Progress 07/01/21 to 02/28/22

Outputs
Target Audience:The target audience for this project includes farmers, manufacturers looking to decrease their dependence on petroleum-based plastics, and consumers who have become increasingly skeptical of GMO products. Farmers will be able to expand the use of agricultural land since pennycress can be "sandwiched" between alternating soybean and corn crops. This will also increase their ability to maintain productive land management creating another income source for our nation's farmers. Meredian Bioplastics research team is seeking to utilize the pennycress oil produced by Dr. David Marks' team at the University of Minnesota as a carbon source to produce our proprietary PHA. Our research hopes to confirm that the oil produced by the double-mutant strain will provide a carbon source comparable in performance to canola oil. Once this is established, manufacturers who are seeking to decouple the use of petroleum-based plastics will have options for using biodegradable polymers to meet their plastic needs. Additional benefits include pennycress oil as a non-edible oil so that the food chain supply will not be depleted by the diversion of oils such as canola or peanut. Dr. Marks' hybridization methods have produced a plant that is completely free of GMO modifications; this too, increases the target market to companies and consumers who are increasingly skeptical of genetically modified foods and products. Changes/Problems:The research progressed as expected asoutlined in our original grant proposal. I contacted our Program Director, David Songstad, with the following information and he advised me to include this in our REEport. Our total funding was not utilized due to two factors: 1) We had a 15,000.00 set aside for Dr. Marks at the University of Minnesota for growing, pressing, and providing the pennycress oil to us. Midway through the project, it was discovered that Dr. Marks would be unable to accept these funds due to a possible conflict of interest with the University and other research obligations, so these funds were left unspent. 2) We also had 37,041.07 unspent of our 99,488.00 funds. We were very conscientious of time spent and billing appropriately. This was our first federally funded project and we simply overestimated the employee time needed. The research was completed as proposed and all objectives were met. What opportunities for training and professional development has the project provided?Meredian Bioplastics usually has university as well as high school groups in for touring to discuss future job opportunities and the research underway at our company. However, given the CDC guidelines during the COVID pandemic, our facilities have limited this type of education. We are hopeful to renew these training and educational opportunities as restrictions continue to be lifted. Professional development has been enriched by our affiliation with the University of Minnesota and the exchange of research data as this project moves forward. Meredian also is a training facility for the University of Georgia, accepting engineering interns on a regular basis. How have the results been disseminated to communities of interest?At this point, our results have been discussed with our partners at the University of Minnesota and potential commercial partners such as Mars and PepsiCo. These companies are very interested in the outcome of this research and the possibility of biodegradable polymers produced from non-edible oils such as pennycress. What do you plan to do during the next reporting period to accomplish the goals?Our goals for this first-year funding have been met. We are hopeful to receive funding for Phase 2 of this grant to continue our study of pennycress oil in a scale-up capacity. We will be submitting a proposal for Phase 2 funding in April 2022.

