Progress 09/01/22 to 08/28/24

Outputs
Target Audience:During this reporting period, our efforts successfully engaged the following target audiences: Industry Stakeholders: Consumer Products Companies: These companies, with a strong emphasis on Environmental, Social, and Governance (ESG) initiatives, are critical as they create demand for innovative products. We targeted companies such as Pepsi, Clorox, and Amazon to gather voice-of-the-customer feedback, focusing on their product preferences and performance requirements. Waste Management / Anaerobic Digestion Businesses: These companies have potential interest in value-added uses of AD output. Our outreach aimed to gauge their interest in adopting ILB's technology. Plastics Manufacturing Companies: We targeted companies involved in bio-based plastics production, as they represent a potential market for new suppliers. Larta Inc. identified approximately 45 companies across these categories, which were reviewed and confirmed by ILB. Suitable points of contact for primary market research interviews were established, with the initial list now complete, though expansion is planned as research progresses. Educational Outreach: Undergraduate Students in Food Engineering: We collaborated with over ten universities across various states in the USA to engage food engineering undergraduates through an experimental teaching initiative. Due to lab space restrictions and the COVID-19 pandemic, a take-home experiment was implemented, focusing on the water vapor transmission rate (WVTR) of polymer packaging materials. Students received materials and manuals via mail, conducted the experiments at home, and submitted their results online. Research Scholars: We organized a workshop targeting scholars in the Department of Biological Systems Engineering at Washington State University, focusing on smart packaging. The workshop covered topics such as evaluating insulating packaging performance and preparing pH-responsive intelligent packaging. Outcomes: Our outreach efforts successfully reached and engaged a diverse group of participants, including undergraduate students majoring in food engineering and research scholars from fields like food science, chemistry, and polymer engineering. These efforts provided formal educational experiences through hands-on experiments and workshops, contributing to the development of knowledge and skills in intelligent packaging materials. In summary, the target audiences reached during this reporting period include key industry stakeholders and educational participants, with efforts focused on delivering science-based knowledge through innovative teaching methodologies and experiential learning opportunities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has created several valuable opportunities for training and professional development for team members involved: Recruitment and Skill Development: Matrika Bhattarai: A recent PhD graduate from Ohio University, specializing in Molecular Cellular Biology, has joined ILB LLC as a research scientist. Matrika is being trained in advanced genetic engineering, focusing on the modification of Y. lipolytica for PHB production using acetate. Additionally, he is gaining hands-on experience in engineering practices related to the integration and upscaling of hypothermophilic anaerobic acidification and yeast fermentation techniques. Masoud Tourang: A PhD student at Washington State University (WSU), Masoud is being trained to deepen his expertise in microbiology and chemistry. This training is crucial for his future work in bioremediation and biorefinery, enhancing his skill set in these interdisciplinary areas. Sara Sarkhosh: A technician with a Master's in Agricultural Biotechnology, Sara is focusing on fermentation and food bioprocess engineering. Her role in converting food waste into VFAs and PHB bioplastics is providing her with practical experience and skill development in bioprocessing. Postdoctoral Training and Collaboration: Dr. Chandra Dev and Dr. Jinlong: Both postdoctoral research associates at WSU have been integrated into the project, with Dr. Chandra Dev specializing in genetic engineering and fermentation, and Dr. Jinlong in polymer science. Dr. Jinlong has further expanded the team's knowledge of PHB product quality, including its chemical and physical properties. Their involvement in this project provides ongoing professional development through collaborative research and knowledge exchange. Mentorship and Weekly Collaboration: The entire team, including Matrika Bhattarai, Dr. Chandra Dev, Dr. Jinlong, Masoud Tourang, and Sara