Cyanobacterial hydrogen production – A step towards clean environment

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Review

Cyanobacterial hydrogen production e A step towards clean environment Archana Tiwari 1, Anjana Pandey* Nanotechnology and Molecular Biology Laboratory, Centre of Biotechnology, University of Allahabad, Allahabad 211002, U.P.,India

article info

abstract

Article history:

Environmental pollution and exhaustive depletion of non-renewable energy sources

Received 9 August 2011

demand the exploration of alternate energy sources. Hydrogen has been crowned as future

Accepted 23 September 2011

fuel by virtue of its immense potential. Many microorganisms mediate hydrogen produc-

Available online 22 October 2011

tion. Cyanobacteria are excellent biological means of hydrogen production. This review highlights the significant progress achieved in cyanobacterial hydrogen production

Keywords:

methods. The role of key enzymes catalyzing hydrogen production and the various

Hydrogen production

parameters influencing the path of increased hydrogen productivity has been discussed.

Cyanobacteria Molecular hydrogen

Recent approaches towards enhanced hydrogen production like genetic engineering, alteration in nutrient and growth conditions, entrapment in reverse micelles, combined culture and metabolic engineering have been emphasized. Improvisation in hydrogen production methods mediated by microbes will pave the path for commercialization of molecular hydrogen as environmental friendly energy source. Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Exploration of alternate energy sources is the need of today, considering the environmental pollution and exhaustive depletion of non-renewable energy sources. Hydrogen, the most abundant element in the universe has the potential to serve the purpose of fuel and is ecofriendly. Hydrogen can serve as an excellent fuel because it is renewable, does not pollute the environment by evolving carbon dioxide, liberates large amounts of energy per unit weight in combustion (122 kJ/ g) and is easily converted to electricity by fuel cells [1,2]. Numerous virtues of hydrogen have crowned it as future fuel. The photoconversion of water to hydrogen is an excellent solution. Three ways to achieve photoconversion of water to hydrogen are:

(a) The use of photochemical fuel cells, (b) By applying photovoltaics, or (c) By photosynthetic microorganisms [3e5]. The production of hydrogen is ubiquitous, natural phenomenon under anoxic or anaerobic conditions. The capacity of certain microorganisms to metabolize molecular hydrogen was discovered at the end of the 19th century [6] and later identified to be catalyzed by a hydrogenase [7]. Microorganisms are capable of producing hydrogen via either fermentation [8,9] or photosynthesis [10e12]. The biological species involved in hydrogen production are green algae, heterocystous and non-heterocystous cyanobacteria, photosynthetic bacteria and fermentative bacteria [13]. Hydrogen production by biological means is advantageous since the

* Corresponding author. Tel.: þ91 532 2271977. E-mail addresses: [email protected] (A. Tiwari), [email protected] (A. Pandey). 1 Present address: Guru Nanak Girls College, Ludhiana, Punjab, India. 0360-3199/$ e see front matter Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.09.100

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energy requirement and investment cost is low. Biological hydrogen production involves fermentative hydrogen production by anaerobic bacteria and photobiological hydrogen production by photosynthetic bacteria, cyanobacteria and green algae. This review highlights the advancements achieved in enhancing cyanobacteria mediated hydrogen production and the various strategic approaches. Biological Hydrogen Production

Photodecomposition of organic compounds Photosynthetic Bacteria Fermentative Hydrogen Production Fermentative Bacteria Biophotolysis of Water Algae & Cyanobacteria Hybrid Systems Photosynthetic Bacteria &Fermentative Bacteria

1.1.

Hydrogen production by cyanobacteria

Cyanobacteria are unique prokaryotes with diverse range of properties. They are potential microbial species for hydrogen production via direct and indirect photolysis [14]. They are ideal microbes for photobiological H2 production, since they require the simplest nutritional conditions. For example, even the nitrogen and carbon dioxide available in open air is sufficient for their growth while they can use water as reductant and a source of electrons. Moreover, simple mineral salts present in natural water along with sunlight can act as source of energy for them. Hydrogen production has been studied in a very wide variety of cyanobacterial species and strains. Hydrogen production occurs within at least 14 genera of cyanobacteria, under a vast range of culture conditions [14]. In addition to the advantages of cyanobacterial hydrogen production there are some obstacles namely inhibition of enzymes directly involved in hydrogen production by oxygen, H2 consumption by an uptake hydrogenase, and an overall low productivity

