Photosynth Res DOI 10.1007/s11120-015-0152-7

REVIEW

Relevance of nutrient media composition for hydrogen production in Chlamydomonas David Gonzalez-Ballester1 • Jose Luis Jurado-Oller1 • Emilio Fernandez1

Received: 23 March 2015 / Accepted: 29 April 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Microalgae are capable of biological H2 photoproduction from water, solar energy, and a variety of organic substrates. Acclimation responses to different nutrient regimes finely control photosynthetic activity and can influence H2 production. Hence, nutrient stresses are an interesting scenario to study H2 production in photosynthetic organisms. In this review, we mainly focus on the H2-production mechanisms in Chlamydomonas reinhardtii and the physiological relevance of the nutrient media composition when producing H2.

Keywords Hydrogen
Algae
Chlamydomonas
Nutrient stresses

Introduction Hydrogen gas (H2) is a promising option for fulfilling the world’s energy requirements and replaces non-renewable and carbon-based fuels. The use of H2 as a future energy fuel has great potential because it is a clean fuel that generates only water vapor when used and because has the highest energy content per unit weight (122 kJ/g) (Das and Veziroglu 2008). Current commercial world H2 production is based mainly on the use of fossil fuel through steam methane reforming (SMR) technologies (Lam and Lee 2011), which is a process that requires very elevated energy

& David Gonzalez-Ballester [email protected] 1

Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias, Universidad de Co´rdoba, Campus de Rabanales, Edif. Severo Ochoa, 14071 Co´rdoba, Spain

inputs (high temperatures and pressures) and do not alleviate the used of carbon-based non-renewable fuels. Over the last few decades, biological H2 production has shown great promise for generating large-scale sustainable energy. Some bacteria, cyanobacteria, and algae are capable of producing H2 from water, solar energy, and a variety of organic substrates. However, biological processes are not yet economically viable for large-scale economic applications in part due to the low yields obtained so far. Many publications have arisen in the last years covering the details of how these microorganisms produce H2. They can perform photosynthesis and/or fermentation to attain H2 production, with fermentation providing the option of utilizing different wastes (Yasin et al. 2013; Rittmann et al. 2015). However, photo-H2 production is a more promising alternative to obtain H2 from sunlight without dependence or competition with other feedstocks. Advances have been made in recent years to improve the H2 yield in algae through physiological manipulations and through metabolic and genetic engineering (Beer et al. 2009; Eroglu and Melis 2011; Srirangan et al. 2011; Dubini and Ghirardi 2015). Here, we will mainly focus on the H2-production mechanisms in Chlamydomonas reinhardtii (Chlamydomonas throughout) and the physiological relevance of the nutrient media composition when producing H2. This alga has become a model organism due to its sequenced genome, the genetic tools, and the wealth of information available (Merchant et al. 2007; Harris and Witman 2008). Multiple ‘‘omics’’ studies under different conditions have been published, which renders Chlamydomonas even more compelling as a model organism for biofuel studies in general and H2 in particular (Miura et al. 2004; Bolling and Fiehn 2005; Merchant et al. 2007; Mus et al. 2007; May et al. 2008; Matthew et al. 2009; Chen et al. 2010; Doebbe

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et al. 2010; Gonzalez-Ballester et al. 2010; Terashima et al. 2010; Castruita et al. 2011; Subramanian et al. 2014). Moreover, Chlamydomonas is able to grow heterotrophically using acetate as sole carbon source and also possesses a very versatile fermentative metabolism (Mus et al. 2007; Dubini et al. 2009; Catalanotti et al. 2012; Magneschi et al. 2012; Yang et al. 2014) which gives this alga the potential for producing biofuels under autotrophic or heterotrophic conditions.

