Carbon Dioxide Sequestration by Biological Processes

Abstract

Steep rise in the earth’s atmospheric CO₂ concentration is the main concerned problem nowadays. Presently, the earth’s atmosphere contains nearly 397.34 ppmv of CO₂ which is much higher than the natural range of CO₂ (180–300 ppmv) that existed over millions of years. Anthropogenic CO₂ emission exceeds the natural sink of CO₂ such as CO₂ solubility in ocean, photosynthetic removal of CO₂ by ocean phytoplanktons, and terrestrial plants. Increase in CO₂ may be correlating with the rise in global mean temperature. This in turn is associated with melting of glaciers, rise in sea level, ocean acidification, unpredicted climate changes, etc. Rise in sea level and temperatures has been observed in the past also, but the rates of increase in these parameters are the main indicator of the effect of corresponding increase in CO₂ concentration. The present chapter summarizes the role of various biological processes available for CO₂ mitigation. Use of terrestrial plants, algae, cyanobacteria, carbonic anhydrase (CA) enzyme, and various other bacteria in CO₂ sequestration has been extensively discussed. Chemical fixation of CO₂ in the form of carbonates is a safe and permanent natural process. CA catalyzes the CO₂ hydration reaction and accelerates the transformation of CO₂ into solid carbonates manifold. Terrestrial plants for CO₂ sequestration are limited only to lower range of CO₂ concentration. Forest fertilization has been found to increase the CO₂ mitigation two- to threefold. Contrary to terrestrial plants, algae and cyanobacteria can be used at higher CO₂ concentration with higher photosynthetic efficiency.

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1 Introduction

Carbon dioxide is one among many greenhouse gases such as methane, ozone, NO_x, and water vapor. Greenhouse gases act as blanket, which retain the outgoing sun’s heat (infrared rays) into the earth’s atmosphere. In absence of greenhouse gases, earth would be colder. Contrary to this, increase in any one of the greenhouse gases will result in extra trapping of heat, causing global warming. CO₂ constitutes very small portion of the gases present in the earth’s atmosphere. Natural range of CO₂ concentration over the last 650,000 years was in the range of 180–300 ppmv. However, presently CO₂ concentration has crossed the natural range and reached as high as 397.34 ppmv in March 2013. Moreover, the Keeling curve reveals progressively faster rise in CO₂ concentration after industrialization [1]. Rate of increase in atmospheric CO₂ was 1.94 ppm/year in 2011, which was more than twice the estimated value in 1959 [1]. Temperature contributes significantly to make planet habitable for the living beings. Therefore, trace amount of CO₂ in the earth’s atmosphere is required for maintaining stable temperature. Increase or decrease of few degrees of global temperature can have a devastating effect on earth. Global atmosphere and ocean temperature have increased by 0.6 °C and 0.3 °C, respectively, in the last century despite the fact that solar output witnessed decline in the same period [2]. Rise in temperature is the reason for climate change and melting of glaciers, thus causing rise in the sea level. In addition, global warming will cause increase in soil microbe’s respiration and further addition of greenhouse gas CO₂ [3]. In the last century, global sea level rose by 17 cm [2]. Nearly 2 billion tons of CO₂ is being absorbed in the upper layer of ocean per year. Increase in CO₂ absorption resulted in the increase of acidification that adversely affects the aquatic life [2]. The International Panel on Climate Change (IPCC) forecast increase in CO₂ concentration up to 570 ppmv by the end of the twenty-first century. This will cause nearly a rise of 1.9 °C of mean global temperature, causing an increase in mean sea level by 38 cm [4]. It is disputable to link rise in temperature with increase in CO₂ concentration. Some reports suggest present temperature rise is the result of natural cycle of rise and fall in temperature over the earth’s history. However, some researchers believe present rise in temperature is due to an increase in CO₂ concentration. It becomes more important as some reports reveal unprecedented rate of increase in temperature is contrary to natural cycle of earth temperature observed so far [2].

CO₂ is being injected into the atmosphere from natural as well as artificial sources. Natural sources of CO₂ are volcanic eruptions, decomposition of organic matters, and autotrophic and heterotrophic respiration [5]. However, there is a natural mechanism of CO₂ removal such as oceanic and terrestrial CO₂ removal from the atmosphere. Imbalance in natural CO₂ addition and removal from the atmosphere is due to anthropogenic emission of CO₂ by human activity despite the fact that natural processes remove 50 % of the anthropogenic CO₂ emissions. Therefore, increasing consumption of fossil fuels is the main matter of concern due to the anthropogenic CO₂ emissions. Total annual anthropogenic CO₂ emission due to fossil fuel consumption is 29 Gt per year [6]. Today, power plants, cement, steel manufacturing industries, transportation, and household usages are dependent on fossil fuels [7]. Coal-fired power plants consume nearly one third of the total fossil fuel consumption and the remaining from fossil fuel usages in other sectors such as transportation, industry, and homes [2, 8]. Increase in human population and modernization are the reasons for the booming of these sectors.

In this scenario, it is necessary to adopt some strategies to sequester CO₂ from the earth’s atmosphere. CO₂ sequestration strategies can be divided into abiotic and biotic categories. Abiotic categories involve scrubbing, mineral carbonation, and geological and ocean injection. Several techniques are available to separate CO₂ from the flue gas. Further, they can be stored at various locations such as oceans, deep aquifers, and depleted oil and gas reservoirs [9]. However, these methods can pose potential threat for safety and environmental impact due to accidental leakage especially in long term [10]. Biotic categories include CO₂ sequestration in oceanic, terrestrial, and secondary carbonates. There are several natural phenomena occurring in the ocean, which ultimately remove atmospheric CO₂ [11]_. Oceanic CO₂ at the surface is in equilibrium with atmospheric CO₂. CO₂ dissolved in water forms weak acid, which reacts with carbonate anions and water to form bicarbonate. Continuous supplies of cations are required to maintain the buffering capacity of bicarbonate system. CO₂ solubility gradient is another phenomenon by which large amount of CO₂ is being sent to the bottom of ocean. CO₂ is more soluble in cold and saline water. Therefore, cold dense water masses at higher latitude especially at the North Atlantic and Southern Ocean confluence sink into the interior of ocean carrying huge amount of CO₂ [11]. This eventually gets trapped by less dense water at the top for several hundred years. Further, oceanic phytoplanktons play a vital role by absorbing atmospheric CO₂ at the surface of the ocean during photosynthesis. Nearly 25 % of carbon fixed by these processes sinks to the bottom of the ocean. It has been estimated that 11–16 Gt of carbon is removed from the atmosphere by this process [11]. Oceanic phytoplanktonic photosynthesis lowers the atmospheric CO₂ by 150–200 ppmv [11]. CO₂ is also absorbed naturally such as through sedimentation. CO₂ conversion into bicarbonate rocks is a natural process. Nearly 2 Gt of carbon is sequestered in the soil through burial of decomposed organic material from plants, animals, biomass, and agriculture by microbes [6]. However, most of those are released back into the atmosphere during soil erosion and oxidation. Biochar is another promising material helpful in CO₂ removal. Biochar is charcoal, which is produced when smoldering biomass is burnt in limited oxygen producing little heat. The advantage of biochar in CO₂ sequestration is that it gives stability to carbon present in the biomass against microbe’s oxidation [6] and, thus, prevents the release of CO₂ into the atmosphere.

Land-based plants consume CO₂ in photosynthesis and release CO₂ during respiration. However, there is net CO₂ sequestration from the atmosphere. Plants are not capable of utilizing the concentrated CO₂ present in flue gas. It has been estimated that afforesting 22 % of earth's terrestrial surface, which is equal to present forested land, is required to sequester CO₂ emitted due to fossil fuel usages [12]. Other reports say only 345 million hectares is available for afforestation, which will result in 1.5 Gt C annually [6]. This can lead to little less than 20 % reduction in anthropogenic CO₂ emission. However, afforestation in total available area is not possible due to huge pressure on land for other purposes. Each year nearly 10 % of total atmospheric CO₂ is fixed into carbohydrates by the photosynthetic process [13]. Total global CO₂ removal by land-based plants and oceanic phytoplanktons are 403 Gt CO₂ (equivalent to 110 Gt carbon) and 385 Gt CO₂, respectively [8]. Microalgae, seaweeds, and higher aquatic plants are other alternatives for CO₂ sequestration because of their photosynthetic ability. However, the latter two are not promising because of low CO₂ and other nutrient mass transfer [14]. They require high amount of energy input for mixing. However, seaweeds are more promising compared to higher aquatic plants because it is difficult to achieve proper water exchange at the submerged plants and their dense stands [14]. Therefore, seaweeds are grown near the seashore and shallow ocean systems. Carbon is provided by seawater exchange system but requires high amount of energy for water pumping.

