Progress 09/01/19 to 06/30/22
Outputs
Target Audience:The target audiences for this project are US growers, greenhouses, plastic product manufacturers, consumer brands, retailers, waste management stakeholders, lignin suppliers, biopolymer suppliers, US regulators, and the general public (especially those concerned with the sustainability of our food system). We want US growers and greenhouses to see how biodegradable and compostable materials can be used in their operations and improve the economics and sustainability of their business by reducing the time and money they spend disposing of plastic waste. For plastic product manufacturers, consumer brands, retailers, and their supply chain partners, we want to share our insights into bioplastic degradation with them so that we can work with these companies to provide better materials for our farmers. As part of our supply chain, we want lignin and biopolymer suppliers to see our results in this project and work with them to improve future versions of our products. For US regulators and waste management stakeholders, we want to share these results so that US policies and practices reflect the best science. Current regulations restrict the use of biodegradable and compostable materials, especially for USDA Organic growers. As we learn more about how bioplastics degrade, we want to share these findings with regulators so that US policies reflect this change in knowledge. For the general public, we want to share our findings so they see the US government is funding work to investigate more sustainable materials and practices in our food system, like adopting biodegradable plastics that mitigate the use of single-use petroleum based plastics that are bound for landfills. Changes/Problems:Major changes We experienced significant delays and challenges during the last 2 years due to COVID which forced us to shift focus during Phase I. Without access to facilities to conduct experiments, we had to apply for multiple no-cost extensions to complete the proposed work. Fortunately, we used this time and support from the TABA award to engage with commercial partners to better understand the needs and specifications for this technology. Additional Findings While the challenges presented above regarding accurate and precise measurement of enzymatic and microbial hydrolysis remained throughout the project, we did start to identify and validate additional mechanisms that influenced degradation kinetics of polymers in soil. In particular, we found and confirmed in a parallel Phase I SBIR from USDA, as well as with primary feedback from consumers using biodegradable products in soil environments, that degradation kinetics were severely reduced in nitrogen limited soils. Our subaward PI and key collaborator on this grant, Dr. Jennifer DeBruyn, has published on this topic during the last few years as part of a separate initiative evaluating the performance of biodegradable mulch film degradation in on-farm trials. Her work highlighted not only degradation rates, but the density and speciation of microbial colonies that populated the surface of those materials. Using this information and our parallel work, we began to explore the concept of adding key microbial nutrients - namely nitrogen - directly to our compounded polymers in an effort to prepare for future degradation testing and kinetics determination in N-limited environments. The exploration of nitrogen additives directly compounded into polymers, including mobius' lignin-based degradable polymers, will also provide useful data on how lignin impacts the release of these nitrogen compounds from said polymers. We plan to explore this mechanism of degradation control and nitrogen release, along with other opportunities to embed beneficial compounds into our lignin-based polymers, in our Phase II SBIR application. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?We have been in contact with industry stakeholders throughout our Phase I project and have shared some of the preliminary results on our nutrient release and plant growth trials. In parallel with our Phase I project, we have been communicating with these stakeholders and customers to better understand the specifications that are required for a minimum viable product, such as mechanical properties, manufacturing properties, and nutrient release profiles. We plan to continue these conversations during Phase II and to disseminate more information after we have sufficiently protected our intellectual property. Business Development & Commercialization In addition to the technical goals of this project, we accomplished several goals through the TABA Program. We employed Larta Inc to perform market research, a patentability assessment, and review our strategy for commercialization. One of the key takeaways from these activities was that the route to create a robust intellectual property strategy for this technology would be extremely challenging, mostly due to the fact that we are using wild-type enzymes and commercial compost teas. In addition, through the customer discovery and stakeholder outreach conversations we had through this project and our parallel USDA Phase I Award, we saw an opportunity to apply the findings from this project toward controlled-release fertilizer applications. Moving forward as we prepare our USDA Phase II SBIR proposal, we will be targeting these applications for this technology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Project Goals During the project, we identified