1. Scalability of the Experiment:

• a. Recombinant CA Production in Pichia pastoris:

  • i. Industrial Feasibility: Using P. pastoris allows for high-yield expression of the CA enzyme with post-translational modifications essential for functionality. This yeast can grow to high cell densities in bioreactors, making it an ideal host for large-scale production.
  • ii. Cost Efficiency: Once the genetic transformation is complete, P. pastoris can produce CA with minimal setup changes, significantly reducing production costs compared to continuous plant-based extraction.
  • iii. Reproducibility: This method ensures consistent enzyme quality and quantity across batches, which is critical for scalability.

• b. Plant-Based Extraction:

  • i. Challenges in Scale-Up: The extraction from spinach involves labour-intensive steps like washing, blending, centrifugation, and dialysis. Spinach quality variation could impact enzyme yield and activity, limiting its scalability.
  • ii. Raw Material Sourcing: Ensuring a continuous supply of fresh spinach at an industrial scale could add logistical challenges and costs.
  • iii. Suitability: This method may be more suited for laboratory or small-scale applications where fresh plant material is easily accessible.

• c. Magnetite Nanoparticle synthesis:

  • i. Advantages of Coprecipitation Method:
    • 1. Simplicity and Efficiency: The method’s simplicity makes it well-suited for scale-up, as the core process of mixing, reaction, and separation can be easily adapted for larger volumes.
    • 2. Consistent Particle Quality: By controlling reaction conditions, particle size and magnetic properties are kept uniform, essential for industrial-scale consistency.
    • 3. Low Energy and Cost: The process operates at ambient or slightly elevated temperatures, reducing energy requirements compared to high-temperature methods.
  • ii. Challenges in Scaling:
    • 1. Reaction Environment Control: Scaling up would require maintaining an inert atmosphere to prevent oxidation. This would necessitate a closed or controlled reactor system, especially for large-scale production.
    • 2. Magnetic Separation Logistics: At a larger scale, magnetic separation of nanoparticles from reaction mixtures would require stronger magnets and possibly multi-stage separation to ensure efficiency and minimise loss.
    • 3. Waste Management: The need for repeated washing steps using deionised water could generate significant wastewater at an industrial scale. Efficient recycling and waste management systems would be essential to keep the process cost-effective and sustainable.

• d. Conclusion:

  • The recombinant production method in Pichia pastoris is considerably more scalable and industrially viable than plant-based extraction. Furthermore, the coprecipitation method is inherently scalable, with some adaptations required for inert gas maintenance, large-scale magnetic separation, and efficient water use management.

2. Cost and Economics of the Process:

• a. Recombinant Method:

  • i. Initial Investment: Genetic engineering and transformation require specialised equipment and reagents (e.g., plasmids, restriction enzymes, selective media), which might be costly initially. Since all the equipment was in our institute, we did not include them above. We suggest that the industry build an on-site biotechnology lab. The initial investment can vary from Rs 50 Lakhs to Rs 1 Crore. This minimises the risk of damage or loss in transporting recombinant P. pastoris from different locations. Many of the chemicals, buffers, and reagents were available in the lab, due to which our budget decreased substantially. The best approximation of the cost required is up to Rs 3 Lakhs.
  • ii. Economic Long-Term Production: After the initial setup, this method becomes cost-efficient. With P. pastoris, high enzyme yields reduce the cost per unit of CA produced, making it viable for industrial CO₂ sequestration.
  • iii. Reduced Chemical Dependency: This approach avoids recurring expenses for plant-based reagents like ammonium sulphate, making it more sustainable.

• b. Plant-Based Extraction:

  • i. Variable Cost: Dependence on fresh spinach for raw materials introduces variability in cost. Storage, handling, and extraction of enzymes require consistent replenishment of chemicals and buffers, impacting the cost-effectiveness. At the lab scale, procuring the raw material is cheap as it is available in any vegetable market. Procuring the same at the industrial level is difficult. The equipment (like dialysis membrane, centrifuge, stirrer) can cost around Rs 50 thousand to 2 Lakh. Even though fewer reagents are required initially, it will be counted as a recurring expense (up to Rs 50 thousand). The total amount can double or triple depending on the enzyme's production scale.
  • ii. Labor and Equipment: This method requires extensive manual processing steps, which may increase labour costs and limit economic efficiency.

