Efficient H2 production via Chlamydomonas reinhardtii - Cell Press

Opinion

Efficient H2 production via Chlamydomonas reinhardtii Maria G. Esquı´vel1, Helena M. Amaro2, Teresa S. Pinto1, Pedro S. Fevereiro3 and F. Xavier Malcata3,4 1

Instituto Superior de Agronomia (ISA)/Centro de Botaˆnica Aplicada a` Agricultura (CBAA), Calc¸ada da Tapada, 1349-017 Lisboa, Portugal 2 Centro Interdisciplinar de Investigac¸a˜o Marinha e Ambiental (CIMAR/CIIMAR), Rua dos Bragas no. 289, 4050-123 Porto, Portugal 3 Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Avenida da Repu´blica, 2780-157 Oeiras, Portugal 4 Instituto Superior da Maia (ISMAI), Avenida Carlos Oliveira Campos, 4475-690 Avioso S. Pedro, Portugal

Molecular hydrogen (H2) obtained from biological sources provides an alternative to bulk chemical processes that is moving towards large-scale, economical generation of clean fuel for automotive engines. This opinion article examines recent improvements in H2 production by wild and mutant strains of Chlamydomonas reinhardtii – the green microalga currently considered the best eukaryotic H2 producer. Here, we review various aspects of genetic and metabolic engineering of C. reinhardtii, as well as of process engineering. Additionally, we lay out possible scenarios that would lead to more efficient research approaches in the near future, as part of a consistent strategy for sustainable biohydrogen supply. Current status of hydrogen production Biofuels have attracted worldwide interest and have been discussed in various contexts including food availability, water resources, carbon emissions and environmental sustainability. Molecular hydrogen (H2) is arguably an ideal future transportation fuel because it is easily converted to electricity in fuel cells and liberates a large amount of energy per unit mass; this occurs in a nonpolluting manner, with its source water being, in general, widely available at essentially no cost. In addition, H2 can be produced from water at the expense of solar energy by certain microalgae, which have an ability to reduce free protons into H2 via action of specific enzymes (called hydrogenases). Chlamydomonas reinhardtii, a soil-dwelling microalga, is considered to be one of the best eukaryotic producers of H2. Based on available experimental information, including genomics, it is clear that this photosynthetic microorganism possesses a remarkable metabolic complexity that includes not only aerobic respiration but also a great many alternative fermentation circuits, with changes in the rates of accumulation of organic acids, ethanol, CO2 and H2 [1– 7]. Such a metabolic flexibility permits acclimation to hypoxic and anoxic conditions, under essentially uncontrollable external environments – a desirable property that, coupled with its fully sequenced genome [1], could eventually permit high production yields of H2 from water following cell and process optimization. For these reasons, C. reinhardtii is the crucial model organism for investigatCorresponding author: Malcata, F.X. ([email protected]).

ing hydrogen metabolism in photosynthetic eukaryotes [4,8], and thus the focus of this opinion article. Hydrogen release by C. reinhardtii when exposed to light is an ephemeral phenomenon, which was first reported in the early 1940s [9]. Interest renewed in the past decade after sustained H2 production was achieved by inducing sulfur repletion in a culture medium containing acetate, a carbon source used as a culture switch from aerobic to anaerobic state [10]. The potential usefulness of this model microorganism was boosted by the completion of its genome sequencing in 2007 [1]. Despite these efforts, C. reinhardtii has failed to meet with commercial success, owing to a combination of several biochemical and engineering shortcomings. For example, oxygen inhibits transcription and action of hydrogenase(s), so anoxia is required for hydrogen production; however, this is a crucial issue that delays provision of electrons and protons from water via the photosynthetic electron transport chain for hydrogenase(s) – because one of the electron transport complexes, Photosystem II (PSII), is responsible for oxygen evolution in photosynthesis, and consequently constrains the overall hydrogen productivity. Moreover, preliminary economic assessments indicate that bioenergy from microalgae should attain an efficiency of at least 10% in terms of solar energy conversion to be competitive with other modes of H2 production (e.g. biomass gasification or photovoltaic electrolysis) [11]. In this opinion article, we provide a brief biochemical rationale for C. reinhardtii H2 production and address methodological shortcomings. We also provide specific suggestions for future research efforts, such as improvement of the underlying relevant enzymes, exploitation of metabolism under limiting and nonlimiting sulfur supply, use of ‘anaerobic oxygenic photosynthesis’ (i.e. when oxygen generated in photosynthesis is uptaken by cell respiration, thus causing anaerobiosis in the light [12]), radiant energy absorption and finally optimization of bioreactor operating conditions. Biochemical pathways involved in hydrogen production Hydrogen can be produced using light energy, through reduction of free protons into molecular H2 catalyzed by ferredoxin (Fd)-dependent Fe-hydrogenases (Box 1).

