Internal carbon sequestration has taken many different forms throughout history. Even before the evolution of eukaryotic plants utilizing photosynthesis and light to convert CO
2 and energy from light to compose simple sugars, single-celled organisms had already developed mechanisms to capture atmospheric CO
2 and transform it into essential compounds for the cell’s development. These primitive mechanisms, especially those in microorganisms like acetogens and methanogens, have shown to be highly efficient, utilizing unique proteins and metabolic pathways for carbon sequestration [
1]. Furthermore, microorganisms, especially microalgae and cyanobacteria, exhibit significant advantages over higher plants in their capacity for CO
2 fixation as they can yield higher solar energy retention and the potential for year-round growth compared to their more complex plant counterparts [
25]. While microalgae are well-recognized for their CO
2 fixation capabilities, bacteria present advantages that cannot be overlooked [
26]. Microalgae cultivation can be subject to biocontamination over prolonged use from fungal and bacterial species and often run into issues pertaining to even distribution of sun exposure over larger microalgae ponds due to their preferred growth environments, vastly limiting their ability to be utilized on an industrial scale without major alternations to the water infrastructure the microalgae is grown on. Bacteria and some yeasts, on the other hand, have been widely used in biotechnology industry due to inherent compatibility to produce chemicals and their rapid growth rates and life cycles. Further, they are more inclined to accept DNA during genetic modification in the form of plasmids and genomic alternations. This ability allows bacteria and yeast to have DNA introduced into their cells of enzymes to complete metabolic pathways previously incompletely represented in the cells and allow production of specialized products, including bio-alcohols and essential fatty acids. Through this biotechnological approach, CO
2 can be directly converted into value-added products, offering an advantage over traditional methods like catalytic conversion, which demand energy-intensive conditions [
24].
In this section, we will provide an overview of the one-step strategy for directly using CO2 as the feedstock for biomanufacturing, which includes (1) natural CO2 fixation pathways, (2) synthetic CO2 fixation pathways, (3) host selection and reducing power required for biomanufacturing with CO2, and (4) Using microbial electrosynthesis to utilize CO2 for biomanufacturing.
2.1.1. Natural CO2 fixation pathways
Several pathways facilitate the assimilation of atmospheric CO
2 into organic materials, as shown in
Figure 2. Among all natural CO
2 fixation pathways, the Calvin-Benson-Bassham (CBB) cycle dominates, and is responsible for 90% of global CO
2 uptake, primarily driven by the enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) [
27]. This enzyme catalyzes the transformation of ribulose 1,5-bisphosphate (RuBP) into 3-phosphoglycerate (3-PGA), but its efficiency is occasionally halved due to its tendency to favor O
2 during photorespiration [
28]. Additionally, pathways such as the Wood-Ljungdahl (WLP), reductive glycine pathway (rGlyP), reductive tricarboxylic acid (rTCA) cycle, 3-hydroxypropionate bi-cycle (HP), 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle, and dicarboxylate/4-hydroxybutyrate (DC/HB) cycle play significant roles in CO
2 utilization [
29]. These processes, predominantly in autotrophic microorganisms, often lead to vital metabolites like pyruvate or acetyl-CoA, each with unique energy efficiency concerning ATP consumption [
30].
Calvin-Benson-Bassham (CBB) Cycle: The CBB cycle stands as the premier identified CO
2 biofixation route and remains the primary carbon fixation method in nature. Since it shares numerous metabolites and enzymes with the pentose phosphate pathway (PP pathway), leading to its alternate naming as the reductive pentose phosphate pathway. Found in a variety of organisms such as plants, algae, cyanobacteria, and specific chemoautotrophic microorganisms, this cycle fundamentally operates through the enzymatic action of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). This enzyme facilitates the electrophilic addition of CO
2 to ribulose 1,5-bisphosphate (RuBP), producing two molecules of 3-phosphoglycerate (3-PG). Interestingly, RuBisCO can also introduce oxygen (O
2) instead of CO
2, forming 3-PG and glyoxylate. This alternative incorporation initiates photorespiration, a series of reactions that release rather than assimilate CO
2. As the cycle progresses, 3-PG is subsequently converted to glyceraldehyde 3-phosphate (G3P) with the assistance of enzymes from the gluconeogenic pathway. From there, some G3P molecules fuel central carbon metabolism, while others contribute to the regeneration of RuBP, which is crucial for ongoing CO
2 fixation [
31]. While RuBisCO's central role in the CBB cycle is undeniable, its efficiency is often questioned. Known for its limited catalytic activity, RuBisCO prefers O
2 over CO
2, complicating efforts aimed at engineering it for enhanced kinetics largely due to the intricate nature of its substrate-binding pocket [
32]. However, efforts to enhance the cycle's efficiency have centered on engineering RuBisCO. For instance, a heterologous cyanobacterial RuBisCO, thanks to its carbon fixation efficiency, was successfully overexpressed in
Ralstonia eutropha (
Cupriavidus necator), bolstering autotrophic growth and CO
2 fixation capabilities [
33]. Furthermore, a comprehensive in vitro examination of 143 RuBisCO enzyme activities unveiled a promising type-II RuBisCO variant from
Gallionella sp., which is iron oxidizing chemolithotrophic bacteria [
34]. Such advancements underscore the potential to amplify CO
2 assimilation rates by harnessing superior RuBisCO variants.