Sarkhosh, actively participates in weekly meetings with mentors Dr. Shyam S. Sablani, Dr. Shulin Chen, and Dr. Liang Yu. These sessions are designed to discuss project advancements, receive expert guidance, and foster a collaborative learning environment. This structured mentorship provides continuous professional development and ensures that each team member is supported in their research and skill enhancement. These training and professional development activities are integral to the project, equipping the team with advanced knowledge and practical skills in genetic engineering, bioprocessing, and polymer science, and fostering a collaborative research environment. How have the results been disseminated to communities of interest?Our efforts in converting organic waste into PHB bioplastics have been effectively disseminated to various communities of interest through a strategic outreach initiative. The primary goal of this initiative is to enhance public understanding and stimulate interest in science, technology, and related fields. To achieve this, we have executed a series of targeted outreach activities: Conference and Workshop Presentations: We have actively presented our research breakthroughs in PHB bioplastics at conferences and workshops across Washington state and the broader United States. These events serve as critical platforms for sharing our findings with a wide audience, fostering knowledge exchange, and receiving feedback from experts and peers in the field. Community Engagement through SP3NW Collaboration: Our collaboration with SP3NW (Strategic Partnerships for Economic Engagement in the NorthWest) has been instrumental in expanding the reach of our research. We have conducted engaging talks and interactive sessions aimed at individuals who might not typically encounter scientific research. These sessions are designed to increase awareness of the practical impact of our work and to connect with diverse audiences beyond the traditional scientific community. SP3NW, based in Spokane's University District and established in December 2020, is dedicated to fostering entrepreneurship and innovation. Their support includes providing physical office spaces, wet labs, educational programs, and executive guidance, all of which contribute to the growth of new ventures. Through SP3NW, we have connected with resources and advisors from Washington State University Health Sciences Spokane, Eastern Washington University, Gonzaga University, Whitworth University, and Community Colleges of Spokane. Public Awareness and Community Impact: Through our outreach efforts, we are not only advancing scientific understanding of bioplastics but also contributing to the enrichment of local communities. By presenting our research in accessible formats and engaging directly with the public, we aim to inspire interest in careers in science, technology, and the humanities, and to demonstrate the real-world applications and benefits of our research. These outreach activities have been central to disseminating our research results, reaching a broad audience, and enhancing public understanding of the potential of PHB bioplastics in sustainable development. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Major Activities Completed / Experiments Conducted Objective 1: Enhancing Cell Factory for PHB Production Genetically modified Y. lipolytica to redirect its lipogenesis pathway towards PHB synthesis, incorporating modifications into stable genetic loci to ensure strain stability and increase PHB content in cell biomass. Objective 2: Bench-Scale Optimization and System Integration Optimized and integrated anaerobic digestion and fermentation systems at bench-scale, converting waste into PHB. Key activities included pH optimization, substrate concentration adjustments, employing a co-substrate strategy, and improving PHB extraction during recovery. Objective 3: Pilot Testing of Integrated System Conducted pilot testing with anaerobic digestion in a 50L reactor and subsequent fermentation in a 20L stainless steel fermenter. Optimized the process for consistent biopolymer production at pilot scale, using dairy manure and hyperthermophilic seed culture. Objective 4: Product Evaluation and Techno-Economic Analysis (TEA) Task 1: Prepared and evaluated PHB sheets, measuring water vapor and oxygen transmission rates and heat-sealing properties. Task 2: Developed and tested PHB-coated papers for water and oil resistance. Task 3: Characterized PHB powders and prepared solution casting films, assessing thermal stability and dielectric properties. These activities advanced the project's goals by optimizing PHB production, testing the integrated system at pilot scale, and evaluating the final product's properties. 