Biophotolysis-Based Hydrogen Production by Cyanobacteria

Abstract

Hydrogen is the most abundant element in the universe, comprising approximately 75% of all matter by weight, molecular hydrogen (H₂) exists in only trace amounts within the Earth’s atmosphere. As a gaseous and carbon-free fuel, hydrogen can be combusted with water and therefore is regarded as a clean nonpolluting fuel. Hydrogen is produced by steam reformation of methane, gasification of coal and biomass, and metabolic pathway of special type of microorganisms, commonly known as biological hydrogen production. Biological hydrogen evolution provides a sustainable and environmentally friendly way to produce clean energy from renewable resources. Biological hydrogen production processes are mostly controlled by either photosynthetic or fermentative organisms. Hydrogen can be produced biologically by direct biophotolysis, indirect biophotolysis, photofermentation, dark fermentation, combination of these processes (such as integration of dark- and photofermentation, etc.) or by water–gas shift reaction. Among a selection of biological systems, cyanobacteria have become a major source as potential cell factories for hydrogen production. They are highly promising microorganisms for biological photohydrogen production. Cyanobacteria grow by photosynthesis, and essentially contain chlorophyll and various carotenoids whose main functions are light-harvesting and photoprotection. They produce chlorophyll a, and most also have characteristic pigments called phycobilins, which function as accessory pigments in photosynthesis. Cyanobacteria produce hydrogen gas using nitrogenase and/or hydrogenase. This study explains the potential of cyanobacteria to produce biohydrogen and focuses on biophotolysis-based hydrogen production by cyanobacteria.

5.1 Introduction

New energy sources have emerged as a result of reduction in fossil energy resources. Hydrogen gas is one of the potential future energy sources that substitutes fossil fuel resources. It is clean fuel with no carbon dioxide emissions and can easily be used in fuel cells for the generation of electricity, liberates a large amount of energy per unit mass and renewable (Demirbas 2009). Molecular hydrogen has the highest energy relative to the molecular weight among the known gaseous fuels (120 M J kg⁻¹ against 50 M J kg⁻¹ for natural gas) and is the only carbon-free fuel which ultimately oxidizes to water as a combustion product (de Poulpiquet et al. 2014). Hydrogen can be produced from fossil fuels and biomass: coal gasification, steam reforming, partial oxidation of oil. In addition to hydrogen production from H₂O through nonbiological methods: thermal and thermochemical processes, electrolysis, and photolysis. H₂ can be produced biologically. Hydrogen is produced by many microorganisms’ reactions which are linked to their energy metabolism. All processes of biological H₂ production are dependent on the presence of H₂-producing enzymes. It was found that all the enzymes contain complex metalloclusters as active sites and that the active sites of the enzyme units are synthesized in a complex process involving auxiliary enzymes and protein maturation steps (Azwar et al. 2014). Biohydrogen can be produced by both autotrophic and heterotrophic microorganisms. Some important biological hydrogen production processes are dark fermentation (with obligate or facultative anaerobe microbes), photofermentation (with photoheterotrophic bacteria), hybrid system, biophotolysis of H₂O using green algae and cyanobacteria (Chaubey et al. 2013) and water–gas shift reaction (Fig. 5.1). All processes are controlled by the hydrogen-producing enzymes, such as hydrogenase and nitrogenase (Holladay et al. 2009).

Fig. 5.1

Biological pathways to produce hydrogen

5.2 Biohydrogen Production

Biological production of hydrogen (biohydrogen), is conceived of as a fuel which is produced via microbial metabolism, resembling bioethanol or biogas. Biological systems provide a wide range of approaches to generate hydrogen and include direct biophotolysis, indirect biophotolysis, photofermentations, dark fermentation, and hybrid system (Chaubey et al. 2013) and water–gas shift reaction (Holladay et al. 2009). Hydrogen metabolism is primarily the domain of bacteria and microalgae. Table 5.1 summarizes various biological hydrogen production processes with general overall reactions involved therein. Dark fermentation is carried out under anoxic conditions (i.e., no oxygen present as an electron acceptor). Carbohydrates including glucose, amino acids, fatty acids supply many anaerobic microorganisms such as heterotrophic obligate anaerobes (e.g., Clostridium sp.) and facultative anaerobes (e.g., Enterobacter sp.) with both carbon and energy, resulting in the production of H₂, CO₂ with CH₄ or H₂S and other reduced end products.

Table 5.1 Different biohydrogen production processes with general overall reaction

In anaerobic environments, protons (H⁺), which are reduced to molecular hydrogen (H₂), need to act as an electron acceptor. In the dark fermentation of glucose, it is first converted to pyruvate producing adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and the reduced form of nicotinamide adenine dinucleotide (NADH). Pyruvate is then further converted to acetyl coenzyme A (acetyl-CoA), carbon dioxide (CO₂), and H₂ by pyruvate ferredoxin oxidoreductase and hydrogenase (Ghimire et al. 2015). Pyruvate may also be converted to acetyl-CoA and formate using the pyruvate-formate hydrogenlyase (PFHL) enzyme complex which may be further converted into H₂ and CO₂ by enteric bacteria such as Escherichia coli. The hydrogen yield by strict anaerobes such as the many clostridial species via the pyruvate–ferredoxin oxidoreductase (PFOR) coupling with a hydrogenase (Turner et al. 2008).