0167-7799/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2011.06.008 Trends in Biotechnology, December 2011, Vol. 29, No. 12

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Opinion Box 1. Basic mechanism of H2 photoproduction The H2 photoproduction in green algae can follow two processes, direct and indirect, both of which use reduced ferredoxin (Fd) as electron donor for the hydrogenases [11]. In the former, photooxidation of water occurs, and the activities of both Photosystem II (PSII) and Photosystem I (PSI) are needed to supply reductants (or electrons) to Fd via the photosynthetic electron transfer chain. Conversely, the indirect pathway involves oxidative carbon metabolism (e.g. starch degradation), and NADP-plastoquinone oxidoreductase (NPQR) and PSI activities are required to supply the reductants. Both electron sources (i.e. water and starch) can eventually be used, but the contributions of each one depend on the type of strain, culture conditions, extent of damage of PSII and specific metabolic constraints. In the earliest studies, H2 production was induced by anaerobic incubation of cells in the dark, followed by illumination to detect activity. The hydrogenases were expressed under anaerobic conditions, and the direct pathway for transport of electrons to Fd was pursued in the light period; however, this process exhibited a short lifetime due to O2 production by PSII [4].

In C. reinhardtii, these enzymes are encoded by HydA1 and HydA2 genes, and take advantage of the low potential electrons involved in the photochemical reactions of photosynthesis [4,13,14]. Unfortunately, hydrogenases are catalytically inactivated by O2 [4,15], which diffuses into the core of the protein, and reacts with the catalytic 4Fe-4S cluster leading to inhibition [16]. Furthermore, O2 downregulates hydrogenase transcription [11]. After its initial success [10], sulfur deprivation became the inducing method of choice for H2 production; in essence, Box 2. Applied strategies for enhancement of H2 photoproduction The underlying principle here is that solar-powered H2 production by green algae can be divided into two stages: an aerobic stage, when cells are cultivated photoautotrophically to produce biomass (synthesis of carbohydrates from CO2); and an anaerobic phase, induced by sulfur deprivation, which promotes H2 production due to starch catabolism [44]. An important remark is that standard Tris, acetate and phosphate (TAP) medium, or its sulfur-deprived counterpart, used to grow C. reinhardtii contains acetate – which can be used by this species as a carbon source both for growth and respiration (i.e. photoheterotrophic H2 production). Acetate is essential for the establishment of anaerobic conditions, unless PSII activity is rapidly diminished by applying light stress to the cells grown in dimmed light. However, to produce H2 via a pure photoautotrophic route, PSII is often (but not necessarily) inhibited by DCMU added at optimal starch accumulation, or by removing O2 using N2 bubbling [52]. It is generally accepted that the electron transport by the two photosystems and via the hydrogenase pathway for production of 1 mol H2 requires absorption and utilization of a minimum of 5 mol photons by the photosynthetic apparatus [53]. Beyond sulfur deprivation to improve inactivation of PSII, another important factor precluding the practical application of algae for H2 production is their low light utilization efficiency in mass culture [41]. Irrespective of the mutant(s) used, light has to be harvested as source of energy for the photosynthetic release of H2 by microalgae; however, the rate of photon absorption by common chlorophyll antenna in the chloroplasts of the top cell layers in ponds and photobioreactors usually exceeds the rate of photosynthesis, so up to 80% of the light absorbed is actually wasted [54]. As a consequence, mutual shading occurs that could be detrimental in dense cultures. By contrast, intense incident light might cause photoinhibition at low culture densities [43]. Previous evidence has revealed that the minimum number of chlorophyll molecules is 37 for PSII and 95 for PSI [43]. Finally, for the so-called light limited regime, reduction of the antenna size would lead to a better efficiency in light capture only if the efficiency of photosynthesis could be increased.

