15: Fixing Carbon Fixation

Biological Carbon Fixation

Nature has produced many enzymes that can fix atmospheric CO₂, and, of course, everyone knows that photosynthesis is the source of most fixed CO₂ in the biosphere.  Plankton, cyanobacteria, algae, and plants are key in removing atmospheric CO₂ through photosynthesis.  Yet, we can't plant enough trees to reduce CO₂ in the atmosphere in the next decade to avoid some of the worst consequences of anthropogenic climate change.  Old-growth trees (mostly gone or under significant stress) are best at removing CO₂.  New trees would take decades of growth before their effect on carbon drawdown would be consequential.  We also need to fix carbon dioxide to decrease atmospheric CO2 and make more food!

Carbon (about 100 petagrams) is sequestered yearly in net primary production (carbon fixation into biomolecules). This is split almost equally between land and ocean organisms.  The key enzyme in this process is Rubisco in the C3 (Chapter 20.4) and C4/CAM (Chapter 20.5) pathways.  The enzyme is large containing many large subunits and an equivalent number of small ones.  A chaperonin is required for folding.  It is also a very slow enzyme with a k_cat of around 2-10 CO₂/sec. In addition, it can bind another substrate, O₂, and engage in a competing reaction of photorespiration as described in Chapter 20.4.   

Figure \(\PageIndex{1}\) shows an interactive iCn3D modelof ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus PCC6301 (1RBL)

This structure is a hetero 16-mer consisting of 8 small and 8 large chains. The small chains are gray, and the large ones are different colors. Each large subunit contains a bound reaction intermediate analog, 2-carboxyarabinitol 1,5-bisphosphate.

This chapter will focus on improving and designing new ways to capture carbon dioxide to reduce its concentration in the atmosphere.  Don't lose sight that the best way to deal with anthropogenic climate change is to drastically decrease the burning of fossil fuels.

Naturally occurring pathways to fix carbon

(Much of this immediate section derives from the following reference: Natural carbon fixation. Sulamita Santos et al.,  Natural carbon fixation and advances in synthetic engineering for redesigning and creating new fixation pathways, Journal of Advanced Research, Volume 47, 2023, Pages 75-92, ISSN 2090-1232, https://doi.org/10.1016/j.jare.  Creative Commons license

Six naturally occurring pathways fix carbon. These are illustrated in Figure \(\PageIndex{2}\) below.  

Figure \(\PageIndex{2}\):  Natural carbon fixation. Sulamita Santos et al.,ibid.

Panel (A) shows the CBB cycle, which we discussed in great detail in Chapter 20.4. The enzymes involved are ribulose-1,5-bisphosphate carboxylase/oxygenase, 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and ribulose-phosphate epimerase.

Panel (B) shows the reverse (reductive) TCA cycle, which we discussed in Chapter 16.4 in the section on the α-ketoacid pathway—a primordial, prebiotic anabolic "TCA-like" pathway. The enzymes include ATP-citrate lyase, malate dehydrogenase, succinyl-CoA synthetase, ferredoxin (Fd)- dependent 2-oxoglutarate synthase, isocitrate dehydrogenase, and PEP carboxylase. 

Panel (C) shows the Wood–Ljungdahl (or reductive Acetyl-CoA) cycle, which we discussed in Chapter 30.1.  At the top of the pathway are the acetogens Archaea, and at the bottom are the methanogens Archaea.  The enzymes include MPT-methylene tetrahydromethopterin, MFR-methanofuran, THF, tetrahydrofolate. 

Panel (D) shows the 3-hydroxypropionate (3HP) cycle.  The enzymes include acetyl-CoA carboxylase, propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, succinyl-CoA:(S)-malate-CoA transferase, trifunctional (S)-malyl-CoA, -methylmalyl-CoA, mesaconyl-CoA transferase, mesaconyl-C4-CoA hydratase. 