Photofermentation is performed under anaerobic conditions. Photosynthetic bacteria like Rhodobacter sphaeroides O.U001, Rhodobacter capsulatus, R. sphaeroides-RV, Rhodobacter sulfidophilus, Rhodopseudomonas palustris, and Rhodospirillum rubrum using light as energy source and organic acids (e.g., acetate, lactate, butyrate, maltate, etc.) and carbon sources like glucose, sucrose, succinate in the presence of nitrogenase enzyme for synthesizing hydrogen (Argun and Kargi 2011). The general reaction is given as follows when acetic acid is present in the fermentation medium:

$${\text{CH}}_{ 3} {\text{COOH}} + {\text{ 2H}}_{ 2} {\text{O}}\,\mathop{\longrightarrow}\limits\limits^{\text{Light Energy}}\, 4 {\text{H}}_{ 2} + 2 {\text{CO}}_{ 2}$$

(5.9)

These bacteria are able to use simple organic acids, like acetic acid as electron donors. These electrons are transported to the nitrogenase (N₂ase) by ferredoxin (Fd) using energy in the form of ATP (Hallenbeck and Benemann 2002) (Fig. 5.2). The optimum growth temperature and pH for the photosynthetic bacteria are in the range of 31–36 ℃ and 6.8–7.5, respectively. Hydrogen production rates vary depending on the light intensity, carbon source, and the type of microbial culture. Suitable light intensities for this process were reported to be between 6 and 10 klux. The activity of the nitrogenase is inhibited in the presence of oxygen, ammonia (Argun and Kargi 2011).

Fig. 5.2

Photofermentations (Hallenbeck and Benemann 2002)

Hybrid system consisted of two stages, dark fermentation followed by photofermentation. Thus, in this system, the light-independent bacteria and photosynthetic bacteria provide an integrated system for maximizing the H₂ yield. In such a system, the anaerobic fermentation of carbohydrate (or organic wastes) produces intermediates, such butyrate and acetate with a small amount of propionate, which are then converted into H₂ by the photosynthetic bacteria in the second step in a photobioreactor (Chaubey et al. 2013; Nikolaidis and Poullikkas 2017). The water–gas shift reaction, an exothermic reaction, increases the concentration of hydrogen gas in the product gas through the conversion of CO into CO₂ by steam. Certain photoheterotrophic bacteria, such as Rhodospirllacae (Holladay et al. 2009) and the gram-positive bacteria, such as Carboxydothermus hydrogenoformans (Lazarus et al. 2009) are capable of performing water–gas shift reaction at ambient temperature and atmospheric pressure. These bacteria can survive in the dark by using CO as the sole carbon source to generate adenosine triphosphate (ATP) coupling the oxidation of CO with the reduction of H⁺ to H₂ (Holladay et al. 2009). Water–gas shift reaction can be applied as follows:

$${\text{CO }} + {\text{ H}}_{ 2} {\text{O}} \to {\text{CO}}_{ 2} + {\text{ H}}_{ 2}$$

(5.10)

Under anaerobic conditions, CO induces the synthesis of several proteins, including CO dehydrogenase, Fe–S protein and CO-tolerant hydrogenase. Electrons produced from CO oxidation are conveyed via the Fe–S protein to the hydrogenase for hydrogen production (Lazarus et al. 2009). Biophotolysis is regarded as an activity performed in the presence of light in biological systems. It is comprised of direct biophotolysis and indirect biophotolysis. Direct biophotolysis is similar to the processes found in plants and algal photosynthesis. In this process, solar energy is directly converted to hydrogen via photosynthetic reactions. This is an attractive process since solar energy is used to convert a readily available substrate, water, to oxygen and hydrogen (McKinlay and Harwood 2010). In indirect biophotolysis, compounds such as starch and glycogen accumulated during CO₂ fixation are degraded to produce H₂ by an anaerobic fermentation process. This process can be done in the dark or in the light condition with cells that have impaired O₂-evolving photosystems II (Huesemann et al. 2010). Table 5.2 summarizes the relative advantages and disadvantages some important biological hydrogen production processes.

Table 5.2 Comparison of hydrogen production by some important biological hydrogen production processes (Demirbas 2009; Nath and Das 2004)

5.3 Cyanobacteria

Cyanobacteria are oxygen-evolving, photosynthetic prokaryotes that can grow in air (Nitrogen and Carbon dioxide as N and C source), H₂O (electrons and reductant source) and simple mineral salts with light as the energy source. These bacteria have been shown to posses multiple hydrogen-producing enzymes and are capable of both dark- and light-driven hydrogen productions in a variety of configurations. The cyanobacteria are morphologically and developmentally one of the most diverse groups of prokaryotes. The cell wall of cyanobacteria contains peptidoglycan and is structurally similar to that of gram-negative bacteria that obtain their energy through photosynthesis. These organisms were the first oxygen-evolving phototrophic organisms on Earth, and over billions of years converted the once anoxic atmosphere of Earth to the oxygenated atmosphere that we see today. They are a significant component of the freshwater and marine and an important primary producer in many areas of the ocean, but are also found in habitats other than the marine environment; in particular, cyanobacteria are known to occur in both freshwater and hypersaline inland lakes (Hallenbeck 2012). Cyanobacteria have evolved heterocysts and nonheterocysts. Heterocysts provide the anaerobic environment required for the activity of the oxygen-sensitive nitrogenase enzyme complex.

The morphology of these bacteria varies from unicellular to filamentous or colonial forms and there is considerable variation within these morphological types (Hallenbeck 2012). Cyanobacterial cells range in size from 0.5 μm in diameter to cells as large as 100 μm in diameter. They have specialized membrane systems that increase the ability of cells to harvest light energy. Photosynthetic complexes takes place in specialized regions of the plasma membrane which are also, for analogy with eukaryotes, called thylakoids, but lack the characteristic morphological structure of the latter. In the thylakoid membrane, a complex and multilayered photosynthetic membrane system containing photopigments and proteins that mediate photosynthesis. In most unicellular cyanobacteria, the thylakoid membranes are arranged in regular concentric circles around the periphery of the cytoplasm (Hazra and Kesh 2017).