Wood-Ljungdahl Pathway (WLP): The WLP, referred to as the reductive acetyl-CoA (rAc-CoA) pathway, is an exemplar of efficient non-photosynthetic carbon fixation. Requiring only one ATP molecule to produce pyruvate is notably more energy-conserving than the CBB cycle, which expends seven ATPs for the same result [
5]. The WLP, primarily recognized in acetogens, operates exclusively under anaerobic conditions [
35]. This is attributed to the oxygen sensitivity of its key enzymes: carbon monoxide dehydrogenase (CODH). In the rAC-CoA pathway, two CO
2 molecules are reduced and converted into acetyl-CoA. A bifunctional enzyme, CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), operates this by catalyzing both the reduction of CO
2 to CO and the subsequent condensation of coenzyme A, methyl group, and CO to produce acetyl-CoA. The same rTCA cycle pathways then assimilate this acetyl-CoA further. Microbes utilizing the rAC-CoA pathway often produce acetate or methane as end products [
36].
Reductive Glycine Pathway (rGlyP): The initial CO
2 assimilation steps in WLP parallel the reductive glycine pathway (rGlyP), wherein formate dehydrogenase reduces CO
2 by employing formate dehydrogenase (FDH) to formate, which is subsequently incorporated into the THF cycle to yield 5,10-methylene-THF. This is then reduced further to generate 5-methyl-THF in WLP. Eventually, a combination of this methyl group with CoA and CO produces acetyl-CoA. rGlyP, instead, employs glycine cleavage/synthase system (GCS) to incorporate CO
2 and ammonium into 5,10-methylene-THF to produce l-glycine and recycle THF back [
30]. Highlighting their potential in microbial CO
2 utilization, the WLP and the rGlyP stand out for their ATP efficiency in carbon fixation [
37]. The most important advantage of the rGly pathway over WLP is that rGlyP can be operate both in aerobic and anaerobic microorganisms. [
38]. Strategies such as overexpressing the essential enzymes can further augment CO
2 assimilation efficiency. For instance, enzymes related with THF cycle overexpression in
Acetobacterium woodii to create a more productive WLP resulted in a 14% rise in acetate production [
39]. Similarly,
Eubacterium limosum, when introduced with the GCS, exhibited an improved growth rate and acetate production [
40]. Taking it further, even heterologous microbes like
E. coli have been successfully engineered with WLP and rGlyP, broadening their carbon conversion capabilities [
41,
42].
Reductive Tricarboxylic Acid Cycle (rTCA): Initially discovered in the green sulfur bacterium
Chlorobium limicola, the rTCA functions as the reverse counterpart to the traditional TCA (or Krebs cycle), primarily in strictly anaerobic or microaerobic autotrophic eubacteria [
43]. Key to the rTCA cycle's operation are various enzymes, such as fumarate reductase, 2-oxoglutarate: ferredoxin oxidoreductase (OGOR), ATP-citrate lyase, pyruvate: ferredoxin oxidoreductase (PFOR), and either PEP or pyruvate carboxylase [
44]. The rTCA cycle begins with the reductive carboxylation of acetyl-CoA to pyruvate by PFOR, which uses reduced ferredoxin as an electron donor in an oxygen-sensitive process. From pyruvate, several transformations lead to acetyl-CoA and oxaloacetate, completing the cycle. Interestingly, in thermophilic species such as
Hydrogenobacter thermophilus, there's a unique conversion of 2-oxoglutarate to isocitrate by enzymes that avoid the accumulation of the unstable intermediate succinyl-CoA [
45]. Another variation, the reverse oxidative tricarboxylic acid (roTCA) cycle, is akin to the rTCA cycle but stands out in its enzyme utilization for citrate cleavage, which results in an energy-efficient pathway, despite its thermodynamic challenges [
46]. Both cycles highlight the intricate ways organisms assimilate carbon dioxide, ultimately contributing to pyruvate biosynthesis. Although studies on the rTCA cycle's application in metabolic engineering remain limited, emerging research, such as one involving
E. coli, has shown promising results in recycling CO
2 and optimizing the production of acetate and ethanol [
47].
