Progress 09/01/20 to 04/30/22
Outputs
Target Audience:This project addresses the Biofuels and Biobased Products (8.8) Topic of USDA NIFA SBIR Program. As such, it responds to the USDA priorities, including 1. Addressing climate change via climate-smart agriculture and forestry, and 3. Creating more and better market opportunities. Furthermore, this application is directly related to one of USDA's Strategic Goals, Strategic Goal 4: Facilitate Rural Prosperity and Economic Development. More specifically, we target to build an innovative Waste to Bioproduct (W2B) technology platform using organic wastes for low-cost production of biodegradable polymers, which have wide applications and an enormous market opportunity. This project will contribute to agriculture and rural development by creating new economic development opportunities, achieved by overcoming economic barriers of biobased products and shifting from the dependence on petroleum as the raw material to the use of renewable waste biomass. Our W2B technology is designed to convert the wastes to biodegradable plastics so that "to kill two birds by one stone". This approach is especially appealing to customers and companies who are passionate or committed to creating a circular economy. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this project, we have synergized the strengths of small business company and public university to offer the unique opportunities for training and professional development. The opportunities included the collaborative research, inter-laboratory training, and other outreach and extension. We have reached out to the industries, including food processing companies and commercial users of plastics. In addition to the research activities, the student and staff have gained the training experience and professional development. How have the results been disseminated to communities of interest?We have identified and reached out to the three types of customers who are interested in our technology: downstream users, existing manufacturers, and waste management companies. We have reached out to the representing companies and they showed interest in our technology. One of the companies seeks packaging technology innovation to reduce food waste in future has also showed interests in our technology. As interest in biodegradable plastics increases, plastics manufacturers have started looking for more sustainable products to meet the growing market demand and to retain their competitive edge. Additionally, existing bioplastic producers will look for new ways to enhance their technology portfolio and to reduce cost. The other type of our potential customer is food processers and waste management facilities who typically have well defined waste streams with relatively homogeneous composition. We have initiated the discussions with the companies which manage municipal organic wastes at their respective composting facilities. More values are added downstream by others when the bioproduct is made into consumer products. If succeeds, our technology should be attractive to these facilities as potential customers. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our Phase I progress in achieving performance targets is summarized according to the proposed two objectives. Objective #1. Metabolic engineering of yeast strains for overproducing biopolymer and recovery of produced biopolymer from recombinant yeast cells After genetic modifications, the final strain could produce the biopolymer molecule with a very high titer (g/L) and with over 35% of dry cell weight (DCW) under shake flask culture conditions. More than ten generations of the strains have been iteratively developed and further tested for their performance in producing the targeted product. The copolymer could also be produced by the strains from the mixture substrates. The engineered strain showed a nine-fold increase in titer for production of biopolymer under similar flask culture conditions compared with a previously engineered strain. Reaching our target indicated that we have developed one of the most productive strains reported for this product. We developed a novel process for the recovery of biopolymer to replace the traditional energy-intensive solvent-based extraction. Using our approach, the purity and recovery rate reached over 90% and 85%, respectively. We are working on the characterization of the produced biopolymers using the facility at Composite Materials & Engineering Center (CMEC) at Washington State University (WSU). Objective #2. Maximizing biopolymer production from wastes by fermentation The titer of product reached 20-40 g/L by the engineered strain in a 1-liter fermentor with pH control. The food waste was collected from the cafeteria, and further treated in a 7.5-liter bioreactor with a 5-liter working volume. Techno-economic analysis (TEA) was conducted to evaluate the economics of our platform technology. The mass and energy balance of waste treatment and fermentation processes were calculated with the Aspen PlusTM process engineering software. The development of waste treatment process and fermentation model was based on data from literature and our results from Phase I as the baseline. The cost values were estimated using Aspen Process Economic Analyzer v8.8. We also analyzed the investment and capital cost by model simulation. The TEA model will be further tailored in Phase II project for system design and analysis.