Panel (E) shows the hydroxypropionate/4-hydroxybutyrate (HP/HB) and the dicarboxylate/4-hydroxybutyrate (DC/HB) cycles.  The enzymes include pyruvate synthase, PEP-carboxylase, malate dehydrogenase, fumarate hydratase/reductase, acetyl-CoA/propionyl-CoA carboxylase, 3-hydroxypropionate-CoA ligase/dehydratase, methylmalonyl-CoA mutase, succinyl-CoA reductase, 4-hydroxybutyrate-CoA ligase, crotonyl-CoA hydratase, acetoacetyl-CoA-ketothiolase.

Table \(\PageIndex{1}\) below shows a comparative description of the natural and synthetic carbon fixation pathways.

Sulamita Santos et al.,  Natural carbon fixation and advances in synthetic engineering for redesigning and creating new fixation pathways, Journal of Advanced Research,
Volume 47, 2023, Pages 75-92, ISSN 2090-1232, https://doi.org/10.1016/j.jare.  Creative Commons license

Plants and photoautotrophic microorganisms fix CO₂ by the Calvin cycle using NADPH and ATP, producing O₂.  Some anaerobic photosynthetic bacteria don't produce O₂.  In chemolithoautotrophic microorganisms, energy sources (i.e., electron donors like H₂, H₂S, sulfur, Fe²⁺, nitrite, and NH₃ found in bedrock - the lithosphere) other than NADPH, ATP, and light can be used to drive CO₂ uptake. For chemoorganotrophic microbes, reduced organic molecules such as sugars and amino acids serve as electron (donor) sources.  

Genetic engineering and synthetic biology are now employed to improve preexisting enzymes and create new pathways for carbon capture.

As a simple example, people are trying to engineer carbonic anhydrase, which is used to convert CO₂ to HCO₃^-, for transport to the lungs, where it is converted back to CO₂ and released.  It is a diffusion-controlled enzyme with a k_cat/K_M reported as high as 8 x 10⁷ M^-1s^-1, so how can it be made better?  One way is to engineer thermal stability into the enzyme. A critical problem for the forward reaction is that the enzyme is readily reversible, so bicarbonate, HCO₃^-, is a competitive inhibitor of the forward reaction. In addition, the enzyme can be engineered to be more stable at higher pH to allow product (HCO₃^-) removal by the addition of OH^- in the process of mineralization, as shown in the equation below,

HCO₃^- + OH^- →  CO₃^2- + H₂O

where the carbonate anion can precipitate in the presence of divalent cations like  Ca²⁺, Mg²⁺, and Fe²⁺.  Here is a link to a Literature-based Guided Assessment on thermoengineering of carbonic anhydrase.  

Now, let's explore using new pathways created by synthetic biology to capture carbon.  Some of these pathways are engineered to produce reactants (feedstocks) for biofuels, which we discussed in-depth in previous Chapter 32 sections for chemical synthesis.  We'll focus on the CETCH pathway, the reductive glyoxylate and pyruvate synthesis (rGPS) cycle, and the malyl-CoA-glycerate (MCG) pathway.

CETCH (Crotonyl-CoA-EThylmalonyl-CoA-4Hydorxybutyl-CoA) pathway

To engineer a new pathway, Swander et al identified efficient carboxylases from known ones (acetyl-CoA carboxylase, Rubisco, propionyl-CoA carboxylase, PEP carboxykinase, 2-oxoglutarate carboxylase, and pyruvate carboxylase), all of which we have discussed in previous chapters.  They created new pathways, calculated free energy and ATP/NADPH requirements, and then optimized the pathways.  They chose CoASH–dependent carboxylases and enoyl-CoA carboxylases/reductases.  Figure \(\PageIndex{3}\) below shows the reaction of one key carboxylase.  

Figure \(\PageIndex{3}\): Carboxylase used in the CETCH pathways to fix CO₂  

The pathway was named CETCH (Crotonyl-CoA-EThylmalonyl-CoA-4Hydorxybutyl-CoA) which catalyzes this next reaction in cell lysates (in vitro):

2CO₂ + 3NAD(P)H + 2ATP + FAD → glycolate + 3NAD(P) + 2ADP +2P_i + FADH₂

The rate of CO_2­ fixation by the CETCH pathways was similar to the rate of the Calvin cycle rates in cell lysates. 

