Week 1 HW: Final Project Ideas

(1) What would you like to biomanufacture and why?

I would like to produce second-generation biobutanol from de-oiled citrus waste sourced from CENADA (National Centre for Food Supply and Distribution). This biofuel will be produced via ABE (Acetone-Butanol-Ethanol) fermentation, supported by automated systems. This project aims to generate alternatives to fossil fuels given current geopolitical disruptions in oil distribution, while supporting Costa Rica’s goals under the National Decarbonization Plan.

(2) What living system would you use?

Solventogenic anaerobic bacteria from the genus Clostridium. Alternatively, non-model organisms or genetically modified strains could be evaluated.The process involves the following steps: Hydrolysis: Breakdown of the citrus substrate to release fermentable sugars and pectin-derived galacturonic acid.Anaerobic Fermentation (Two Phases): Acidogenesis: Converts input sugars into organic acids (acetic and butyric acids). Solventogenesis: Reassimilates these acids—counteracting environmental pH drop—and converts them into neutral solvents (acetone, ethanol, and biobutanol). Product Recovery: Generation of refined biobutanol, coproducts (acetone and ethanol), and biogas (CO2 and H2).

(3) How would you use automation?

Automation for Research: High-throughput microdroplet screening to test strain tolerance to residual d-limonene (ensuring concentrations remain below critical limits) and high butanol concentrations. Automation ensures rapid prototyping, reducing time and capital investment in the initial R&D phase. Automation for Test Production: Automated pipetting and media preparation using platforms like Opentrons and Cloud Labs. This allows optimization of hydrolysis ratios and nitrogen supplementation. Furthermore, growth kinetics and solvent production can be monitored in real time across multiple parallel micro-reactors.

(4) How much would you need to make, and how long would it take?

(a) Production Quantity:CENADA generates an average of 10,000 kg/day of waste. At ~20% dry matter, this yields approximately 2,000 kg/day of dry substrate. Assuming 50% of this substrate converts to fermentable sugars via hydrolysis, and applying a 30% theoretical yield for ABE fermentation (60% of which is butanol), estimated potential yield is 180 kg of biobutanol per day.

(b) Timeline: Prototyping: Evaluating 96 to 384 parallel hydrolysis and fermentation conditions takes 24–48 hours post-plate preparation, based on standard bacterial growth kinetics.Batch Fermentation: Each ABE batch requires 60–72 hours due to sequential acidogenic and solventogenic phases.Continuous Operation: Because butanol concentrations above 1.5% inhibit Clostridium, an in-situ continuous extraction system (gas stripping or pervaporation) will be used. This will allow the reactor to run in extended 5 to 7-day operational cycles based on demand.

(5) What don’t you know yet?

The three primary technical bottlenecks and assumptions in this biomanufacturing system are:

Tolerance to Residual Inhibitors: Although d-limonene can be reduced to 0.05%, experimental testing is still required to determine strain sensitivity to residual flavonoids and freed galacturonic acid.

Carbon Catabolite Repression (CCR): Determining whether the selected strain can simultaneously metabolize glucose, fructose, xylose, and galacturonic acid without glucose suppressing the uptake of other sugars.Energy Costs of Continuous Separation: Evaluating the economic viability and energy consumption of in-situ separation systems (pervaporation/gas stripping) scaled to production volumes.

Citation Statement AI Client Used: Gemini

Prompt:“Can you ensure the following text follows a correct grammatical structure, there are no typos and correctly explains the process with a clear and concise language considering this text has been manually translated: [Insert raw text]

Sources Aditiya, H. B., Mahlia, T. M. I., Chong, W. T., Nur, H., & Sebayang, A. H. (2016). Second generation bioethanol production: A critical review. Renewable and Sustainable Energy Reviews, 66, 631–653. https://doi.org/10.1016/j.rser.2016.07.015

Bayona, M. (2021). Pretratamientos físico-químicos de residuos agrícolas para la obtención de biocombustibles [Master’s thesis, Universidad de Zaragoza].Boluda Aguilar, M. (2013). Producción de bioetanol mediante fermentación de subproductos cítricos pretratados con steam explosion [Doctoral dissertation, Universidad Politécnica de Cartagena].

El Economista. (2021, October 26). Desperdicio de alimentos en Costa Rica se estima en 365 000 toneladas anuales. https://www.eleconomista.net/actualidad/Desperdicio-de-alimentos-en-Costa-Rica-se-estima-en-365000-toneladas-anuales-20211025-0016.htmlLi, L., Wang, Y.,

Wang, N., Chen, X., Li, B., Shi, J., & Li, X. (2021). Process optimization of acetone-butanol-ethanol fermentation integrated with pervaporation for enhanced butanol production. Biochemical Engineering Journal, 173, 108070. https://doi.org/10.1016/j.bej.2021.108070

Lizano Orozco, K. (2026). Valorización de residuos cítricos del CENADA mediante extracción de aceites esenciales como pretratamiento integrado para la producción de bioetanol: análisis de prefactibilidad bajo condiciones de Costa Rica [Project in development, Universidad Nacional de Costa Rica (UNA)].