Extraction

Overview

After passing through the external cell separator, the liquid stream from the bioreactor will contain dissolved salts, PHB granules, unused volatile fatty acids and other solutes. Commercially, PHB extraction is most commonly carried out using organic solvent extraction. However, the disadvantages of solvent extraction include large volumes of toxic and volatile solvents such as chloroform (Kunasundari & Sudesh, 2011). Therefore, solvent extraction, would not be feasible on Mars due to their toxicity and the large volumes of solvents required, since they would have to be shipped.

We partly tackled the challenge of PHB extraction by engineering our PHB-producing E. coli to secrete PHB granules. However, the next challenge was retrieving the secreted PHB granules out of the media. Based on a literature review, we anticipated PHB granules to be 20-60 nm in size (Rahman et al., 2013). Given the size of the particles, we expected that the first step in the extraction process would be agglomeration or flocculation to obtain larger particles, which could then be separated using centrifugation or sedimentation. Following that, we explored a number of methods to agglomerate the PHB nanoparticles and subsequently separate them from the medium including dissolved air flotation, chemical coagulation, and electrocoagulation.

Design Options

Chemical Coagulation and Centrifugation

One of the first design options we tested in the lab was chemical coagulation using calcium chloride. It was shown in the literature that PHB particles have an isoelectric point of pH 3.5. Therefore, when in our supernatant which had a pH of 5, PHB would be negatively charged (van Hee, Elumbaring et al., 2006). By supplying positively-charged ions to the solution, it would be possible to neutralize the charge of PHB nanoparticles and have them agglomerate to form larger particles. Previously, experiments with PHB released from E. coli cells found that dications, especially calcium ions, work well to agglomerate PHB (Resch et al., 1998). Therefore, we used calcium chloride as the coagulant in our lab experiments. We demonstrated that the addition of calcium chloride followed by centrifugation increases the amount of PHB recovered as opposed to centrifugation by itself. More about the methods of these experiments can be found here.

The large-scale process that we envisioned would have had the liquid stream from the PHB fermentation unit pass into a mechanical flocculator, where it would be mixed with calcium chloride before being passed into a centrifugation unit. The PHB would be settled out and the remaining liquid could be used to recover water. However, we encountered several setbacks when planning to use this process on Mars. Firstly, there was the issue of obtaining calcium chloride on Mars. It would either have to be shipped to Mars or produced in situ. Currently, there has been some investigation into producing metal oxides and oxygen from Martian soil (Badescu, 2014). This process could potentially be modified to produce calcium chloride; however, that investigation was beyond the scope of this project.

Unfortunately, a crucial problem with the flocculator and centrifugation design was the potential of the calcium ions remaining bound to the PHB particles. We spoke to Dr. Nashaat Nassar, a professor in the Chemical Engineering department at the University of Calgary, and he confirmed that this would likely be the case. He suggested we wash the recovered PHB with acidified water to remove the calcium chloride. However, this introduced a number of challenges including risk of resuspending the PHB once again and breaking up the PHB agglomerates, essentially undoing all the work we did to separate the PHB.

Therefore, for these reasons we decided not to pursue chemical coagulation as our method of separation.

Electrocoagulation

Next, we looked at electrocoagulation (EC). In principle, it works similarly to chemical coagulation, but in this case the ions causing the coagulation are supplied by sacrificial anodes which are usually made from iron or aluminum (Tian et al., 2017). EC has a number of advantages such as use of simple equipment, easy operation, a shortened reactive retention time and no chemical additions (Ozyonar & Karagozoglu, 2017).

Our lab experiments with electrocoagulation which is described here, and showed that while it was possible to settle out PHB using electrocoagulation, we obtained a lot of sludge when from the synthetic feces present in the sample. It proved very difficult to extract PHB from this sludge. Since electrocoagulation was not selective for PHB in our synthetic feces samples we decided not to pursue electrocoagulation for our process on Mars.

Dissolved Air Flotation

Literature has demonstrated the successful use of dissolved air flotation (DAF) to separate PHB with an overall yield of 86% in lab-scale experiments (van Hee, Elumbaring et al., 2006). In DAF, air bubbles are created by injection of air-oversaturated water. With our proposed process, compressed Martian air will be passed into a dissolved air vessel where it will be dissolved in water before being injected into a flotation column. The fluid stream from the bioreactor will also be fed into the flotation column. In it, the passage of air bubbles would cause PHB nanoparticles to float up to the top. Eventually, the PHB-rich top phase will be removed and passed into a dryer, similar to ones currently being used in the industrial production of PHB.

DAF has several advantages, the most significant of them being that it does not use up large quantities of toxic chemicals (Kunasundari & Sudesh, 2011). Every other design option we examined would require shipment of chemicals such as coagulants or solvents. DAF would also work well in conjunction with the secretion system which produces PHB nanoparticles that would otherwise need to be agglomerated before separation via either sedimentation or centrifugation. However, there are also several drawbacks with DAF. First of all, it would have to run in batch mode, perhaps even requiring several consecutive flotation steps (van Hee, Elumbaring et al., 2006). The process also has not been used in scaled-up applications yet and PHB yield is comparatively low when compared to solvent extraction. We would also need to recycle the water we recover in our process into the dissolved air vessel to ensure it is an efficient process fit for space colonization.

Recommendations for further experiments would be optimizing batch duration and designing a pilot large-scale system. When designing a large-scale system, experiments with increasing pressure in the dissolved air vessel could be conducted, since a higher pressure would lead to higher dissolved air concentration and increase the volume fraction of air bubbles (van Hee et al., 2006). This, in turn, could potentially improve the yield of PHB. The batch flotation system could also be turned into a continuous process with the PHB-rich top phase being removed continually. It is expected that in continuous flotation that the top phase PHB concentration will be higher than in batch flotation (van Hee et al., 2006).

ESM Estimation for Dissolved Air Flotation System

Based on the experiments we conducted in the lab and literature search we decided to implement the DAF system in our final process on Mars. We then calculated ESM for the DAF to determine feasibility on Mars.

The air compressor was modeled after small commercial models (The Home Depot, 2017) that would be able to supply the pressure that we anticipate would be required, based on lab-scale experiments described in (Okada, Akagi, Kogure & Yoshioka, 1990).

Similarly, we modeled the flotation column and dissolved air vessel for our system based on the volumes described in (Okada, Akagi, Kogure & Yoshioka, 1990).

For the drying machine, we scaled up the system used to dry PHB in (Godoi, Pereira & Rocha, 2011) using a commercially available machine as a reference (2017).