Among major barriers to optimal H2-production yields in algal cultures are high sensitivity of algal [Fe—Fe]- hydrogenases to O2 inactivation, low light saturation levels of photosynthesis, competition for reductant from alternative metabolic pathways, state transition and establishment of cyclic electron flow around PSI, and the reversible nature of the hydrogenase— driven reaction. All these barriers have been exten­sively studied by different research groups in the last few years.

Clearly, the O2 sensitivity of [Fe—Fe]-hydrogenases is the major barrier preventing the application of green algal H2 photoproduction in commercial systems. Several approaches for solving the O2-sensitivity issue have been suggested: (1) identifying and implementing mutations, which narrow the channel(s) of the [Fe—Fe]-hydrogenase enzyme for blocking access of O2 molecules to the catalytic center (Cohen et al., 2005; Posewitz et al., 2009); (2) selecting for O2-tolerant en­zymes through random mutagenesis (Nagy et al., 2007; Stapleton and Swartz, 2010); (3) introducing enzymes from other organisms, which are more stable to O2 inac­tivation, into algal cells. None of these approaches have yet resulted in a mutant with improved O2 tolerance. Nevertheless, Stapleton and Swartz (2010) applying the directed evolution approach identified a version of C. reinhardtii HydA1 with a fourfold increase in catalytic activity as compared to the wild-type enzyme.

Low light-utilization efficiency in mass cultures is another important factor precluding the use of H2- producing green algae in practical applications (Torzillo et al., 2003). In algal suspensions, light intensity decreases with the depth of the culture. The light atten­uation is more pronounced in dense cultures, where shading limits the productivity of inner parts of the cul­ture. On the contrary, algae in the upper layers suffer from photoinhibition, which is more pronounced under high light intensities. The latter significantly limits application of high light intensities for improving the overall algae productivity. The problem can be addressed in part by immobilizing algae in thin layers or films. Immobilization fixes algal cells within a controllable volume and allows uniform light distribu­tion to the cells that makes light utilization per volume basis more efficient. Indeed, immobilization of sulfur — deprived C. reinhardtii cultures on glass fiber matrices demonstrated significant improvements both in the volumetric rate of H2 photoproduction and in the dura­tion of the process (Laurinavichene et al., 2006). This technique used the property of microalgae to form biofilm on the glass surface. The attachment of cells occurred through natural colonization that, if required, can be accelerated by activating glass fibers with 3-(2-aminoethyl-aminopropyl)-trimethoxysilane (Tsygankov et al., 1994). Later studies of immobilized algae with either a constant flow of medium containing micromolar sulfate concentrations or cycling of immobi­lized cells between minus and plus sulfate conditions improved the duration of H2 production up to at least 3 months (Laurinavichene at al., 2008). However, due to irregular colonization of glass fibers by the algal cells, the system showed significant physical and physiolog­ical heterogeneities in different parts of the matrix, resulting in irregular light and nutrient distributions, and decreasing the overall performance of H2 photopro­duction. In order to improve the light absorption proper­ties of immobilized microalgae, Kosourov and Seibert

(2009) entrapped cells within thin alginate films. This technique produced films with uniform distribution of algal cells within the matrix that had very high cell densities (up to 2000 mg total Chl per ml of the matrix). As a result, the light conversion efficiency in alginate films at ~ 60 mE/m2 s PAR (photosynthetic active radia­tion) achieved 1.5% for the period of the maximum H2- production rate and was close to 1% for the whole period of nutrient deprivation.

Another approach for improving light utilization effi­ciency in mass algal cultures is to find or generate algal mutants with a small chlorophyll antenna size. Strains with the truncated antennae allow greater transmittance of irradiance through the ultrahigh cell density culture without significant dissipation of light energy and, as a result, have a higher photosynthetic productivity in out­door conditions. Recently, C. reinhardtii mutants with truncated chlorophyll antennae were generated and characterized (Polle et al., 2000, 2003). These mutants have shown promise in increasing the light utilization efficiency and the overall productivity in mass cultures (Polle et al., 2002, 2003), but suspensions have not

established anaerobiosis and so have failed to produce H2 gas under sulfur-deprived conditions. Despite this, nutrient-deprived mutants with truncated chlorophyll antennae produced H2 after immobilization within thin alginate films (Table 21.1). These mutants showed higher efficiency of H2 photoproduction than the parental CC-425 strain under saturating light conditions (Kosourov et al., 2011).