Hydrogen production pathways in green algae Chlamydomonas possesses two chloroplast hydrogenases (HYDA1 and HYDA2) that can evolve H2 under anoxia. The ferredoxin enzyme FDX1 (or PETF) is the sole natural electron donor for both HYDA (Roessler and Lien 1984; Winkler et al. 2010; Meuser et al. 2012; Peden et al. 2013). Under light, HYDA1 accounts for most of the H2 produced in Chlamydomonas, while HYDA2 contribution is about 25 % of the total H2 photoproduction (Meuser et al. 2012). No significant differences in H2 production have however been found between hyda1 and hyda2 mutants in the dark (Meuser et al. 2012). Three different metabolic pathways have been described in Chlamydomonas for H2 production. Two of them are linked to the photosynthetic electron chain (PEC) and are termed PSII-dependent and -independent pathways. A third pathway is linked to the fermentative metabolism. FDX1 is the common branch point of the electrons flow, for the three metabolic pathways, prior its donation to HYDA. The PSII-dependent pathway is also known as the direct-photolysis pathway where H2 photoproduction is achieved from H2O splitting, and the concomitant release of O2. The resultant electrons are transferred through the photosynthetic apparatus via linear electron transfer (LET) involving both photosystems (PS). Under normal photosynthetic conditions, FDX1 accepts the electrons from PSI and most of them go to ferredoxin NADP? oxidoreductase (FNR) to produce NADPH for CO2 fixation. However, under light anaerobic conditions, FDX1 can redirect the electrons to the HYDA to release H2 (Roessler and Lien 1984; Happe and Naber 1993; Winkler et al. 2010; Peden et al. 2013). The main drawback of this pathway is the incompatibility of simultaneous production of O2 and H2, since algal HYDA is strongly inhibited by O2, which normally makes the PSII-dependent H2 production pathway a transient process. However, some physiological approaches (e.g., sulfur depletion) can expand the sustainability of this process (Melis et al. 2000). The PSII-independent pathway refers to the indirect H2 photoproduction that utilizes electrons from NAD(P)H,

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which are transferred directly to the PQ pool, bypassing PSII. Subsequently, the electrons are transported through the photosynthetic chain reaching PSI, FDX1, and HYDA (Mus et al. 2005; Baltz et al. 2014). The non-photochemical reduction of the PQ pool is mediated by NDA2, a type II NADH dehydrogenase in Chlamydomonas (Jans et al. 2008; Desplats et al. 2009; Mignolet et al. 2012; Baltz et al. 2014). It is generally assumed that the glycolytic degradation of glucose (or starch) is the most common source of PQ reductants (Gibbs et al. 1986; Chochois et al. 2009). The PSII-independent pathway does not require PSII activity and consequently O2 and H2 productions are not linked, which reduces the HYDA inhibition by O2. However, H2 production rates obtained via the PSII-independent pathway are around 10 times lower than those obtained via PSII-dependent pathway (Cournac et al. 2002; Antal et al. 2009; Chochois et al. 2009). It has been proposed that several metabolic factors limit PSII-independent H2 production, such as molecular processes competing with HYDA for electrons like the photosynthetic cyclic electron transfer (CET) or plastid terminal oxidase (PTOX) activity (Cournac et al. 2002; Alric 2014), or the capacity to accumulate and breakdown internal reserves, such as starch, with the subsequent reduction of the PQ pool (Chochois et al. 2009, 2010). The third H2 production pathway is linked to the fermentative metabolism, and dark anoxia is normally used to study the contribution of this pathway (Florin et al. 2001; Mus et al. 2005; Ghirardi et al. 2009). In this case, HYDA requires electron transfer from the decarboxylation of pyruvate to acetyl-CoA mediated by the pyruvate ferredoxin oxido reductase (PFR), which reduces FDX1 (Noth et al. 2013; van Lis et al. 2013). Fermentation-derived H2 production is however quantitatively less important than H2 photoproduction, although it is part of the cell acclimation process to survive and balance its redox status under prolonged anaerobiosis. Chlamydomonas, as many others photosynthetic algae, can catabolize endogenous carbohydrates, such as starch, and other metabolites using diverse fermentative pathways that allow them to generate the ATP necessary to drive metabolic and energy-requiring processes during anoxia (Gfeller and Gibbs 1984; Gibbs et al. 1986; Catalanotti et al. 2013). Chlamydomonas shows a high metabolic flexibility and is a rare example of a eukaryote that has homologs of all four predominant enzymes used in the fermentative metabolism of pyruvate. These include pyruvate formate lyase (PFL), PFR, lactate dehydrogenase (LD), and pyruvate decarboxylase (PDC) (Atteia et al. 2006; Mus et al. 2007). Hence, PFL, LD, and PDC compete with PFR for the pyruvate and are in part responsible of the low H2 production rates observed during dark anoxia. Recently, it was shown that oxaloacetate, in addition to pyruvate, could be used as a substrate by PFR,

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thus contributing with reductants for H2 production (Noth et al. 2013).