In a more expansive approach, Gleizer used synthetic biology to change E. Coli from a heterotroph to an autotroph in which its biomass (carbon reservoir) came from CO₂.  Formate (HCO₂^-) was used as a source of reducing power (electrons) as it was oxidized by an added formate dehydrogenase to produce NADH for the autotropic fixation of CO₂ by adding Calvin cycle enzymes.  Using isotopically labeled ¹³CO₂ to follow carbon flow, after 10 generations and evolution, the cells were completely autotrophic through fixation of CO₂.  To accomplish this, they knocked out genes for phosphofructokinase (glycolysis) and glucose-6-phosphate-dehydrogenase (pentose-phosphate pathway) to impair these main metabolic pathways. They added carbonic anhydrase (to interconvert CO₂ and HCO₃^-) and Rubisco and phosphoribulokinase.  As formate was ultimately converted to CO₂, the net effect was not carbon neutral but could be if atmospheric CO₂ was used to make formate (for a feedstock) by electrochemical reduction.

Figure \(\PageIndex{4}\) below shows the next synthetic E. Coli autotrophs reactions.

Figure \(\PageIndex{4}\):  Schematic Representation of the Engineered Synthetic Chemo-autotrophic E. coli.  Shmuel Gleizer et al., Conversion of Escherichia coli to Generate All Biomass Carbon from CO2, Cell, 179 (2019). https://doi.org/10.1016/j.cell.2019.11.009.   Creative Commons license.  

CO₂ (green) is the only carbon source for all the generated biomass. The fixation of CO₂ occurs via an autotrophic carbon assimilation cycle. Formate is oxidized by a recombinant formate dehydrogenase (FDH) to produce CO₂ (brown) and NADH. NADH provides the reducing power to drive carbon fixation and serves as the substrate for ATP generation via oxidative phosphorylation (OXPHOS in black). The formate oxidation arrow is thicker than the CO₂ fixation arrow, indicating a net CO₂ emission even under autotrophic conditions.

Figure \(\PageIndex{5}\) shows that almost 100% of carbon atoms after many generations are labeled with ¹³C (detected by mass spec analysis) derived from ¹³CO₂.   

Figure \(\PageIndex{5}\): Isotopic Labeling Experiments Using ¹³C Show that All Biomass Components Are Generated from CO₂ as the Sole Carbon Source.  Gleizer  et al., ibid.

(A) Values are based on LC-MS analysis of stable amino acids and sugar-phosphates. The fractional contribution of ¹³CO₂ to various protein-bound amino acids and sugar-phosphates of evolved cells grown on ¹³CO₂ and naturally labeled formate showed almost full ¹³C labeling of the biosynthesized amino acids. The numbers reported are the ¹³C fraction of each metabolite, taking into account the effective ¹³CO₂ fraction out of the total inorganic carbon (which decreases due to unlabeled formate oxidation to CO₂). The numbers in parentheses are the uncorrected measured values of the ¹³C fraction of the metabolites. 

Synthetic reductive glyoxylate and pyruvate synthesis (rGPS) cycle and the malyl-CoA-glycerate (MCG) pathways

These pathways were created to synthesize acetyl-CoA, pyruvate, and malate from CO₂ in cell-free systems to free the system from cell growth and regulation requirements and to make it insensitive to oxygen.  These molecules are also intermediates in the created cycle, which could operate continuously for hours at the same or greater rates of CO₂ fixation compared to photosynthesis. The cycle is shown in Figure \(\PageIndex{6}\) below.