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Progress 09/01/20 to 04/30/21
Outputs
Target Audience:To realize the commercialization potential of our technology, we have made significant progress in identification of market opportunities. Based on the market survey, we have developed a discovery list including 15 manufacturers, customers and end-users with potential interests of the target products and the devoloped technology. By working with TechOpp Consulting, Inc. as TABA provider, we have reached out the companiesthrough the online virtual meetings. We have been invited to join the innovation platform provided by another company, and made connection with some of the large corporations. By the end of this SBIR Phase I project, we will reach out all these potential customers and end-users. Changes/Problems:1). More than 10 days for carrying out and getting results for one genetic manipulation; 2). At least 10 flasks culture to be carried out to get a productive strain from genes randomly integrated events; 3). Further incorporation of other variants of genetic manipulations. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?1). Improvement of strains for producing biopolymers (To be finished by February 28, 2021) In our previous studies, we have identified the genes to deliver the targeted product. Their expression cassettes will be further introduced into genomes with various copy numbers. We will check the stability of the strains devoloped. The product formation, cell biomass yield, substrates consumption and cell growth rate will be determined. The efforts will result in construction of an efficient microbial cell factory for producing target. 2). Characterization and recovery of products (To be finished by March 31, 2021) We will characterize the physical properties including the molecular weight of biopolymer, the melting temperature (Tm), apparent heat of fusion (ΔHm), crystallization behavior and the mechanical strength of biopolymers produced. The more advanced testing such as chemical resistance resting and plastic testing for tensile strength and elongation, and specific applications testing including packaging testing will be carried out by using the service available from the experienced polymer characterization companies. The biosynthesized biopolymer will be recovered from yeast cells in a cost-effective and environmentally friendly way. 3). Fermentation optimization for maximizing biopolymer production (To be finished by April 30, 2021) We will optimize the fermentation process to achieve superior product titer and productivity. Control of pH control is very important because it affects the formation of substrate as either weak acids or sodium salts. To achieve high-cell culture density and high-titer production, it is necessary to provide sufficient nitrogen to maintain a specific C/N ratio during the different phases of cell growth and product accumulation. The high-performance fermentation process can be extended for production of biopolymers from waste streams by incorporation of waste treatment process.
Impacts
What was accomplished under these goals?
1). Design of parallel pathways compartmentalized in yeast organelles for biosynthesis of target product and one novel product; 2). DNA Constructs development for pathway engineering; Several important factors including promoter strength and copy number have been considered to construct an efficient pathway for producing target. In addition, pathway compartmentalization has been employed as an advanced metabolic engineering strategy for expression of the enzymes in both cytosol and other yeast compartments for product biosynthesis. 3). Selection of genes and optimization of their expression levels We investigated the performance of strains bearing the replicable plasmid for producing biopolymers. These results provided essential clues to metabolically engineer yeast for more efficiently producing biopolymers. 4). Development of stable and productive strains The expression cassettes containing target genes have been cloned into the vectors containing selection marker flanked with loxp sites, which can be removed by introducing the vector expressing Cre recombinase. Once identification of the function and optimal expression level of target genes, expression cassettes were integrated into the yeast genome with variable copy number. 5). Optimization of biopolymer detection method So far for detection of biopolymers, one of the common methods is lyophilization of the cell biomass and then accomplishment of methanolysis. There are some advantages such as enhancing the stability of a dry powder as well as the product stability in a dry state for removal water from the biological samples by using lyophilization. However, lyophilization process is energy-intensive and requires the special instrument, freeze drying machine. Alternatively, we have evaluated the methods for drying samples at 55°C for 48 hours and 105°C for 12 hours. No big difference of treatment by lyophilization or heating at 55 °C for 48 h was observed, although lyophilization of samples led to a litter higher content of product detected by GC-FID or GC-MS. However, heating samples at 105°C greatly reduced the content of biopolymers for measurement. According to these results, we used the approach for drying the cell biomass at 55°C for 48 hours as an alternative method for lyophilization, and then carried out methanolysis of the samples. 6). Organic waste treatment We collected dairy manure from Knott Dairy Center at Washington State University (WSU). Different approaches for treatment of dairy manure were carried out to evaluate the process performance. 7). Production of biopolymers from the substrate by using fermentor We carried out biopolymer production in 7.0-L fermentor by cultivation of the engineered strains. We also made substantial progress towards building a novel culture system.
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