3-Hydroxypropionate (3HP) Bi-Cycle: The 3HP bi-cycle, or Fuchs-Holo bicycle, was first discovered in the thermophilic phototrophic bacterium
Chloroflexus aurantiacus [
48]. This cycle is considered unique due to its two cyclic CO
2 assimilation pathways that collaboratively share initial reactions for CO
2 assimilation, forming a complex bicyclic system. Distinctively, acetyl-CoA is first carboxylated to malonyl-CoA and, through a sequence of reactions involving characteristic intermediates like 3-hydroxypropionate and (S)-malyl-CoA, leads to the formation of propionyl-CoA and glyoxylate. The latter subsequently enters a second cycle, culminating in the formation of pyruvate. The 3HP bicycle consumes approximately 2.3 mol ATP to reduce 1 mole of CO
2 to pyruvate, similar to the CBB cycle [
49].
3-Hydroxypropionate/4-Hydroxybutyrate (HP/HB) Cycle and Dicarboxylate/4-Hydroxybutyrate (DC/HB) Cycle: Beyond the 3HP bi-cycle, other carbon fixation pathways like the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle and the dicarboxylate/4-hydroxybutyrate (DC/HB) cycle have also been identified. Notably, the HP/HB and DC/HB cycles, prevalent in certain archaea, demonstrate higher energy efficiency in anaerobic environments, with the DC/HB cycle being particularly efficient, requiring only 1.6 mol ATP to reduce one mol CO
2 to pyruvate [
49]. From an evolutionary perspective, the capability of the 3HP bicycle and the HP/HB cycle to assimilate bicarbonate rather than CO
2 is notable. This adaptability likely stems from the higher intracellular concentration of bicarbonate compared to CO
2. This feature and oxygen tolerance potentially contribute to their evolutionary survival [
45]. From an application standpoint, there have been attempts to harness these pathways for biotechnological purposes. The 3HP bi-cycle’s key enzymes, such as propionyl-CoA synthase and malonyl-CoA reductase, have been leveraged to construct efficient cell factories for 3-hydroxypropionic acid [
50]. Similarly, parts of the HP/HB cycle have been expressed in
Pyrococcus furiosus, highlighting advancements in CO
2 reduction to acetyl-CoA [
51]. However, attempts to fully recreate and utilize these pathways in common microbial hosts like
E. coli have faced challenges [
5].
2.1.2. Synthetic CO2 fixation pathways
Synthetic CO
2 fixation pathways have garnered significant attention as potential alternatives to enhance carbon assimilation efficiency, transcending the inherent constraints observed in natural pathways. The focus lies in developing pathways with optimized thermodynamic and kinetic properties while overcoming difficulties associated with key enzymes like RuBisCO [
32,
34]. One noteworthy example is the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle. Assembled using 17 enzymes derived from nine distinct organisms, the CETCH cycle has displayed a greater rate of CO
2 fixation and a reduced ATP requirement compared to the CBB cycle [
27]. Its efficiency is partly attributed to the use of the enoyl-CoA carboxylase/reductase enzyme, which showcases high carboxylation activity. However, translating the in vitro success of the CETCH cycle into in vivo applications remains a challenge [
45].
Another synthetic CO
2 assimilation route is the Gnd-Entner-Doudoroff (GED) pathway. By inducing specific gene deletions in
E. coli, researchers demonstrated the energy-efficient reductive carboxylation of ribulose-5-phosphate via this pathway. Despite its potential, the complete cyclic GED pathway has only been partially shown
in vivo [
52]. Another advancement was made when researchers synthesized starch from CO
2 and hydrogen in a cell-free system. This process coined the artificial starch anabolic pathway (ASAP), comprised 11 core reactions, and showcased an impressive CO
2-to-starch conversion rate. This rate was approximately 8.5 times faster than starch synthesis observed in corn [
53]. Since pathway length also generates problems for energy efficiency, novel pathways like the POAP cycle and the ICE-CAP pathway have been proposed [
54]. The POAP cycle, comprising merely four steps, potentially offers a more streamlined and efficient approach to carbon sequestration. The ICE-CAP pathway, on the other hand, utilizes CO
2 alongside high-energy C1 compounds, such as methanol or formaldehyde, obviating the need for ATP and cofactors like NAD(P)H [
55].