Cyanobacteria constitute a vast potential resource in varied applications such as mariculture, food, feed, fuel, fertilizer, medicine, industry, and in combating pollution. These bacteria are oxygenic phototrophs and therefore have both type I and type II photosystems. All species are able to fix CO₂ by the Calvin cycle, many can fix N₂. Cells harvest energy from light and fix CO₂ during the day. During the night, cells generate energy by fermentation or aerobic respiration of carbon storage products such as glycogen. While CO₂ is the predominant source of carbon for most species, some cyanobacteria can absorb simple organic compounds such as glucose and acetate if light is present, a process called photoheterotrophy. A few cyanobacteria, mainly filamentous species, can also grow in the dark on glucose or sucrose, using the sugar as both carbon and energy source. Finally, when sulfide concentrations are high, some cyanobacteria are able to switch from oxygenic photosynthesis to anoxygenic photosynthesis using hydrogen sulfide rather than water as electron donor for photosynthesis (Singh et al. 2011). Many cyanobacteria have the ability to fix atmospheric nitrogen. Nitrogen fixation of cyanobacteria is catalyzed by the enzyme nitrogenase, which is sensitive to oxygen and is irreversibly inactivated in the presence of free oxygen. N₂ fixation is restricted to specialized cells (heterocysts) (Aryal and Sherman 2017).

Hydrogen production has been studied in a range of cyanobacterial species and strains. Hydrogen production happens in at least 14 cyanobacteria genera, in a wide variety of culture conditions (Kufryk 2013). Hydrogen production is affected by diverse parameters in various ways, for example, environmental conditions and Intrinsic factors affecting hydrogen production. Light, temperature, salinity, nutrient availability, gaseous atmosphere as environmental conditions can make a contribution to hydrogen production. To have optimum hydrogen production, different cyanobacterial species are required (Tiwari and Pandey 2012).

5.4 The Photosynthetic Pigments of Cyanobacteria

Cyanobacteria are photosynthetic bacteria found in diverse environments including freshwater, oceans and terrestrial habitats. They are major contributors to the global oxygen cycle, carbon- and nitrogen fixation (Tóth et al. 2015). Cyanobacteria are a rich source of pigments such as chlorophyll a, carotenoids, and phycobiliproteins.

5.4.1 Chlorophylls

Chlorophylls (Chls) are the essential molecules of oxygenic photosynthesis. Cyanobacteria contain chlorophyll a (a few species contain chlorophyll d and chlorophyll f). Chl a is the essential molecule for cyanobacteria, excluding the Chl d–containing cyanobacterium, Acaryochloris marina. A. marina is the only cyanobacterium reported that uses Chl d as its major photosynthetic photopigment. It is found in filtered light environments in various ecological niches. The advantage of using Chl d and long wavelength absorbing chlorophylls in oxygenic photosynthetic organisms is intriguing due to its unique absorption properties and its potential for increased photosynthetic efficiency. Chlorophyll f was recently found within a filamentous cyanobacterium and has a maximum Q_Y absorption peak at about 707 nm (in methanol) (Vinyard et al. 2013). Chlorophyll a is the predominant light-absorbing pigment of Photosystem I (PS I), while the phycobilins are the predominant energy collectors of PS II, passing absorbed energy to the photosynthetic reaction center through relatively small number of chlorophyll a molecules (Wiwczar et al. 2017).

5.4.2 Carotenoids

In cyanobacteria, carotenoids are also associated with proteins devoid of chlorophyll. They have two main functions: carotenoids serve as light-harvesting pigments in photosynthesis and they protect chlorophyll against photooxidative damage. However, excess light can be lethal for photosynthetic organisms because it can catalyze photooxidation reactions that can produce toxic forms of oxygen, such as singlet oxygen. In cyanobacteria the most abundant Cars are β-carotene and various xanthophylls, such as synechoxanthin, canthaxanthin, caloxanthin, echinenone, myxoxanthophyll, nostoxanthin and zeaxanthin (Zakar et al. 2016).

5.4.3 Phycobiliproteins

Phycobiliproteins assemble into aggregates called phycobilisomes that attach to cyanobacterial thylakoids. In cyanobacteria, the phycobilisomes (PBSs), serve as light-harvesting antennae for the photosynthetic complexes. In phycobilisomes the phycobilin pigments (phycocyanobilin, phycourobilin, phycoerythrobilin, phycobiliviolin) attached to phycobiliproteins are responsible for light-harvesting. Phycobiliproteins are associated with the photosynthetic apparatus. They are usually divided into three separate groups based on their color and absorption properties (Stadnichuk et al. 2015). One class of phycobiliproteins, phycocyanins, are blue and, together with the green chlorophyll a, are responsible for the blue-green color of most cyanobacteria (Hazra and Kesh 2017).

Phycocyanins absorbs most strongly at 620 nm. Some cyanobacteria produce phycoerythrin, a red phycobiliproteins that absorbs most strongly at wavelengths around 560 nm, and species producing phycoerythrin are red or brown. A third phycobiliprotein, called allophycocyanin, absorbs at about 650 nm. Phycobilisomes are arranged in rows, often parallel to each other. They are arranged such that the allophycocyanin molecules are in direct contact with the photosynthetic membrane. Allophycocyanin is surrounded by phycocyanin or phycoerythrin (or both, depending on the organism). The energy transfer occurs from phycocyanin and phycoerythrin to allophycocyanin which is positioned closest to the reaction center chlorophyll and transfers energy to it. Phycobilisomes facilitate energy transfer to cyanobacterial reaction centers, allowing cyanobacteria to grow at lower light intensities than would otherwise be possible (Stadnichuk et al. 2015).