H2 photoproduction in green algae competes with a number of different metabolic pathways for the reduc — tant originated in photosynthesis (Hemschemeier and Happe, 2011). Here, CO2 fixation is one of the most important. The affinity of Fd to FNR is very high and on the order of 0.6 gM (Kurisu et al., 2005), while the af­finity of Fd to hydrogenase enzyme is only about 10 gM (Roessler and Lien, 1984). It is clear that electrons in healthy algal cells will be preferably directed toward reduction of NADP+ and, hence, toward CO2 fixation. Sulfur-deprived algae, however, inactivate Rubisco, the key enzyme of CO2 fixation, by the time of the establish­ment of anaerobiosis in the photobioreactor (Zhang et al., 2002). Zhang and coauthors showed that only about 3% of this protein is present in cells during the H2 production stage. This finding suggests that the photosynthetically generated reductants in sulfur — deprived algae are preferably used for generation of H2, but not for CO2 fixation. It is important to note here, that according to Hemschemeier et al. (2008) the Rubisco-deficient C. reinhardtii, CC-2803 strain produces H2 gas even under sulfur-replete conditions (Table 21.1). H2 evolution in this strain is almost completely depen­dent on electron flow from PSII. This finding shows that flow of electrons in engineered green algae can be successfully redirected toward H2 photoproduction. The competition for the photosynthetically generated reductant from other metabolic pathways is less studied. There has been some evidence for the competition from nitrate reductase (Aparicio et al., 1985). However, the wild-type CC-124 and 137C strains of C. reinhardtii that are commonly used in sulfur-deprivation experiments (Table 21.1) carry the nitl and nit2 mutations and cannot grow on nitrate. Therefore, the question about possible competition for the reductant between hydrogenase and nitrate reductase should be studied in detail using the Sager’s line of C. reinhardtii wild-type strains (Praschold et al., 2005).

A novel approach for preventing competition for the reductant from other metabolic pathways is formation of a fused complex of Fd and hydrogenase. In vitro analysis of such a complex showed that replacing the hydroge — nase with the Fd/hydrogenase fusion switches the bias of electron transfer from FNR to hydrogenase and re­sults in an increased rate of H2 photoproduction (Yacoby et al., 2011). This experiment indicates that the idea of the formation of a fused Fd/hydrogenase complex is promising, but should be checked in the C. reinhardtii mutant in vivo.

Another barrier for the industrial H2 photoproduc­tion system involves the redirection of photosynthetic electron flow from linear to cyclic and production of ATP, which results in a nonproductive pathway and decreased H2 production under anaerobic conditions. In green algae, this process also involves phosphoryla­tion and dissociation of PSII-light-harvesting antenna and results in the so-called state 1 to state 2 transitions that lead to higher excitation of PSI over PSII. A prom­ising approach for prolongation of H2 production in algae has recently been proposed by Kruse et al.

(2005) . They generated the mutants affected in state tran­sition. These mutants are blocked in state 1 that inhibits cyclic electron flow around PSI. One of these mutants, stm6, accumulated larger starch reserves under sulfur deprivation and produced almost five times more H2 gas than the wild type (Table 21.1).

H2 photoproduction in green algae is driven by the bidirectional [Fe—Fe]-hydrogenase enzyme that cata­lyzes not only the forward (H2 photoproduction) but also the reverse (H2 uptake) reaction. Under high H2 partial pressure in the photobioreactor, the rate of reverse reaction is significant (Kosourov et al., 2012). The authors showed that the decrease in H2 partial pres­sure improves significantly the yields and rates of H2 photoproduction in algal cultures. They also suggested the existence in sulfur-deprived algae of H2-uptaking pathways, either photoreduction or oxy-hydrogen reac­tion. The possibility of photoreduction in nutrient — deprived algae is questionable because of the significant degradation of the Rubisco enzyme by the time H2 photoproduction begins (Zhang et al., 2002). Therefore, it is most likely that nutrient-deprived algae utilize H2 gas through the indirect oxy-hydrogen reaction involving the chlororespiration pathway from [Fe—Fe]- hydrogenase(s) to O2 through Fd, NADP+/NADPH and the PQ-pool. If H2-uptaking pathway(s) does exist, H2 photoproduction in green algae can be further improved by downregulating this pathway(s).

Acknowledgments

This work was financially supported by the Academy of Finland Center of Excellence project (118637) and by the Kone foundation (YA, SNK).