2) Data Collected Objective 1: Strain Growth and PHB Production Monitored the growth of modified and basic yeast strains in YNB media with 40 g/L glucose over 4 days. Measured growth by cell dry weight (CDW) and quantified PHB production as a percentage of CDW. Objective 2: System Integration and Fermentation Monitoring Used two series-connected fermenters to optimize system integration. Monitored yeast growth daily via optical density at 600nm. Analyzed substrate consumption using HPLC and tracked biopolymer production with GC-MS, calculating PHB yield per dry cell weight. Dried yeast biomass at 60°C to determine dry cell weight. Objective 3: Biogas and VFA Analysis, Pilot-Scale Fermentation Quantified biogas production with a wet-tip gas meter linked to the AAR and measured pH daily. Collected AAR effluent and gas samples every two days. Analyzed VFAs using Agilent GC-FID and determined the effects of hydrothermal treatment via PY-GCMS, focusing on lignin subunit changes. Quantified pilot-scale fermentation growth by dry biomass, with parameters automatically controlled. Extracted and weighed the final product, calculating yield per biomass. Objective 4: PHB Product Evaluation Task 1: Prepared PHB sheets by melting compounding and measured heat sealing properties, water vapor, and oxygen transmission rates. Task 2: Prepared PHB-coated paper via spray coating, using chloroform as a solvent and ethyl cellulose (EC) as a binder. Evaluated water and oil resistance. Task 3: Analyzed thermal properties of PHB powders using DSC and TGA. Prepared solution-cast PHB films and measured dielectric properties. 3) Summary Statistics and Discussion of Results Objective 1: Growth and PHB Production in Yeast Strains The sequentially modified strain exhibited a slightly higher growth rate and significant PHB accumulation, reaching 40% of CDW, indicating a marked improvement in PHB production compared to the basic strain. Objective 2: Acetate Utilization and PHB Production The engineered strain demonstrated substantial potential for utilizing acetate for PHB production, producing 37.5% dry cell weight (%DCW) PHB on acetate media, compared to 7.3% DCW on glucose media. Achieved a maximum biomass yield of 0.48 g/g and 20% DCW PHB when yeast was grown on anaerobic digestion (AD) effluent. Ongoing optimization aims to further increase PHB yields. Objective 3: VFA Concentration and PHB Production Maintained VFA concentration at 26.1 g/L, producing 10 L of biogas daily with 40% methane content. Total solids (TS) measured at 6.28% and volatile solids (VS) at 4.61%, with pH ranging from 6.2 to 6.7. Hydrothermal treatment at 180°C significantly improved lignocellulosic biomass breakdown, increasing VFA concentrations and enhancing organic waste solubilization. In the fermenter, initial substrate concentrations of 50 g/L glucose and 10 g/L sodium acetate led to high biomass and PHB yields, with yeast biomass exceeding 20 g/L in 5 days. Objective 4: PHB Product Evaluation Task 1: Produced heat-sealable PHB sheets with a water vapor transmission rate (WVTR) of 12.11 g/m²/day and an oxygen transmission rate (OTR) of 27.44 cc/m²/day. Task 2: Developed PHB and ethyl cellulose (EC) blend coatings that significantly improved water resistance. The 35 wt% EC blend also enhanced vegetable and peanut oil resistance. Task 3: Determined PHB powders' glass transition temperature at 2°C and melting temperature at 177°C, with degradation starting at 247°C and peaking at 284°C. PHB films exhibited dielectric properties with an ? value of 1.1036 and a ? value of 0.001. 4) Key Outcomes or Other Accomplishments Realized Objective 1: Strain Development Developed a robust yeast strain accumulating PHB at over 40% of CDW and engineered a genetically stable strain for PHB production. Enhanced acetate tolerance, allowing growth in up to 72 g/L acetate. Objective 2: Fermentation Optimization Validated a cost-effective co-substrate fermentation strategy using acetate and glucose, increasing growth rates and PHB content. Identified optimal pH and substrate concentrations for maximizing biomass productivity and PHB production. Demonstrated the effective functioning of an integrated process from anaerobic digestion to PHB production. Objective 3: Pilot System Establishment Established a pilot system integrating hyperthermophilic anaerobic acidification with yeast fermentation, achieving consistent VFA production with a stable concentration of 26.1 g/L from dairy manure. Demonstrated the effectiveness of hydrothermal treatment in increasing VFA production. Objective 4: Product Evaluation Produced PHB sheets with an OTR of 27.44 cc/m²/day and a WVTR of 12.11 g/m²/day. Developed PHB and EC blend coatings that improved water and oil resistance. Determined PHB powders' thermal properties and evaluated PHB films' dielectric properties.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Masoud Tourang, et. al. Polyhydroxybutyrate (PHB) Biosynthesis by an Engineered Yarrowia lipolytica train Using Co-Substrate Strategy. Fermentation 2023, 9(12), 1003; https://doi.org/10.3390/fermentation9121003