One computational study, utilizing a repository of around 5,000 known enzymes, unveiled the Malonyl-CoA-Oxaloacetate-Glyoxylate (MOG) pathways. These proposed pathways, which display ATP efficiency over the conventional CBB, might be revolutionary. They use rapid carboxylases and are oxygen-tolerant. However, some enzymes in MOG pathways are thermally sensitive, and their end-product, glyoxylate, when integrated into central metabolism, could revert to CO
2, causing this study performed only in
in silico [
56]. Nevertheless, designing and implementing synthetic pathways isn't without its challenges. When introduced into diverse microbes, these synthetic pathways can disrupt the metabolic balance, necessitating further optimization to realign central metabolic fluxes. Despite this, the capabilities of these synthetic pathways, especially when combined with other technological advancements like biocompatible semiconductor materials or cell-free systems, offer promising avenues for the future of carbon sequestration and utilization [
57].
2.1.4. Microbial electrosynthesis
As shown in
Figure 3, microbial electrosynthesis (MES) is an innovative bioelectrochemical approach that leverages electroactive microorganisms to convert renewable electrical energy into value-added products [
91,
92]. Rooted in bioelectrochemical systems (BES) principles, MES offers a sustainable route to harness CO
2 for the synthesis of biofuels and commodity chemicals, some of which include methane, acetate, formic acid, and ethanol, among others, potentially mitigating the detrimental impacts of CO
2 emissions [
93]. At its core, MES operates by utilizing a biofilm on an electrode as a catalyst, which contrasts with traditional methods that employ chemical catalysts [
24].
The MES architecture is intricate [
94]. The anodic chamber operates abiotically, where water undergoes splitting to generate protons, electrons, and oxygen. Electrons generated in this chamber are channeled towards the biocathode via an external circuit when an external voltage is applied to the electrochemical cell. Conversely, electrophilic bacteria, primarily acetogens, inhabit the cathodic chamber, which maintains anaerobic, biotic conditions. CO
2 acts as an electron acceptor in the MES system, undergoing fixation and conversion at the cathode [
95]. Certain electroactive microbes have demonstrated the ability to shuttle electrons intra- and extra-cellularly in this environment [
96]. Herein, specialized microbes like
Sporomusa species and engineered strains of
Clostridium have exhibited the potential to generate biofuels directly from CO
2 [
97,
98]. A classic example demonstrates an acetate production rate of 142.2 mg/L/d and a carbon conversion efficiency of 84% when utilizing enriched mixed homoacetogenic bacteria [
99]. Notably, other microbes such as
Clostridium scatologenes ATCC 25,775 employ the WLP pathway for CO
2 fixation, generating acetic acid, butyric acid, and ethanol by using H
2 as reducing power [
100].
The true potential of MES lies in its scalability and flexibility. The efficiency and spectrum of products from MES can be influenced by adaptive measures like improved electrode materials, specialized bioreactor designs, and genetically engineered biocatalysts [
101]. Indeed, bioreactor optimization, which included strategies like increasing biomass retention and media dilution rate, showcased an acetate production with a titer of 13.5 g/L [
102]. Beyond acetate, MES also promises the generation of other valuable bioproducts like butyrate, caproate, and polyhydroxybutyrate (PHB) [
103,
104,
105].
However, MES also faces challenges for more wide applications. Current systems grapple with issues like low CO
2 conversion rates, high energy input, and the nuances of maintaining effective microbial communities [
106]. Fortunately, recent innovations have exhibited promise to enhance system efficiency. For instance, thermal conditions have been found to influence these processes;
Moorella thermoautotrophica exhibited an enhanced rate of acetate and formate production at 55°C as opposed to 25°C [
107]. The microbes' biodiversity in MES also plays a pivotal role in its efficiency. Notably, autotrophic sulfate-reducing bacteria (SRM) have displayed potential as excellent biocatalysts, elevating the performance of BES in CO
2 fixation [
108]. These bacteria hold the potential to improve hydrogen production and water sulfate removal. In a recent study, a co-culture of
Desulfopila corrodens and
Methanococcus maripaludisco magnified methane production twenty-fold compared to
M. maripaludisco alone [
109]. Electro-catalyst-assisted MES systems have been developed with electrical-biological hybrid cathodes to improve product rates and variety. Here, Zn-based electrodes have outperformed others; one system achieved an acetic acid production rate of 1.23 g/L [
110].
Overall, the CO
2 bioelectrorefinery concept, as heralded by MES, is an embodiment of a circular bioeconomy, envisioning an integration of CO
2 capture, renewable energy, and sustainable production of chemicals and fuels [
111]. While strides have been made, the commercial realization of MES awaits advancements in electrode materials, microbial communities, and process optimization to rival traditional biomass-based processes. Nevertheless, the trajectory of MES research promises a sustainable and innovative path to a cleaner, greener future [
112].