Towards the selection of a produced water enrichment for biological gas hydrate inhibitors.

Economic concerns associated with the recovery of non-conventional hydrocarbon reserves include unexpected ice as well as ice-like gas hydrate formation. Antifreeze proteins (AFPs) inhibit ice growth, and experiments with fish, plant, and insect AFPs have shown promise of effective gas hydrate inhibition in lab-scale experiments. If produced on an industrial scale, AFPs could provide a more environmentally friendly alternative to kinetic inhibitors, but a large-scale production of these AFPs is not currently feasible. We believe that these difficulties could be surmounted by the production of microbial AFPs, but to date, only a few such proteins have been identified and purified, and none of these are associated with hydrocarbon reserves. Here, we have used ice-affinity and freeze-thaw stress to select microbes derived from oil and gas formation water, or produced water, as a source of anaerobic microbial communities. Ice-affinity successfully incorporated anaerobic bacteria under aerobic conditions, and the mixed culture had ice-associating properties. Under these conditions, ice-affinity selection does not result in cultivatable isolates, but similar, cultivable microbes were obtained following freeze-thaw selection under anaerobic conditions. Since these mixed cultures inhibited the growth of ice crystals, they also have the potential to inhibit hydrate growth. Overall, freeze-thaw selection provides a promising first step towards the isolation of microbes capable of the inhibition of ice and gas hydrate growth, for possible application for energy exploration and recovery at high-latitudes and in-deep, cold waters.