Use of microalgae, cyanobacteria, and other bacteria is another alternative available for CO₂ sequestration. A large number of microbes are known capable of utilizing CO_2. Except microalgae, all require some inorganic reducing agent such as H₂, H₂S, NH₃, etc. [14]. Photosynthetic efficiency of algae is 10 times higher than that of terrestrial plants. They are efficient in growing at relative higher concentration of CO₂ compared to plants. This makes them a suitable candidate for CO₂ sequestration from the flue gas. Cultivation of algae requires less water, and they can be grown in non-fertile land. Further, their biomass can be used for food and feed supplements, for the production of biofuels such as biohydrogen, bioethanol, biodiesel, and industrially important biomolecules and biofertilizers [7, 15, 16].

Use of carbonic anhydrase (CA) is another thrust area for CO₂ sequestration. Carbon content of earth’s lithosphere is 42 % w/w in the form of CaCO₃ and other carbonates which indicates transformation of gaseous CO₂ into solid carbonates is a geological stable process and has potential to exploit for CO₂ sequestration [17]. One of the reactions, hydration of CO₂ to carbonic acids, is the rate limiting step in CO₂ mineralization reaction [18]. CA is the catalyst of this reaction, and the use of this enzyme has been found to enhance the rate of reaction manifold [19]. Advantages of using CA for CO₂ sequestration are eco-friendliness, cost-effectiveness, simple process, and abundance of CA enzymes among microorganisms [20].

Thus, the present study aims to summarize various biological processes such as use of plants, microalgae, cyanobacteria, and other non-photosynthetic microbes for CO₂ sequestration. It also attempts to highlight different pathways occurring in biological systems along with the use of CA for this cause.

2 Biological Processes of CO₂ Sequestration

2.1 CO₂ Sequestration by Plants

The amount of CO₂ required for the terrestrial plants and crops is very less, nearly equal to atmospheric CO₂ concentration. Limited number of reports are available, demonstrating the long-term effect of CO₂ concentration on plants because of high amount of time and space required along with variation in experimental conditions. Therefore, it is not clear that the rise in CO₂ concentration will increase the CO₂ sequestration in existing forests [21]. Most suitable CO₂ concentration for plants obtained in greenhouses was nearly three times (0.1 %) the atmospheric CO₂. An experiment conducted by Norby et al. (2005) by analyzing four Free Air Carbon dioxide Enrichment (FACE) studies on forest reported that there will be 23 % increase in the plant productivity at the predicted level of CO₂ in 2050 compared to that of the present atmospheric CO₂ concentration [22]. However, they cautioned that increment in productivity may not increase the long-term CO₂ sequestration. Most of the trees in forest (~95 % of all higher plants) are C3 type having positive photosynthetic response to elevated CO₂ concentration [21, 23, 24]. Nearly one third of the global land is covered by forest. In another report, on average, 60 % and 27 % enhancement of photosynthesis and growth, respectively, for trees have been reported [25]. Elevated CO₂ concentration decreases the stomata aperture opening and reduces the water loss. Trees planted at CO₂ spring in Italy were found growing at similar rate compared to that of normal atmospheric CO₂ concentration [26]. Respiration rate has been found to decline in elevated CO₂ environment with little improvement in the photosynthetic ability of the plants. Thus, it is impractical to use terrestrial plants and crops for CO₂ sequestration from highly rich CO₂ stream of flue gas emitted from power plants [14]. The effect of temperature, one of the associated parameters with CO₂ concentration, has also been studied. Net CO₂ removal rate by plants was found to increase with temperature [27]. Further increase in temperature from optimum resulted in decreased CO₂ sequestration rate. Therefore, CO₂ sequestration rate by green plants varies from day to day and season to season. It was observed that in CO₂-enriched atmosphere, plants shift the optimum temperature of CO₂ sequestration to higher level. Moreover, at this condition, process was found to be less sensitive to increase in temperature [27]. Despite the fact that plants have limited applicability in CO₂ sequestration at highly CO₂-rich environment, afforestation has a number of advantages. Firstly, nearly 30–40 % of captured carbon from atmosphere gets stored in depth of soil system through plant roots [28]. Secondly, cultivated agricultural crops contribute large amount of atmospheric CO₂ removal (in the order of 190 t ha⁻¹ C) into the soil system acting as perpetual sink [28]. Thirdly, the lack of proper nitrogen fertilizer for land-based plants is one of the limiting factors. Therefore, CO₂ removal rate by plants can be enhanced significantly by the use of nitrogen fertilizer [8].

2.1.1 Mechanism of CO₂ Fixation

Each leaf cell contains nearly 50 chloroplasts [13]. Stomata are the entry sites of CO₂ into the leaf where it interacts with the RuBisCO present in chloroplasts and gaseous CO₂ reduced into carbohydrates. Stomata of leaves have been found unresponsive to higher CO₂ concentration. RuBisCO is the key enzyme of the Calvin cycle for all the three types of the plant C3, C4, and CAM. K _m of CO₂ for the C3 plants is 15–25 μM, and carboxylase activity of RuBisCO operates below the K _m of CO₂ and is not more than 25–30 % (equilibrium concentration of CO₂ in water and air is nearly 10 μM) of its maximum capacity (24, 29). However, C3 plants do not concentrate CO₂ using carbon concentrating mechanisms (CCMs) probably because of having large amount of RuBisCO and the presence of highly active β type of CA in the thylakoid stroma (Table 12.1). RuBisCO has both oxygenase and carboxylase activity. K _m for oxygenase activity is 700 times lower than that of carboxylase activity. Therefore, increase in atmospheric CO₂ concentration is associated with not only the increase in CO₂ assimilation but also the reduction in photorespiration consuming significant amount of oxygen [24]. However, at higher CO₂ concentration, rate of photosynthesis is limited by the regeneration of the ribulose-1,5-bisphosphate (RuBP). Triose phosphate produced during Calvin cycle releases inorganic phosphate P_i which is essential for ATP synthesis and RuBP regeneration from phosphorylated intermediates. Therefore, under these circumstances, photosynthesis is called P_i or triose phosphate limited use limited [24]. Fixed CO₂ can have three fates: (a) some of the CO₂ releases back into the atmosphere through respiration; (b) some of the CO₂ transfers to the soil through root exudates, root death, litter fall, and coarse woody debris which after decomposition releases back the CO₂ into the atmosphere; and (c) some of the CO₂ gets stored in the wood [21]. Contrary to C3 plants, C4 plants have the ability to grow efficiently in stress condition such as high temperature, low water availability, high irradiance, and saline soils. C4 plants on land have thick-ended walls of bundle-sheath cells, which help them in preventing the leakage of CO₂ outside [30]. CCM activates in plants by spatial separation of CO₂ fixation by Calvin cycle. Normally PEP carboxylase catalyzes the CO₂ fixation in the mesophyll cells. C4 acids transported to bundle-sheath cells are decomposed to gaseous CO₂ by the action of NADP-malic enzyme and other decarboxylases. Both these processes are temporarily separated in CAM plants. Detailed discussions on Calvin cycle and CCMs in C4 and CAM plants are discussed in another section.

Table 12.1 Comparative study of CCMs occurring in plants (C3 and C4), microalgae, and cyanobacteria [29, 47]

2.1.2 Oceanic Fertilization

Oceanic fertilization can be defined by adopting a practice of supplementing limiting nutrients to the phytoplanktons, causing increase in photosynthetic efficiency and CO₂ removal rate. For example, micronutrient Fe was added in 10 × 10 km patches of ocean, and 30 times increase in chlorophyll concentration with increase in 100,000 kg of carbon fixation was found [4]. However, its practice should be adopted cautiously as it can negatively interfere with the marine ecosystem. In addition, sinking organic matters on decomposition may produce other stronger greenhouse gases such as methane and NO_x [31].

2.1.3 Forest Fertilization

The study conducted by Oren et al. (2001) revealed that the enriched CO₂ concentration (550 ppmv) had only marginal positive effect on the biomass carbon increment. However, synergistic gain was observed when plants were grown in enriched CO₂ as well as nutrient condition. The gain was threefold and twofold larger at the poor site and at the moderate site, respectively. Fertilizing at higher CO₂ concentration is more prominent than the ambient CO₂ concentration [32]. Further, application of fertilizer in paddy soils has been found favoring the growth of autotrophic microorganisms, resulting in increased CO₂ sequestration [33].

2.2 CO₂ Sequestration by Microalgae and Cyanobacteria

2.2.1 Microalgae

Photosynthesis in microalgae takes place in chloroplast. Apart from other pigments such as β-carotene and xanthophylls, they have chlorophyll a and chlorophyll b as the major pigments, which give them bright green color. Light requirement of the typical algae is lower than the higher plants [34]. Light conversion efficiency and productivity are proportional to the increase in light intensity till it attains saturation light intensity [35]. Saturation light intensity of algae such as Chlorella and Scenedesmus sp. is of order 200 μ mol m⁻² s⁻¹ [36]. Photosynthesis is coupled with release of O₂ as by-product. High dissolved oxygen (DO) in the culture (>35 mg L⁻¹) has inhibitory effect on the photosynthesis [7, 37]. In the closed photobioreactor, DO level has been found to increase as high as 400 %, thus severally affecting the photosynthetic process [38]. Further, cations dissolved in medium also negatively affect the photosynthetic process. Algae carry net negative charge in their surface and hence are a potential adsorber of polyvalent cations present in the surrounding medium. Adsorption of polyvalent cations on the surface of algae causes morphological changes in the cell morphology or can replace/block the prosthetic metal atoms in the active site of relevant enzymes leading to photosynthesis inhibition [34].