Figure \(\PageIndex{6}\):  The rGPS–MCG cycle with acetyl-CoA as the end product. Luo, S., Lin, P.P., Nieh, LY. et al. A cell-free self-replenishing CO₂-fixing system. Nat Catal 5, 154–162 (2022). https://doi.org/10.1038/s41929-022-00746-x .  Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/

The rGPS cycle consists of the reductive glyoxylate synthesis (rGS) pathway (blue) and the reductive pyruvate synthesis (rPS) pathway (green). The malyl-CoA-glycerate (MCG) pathway (orange) consists of the rGS and glycerate pathways. The red arrow indicates the carboxylation reaction. Gcl, glyoxylate carboligase; Tsr, tartronate semialdehyde reductase; Gk, glycerate 2-kinase; Eno, enolase and 2PG, 2-phospho-d-glycerate

Microbial electrosynthesis from CO₂

We mentioned above that if the formate used in the CETCH pathway could be synthesized through electrochemistry from CO₂, then the pathway would be truly carbon neutral. New microbial electrochemical methods are being designed to synthesize various small molecules that could serve as feedstocks for chemical synthesis in industry.  The carbon in CO₂ has an oxidation number of +4, while the carbon in formic acid has an oxidation number of +2.  Hence, two electrons must be added electrochemically to make the CETCH pathway truly carbon neutral.  Bigger electrochemical reductants of CO₂ require more electrons.  Figure \(\PageIndex{7}\) below shows how key feedstocks could be made electrochemically from CO₂ and how they are usually made in industry.  

Figure \(\PageIndex{7}\) below.  :  Overview of the Main Products Formed from Microbial Electrosynthesis (MES) From CO₂, Along With the Main Industrial Methods to Manufacture These Products.  Jourdin et al., Trends in Biotechnology, April 2021, Vol. 39, No. 4 https://doi.org/10.1016/j.tibtech.2020.10.014.  Creative Commons Attribution (CC BY 4.0)

These feedstocks could be made by reductive electrosynthesis using electrons from the oxidation of water through electrolysis.  Released electrons  (oxidation number of O in water is -2 and 0 in O₂) move to a biocathode to reduce CO₂, as illustrated in Figure \(\PageIndex{8}\) below. 

Figure \(\PageIndex{8}\):  Reactor configurations for MES-based CO₂ conversion: (a) H-type, (b) single chamber, (c) dual chamber, (d) continuous stirred tank, and (e) schematic of the electron transfer mechanism.  G. S. Lekshmi et al., Microbial electrosynthesis: carbonaceous electrode materials for CO₂ conversion.  Mater. Horiz., 2023, 10, 292-312.  DOI: 10.1039/D2MH01178F.  Creative Commons Attribution-Non Commercial 3.0 Unported Licence

The biocathode consists of a biofilm of cells printed onto a graphene, graphite, or carbon nanotube-laden support (all carbonaceous).  

Marine CO₂ Removal Through Geoengineering

As the climate worsens, more climate scientists are contemplating improving the oceans' ability to capture CO₂. Both inorganic and biological methods have been proposed. These methods will likely have unintended consequences, affecting biodiversity and the natural mechanisms for carbon sequestration. Hence, they are both controversial.

Inorganic:  

Here is a review from Chapter 32.3:  The Carbon Cycle and Carbon Chemistry

The reversible movement of CO₂ from the atmosphere to the oceans, CO_2 atm ↔ CO_2 ocean, depends on the difference in the partial pressures of CO₂ (ΔpCO₂) in the atmosphere and surface waters.  The reaction is also driven to the right by the removal of CO₂ (aq) as it forms carbonic acid (H₂CO₃), which then forms bicarbonate (HCO₃^–) and carbonate (CO₃^2–). These coupled reactions chemically buffer ocean water, thus regulating ocean pCO₂ and pH.

pCO₂ is not homogenous in ocean surface waters and varies with different conditions of current and temperature.  CO₂ can be more readily released from upwellings of nutrient-rich and warm waters, especially in the tropics.  In cold Northern waters and the Southern Ocean, where water sinks, it is taken up from the atmosphere (again, CO₂ is more soluble in cold water). 

