For a long time, enzymes are being used as biocatalysts in the field of biochemical engineering. Enzymes are nature’s catalysts. They are highly specific and provide predictive yield. But enzymes are usually unstable at high pH and high temperatures. Generally, these enzymes are employed in reactions at moderate pH and temperature (Polizzi et al., 2007). However, extensive research is being conducted to improve their stability.

Immobilization of the enzymes on solid support or in polymer matrices is one of the techniques employed to enhance its stability, reusability, and recovery efficiency, but usually at the cost of reduced activity. Their activity gets reduced because of presence of diffusivity limitations. However, in some cases, the activity might increase depending on the stability conferred to it at the molecular level (Oviya et al., 2012). One of the ways to tackle this limitation is to immobilize our enzymes on nanoparticles as they offer high surface area per unit volume, which improves the rate of mass transfer. Nanoparticles are nano-objects whose length, breadth and width are in the range of nanoscale. Based on their size, they can be classified into 3 types; size is more than 500 nm, between 100 to 500 nm and between 1 to 100nm. Nanoparticles are available in various shapes and structures. They can be synthesized using methods like chemical reduction, coprecipitation, seeding, microemulsion, hydrothermal method and sono-electrodeposition. Different properties are exhibited based on the composition of the nanoparticles (Nam & Luong, 2019). For instance, gold nanoparticles are being employed in the biomedical field due to its biocompatibility, low toxicity, and optoelectronic properties. Additionally, they exhibit a range of colors as their size increases (Yeh et al., 2012).

Figure 1: Various Structures of Nanoparticles

Magnetite nanoparticles exhibit specific magnetic properties and are being employed in fields of chemical industry, agriculture, engineering, medicine, pharmaceutics, and new energy sciences (Dudchenko et al., 2022). One of the major advantages of using magnetite nanoparticles is that it simplifies the downstream processing at industrial scale (Vaghari et al., 2016).

According to reports published by the IEA (International Energy Agency), global energy-related CO₂ emissions grew by 0.9% or 321 Mt (metric tons) in 2022, reaching a new high of over 36.8 Gt (giga tons). Furthermore, the emissions from energy combustion increased by 413 Mt (Energy Agency, 2022). Carbon dioxide is a greenhouse gas (GHG). The Emissions Gap Report 2024 estimates a temperature increase of 2.6 to 3.1 ֯C globally over the course of this century if countries all over the globe fail to comply with the norms and regulations regarding the release of greenhouse gases (Emissions Gap Report 2024, n.d.). These gases do not let the infrared radiation escape from the Earth’s atmosphere and eventually lead to an increase in the temperature of our planet i.e. global warming. This effect is called as the greenhouse gas effect.

Figure 2: Rise of Temperatures on Earth since industrial revolution (obtained from literature survey)

Other greenhouse gases include methane, water vapor, nitrous oxide etc. Increased levels of emissions of carbon dioxide gas into the atmosphere has led to elevated temperature levels (Kweku et al., 2018). Hence, we need to understand and find ways to capture and utilize this CO₂.

Carbon dioxide capture technologies are available in the market but are very costly. These technologies contribute to 70- 80% of all the activities that take place in the CCS (carbon dioxide and storage) cycle. The existing processes have their own pros and cons and some have not been operated at large scales yet. Technologies such as absorption, adsorption, chemical looping combustion, membrane separation etc. have been implemented for CO₂ separation. CO₂ is transported using pipelines, road tankers, ships etc. depending on the volume of the gas. CO₂ can either be sent to a geological storage site or be utilized in different industrial processes (like mineralization, methanol production, enhanced oil recovery etc.), agriculture and energy production (Leung et al., 2014). One of the important means for CO₂ capture is conversion into valuable industrial products like methanol (Jadhav et al., 2014), syngas (Al–Swai et al., 2021), methane (Alqarni et al., 2021), hydrocarbons and oxygenates (Gurudayal et al., 2017), etc. Conversion of CO₂ to biomass is gaining popularity as well (Liu et al., 2024).