5.5 Photosynthesis and Biophotolysis

Photosynthesis has recently gained considerable attention for its potential role in the development of clean and renewable energy sources. Light energy can also be converted into H₂ chemical energy using cyanobacteria, obtaining electrons from water. For hydrogen production, either hydrogenase or nitrogenase enzymes can be used (Savakis and Hellingwerf 2015). Cyanobacteria carry out oxygenic photosynthesis, so named because oxygen is generated when light energy is converted to chemical energy. They convert light energy to chemical energy by means of two large protein complexes located in the thylakoid membranes: photosystems I (PSI) and photosystems II (PSII). They are built around a scaffold, which takes an absorbed photon of light and uses this to drive an electron across the membrane along a chain of cofactors, forming a primary reductant and a primary oxidant molecule. And from there an electron transport chain carries out the fixation of energy as ATP and NADPH. In the subsequent dark reactions, NADPH and ATP are used to convert CO₂ to carbohydrates. Central to this process, and to all other phototrophic processes, are light-absorbing pigments. When light energy is transmitted to the reaction center P700 chlorophyll pair through the photosystem I antenna; absorbing the energy, P700 releases the electrons. The term P700 implies that the chlorophyll pair absorb light most efficiently at a wavelength of 700 nm. This allows it to donate its released electron to a particular acceptor, which can probably be a peculiar chlorophyll a molecule or an iron-sulfur protein. Ultimately, ferredoxin accepts the electron and then there are two directions available for it to travel. One direction is the cyclic pathway in which the electron moves through a series of electron carriers and return to the oxidized P700. PSI comprises the primary electron donor P700 dimer of Chl a. and five electron acceptors: the primary acceptor chlorophyll a (A₀), the secondary phylloquinone molecule (A₁), the tertiary and the terminal acceptors Fe4S4 clusters F_X, F_A, and F_B, respectively. Upon excitation of P700 to its lowest excited singlet state (P700), an electron is transferred from P700 to A₀ and further to A₁ on a ₀ picosecond time scale, then further to F on a X nanosecond time scale and finally to F_B/ and or F_A with not yet well established kinetics, illustrates the linear (noncyclic) electron transfer pathway.

The pathway is termed cyclic because the electron from P700 returns to P700 after traveling through the photosynthetic electron transport chain. PMF is formed during cyclic electron transport in the region of cytochrome b6 at the inner side of the membrane and used to synthesize ATP. The electron carried by cytochrome c6 is provided by PSII by way of a pool of plastoquinones and the cytochrome b6/f complex.

This process is referred to as cyclic photophosphorylation because electrons move in a cyclic manner and ATP is formed. Cyclic photophosphorylation is only observed in photosystem I. The second direction is known as the noncyclic pathway, in which electrons can also move and it involves both photosystems. As stated above, the electrons are released from P700 and transferred to ferredoxin. In the noncyclic pathway, however, the photosynthetically produced reductant, either ferredoxin or NADPH, directly reduces hydrogenase. Thus, in this process, hydrogen production is strictly light-dependent. However, many cyanobacteria can also use nitrogenase (McKinlay and Harwood 2010). Electrons are transferred to oxidized P700 and ATP is generated in this process. Light is absorbed in shorter wavelengths (680 nm) by photosystemII and its energy is transmitted to the particular chlorophyll pair P680. Photosystem II (PSII) is the multicomponent enzyme of cyanobacteria that catalyzes the light-driven oxidation of water to molecular oxygen. In cyanobacteria, PSII is found throughout the multisubunit membrane–protein complex located in the thylakoid membranes (Vinyard et al. 2013)

Light energy is absorbed by the photosystem II antenna, leading to electron release from P680. This can then reduce pheophytin a. Pheophytin a is a type of chlorophyll a in which the central magnesium is substituted by two hydrogen atoms. Afterward, electrons move to the plastoquinone pool, reach the electron transport chain and finally get to P700. Although P700 has been reduced, P680 must also be reduced if it is to accept more light energy. Thus, H₂O can be used to donate electrons to P680 resulting in the release of oxygen. These reactions result in the conversion of light energy into biologically useful chemical energy and the evolution of molecular oxygen. Because electrons flow from water to NADP with the aid of energy from two photosystems, ATP is synthesized by noncyclic photophosphorylation (Shevela et al. 2013).

5.5.1 Direct Biophotolysis

Direct biophotolysis has only been reported in green algae and cyanobacteria. This process deals with photosynthetic reaction in which light energy is converted into chemical energy. Thus, in this process, hydrogen production is strictly light dependent. In direct biophotolysis, the photosynthetically produced reductant, either ferredoxin (Fd) or NADPH, directly reduces hydrogenase (H2ase) (Nagarajan et al. 2017) (Fig. 5.3). This enzyme is very sensitive to O₂. Identifying or designing an oxygen-tolerant hydrogenase would be the most direct route to improving hydrogen yields (Ducat et al. 2011). Thus, direct biophotolysis, although theoretically attractive as a hydrogen production process, suffers from the major limitations of oxygen sensitivity and low light conversion efficiency. The ways to overcome this problem is the use of a hydrogenase engineered to be insensitive to oxygen inactivation and use of oxygen absorbers. In addition, this method requires genetic manipulation of light antenna and optimization of light input into photobioreactor (Shaishav et al. 2013).