As we discussed in Chapter 31.1, the ocean chemistry of CO₂ determines, in large part, the levels of atmospheric CO₂.  The coupled reactions of CO₂ in the oceans are shown below.

\begin{equation}
\mathrm{CO}_2(\mathrm{~g}, \mathrm{~atm}) \leftrightarrow \mathrm{CO}_2(\mathrm{aq}, \text { ocean) }
\end{equation}

\begin{equation}
\mathrm{CO}_2(\mathrm{aq} \text {, ocean })+\mathrm{H}_2 \mathrm{O}(\mathrm{I} \text {, ocean }) \leftrightarrow \mathrm{H}_3 \mathrm{O}^{+}(\mathrm{aq})+\mathrm{HCO}_3^{-}(\mathrm{aq})
\end{equation}

\begin{equation}
\mathrm{H}_2 \mathrm{O}(\mathrm{I})+\mathrm{HCO}_3^{-}(\mathrm{aq}) \leftrightarrow \mathrm{H}_3 \mathrm{O}^{+}(\mathrm{aq})+\mathrm{CO}_3{ }^{2-}(\mathrm{aq} \text {, sparingly soluble })
\end{equation}

These reactions should be familiar to all chemistry students and were presented previously in Chapter 31.1 and in Chapter 2.  A significant contributor to ocean bicarbonate is the weathering of rocks like limestone. and marble, both forms of CaCO₃. The relevant reactions are shown below.  

\begin{equation}
\begin{aligned}
&\mathrm{CaCO}_3(\mathrm{~s})+\mathrm{H}_2 \mathrm{O} \leftrightarrow \mathrm{Ca}^{2+}(\mathrm{aq})+\mathrm{CO}_3{ }^{2-}(\mathrm{aq}) \\
&\mathrm{CO}_3{ }^{2-}(\mathrm{aq})+\mathrm{H}_2 \mathrm{O} \leftrightarrow \mathrm{HCO}_3{ }^{-}(\mathrm{aq})+\mathrm{OH}^{-}(\mathrm{aq})
\end{aligned}
\end{equation}

CO₂ is nonpolar and not very soluble in water.  Either is CO₃^2- in the presence of divalent cations like Ca²⁺. However, HCO₃^- is and can be considered a "soluble" form of carbon.  This soluble form from terrestrial weatherings ends up in rivers and eventually enters the ocean.  It is also the form of carbonate that is transferred into cells by anion transporters for eventual shell formation. HCO₃^- is also a chief regulator of both blood and ocean pH.  Weathering is slow compared to anthropogenic emissions of CO₂ from fossil fuel use, but it is nevertheless a key player in the carbon cycle and the regulation of ocean pH. 

The same weathering reactions on silicate rocks lead to the transfer of silicate ions into rivers and the ocean, where they can be used by diatoms for the formation of  CaSiO₄ shells. As the oceans take up more CO₂, they become more acidic, which leads to the equivalent of "weathering" of shells of living organisms, leading to their potential death. Silicon is directly underneath carbon in the periodic table, so this simplified reaction is analogous to those we see with CO2 and its inorganic ions.

\begin{equation}
\mathrm{H}_4 \mathrm{SiO}_4=\mathrm{SiO}_2+2 \mathrm{H}_2 \mathrm{O}
\end{equation}

H₄SiO₄ is silicic acid.

With this background in mind, you can understand the proposed method of adding crushed limestone (CaCO₃) to the rivers (which drain into the oceans) to remove atmospheric CO₂. As the solid crushed limestone dilutes, it gets solubilized. As CO₃^2- is a reasonable base, the water would become more alkaline.  It would mostly get converted to HCO₃^- which can be transported into river shell-forming organisms.  The rest enters the oceans where it can be transported into organisms and used to make CaCO₃ shells, eventually sinking to the depths where the carbon would be sequestered for 1000s of years.   This would have the added benefit of decreasing the acidification of rivers.  Whether this process could be run at the scale needed to reduce atmospheric CO2 significantly is unclear.

Biological

This method involves adding Fe²⁺, usually iron (II) sulfate (FeSO₄), to the oceans to promote phytoplankton growth. This process has been called ocean iron fertilization (OIF). One problem with FeSO₄ is that it rapidly oxidizes to insoluble iron (III) species in a temperature-dependent process. It must be transported to sufficient depths so it doesn't return to the surface. Another problem is the adverse effects of nutrient removal on other marine organisms.