Progress 09/01/20 to 04/30/21

Outputs
Target Audience:The Phase I project focused on the development of a processing method for Stony Creek's biochemicallystabilized dry biomass. While much of the work was directly useful to Stony Creek Colors specifically, as the company is the only consumer of the stabilized biomass, the impacted audience is much larger. This primarily includes farmers and farming communities traditionally outside the reach of Stony Creek's farmer network, due to geographic constraints. By proving the extraction process to be viable, sustainable, and replicable, the company's sourcing geography can now be expanded to all growing areas as long as a drying facility can be supported nearby. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the course of this project, reseachers were able to expand theirexperience and understanding in functional areas related to bacterial isolation and protein determination as weel as membrane filtration. Throughout the tasks, research technicians received instruction and learning from senior research personnel, providing opportunities to pursue new areas of research and expanding their skillsets. How have the results been disseminated to communities of interest?The final technical report has been shared with factory and field operations personnel at Stony Creek Colors who will be responsible for implementing the finding into 2021 operational practices and equipment design. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Regarding the extraction parameters for dry leaf biomass, optimal soak environment conditions were established for pH, temperature, salinity, and time to yield maximum indican extraction with minimal coextracted contaminants. This was confirmed through post-extraction conversion of the indican to indigo as well as via UV-Vis spectral analysis. For the hydrolysis step, multiple endogenous and commercial enzymes were evaluated, but during these experiment, Stony Creek researchers were able to isolate and culture a bacterial strain with glucosidasic activity which outperformed even the native beta-D-glucosidase. This bacteria was cultured and scaled, and functional constraints were established relating to its growth and activity. Ultimately this bacteria was used in Stony Creek's dry leaf extraction factory pilot runs, outperfroming the corresponding freshleaf batches by significant margins. Trials were further performed around concentrating indican - and simultaneously treating process water for recycle - using reverse osmosis. The proof of concept was successful, and this will be a focus of subsequent Phase II experimentation using nano- and ultra-filtration. The water re-use, precursor concentration, and soak condition experiments were successful enough to allow for an off-site preprocessing scheme. This would in turn allow for leaf biomass to be soaked at the dryer site, concentrated to an indican syrup, and shipped to the dye production facility in lieu of transporting low bulk-weight leaf material from remote farms. The separation technology also enables continuous extraction opportunities using less water per pound of biomass while also allowing for a more complete extraction of the indican.

Publications