Physiological strategies employed to sustain H2 production All the strategies employed to achieve and sustain algal H2 production share few common ideas. First, establishment of anaerobic/hypoxic condition in the cultures is a prerequisite to produce H2. Induction of anaerobiosis in photosynthetic organisms is not a trivial task, especially under light conditions where O2 photoproduction takes place. In general, all strategies must fulfill at least one of the two following requirements: reduced levels of photosynthetic activity and increased rates of mitochondrial respiration. When mitochondrial respiration activity exceeds photosynthetic activity, (and more specifically PSII activity) the O2 levels in a culture drop to values compatible with induction of HYDA genes expression and proteins activities. Although hypoxia/anoxia is the first requirement to produce H2 in algae, it is not sufficient. Hence, cell metabolism, especially chloroplast metabolism, has to provide HYDA efficiently with substrates, which essentially are H? and electrons. Thus, a highly reduced state of the PEC could facilitate the transfer of electrons to FDX1 and HYDA1 for H2 production in illuminated cultures. Accumulation of reductants equivalents in the chloroplast, such as NAD(P)H, can also contribute to non-photochemical reduction of the PQ pool and in turn of the PEC. Finally, but not least, to achieve long-term H2 production, the previous conditions (low-O2 concentration, reduced state of the PEC, and accumulation of reducing equivalents in the chloroplast) need to be sustained in time. Again, this is not a trivial task since either prolonged anoxia or redox imbalance can affect cell viability. In order to produce H2 in green algae, several physiological strategies have been employed. Among them, nutrient stress approaches are widely used by the scientific community.

Nutrient stress conditions In general, macronutrient stress in Chlamydomonas results in decline of cell growth, photosynthetic activity, carbon fixation rates, and overall anabolic metabolism, whereas the catabolism of some metabolites, such as starch, lipids, or proteins, can be promoted (Wykoff et al. 1998; Grossman 2000; Ferna´ndez and Galvan 2007; Irihimovitch and Yehudai-Resheff 2008; Gonzalez-Ballester et al. 2010; Antal et al. 2011; Johnson and Alric 2013). All these factors can benefit H2 production.

Reduction of carbon fixation rates and of anabolic metabolism, especially nitrogen (N) and sulfur (S) assimilation pathways under S and N deficiency, respectively, decreases the demand for electrons and NAD(P)H (Winkler et al. 2010). This prevents the re-oxidation of the PEC, making photo-derived electrons potentially available for HYDA. Moreover, during most nutrient stresses, the algal photosynthetic machinery undergoes rearrangements and activates the so-called non-photochemical-quenching mechanisms (NPQ) to cope with the excess of light that the cells no longer can efficiently utilize (Harris and Witman 2008). Among other consequences, the PSII activity declines and so does the O2 photoproduction. Finally, prolonged nutrient stress can cause the mobilization of internal reserves as sources of ATP with the concomitant generation of reduced equivalents (predominantly NADH and NAD(P)H). Indeed, the mobilization phase of internal metabolites (e.g., starch and proteins) is often reported to be coordinated with the H2-production phase (Bamberger et al. 1982; Aparicio et al. 1985; Zhang et al. 2002; Batyrova et al. 2012; Volgusheva et al. 2014). Yet, even though there are general responses for all macronutrient stress, each specific macronutrient stress has its own metabolic particularities or specific responses that make them more or less suitable for H2 production.

Sulfur deficiency Sulfur (S) deficiency is the most employed and studied strategy to sustain H2 production in Chlamydomonas since Melis et al. (2000) reported that Chlamydomonas cultures were capable of producing H2 in medium devoid of S. S deficiency has a very broad impact in cell metabolism (Bolling and Fiehn 2005; Timmins et al. 2009; Chen et al. 2010; Gonzalez-Ballester et al. 2010; Toepel et al. 2013). Among other consequences, PSII centers undergo a rapid degradation and electron flow is severely inhibited at the PSII level, whereas PSI activity is essentially unchanged in S-starved cells (Wykoff et al. 1998; Melis et al. 2000; Zhang et al. 2002). Moreover, the rate of photosynthetic O2 evolution in Chlamydomonas declines by 75 % after 1 day of S starvation (Wykoff et al. 1998), and transition to state 2 and CET is promoted, which further reduces PSII activity and O2 evolution (Wykoff et al. 1998; Finazzi et al. 2002). On the other hand, mitochondrial activity is not severely affected (Melis et al. 2000; Zhang and Melis 2002), which favors a rapid depletion of O2 from sealed cultures when acetate is present in the media; it usually takes between 1 and 3 days for mixotrophic light-saturated S-depleted cultures to become anaerobic (Kosourov et al. 2002; Antal et al. 2003; Fouchard et al. 2005). All these factors help H2 production.