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it is a two-stage indirect biophotolysis process, in which O2 and H2 production are out of phase in time [17] (Box 2). During the first 24 h of sulfur depletion under illumination, cells accumulate energy in the form of starch, and the rate of photosynthesis declines owing to a gradual drop in PSII activity; this results in anoxia, which consequently induces hydrogenase activity. PSII inactivation is associated with the photoinduced susceptibility of D1, the main protein in the reaction center core, or D1/D2 heterodimer, of that photosystem. Under normal conditions, damaged D1 is replaced via a turnover protein process; however, in the absence of sulfur, protein synthesis is blocked, thus enabling the self-repair of D1. Subsequently, cells undergo a second metabolic switch that breaks down starch, and consequently supplies electrons to hydrogenase via the plastoquinone-mediated indirect pathway [17]. In conclusion, when sulfur deprivation is imposed on a microalgal culture in acetate medium in the light, a sequence of events is triggered: (i) higher synthesis of starch; (ii) gradual drop in PSII activity; (iii) induction of anoxia; (iv) expression of hydrogenase; (v) degradation of starch; and (vi) release of H2. In recent years, the photo evolution of H2 upon sulfur deprivation has been monitored in a stepwise manner [12,18–20], as well as studied using transcriptomics [21] and proteomics [22]; for more information on these issues, we refer the reader to recent reviews [8,23] that discuss the hydrogen metabolic network in microalgae under sulfur deprivation. Note, finally, that a pure autotrophic protocol to produce hydrogen does not strictly require sulfur deprivation or PSII inhibition, for example via 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which disturbs the photosynthetic capacity of the cells [24]. Genetic transformation appears to be a promising route in attempts to improve H2 production. A list of favorable mutants is provided in Table 1, targeting hydrogenase, sulfate permease and ribulose-1,5-bisphosphate carboxylase (RuBisCO) enzyme, as well as PSI and PSII photosystems, starch reserves and respiration rates. Mutants bearing deficient H2 production (listed in Table 2) are also important, because they provide valuable information on the intrinsic features and regulatory mechanisms of H2producing pathway(s), and for fundamental validation of molecular strategies. Attempts to improve hydrogen production Improvements of hydrogenases Increasing hydrogenase tolerance to O2 is one of the first challenges towards commercial feasibility of microalgal H2 production. Recent in silico and in vitro studies have been conducted with the goal of engineering hydrogenases for a restricted accessibility of oxygen to their catalytic site, thus resulting in development of O2-tolerant phenotypes in H2producing strains [4,25–27]. Another approach is to maintain anaerobic conditions during photosynthesis via reducing O2 evolution by PSII. However, inhibiting the performance of PSII via mutations in the D1 protein concomitantly produces a decrease in the rate of H2 release [28]. This occurs even when alternate expression and repression of D1 is achieved, via a nuclear factor (Nac2) that regulates expression of D1 under control of the cytochrome c6 promoter – which is, in turn, repressed in the

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Table 1. Mutants of C. reinhardtii with enhanced H2 production: major features and mode of action Target Hydrogenase

Strain hemH-lba

D102KH T99KH

Mutation hemH gene from Bradyrhizobium japonicum and lba gene from Glycine max transferred into chloroplast of C. reinhardtii [FeFe] hydrogenases

Sulfate permease

antisulP

Repression of SulP expression

RuBisCO

CC-2803

State transition process

Stm6

Deficient in large subunit of RuBisCO Blocked cyclic electron transport

PSII

L159I-N230Y

D1 mutant

Stm6Glc4T7

Small PSII antenna size

Stm6Glc4

Stm6 cell line, expressing Chlorella kessleri HUP1 hexose transporter

std3 sda6

Altered aerobic starch degradation

apr1

Altered P/R ratio

Starch reserves

Respiration

Phenotype # Growth of transgenic algae vs. wild type = chlorophyll content of two cultures Transgenic cultures with more rapidly consumed O2 and increased H2 output, compared with controls in both S-free and S-containing medium

H2 production 4-fold increase in H2 production vs. wild type

Refs [50]

# Binding free energies " Association rate with FDX1 # Rates of light-saturated oxygen evolution # Levels of RuBisCO in chloroplast # Steady state levels of PSII D1 reaction center protein Production of H2 in presence of S; light sensitive " Starch reserves # Dissolved [O2]

Promising targets for improving H2 production " H2 production in strains with attenuated sulfate uptake and H2O oxidation capacities

