least of which is to overcome thehurdle of sufficient production of these AFPs or alternatively AFPmimics. One can only hope, however, that in another 15 years, ourefforts will have paved the way for useful solutions in the field.

AcknowledgementsNatural Sciences and Engineering Research Council of Canada

and National Research Council are acknowledged for fundingmuch of the research described here. We especially thank all ourtalented students at Queen’s University and The University ofBritish Columbia who have worked so diligently over the years, aswell as valued collaborators at those institutions and at NRC.Dr. N. Wu is thanked for providing information from the ICGHcommittee. Lastly, we want to acknowledge our enormous debt ofgratitude to John Ripmeester, without which none of this wouldhave been possible.

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Fig. 9. The �-helical fish Type I AFP (AFP I) is shown as a transparent cyan blue ribbon. Amino acid residues are shown by lines and those onthe presumed hydrate-binding surface are emphasized by thick bars. Methyl groups of the amino acids believed to be involved in binding aredesignated by small spheres, with the colour scheme as for Fig. 1. (See online version for colour.)

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