[15]. Cyanobacteria have some unique strategies to overcome some obstacles in the path of hydrogen production. Cyanobacterial species capable of hydrogen production are categorized into three groups e heterocystous, nonheterocystous and marine (Table 1). Heterocystous cyanobacteria are specialized for nitrogen fixation by possessing unique structures called heterocysts. The heterocyst provides a microanaerobic environment suitable for the functioning of nitrogenase since it lacks photosystem II activity, it has a higher rate of respiratory oxygen consumption, and it is surrounded by a thick envelope that limits the diffusion of oxygen through the cell wall [16,17]. In non-Heterocystous cyanobacteria temporal separation between photosynthetic oxygen evolution and nitrogen fixation seems to be the most common strategy adopted by non-heterocystous cyanobacteria. Cyanobacteria have unique feature of oxygenic photosynthesis. Oxygenic photosynthetic cyanobacteria normally absorb sunlight and store the energy in the form of polysaccharide glycogen and these storage biomolecules are mobilized, as required, to produce the energy needed to drive microbial metabolism. The photosynthetic apparatus is represented by isolated and freely lying photosynthetic lamellae. Chlorophyll a and several accessory pigments (phycoerythrin and phycocyanin) are embedded in these photosynthetic lamellae. The major photosynthetic pigment in cyanobacteria is Chlorophyll a. They use water as electron donor and oxygen is evolved as by-product. Carbon dioxide is reduced to carbohydrate through Calvin cycle. However, under certain conditions, electrons on the reducing side of PSI can be diverted at the level of ferredoxin (Fd) to another pathway (Fig. 1), employing an [FeFe]-hydrogenase (H2ase) to evolve H2 gas. During photosynthetic H2 production, two protons and two electrons are recombined in a reaction catalyzed by the hydrogenase enzyme to yield one H2 molecule. The rate of hydrogen production varies from species to species within cyanobacterial strains (Table 2).

2. Role of enzymes in cyanobacterial hydrogen production In cyanobacteria, hydrogen production is accomplished by the catalytic action of enzymes (Fig. 2). Cyanobacteria use two distinct enzymes to generate hydrogen gas:

Table 1 e Hydrogen producing cyanobacteria. Heterocystous cyanobacteria

Marine cyanobacteria

Non-Heterocystous cyanobacteria

Anabaena flos-aquae Anabaena cylindrica Anabaena variabilis Anabaena azollae Anabaena sp. PCC 7120 Nostoc muscorum Nostoc linckia Nostoc commune Anabaenopsis circularis

Oscillatoria brevis Oscillatoria limosa Oscillatoria sp. Miami BG7 Calothrix scopulorum Calothrix membranacea Cyanothece 7822 Anabaena cylindrica B-629

Synechococcus sp. Microcystis sp. Gloebacter sp. Synechocystis sp. Aphanocapsa montana Gloeocapsa alpicola CALU 743 Chroococcidiopsis thermalis CALU 758 Mycrocystis PCC 7806 Microcoleus chthonoplasts

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Fig. 1 e Abbreviated Z-scheme for the light reactions of oxygenic photosynthesis showing electron transport pathways to carbon fixation and hydrogen production.

I) Nitrogenase: This enzyme is found in the heterocysts of filamentous cyanobacteria when they grow under nitrogen limiting conditions, catalyzing the production of hydrogen concomitantly with the reduction of N2 to NH3. II) Hydrogenase a) an uptake hydrogenase, catalyzing the consumption of hydrogen produced by the nitrogenase, and b) a bidirectional hydrogenase, which has the capacity to both take up and produce hydrogen. In N2-fixing strains, the net H2 production is the result of H2 evolution by nitrogenase and H2 consumption mainly catalyzed by the uptake hydrogenase.

(Fe Protein, encoded by nifH ). Dinitrogenase is a a2b2 heterotetramer, having molecular weight of about 220e240 kDa respectively, breaks apart the atoms of nitrogen. Dinitrogenase reductase is a homodimer of about 60e70 kDa and plays the specific role of mediating the transfer of electrons from the external electron donor (a ferredoxin or a flavodoxin) to the dinitrogenase [35e37]. Depending on the metal content, there are three types of dinitrogenase found in Nitrogenase enzyme. i) Type I e contains molybdenum (Mo) ii) Type II e contains vanadium (V) [38e40] iii) Type III e contains iron (Fe) [41,42].

2.2. 2.1.

Hydrogenases

Nitrogenase

It is responsible for nitrogen fixation [34] and is distributed mainly among prokaryotes, including cyanobacteria. Molecular nitrogen is reduced to ammonium with consumption of reducing power and ATP [15]. 16ATP þ 16H2 O þ N2 þ 10Hþ þ 8e /16ADP þ 16Pi þ 2NHþ 4 þ H2 The reaction is substantially irreversible and produces ammonia. Hydrogen production catalyzed by nitrogenase occurs as a side reaction at a rate of one-third to one-fourth that of nitrogen fixation, even in a 100% nitrogen gas atmosphere. Nitrogenase itself is extremely oxygen-labile. However, cyanobacteria have developed mechanisms for protecting nitrogenase from oxygen gas and supplying it with energy (ATP) and reducing power (Fig. 3). The most successful mechanism is the localization of nitrogenase in the heterocysts of filamentous cyanobacteria. Hydrogen is produced as a by-product of fixation of nitrogen into ammonia. A Nitrogenase enzyme consists of two parts: one is dinitrogenase (MoFe Protein, encoded by the genes nifD and nifK, a and b respectively) and the other is dinitrogenase reductase