[27]

" H2 production

[38]

" 9-fold increase in H2 production relative to parental, cell wall-less strain " 10-fold increase in H2 production vs. wild type " 5-fold increase in H2 production vs. CC-124 Promising target for improving H2 production

[30]

# Chlorophyll content " Quantum yield of photosynthesis " Respiration rate " Period of H2 production " Photon conversion efficiency " Growth rates under high light conditions " Photosynthetic efficiency " Densities when grown in large scale Ability to import glucose from medium and use thereof to enhance bio-H2 production " Growth rates under high light conditions Remarkable metabolic differences under anaerobic H2 photoproduction phase, compared with wild type (CC406) " Residual starch amounts (from higher starch accumulation or slower starch degradation, or both) " Respiration rate # Photosynthesis rate

presence of Cu2+ [29]. Addition of these cations to the medium inhibits the photosynthetic activity, so anoxia is reached; hence, H2 production takes place only for a short period. These results suggest that PSII activity is indispensable for accumulation of the starch reserves required for cell growth, and that biomass has to be periodically replenished for sustained bioreactor operation. By contrast, reduction of PSII activity limits the use of water as an electron source for H2 production. Table 2. C reinhardtii mutants with deficient H2 production Target Hydrogenase

Starch catabolism PSII PSI/PSII

Mutant strain M214KH E221KH hydEF-1 76D4 141F2 104G5 155G6 std1 sda3 D1-R323 Cy6Nac2.49 tla1

Refs [27] [27] [3] [25] [25] [25] [25] [33] [28] [29] [40]

[34]

[51]

[32]

" 150% increase in H2 production vs. Stm6 strain

[31] [6]

" H2 production vs. wild type only obtained with two strains std3 and sda6 "2- to 3-fold in H2 production vs. wild type

[33]

[36]

Improvements in metabolism under sulfur deprivation PSII activity and inactivation in C. reinhardtii throughout sulfur deprivation has been monitored because its residual activity (i.e. supplying electrons for the hydrogenase) is crucial for sustained H2 evolution [10,18,28]. The anaerobic conditions are maintained by respiration, which is not affected by sulfur deficiency [10,18]. When starch is degraded, it supplies electrons to hydrogenase through the plastoquinone pool [17], which justifies the use of starch metabolism to control H2 production. A hydrogen-efficient C. reinhardtii strain ‘stm6’ has been identified by random screening [30]. In this strain, the cyclic electron transfer is inhibited, and the algae exhibit a high rate of O2 consumption in respiration and an extra accumulation of starch, thus leading to 9-fold increase in H2 production relative to its parental strain, with a maximum production rate of 4 ml H2/l culture/h. The ‘stm6’ strain has served as a basis towards further improvements: the first new derivative, ‘stm6Glc4’, has an inserted hexose symporter system to supply sugars that could furnish additional electrons to plastoquinone, thus enhancing hydrogen production relative to the initial strain (6 ml H2/l culture/h) [31]. The ‘stm6Glc4T7’ strain is a result of further genetic engineering, aimed at reducing the 597