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Chlamydomonas cultures subjected to S deprivation undergo two physiological phases. First, shortly after the initiation of S deprivation and under aerobiosis, about 30–75 % of PSII is irreversibly inactivated, which favors the net consumption of O2 from the cultures when acetate is present (Wykoff et al. 1998; Antal et al. 2003, 2011; Volgusheva et al. 2013). Once O2 is totally consumed and cultures became anaerobic, there is a rapid and almost complete loss of activity of the remaining PSII centers, which is probably linked to a completely reduced state of the PQ pool (Antal et al. 2011; Volgusheva et al. 2013). However, this second tier of PSII inactivation is not irreversible and these PSII centers can undergo gradual reactivation as soon as H2 production begins. This is likely because HYDA1 activity leads to the re-oxidation of the photosynthetic e- transport chain, causing a partial reactivation of PSII activity (Antal et al. 2003, 2011; Volgusheva et al. 2013). However, there is a mutual negative feedback between PSII and HYDA1 activities because of the inhibitory effect of the O2 produced by the PSII on HYDA1. The PSII inhibitor DCMU (3-(3,4-dichlorophenyl)-1,1dimethylurea) significantly reduces H2 photoproduction in S-deprived cells (Kosourov et al. 2003; Hemschemeier et al. 2008; Antal et al. 2009; Chochois et al. 2009). This has been used to propose that the low residual PSII activity is the predominant H2 production pathway during S deprivation. It has been estimated that about 90–60 % of H2 production is PSII-dependent (Kosourov et al. 2003; Fouchard et al. 2005; Hemschemeier et al. 2008; Antal et al. 2009, 2011), and that the PSII centers that remain active during the anaerobiosis phase (Makarova et al. 2007; Antal et al. 2011; Volgusheva et al. 2013) contribute to sustain the H2 production observed during S deficiency. In addition to the PSII-dependent H2 production, there is also PSII-independent production in S-depleted cultures that contributes about 20–30 % of the total H2 production under S deficiency. Evidence of this was demonstrated by Mignolet et al. (2012) who reported that a NDA2 mutant accounts for a 30 % loss in the H2 production in S-depleted cultures. Also, increased rate of electrons flow from stromal reductants, such as NAD(P)H, to the PQ pool has been directly observed (Antal et al. 2006). The degradation of the previously accumulated starch has been proposed as the main source of ATP and NAD(P)H (Melis et al. 2000; Chochois et al. 2009). Finally, a minor H2 production derived from fermentative metabolism during S deprivation cannot be ruled out (Antal et al. 2009). Evidence of fermentative metabolism in illuminated S-deprived cultures is revealed by the accumulation of formate, and ethanol, suggesting the possible existence of fermentative-derived H2 production coupled to the oxidation of pyruvate to acetyl-CoA (Kosourov et al. 2003; Timmins et al. 2009).

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Nitrogen deficiency Nitrogen (N) deficiency is able to elicit H2 photoproduction although this phenomenon has been substantially less studied than H2 production under S deprivation (Aparicio et al. 1985; Philipps et al. 2012). Similarly to S deficiency, N deficiency results in the decline of the photosynthetic activity, triggers the transition to state 2, and leads to the accumulation of starch and lipids in the presence of acetate (Martin and Goodenough 1975; Peltier and Schmidt 1991; Work et al. 2010). However, N deprivation induces a much slower decline in PSII activity, which leads to a more prolonged aerobic phase and a delayed H2 production relative to S deficiency (Philipps et al. 2012). According to Philipps et al. (2012), the H2 production rates are also lower than those obtained under S deficiency. Intriguingly, Aparicio et al. (1985) reported no advantage in H2 production when comparing N-depleted vs ammonium-containing cultures. Hence, H2 production under N deficiency in Chlamydomonas needs to be better studied. Notably, DCMU does not significantly influence H2 yields in N-depleted cultures, indicating a minor contribution of the PSII-dependent pathway on H2 production, unlike S-depleted cultures (Philipps et al. 2012). Interestingly, N-depleted cells can accumulate starch and lipids more efficiently than S-depleted cells, although the starch reserves are not mobilized during the H2 production phase indicating that starch degradation does not limit H2 production during N starvation (Philipps et al. 2012). Moreover, cytochrome b6f complex is selectively degraded upon nitrogen deprivation (Bulte´ and Wollman 1992; Philipps et al. 2012), affecting both LET and CET, and compromising PSII-dependent and independent H2 photoproduction in N-depleted cultures. On the contrary, the H2 production in dark N-depleted and low light N-depleted cultures is similar (Aparicio et al. 1985), but significantly higher than in dark S-depleted cultures (Philipps et al. 2012). Thus, it is proposed that fermentative H2 production plays an important role in N-depleted cells. As suggested by Aparicio et al. (1985), protein degradation could be the major source or reductive equivalents under N deficiency; this suggestion is based on the observation that cells excrete ammonium during H2 production even in N-depleted cultures. Hence, the pyruvate resulting from protein degradation could contribute to H2 production via PFR in N-depleted cultures. Phosphorous deficiency The effects of phosphorous (P) deprivation in H2 production in Chlamydomonas and Chlorella have been studied by Batyrova (2012, 2015). In the case of Chlamydomonas, P deficiency causes a decline in PSII activity and a switch to state 2, which results in the establishment of culture

