SecondCircle ($45M for fermenting CO2 into biofuel)

SecondCircle, a biotech company founded in Denmark in 2020, has developed a gas fermentation technology that uses thermophilic bacteria in a bioreactor to transform carbon dioxide (CO₂) from flue gasses into biofuels like acetate, acetone, and ethanol. This process not only cuts down on CO₂ emissions, but it also makes green, sustainable fuels that can replace chemicals made from traditional fossil fuels.

Challenges: carbon emissions and ferment CO2

Carbon emissions

Since the early 1900s, carbon dioxide (CO₂) levels in the atmosphere have increased by 50% due to human activities. When fossil fuels (such as coal, oil, and natural gas) are burned for energy production, transportation, and industrial processes, CO₂ is released into the atmosphere. This excess CO₂ acts as a greenhouse gas, trapping heat and causing the air and ocean temperatures to rise. CO₂ emissions play a crucial role in driving climate change.

This warming effect has caused the global average temperature to rise by about 1.1 ºC since the pre-industrial period. This has led to rising in the frequency and intensity of extreme weather events, melting of polar ice caps and glaciers and rising sea levels, shifts in species ranges and increased risk of species extinction, agriculture and food security, and ocean acidification.

To mitigate these impacts, the Paris Agreement aims to limit global warming to well below 2 ºC above pre-industrial levels. The Intergovernmental Panel on Climate Change (IPCC) estimates that a “carbon budget” of about 500 GtCO₂, which corresponds to about ten years at current emission rates, provides a 66% chance of limiting global warming to 1.5 ºC.

Negative emissions technologies (NET) can help companies, sectors, or countries remove more CO₂ from the atmosphere than they emit. Examples of NETs include Direct Air Capture (DAC), enhanced weathering, and Ocean Alkalinity Enhancement. According to climate models, a significant deployment of NETs will be needed to prevent catastrophic ocean acidification and global warming beyond 1.5 ºC.

Moorella thermoacetica for CO₂ fixation

Acetogenic bacteria grow by consuming gaseous CO₂ (or CO). The process fixes CO₂. Thermophilic bacteria for CO₂ fixation at typical temperatures between 45 ºC and 80 ºC are of particular interest because they reduce risk of contamination with unwanted microorganisms, use less energy for cooling the fermentation system, have a faster production rate due to the advantageous thermodynamics, and lower capital and operational expenditures.

Moorella thermoacetica is a type of thermophilic bacteria that can turn CO₂ gas into acetone and other useful chemicals. So, they have attracted significant interest in the applications in clean technology, renewable energy, and the production of fuels and other valuable chemicals.

Moorella thermoacetica is very good at fixing CO₂, but its growth rate is slow, which makes it less effective at fixing CO₂. Moorella thermoacetica is also sensitive to fluctuations in the gas supply and gradients in the industrial production bioreactor. If there aren’t enough nutrients or CO₂ gas in the bioreactor, Moorella thermoacetica can die or sporulate. This makes subcultures inactive and decreases the overall efficiency significantly.

Therefore, Moorella thermoacetica needs to be able to live for a longer time and recover faster when nutrients become available so that it can fix CO₂ more efficiently.

SecondCircle Technology

SecondCircle has developed a biotechnology to increase the growth of a Moorella thermoacetica by deleting SpoOA gene and/or introducing genes to express a variant of SinR in the bacterium. This gene modification has increased the growth rates and decreased the lag phase duration of the Moorella thermoacetica, increasing their CO₂ fixation and biochemical production efficiency.

Thermophilic bacteria fix CO2 to produce biofuel.

How SecondCircle technology work

The growth of a bacterial culture can be divided into four distinct phases: lag phase, log phase, stationary phase, and death phase.

Lag phase

This is the first stage of bacterial growth, when the number of bacteria stays the same as they get used to the conditions of the growth medium. They increase in size and synthesize enzymes, RNA, and other molecules necessary for cell division and population growth under their new environmental conditions. How long the lag phase lasts depends a lot on where the bacteria came from and how healthy the bacteria cells are.

Log phase

In this phase, the population of bacteria grows logarithmically.

Stationary phase

In this phase, the bacterial population stays the same because the nutrient supply is depleted, so growth stops.

Death phase

This is the last stage of growth, when all the nutrients have been used up and the number of cells starts to go down.

SecondCircle has increased the growth of a Moorella species bacteria by modifications of the genes that code for SpoOA and SinR to make Moorella thermoacetica grow faster. This makes the bacteria more efficient at fixing CO₂ and producing biochemicals.

SpoOA (Stage 0 sporulation protein A homolog) is a protein that regulates sporulation, the process by which bacteria form spores in response to environmental stress. SpoOA gene deletion can prevent sporulation.

The SinR (HTH-type transcriptional regulator SinR) is a protein capable of binding to specific DNA sequences to regulate gene bacterial expression in response to nutrient depletion at the end of vegetative growth. For example, the SinR can function as a repressor of SpoOA.

SecondCircle has introduced genetic modifications into the bacterium that allow it to express a variant of SinR, which aids bacteria in adapting more quickly to the new environment, potentially shortening the lag phase boosting their growth rates.

The SinR variant has at least 90% sequence identity with SEQ ID NO: 2 and an amino acid other than valine (V) at the position corresponding to position 198 in SEQ ID NO: 2. The amino acid at the position corresponding to position 198 in SEQ ID NO: 2 is phenylalanine (F).

A V198F mutation in SinR decreases the duration of Moorella thermoacetica’s lag phase (it leads to a more rapid recovery from resting state) upon inoculation into fresh medium following a longer incubation period. Moreover, the V198F in Moorella thermoacetica SinR may affect the protein’s stability, affinity for the anti-repressor SinI, and/or ability to oligomerize.

Advantages of SecondCircle technology

SecondCircle’s biotechnology has the following the advantages:

The application of Moorella thermoacetica has a low risk of contamination, higher conversion rates, no requirement for cooling water, and significantly low capital and operational expenditures.

Cells which are able to recover faster after being in a stressful situation are highly advantageous for use in bioreactors, as this allows a larger degree of fluctuation and gradients (nutrients, pH, and CO₂) in the bioreactor.

No genes or operons will need to be overexpressed, which would result in an increased metabolic burden. These engineered bacteria will maintain a high metabolic activity throughout the fermentation.

Applications of SecondCircle technology

SecondCircle has increased the growth of Moorella thermoacetica through genetic modification, resulting in enhanced fixation of CO₂ and biofuel production. The genetically modified Moorella thermoacetica also contains enzymes that prompt the production of the selected biochemical, such as acetone.