Opinion pigments of the antenna complex (i.e. the chlorophylls and carotenoids that collect light for photosynthesis) [32], and consequently can increase the efficiency of H2 production in mass cultures at high light intensities. Conversely, mutants exhibiting high starch catabolism show a reduction in H2 production [33]. Mimicking sulfur deprivation might constitute an approach to speed up culture transition to the H2 production phase. Repression of the sulfate transporter gene SulP has resulted in mutants with reduced sulfur uptake that simulate sulfur deprivation [34]; they also exhibited a high H2 production under anoxia. In either case, cells under sulfur limitation will eventually become stressed, so H2 production cannot be assured for long; this constraint could be partially alleviated by cell immobilization, because it allows easier replacement of the medium [35]. To this end, approaches that do not resort to sulfur deprivation should be sought. The newly metabolomic study of anaerobic H2 photoproduction, under sulfur deprivation [6], with one of the best hydrogen producing strains, ‘stm6glc4’ [6,31], showed the involvement of different pathways in the synthesis of fatty acids, neutral lipids and fermentation products [6], and consequently opened new perspectives. Those compounds are potential targets for metabolic engineering, in attempts to store energy and supply substrate(s) for the hydrogenases. The upgrading of tagged mutant collections will provide a much more detailed picture of those metabolic pathways, as well as new insights about metabolite exchanges between organelles. Improvements in metabolism under sulfur repletion Strategies to induce H2 production without the stressful impact of nutrient deprivation are being widely studied as the platforms of choice for research in this field [2,12,36]. One strategy has focused on attenuation of the photosynthesis/respiration (P/R) ratio that could induce anoxia. The screening yielded a strain (‘apr1’) showing the lowest P/R ratio, and resulted in 2- to 3-fold increase in H2 production when compared with the wild type [36]. Research efforts have also taken advantage of the great flexibility of C. reinhardtii fermentative metabolism that involves hydrogenase activity. In the laboratory, fermentative pathways became active in C. reinhardtii by sparging cultures with an inert gas. Interestingly, the induction of anoxia by sulfur-replete cells exposed to an anaerobic atmosphere differs significantly in gene expression and protein level from sulfur-deprived cultures [23]. Recently, the extensive fermentative pathways in this organism started to be elucidated at the level of transcriptome, proteome [37] and metabolome [2,3]. These pieces of information could potentially be utilized to optimize production of H2, but there are still several aspects of the anoxia acclimation network that are not fully understood. The generation of mutations that block each of these pathways will provide valuable information to apply new strategies for balancing ATP production with less reducing equivalents, and for rerouting electrons through the alternative fermentation pathways for H2 production [4,7]; however, accurate models will have to account for the diversity of anaerobic metabolic processes available in C. reinhardtii. 598

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Finally, H2 photoproduction was observed when CO2 fixation is impaired in sulfur-containing media [38]. In this case, the major photosynthetic electron sink, the Calvin cycle, is inhibited, and the hydrogenase pathway (a competitor electron sink) is in turn activated. Hence, the enzyme RuBisCO, which is directly responsible for CO2 fixation, could be engineered by site-directed replacement of structurally relevant residues towards slowing down the Calvin cycle (which competes with the hydrogenase for photosynthetic reductant from Fd). To be a suitable target for biotechnological optimization of H2 production, the resulting enzyme should have a lower level of expression, a weaker structure and a faster turnover [39]. Improvements in light capture For commercial production of H2 via microalga-mediated processes, the operating costs are to be affordable so as to compete with other sources of renewable energy. The most important factor for low cost generation of H2 and biomass is the photosynthetic productivity and light utilization efficiency attainability [40]. Therefore, truncation of the antenna size in a C. reinhardtii mutant ‘T7’ demonstrated that a 10–17% reduction resulted in nearly 50% increase in photosynthetic efficiency at saturating light intensity relative to the parental strain. Beckman et al. [32] provided further evidence that a controlled reduction of the PSII antenna size by less than 20% is sufficient to bring about a remarkable increase in photosynthetic efficiency of PSII, and an enhanced insensitivity against light photoinhibition; however, a saturating light intensity was assumed, which does not correspond to the conditions normally prevailing in photobioreactors. In another study led by Kosourov et al. [41], both a mutant ‘tla1’ and its CC-425 parental strain were able to produce H2 under several light conditions. Although the CC-4169 mutant exhibited lower maximum specific rates of H2 production at low and medium light intensities (i.e. 19 and 184 mE/m2/s), it exhibited a 4-fold and an 8.5-fold higher maximum specific rate at 285 and 350 mE/m2/s, respectively, when immobilized in 300 mm-thick alginate films and deprived of sulfur and phosphorus – for approximately the same cell density as the parental strain; in addition, its light energy conversion to H2 was also higher (0.08  0.04%). Hence, the mutation holds a promise for future application if the accompanying photoinhibition response can be appropriately addressed. The H2 production by C. reinhardtii has also been studied as a function of spectral irradiation; the maximum solar energy conversion efficiency was 0.061% in a flat panel photobioreactor, characterized by an optical thickness of 20 cm [42]. By contrast, cultures grown using increasing light levels – instead of exposure to constant light, underwent higher increases in culture density and H2 release [43]. Finally, a mutant of C. reinhardtii, known for its efficiency of photon to H2 conversion (2% at 20 W/m2, in the presence of acetate and absence of sulfur), was grown under a novel LED lighting system that promoted a homogeneous light distribution inside the reactor at moderate cell densities. Together with a novel self-desulfurization method applied by the end of the anaerobic stage, hydrogen fractions of 90% could be reached [44].