They occur as two distinct types in different cyanobacterial species. One type of them, uptake hydrogenase (encoded by hupSL) [4], has the ability to oxidize hydrogen (Fig. 4) and the other type of hydrogenase is reversible or bidirectional hydrogenase (encoded by hoxFUYH ) and it can either take up or produce hydrogen. Uptake hydrogenase enzymes are found in the thylakoid membrane of heterocysts from filamentous cyanobacteria, where it transfers the electrons from hydrogen for the reduction of oxygen via the respiratory chain in a reaction known as oxyhydrogenation or Knallgas reaction. The enzyme consists of two subunits. The hupL-coded protein is responsible for the uptake of hydrogen and the smaller subunit that is coded by hupS looks after the reduction affair. The hydrogen formed is usually re-oxidized by an uptake hydrogenase via a Knallgas reaction and hence there is no net H2 production in strains with uptake hydrogenases under ambient conditions. Therefore, it is counterproductive when the goal is to produce hydrogen on a commercial scale. The reaction catalyzed by the uptake hydrogenase takes the following form:

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Table 2 e Rates of hydrogen production in cyanobacteria. Cyanobacterial strain Anabaena sp. PCC 7120 Anabaena cylindrica IAMM-1 Anabaena cylindrica IAMM-58 Anabaena cylindrica UTEX B-629 Anabaena flos-aquae UTEX 1444 Anabaena flos-aquae UTEX LB 2558 Anabaenopsis circularis IAM M-13 Nostoc muscorum IAM M-14 Nostoc linckia IAM M-30 Nostoc commune IAM M-13 Anabaena variabilis AVM13 Anabaena variabilis PK84 Anabaena variabilis PK84 Anabaena variabilis PK84 Anabaena variabilis ATCC 29413 Anabaena variabilis ATCC 29413 Anabaena variabilis ATCC 29413 Anabaena variabilis 1403/4B Anabaena azollae Anabaena variabilis PK17R Anabaena variabilis SPU 003 Synechococcus PCC 6830 Synechococcus PCC 602 Synechococcus PCC 6307 Synechoccus PCC 6301 Microcystis PCC 7820 Gloebacter PCC 7421 Synechocystis PCC 6308 Synechocystis PCC 6714 Gloeocapsa alpicola CALU 743 Chroococcidiopsis thermalis CALU 758 Mycrocystis PCC 7806 Microcoleus chthonoplasts Anabaena cylindrica B-629 Oscillatoria brevis B-1567 Calothrix scopulorum 1410/5 Calothrix membrnacea B-379 Oscillatoria sp. Miami BG7 Oscillatoria limosa Cyanothece 7822

Maximum H2 evolution

Reference

2.6 mmol/mg chl a/h 2.1 mmol/mg chl a/h 4.2 mmol/mg chl a/h 0.91 mmol/mg chl a/h

[18] [18] [18] [18]

1.7 mmol/mg chl a/h

[18]

3.2 mmol/mg chl a/h

[18]

0.31 mmol/mg chl a/h

[18]

0.60 mmol/mg chl a/h 0.17 mmol/mg chl a/h 0.25 mmol/mg chl a/h 68 mmol/mg chl a/h 32.3 mmol/mg chl a/h 167.6 mmol/mg chl a/h 0.11 mmol/mg chl a/h 45.16 mmol/mg chl a/h

[18] [18] [18] [19] [20] [21] [22] [21]

0.05 mmol/mg dry wt/h

[23]

39.4 mmol/mg chl a/h

[20]

20 mmol/mg chl a/h 38.5 mmol/mg chl a/h 59.18 mmol/mg chl a/h 5.58 nmol/mg dry wt/h 0.26 mmol/mg chl a/h 0.66 mmol/mg chl a/h 0.02 mmol/mg chl a/h 0.09 mmol/mg chl a/h 0.16 mmol/mg chl a/h 1.38 mmol/mg chl a/h 0.13 mmol/mg chl a/h 0.40 mmol/mg chl a/h 0.58 mmol/mg protein

[24] [21] [21] [25] [26] [26] [26] [25] [25] [26] [26] [26] [27]

0.7 mmol/mg chl a/h

[28]

11.3 nmol/mg prot/h 1.7 nmol/mg prot/h 0.103 mmol/mg dry wt/h 0.168 mmol/mg dry wt/h 0.128 mmol/mg dry wt/h 0.108 mmol/mg dry wt/h 0.250 mmol/mg dry wt/h 0.83 mmol/mg chl a/h 0.92 mmol/mg chl a/h