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anaerobiosis in the presence of acetate (Wykoff et al. 1998). However, the inactivation of PSII centers is not as fast as under S deficiency, and O2 evolution declines more slowly relative to S-starved cells. Among other factors that may contribute to a slow inactivation of PSII center under P conditions is the fact that, as opposed to S or N, Chlamydomonas cells can store P reserves in form of polyphosphate (Siderius et al. 1996; Komine et al. 2000). These P reserves can be used for days before the cells suffer a real P deficiency. Batyrova et al. (2012) managed to bypass this problem by using very low concentrations of cells, washing and re-suspending then in—P media. Similarly to S-deprived cultures, acclimation of algal cells to P-deprived conditions is accompanied by the accumulation of starch during the aerobic phase and its degradation during the H2 production phase. However, there is no evidence that reductants derived from starch provide electrons to the HYDA via the PSII-independent pathway. Indeed, the relative contributions of PSII-dependent and PSII-independent pathways during H2 production in P-depleted Chlamydomonas cultures are not yet known. Other nutrient deficiencies Very recently, it was reported that Chlamydomonas cells depleted of magnesium (Mg) can sustain H2 evolution, which interestingly is higher when compared to that obtained in S-depleted media (Volgusheva et al. 2014). Mg depletion leads to a 20 % decrease of the PSII activity and to an increase of starch accumulation, whereas mitochondrial respiration is not affected. The H2 production under Mg deficiency is mainly linked to the PSII-dependent pathway since DCMU inhibits 74 % of the H2 production under this condition. There are no molecular evidences that can explain why Mg deficiency can enhance H2 production. Media depleted in some elements, in addition to S, P, N, and Mg, have been studied in the context of H2 production in the alga Scenedesmus obliquus (Papazi et al. 2014). From all the elements under study, potassium (K) deficiency leads to the highest H2 production. Other nutrient deficiencies, such as cooper, manganese, and S, also promote H2 production in Scenedesmus obliquus. Interestingly, Mg did not enhance H2 production compared to control conditions. In the case of K deficiency, a decrease of D1 protein was observed, whereas PSaA protein of photosystem I was accumulated, when compared to control cultures.

Reduced carbon source supplementation Several decades ago, it was observed that H2 photoproduction in some algal species adapted to light anaerobiosis is enhanced by the presence of acetate in the media (Jones

1963; Healey 1970; Klein and Betz 1978; Bamberger et al. 1982; Gibbs et al. 1986). These former studies established that under illumination, algae can consume the acetate available in the medium and release H2 and CO2; in the dark, however, acetate has no effect on H2 production and acetate uptake is not detected. The addition of acetate to Chlamydomonas cultures decreases photosynthesis efficiency, net O2 evolution, and CO2 fixation, as well as promotes the transition from state I to state II and mitochondrial respiration (Asada and Miyake 1999; Endo and Asada 1996; Ge´rin et al. 2014; Heifetz et al. 2000). All these factors help the establishment of anoxia in sealed cultures under moderate/low illumination. Repression of CO2 fixation may also contribute to H2 production by reducing the competition for electrons, for HYDA, at the level of FDX1 (Hemschemeier et al. 2008). In nutrientreplete medium, it is still unresolved, whether the presence of acetate favors the PSII-dependent H2 production since the effect of the addition of DCMU is unclear. Some works revealed no impact on H2 production (Healey 1970), whereas others observed a substantial inhibition of both H2 production and acetate uptake (Gibbs et al. 1986, Bamberger et al. 1982). Since DCMU blocks both PSII activity and acetate uptake, and consequently any starch accumulation derived from acetate assimilation, it is not easy to discriminate which H2 photoproduction pathway is acting when this inhibitor is used; DCMU may result in an overestimation of the role of the PSII-dependent pathway in nutrient-replete mixotrophic conditions. On the other hand, it has been proposed that acetate contributes to PSII-independent H2 production by promoting the accumulation of starch during aerobic conditions; starch would be later mobilized during anoxia and supply with NAD(P)H to the chloroplast (Klein and Betz 1978; Bamberger et al. 1982; Gibbs et al. 1986). In addition to the PSII-independent pathway linked to the mobilization of the starch previously accumulated, other alternatives have been proposed to explain H2 production in media with acetate (Willeford and Gibbs 1989; Willeford et al. 1989). Acetate dissimilation/photoassimilation requires the participation of the tricarboxylic acid (TCA) and glyoxylate cycles (Gibbs et al. 1986). Studies on enzyme localization and proteomics (Willeford and Gibbs 1989; Willeford et al. 1989; Atteia et al. 2009; Rolland et al. 2009) have helped to identifying the enzyme localization of these pathways, which requires the participation of enzymes localized in the cytosol, the mitochondria, and the chloroplast. The localization of two enzymes involved in acetate assimilation (succinate dehydrogenase and malate dehydrogenase) in the chloroplast may provide this organelle with the reductive equivalents needed to feed the PQ pool (Willeford and Gibbs 1989; Willeford et al. 1989). Moreover, an important contribution of acetate in the