2.2.2 Cyanobacteria

Cyanobacteria is a gram-negative bacteria. Compared to other gram-negative bacteria, the cell wall of cyanobacteria has thicker peptidoglycan layer [39]. Cyanobacteria lack organelles in the cell. Respiratory chain is located in thylakoid membrane and plasma membrane. Photosynthesis takes place in thylakoid located in the cytoplasm, and photosynthetic electron transport takes place in its membranes. The photosynthetic apparatus of cyanobacteria is similar to the chloroplast of green algae and plants. However, major difference is in the antenna system. Cyanobacteria lack chlorophyll b and depend primarily on chlorophyll a and specialized protein complex known as phycobilisomes for harvesting light energy. Light harvesting complex can be lipophilic as well as hydrophilic in nature. Lipophilic pigments such as chlorophyll a and carotenoids are located within the thylakoid membranes whereas hydrophilic antenna pigments are housed in phycobilisomes attached outside of thylakoid membranes. Examples of hydrophilic pigments are allophycocyanin (APC), phycocyanin (PC), phycoerythrin (PE), or phycoerythrocyanin (PEC) [40]. Similar to green algae and plants, cyanobacteria carry out oxygenic photosynthesis, releasing O₂. However, in anaerobic condition, they can also carry out anoxygenic photosynthesis using PSI. Sources of electrons are smaller organic compounds such as succinate, hydrogen sulfide, and thiosulfate instead of water [41]. Anoxygenic photosynthesis results in generation of ATP by cyclic photophosphorylation around the PSI.

2.2.3 Photosynthesis: Key for CO₂ Sequestration

Photosynthesis, the key biological process occurring in plants and a wide number of microorganisms such as algae and cyanobacteria, helps in mitigating atmospheric CO₂. Photosynthesis occurs in the chloroplasts of plants and algae. Chloroplasts contain the thylakoid vesicles arranged in stacks, containing photosynthetic apparatus. Contrary to plants and algae, prokaryotic cyanobacteria do not have fixed organelles for keeping photosynthetic apparatus. They are located in cytoplasm as free and isolated photosynthetic lamellae [42]. Biological system uses photosynthesis for harnessing solar energy for the preparation of food with the help of CO₂ and water. Photosynthesis consists of two distinct processes: light-dependent process and light-independent process. In light-dependent process, they conserve light energy in the form of ATP and NADPH with the help of chlorophyll and light harvesting complex. Light-independent process utilizes ATP and NADPH for the fixation of CO₂ into triose phosphates, starch, sucrose, and other derived products. Photosynthetic apparatus of plants, algae, and cyanobacteria are similar in nature. Photosynthetic apparatus are found in the thylakoid membranes consisting of protein complex, electron carriers, and lipid molecules. The electron carriers are arranged in the shape of 90° tilted English alphabet Z. Two reaction centers, PSII and PSI, help in exciting the electrons by absorbing the light of 680 nm and 700 nm, respectively (Fig. 12.1). Two reaction centers are connected with series of electron carriers such as plastoquinone, cytochrome b₆f complex, and plastocyanine arranged in fixed increasing order of redox potential. Electron carriers between two centers help in transferring excited electrons at PSII to pass to PSI smoothly. Electrons at PSII come from breaking of water molecules into oxygen molecules, protons, and electrons. Two moles of water dissociates into four moles of protons and electrons and one mole of oxygen molecule (Eq. 12.1) [42].

Fig. 12.1

Schematic diagram of Z scheme of photosynthetic process involving PSII and PSI. Solid and dashed lines show the noncyclic and cyclic flow of electrons. Abbreviation: PQ _A plastoquinone, PQ _B second quinine, Chl a _o chlorophyll a, Chl a ₁ phylloquinone, PC plastocyanin [53]

$$ 2{\mathrm{H}}_2\mathrm{O}\to 4{\mathrm{H}}^{+}+4{\mathrm{e}}^{-}+{\mathrm{O}}_2 $$

(12.1)

Protons accumulate into the lumen of thylakoid membranes, generating proton gradient across the membrane. Concentration gradient drives protons outside thylakoid membrane through ATP synthase producing ATP. Thus, ATP synthase produces not only energy for the cell but also avails protons for the reduction of NADP⁺ to produce NADPH. At PSII, light energy causes charge separation between P680 and pheophytin, creating P680+/Pheo−. Pheophytin transfers the electrons to a permanently bounded molecule (QA) to photosystem II. At QA site plastoquinone receives single electron instead of two. QB and QA sites differ from each other as the former requires two electrons to reduce instead of one, which is the case of the latter, causing two turnovers for the complete reduction of plastoquinone at QB. Due to close proximity of QB site, protons added to plastoquinone during its reduction come from the outside aqueous phase of the membrane [13]. At photosystem I, most of the antenna chlorophyll molecules are attached to the reaction center proteins [13]. Plastoquinone then transfers their electrons to next electron carrier cytochrome b₆f complex with simultaneous release of protons into the lumen. Cytochrome b₆f complex is attached with membrane-bound protein complex. Plastocyanin (PC) acts as a last protein carrier delivering electrons to PSI. PSI again excites the electrons received from electron carriers. Here, excited electrons are used to reduce ferredoxin (Fd), a protein loosely attached with thylakoid membranes from outside. Reduced Fd interacts with ferredoxin NADP⁺ reductase (FNR) and the latter catalyzes the reduction of NADP⁺ to NADPH as shown in (Eq. 12.2).

$$ 2{\mathrm{Fd}}_{\mathrm{red}}+2{\mathrm{H}}^{+}+{\mathrm{NADP}}^{+}\to 2{\mathrm{Fd}}_{\mathrm{ox}}+\mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+{\mathrm{H}}^{+} $$

(12.2)

Each of the photosystem contributes one photon to the transfer of one electron. Therefore, two photons are required for the transfer of one electron along the electron carriers to the NADP⁺. Two moles of water and eight photons are required to produce two moles of NAD(P)H as shown in (Eq. 12.3).

$$ 2{\mathrm{H}}_2\mathrm{O}+2\ {\mathrm{NADP}}^{+}+8\ \mathrm{photons}\to {\mathrm{O}}_2+2\mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+2{\mathrm{H}}^{+} $$

(12.3)

Reduction of one molecule of CO₂ requires two moles of NAD(P)H. Energy content in photon is 480 KJ/mol, whereas energy gathered after reduction of one reduced CO₂ molecule is equivalent to 1,400 KJ. Therefore, theoretical maximum efficiency of the process is 36.4 %.

2.2.4 Carbon Concentrating Mechanisms (CCMs)

Cyanobacteria and algae behave like C3 plants but have much less affinity for CO₂ [29]. However, they develop CCMs similar to C4 and CAM plants, causing better photosynthetic efficiency compared to C3 plants [30]. Their RuBisCO have oxygenase as well as carboxylase activities, depending upon the concentration of local CO₂ in the compartment housing RuBisCO. Carboxylase activity of RuBisCO activates under high CO₂ pressure, whereas low concentration of CO₂ forces it to undergo oxygenase activity. Oxygenase reaction has many disadvantages. Firstly, glycolate 2-phosphate is the end product of oxygenase activity of RuBisCO (Eq. 12.4). It has no use to algal cells; therefore, significant amount of cellular energy is wasted by consuming it. Secondly, it releases previously fixed CO₂, during the carboxylase activity of RuBisCO, which causes the loss of nearly 50 % of algal biomass [43]. Metabolism of glycolate 2-phosphate produces glycine, which on condensing with another glycine molecule produces serine, resulting in the loss of CO₂ [44]. Thirdly, loss of fixed carbon further negatively interferes with the regeneration of RuBP, required for the smooth functioning of the cycle.

$$ \mathrm{Ribulose}\ 1,5\ \mathrm{bisphosphate}+{\mathrm{O}}_2\to \mathrm{glycerate}\ 3\hbox{-} \mathrm{phosphate}+\mathrm{glycolate}\ 2\hbox{-} \mathrm{phosphate} $$

(12.4)

