Week 1 HW: Final Project Ideas

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1. What would you like to biomanufacture and why?

I want to make drop-in renewable diesel consisting primarily of C15 and C17 terminal alkenes and alkanes, synthesized directly from partially dewatered cheese whey permeate (a high-lactose dairy processing waste stream). Instead of buying expensive enzymes to break down the feedstock, engineered E. coli will hydrolyze lactose in the permeate using its native lac operon, specifically lactose permease (lacY) and β-galactosidase (lacZ). The bacteria then will turn those sugars into fatty acids using native Enoyl-ACP reductase (FabI). An added decarboxylase (like UndB or CvFAP) will cleave the carboxyl group, leaving clean diesel hydrocarbons.

My motivation is to fix the practical bottlenecks that kill most biofuel projects: expensive feedstocks and costly enzymes. For example, kelp and plant wastes require complex, expensive cocktails of heterologous enzymes to liberate simple sugars. Used cooking oil (UCO) avoids that, but fierce international competition from industrial refiners has pushed UCO prices to $800–$1,200+ per ton. Cheese permeate is cheap, abundant, and comes directly out of dairy plants in huge volumes. Dairy processing generates massive quantities of cheese whey (~9 L of liquid whey per 1 kg of cheese produced). It is often a wastewater liability for dairy farms, so using it creates a clean win for both waste management and affordable fuel production.

Biomanufactured renewable diesel is designed for heavy transport such as freight trucks, cargo ships, and farm machinery that cannot easily switch to electric batteries. It matters because it is a true drop-in replacement for regular diesel, meaning it works in existing engines and pipelines without any modifications. By coupling mechanical pre-concentration at the dairy facility with a streamlined synthetic biology route, this project demonstrates a scalable, carbon-neutral circular economy model that turns agricultural waste into high-density liquid fuel.

2. What living system would you use?

I will use a microbial system for this project, namely E. coli BL21.

What goes in:

  • Feedstock: Dewatered cheese whey permeate wet cake, providing lactose as the core carbon and energy source.
  • Growth Medium: Water, basic mineral salts, and a nitrogen source to support cell growth.

What biological processes happen:

  1. Lactose Import and Breakdown: E. coli imports lactose through its cell wall using its native lactose permease (LacY) and splits it into glucose and galactose using its native β-galactosidase enzyme (LacZ).
  2. Fatty Acid Elongation: The cell burns those simple sugars through glycolysis to generate energy and building blocks, driving its native fatty acid cycle (boosted by overexpressed FabI and a modified ‘TesA thioesterase) to produce C16 and C18 free fatty acids.
  3. Decarboxylation: UndB targets those free fatty acids and strips off their carboxyl group as carbon dioxide, converting them directly into C15 and C17 fuel molecules.

What comes out:

  • Renewable Diesel: C15 and C17 terminal alkenes. Because fuel and water do not mix, the diesel naturally floats to the top of the fermentation liquid, making it easy to skim off.
  • Byproducts: CO2 gas and bacterial cell mass which can be dried and further processed into animal feed or fertilizer.

Assumptions:

  • Functional Lac Operon: The E. coli strain has a fully functional lac operon and has not been genetically altered to disable lactose consumption.
  • Phase Separation: The synthesized diesel hydrocarbons will separate from the water-based broth without accumulating inside the cell walls to toxic levels before floating out.

3. How would you use automation?

I would integrate a desktop liquid-handling robot, like an Opentrons OT-2, to automate high-throughput strain screening and media optimization.

  • Balancing Enzyme Expression: Expressing ‘TesA, FabI, and UndB together inside E. coli requires precise fine-tuning. If ‘TesA works too fast, free fatty acids accumulate to toxic levels and kill the bacteria. If UndB is under-expressed, the cells waste energy making fats they never convert into diesel. An Opentrons robot can rapidly assemble and test hundreds of genetic variants, combining different promoter strengths and ribosome binding sites, to find the exact expression ratio that maximizes fuel output without killing the cells.
  • Optimizing Whey Permeate Recipes: Cheese permeate is an agricultural waste, meaning its lactose, mineral, and protein concentrations vary from batch to batch. The robot can automatically pipette dozens of different combinations of permeate syrup, water, and trace salts into microplates to quickly identify the ideal growth medium for maximum yield.

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

a) How much?

A regional circular-economy model producing enough renewable diesel to fuel a fleet of 10 heavy-duty milk tanker trucks for 1 year.