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production of H2 through the fermentative pathway cannot be ruled out. Recently, it has been shown that the pyruvate: ferredoxin oxidoreductase (PFR) enzyme of Chlamydomonas possesses an important affinity for oxaloacetate (Noth et al. 2013), which opens the possibility that acetate, via glyoxylate cycle, might also be coupled to fermentative H2 production.

Optimization of medium composition for H2 production in Chlamydomonas From studies in Chlamydomonas under autotrophic conditions, it is concluded that H2 production is lower relative to mixotrophic conditions (Fouchard et al. 2005; Tsygankov et al. 2006; Kosourov et al. 2007; Tolstygina et al. 2009; Degrenne et al. 2011), which reveal the importance of the presence of acetate in the medium composition. Optimization of nutrient medium composition (acetate, N, and P) for H2 production in Chlamydomonas under S-depleted conditions has been already studied (Jo et al. 2006). Authors concluded that under mixotrophic S-depleted conditions, 9.20 mM of ammonium, 2.09 mM of phosphate, and pH 7.00 were optimal conditions for H2 production; concentrations of ammonium and phosphate are 1.23 times and 2.09 times higher, respectively, than regular Tris–Acetate-Phosphate (TAP) media. However, from the work of Jo et al. (2006), no clear conclusions can be obtained regarding the physiology of H2 production in Chlamydomonas. Optimization of the salinity in medium devoid of S has been assayed by Zhang and Melis (2002). Authors concluded that H2 production in cultures with 10 mM NaCl was enhanced by 30–40 % compared with control cultures. Also, the effect of the extracellular pHs when producing H2 has been studied in Chlamydomonas S-depleted cultures (Kosourov et al. 2003); authors concluded that the optimum pH is 7.7, and that the pH profile of H2 photoproduction correlates with that of the PSII activity during S deprivation. Noteworthy, medium containing either nitrate or nitrite suppresses H2 production (Aparicio et al. 1985). This fact likely reflects the high NADH and electron demand of nitrate reductase and nitrite reductase, respectively, which contributes to the re-oxidation of the PEC and competes with HYDA for electrons. Improvement of the sustainability and efficiency of H2 production under S deficiency using medium with different initial low S concentrations in mixotrophic (Fouchard et al. 2009) and autotrophic (Degrenne et al. 2011) cultures have been already performed. Similarly, continuous flow and readdition to the media of low S concentrations have been already assayed to optimize H2 production (Kosourov et al. 2002; Laurinavichene et al. 2008; Tamburic et al. 2013).

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Interestingly, the simultaneous deprivation of S and P does not result in any improvement of H2 production relative to when single S deficiency is used (Kosourov and Seibert 2009; Batyrova et al. 2012).