[29] [25] [30] [30] [30] [30] [31] [32] [33]

hoxYH gene. Maturation of reversible hydrogenases requires the action of several auxillary proteins collectively termed as hyp (products of genes: hypF, hypC, hypD, hypE, hypA, and hypB) [45]. Unlike uptake hydrogenase, reversible hydrogenases are helpful in hydrogen production. While the uptake hydrogenase is present in all nitrogen-fixing strains tested so far, the bidirectional enzyme is distributed among both nitrogenfixing and non-nitrogen-fixing cyanobacteria (although it is not a universal cyanobacterial enzyme). Individual strains may harbour one or both hydrogenases and none to several nitrogenases. According to the metal composition of the active site, hydrogenases are classified into three major groups [46]. I) NieFe hydrogenases, II) Fe hydrogenases and III) Metal-free hydrogenases. The presence and physiological roles of hydrogenases in nitrogen-fixing cyanobacteria remains controversial, but ’uptake’ hydrogenase appears to consume and re-use hydrogen gas, resulting in a decrease in net hydrogen production (Fig. 2). Asada & Kawamura [47] reported aerobic hydrogen production by a nitrogen-fixing Anabaena sp., believed to be an uptake hydrogenase-deficient strain. An uptake hydrogenase, with the evident function of catalyzing the consumption of the hydrogen produced by nitrogenase, has been found in all nitrogen-fixing cyanobacteria examined so far [30,48,49]. Consequently, the production/selection of mutants deficient in H2 uptake activity is necessary. Moreover, the nitrogenase has a high ATP requirement and this lowers considerably the potential energy conversion efficiency. On the other hand, the bidirectional hydrogenase requires much less metabolic energy, but is extremely sensitive to oxygen. An efficient photoconversion of water to H2 by cyanobacteria is certainly influenced by many other factors, and only an extensive knowledge within this field can lead to the improvement of the H2 production rates.

3. Factors affecting hydrogen production in cyanobacteria Many factors play a significant role in hydrogen production by cyanobacteria. These factors may be categorized as intrinsic or environmental.

References

H2 /2Hþ þ 2e The biological role of bidirectional or reversible hydrogenase is poorly understood and thought to control ion levels in the organism. Reversible hydrogenase is associated with the cytoplasmic membrane and likely functions as an electron acceptor from both NADH and H2 [43]. The reversible hydrogenase is a multimeric enzyme consisting of either four or five different subunits apparently depending on the species [43,44]. Molecularly it is a [NieFe]-hydrogenase of the NAD(P)þ reducing type consisting of a hydrogenase dimer coded by

I. Role of Intrinsic factors on Hydrogen Production Metabolic potential of microorganisms: On the basis of type of Cyanobacterial species selected for production, the efficiency of hydrogen production varies. The acumen of microbes to convert light energy to chemical energy of hydrogen by photosynthetic microorganisms is reported to be low.

[50]

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(continued)

(continued) References

Role of uptake hydrogenase: The production of hydrogen is affected by the consumption of hydrogen produced by uptake hydrogenase. Deletion of this gene by mutation is reported to enhance production. Presence of molecular oxygen: Molecular oxygen is inhibitor for the activity of enzymes hydrogenase and nitrogense. This problem can be encountered by the aid of biotechnological tools. Under aerobic outdoor conditions operated for 4 months, a maximum rate of 80 ml of hydrogen per reactor (4.35 L) per hour was obtained from a batch culture on a bright day. II. Role of environmental factors on hydrogen production Light: There is variation in light requirement by cyanobacterial species. Some cyanobacteria produce hydrogen in presence of light. While some produce in both light and dark condition. Nitrogen-fixing cells of A. variabilis SPU 003 have the capacity to produce hydrogen mainly in darkness. Temperature: Variation in optimum temperature requirement exists, depending upon the species of cyanobacteria. The optimum range starts from 30  C and ranges to 40  C. Nitrogen source: A number of nitrogenous compounds affect hydrogen production. Nitrate, nitrite and ammonium inhibit nitrogenase activity in Anabaena variabilis SPU 003 and A. cylindrical. In general, the nitrogenase activity is inhibited by nitrogenase sources. Molecular Nitrogen: The production of hydrogen by cyanobacteria is inhibited by presence of molecular nitrogen because molecular nitrogen is competitive inhibitor for hydrogen production. Carbon Source: The activity of enzyme nitrogenase is affected by carbon source. In presence of simple organic compounds increase hydrogen production as electron donation by cofactor compounds enhance nitrogenase activity. Addition of various sugars stimulated the production of hydrogen, with mannose giving the highest rate, 5.58 nmol of hydrogen produced per mg dry weight per hour. Oxygen: Hydrogen producing enzymesnitrogenase and hydrogenase are very sensitive to oxygen, so these enzymes function in anaerobic ambience. Sulphur: The starvation of sulphur enhances hydrogen production as reported in Gloeocapsa alpicola.

