Week 1 HW: Final Project Ideas

HTBAA Homework #1 Committed listener | Marco Arias Nuñez

What you would like to biomanufacture and why?

The focus that it’s going to be planned is a food-packaging film composed of polyhydroxybutyrate (PHB), that will be obtained through fed-batch fermentation of Cuprivavidus necator H16. The product is going to be designed for dry and high-fat foods, with the idea of replacing a percentage of the petroleum-based plastics that are commonly used in this industry. PHB has demonstrated to prevent quality loss in packaged foods, however it has a stiff and brittle nature so it must be complimented with PLA and ATBC to manufacture an alternative that addresses the environmental impact of plastics based on fossil fuels (Atares, et al., 2024). This project connects fermentation, sustainability, and the food and beverages industry. It can be a suitable replacement for single use plastics that contaminate the planet.

What living system would you use?

The proposed manufacturing chassis is Cuprivavidus necator H16, a non-engineered bacterial strain that naturally produces and stores PHB as intracellular granules. The system’s carbon source would be pretreated waste cooking oil, to further increase the project’s environmental impact. C. necator H16 would grow using fed-batches to boost the production of PHB utilizing the native PhaA, PhaB and PhaC enzymes. The main output of the process includes the PHB rich biomass, carbon dioxide, fermentation broth and residual nutrients (Loan, et al., 2022). The downstream process would be focused on the recovery and purification of PHB, which will be further melt-blended with commercially sourced PLA and ATBC. The proposed formula for the product is 75% of PLA. 25% of PHB and 10 parts of ATBC per 100 parts of PLA/PHB (Arrieta, et al., 2014).

How would you use automation?

In the supposition of a pilot scale, the PLC-SCADA bioreactor automation system integrated with process analytical technology (PAT). The system would be designed to automate the aerobic fed-batch fermentation process, utilising load-cell-controlled feed tanks, variable-speed pumps, and online sensors for pH, temperature, dissolved oxygen, foam, biomass, and exhaust-gas composition. Feedback loops would ensure regulation of the pH level through the addition of acid or base, while maintaining dissolved oxygen levels through a series of agitation, airflow, and oxygen enrichment processes. The carbon feed, which is pretreated waste cooking oil, will be delivered automatically according to biomass growth, oxygen demand, and a predetermined feeding profile. Automated feed control is of particular importance because the feeding strategy has a significant impact on biomass growth and PHB accumulation in C. necator (Rathore, 2021). In addition, I would incorporate an automated sterile sampler connected to at-line analytics. Samples would be collected at predetermined intervals to measure biomass, residual nitrogen, oil, and intracellular PHB, while the SCADA platform would record and visualise all process data. At-line flow cytometry with a PHB-selective fluorescent stain is an efficient way of obtaining faster estimates of intracellular polymer accumulation. In order to validate these estimates, periodic GC or HPLC measurements are required. This automation would reduce manual sampling and contamination risk, improve batch-to-batch reproducibility, and provide the data needed to determine when to switch from biomass growth to nitrogen-limited PHB accumulation and when to harvest the pilot-scale reactor. PAT platforms have previously monitored microbial PHB fermentations using a combination of online sensors, automated sampling, off-gas analysis and at-line flow cytometry (Karmann, et al., 2017).

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

With the help of the ai tool perplextity, the estimates were made for 100,000 final units utilizing two fed-batches of 1000L. The following was the prompt used: Role & Context: Act as a Bioengineering and Bioprocess Specialist. Perform a rigorous, back-of-the-envelope engineering calculation to evaluate the manufacturing feasibility of our product at a realistic target scale. Production Target & System Constraints: Target Output: 100,000 final units/packages. Bioreactor / Feed Setup: 2 fed batches of 1,000 L each (total working volume: 2,000 L). Task Requirements: (a) How much? Define and justify a project-relevant scale corresponding to the production target of 100,000 units/packages. Calculate the approximate mass/volume yield required per unit based on the total 2,000 L feed capacity. Clearly show all step-by-step calculations, explicitly including dimensional units, engineering assumptions (e.g., downstream recovery yield, volumetric productivity, density), and standard industry benchmarks/sources for key parameters. (b) How long? Using our living system framework and automation setup, estimate the total time required to manufacture this batch quantity. Incorporate key bioprocess parameters: growth kinetics, batch/fermentation time, turnaround/cleaning time (CIP/SIP), downstream processing (DSP) throughput, and parallel reactor capacity. Contrast early-stage prototyping automation (e.g., high-throughput screening, microfluidics) with full-scale manufacturing automation if applicable. Present a detailed step-by-step engineering calculation showing all math and kinetic assumptions. Formatting & Output Constraints: Provide a comprehensive Reasoning & Methodology section explaining the logic and equations behind each calculation. Format all references, citations, and parameter sources in strict APA 7th Edition style. And after corroborating the results, the following are the estimations to produce 100,00 final units. Film needed The two pouch faces require:

The mass of one pouch is estimated from area, thickness, and density:

Therefore:

The manufacturing target is therefore 625 kg of accepted finished film. Formulation requirement The formulation contains 75 parts PLA, 25 parts PHB, and 10 parts ATBC, for a total of 110 parts. The final mass fractions are therefore:

For 625 kg of accepted film, the theoretical content is:

Table 1. Theoretical content for proposed formula

This feed would contain approximately 473.5 kg PLA, 157.8 kg purified PHB, and 63.1 kg ATBC. Fermentation requirement Loan et al. reported fed-batch production by Cupriavidus necator H16 on waste cooking oil with 135.1 g/L cell dry weight, 76.9 wt% PHB, 1.73 g/L·h productivity, and 0.8 g PHB/g oil yield. The implied PHB titer is

Because PHB recovery is assumed to be 85%, intracellular PHB production must be:

Required broth volume is therefore:

The theoretical volume is about 1.79 batches at 1,000 L working volume, so practical scheduling requires two complete 1,000 L batches. Each batch would contain about 103.9 kg intracellular PHB and yield approximately 88.3 kg purified PHB after 85% recovery. Two batches would provide about 176.6 kg purified PHB, leaving roughly 18.8 kg as contingency for analytical sampling, extraction variability, or additional film trials. At the reported yield of 0.8 g PHB/g waste cooking oil, the expected oil consumption is:

A real campaign would charge somewhat more than 232 kg because substrate remaining in the reactor, transfer losses, and feedstock variability are not included in the published yield-based estimate. Fermentation time Using the literature titer and productivity, the approximate productive fermentation time is:

For a single 1,000 L reactor, a reasonable cycle is therefore: Fed-batch fermentation: 60 h Automated cleaning, sterilization, charging, and inoculation: 12 h Total reactor cycle: 72 h Two sequential batches require:

Downstream and film conversion If downstream recovery is performed on the first batch while the second fermentation is running, only the last batch adds a full 24 h downstream delay after the reactor campaign. The formulation feed of 694.4 kg would require:

Allowing approximately 4 h for film extrusion, the overlapped campaign time is:

Adding a 15% engineering contingency for sampling, equipment checks, feed delays, quality-control holds, and minor downtime gives:

The practical estimate is therefore 8–10 days. If downstream recovery cannot overlap fermentation, the base schedule becomes approximately 196 h, or 8.2 days before contingency, corresponding to approximately 9–10 days after contingency. Automation strategy Manufacturing would move to a 1,000 L stirred-tank reactor controlled by PLC–SCADA and Process Analytical Technology. The pilot system would automate gravimetric oil feeding, pH control, dissolved-oxygen cascade control through agitation/air/oxygen, temperature, foam suppression, sterile sampling, alarms, and batch records. This distinction matters: high-throughput microbioreactors accelerate Design of Experiments, whereas the PLC–SCADA pilot reactor supplies the mass required for the 625 kg film campaign.

What don’t you know yet?

