Our visit to the ABPDU made it evident that our biological dyeing process would need to be economically competitive with the current industrial process. As a result, we have made it a goal to optimize our system for future scale up. In addition, after interviewing an executive in the dyeing industry, we learned that there may be market interest for new eco-friendly technologies.

Here, we show a schematic of our envisioned scale up. Given the potential cost benefits of anaerobic fermentation, we have worked to ensure that our engineered indican-producing bacteria would be grown anaerobically. The dyeing agent (indican) would be isolated from cell culture by lysing and centrifuging the mixture. Fabric would be dipped into indican and subsequently exposed to B- glucosidase, from a second reactor.

Visiting the ABPDU confirmed our suspicion that removing steps in our scaled process could reduce cost. We identified that secretion of B-glucosidase would allow us to avoid the need for cell lysis. We have built a plasmid meant to express and secrete B-glucosidase in S.cerevisiae.

Prepro tag targets the B-glucosidase fusion for secretion. We are currently working on testing the efficiency of this construct.

Improving indican titer directly impacts the economics of our system. We anticipate that in vivo concentration of our donor molecule (UDP-glucose) can be a limiting factor to indican titer. To address this problem, we have constructed the following plasmids meant to overproduce the endogenous enzymes (PGM, and GalU).

PGM and GalU directly participate in the production of UDP-glucose in E.coli. We have built these plasmids, and we are currently working on testing them for increased indican production.

Even after our process is sufficiently optimized for scale up, several considerations must be taken into account to accurately assess economic feasibility. Here, we have included a list of considerations in developing a economically competitive dyeing methodology. The diagram, adapted from a presentation by Dr. Daniel Klein-Marcuschamer, combines the capital and operating costs associated with developing a bioreactor facility. Capital costs, such as the price of initially constructing a plant, equipment, intellectual property etc., amount to about half of the total costs associated with scale up of processes. Operating costs such as the cost of maintaining the plant, feed chemicals (i.e. tryptophan and sugar), and consumables (i.e nickel resin and lysis beads) will also contribute to the over all affordability of our process. As we optimize our method, we hope to gather data regarding the costs of each of these aspects of scale up in order to assemble a commercially viable dyeing process.

Adapted from lecture by Dr. Daniel Klein-Marcuschamer; Director, Technoeconomic Analysis, JBEI; Manager, Technoeconomic Analysis, AIBN

Klein-Marcuschamer, Daniel. "The Challenge of Enzyme Cost in the Production of Lignocellulosic Biofuels ." Biotechnology and Bioengineering. (2011): n. page. Print.