[28,51]

[16,21,49]

[52e54]

[55,56]

[55,57e59]

[30]

[54,55,60]

[16]

[27]

References Methane: Presence of methane enhances hydrogen production in Gloeocapsa & Synechocystis PCC 6803 during dark anoxic condition. Salinity: Increasing salinity lowers hydrogen production because of diversion of energy reductants for extrusion of sodium ions from within the cells or prevention of sodium ions influx. The production of hydrogen in marine cyanobacteria is unaffected by salinity to some extent. Micronutrients: Many trace elements like Cu, Co, Mo, Zn and Ni show pronounced enhancement in hydrogen production due to their involvement in the enzyme nitrogenase. Nickel adaptation permits normal biomass accumulation while significantly increasing the rate of fermentative hydrogen production in Arthrospira maxima. Reports show that the hydrogenase activity in cell extracts (in vitro) and whole cells (in vivo) correlates with the amount of Ni2þ in the growth medium (saturating activity at 1:5 mM Ni2þ). This and higher levels of nickel in the medium during photoautotrophic growth cause stress leading to chlorophyll degradation and a retarded growth rate that is severe at ambient solar flux. Relative to nickelfree media (only extraneous Ni2þ), the average hydrogenase activity in cell extracts (in vitro) increases by 18-fold, while the average rate of intracellular H2 production within intact cells increases 6-fold. Nickel is inferred to be a limiting cofactor for hydrogenase activity in many cyanobacteria grown using photoautotrophic conditions, particularly those lacking a highaffinity Ni2þ transport system.

[27]

[61]

[55,62,63]

4. Photobioreactors used for cyanobacterial hydrogen production Large-scale production of hydrogen mediated by cyanobacteria requires bioreactors. The bioreactors meant for algal growth are transparent as light is an essential factor, hence termed photobioreactors. The optimal design of algal photobioreactor depends upon the characteristics of the cyanobacterial strain. Light is the most vital parameter and in addition the specialised nutritive needs of the strain have to be taken into consideration. Inside the photobioreactor there is a photic zone, close to the illuminated surface, and a dark zone. The dark zone is due to absorption of light by the algae, and is dependent on the light conditions and the absorption properties of the algae. The position of the light source as well as gas liquid hydrodynamics also affects cyanobacterial growth as well as hydrogen production [64]. Several

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Fig. 2 e Role of nitrogenase and hydrogenase in cyanobacterial hydrogen production [4].

flexibility in volume to surface area ratio and flexibility in shifting the place receiving light [66e71]. Flat panel Photobioreactor consists of a stainless-steel frame and three polycarbonate panels [58]. The reactor comprises of two compartments placed side by side. The front compartment contains the bacterial culture. Water is circulated via a temperature controlled water bath through the hind compartment in order to maintain the desired temperature of the culture. Vertical PBR consists of a transparent column usually made up of high quality glass and surrounded by a water jacket that while allowing maintenance of the temperature with circulating water allows adequate entry of light [72]. Fig. 3 e Nitrogenase mediated hydrogen production in heterocystous cyanobacteria.

5. Recent approaches towards enhancement of hydrogen production yield conditions affect algae growth, including the light source, and hydrodynamics. Some photobioreactors circulate the liquid or aerate the algae with air or an inert gas. Photobioreactors for hydrogen production must fulfil several requirements. The photobioreactors must be a closed system to prevent hydrogen escape and facilitate collection of gas, the design must allow for easy sterilization and it should provide high surface area to volume ratio [65].

5.1. Exploration of growth conditions & nutrient media to increase production Growth conditions and media can enhance the productivity of hydrogen. Many reports have highlighted the importance of systematically exploring nutritional requirements in addition to other parameters. Unicellular non-diazotrophic Cyanobacteria

Photobioreactors used for hydrogen production in cyanobacteria

Tubular PBR Flat Panel PBR Vertical Column PBR

Tubular PBRs consists of long transparent tubes with diameters ranging from 3 to 6 cm, and lengths ranging from 10 to 100 m [66]. The culture liquid is pumped through these tubes by means of mechanical or airlift pumps. There is

Fig. 4 e Hydrogenase-mediated hydrogen production.