Affinity constant (Km) of microalgae and cyanobacteria for CO₂ is high compared to C3 plants, indicating low affinity of RuBisCO for CO₂ (Table 12.1). Under normal atmospheric air, RuBisCO is only half-saturated with CO₂ [7, 29]. The diffusion of CO₂ in aqueous solution is 10,000 times slower than the CO₂ diffusion in air [30]. Poor diffusion of atmospheric CO₂ in aqueous solution and low affinity of CO₂ by algae, cyanobacteria, and some chemoautotrophic bacteria pose a limitation to the carboxylase activity of RuBisCO [45]. Therefore, for maintaining this, it is necessary to saturate the compartment containing RuBisCO with CO₂. Fortunately, algae have carbon concentrating mechanisms (CCMs), which help them to increase the local CO₂ even when there is lower CO₂ concentration outside the algal cells [46]. Contrary to plants, algae and cyanobacteria have single-cell CO₂ concentrating mechanisms. However, they have internal compartments within the chloroplasts (pyrenoid in algae and carboxysome in cyanobacteria). Increase in concentration of CO₂ at close proximity of RuBisCO has many advantages. Firstly, it activates RuBisCO. Secondly, it increases carboxylase activity of RuBisCO, and, thirdly, dissolved inorganic carbon (DIC) influx may help in maintaining internal pH and dissipating excess light energy [47]. CCMs can be divided broadly into two categories: biochemical and biophysical CO₂ pumps. CCMs help in accumulation of large number of intracellular inorganic carbon inside the RuBisCO compartment. However, inorganic carbons are not available for fixation by RuBisCO. They must be converted back into gaseous CO₂ for the action of RuBisCO. CA is a vital enzyme, which overexpresses under low external CO₂ environment. CA acts as a catalyst in the interconversion of inorganic carbon back to gaseous CO₂.

2.2.4.1 Biochemical CO₂ Pump

C4 Mechanism – Biochemical pumps are mostly found in the terrestrial plants. However, some phytoplanktons and macroalgae also have evidence of biochemical pumps. C4 mechanisms have also been found in macroalgae such as Udotea flabellum and Thalassiosira weissflogii [43]. The role of C4 mechanism is to biochemically transport the DIC from the site excessive in it to the site where RuBisCO is active [43]. As the name indicates, CO₂ is stored in a four-carbon compound, oxaloacetate. Phosphoenolpyruvate (PEP) is the carrier molecule, which combines with HCO₃ ⁻ using PEP carboxylase to form oxaloacetate. PEP carboxylase utilizes bicarbonates rather than CO₂; therefore, the gaseous CO₂ entering into the mesophyll cell must be rapidly converted to bicarbonate with the help of carbonic anhydrase [48]. At physiological CO₂ levels and pH, Km (HCO₃ ⁻) of PEP carboxylase and HCO₃ ⁻ concentration in the cytoplasm of mesophyll cells were estimated to be about 8 μM and 50 μM, respectively. Therefore, unlike RuBisCO, PEP carboxylase is always saturated with HCO₃ ⁻ at ambient CO₂ concentration. Therefore, CA is mostly confined to mesophyll cell in the C4 plants compared to chloroplast in C3 plants [49]. Oxaloacetate further readily reduced to malate by the action of NADP-malate dehydrogenase. In plants, malate is transported to bundle-sheath cell where it is decarboxylated by the action of NADP-malic enzyme. At the site of RuBisCO, malate can be decarboxylated to pyruvate and releases gaseous CO₂ for the action of RuBisCO enzyme in Calvin cycle.

CAM Mechanism – CAM plants are usually found in desert area. In cool night, guard cells open to receive CO₂, while in daytime, it is closed to prevent water loss. This mechanism is also proposed in brown macroalgae for the assimilation of photosynthetic inorganic carbon [43]. PEP comes from the starch accumulated during daytime using Calvin cycle. Enzymes and compounds taking part in CAM mechanism are similar to C4 mechanism. However, end storage compound malate is temporarily separated over time rather than spatially as in C4 plants [43]. PEP carboxylase catalyzes the reaction of PEP and HCO₃ ⁻ to form oxaloacetate. NADP-malic dehydrogenase reduces oxaloacetate to malate, which is transported to vacuole having low pH at night. In daytime, whole pathways get reversed back and malic acid transported back to cytosol for the decarboxylation reaction, flooding cytosol with CO₂. Guard cell is closed during the daytime, preventing CO₂ diffusion outside the cell. It is to be noted that PEP carboxylase activity is also under control to prevent wasteful synthesis of C4 compounds during daytime.

2.2.4.2 Biophysical CO₂ Pump

2.2.4.2.1 CCMs in Cyanobacteria

Being simple in structure, algae and cyanobacteria cannot stop the diffusion of CO₂ [30]. Carboxysome is the key for the success of CCMs in cyanobacteria. It acts as a storehouse for CO₂ having limited permeability for CO₂ leakage. The ultimate target of CCMs is to transport DIC to the storehouse, carboxysome. Along with cyanobacteria, carboxysomes are also found in some chemoautotrophic bacteria growing in CO₂ concentration lower than the K _m of RuBisCO [45]. Carboxysomes have mainly icosahedral structures with the diameter of 100–200 nm. The number of carboxysomes present in per cell of cyanobacteria varies from 5 to 20 depending upon the growth conditions and species to species [45].

Most of algae and cyanobacteria examined so far have been found to have transporter for both CO₂ and HCO₃ ⁻. However, few algae can also assimilate either CO₂ or HCO₃ ⁻. CCMs are extensively studied in cyanobacteria compared to microalgae. Active transport occurs across the membrane having low permeability to DIC. In cyanobacteria, transport of CO₂ or HCO₃ ⁻ occurs via plasmalemma or thylakoid membranes. All the DIC removed from the outside by various DIC transporters are delivered only in the form of HCO₃ ⁻ into the cytosol of cell [45]. Bicarbonate concentrating ability of cyanobacteria is nearly five times greater than the microalgae, causing higher photosynthetic and CO₂ consumption efficiency of the former [30]. Outside, DIC including CO₂ penetrate the plasma membrane and reach the closest membrane of the chloroplast with the help of various transporter proteins. At least three mechanisms of active transport have been proposed in cyanobacteria (Fig. 12.2) [29, 49, 50]: (1) HCO₃ ⁻ can be transported to cytosol with the help of ABC-type transporter utilizing energy in the form of ATP, which is a high-affinity, low-CO₂-induced, and sodium-independent mode of HCO₃ ⁻ assimilation; (2) HCO₃ ⁻ transport into cytosol may also be the result of HCO₃ ⁻/Na⁺ symporter or the regulation of pH through Na⁺/H⁺ antiporter; and (3) for the active transport of CO₂, NADH dehydrogenase may have constitutively low or inducible high affinity for CO₂. In either case, CO₂ is first converted to HCO₃ ⁻ by NADHdh at plasmalemma of cyanobacteria and then transported into the cytosol. Intracellular pH of cyanobacteria is near to 8, causing HCO₃ ⁻ as predominant species inside the cytoplasm (equilibrium ratio of HCO₃ ⁻ and CO₂ is near to 100). HCO₃ ⁻ is the charged ion and therefore cannot escape the lipid bilayer of the cyanobacteria [45]. HCO₃ ⁻ then diffuses into carboxysome where it is acted upon by CA enzyme to flood the compartment with gaseous CO₂ for the action of RuBisCO of Calvin cycle [43]. Remaining amount of CO₂ in carboxysome is diffused outward [47]. In addition, it has also been proposed to have direct access of HCO₃ ⁻ to cytoplasm of cyanobacteria. Rate of direct transport of HCO₃ ⁻ across plasma membrane is less significant compared to its active transport [51]. In yet another mechanism, compartment containing high concentration of HCO₃ ⁻ is acidified using proton pump and flooding with CO₂ (Eq. 12.5). In acidic compartment, HCO₃ ⁻ decomposes into CO₂ by the action of CA or proton-driven catalysis of HCO₃ ⁻ to CO₂. The high level of CO₂ then diffuses into the more alkaline compartment housing RuBisCO for the action of RuBisCO [43].