Underlying differences in H2 production under different nutrient conditions Despite the fact that algal cultures depleted of S, N, P, and Mg share very similar physiological responses such as PSII inactivation, switch to state 2, starch accumulation, and O2 depletion, their physiology for H2 production is intrinsically different (Table 1). For example, H2 production in Mg- and S-depleted cultures occurs mainly via the PSIIdependent pathway (Kosourov et al. 2003; Volgusheva et al. 2014), whereas in N-depleted cultures, it could occurs essentially via the fermentation (Aparicio et al. 1985; Philipps et al. 2012). Still, for S and Mg deficiency, about 20–30 % of H2 production is linked to the PSII-independent pathway. Since starch degradation is concomitant with H2 production during S and Mg deficiency, the mobilization of starch reserves has been proposed to contribute to PSII-independent pathway under these conditions. However, in N-depleted cultures, and in spite the fact that starch reserves are higher than in S- or Mg-depleted cultures, the mobilization of starch reserves is not linked to H2 production under this condition, but likely to protein degradation. Another important physiological difference among Mg-, S-, P-, and N-depleted cultures during H2 production relies on the PSII inactivation and O2 depletion from the cultures. These two factors seem to occur much faster under Mg and S deficiency than under P or N deficiency (Wykoff et al. 1998; Philipps et al. 2012; Volgusheva et al. 2014). In the case of P depletion, this could be partially attributed to the internal reserves of P that delay a severe P-stressed situation and help cell survival. Under N deficiency, however, this is not easy to explain. The rapid degradation of PSII centers observed in the absence of S (Melis et al. 2000; Zhang et al. 2002) has been traditionally linked to the inability to repair photo-damage D1 proteins due to the down-regulation of the de novo protein biosynthesis. However, if we assume that this is a non-specific phenomenon, it should be even more prominent in the case of N depletion, since protein turnover in N-depleted cells is even more compromised than in S-depleted cells. Moreover, N depletion leads to a much rapid loss of culture viability than S depletion (Cakmak et al. 2012). These two factors would theoretically favor a rapid PSII inhibition in N-depleted cells. On the other hand, S deprivation causes an 80 % decrease of the PSII activity, whereas in the case of Mg depletion, the PSII activity decreases only by 20 %.

References 1 Kosourov et al. (2003), 2 Antal et al. (2009), 3 Hemschemeier et al. (2008), 4 Melis et al. (2000), 5 Chochois et al. (2009), 6 Wykoff et al. (1998), 7 Philipps et al. (2012), 8 Zhang and Melis (2002), 9 Aparicio et al. (1985), 10 Batyrova et al. (2012), 11 Volgusheva et al. (2014), 12 Morsy (2011), 13 Bamberger et al. (1982), 14 Gibbs et al. (1986), 15 Healey (1970), 16 Noth et al. (2013), 17 Klein and Betz (1978), 18 Willeford and Gibbs (1989), 19 Degrenne et al. (2010), 20 Endo and Asada (1996), 21 Heifetz et al. (2000)

H2production when compared with autotrophic cultures (395–250 %)

H2production when compared with cultures grown in TAP media, bCultures conditions used for comparison are not identical,

Primary? Nutrient replete (TAP)

19 %

12

c13,14

Unclear

74 %11 200 %11 -Mg

(86 %)

a,9

58 %

7

-N

-S

a

Starch mobilization / acetate assimilation16,18 Theoretical (via oxaloacetate derived from acetate assimilation)16

N/A Likely

N/A 40 %b,10 -P

13-15

N/A

N/A

13,14

N/A

Likely high

13,17

Starch mobilization11

Starch mobilization10

Protein degradation

9

Null

20-30 %1-3 60-90 %1-3 100 %1-3

7

Residual2

7,9

Starch mobilization4,5

c

Yes/yes

*211

119 1

19

*111

13

Yes/yes11

*610 C56,10 Yes/yes10

Yes/no

1–31,4,5,7

*47 [8

7

1–31,5,6 Yes/yes3,4

7

Time to reach anoxia (days) Contribution of fermentative H2 production via PFR Contribution of PSII-independent H2 production Contribution of PSII-dependent H2 production H2 production rates (relative to TAP-S)

Table 1 Comparative analysis of H2 production under different nutrient conditions

Possible source of reductants for PSII-independent and fermentative pathways

Starch accumulation/mobilization

Lag H2 production (days)

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Despite this large difference in functional PSII centers, there seems to be no significant differences regarding O2 depletion from the cultures between these two conditions. Regardless of the different culture conditions employed, the large majority of the studies about H2 production in Chlamydomonas have been performed in acetate-containing medium. Mixotrophic condition greatly enhances H2 production in all cases (Fouchard et al. 2005; Tsygankov et al. 2006; Kosourov et al. 2007; Tolstygina et al. 2009), which point out the importance of acetate in the context of H2 production. Nevertheless, little has been done in the last years regarding the precise effect of acetate in the production of H2 in algae independently of other nutrient stresses (Degrenne et al. 2010; Wang et al. 2011).