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Gloeocapsa alpicola under sulphur starvation shows increased hydrogen production [27]. Arthrospira (Spirulina platensis) is reported to produce hydrogen (1 mmole H2/12 h/mg cell dry weight) in complete anaerobic and dark condition [53]. Another nitrogen-fixing cyanobacterium, Anabaena cylindrica, produces hydrogen and oxygen gas simultaneously in an argon atmosphere for 30 days in light limited condition [73]. Symbiotic Cyanobacteria within coralloid roots of the cycads Cycas revoluta (king Sago palm) and Zamia furfuracea showed a significant in vivo hydrogen uptake [74]. Anabaena sp. is able to produce significant amount of hydrogen. Among them nitrogen-starved cells of A. cylindrica produces highest amount of hydrogen (30 ml of H2/lit culture/hour). Hydrogenase-deficient cyanobacteria Nostoc punctiforme NHM5 when incubated under high light for a long time, until the culture was depleted of carbon dioxide shows increase in hydrogen production [29]. In heterocystous cyanobacteria Anabaena variabilis ATCC 29413, the effect of growth media was explored, and it was found that hydrogen production could be increased 5.5-fold with proper nutritional requirements [75]. In addition, supplementation of media with mannose has been shown to increase hydrogen production in A. variabilis SPU 003 by about 78% [54]. In conjunction, the study also showed the importance of salinity and micronutrients on hydrogen production. Reports show that when incubated anaerobically, in the light, in the presence of C2H2 and high concentrations of H2, both Mo-grown A. variabilis and either Mo- or V-grown Anabaena azotica produce large amounts of H2 in addition to the H2 initially added [76]. The additional H2 production mainly originates from nitrogenase with the V-enzyme being more effective than the Mo-protein. This enhanced H2 production in the presence of added H2 and C2H2 should be of interest in approaches to commercially exploit solar energy conversion by cyanobacterial photosynthesis for the generation of molecular hydrogen as a clean energy source.

5.2. Combined cultures: a good solution to create natural conditions in flasks Filamentous cyanobacteria like Nostoc, Anabaena and many other types of cyanobacteria can produce nonphotosynthetic cells called heterocysts where nitrogen fixation takes place. Under natural conditions, cyanobacteria are found associated with many other type of organisms. In such systems, the depletion of oxygen by heterotrophic bacteria and oxygenconsuming reactions of cyanobacteria allow nitrogen fixation by the enzyme nitrogenase [77,78]. Improvement in the potential of H2 production by cyanobacteria coupled with facultative anaerobic bacteria within appropriate reverse micelles has also been reported. The system mimicked the natural conditions by allowing heterotrophic bacteria R. palustris P4 to grow in the vicinity of uptake hydrogenase-negative cyanobacteria (Nostoc and Anabaena) within reverse micelles to enhance both the rate and duration of H2 production [51].

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found to show significant increase in their hydrogen production capability. This is due to the unique properties found within reverse micelles, which include compartmentalization, transparency and their ability to create anaerobic environment. These conditions are highly favourable especially for nitrogenase mediated H2 production [79,80]. Reverse micelles consist of three components: amphiphilic surfactant molecules, water and a non-polar organic phase. The polar heads of the surfactant molecules are directed towards the interior of a water-containing sphere, while the aliphatic tails are oriented in direction of the non-polar organic phase (Fig. 5). The water structure within the reverse micelles may resemble that of water adjacent to biological membranes and it has been suggested that the reverse micellar system reliably mimics the microenvironment that enzymes encounter in the intracellular milieu [81]. One of the most important factors determining the behaviour of enzymes in reverse micelles is the ratio of concentrations of water and the surfactant (wo ¼ [H2O]/[surfactant]) which is directly related to the size of the reverse micelles [82]. The most commonly used surfactants are the single chained anionic sodium dodecyl sulphate (SDS), the cationic cetyl trimethyl ammonium chloride (CTAC) or bromide (CTAB), and the double-chained, anionic N-ethyl hexyl sodium sulfosuccinate (AOT). Higher rates of H2 generation have been reported when entrapment of microorganisms is done within reverse micelles [79,80,83]. Due to dispersion and compartmentalization of each cell within hydrophilic core of reverse micellae the efficiency of biohydrogen production of this system increases various fold in comparison to open systems. The other added advantages of reverse micelles include their optical transparency (providing each cell a proper exposure to light), thermostablity and ability to provide complete anaerobic environment to a nitrogenase active enzyme for producing hydrogen. Results show that in comparison to the unmutated Nostoc with R. palustris (within AOT/isooctane) the coupled system of mutated Nostoc and R. palustris produced H2 by 3.9-fold higher rate, which is 8.6 mmol H2/h/mg protein. Whereas, mutated Anabaena coupled with R. palustris produced 4.8 times higher hydrogen production within (AOT)/isooctane reverse micelles in comparison to the unmutated Anabaena with R. palustris [51].

5.4. Genetic engineering as a tool to increase hydrogen production There exists a variety of genetic tools to metabolically engineer cyanobacteria [85]. Cyanobacteria can achieve spatial

5.3. Reverse micelles: a miniature microenvironment to boost enzyme action Microorganisms (i.e. photosynthetic and heterotrophic bacteria) when entrapped within reverse micelles have been

Fig. 5 e Schematic representation of reverse micelle [84].