Fig. 12.2

Schematic diagram of CCMs occurring in cyanobacteria [29, 43, 49]

$$ \mathrm{Alkaline}\ \left({{\mathrm{HCO}}_3}^{-}\right)\to \kern0.5em \mathrm{Acidic}\ \left({{\mathrm{HCO}}_3}^{-}\to {\mathrm{CO}}_2\right)\kern0.5em \to \kern0.5em \mathrm{Alkaline}\ \left({\mathrm{CO}}_2\right) $$

(12.5)

2.2.4.2.2 CCMs in Algae

Compared to cyanobacteria, CCMs in microalgae are less understood because of more compartments inside the cell and very diverse group of microorganisms [29]. Microalgae composed of one or few cells do not have impermeable cell walls like plants to prevent leakage of CO₂. The challenging task is to prevent the leakage of concentrated CO₂ while allowing other nutrients to come in. The efficiency of acquisition of DIC depends on environmental conditions. For example, acquisition of DIC in low atmospheric CO₂ was found higher compared to high atmospheric CO₂ condition. However, the amount of RuBisCO did not change during adaptation from low to high CO₂ condition. This indicated the existence of transport system for the uptake of DIC into the cells. Cyanobacteria and microalgae can accumulate nearly 100- and 20-fold increase in HCO₃ ⁻ within the cells, respectively, over ambient CO₂ level [29]. DIC accumulation in algae is generally lower than cyanobacteria probably due to higher affinity of RuBisCO present in the former for CO₂. CCMs in microalgae have been hypothesized similar in nature as cyanobacteria by most of the researchers. Similar to carboxysome of cyanobacteria, microalgae also have a compartment (pyrenoid) in chloroplast densely packed with RuBisCO. Accumulation of charged HCO₃ ⁻ ions lessens the chance of leakage. Freshwater and marine green algae are capable of utilizing HCO₃ ⁻. However, in most of the microalgae, CO₂ is main form of carbon entering into the cell and HCO₃ ⁻ in chloroplast. In Chlamydomonas reinhardtii, active transport of CO₂ has been found to have preference over HCO₃ ⁻ [51]. CO₂ uptake in whole cell is due to diffusion, while through the chloroplast, it is mediated by transfer [47]. A range of CAs participate in each of the compartment to maintain the equilibrium between CO₂ and HCO₃ ⁻ (Fig. 12.3). Eventually DIC entering into the pyrenoid is in the form of HCO₃ ⁻, which needs to be converted back to CO₂ to enrich RuBisCO compartment for the carboxylase reaction. Cell membrane of pyrenoid does not allow CO₂ to leak out, allowing sufficient time for RuBisCO to use CO₂ in the Calvin cycle. CA is an important enzyme for the successful operation of CCM. CA in microalgae is of different type and located in different places inside the cell. It has been found in periplasmic space, cytosol, as well as inside the pyrenoid. Direct uptake of HCO₃ ⁻ and CO₂ diffusion facilitated by periplasmic CA across the plasma membrane have been proposed [29]. Periplasmic CA probably helps in the diffusion of CO₂ and supply of HCO₃ ⁻ into the cytosol across the plasma membrane. However, in every case examined so far, electrochemical potential gradient for HCO₃ ⁻ was outward. By examining the HCO₃ ⁻ transport in Ulva, HCO₃ ⁻ uptake in microalgae has been proposed to occur through the HCO₃ ⁻/OH⁻ antiport [47].

Fig. 12.3

Schematic diagram of CCMs occurring in green algae [29, 43, 49]

2.3 Bacteria

The non-photosynthetic bacteria play an important role in global carbon cycle. Advantages of these microorganisms are their ability to adapt to and survive extreme conditions of the environment. However, consumption of large amount of H₂ as electron donor limits their practical application [52]. Enzymes involved in the CO₂ fixation pathway are sensitive to oxygen; therefore, they grow best in anaerobic environment [52]. Their ability to sequester CO₂ under anaerobic environment can be helpful for their application in O₂-deficient but CO₂-rich environment such as under the soil and flue gas [52].

Photosystem of some of the bacteria looks like either PSI or PSII. Lack of PSII deprives bacteria to use electrons of water and evolve oxygen as by-product of photosynthesis. Some simple inorganic or organic molecules substitute the water for the electrons needed to reduce CO₂ into useful simple sugar. They have bacteriochlorophyll, a family of molecules similar to chlorophyll, but absorb light in the range of 700–1,100 nm. Similar to oxygenic photosynthesis, electron transfer results in the generation of proton gradient across the thylakoid membrane. Outward protein gradient drives the proton out through ATP synthase, causing synthesis of ATP. Energy for CO₂ reduction comes from ATP and NADH. Electron carriers are quinone such as ubiquinone, menaquinone, and the cytochrome bc complex, which work similarly to cytochrome b₆f complex present in the oxygenic photosynthetic system [13].

Green gliding bacteria such as Chloroflexus aurantiacus harvest light using chlorosomes similar to green sulfur bacteria. CO₂ fixation in these microorganisms does not involve Calvin or Krebs cycle. They usually do photosynthesis under anaerobic condition. Green and purple bacterial membranes are in the form of lamellae, vesicles, or specialized structures (chlorosomes) where light reaction takes place. Sulfur purple bacteria such as Chromatium vinosum fix CO₂ for their survival using Calvin cycle.

2.3.1 Purple Bacteria

Photosynthetic machinery of purple bacteria such as Rhodospirillum rubrum, Rhodopseudomonas viridis, and Rhodobacter sphaeroides is of pheophytin-quinone type. Pheophytin is similar to chlorophyll but lacks central Mg²⁺ ions. Purple bacteria have single reaction center called P870. Electrons at the reaction center get excited, absorbing light of 870 nm. Excited electrons pass to cytochrome bc1 complex through pheophytin and quinine sequentially [53, 54]. Cytochrome bc1 complex is the hub that pumps the protons to generate proton gradient and electrons back to reaction center via cytochrome c₂ (Fig. 12.4a). Light-driven cyclic flow of electrons enables to produce ATP using ATP synthase. Purple bacteria are of two types: non-sulfur purple, such as Rhodopseudomonas viridis and Rhodobacter sphaeroides, and sulfur purple bacteria, such as Chromatium vinosum. Electron source for non-sulfur bacteria is organic compounds such as malate and succinate, whereas sulfur purple bacteria extract electrons from inorganic sulfur compounds such as hydrogen sulfide. CO₂ is fixed in purple bacteria using Calvin cycle.

Fig. 12.4

Schematic diagram of photosynthetic process involving only PSI in (a) purple bacteria and (b) green sulfur bacteria. Solid and dashed lines show the noncyclic and cyclic flow of electrons. Abbreviation: Q quinine [53]

2.3.2 Green Sulfur Bacteria

Reaction center of green sulfur bacteria such as Chlorobium vibrioforme and Chlorobium thiosulfatophilum is similar to PSI of oxygenic photosynthesis. Similar to phycobilisomes of cyanobacteria, the antenna system of the green sulfur bacteria such as bacteriochlorophyll and carotenoids is contained in complexes known as chlorosomes attached to the surface of the photosynthetic membrane through baseplate containing antenna bacteriochlorophyll a [13]. Reaction center involved in green sulfur bacteria is called Fe–S type. Therefore, green sulfur bacteria are not dependent on the reverse electron flow for the carbon reduction as the reduced ferredoxin uses its electrons to reduce NAD⁺/NADP⁺ using ferredoxin–NAD(P)+ oxidoreductase enzyme. At reaction center, electrons get excited by absorbing light intensity of 840 nm. Excited electrons can follow two pathways: one cyclic flow of electrons back to the reaction center via Q, Cyt bc₁ complex, and Cyt c₅₅₃, and another noncyclic flow of electrons through iron–sulfur protein ferredoxin (Fd) to reduce NAD⁺ to NADPH using ferredoxin/NAD reductase (Fig. 12.4b) [53, 54]. Similar to purple bacteria, protons are pumped by the cytochrome bc1 complex to generate ATP. Electrons at reaction center are replaced by the oxidation of H₂S to elemental S^o and then to SO₄ ²⁻. Electron carriers in green sulfur bacteria are much better placed according to their electronegativity than the purple bacteria which ensure reduction of NAD without the need of reverse electron flow. Green sulfur bacteria reduce free CO₂ by reversing original tricarboxylic acid cycle (Krebs cycle) with the input of energy. Thus, green sulfur bacteria can fix CO₂ even in the absence of RuBisCO. Green sulfur bacteria extract both electrons and hydrogen from sulfur compounds [13].

3 CO₂ Sequestration Pathways

3.1 Calvin Cycle

CO₂ is fixed into carbohydrate in light-independent stage using Calvin–Benson cycle. Proteins participating in CO₂ fixation have been found outside the thylakoid membrane in aqueous phase [13]. CO₂ fixation reaction is catalyzed by the carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) [55]. Calvin cycle can be divided into carboxylation, reduction, and regeneration reaction (Fig. 12.5). In carboxylation reaction, three molecules of CO₂ combine with three molecules of ribulose-1,5-bisphosphate (5 C) using carboxylase activity of RuBisCO enzyme to form six molecules of glycerate 3-phosphate (Eq. 12.6). Glycerate 3-phosphate further reduces to glyceraldehyde 3-phosphate in the reduction step. Reduction reaction is followed by regeneration reaction where ribulose-1,5-bisphosphate, the starting material of Calvin cycle, is regenerated using five molecules of glyceraldehyde 3-phosphate, while the remaining one molecule of glyceraldehyde 3-phosphate is used for the synthesizing biosynthetic material [56].