Future directions Green algae have a great potential for commercial application, including biological H2 production. However, major issues remain unsolved delaying the development of an applied and low cost, alga photobiological H2 producing system. Most of the biochemical factors limiting the scaling of H2 production are known, and some improvements have been achieved through the optimization of cell concentration, medium composition, light intensity, synchronization of cell division, H2 partial pressure, and cell immobilization, as well as by using specific mutants lines (Tsygankov et al. 2002; Kosourov et al. 2003; Laurinavichene et al. 2004; Kim et al. 2006; Giannelli et al. 2009; Kosourov and Seibert 2009; Kosourov et al. 2012; Dubini and Ghirardi 2015). The imposition of nutrient deficiencies is a successful strategy to enhance H2 production in algae cultures. However, nutrient deficiencies cause drastic metabolic changes in cell cultures resulting in loss of cell viability, which greatly affect the potential for large-scale applications. Today, these harmful effects can be partially overcome by using continuous or semi-continuous regimes of cultivation (Fedorov et al. 2005; Oncel and Vardar-Sukan 2009) or by re-addition of nutrients to the medium (Kosourov et al. 2005). However, these protocols either require high-energy inputs, do not support algae biomass and H2 production simultaneously, or reduce the theoretical maximal efficiency of H2 production. Nutrient deficiencies are also excellent scenarios to depict the precise molecular and physiological mechanisms that trigger H2 production. Deconvolution of the precise mechanisms involved in H2 production under different nutrient stresses can help us to understand the rationale behind H2 photoproduction and could allow the designing of specific molecular and genetic manipulations

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leading to optimal H2 production without the need of imposing nutrient stresses. Besides general responses to nutrient limitation, specific acclimation responses to different nutrient stresses may be of great interest for fully understanding the physiological differences related to H2 production. In this sense, regulatory mutants impaired in the specific acclimation responses to S (e.g., sac1, sac3, and snrk2.1) (Davies et al. 1996, 1999; Gonzalez-Ballester et al. 2008), N (e.g., nit2) (Camargo et al. 2007), and P (e.g., psr1) (Wykoff et al. 1999) deficiency, or unable to grow under heterotrophic conditions (Harris and Witman 2008) could be very valuable tools to study H2 production. An interesting example is that of the S-deprivation regulatory mutants (sac1 and snrk2.1), which show a lightdependent bleaching phenotype during S deprivation that results in a fast cell death (Davies et al. 2006; GonzalezBallester et al. 2008). This phenotype has been linked to a failure of the mutant cells to down-regulate photosynthetic electron flow from PSII (Wykoff et al. 1998), which indicates that S-deficient specific photo-adaptive responses are critical for cell survival. In agreement with these data, Malnoe et al. (2014) showed that D1 degradation during S deprivation is a fine-tuned process, controlled by the FstH protease. Authors showed that the D1 degradation in S-depleted cells occurs even in the dark, contrary to what they observed under other macronutrient stresses (-P) or under light photo-inhibition conditions. This observation suggests that D1 degradation in S-depleted cells is not just a non-regulated process consequence of an impairment of the turnover of damaged proteins but also an S-specific acclimation process. It would be interesting to verify if the activity of the FstH protease is somehow under the control of the S-deprivation regulatory elements and if a fsth mutant can impact H2 production in S-depleted cells. Regulation of NPQ processes could be another level of nutrient-specific responses that can differently modulate H2 production. NPQ mechanisms occurring during stress conditions are critical to down-regulate the photosynthetic activity and to avoid photo-damage. Modulation of the light-harvesting complex (LHC) proteins is crucial to regulate the light absorption by photosynthetic organisms. The LHCBM9 transcript, which is barely detectable in Chlamydomonas cells grown in nutrient-replete medium, increased by [1000-fold during S deprivation and is the second most abundant mRNA in S-depleted cells (Gonzalez-Ballester et al. 2010). This large increase in transcript abundance is specific to S deprivation since this phenomenon is neither observed in -N or -P cultures nor in S deficiency regulatory mutants (Gonzalez-Ballester et al. 2010). It has been shown that RNAi lines impaired in LHCBM9 expression undergo a more pronounced oxidative damage caused by singlet oxygen and a diminished

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capacity to photo-produce H2 during S deprivation (Grewe et al. 2014). Similarly, other specific nutrient responses potentially modulating LET, CEF, the ferredoxin/thioredoxin network, and electron dissipating mechanisms such as chlororespiration and Mehler’s reaction, which compete with HYDA for electron (Kruse et al. 2005; Antal et al. 2009; Tolleter et al. 2011), could greatly influence H2 production under different nutrient stresses. Comparative analyses of these processes performed under different nutrient conditions and using specific nutrient regulatory mutants may illustrate the particularities of H2 production between different physiological conditions. Acknowledgments This work was funded by the MINECO (Ministerio de Economia y Competitividad, Spain, Grant no. BFU201129338), supported by the European ‘‘Fondo Europeo de Desarrollo Regional (FEDER)’’ program, the Plan E program (CONV 188/09), the Ramon y Cajal program (RYC-2011-07671), the Junta de Andalucıa grants (P08-CVI-04157 and BIO-502), and the Plan Propio de la Universidad de Cordoba.

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