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: J.L. Zhang, S. S Sablani, H. Liu, S.L. Chen. Comparative Physical Properties of Poly (3-hydroxybutyrate) and Its Application Developments as Sustainable Packaging Materials. Journal of Food Engineering, 2024, submitted.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:We identified the following three types of companies that would be good targets for voice-of-the-customer feedback. Consumer Products companies with strong ESG push, because these companies will create the demand. It would therefore be valuable to understand exactly which products they would prefer, and the required performance of those products. Examples are Pepsi, Chlorox, and Amazon. Waste Management / Anaerobic Digestion businesses that may benefit from a value-added use of the AD output, for example, to understand their level of interest in adopting ILB's technology. Plastics Manufacturing companies, which would be the companies that make bio-based plastics using what amounts to a new supplier. Larta Inc. identified about 45 companies across these three categories and sent those to ILB to confirm that they're suitable or to remove them from the list. After approval of the list, Larta identified suitable points of contact for the upcoming primary market research interviews. The list of contacts is complete and was provided to ILB. This will be further expanded as we work on the Primary market research but is considered complete in terms of having enough to get started. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Within this endeavor, a fresh impetus has been injected by the recruitment of Matrika Bhattarai, a recently graduated PhD candidate from Ohio University, specializing in Molecular Cellular Biology. Matrika has assumed the role of a research scientist at ILB LLC. His profound expertise will be harnessed not only to genetically engineer Y. lipolytica for PHB production via acetate utilization but also to immerse himself in engineering practices. These encompass the integration and upscaling of hypothermophilic anaerobic acidification and yeast fermentation techniques. Complementing this stride, we have also welcomed Masoud Tourang, a dedicated PhD student, and Sara Sarkhosh, a skilled technician, both joining the team at WSU. Masoud's aspirations lie in fortifying his proficiency in microbiology and chemistry, recognizing their pivotal roles in the realms of bioremediation and biorefinery. Sara Sarkhosh, a Master in Agricultural Biotechnology, brings her expertise in fermentation and food bioprocess engineering to the forefront. Her engagement in this project entails the conversion of food waste into VFAs and, ultimately, PHB bioplastics. Throughout the project's duration, these valued members have maintained an active presence in weekly meetings, fostering close collaboration with their mentors, Dr. Shulin Chen and Dr. Liang Yu. These meetings have served as platforms to deliberate over project advancements and solicit expert guidance, ensuring a cohesive and fruitful trajectory. How have the results been disseminated to communities of interest?Our endeavors in the realm of converting organic waste into PHB bioplastics have been actively disseminated among various communities of interest through a well-structured strategy. This initiative aims to amplify public understanding and stimulate curiosity in science, technology, and the humanities. To achieve this, an array of outreach activities has been executed. We have showcased our breakthroughs in PHB bioplastics at conferences and workshops held across Washington state and throughout the United States. These platforms offer an opportunity for researchers to present their discoveries to a broader audience, facilitating not only knowledge-sharing but also receiving invaluable insights from fellow experts in the field. Our collaborations with SP3NW (Strategic Partnerships for Economic Engagement in the NorthWest) have yielded engaging talks and