Fig. 12.5

Schematic diagram of Calvin cycle

$$ \mathrm{Ribulose}\ 1,5\ \mathrm{bisphosphate}+{\mathrm{CO}}_2+{\mathrm{H}}_2\mathrm{O}\to 2\ \mathrm{glyceraldehyde}\ 3\hbox{-} \mathrm{phosphate} $$

(12.6)

Standard free energy for the synthesis of one mole of glucose is equal to 2,870 KJ [13]. Photosynthetic process is further generalized by Van Niel (Eq. 12.7).

$$ {\mathrm{CO}}_2+2{\mathrm{H}}_2\mathrm{A}+\mathrm{light}\to {\left({\mathrm{CH}}_2\mathrm{O}\right)}_{\mathrm{n}}+2\mathrm{A}+{\mathrm{H}}_2\mathrm{O} $$

(12.7)

where A is O and S for oxygenic photosynthesis and anoxygenic photosynthesis (taking H₂S as electron donor), respectively. Later, it was demonstrated by Van Niel that molecular oxygen comes from dissociation of water rather than CO₂. The end product of Calvin cycle, glyceraldehyde 3-phosphate, is used in synthesizing cellular biosynthetic material for immediate energy source and sucrose that is transported to cytosol for storage as starch in chloroplast of green algae and glycogen in cyanobacteria [57]. Considering acetyl-CoA as the end product, seven molecules of ATP and four molecules of NAD(P)H are required to reduce two molecules of CO₂. Thus, Calvin cycle is the most energy intensive pathway for CO₂ sequestration [58].

$$ 2\ {\mathrm{CO}}_2+7\ \mathrm{ATP}+4\ \mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+\mathrm{HS}\hbox{-} \mathrm{CoA}\to \mathrm{Acetyl}\hbox{-} \mathrm{CoA}+7\ \mathrm{ADP}+4\ \mathrm{NAD}\ {\left(\mathrm{P}\right)}^{+} $$

(12.8)

3.2 Wood–Ljungdahl (WL) or Reductive Acetyl-CoA Pathway

Wood–Ljungdahl (WL) or reductive acetyl-CoA pathway is a bidirectional pathway, which is predominant primarily in strict anaerobic bacteria and archaea of phyla Firmicutes and Euryarchaeota, respectively [58]. This pathway is used for chemoautotrophic carbon fixation in acetogens such as Clostridium thermoaceticum and Acetobacterium woodii and methanogens such as Methanobacterium thermoautotrophicum and most autotrophic sulfate reducers such as Desulfobacterium autotrophicum. Wood–Ljungdahl (WL) pathway was found to be the most efficient non-photosynthetic pathway based on the most expensive substrate (i.e., H₂ or electrons), for the production of acetate and ethanol [58]. Reductive acetyl-CoA pathway uses only four moles of H₂ to form one mole of acetate. Two molecules of CO₂ directly combine to form one molecule of acetate. This pathway is different from other six known CO₂ fixation pathways as it does not undergo in cyclic manner. In addition to the use of Fd for reduction reaction, carbon monoxide dehydrogenase and acetyl-CoA synthase are the main enzymes involved in this pathway. These enzymes are very sensitive to oxygen. It has both carbonyl as well as methyl components (Fig. 12.6). Out of two molecules of CO₂, one is reduced to carbonyl group (C=O) catalyzed by CO dehydrogenase, and the other CO₂ is captured on special tetrahydrofolate cofactor and reduced to a methyl group. Carbonyl group bonded with enzyme is combined with the methyl group to form acetyl-CoA by enzyme acetyl-CoA synthase complex. The reducing equivalents for the pathway are obtained by oxidation of molecular hydrogen during autotrophic growth or NADH and reduced ferredoxin during heterotrophic growth [59]. For the formation of one molecule of acetyl-CoA using two molecules of CO₂, WL pathways need one molecule of ATP and four molecules of NAD(P)H (Eq. 12.9) [58]. Among the four pathways discussed above, WL pathway is most efficient based on CO₂ sequestration per ATP consumed. Moreover, ATP consumed in WL pathway is even less than one, as some of the energy is conserved in the membrane gradient in the form of ATP. rTCA cycle comes next to WL pathway in terms of energy efficiency. Both the pathways are found in highly reduced environments in which carbon reduction is more favorable [58].

Fig. 12.6

Schematic diagram of Wood–Ljungdahl (WL) or reductive acetyl-CoA pathway. (Reprinted from Ref. [60]. Copyright 2011, with permission from Elsevier)

$$ 2\ \mathrm{CO}_2+1\ \mathrm{ATP}+4\ \mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+\mathrm{HS}\hbox{-} \mathrm{CoA}\to \kern0.5em \mathrm{Acetyl}\hbox{-} \mathrm{CoA}+1\ \mathrm{ADP}+4\ \mathrm{NAD}\ {\left(\mathrm{P}\right)}^{+} $$

(12.9)

3.3 Reductive Tricarboxylic Acid Cycle (rTCA) or Reverse Citric Acid Cycle

Reductive tricarboxylic acid cycle (rTCA) has been found in bacteria under anaerobic or microaerobic conditions. Bacteria dwelling under these conditions are green sulfur bacteria such as Chlorobium sp. (Chlorobium limicola, Chlorobium thiosulfatophilum, etc.), sulfur-reducing bacteria (Desulfobacter), knallgas bacteria or hydrogen-oxidizing bacteria (Hydrogenobacter, Aquifex), and archaea (Thermoproteus). They utilize this cycle to fix atmospheric CO₂ through anoxygenic photosynthesis [59]. Reductive citric acid cycle is oxidative citric acid cycle running in reverse direction [61]. In TCA cycle, one molecule of acetyl-CoA breaks down to form two molecules of CO₂ and energy. However, in reverse TCA cycle, two molecules of CO₂ are used to synthesize one molecule of acetyl-CoA using 2 ATP and H⁺ from NADH and NADPH (Fig. 12.7a). Two carbon fixing enzymes, pyruvate synthase (pyruvate:ferredoxin oxidoreductase) and 2-oxoglutarate synthase (2-oxoglutarate:ferredoxin oxidoreductase), are the key enzymes of rTCA cycle and were found to be dependent on Fd [61, 62]. Two reactions involving ferredoxin (Fd) significantly help in reversal of some of the reactions, which are otherwise nonreversible in nature. Another enzyme citrate lyase was discovered which is dependent on ATP [63]. ATP-citrate lyase cleaves citrate, a six-carbon compound, into oxaloacetate, a four-carbon compound, and acetyl-CoA. These three enzymes work together to make energetically unfavorable reverse reactions possible. One molecule of succinyl-CoA combines with one molecule of CO₂ to form one molecule of 2-oxoglutarate using 2-oxoglutarate synthase. 2-Oxoglutarate further takes one molecule of CO₂ to form isocitrate using isocitrate dehydrogenase (IDH). Aconitase converts isocitrate to citrate, which is acted upon by citrate lyase to form oxaloacetate and acetyl-CoA. Acetyl-CoA combines with another molecule of CO₂ to form pyruvate using pyruvate synthase. It is followed by synthesis of PEP which combines with another molecule of CO₂ to regenerate oxaloacetate or other intermediates of the cycle in an anaplerotic manner [61]. Shiba et al. (1985) reported direct conversion of pyruvate into oxaloacetate in H. thermophilus by an enzyme pyruvate carboxylase [64]. In one complete cycle, four molecules of CO₂ are fixed to generate one molecule of oxaloacetate, which itself is an intermediate of the cycle. It requires two molecules of CO₂, 2 ATP, and 4 NAD(P)H for the formation of one molecule of acetyl-CoA as the end product as shown in Eq. (12.10) [58].

Fig. 12.7

Schematic diagram of (a) reductive tricarboxylic acid cycle (rTCA) or reverse citric acid cycle shown in green arrow and (b) dicarboxylate/4-hydroxybutyrate cycle shown in red arrow. Black arrows showing reaction pathways are common to both of them (Reprinted from Ref. [60]. Copyright 2011, with permission from Elsevier)

$$ {\mathrm{CO}}_2+2\ \mathrm{ATP}+4\ \mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+\mathrm{HS}\hbox{-} \mathrm{CoA}\to \mathrm{Acetyl}\hbox{-} \mathrm{CoA}+2\ \mathrm{ADP}+4\ \mathrm{NAD}{\left(\mathrm{P}\right)}^{+} $$

(12.10)