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separation of photosynthesis and hydrogen production by the use of specialized cells called heterocysts, which harbour the enzyme nitrogenases requiring ATP to function and can produce hydrogen even under high H2 partial pressures [86]. The heterocysts have reduced O2 pressure and can evolve hydrogen through the use of nitrogenases even while photosynthesis is occurring inside other vegetative cells [4,49]. However, heterocyst formation is only achieved by 5e10% of the vegetative cells [86]. Therefore, an increase in heterocyst formation could result in significantly more hydrogen formation [86]. The incorporation of a heterologous [FeFe]hydrogenase into the heterocysts of cyanobacteria could also potentially provide a way to increase hydrogen production in this organism [87,88]. Determination of the maturation genes in cyanobacteria that are involved in expressing an active heterologous [FeFe]-hydrogenase needs to be explored. In photosynthetic bacteria and cyanobacteria, photohydrogen production is mainly associated with nitrogenase and coupled with ferredoxin or flavodoxin rather than hydrogenase [89]. Therefore, hydrogenase-negative gene has been found to be useful for the H2 generation [90]. The H2 production was investigated using hydrogenase mutants from Anabaena sp. PCC 7120 namely DhupL (deficient in uptake hydrogenase), DhoxH (deficient in bidirectional hydrogenase), and DhupL/DhoxH (deficient in both genes). The results showed that the DhupL and DhupL/DhoxH produced H2 at a rate 4e7 times than that of wild type under optimal conditions [91]. The detailed analysis of Dhup showed that H2producing activity was moderately improved in older cultures when 1% CO2 was added to the bubbling air. An expression vector has been constructed to express a Feonly hydrogenase specifically in heterocysts. This vector was conjugated (bacterial mating) into the cyanobacterium Anabaena. Hydrogen production was examined with a batch culture assay of the transconjugants. Preliminary analysis suggests an increase in hydrogen production [92]. The oxygen-tolerant [NieFe]-hydrogenase genes, hydS and hydL from Thiocapsa roseopersicina have been expressed in the cyanobacteria Synechococcus PCC7942 [93]. Improvement of cyanobacterial hydrogen production can be achieved by expressing a clostridial [FeFe]-hydrogenase in the cyanobacteria Synechococcus elongates [88,94]. But the mechanism by which cyanobacteria are able to express an active [FeFe]hydrogenase, without the co-expression of the appropriate maturation proteins, is still unknown [88,95]. The uptake of hydrogen by the [NieFe]-hydrogenase in cyanobacteria can also be eliminated to increase hydrogen yield [91,96]. Knowledge about the biosynthesis/maturation of the cyanobacterial hydrogenases needs to be gained. Several genes presumably involved in this process, and affecting hydrogenases pleiotropically (hypFCDEAB), have been identified clustered or scattered throughout the genomes of several cyanobacterial strains [4,98e100]. The presence of a single copy of the hyp operon in the genome of cyanobacteria possessing a bidirectional and an uptake enzyme suggests that they might be responsible for the maturation of both hydrogenases [4]. Recently, the genes encoding the putative Cyanobacterial specific C-terminal endopeptidases (hupW and hoxW ) were also identified by analyzing the available genome sequences [45]. In the heterocystous strains Nostoc sp. PCC

7120 and N. punctiforme ATCC 29133/PCC 73102, hupW (encoding the endopeptidase specific for the uptake hydrogenase) is not part of any known hydrogenase cluster. In contrast, in the non-heterocystous Gloeothece sp. ATCC 27152 and Trichodesmium erythraeum IMS101, hupW is located downstream of hupL, and was shown to be co-transcribed with hupSL in Gloeothece sp. ATCC 27152 [101]. The characterization of the hyp gene cluster and hupW in the marine N2-fixing Lyngbya majuscula CCAP 1446/4 has been done (Fig. 6). L. majuscula is a non-heterocystous cyanobacterial strain that contains both the uptake and the bidirectional hydrogenases has been reported [102]. The hydrogenase(s) accessory genes characterized are located in the vicinity of the uptake hydrogenase structural genes, and are co-transcribed under N2-fixing conditions.

5.5.