Fig. 5.3

Direct biophotolysis (Hallenbeck and Benemann 2002)

Direct biophotolysis in cyanobacteria: Cyanobacteria are considered as the microbial species which have the potential to produce hydrogen through direct biophotolysis. These bacteria may possess several enzymes directly involved in hydrogen metabolism: (i) nitrogenase, catalyzing the production of H₂ concomitantly with the reduction of N₂ to ammonia, (ii) uptake hydrogenase, catalyzing the consumption of H₂ produced by the nitrogenase, and (iii) bidirectional/reversible hydrogenase, which has the dual capacity. Under different atmospheric conditions, the first stage for cell growth followed by the second stage for hydrogen evolution. Nitrogen starvation is often applied at the end of the growth stage as efficient metabolic stress to induce the activity of nitrogenase. The atmosphere plays an important role in hydrogen evolution by cyanobacteria and could be a cost factor in large-scale hydrogen production. A N₂-free gas phase such as argon plus CO₂ gives a high hydrogen evolution rate (Demirbas 2009). In case nitrogen is present, for nitrogen reduction, nitrogenase preferably uses the reducing power than hydrogen evolution. The cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena PCC 7120) is a free-living filamentous cyanobacterium originally isolated from a freshwater pond in North America. It is known that this strain contains one molybdenum-nitrogenase, one uptake hydrogenase, and one bidirectional hydrogenase. A mutant strain AMC 414 (Anabaena AMC414) cannot form a functional uptake hydrogenase, i.e., it is effectively a Hup minus (a hydrogen uptake deficient) mutant (Nath and Das 2004).

5.5.2 Indirect Biophotolysis

In the indirect biophotolysis process, reduced substrates (starch in Microalgae and glycogen in cyanobacteria) accumulate during the photosynthetic O₂-production and carbon dioxide fixation stage, and these are then used in a second stage for H₂ production under anaerobic conditions with carbon dioxide evolution (Hallenbeck 2012). This process resembles the anaerobic hydrogen fermentation; however, the endogenous carbon supply is made in vivo over photosynthesis. In this process, H₂O donates the electrons or reducing equivalents to P689 by photoautotrophic cells. Figure 5.4 demonstrates the indirect biophotolysis processes including two stages: photosynthesis for carbohydrate accumulation, and dark fermentation of the carbon reserve for H₂ production (Demirbas 2009). In a typical indirect biophotolysis H₂ is produced as follows:

Fig. 5.4

Indirect biophotolysis (Hallenbeck and Benemann 2002)

$$6 {\text{H}}_{ 2} {\text{O }} + {\text{ 6CO}}_{ 2} \to \, \left( {{\text{C}}_{ 6} {\text{H}}_{ 1 2} {\text{O}}_{ 6} } \right)_{\text{n}} + {\text{ 6O}}_{ 2}$$

(5.11)

$$\left( {{\text{C}}_{ 6} {\text{H}}_{ 1 2} {\text{O}}_{ 6} } \right)_{\text{n}} + {\text{ 12H}}_{ 2} {\text{O }} \to {\text{ 12H}}_{ 2} + {\text{ 6CO}}_{ 2}$$

(5.12)

Hydrogenase and nitrogenase inhibitors are used in an attempt to screen for aerobic hydrogen evolution potential. It has been observed that these inhibitors allow for hydrogen to be released from aerobic cultures in amounts similar to those in argon. Photobiological technology is promising; however, since O₂ is produced together with the H₂, the technology must conquer the hydrogen-evolving enzyme systems’ sensitivity to O_2. To overcome this limitation, the researchers propose two solutions: screening those naturally occurring organisms which are more tolerant of oxygen as well as creating new genetic forms of the organisms that are capable of hydrogen production while oxygen is available. Moreover, a new system was developed through which a metabolic switch (sulfur deprivation) is used to cycle algal cells between a photosynthetic growth phase and a hydrogen production phase (Shaishav et al. 2013).

Indirect biophotolysis in cyanobacteria: Cyanobacteria are a large and diverse group of photoautotrophic microorganisms, which can evolve hydrogen by indirect biophotolysis. They can use either a temporal or spatial separation of photosynthesis from hydrogen evolution in order to perform indirect biophotolysis. The first step fixes CO₂ to produce cellular substances and carbohydrate stores, and the second step produces hydrogen from those stores in dark anaerobic conditions (Gouveia and Passarinho 2017). PhotosystemII utilizes the energy of sunlight in photosynthesis to extract electrons from water molecules. Electrons released upon the oxidation of water are transported to the Fe–S protein ferredoxin on the reducing side of photosystem I. The hydrogenase accepts electrons from reduced ferredoxin and donates them to two protons to generate one H₂ molecule. This process is achieved by differentiation of two cell type “vegetative” cells, which carry out normal photosynthesis and provide the nitrogen-fixing “heterocysts” with the reductant (carbohydrate) required by nitrogenase. In this two-phase process of hydrogen production, both the bidirectional [NiFe]-hydrogenases which use the reduced NAD(P)H as the substrates in hydrogen evolution and nitrogenases can be used. Since these nitrogen-fixing enzymes, nitrogenase, are localized within the heterocyst, they provide an O₂ free environment to carry out the H₂ evolution reactions (Azwar et al. 2014).

5.6 Enzyme Systems for Hydrogen Production in Cyanobacteria

Cyanobacteria may possess three enzymes directly involved in H₂ metabolism: these include an uptake hydrogenase (Hup), a reversible bidirectional hydrogenase (Hox), and nitrogenase. All of these enzymes are oxygen-sensitive (Gouveia and Passarinho 2017). The fundamental aspects of cyanobacterial hydrogenases, and their more applied potential use as future producers of renewable H₂ from sun and water, are receiving increased international attention.