3.4 3-Hydroxypropionate Cycle

CO₂ fixation in phototrophic green non-sulfur bacteria takes place using 3-hydroxypropionate pathway unique to eubacterium Chloroflexus aurantiacus and some chemotrophic archaebacteria such as Sulfolobus metallicus, Ignicoccus hospitalis, Acidianus brierleyi, and Acidianus infernus [60, 65, 66]. Enzymes participating in this pathway are not sensitive to oxygen [60, 67]. Acetyl-CoA is the starting precursor of 3-hydroxypropionate cycle. It combines with HCO₃ ⁻ to form malonyl-CoA with the help of ATP-dependent acetyl-CoA carboxylase (Fig. 12.8a). Acetyl-CoA carboxylase is also an essential enzyme for the synthesis of fatty acid. It is followed by the conversion of malonyl-CoA to 3-hydroxypropionate by malonyl-CoA reductase in an NADPH-dependent reaction. Malonyl-CoA reductase is a bifunctional enzyme, which reduces malonyl-CoA to 3-hydroxypropionate via malonate-semialdehyde as an intermediate using its aldehyde dehydrogenase and alcohol dehydrogenase domain. Reductive conversion of 3-hydroxypropionate to propionyl-CoA is catalyzed by propionyl-CoA synthase. Propionyl-CoA synthase is a trifunctional enzyme and formally requires three enzymatic reactions. In the first step, activation to 3-hydroxypropionyl-CoA is catalyzed by CoA ligase, which is followed by dehydration of 3-hydroxypropionyl-CoA to acrylyl-CoA by an enoyl-CoA hydratase. Finally, acrylyl-CoA is reduced to propionyl-CoA by an enoyl-CoA reductase using NADPH [65, 68, 69]. Propionyl-CoA undergoes carboxylation, catalyzed by ATP-dependent propionyl-CoA carboxylase to form methylmalonyl-CoA. Isomerization of methylmalonyl-CoA takes place in two sequential steps, catalyzed by methylmalonyl-CoA epimerase and methylmalonyl-CoA mutase to form succinyl-CoA. Succinyl-CoA transfers CoA for malate activation and forms succinate and malyl-CoA [65, 68, 69]. Malyl-CoA, a four-carbon compound, breaks down by malyl-CoA lyase to regenerate starting acetyl-CoA and glyxolate. Carboxylations of acetyl-CoA and propionyl-CoA are the main CO₂ fixation reactions. It is to be noted that actual substrate for both the carboxylation reactions is HCO₃ ⁻ rather than CO₂. Each turn of cycle results in net fixation of two molecules of bicarbonate to produce one molecule of glyxolate. Glyoxylate is considered as the initial CO₂ fixation product. Glyxolate is further utilized in the synthesis of the cellular material. Intermediate 3-hydroxypropionate is the characteristic of the cycle, which on reductive conversion produces propionyl-CoA [65, 68, 69]. In 3-hydroxypropionate cycle, six molecules of ATP and four molecules of NAD(P)H are required to reduce two molecules of CO₂ to form one molecule of acetyl-CoA as shown in Eq. (12.11) [58].

Fig. 12.8

Schematic diagram of (a) 3-hydroxypropionate cycle shown in light blue arrow, (b) 3-hydroxypropionate/4-hydroxybutyrate cycle shown in purple arrow. Black arrows showing reaction pathways are common to both of them (Reprinted from Ref. [60]. Copyright 2011, with permission from Elsevier)

$$ {\mathrm{CO}}_2+6\ \mathrm{ATP}+4\ \mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+\mathrm{HS}\hbox{-} \mathrm{CoA}\to \mathrm{Acetyl}\hbox{-} \mathrm{CoA}+6\ \mathrm{ADP}+4\ \mathrm{NAD}\ {\left(\mathrm{P}\right)}^{+} $$

(12.11)

3.5 Other Pathways

3.5.1 Dicarboxylate/4-Hydroxybutyrate Cycle

Dicarboxylate/4-hydroxybutyrate cycle is the newly discovered autotrophic pathway for carbon dioxide fixation in Ignicoccus hospitalis (Desulfurococcales), Thermoproteus neutrophilus, and an anaerobic member of Thermoproteales [70–72]. Evidence of dicarboxylate/4-hydroxybutyrate cycle has been reported by Ramos-vera et al. (2009) in T. neutrophilus [72]. This pathway can also be divided into two parts: the first part involves formation of succinyl-CoA from acetyl-CoA using two inorganic carbons and the second part deals with regeneration of acetyl-CoA from succinyl-CoA. Thus, this pathway has similarity with both reductive tricarboxylic acid cycle (rTCA) and 3-hydroxypropionate/4-hydroxybutyrate cycles (Fig. 12.7b). The first part of the cycle involves the intermediates of reductive tricarboxylic acid cycle (rTCA) and the second part involves the intermediates of 3-hydroxypropionate/4-hydroxybutyrate cycle via the route similar to 4-hydroxybutyrate pathway to succinyl-CoA using pyruvate synthase and pyruvate carboxylase, as carboxylating enzyme. So, the only difference between this and the 3-hydroxypropionate/4-hydroxybutyrate cycle is the way succinyl-CoA is created.

3.5.2 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

Another pathway has been reported in some bacteria called 3-hydroxypropionate/4-hydroxybutyrate cycle. This pathway was found in aerobic autotrophic members of Sulfolobales. 3-Hydroxypropionate/4-hydroxybutyrate pathway was discovered in Metallosphaera sedula which was earlier believed to fix CO₂ using 3-hydroxypropionate cycle. Malyl-CoA lyase, an enzyme used in the regeneration of acetyl-CoA, was absent in the cell extract of M. sedula, resulting in the proposed alternative pathway for the regeneration of starting material of cycle. Therefore, 3-hydroxypropionate and 3-hydroxypropionate/4-hydroxybutyrate cycle share the same steps from acetyl-CoA to succinyl-CoA (Fig. 12.8b). However, the enzymes involved in the reaction steps from acetyl-CoA to succinyl-CoA are not same in both cases. Detailed structure of acetyl-CoA carboxylase participating in 3-hydroxypropionate cycle reveals that the enzyme is composed of four subunits. Contrary to this, acetyl-CoA carboxylase taking part in 3-hydroxypropionate/4-hydroxybutyrate cycle has only three subunits. Similarly, malonyl-CoA reductase of both the cycles is not the same. They differ in the different intermediates through which regeneration of acetyl-CoA takes place from succinyl-CoA. The intermediates between acetyl-CoA and succinyl-CoA in 3-hydroxypropionate/4-hydroxybutyrate cycle are succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, and acetoacetyl-CoA, formations of which are catalyzed by succinyl-CoA reductase, succinate semialdehyde reductase, 4-hydroxybutyryl-CoA synthetase, 4-hydroxybutyryl-CoA dehydratase, crotonyl-CoA hydratase, 3-hydroxybutyryl-CoA dehydrogenase, and acetoacetyl-CoA β-ketothiolase, respectively [60].

4 Enzymes for CO₂ Sequestration

Carbonic anhydrase (CA) is zinc metalloenzyme which catalyzes conversion of free CO₂ into bicarbonates and protons. It is one of the fastest known enzymes catalyzing 10⁴–10⁶ reactions per second [73]. It has been found in a wide number of living beings such as plants, animals, and microorganisms as they have been found growing well in CO₂-rich conditions, and CA was found essential for this purpose [74, 75]. In human body, CA is present in the erythrocyte and converts poorly soluble CO₂ in aqueous solution, such as blood plasma to water-soluble bicarbonate (HCO₃ ⁻) anion [76]. Human isozyme HCA II having molecular mass of 30,000 is the fastest CA known so far, having a hydration rate of 1.4 × 10⁶ molecules of CO₂ per second per molecule of CA [19]. Some of the microalgae which have been found containing CA are Chlamydomonas reinhardtii, Scenedesmus obliquus, Dunaliella tertiolecta, Chlorella saccharophila, Chlorella vulgaris, Chlorella pyrenoidosa, and Chlorococcum littorale [77]. BCA (bovine carbonic anhydrase) has been found stable in wide range of pH (5–10) and temperature (up to 70 °C) [9]. Sulfate and zinc have been found to enhance CA activity.

There are contradictory reports on the effect of CO₂ concentration on the activity of CA in photosynthetic microorganisms such as plants, green algae, and cyanobacteria. In most of the reports, activity of CA isolated from algae is inversely proportional to the CO₂ concentration. It was explained with the fact that RuBisCO can catalyze both oxygenase and carboxylase activities depending upon the nearby CO₂ concentration [29]. CA helps in increasing the concentration of CO₂ near the RuBisCO site. CA can enhance the internal CO₂ concentration up to 1,000 times higher than the external fluid. Therefore, at lower CO₂ concentration, expression of CA increases to maintain carboxylase activity of RuBisCO which in turn reduces atmospheric CO₂ into cellular constituents such as starch, lipid, and protein. However, in few reports, activity of CA initially increases with increase in CO₂ concentration and later starts decreasing with further increase in CO₂ concentration, thus making bell-shaped curve [20]. The reason behind the decreasing trend of CA at higher CO₂ was postulated due to feedback inhibition by bicarbonate and/or decrease in pH at this concentration.

Extracellular and intercellular crude extract of enzyme CA II isolated from Chlorella vulgaris were 72 and 160 mg CaCO₃ per milligram of protein, comparable to 225 mg CaCO₃ per milligram of protein with the purified enzyme from Citrobacter freundii [78].

4.1 Mechanism of CO₂ Captured by CA

Hydration of CO₂ into solid carbonate such as calcium carbonate is a natural process. There are five reactions required for the transformation of CO₂ into minerals [9, 19, 76].