interactive sessions. These initiatives are specially designed to connect with individuals who might not typically encounter scientific research, thus expanding the awareness of our research's real-world impact. SP3NW, headquartered in the University District of Spokane, was officially inaugurated in December 2020. The organization's core mission is to cultivate a flexible infrastructure that bolsters resilient entrepreneurship, identifying, nurturing, and deploying emerging innovations. SP3NW's comprehensive support extends to fostering the establishment of new businesses by providing essential resources like physical office spaces and wet labs. Their educational and experiential programs, coupled with executive guidance, contribute to the growth of these emerging ventures. Additionally, SP3NW grants access to a wealth of resources and advisors hailing from five prominent universities and colleges, including Washington State University Health Sciences Spokane, Eastern Washington University, Gonzaga University, Whitworth University, and Community Colleges of Spokane. Through our combined efforts, we are not only advancing the understanding of bioplastics but also actively contributing to the enrichment of local communities and the broader public sphere. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Major activities completed / experiments conducted; Objective 1:This objective focuses on increasing PHB content in the cell biomass. Y. lipolytica's lipogenesis pathway (which converts acetyl-CoA to fatty acids) is more efficient than the PHB synthesis pathway. Genetic enhancements, including multiple copies of phaA, phaB, and phaC genes, were introduced to boost PHB production. The NphT7 gene was modified to enhance precursor conversion, and phaC was altered to target PHB production in peroxisomes. Additionally, bacterial acetyl-CoA synthase was integrated to improve growth using acetic acid as a substrate. Objective 2:The optimization of fermentation conditions involved the investigation of two primary variables. Initially, the influence of various pH levels-basic, neutral, and acidic-on both the growth rate and PHB accumulation within the produced biomass was explored. Subsequently, the impact of a co-substrate strategy, involving varying ratios of glucose and acetate, was examined to assess its effect on doubling time and PHB content. Objective 3:Enhancing the production of volatile fatty acids (VFAs) in a pilot-scale anaerobic acidification reactor (AAR) involved the utilization of a 50-L glass reactor, operating under hyperthermophilic conditions (70°C). To facilitate the process, the agitator speed was adjusted within the range of 200 to 300 rpm.A 20-L stainless steel fermentor/bioreactor (BIOFLO 410, New Brunswick Scientific) has been acquired and is presently undergoing preparation for the production of poly(3HB-co-3HP) (PHB). Objective 4: A preliminary TEA was conducted. 2) Data collected; Objective 1. The newly sequentially modified strain and the basic strain were grown in YNB media containing 40g/L glucose to measure the growth by cell dry weight (CDW) and the amount of PHB products as a percentage of CDW to compare the efficiency and productivity of the sequentially modified strain. Both cultivated on YNB medium with 40 g/L glucose, their development in terms of CDW over a 4-day incubation period was examined. Additionally, the percentage of produced PHB content in relation to CDW over the same 4-day period was assessed. Objective 2. To conduct the experimental evaluation of pH's impact on both growth rate and PHB content, a New Brunswick Bioflo 110 fermenter was utilized, operating at three distinct pH levels: 5.5, 7, and 8.5. The fermentation was carried out using YNB as the medium and 50g/L glucose as the carbon source. To investigate the influence of the co-substrate strategy involving glucose and acetate, a series of flask experiments were undertaken. Experiments encompassed various concentrations of glucose (ranging from 5 to 150g/L) and sodium acetate (ranging from 20 to 100g/L). Objective 3. Biogas was quantified through a wet-tip gas meter interlinked with the AAR. pH measurements were conducted daily utilizing a pH meter. Every two days, samples of both AAR effluent and gas were procured. Subsequently, the produced VFAs underwent comprehensive analysis via the Agilent GC-FID instrument. Objective 4: Foodwaste tipping fee is $60/ton (30%TS). AD fertilizer is $20/ton. PHB is $5.69/kg. Chloroform was used to extract the polymer and the price is $0.48/kg. The purification process is referred to in the publication. The cost values were estimated by Aspen Process Economic Analyzer v8.8. 