Opinion Improvements of processing conditions In addition to the crucial roles played by the quality and quantity of light supplied, other parameters such as pH, temperature, stirring rate, cultivation mode and cell localization (and their mutual interactions) also affect H2 production. The maximum production of H2 (i.e. 9.4 mmol/mg chlorophyll/h) was reported at pH 7.7 at the onset of sulfur deprivation [19]. A higher initial extracellular pH extends the lag period before C. reinhardtii starts producing H2, probably because of a delay in establishing an anaerobic environment under alkaline conditions. Temperature also plays a significant role upon the aforementioned lag period and on the total amounts of H2 that eventually accumulated [12]. Marine-type impellers led to good stirring results, yet a combination of airlift systems encompassing bubbling of an inert gas (e.g. argon or nitrogen) in helical and flat-plate bioreactors appeared to be the most suitable configuration to achieve high rates of both high CO2 fixation and H2 production – in addition to promoting dilution of the hydrogen stream [45]. (Note that in the absence of CO2 limitation – as arising from resistance to mass transfer, sequestration of that gas would in general relate to the biomass only.) Another interplay of processing conditions, namely light intensity, chlorophyll concentration and culture mixing, was studied upon productivity of H2 by C. reinhardtii [46]: the best rates were achieved by the wild type under a light intensity of 140 mE, with 2.4  108 cells/l. Batch photomixotrophic cultures grown under sulfur deprivation produced an average of 3.7 ml H2/l culture/ day (or 0.39 ml H2/mg chlorophyll/day) under reference conditions, with a 2-day lag time and a maximum production by 4 days, which declined considerably after 1 week [47]. Semicontinuous cultivation was possible for up to 127 days and yielded an average of 9.1 ml H2/l culture/day (or 1.3 ml H2/mg chlorophyll/day), with the amount and frequency of dilution correlating directly with H2 production. Note that most authors tend to refer the rate of release of H2 to volume of the culture rather than microalgal biomass, because sulfur-free media cause major metabolic changes (e.g. protein synthesis during the initial stress, followed by sudden protein loss) and morphological changes (e.g. cell size increase, followed by decrease); furthermore, some mutant strains possess cell walls and others do not, so the differences in fresh biomass make gravimetric data hardly comparable. Following sulfur deprivation, an increased productivity of C. reinhardtii, immobilized on a glass fiber matrix within alginate films, was observed – with an extension of the H2 production phase, in addition to higher cell densities (up to 2000 mg chlorophyll/ml matrix), higher specific rates of H2 released (up to 12.5 mmol/mg chlorophyll/h) and light conversion efficiencies to H2 above 1%; a surprisingly high resistance to inactivation by atmospheric O2 was recorded, so easy scale-up is probable [48,49]. Therefore, cell localization in the bioreactor should not be overlooked in optimization efforts. Concluding remarks Full rational manipulation of the microalgal metabolism is still currently unachievable, and therefore constitutes part

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of the future efforts necessary. Our fundamental understanding of the pathways and intrinsic controls leading to H2 release is, at the moment, often insufficient to permit accurate predictions of the metabolic consequences of genetic transformation. In general, the mechanisms involved in regulation of gene expression have not yet been fully elucidated, even in the specific case of C. reinhardtii, and new molecular biology tools are needed to generalize and standardize genetic modifications in microalgae. Such modifications might include efficient nuclear transformation, availability of promoter or selectable marker genes and stable expression of transgenes. Additionally, the problem of light capture is seminal, because light provides the energy required for water oxidation – which supplies the electrons required for H2 production. Genetic manipulation towards reduction of the chlorophyll antenna size appears promising, but research is still required on innovative reactor configurations, including optical fiber-based devices. Furthermore, construction parameters, such as transparency and durability of the material, and operating parameters, such as rate of gas exchange, should also be thoroughly addressed in the future. Further research efforts are consequently needed in the field of H2 production to develop better strategies for sustained bioproduction of H2, a clean fuel produced primarily from two of the (still) most abundant resources: water and sunlight. Acknowledgements This work received partial financial support from project MICROPHYTE (ref. PTDC/EBB/102728/2008), funded by Programa Operacional de Cieˆncia, Tecnologia e Inovac¸a˜o – POCI 2010 with the support of Fundo Social Europeu – FSE (Portugal), under the coordination of author F.X. Malcata.

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