• 1. Dissolution of gaseous CO₂ in liquid (Eq. 12.12)

• 2. Hydration of aqueous CO₂ into carbonic acids (H₂CO₃) (Eq. 12.13)

• 3. Dissociation of carbonic acid into bicarbonate ions (HCO₃ ²⁻) and protons (Eq. 12.16)

• 4. Dissociation of bicarbonate ions into carbonate ions (CO₃ ⁻) (Eq. 12.17)

• 5. Reaction of carbonate ions and calcium to form solid calcium carbonate (Eq. 12.18)

4.1.1 CO₂ Dissolution

$$ {\mathrm{CO}}_{2\left(\mathrm{g}\right)}\leftrightarrow {\mathrm{CO}}_{2\left(\mathrm{aq}\right)} $$

(12.12)

4.1.2 Carbonic Acid Formation

$$ {\mathrm{CO}}_{2\left(\mathrm{aq}\right)}+{\mathrm{H}}_2\mathrm{O}\leftrightarrow {\mathrm{H}}_2{\mathrm{CO}}_3 $$

(12.13)

$$ {K}_2=\frac{k_2}{k_{-2}}=\frac{6.2\times {10}^{-2}{\mathrm{s}}^{-1}}{23.7\;{\mathrm{s}}^{-1}}=2.6\times {10}^{-3} $$

Where k ₂, k ₋₂ and K ₂ are forward rate, reverse rate and equilibrium constants respectively. As shown in Eq. (12.13), hydration of CO₂ to carbonic acids is the rate limiting step having very less forward reaction constant 6.2 × 10⁻² s at 25 °C [18]. However, catalysis by CA increases the rate of reaction manifold showing the high substrate specificity of this enzyme [19]. Enzyme catalyzes the CO₂ hydration reaction in the following two half reactions (Eqs. 12.14 and 12.15) [79]:

$$ \mathrm{E}-\mathrm{Zn}-{\mathrm{H}}_2\mathrm{O}\leftrightarrow \mathrm{E}-\mathrm{Zn}+{\mathrm{OH}}^{-}+{\mathrm{H}}^{+} $$

(12.14)

$$ \mathrm{E}-\mathrm{Zn}-\mathrm{OH}+{\mathrm{CO}}_2\leftrightarrow \mathrm{E}-\mathrm{Zn}-{{\mathrm{CO}}_3}^{-}+{\mathrm{H}}^{+}\to \mathrm{E}-\mathrm{Zn}-{\mathrm{H}}_2\mathrm{O}+{{\mathrm{CO}}_3}^{-} $$

(12.15)

The above reaction was found dominating when the pH is higher than 10 while it was found negligible at pH less than 8 [80].

4.1.3 Bicarbonate Formation

$$ {\mathrm{H}}_2{\mathrm{CO}}_3\leftrightarrow {\mathrm{H}}^{+}+{{\mathrm{H}\mathrm{CO}}_3}^{-} $$

(12.16)

$$ {K}_3=\frac{k_3}{k_{-3}}=\frac{8\times {10}^6\;{\mathrm{s}}^{-1}}{4.7\times {10}^{10}\;{\mathrm{s}}^{-1}}=1.7\times {10}^{-4} $$

Where k ₃, k ₋₃ and K ₃ are forward rate, reverse rate and equilibrium constants respectively. Bicarbonate formation reaction is diffusion controlled and very fast in nature.

4.1.4 Carbonate Formation

$$ {{\mathrm{H}\mathrm{CO}}_3}^{-}\leftrightarrow {\mathrm{H}}^{+}+{{\mathrm{CO}}_3}^{2-} $$

(12.17)

$$ {K}_4=\frac{k_4}{k_{-4}}=\frac{8\times {10}^6\;{\mathrm{s}}^{-1}}{4.7\times {10}^{10}\;{\mathrm{s}}^{-1}}=4.69\times {10}^{-11} $$

Where k ₄, k ₋₄ and K ₄ are forward rate, reverse rate and equilibrium constants respectively.

4.1.5 Calcium Carbonate Reaction

$$ {\mathrm{Ca}}^{2+}+{{\mathrm{Ca}\mathrm{CO}}_3}^{2-}\leftrightarrow {\mathrm{Ca}\mathrm{CO}}_3\downarrow $$

(12.18)

Calcium carbonate precipitates quickly at the saturation concentration of calcium and carbonate ions. Therefore, continuous supply of carbonate ions by hydration of CO₂ and water is requisite [19]. CA was also found to enhance the calcium carbonate precipitation reaction [9]. However, contrary to enhanced hydration reaction, higher concentration of CA did not increase CaCO₃ precipitation reaction [9].

Some of the approaches which have been applied for improving the performance of CA are (1) isolating CAs from thermophilic microorganisms, (2) use of protein engineering to create thermotolerant enzymes, and (3) immobilizing the enzyme for stabilization and confinement to cooler regions and process modification that minimize the stresses such as cooling of the flue gas [81].

4.2 Immobilization of CA

Use of CA in aqueous solution has many disadvantages such as reusability and recovery. Immobilization of the CA in the nanoparticles significantly improves the catalytic property, storage, and thermal stability of the enzyme. Immobilized BCA and HCA enzymes were found retaining nearly 90 % of enzymatic activity for more than 20 cycles. Carbonic anhydrase (CA) can be immobilized on functionalized and metal nanoparticles confined mesoporous silica for CO₂ hydration and its sequestration to CaCO₃ [76, 82]. Surface-modified magnetic nanoparticle is one of such attractive templates for enzyme immobilization. Enzyme can be easily recovered from a reaction medium by applying a static magnetic field near the immobilized CA in the reactor [76, 82]. Inert materials such as chitosan and sodium alginate are widely used for immobilization of enzymes and microorganism. Whole cells of Pseudomonas fragi, Micrococcus lylae, and Micrococcus luteus 2 were immobilized on different biopolymer matrices [83]. Bovine carbonic anhydrase (BCA) was covalently immobilized by Vinoba et al. (2012) onto OAPS (octa(aminophenyl)silsesquioxane)-functionalized Fe₃O₄/SiO₂ nanoparticles by using glutaraldehyde as a spacer [76]. Immobilization of CA can be done in many ways such as adsorption on surfaces, entrapment within matrices, and cross-linking within polymeric scaffold [80]. Polyurethane foam is a highly porous hydrophilic polymeric material where enzyme immobilization was easy and fast. CA had 100 % activity over time, and thus reusability was not a concern [80].

4.3 Application of CA

Bovine CA has been proposed to inject into wellbore of geological formations to prevent CO₂ leakage through it [84]. CO₂ sequestration by mineral carbonation can use CA to make the process feasible at large scale. Alkaline silicates are abundant and high enough to sequester all the CO₂ emitted from total fossil fuels. Alkaline silicates can dissolve to provide cations in acidic conditions. However, high alkalinity is required for the increase of the rate of gaseous CO₂ dissolving into the carbonate ions CO₃ ²⁻. Therefore, enhancing the release of divalent cations from the alkaline silicates and enhancing alkalinity are some of the challenges of the process. Increase in alkalinity increases the rate of dissolution of CO₂ into the carbonate ions CO₃ ²⁻, and acidic conditions release the divalent cations required for the formation of carbonates [17]. Denitrification, methane production, and sulfate reduction are some of the alkalinity-producing metabolic processes. Therefore, these processes enhance the carbonation process. Integrating acid-producing process for silicate dissolution and alkaline-producing process for carbonate precipitation together can be used for CO₂ mitigation using biological mineral carbonation. Dupraz et al. (2009) has experimentally shown the use of Bacillus pasteurii as a model carbonate precipitating bacteria on the geological sequestration of CO₂ and its transformation into solid carbonate phases [85].

4.4 Challenges in Use of CA for CO₂ Sequestration

Enzyme works better at optimum temperature, pH, and enzyme concentration [83]. At lower pH, carbonates prefer to be in dissolved state than the precipitated form, while at higher pH, carbonate ions form but concerns about economical and environmental aspect arise [19]. Lifetime and activity of enzyme greatly depend upon pH, temperature, other ions such as CN⁻, and higher concentration of SO_x and NO_x [19]. Higher cost of enzyme and its large-scale production are other bottlenecks to overcome to make the process successful in reality.

5 Conclusions

Imbalance between CO₂ emission and sink is the reason for steep rise in the earth’s atmospheric CO₂ concentration. This may be the reason for the rise in global mean temperature, causing melting of glaciers, rise in sea level, ocean acidification, unpredicted climate changes, etc. Several biological processes are available for CO₂ mitigation. Carbonic anhydrase (CA) enzyme, algae, cyanobacteria, bacteria, and terrestrial plants are some of the biological methods used for CO₂ sequestration. However, these processes are slow and limited in application. Identifying limiting parameters and application of technology can accelerate the natural processes manifold. For example, CO₂ hydration, the limiting step in the transformation of gaseous CO₂ to solid bicarbonates, can be accelerated by catalyzing the reaction using CA. Forest and oceanic fertilization can be applied to enhance the photosynthetic efficiency of terrestrial plants and oceanic phytoplanktons, respectively. Similarly, algae and cyanobacteria can be exploited for CO₂ sequestration as they can be grown efficiently at higher CO₂ concentration with higher photosynthetic efficiency.