Figure 3: Research on CO₂ to biomass process (Liu et al., 2024)

Enhancement of the current CO₂ capture systems using enzymes as catalysts is proving to be very helpful. The use of carbonic anhydrase (CA) as a CO₂ sequestering agent is gaining importance. The mechanism of action of CA is shown below:

Figure 4: Mechanism of action of CA enzyme (obtained from literature survey) (HN, 2017)

Extraction of enzymes is not easy. Most companies tend to buy the enzymes directly. Using the principles of genetic engineering, the process of enzyme production can be established and utilized. Obtaining the enzyme directly from the organism is not feasible and sustainable at an industrial scale. CA obtained from extremophiles is becoming popular due its high thermal stability (Savile & Lalonde, 2011), but obtaining such extremophiles and/ or their enzymes is challenging and costly. Furthermore, the protocols established in literature for the synthesis of nanoparticles are inconsistent.

The BIOMOD team at ICT Mumbai decided that we need to harness the untapped potential of CA as a sequestering agent by establishing the protocol for the synthesis of the CA enzyme and carboxyl group functionalized magnetite nanoparticles. We further plan to study different CA enzymes using the established protocol as a part of our Future.

Our aim for BIOMOD 2024 is to develop an economical CCUS technology to convert CO₂ to valuable biomass using carbonic anhydrase-immobilized magnetite nanoparticles. Given the amount of time and funding, the major challenge was where to get the enzyme from. After a literature survey, we found out that the commonly used baker’s yeast contains CA gene namely NCE103. We extracted the CA gene from Saccharomyces cerevisiae and overexpressed it in Pichia pastoris with the help of Escherichia coli with certain modifications in the plasmid. Details of the experimentation are given here.

Further down the road, we realized that the CA that we obtained was unstable with a half-life of 28 minutes (Genome Database, n.d.).

Since the enzyme we extracted was not stable, we extracted the CA enzyme from spinach for our purpose. Simultaneously, we synthesized magnetite nanoparticles, functionalized them with citric acid, and immobilized the enzyme onto them. Escherichia coli was used as a source of biomass and immobilized nanoparticles were added to the culture. Details of the experimentation are given here. Thus, we were successful in showing a significant increase in the biomass growth rate using these immobilized nanoparticles. We were also able to establish the reusability of the system after magnetic separation.

As is the case with the novelty, there are a lot of things that need to be proven to establish the originality completely. As far as we are aware, we are one of the few group of people who have tried to streamline and enhance the protocols and develop a proof of concept for the optimization of carbon dioxide capture. As such, there needs to be a lot of rigorous scientific experiments that need to be conducted to really be able to justify this protocol, experiments that would need a considerable amount of funding and cutting-edge research facilities that are reserved for highly ambitious research groups.

BIOMOD, on the other hand, gives a relatively short amount of time when compared to a traditional research project on subjects such as these. As such, we have focused our attempts at providing the foundational truth of our idea and have listed the points below as our criteria for success in BIOMOD 2024.

The primary challenges to the feasibility of our project are related to time constraints and costs. With the funding constraints this year, we had a hard time arranging chemicals and reagents needed for research. Synthetic biology projects often require long development cycles, including design, build, test, and iteration phases. Given the time constraints it was difficult for us to order extremophile cultures and extract their CA gene. On the other hand, baker’s yeast was a practical choice. Even the nanoparticle synthesis team had to come up with many makeshift solutions to make the project cost-effective. We also conducted a 2-day hands-on workshop where participants from different colleges got a chance to synthesize their own nanoparticles and immobilize enzymes on them respectively. In this process, we were able to impart our learnings to curious students and made a small profit out of it. To know more about the workshop, click here.

To boost productivity, the project has been executed with a degree of fluidity with respect to the individual roles of the team members. Although a focus was placed on assigned tasks by the team members, they could also intervene and take on another role in case another member was not available or there was an overload of work. This element aided in speeding up the process and kept everyone in the loop about the progress of the project. Furthermore, we also held sessions with our mentor to keep him informed of our development and ask for his help not only in research issues but also for practical aspects, such as when DNA could be ordered and what reagent was in stock.

To manage time and workload, we adopted a flexible team structure, with everyone ready to step into different roles as needed. This approach helped us tackle issues as they arose and kept everyone on the same page. Regular check-ins with our mentor were also key, letting us troubleshoot research challenges and plan around practicalities like when we could order DNA or which reagents were in stock. Overall, we’ve found ways to stay adaptable and resourceful, which has helped us stay on track.