Grantee Research Project Results
Final Report: Co-production of Indigoid Dyes and Bioplastics Using Byproducts of Dairy Processing
EPA Contract Number: 68HERC23C0031Title: Co-production of Indigoid Dyes and Bioplastics Using Byproducts of Dairy Processing
Investigators: Rogers, Kobe
Small Business: Ourobio
EPA Contact: Richards, April
Phase: I
Project Period: December 1, 2022 through May 31, 2023
Project Amount: $100,000
RFA: Small Business Innovation Research (SBIR) Phase I (2023) RFA Text | Recipients Lists
Research Category: SBIR - Air and Climate , SBIR - Sustainability , SBIR - Homeland Security , SBIR - Water
Description:
The impacts of accelerating human industrial activities on the environment and climate are becoming increasingly clear. Studies suggest that the production of geologically novel entities, such as plastics and chemical contaminants like PCBs, has outpaced our societal ability to monitor and assess the safety of these materials and their associated manufacturing processes. Without swift action to reduce waste generation and persistence in the environment, we will continue to experience accelerating climate change and risk collapse of ecosystems that play a critical role in supporting life on Earth. Colorants used in plastics present a particular threat to human and environmental health, as their production is responsible for PCB byproducts that find their way into the colorants themselves, waterways, and waste management systems. This is particularly true of colorants used alongside biodegradable plastics, and as more companies make the switch to biobased/biodegradable plastics, greater access to bio-based dyes will be needed to enable the production of, brightly-colored, brandable bioplastics with no risk of environmental or human toxicity.
Ourobio is a young synthetic biology, biomaterials, and circular economy company. We develop engineered microorganisms to turn industrial byproducts into low-footprint, performance-enhancing biodegradable plastic additives. Our technology is unique in its ability to convert waste-based feedstocks into complementary bio-based, biodegradable products in a single fermentation process, and our proof of concept uses dairy industry byproducts to co-produce bio-based, biodegradable plastic resins and colorants - lowering the cost, footprint, and difficulty of producing marketable/brandable, fully bio-based biodegradable products and packaging.
During the Phase I award period, recombinant E. coli was evaluated for its ability to co-produce PHB with indigo and four indigoid derivatives from whey. Both solvent-based and aqueous-based extraction methods were developed to efficiently extract PHB and indigoid compounds at high purities from the remaining biomass, and material properties such as degradation temperature, color fastness, and compostability were evaluated.
Summary/Accomplishments (Outputs/Outcomes):
During the Phase I timeline, a proof of concept for producing blue, red, and purple polyhydroxybutyrate (PHB) from cheese whey in recombinant E. coli strains was demonstrated. In addition to indigo, the production of two new indigoid pigments in parallel to PHB was confirmed. Poor expression of recombinant enzymes needed to synthesize the remaining indigoid compounds resulted in a lack of pigment formation. From whey, PHB production achieved the target yield, reaching 2 g/L, and indigo surpassed the target yield, reaching an average concentration of 559 mg/L. Both solvent and aqueous extraction of indigoids and PHB were successful, resulting in high yield (>80%) and purity (>95%) for both compounds. Material testing revealed low chemical and UV resistance of the PHB-bound indigo pigment containing 1% (w/w) indigo, but a significantly higher UV resistance for samples containing >1% (w/w) indigo pigment. Initial scale-up with Pow.bio showed an increase in biomass accumulation, with similar indigo yields to benchtop experimentation, suggesting that the process can be accurately scaled.
Conclusions:
Technical
From the research findings, PHB and indigoid compounds can be successfully produced from whey, and in higher than expected yields. This indicates high potential for scale-up and commercialization after strain, media, and bioprocess optimization that will be conducted in the EPA SBIR Phase II award. Due to poor expression of enzymes responsible for the production of certain indigoid derivatives, three target pigments capable of being produced have been selected for optimization and scale-up in the EPA SBIR Phase II award. Additional research will be conducted to identify new enzymes with identical functions in an effort to expand color offerings beyond red, blue and purple. The extraction methods developed are capable of extracting the pigment and biopolymer together or separately, allowing us to serve a wider variety of potential customers interested in pigments, biopolymers, or the combination. Based on material testing performed on PHB-indigoid compounds, the poor chemical and UV stability of PHB compounds with low indigoid concentrations indicates that further material formulation may be required to stabilize the pigment for product applications exposed to excessive sunlight or harsh chemical conditions. However, the results also showed acceptable stability for more common applications like food-service goods and cosmetics.
Commercial
There is an immediate and growing need for more sustainable means of repurposing dairy byproducts, as proper management and disposal of these byproducts/waste streams are a significant burden and expense for dairy processors. As a result, we will be able to secure pilot volumes of whey at little/no cost and have the potential to locate pilot infrastructure on site with or within proximity of a creamery. There is also a growing demand for biodegradable plastics and natural colorants across all industries. PHAs' versatility as a class of polymers and bioplastic additives position it for success in a wide range of product applications, and sustainable colorants have the potential to help accelerate their adoption by helping improve brand recognition.
Our product offering is distinct from yet complementary to other companies in the PHA and bioplastic additives industry, and has large impact, fundraising, and revenue potential as a result. A kilogram scale pilot is a critical next step and will lend itself to getting more user feedback, validating that our material is comparable to/better than what is available on the market, providing significant sample volumes for continued formulation and compounding with our partners, and identifying inefficiencies that occur when scaling a manufacturing process.
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.