Metabolic pathway engineering strategies

Metabolic engineering is the redirection of metabolic pathways for enhanced production of existing natural products, production of unnatural products, or degradation of unwanted molecules (e.g. environmental contaminants). Metabolic engineering joins systematic and quantitative analysis of pathways using molecular biology, modern analytical techniques, and genomic approaches [96]. After the advent of this new field of metabolic engineering, significant advancements have been achieved in varied areas [103e107]. In metabolic engineering, the most commonly used method is the knockout of particular chromosomal genes [108e110]. Mutations were induced earlier by physical methods like by ultraviolet radiation or by chemical methods and later on screened [111]. However, this approach does not assure generation of useful mutation each time and consumes time. Fortunately, with the advent of modern molecular microbiology and the availability of entire genome sequences, this method has been largely replaced by the directed disruption of chromosomal genes [110,112]. Significant progress in computational methods and molecular biology techniques are necessary for the effective implementation of metabolic engineering [85,106,112e114]. The computational frameworks that can be used to find the optimum modulation and deletion strategies for overproduction of fuels and chemicals [115e117] are: OptKnock, OptStrain, and OptReg Studies on the effects of nutrient limitation and substrate utilization have revealed new regulatory mechanisms and

Fig. 6 e Physical map of the Lyngbya majuscula CCAP 1446/4 genome region containing the hyp and hup genes [97].

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alternative metabolic pathways in hydrogen producing organisms [118,119]. A combination of systematic experimental design, gene knockouts, nutritional studies, heterologous expression, and artificial environments will be needed in order to achieve biohydrogen production levels that can compete with existing non-renewable production methods. Complex regulatory networks that exist in hydrogen producing systems need to be explored by more studies addressing the effects of nutrients and micronutrients on hydrogen production. Engineered microorganisms will be optimized to produce high-yields of hydrogen and thus an alternate source of energy in form of hydrogen will save planet earth from harmful effects of non-renewable fuels and help conserving these fuels.

147

cyanobacterium Cyanothece has been developed as a model organism for photobiological hydrogen production. Deep understanding of its metabolism specifically, using genome sequencing, transcriptomics, proteomics, metabolomics, mutagenesis, biochemical analysis and physiological approaches, has been employed. Cyanothece sp. ATCC 51142 exhibited exceptionally high rates of hydrogen production under aerobic conditions. Hydrogen production rates were significantly enhanced by growing Cyanothece cells in the presence of glycerol or high concentrations of CO2. This strain was capable of producing hydrogen at the rate of over 400 mmoles H2/mg Chl/hr–the highest rate yet reported in cyanobacteria. The high rate of hydrogen production in this strain is largely mediated by a very active nitrogenase [122].

5.6. Nanotechnology: nanolipoprotein particles for hydrogen production Nanolipoprotein particles (NLP) have been developed to solubilize and isolate membrane bound hydrogenases; these constructs are less sensitive to oxygen (www.llnsllc.com). Hydrogenases isolated within NLPs retain their functional proton to hydrogen conversion activity. The LLNL researchers have highlighted the extremophile hydrogenase incorporation into NLPs [120]. The information on membrane bound protein isolation using NLPs has been well described [121]. The nanolipoprotein particles also provide the potential for immobilization, in nanopore membranes. Such sequestration might enable hydrogen production from biomass to nearly theoretical yields.

5.6.1.

Advantages

 NLP method provides for rapid, easy isolation, solubilization and stabiliziation of functional hydrogenases for hydrogen production.  Hydrogenases have higher selectivity, lower temperature requirements, and higher abundance than inorganic catalysts currently used in fossil fuel based production processes.  Immobilization in NLPs introduces the capability to use high oxygen sensitive membrane bound hydrogenases.  NLP-hydrogenases can be immobilized on dense, high surface area materials for modular, continuous hydrogen production and direct hydrogen storage interfacing.

5.6.2.

6.

Conclusion

Hydrogen is a unique fuel with numerous properties. In the present world scenario, the prices of fossil fuels are increasing day by day, the availability of non-renewable fuels is depleting and pollution is causing major harm to the environment and health of living organisms. There is a crucial need for using new ecofriendly and renewable fuels. Hydrogen production mediated by microbes possesses immense potential. The microbes are easily cultivated on nutrient media and their growth rate is very fast. The cost of production is thus very low and requires use of simple photobioreactors. The limitations in the path of increased hydrogen productions need to be resolved. The significant progress in genome sequencing and high-throughput expression has enhanced the acumen to engineer microbes for specific metabolic tasks. The new methodologies can pave the path for utmost production of hydrogen. Tremendous progress has been made in numerous fields pertaining to hydrogen productivity enhancement, but more efforts will result in the early commercialization of this ecofriendly fuel.

Acknowlegement The author Dr. Anjann Pandey thankfully acknowledges the funding support received from Ministry of New and Renewable Energy Sources (MNRE), GOI, New Delhi, India.

Potential applications

This invention can be developed for hydrogen generation useful to industry and research. Other near-future uses include distributed hydrogen power applications such as fuel cell electrodes in mobile hydrogen power generators. In line with DOE goals, this invention could be the basis for centralized hydrogen production for scaled power distribution. The invention is protected by a US Patent application and is being further developed.

5.7. Exploration of new strains for enhanced production of hydrogen New cyanobacterial strains having potential of hydrogen production can be explored for enhanced productivity. The

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