• c. Magnetite Nanoparticle synthesis:

  • i. Materials Cost:
    • 1. Iron Salts and NaOH: The primary reagents (iron salts and ammonium hydroxide) are relatively inexpensive and widely available, making them cost-effective at scale. We bought 500 gm ammonium sulphate lab grade at Rs 360. The cost is significantly reduced if the chemical is ordered directly from the manufacturer in bulk. The same applies to the order of ferrous salts.
    • 2. Inert Atmosphere: Using an inert gas like nitrogen could add to operational costs, especially in larger-scale reactors, but closed systems or recirculating inert gases could mitigate this. Commercial-grade Nitrogen gas costs Rs 90/ cubic metre. Nitrogen storage in low-pressure cylinders will cost around Rs 1 to 2 Lakhs per cylinder.
  • ii. Equipment and Setup:
    • 1. Reactor Requirements: At larger scales, reactors with effective agitation and precise temperature/pH control will be necessary to maintain consistent particle quality. Manufacturing and construction costs are dominant. This may vary from vendor to vendor. It could range from Rs 5 Lakhs to 5 Crores.
    • 2. Magnetic Separation: Enhanced magnetic separation equipment will be needed as volume increases. Scalable magnetic separators exist but would be a considerable investment if consistently high yield and purity are desired. Starting prices of small-sized magnetic separators are Rs 2 Lakhs.

• d. Conclusion:

  • While initial costs for recombinant production are high, the long-term cost savings and consistency in enzyme output make it more economically viable than the plant extraction method. The economic feasibility of the coprecipitation method process at scale is favourable due to low-cost reagents and manageable equipment requirements. However, investment in inert gas control and advanced separation systems is necessary.

• e. Comparing Alternative Methods of MNP Synthesis:

  • The coprecipitation method has advantages over other MNP synthesis methods, particularly for scalability:
  • i. Thermal Decomposition: This alternative method offers excellent control over particle size and morphology but requires higher temperatures and organic solvents, which are more costly and less environmentally friendly at scale.
  • ii. Solvothermal Synthesis: Although highly effective, this method also relies on high-pressure systems, which are complex to scale up and costly in terms of energy and equipment.

Hence, the coprecipitation method is favourable for large-scale operations, balancing cost, simplicity, and control over particle quality, making it better suited for industrial applications.

3. Activity of Enzyme Expected:

• a. Recombinant Production:

  For our future experiments, we will screen more CA genes, which will be more stable.

  • i. High Stability and Activity: The yeast-based enzyme production is expected to yield a highly active enzyme with consistent performance.
  • ii. Improved Immobilization: Immobilizing CA on carboxyl-functionalized MNPs enhances stability and reusability. This approach demonstrates improved catalytic activity for CO₂ conversion in controlled settings, making it suitable for sustained sequestration.

• b. Plant-Based Extraction:

  • i. Lower Stability: CA extracted from spinach has lower thermal and pH stability, affecting its suitability for CO₂ sequestration processes involving varied environmental conditions.
  • ii. Limited Reuse Potential: Immobilization might improve its utility somewhat, but variability in the enzyme's initial activity could limit efficiency.

• c. Conclusion:

  • Recombinant CA from P. pastoris is expected to deliver higher, more consistent enzyme activity suitable for industrial applications.

4. CO₂ Capture Results and Efficacy Analysis:

The documents indicate CO₂ capture efficacy was tested using CA immobilised on MNPs. Growth curve analyses with E. coli cultures showed enhanced CO₂ utilisation when CA-MNPs were introduced, increasing cell density, and demonstrating the enzyme's catalytic efficiency in converting CO₂. This immobilised system shows promise for scalability, with a stable enzyme system that could maintain activity across multiple cycles in industrial applications. Using magnetite nanoparticles also supports easy recovery and reuse of the immobilised enzyme, reducing operational costs in CO₂ sequestration processes.