Fleet Consumption Calculations
  • Distance Traveled: 160,000 km/year per truck.
  • Fuel Consumption Rate: Heavy-duty regional transport trucks consume an average of 36 liters per 100 km (0.36 L/km) under loaded conditions.
  • Annual Fuel Volume per Truck: Volume per truck = (160,000 km × 36 L) / 100 km = 57,600 liters/year
  • Total Target Volume (V_target): V_target = 57,600 L/truck × 10 trucks = 576,000 liters/year
  • Total Target Mass (M_target): Using a diesel fuel density of 0.78 kg/L: M_target = 576,000 L × 0.78 kg/L = 449,280 kg (approx. 449.3 metric tons/year)
Substrate Demand (Whey Permeate)
  • Biological Conversion Yield (Y_P/S): Assuming a realistic metabolic engineering yield of 0.15 g diesel per g lactose (15% conversion efficiency by weight).
  • Lactose Substrate Required: Lactose Mass = 449,280 kg diesel / 0.15 g/g = 2,995,200 kg lactose/year (approx. 2,995 metric tons)
  • Raw Whey Permeate Required: Raw liquid cheese permeate averages 4.5% lactose (45 g/L or 0.045 kg/L). Raw Permeate Volume = 2,995,200 kg / 0.045 kg/L = approx. 66.56 million liters/year

b) How long?

1. Prototyping Phase
  • Tools: High-throughput automated liquid handlers (Opentrons OT-2) and acoustic cloud platforms (Ginkgo Nebular).
  • Duration: 3 to 6 months to construct plasmid libraries, sequence-verify strains, and optimize microplate fermentation conditions (0.2 mL to 10 mL scale).
2. Industrial Manufacturing Parameters
  • Target Fermentation Titer: 15 g/L diesel hydrocarbons.
  • Volumetric Productivity: 0.3 g/L/h.
  • Batch Duration: Fermentation Time = 15 g/L / 0.3 g/L/h = 50 hours Adding 22 hours for clean-in-place (CIP), sterilization, and seed culture turnaround yields 72 hours (3 days) per batch.
  • Annual Operating Days: 330 days/year (35 days reserved for plant maintenance).
  • Batches per Reactor: Batches/Year = 330 days / 3 days/batch = 110 batches/year
3. Bioreactor Sizing & Fleet Calculation
  • Reactor Size: Standard industrial 100 m³ stirred-tank fermenters operating at an 80 m³ (80,000 liters) working volume.
  • Yield per Batch per Reactor: Yield/batch = 80,000 L × 15 g/L = 1,200,000 g = 1.2 metric tons diesel
  • Annual Output per Reactor: Annual Output = 1.2 metric tons/batch × 110 batches = 132 metric tons/year
  • Parallel Bioreactors Required: Reactors = 449.3 metric tons / 132 metric tons/reactor = 3.4 We need 4 bioreactors (100 m³ each).
Production Timeline

Once the optimized strain is deployed, running 4 parallel 100 m³ fermenters continuously will manufacture the required 449.3 metric tons (576,000 liters) of renewable diesel over 1 operational year (330 operating days).

5. What don’t you know yet?

  1. Decarboxylase expression and catalytic turnover
  • The Bottleneck: UndB is an iron-dependent, membrane-bound protein. Expressing it at high levels inside E. coli often causes severe membrane stress, poor protein folding, and low catalytic turnover (k_cat).
  • What needs to be measured & determined: Measure the in vivo catalytic turnover rate (k_cat) and substrate conversion efficiency of UndB when converting free fatty acids to alkenes in E. coli. Determine the maximum expression level possible before membrane stress inhibits cell growth.
  1. Cellular toxicity and spontaneous phase-separation
  • The Assumption: The process assumes that generating free fatty acids (FFAs) via ‘TesA and accumulating C15/C17 diesel hydrocarbons will not poison the bacteria, and that the diesel will passively exit the cells and float to the surface. In reality, intracellular FFAs uncouple the proton motive force across cell membranes, and hydrophobic alkenes can get trapped in the cell membrane, dissolving it before reaching the target 15 g/L titer.
  • What needs to be measured & determined:
  • Toxicity limits: Determine the Minimum Inhibitory Concentration (MIC) of both free fatty acids and target C15/C17 alkenes on the engineered E. coli strain.
  • Secretion / Separation efficiency: Quantify what percentage of produced diesel spontaneously passes through the cell envelope into the water phase versus what percentage stays trapped in the bacterial membrane. Determine if a biocompatible organic solvent layer (e.g., dodecane overlay) or active effluent skimming is necessary to keep cells alive.
  1. Salt, ash, and inhibitor toxicity in concentrated permeate
  • The Bottleneck: Dewatering cheese permeate increases lactose concentration, but it also concentrates minerals, ash (Ca2+, Mg2+, phosphates), lactic acid, and residual sanitizers from dairy plant equipment wash cycles. High mineral/salt concentrations create high osmotic pressure that can stunt E. coli growth and reduce metabolic yield.
  • What needs to be measured & determined:
  • Chemical profiling: Run analytical testing (HPLC/ICP-MS) on real-world industrial MVR permeate syrup samples to establish exact salt, ash, and inhibitor baseline levels.
  • Osmotic tolerance threshold: Perform growth and yield assays in microplates using varying concentrations of real permeate syrup (from 5% up to 20% lactose w/v) to measure at what salt concentration E. coli growth or diesel synthesis degrades.