5.6.1 Nitrogenase

Nitrogenase is composed of two distinct proteins (Fig. 5.5). The smaller subunit (dinitrogenase reductase, Fe-protein or protein 2) has the specific role of transferring electrons from external donors to the dinitrogenase. Dinitrogenase reductase is a homodimer, composed of a single [4Fe–4S] cluster bound between identical ~64 kDa subunits. The Fe₄S₄ cluster is redox-active, and is similar to those found in small molecular weight electron carrier proteins such as ferredoxins or flavodoxin (in cyanobacteria, a ferredoxin-type FdxH or FdxN). It is the only known redox-active agent capable of obtaining more than two oxidative states and transfers electrons to the MoFe-protein. The larger subunit (dinitrogenase, MoFe-protein or protein 1) is a protein, usually of molecular mass about 240 kDa, that binds and reduces N₂ or other substrates. The MoFe-protein is an α2β2 heterotetramer. Each unit contains two types of clusters, a P cluster and a MoFe-cofactor. The P cluster consists of a [4Fe–4S] and a [4Fe–3S] that functions as a conduit for electron transfer, from the Fe-protein (in conjunction with ATP hydrolysis) to the MoFe-cofactor. Both the [4Fe–4S] and P clusters are inactivated by O₂, the [4Fe–4S] cluster is much more susceptible and irreversibly damaged in vitro. The structural genes nifHDK encodes the Mo nitrogenase. nifH codes for the structural unit of dinitrogenase reductase, and nifD and nifK for the structural units of dinitrogenase (Bothe et al. 2011).

Fig. 5.5

Nitrogenase (Hallenbeck 2012)

Dinitrogenase catalyzes the formation ammonia from nitrogen (Torzillo and Seibert 2018). Alternative nitrogenases have been found that are homologous to the described enzyme, yet have vanadium or iron substituting for molybdenum. V-nitrogenase and the Fe-nitrogenase are encoded by the vnfHDGK and anfHDGK, respectively (Bothe et al. 2011). Of the three known closely related types of nitrogenases (the Mo-, V- and Fe-only enzymes), both the Mo- and the V-nitrogenase have been reported for cyanobacteria. The V-nitrogenase is less effective than the Mo-enzyme in catalyzing the reduction of both N₂ and C₂H₂ but consequently produces more H₂ (Hallenbeck 2012). N₂ fixation is regarded as the natural function of nitrogenase, and during this process, some H₂ evolution occurs, but in case N₂ is absent, this happens in much larger quantities. What is even worse is the fact that nitrogenases use large quantities of metabolic energy (ATP) when H₂ is produced, which leads to situations in which the energy needed to evolve H₂ is doubled, in comparison to the hydrogen produced via hydrogenases. Hence, nitrogenases do not have practicality for biohydrogen production. Providing that the ineffective nitrogenase is substituted by preferably a [Fe–Fe] hydrogenase, nitrogen-fixing bacteria can contribute to biological H₂ production (Bothe et al. 2011). Many cyanobacteria can fix N₂. The nitrogenase enzyme, itself, is extremely oxygenlabile. Cyanobacteria have a well-developed mechanism for the protection of nitrogenase from oxygen gas that can simultaneously supply both ATP and reducing power. The most successful strategy has been developed by heterocysts of filamentous cyanobacteria. Nitrogenase enzyme is localized in the heterocysts. In, filamentous heterocystous cyanobacteria, up to 10% of the cells in the filament may differentiate into heterocysts. These cells have heavy walls that limit influx of O₂ and other gases, and in differentiating from vegetative cells they lose photosystem II that generates O₂.

Vegetative cells in filamentous cyanobacteria carry out oxygenic photosynthesis. Organic compounds produced by carbon dioxide reduction are transferred into heterocysts. The heterocysts, in turn, use this photosynthate to fix N₂, and they export fixed nitrogen to the vegetative cells. Nitrogenase requires ATP and a source of reducing power to reduce. N₂ or other substrates. ATP can be provided by anoxygenic photosynthesis by Photosystem I in heterocysts (Hallenbeck 2012).

Hydrogen is produced as a byproduct of fixation of nitrogen into ammonia (Azbar 2015). The reaction consumes ATP and has the general form:

$$1 6 {\text{ATP }} + {\text{ N}}_{ 2} + {\text{ 8H}}^{ + } + {\text{ 8e}}^{\_} \mathop{\longrightarrow}\limits\limits^{\text{Nitrogenase}} 1 6 {\text{ADP }} + {\text{ 16 Pi }} + {\text{ 2NH}}_{ 3} + {\text{ H}}_{ 2}^{{}}$$

(5.13)

Cyanobacteria are stimulated in the presence of light to N₂-fixation. Reducing equivalents for the reduction of ferredoxins can be generated by several pathways. In heterocysts, in the light, ferredoxin can be reduced via photosystemI. Alternatively, either NAD(P)H and a dehydrogenase or H₂ and uptake hydrogenase can feed in electrons at the plastoquinone site (or close to it). In darkness, ferredoxin can be reduced by NAD(P)H or pyruvate (Fig. 5.6) (Bothe et al. 2010, 2011).