additional tasks and subtasks to support these specific aims. Notably, we needed to evaluate the activity of commercially available enzymes and compost teas to select candidates for work plan experiments. We also needed to optimize a method to evaluate changes in the chemical structure of polymers by enzymes and compost tea. One major challenge was that we were unable to complete the work outlined in Specific Aim 3, due to the difficult nature of observing the system in vermiculite. But this setback created new opportunities for us to investigate. We experienced significant delays and challenges during the last 2 years due to COVID, which limited our access to laboratory facilities. We used this as an opportunity to focus on business development, customer discovery, market research, and patentability analysis, with support from the TABA program. Through these activities, we identified slow-release and controlled-release fertilizer products as a potential application of this innovation. Accomplishments and Results Below are our Accomplishments & Results from the Phase I project, broken down by Specific Aim and Other Milestones. Specific Aim 1: Assess the hydrolytic power of singular and mixed aqueous enzymatic treatments on biodegradable polymer substrates For the first step in this aim, we evaluated 5 commercially available enzymesfor their ability to hydrolyze biopolymers. To evaluate the activity of these enzymes, we used a Fluorescein Diacetate Assay (FDA). This method was conducted over 28 days and measured the fluorescence output (490 nm). In parallel with the evaluation of commercially available enzymes, we also evaluated commercially available compost teas for their activity and composition. We started with 5 commercially available compost tea kits (a bacterial compost tea, a fungal compost tea, a mixed fungal/bacterial compost tea, an Arbico compost tea, and a True Value compost tea). To evaluate these compost teas, we characterized their pH, carbon:nitrogen ratio, total organic carbon, electroconductivity, total dissolved solids, dissolved oxygen, and activity (using the FDA Assay described above). From this analysis, we selected the Fungal compost tea and Old Truck as candidates for later experiments. At this stage, we also started work on developing Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) methods to measure polymer hydrolysis from enzymes and compost tea. For the hydrolysis experiments, we added individual and mixed enzymes in solution to 96 well plates that contained milled polymers, including commercial polymers labeled asbiodegradable in soil,Kraft Lignin polymer alloys (formulated by mobius) targeted for soil degradation, low-density polyethylene (LDPE, as a negative control), polylactic acid (PLA) as a polymer that is not readily soil degradable but instead industrially compostable, and cellulose (as a positive control). To evaluate the effect of enzymes on polymer hydrolysis, we used FTIR and NMR to measure changes in chemical structure for the polymers. Our hypothesis was that these enzymes had an affinity for hydrolyzing ester bonds found in most commercial biodegradable or compostable materials. In the initial hydrolysis experiments, we could not clearly identify the differences in polymer hydrolysis rates. We continued investigating this under the second Specific Aim. Specific Aim 2: Determine the degree and rate of biodegradation and mineralization of biodegradable polymers in an aqueous enzyme-enhanced compost tea As mentioned above, when we first conducted the polymer hydrolysis experiments with enzymes or compost tea, we could not identify clear or significant changes in the rates of hydrolysis or changes in the chemical structure of the polymers. Before continuing with our planned work plan, we wanted to further investigate and refine the analytical methods we were using. The primary method we used to identify changes in chemical structure was FTIR. We took the FTIR spectra data collected in Aim 1 and applied various statistical methods on the data to identify trends and differences in the data, including Univariate Analysis. Multivariate Analysis, Principal Component Analysis, Linear Discriminant Analysis, and Partial Least Squares Analysis. In some of the analyses, we observed clear trends, such as clusters of data at specific time points during the hydrolysis experiments, but these did not clearly align with the experimental variablesSimilar to the FTIR data with enzymes, we also observed unclear results from the compost tea experiments. In the FTIR spectra, we observed several significant changes in chemical structures that clearly aligned with specific compost teas. Specific Aim 3: Determine the degree and rate of biodegradation and mineralization of biodegradable polymers treated with an enzyme-enhanced compost tea in a vermiculite- based artificial soil There were two challenges that prevented us from completing this specific aim. First, the challenges and delays we faced from COVID-19 reduced the amount of time we had to complete the work plan tasks. Also, due to the complexity of the system we are studying, we were unable to replicate these experiments in artificial soil conditions. We instead used the results from the previous experiments to inform upon our polymer formulations and specifications that may be beneficial for product applications, notably biodegradable horticultural containers and controlled-release fertilizers.
Publications
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