The main assumptions of the process are that the reported yield of PHB at lab scale can be reproduced on pilot scale using waste cooking oil. Due to the impurities present in the oil, emulsification, oxygen transfer limitations, feeding rate and nutrient distribution. Despite the findings of one fed-batch study reporting 135.1 g/L cell dry weight, 76.9% PHB content, 1.73 g/L·h productivity, and a yield of 0.8 g PHB/g waste cooking oil, it is important to note that these results cannot be assumed to be valid at pilot scale without validation (Zhang, et al., 2022). The hypothesis would be deemed to be technically viable if the pilot process consistently achieves a sufficiently high PHB titer and productivity without oxygen limitation, excessive foaming, oil accumulation, or unstable feeding. The second bottleneck involves the recovery of intracellular PHB at a high yield and purity without compromising its molecular weight or resorting to an environmentally burdensome solvent process. The preceding scale estimate presupposed an 85% recovery, yet this figure must be substantiated through experimental means, as extraction conditions exert a considerable influence on recovery, purity, thermal stability, and polymer molecular weight. Green-solvent methods have achieved recoveries of more than 90% and purities approaching 99% at small scale; however, pilot-scale solvent recycling and process economics remain uncertain. The third unknown is whether the PLA/PHB/ATBC formulation can simultaneously provide flexibility, mechanical strength, barrier performance, processability, and food-contact safety. The proposed initial formulation—75:25 PLA:PHB plus 10 parts ATBC per 100 parts polymer—corresponds to normalized mass fractions of 68.18% PLA, 22.73% PHB, and 9.09% ATBC. The incorporation of PLA-PHB blends has been demonstrated to enhance the barrier and mechanical properties of the resultant blends. Conversely, the addition of ATBC has been shown to mitigate brittleness. However, it is noteworthy that the utilization of excessive plasticisers may compromise the strength of the blends or result in migration into the food product. In experimental film trials, a comparison of several ATBC levels should be made, with the following parameters to be measured: tensile strength, elastic modulus, and elongation at break. The following investigation focuses on the rates of oxygen and water vapor transmission. The following parameters are to be considered, film thickness, transparency, puncture resistance, and heat-seal strength. The following investigation focuses on the thermal processing window and degradation during extrusion. The present study investigates the overall and specific migration of ATBC using aqueous, acidic, and fatty-food simulants. The complete film formulation must demonstrate compostability and disintegration (Arrieta, et al., 2014).

References:

Arrieta, M. P., Castro-López, M. del M., Rayón, E., Barral-Losada, L. F., López-Vilariño, J. M., López, J., & González-Rodríguez, M. V. (2014). Plasticized poly(lactic acid)–poly(hydroxybutyrate) (PLA–PHB) blends incorporated with catechin intended for active food-packaging applications. Journal of Agricultural and Food Chemistry, 62(41), 10170–10180. https://doi.org/10.1021/jf5029812pubmed.ncbi.nlm.nih Atarés, L., Chiralt, A., González-Martínez, C., & Vargas, M. (2024). Production of polyhydroxyalkanoates for biodegradable food packaging applications using Haloferax mediterranei and agrifood wastes. Foods, 13(6), 950. https://doi.org/10.3390/foods13060950pubmed.ncbi.nlm.nih Karmann, S., Follonier, S., Egger, D., Hebel, D., Panke, S., & Zinn, M. (2017). Tailor-made PAT platform for safe syngas fermentations in batch, fed-batch and chemostat mode with Rhodospirillum rubrum. Microbial Biotechnology, 10(6), 1365–1375. https://doi.org/10.1111/1751-7915.12727pubmed.ncbi.nlm.nih Loan, T. T., Trang, D. T. Q., Huy, P. Q., Ninh, P. X., & Thuoc, D. V. (2022). A fermentation process for the production of poly(3-hydroxybutyrate) using waste cooking oil or waste fish oil as inexpensive carbon substrate. Biotechnology Reports, 33, e00700. https://doi.org/10.1016/j.btre.2022.e00700pmc.ncbi.nlm.nih+1 Rathore, A. S., Mishra, S., Nikita, S., & Priyanka, P. (2021). Bioprocess control: Current progress and future perspectives. Life, 11(6), 557. https://doi.org/10.3390/life11060557pubmed.ncbi.nlm.nih Zhang, L., Jiang, Z., Tsui, T.-H., Loh, K.-C., Dai, Y., & Tong, Y. W. (2022). A review on enhancing Cupriavidus necator fermentation for poly(3-hydroxybutyrate) (PHB) production from low-cost carbon sources. Frontiers in Bioengineering and Biotechnology, 10, 946085. https://doi.org/10.3389/fbioe.2022.946085