3) Summary statistics and discussion of results Objective 1. The results indicate that the sequentially modified strain displayed a slightly higher growth rate in comparison to the basic strain. Notably, the sequentially modified strain exhibited a significant accumulation of PHB, reaching 40% of CDW, showcasing a substantial enhancement in PHB production. Objective 2. In terms of pH effects, the data collected underscores the significant impact of pH, particularly on growth rate. The findings reveal that the optimal pH for achieving the highest biomass productivity is acidic (pH 5.5), resulting in a growth rate of 0.159 h-1and a maximum OD600reading of 21.29. Notably, elevating the pH to 8.5 demonstrates a marked negative influence on the growth rate, with an observed rate of 0.077 h-1- less than half that of pH 5.5. Nevertheless, pH 8.5 still yielded an OD600value (20.48) comparable to that of neutral and acidic pH. Interestingly, the highest PHB production occurred under neutral conditions (pH 7) rather than the acidic environment; specifically, PHB content reached 31.6% of CDW at pH 5.5, while it significantly rose to 41.49% of CDW at pH 7. Turning to the co-substrate strategy, the outcomes reveal that the most robust growth rate was attained at the lowest concentrations of both substrates. An inhibitory trend emerged with higher concentrations of glucose and sodium acetate, hampering the growth of the modified strain. The combination of 5g/L glucose and 20g/L sodium acetate resulted in an enhanced growth rate of 0.163 h-1- surpassing the growth rate observed when glucose was the sole substrate. Moreover, data on PHB content indicated a substantial increase, reaching 49% of CDW. However, elevating the concentrations of both glucose and sodium acetate exhibited inhibitory effects on the modified strain. Notably, within flasks containing 150g/L glucose and 100g/L acetate, detectable growth was absent during the initial 6 days. Similarly, increasing sodium acetate alongside 50g/L glucose not only reduced biomass productivity, but also negatively impacted PHB content. For instance, the use of 20g/L sodium acetate in conjunction with 50g/L glucose led to a PHB content exceeding 40%. However, raising sodium acetate concentration to 50g/L resulted in a notable decrease in PHB content to 20.5% of CDW. Objective 3. The VFA concentration was upheld at 26.1 g/L, yielding a daily biogas output of 10 L, containing 40% methane. The total solids (TS) and volatile solids (VS) were measured at 6.28% and 4.61% respectively, while the pH fluctuated within the range of 6.2 to 6.7. Notably, the conversion of soluble chemical oxygen demand (sCOD) to VFA was calculated at 1.22. Objective 4: Profitability index (PI) over 1 indicates a profitable plant. A plant scale of approximately 16 tons/day of food waste is expected to be profitable. Total Project Capital Cost is estimated at 10.98 million USD. If PHB is $3.00/kg, it is estimated that the plant scale of around 60 tons/day of food waste could be profitable. The total Project Capital Cost would be 21.53 million USD. 4) Key outcomes or other accomplishments realized. Objective 1. Significant achievements in the strain development phase include: · The successful creation of a strain capable of accumulating PHB at levels exceeding 40% of the CDW. · Substantial enhancement of acetate tolerance within the modified strain, achieving an impressive capacity to thrive in the presence of up to 72 g/L acetate. Objective 2. During the fermentation optimization phase, notable accomplishments were achieved, including: · The successful validation of a co-substrate approach involving acetate and glucose. This strategic combination proved to be a cost-effective fermentation strategy, yielding both a heightened growth rate and increased PHB content. · Identification of optimal pH conditions crucial for maximizing biomass productivity and enhancing PHB production. · Determination of the optimal concentration levels for both substrates utilized in the fermentation process. Objective 3: The establishment of a pilot system encompassing hyperthermophilic AAR (at a 50-L scale) alongside yeast fermentation (using a 20-L vessel) has marked a significant achievement. The AAR has successfully generated a consistent VFA output, with a stable concentration of 26.1 g/L, employing dairy manure as feedstock. Objective 4: The preliminary TEA evaluation indicates that a plant operating at a scale of approximately 16 tons/day of food waste could yield profitability atthe price of $5.69/kg.

Publications