Impacts
What was accomplished under these goals? Objective #1- (See above)Samples of each oil were sent to Anresco for gas chromatography (GC) analysis. Results for Objective #1 The fatty acid profile obtaineddemonstrates that while the monounsaturated fat content is similar, the actual fatty acids present are very different. The wild-type pennycress oil has 50% less oleic acid than the high oleic version of pennycress. The wild type also has an approximately 38% higher ratio of erucic acid than the high oleic version. The differences in these two fatty acid concentrations have an impact on the utilization of these two carbon sources in the production of polyhydroxyalkanoates (PHAs). The fatty acid profile of the high oleic pennycress oil is similar to the profile of canola oil. The secondary fatty acids are all comparable to those seen in commodity canola. The fatty acid distribution is indicative of a triacylglycerol mixture that provides the necessary carbon for the optimal fermentation process of Meredian's proprietary bacterial strains. Objective #2 - (See goals above) Bench-scale fermentation was set up using two of Meredian's proprietary microorganisms. Seven three-baffled 250 milliliter flasks were prepared with minimal media for each bacterial strain. Fifteen percent of the total volume in three flasks was high oleic pennycress (HOP) oil, three flasks contained fifteen percent wild-type (WT) pennycress and one flask contained fifteen percent canola oil. All flasks were autoclaved at 121°C at 17 psi for 25 minutes. Ten percent of a proprietary microbial strain was added to each flask. The flasks were incubated on a rotary shaker at 300 rpm, 30° for 72 hours. The bacterial cells were separated from the media by centrifugation. The PHA was enzymatically extracted, cleaned, and dried. The PHA powder was sent to the analytical lab for analysis. The steps taken for strain #1 and strain #2 were identical. Results for Objective #2 The strain that performed the bestis Meredian's higher mole percent strain. Following fermentation, the broth from the flasks was processed without organic solvents. The powdered PHA from each flask was analyzed using gel permeation chromatography (GPC) for molecular weight, nuclear magnetic resonance (NMR) for mole percent and Fourier-transform infrared spectroscopy (FTIR) for percent cell protein. The data are seen in Table 4 below. Sample GPC* NMR** FTIR (CP%)*** HO Pennycress #1 Average mol wgt increased by 20% v. canola Expected mole % Equivalent clean material HO P Pennycress #2 Average mol wgt increased by 20% v. canola Expected mole % Equivalent clean material HO Pennycress #3 Average mol wgt increased by 20% v. canola Expected mole % Equivalent clean material WT Pennycress# 1 Approximately ½ molecular weight of HO 50% increased mole % Presented challenges in our DSP process WT Pennycress #2 Approximately ½ molecular weight of HO 50% increased mole % Presented challenges in our DSP process WT Pennycress# 3 Approximately ½ molecular weight of HO 50% increased mole % Presented challenges in our DSP process Canola Control Performed as expected Expected mole % Performed as expected Table 4 - Analytical Data for Pennycress Oils from Shake Flasks *Molecular weight is determined by gel permeation chromatography (Shimadzu ABC 830-11). **The mole percent C6 (3-hydroxyhexanoate) is determined by nuclear magnetic resonance (Varian (Agilent); Model MIR1012W054. ***Percent cell protein data are determined by Fourier Transform Infrared Spectroscopy ThermoScientific Model #Nicolet I S10. Based on cell pellet size, which is indicative of titer levels in shake flasks, it was determined that Meredian's higher mole percent strain would be utilized in the scale-up to ten-liter vessels. Objective #3 - (See goals above) Note: In the proposal, Meredian planned to run ten-liter vessels; however, due to oil constraints, five-liter fermentations were performed. Fermentation began with two seed flasks prepared using frozen stock and two-liter flasks, set to shake incubate at 30° C and 300 rpm for 17.5 hours. Production media was made then transferred into the two prepared five-liter vessels. pH and DO probes were checked, cleaned, then calibrated according to proper guidelines. The vessels were then moved into the Steris autoclave and heated for 45 minutes with a target of 121C (no-load probe). After autoclaving, the vessels were positioned onto Bioflo 3000, stations 3 and 4 (see figure 1). The vessels were allowed to cool to a safe handling temperature before attaching all lines and probes. Once final preparations were completed, and the 17.5-hour mark was reached on the incubating seed flasks, the broth cultures were aseptically transferred into all vessels. The appropriate run conditions were set and activated. The two five-liter vessels were then allowed to ferment. At 48 hours, the final samples were taken and sent to the Analytical lab for analysis. Results for Objective #3 The BioFlo experiments ran as expected. Adjustments to volumes were made proportionately to account for a limited supply of high oleic pennycress oil. Due to unforeseen circumstances, Meredian was not able to obtain the high oleic cold-pressed oil from the previous year's crop. An increase in the number of fields planted in 2021, will increase the supply of high oleic pennycress for Phase II studies, if funded. The BioFlo analytical data is presented as a section of the Objective #4 results, see Table #5. Objective #4 -(See goals above) The polyhydroxyalkanoate (PHA) produced from both the high oleic pennycress oil and the wild-type pennycress oil was extracted from the cell culture broths of the BioFlo vessels without the use of organic solvents. The powdered PHA was then analyzed by GPC, NMR, and FTIR. The results are shown below in Table #5. Results for Objective #4 Sample GPC* Titer g/L PHA NMR** Mole % C6 FTIR (%cp) remain protein High Oleic PC 830kD 53% of canola 1.22% of canola Equivalent to canola Wild Type PC 848kD±158 94% of canola 94% of canola Equivalent to canola Canola 557±275kD Expected yield Expectedmole% Expected level Table #5 *Molecular weight is determined by gel permeation chromatography (Shimadzu ABC 830-11). **The mole percent C6 (3-hydroxyhexanoate) is determined by nuclear magnetic resonance (Varian (Agilent); Model MIR1012W054. ***Percent cell protein data are determined by Fourier Transform Infrared Spectroscopy ThermoScientific Model #Nicolet I S10. Objective #5 - (See goals above) The properties and titers of the PHA produced from the wild-type and high oleic pennycress oils are summarized in Table 5. Due to a limited supply of the high oleic pennycress oil, there was only one run in a BioFlo vessel, while the wild-type pennycress oil was run a total of five times. The canola control was run twice for comparison. Results for Objective #5 The PHA derived from the high oleic pennycress oil demonstrated a decrease in titer of 53% however, there was an increase in the amount of hydroxyhexanoate that incorporated into the polymer compared to PHA produced from canola oil. The PHA produced from the wild-type pennycress demonstrated 94% of the mole percent hydroxyhexanoate incorporated compared to canola and produced PHA at a rate that was 94% of the expected yield for canola. Both pennycress oils downstream processed well, indicating that Meredian's isolation and extraction techniques currently in use also work for these carbon feedstocks.

Publications