Analytical approaches to photobiological hydrogen production in unicellular green algae

Abstract

Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of "anaerobic oxygenic photosynthesis", whereby photosynthetically generated oxygen is consumed by the cell's own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular "hydrogen metabolism" process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H(2) production in Chlamydomonas can be monitored and regulated.

Fig. 1

Schematic of photosynthetic electron transport in the unicellular green alga C. reinhardtii during normal photosynthesis (a) and H₂ production during S deprivation (b). S depletion causes a drastic decrease of photosystem II (PSII) activity (indicated by the dotted line of the PSII symbol). In addition, the light harvesting complexes (LHCII) antennae are partially transferred to photosystem I (PSI) (state 2 transitions). The decreased O₂ evolution at PSII results in anaerobic conditions in a respiring, sealed algal culture, so that the hydrogenase (HYD) can become active. Besides residual PSII-activity, the oxidative degradation of organic substrates such as starch is an important electron source for H₂ production. The electrons derived from the latter process are probably transferred into the photosynthetic electron transport chain (PETC) by a plastidic NAD(P)H-dehydrogenase (NDH). The modified PETC of S-depleted algae allows the electron transport to continue so that the cells can generate ATP through photophosphorylation. Further abbreviations: ATP synthase (ATPase), cytochrome b ₆ f complex (Cytb ₆ f), ferredoxin (Fdx), ferredoxin-NADPH-reductase (FNR), plastidic terminal oxidase (PTOX), plastocyanine (PC), plastoquinone (PQ)

Fig. 2

a Development of in vitro hydrogenase activity in a concentrated C. reinhardtii culture sparged with Ar starting at 0 min. Samples of 200 μl containing the algal suspension were removed from the shaded incubation flask at the depicted time points and injected into an in vitro assay reaction mixture containing Triton X-100 used for cell lysis, and sodium dithionite reduced methyl viologen as an efficient, in vitro electron donor to FeFe-hydrogenases. After 15 min of incubation in a shaking water bath at 37°C, the headspace within the reaction vessel was analyzed by gas chromatography (GC). The detected amounts of H₂ were related to the chlorophyll concentration of the cells as an indication for the cell density and the incubation time. b Edge rolls bottles sealed with red rubber Suba Seals. The two left-side bottles contain aliquots of a C. reinhardtii culture (in vivo assays), the two right-side vessels are filled with in vitro assay reaction mixture having the typical deep blue color of reduced methylviologen

Fig. 3

a Development of the concentrations of H₂ (●), O₂ ( ), and CO₂ (○) as measured by MS in the headspace of an S-depleted C. reinhardtii culture incubated in squared glass bottles sealed with Suba seals upon one-site illumination as illustrated by the photograph in (b) (Hemschemeier 2005)

Fig. 4

a Schematic of a measuring chamber connected to the vacuum of an MS as is set-up in the CEA Cadarache. An aliquot (ca. 1.5 ml) of the algal suspension is injected into the measuring chamber of the Hansatech type where it is stirred by a little stir bar (not shown). Light can be applied by a fiber optic cable. Inhibitors such as DCMU can be applied by a syringe through the capillary of the lid. The bottom of the chamber is sealed by a thin gas-permeable Teflon membrane supported by a stainless steel frit. Gases dissolved in the cell suspension (indicated by white circles) can diffuse through the membrane and enter the ion source of the MS by a vacuum line. The addition of heavy isotopes can be applied to differentiate between respiration (uptake of ¹⁸O₂) and oxygenic photosynthesis (production of ¹⁶O₂), as well as between CO₂ assimilation (uptake of ¹³CO₂) and respiratory CO₂ production (¹²CO₂). The metabolism of D₂ is an indicator of the hydrogen metabolism and the hydrogenase turnover rate. b Schematic graph of the effect of DCMU on the in vivo H₂ -production rate of S-depleted C. reinhardtii cells as recorded utilizing the MS system depicted in (a). A stable H₂ graph indicates the instantaneous H₂ evolution rate of an illuminated, S-deprived algal culture. To define the contribution of photosynthetic water splitting to the electron supply of the hydrogenase, DCMU is added. The difference of the H₂-production rates before and after the addition of the PSII inhibitor is equivalent to the fraction of H₂ which is generated with electrons provided by PSII. To determine the low rate of dark H₂ production, light is turned off after the H₂ graph has stabilized.

Fig. 5

H₂-production measurements of S depleted green algae in the laboratory using gas traps. The gases produced by the algae are collected in inverted graduated cylinders via the water displacement method. Samples of the gas can be removed utilizing syringes with long and bended needles. As the cells pass into the H₂-producing phase, yields of H₂ can be measured directly from the volume of the water displaced in the graduated cylinders

Fig. 6

a Schematic process of using chromogenic sensors coated with thin layers of platinum and tungsten oxide to identify C. reinhardtii transformants having defects in the H₂-evolution pathway. The transformant colonies are grown until they form a dome-shaped colony of about 5 mm in diameter and are transferred into an anaerobic glove box in the dark to induce hydrogenase gene expression and activity, respectively. After 12 h, the chromogenic films are placed directly on the colonies. A short (about 3 min) illumination of the algae results in a sudden H₂ evolution depending on PSII activity. The H₂ gas is split by the platinum layer so that the H-atoms can interact with the tungsten oxide causing a blue color (shown in grayshade in b; photograph courtesy of Irene Kandlen). Algal clones with reduced or no H₂-production activity can be identified by a less-pronounced or absent coloration (marked by a white circle in b)

Fig. 7

Photograph of a 48-well plate after treating the wells according to the Winkler test. A deep blue color indicates that normal amounts of O₂ were dissolved in the culture medium, whereas the O₂ concentration was lower or very low in the light-blue or uncolored wells, respectively (photograph courtesy of Thilo Rühle)