Fig. 5.6

Electron donation to nitrogenase in cyanobacteria (Bothe et al. 2011)

5.6.2 Hydrogenase

Several organisms which are capable of producing H₂ can also consume it. Hydrogenase enzyme plays a fundamental role in the metabolism of H₂. The following reaction is done through hydrogenase:

$$2 {\text{ H}}^{ + } + {\text{ 2 e}}^{\_} \leftrightarrow {\text{H}}_{ 2}^{{}}$$

(5.14)

Such a reaction have the capability to be reversible and its direction relies upon the redox potential of the components which can have interaction with the enzyme. Hydrogenases will play the role of H₂ uptake enzyme if an electron acceptor is present, while in conditions where an electron donor is available, H₂ will be produced by the enzyme. Considering metal content, hydrogenases can be categorized to Ni–Fe and Fe–Fe hydrogenases. The two types of enzymes differ in subunit composition, electron carrier specificity, sensitivity to O₂ inactivation (the [Fe–Fe] is commonly more sensitive) (Lee et al. 2010). Whereas Ni–Fe hydrogenases are typically coupled to NAD(P)H, with a reducing potential of approximately 320 mV, many Fe–Fe hydrogenases are partnered with the electron-carrying protein ferredoxin, which can bear electrons with significantly lower reducing potentials (Khanna and Lindblad 2015). Ni–Fe hydrogenases have typically contribution in H₂ uptake reactions, but is also able to play a role in H₂ evolution, while [Fe–Fe] hydrogenases contribute more often to H₂ evolution, and their particular H₂ evolution rates are more rapid than that of the [Ni–Fe] enzymes to the extent of over a hundred times. As a result, they are logically an appropriate alternative for biohydrogen production. Typically, the [Fe–Fe]-hydrogenases are present in strictly anaerobic bacteria, but are also found in some aerobic cyanobacteria and green algae. They include iron-sulfur centers which bind cyanide and carbon monoxide. This structure is unique for enzyme active sites. Cyanobacteria possess two functionally different types of [NiFe]-hydrogenases, an uptake and a bidirectional enzyme (Lee et al. 2010).

5.6.2.1 Uptake Hydrogenase

The uptake hydrogenase encoded by hupL and hupS is believed to be (mainly or exclusively) confined to heterocysts where it recycles the electrons lost as H₂ during the N₂-fixation process (Fig. 5.7). Filamentous cyanobacteria’s thylakoid membrane of heterocysts includes these enzymes. Membrane-bound uptake hydrogenase has the capacity to recycle hydrogen and thereby regain reductant. The uptake hydrogenase has been suggested to be particularly active in heterocysts, the site for nitrogen fixation, compared to in the vegetative cells. No uptake hydrogenase activity could be observed when cells were grown in the presence of combined nitrogen, i.e., without heterocysts.

Fig. 5.7

Hup hydrogenase (Hallenbeck 2012)

$${\text{H}}_{ 2} \to 2 {\text{H}}^{ + } + {\text{ 2e}}^{-}$$

(5.15)

Uptake hydrogenases in heterocysts have several operates, which can function simultaneously. As a result of oxy-hydrogen reaction, uptake hydrogenase can protect nitrogenase by reducing intracellular O₂ levels and also meet the energy requirement of nitrogenase by providing ATP (Hallenbeck 2012).

5.6.2.2 Bidirectional/Reversible Hydrogenase (Hox)

Bidirectional/reversible hydrogenase catalyzes both H₂-production and H₂ consumption in the presence of suitable electron donors/acceptors (Fig. 5.8). This enzyme is widely distributed among cyanobacteria, and is not linked to the presence of nitrogenase.

Fig. 5.8

Hox hydrogenase (Hallenbeck 2012)

$${\text{H}}_{ 2} \leftrightarrow 2 {\text{H}}^{ + } + {\text{ 2e}}^{-}$$

(5.16)

The cytoplasmic membrane is found to be in association with bidirectional/reversible hydrogenase. This enzyme catalyzes a physiologically reversible reaction that inter-converts protons and electrons with hydrogen gas, interacting with the redox partner NAD(P)H as the electron donor (or the oxidized forms as the electron acceptor), as shown below:

$$2 {\text{H}}^{ + } + {\text{ NAD}}\left( {\text{P}} \right){\text{H}} \leftrightarrow {\text{H}}_{ 2} + {\text{ NAD}}\left( {\text{P}} \right)^{ + }$$

(5.17)

Because the bidirectional/reversible hydrogenase is capable of hydrogen evolution in cyanobacteria without the assistance of ATP, there has been much interest in how this enzyme functions physiologically and how it can be used to generate hydrogen gas from only sunlight and water. The bidirectional/reversible hydrogenase is known as a multimeric enzyme composed of four or five various subunits which evidently rely on a variety of species. As far as the molecular structure is concerned, it is a [NiFe]-hydrogenase of the NAD(P)H which contains a hydrogenase dimmer coded via hoxYH gene. The activity of a number of auxillary proteins concertedly known as hyp (products of genes: hypF, hypC, hypD, hypE, hypA, and hypB) is required for the maturation of bidirectional/reversible hydrogenases. Bidirectional/reversible hydrogenases, contrary to uptake hydrogenase, can assist hydrogen production (Azwar et al. 2014; Hallenbeck 2012). Cyanobacteria differ greatly on the conditions required to elicit their Hox activity. Activity appears to be constitutive in some organisms, whereas in others the activity is partially or entirely dependent on a dark anaerobic adaptation period. When dark anaerobic fermentation processes take place, using protones as terminal electron acceptors, this enzyme may contribute to catalyze production of H₂. When the cells are incubated in anaerobic/microaerobic conditions, the bidirectional/reversible hydrogenase activity noticeably enhances (Azwar et al. 2014).

5.7 Role of Environmental Conditions on Hydrogen Production in Cyanobacteria

Light: Various amounts of light is required for hydrogen production in different cyanobacterial species. Hydrogen is produced by Spirulina (Arthrospira platensis) under anaerobic conditions, both with presence of light and without it, while several other species produce hydrogen merely in conditions where light is available (Aoyama et al. 1997). Hydrogen production in A. variabilis SPU 003 have occurred under in the darkness.

Temperature: The optimum temperature needed for hydrogen production varies greatly considering what the microorganism is. The optimum temperature for hydrogen production for most species of cyanobacteria is between 30 and 40 ℃.

Nitrogen source: Several inorganic nitrogenous compounds have been found to influence hydrogen production. It has been reported that NO₂⁻, NO₃⁻, and NH₄⁺ inhibit nitrogenase in Anabaena variabilis SPU003 and Anabaena cylindrical.

Molecular nitrogen: Molecular nitrogen (N₂) is considered as a competitive inhibitor in the production of hydrogen and its removal is often essential when hydrogen is intended to be produced. When N₂ is present, hydrogen production can probably be remarkably inhibited.

Carbon source: It is also found that carbon sources noticeably affect hydrogen production through influencing nitrogenase activity. When a variety of carbon sources are available, it causes electron donation capabilities by the cofactor compounds to nitrogenase to be varied and thus affecting hydrogen production.

Oxygen: Since nitrogenase and hydrogenase as two hydrogen-producing enzymes show sensitivity to oxygen, the anaerobic ambience is appropriate for them to function.

Sufur: In a number of cyanobacterial species (for example, Gloeocapsa alpicola), the rate of hydrogen production is raised by Sulfur starvation.

Methane (CH₄): Over dark anoxic incubation, hydrogen production (up to four times) is augmented by methane in Gloeocapsa and Synechocystis PCC 6803.

Salinity: Hydrogen production is certainly affected by salinity in cyanobacteria. Generally, freshwater cyanobacteria indicate that the rate of hydrogen production is reduced when salinity increases. This may be attributed to the energy distribution and the reductants responsible for the extrusion of sodium ions from the cells or the prohibition of sodium ions influx.

Micronutrients: Hydrogen production is influenced by trace elements including cobalt, copper, molyblednum, zinc, and nickel effects. Most of these metals may lead to remarkable augmentation of hydrogen production and this is presumably the consequence of their involvement in the nitrogenase enzyme.

5.8 Role of Intrinsic Factors Affecting Hydrogen Production

Metabolic potential of microorganisms: Hydrogen production occurs more efficiently by heterocystous cyanobacteria than cyanobacteria with vegetative cells. Such cyanobacteria are involved in concurrent oxygen and hydrogen production which is in conjunction with Carbon dioxide fixation.

Role of uptake hydrogenase: The action of the uptake hydrogenase results in loss of much of the hydrogen produced. Hence, it is assumed that the omission of those genes which are in charge of coding uptake hydrogenase leads to the increase of hydrogen production in the cyanobacteria species that contain uptake hydrogenase.

Presence of molecular oxygen (O₂): The molecular oxygen inhibits hydrogenase and nitrogenase activities. Nonetheless, the reduction or elimination of molecular oxygen through technical interdisciplinary solutions which are innovative and accessible can be carried out and thus increase hydrogen production (Tiwari and Pandey 2012).

5.9 Conclusion and Future Prospect

Hydrogen gas, a clean energy source with high energy yield, is considered to be a promising future fuel. Eukaryotic algae and cyanobacteria have been the primary organisms of interest for this strategy of fuel production. Both can grow much faster than plants and do not need to be grown on arable land (Dismukes et al. 2008). Hydrogen which is produced biologically is advantageous to the hydrogen produced through other conventional processes. The main processes for biological hydrogen production are direct biophotolysis, indirect biophotolysis, photofermentation, and dark fermentation. Cyanobacteria and microalgae are the only organisms known so far that are capable of both oxygenic photosynthesis and hydrogen production. As genetic modification can be performed easily via molecular techniques on both unicellular and heterocystous forms of cyanobacteria and they do not have complex nutritional requirements, cyanobacteria are regarded as one of the ideal candidates for photobiological H₂ production.

They can grow using air, water, and mineral salts, with light as their only source of energy. The simplest and most effective process would be to provide a direct transfer of electrons from water to hydrogen-evolving enzyme which results in simultaneous evolution of oxygen and hydrogen (so-called direct biophotolysis). Indirect biophotolysis processes are the paths followed by cyanobacteria. In this system, photosynthesis (O₂ evolution and CO₂-fixation) and N₂-fixation (thus H₂ production) are either spatially or temporally separated from each other. When analyzing the hydrogen metabolism in nitrogen-fixing cyanobacteria in detail, three enzymes should be considered, nitrogenase, evolving hydrogen during nitrogen fixation, an uptake hydrogenase, recycling the hydrogen produced by nitrogenase, and a bidirectional/reversible hydrogenase that catalyzes both hydrogen production and consumption (Hallenbeck 2012). Previous studies denoted that hydrogen production by cyanobacteria can be an effective procedure providing that a range of beneficial uses of the produced hydrogen are recommended. Numerous applications exist, for example, food and chemical industries, in which the process of biological hydrogen production by cyanobacteria can be well exploited. The cyanobacteria that produce biohydrogen only need to be purified to be used in the industry or in fuel cells. Rigorous hydrogen production is needed for such processes. Cyanobacterial hydrogen production is more economical than the traditional large-scale hydrogen production. Directing the cyanobacterial hydrogen produced in a photobioreactor is easily done to separate compartments which contain the substrate for hydrogen production and particular catalysts (Savakis and Hellingwerf 2015; Sharma et al. 2011).