1. Introduction
The ability of green algae to produce hydrogen in the light was discovered nearly 75 years ago by Gaffron and Rubin, 1942 [
1]. Since then, a variety of phototrophic microorganisms, including numerous species of
Chlamydomonas, have been shown to produce H
2.
Chlamydomonas reinhardtii (
C. reinhardtii) uses light for input energy and CO
2 as a feedstock to oxidize water to produce organic carbon compounds and nicotinamide adenine dinucleotide phosphate (NADPH) for energy. This creates a paradox for the production of biohydrogen from
C. reinhardtii since photosynthesis provides the building blocks for hydrogen gas production (protons and electrons) but also generates oxygen, an inhibitor of hydrogen gas production. Oxygen both inactivates the [Fe–Fe] hydrogenase enzyme that catalyzes H
2 production, and inhibits the transcription of the genes encoding the [Fe–Fe] hydrogenase enzyme [
2]. Hence in nature, the biological production of hydrogen is limited to a short burst that occurs when dark-adapted cultures are exposed to light. During the dark period, photosynthesis does not occur and the residual dissolved oxygen present in the surrounding aqueous environment is consumed by mitochondrial respiration [
3]. This leads to the transcription and translation of the hydrogenase enzyme, which when active, produces hydrogen for only a brief moment in response to light until oxygen is produced to inhibitory concentrations. Therefore, in order to produce biological hydrogen using
C. reinhardtii, specific strategies are required that separate photosynthesis and hydrogen production in time and/or space.
One approach to separate photosynthesis and hydrogen production in time involves the use of nutrient-deprivation. It has been shown that the absence of essential macronutrients and micronutrients in the culture environment, such as sulfur, nitrogen, phosphorus or magnesium leads to gradual inactivation of Photosystem II (PSII) [
4,
5,
6,
7]. Under these conditions, oxygen evolution ceases and residual oxygen is depleted through respiration. This process leads to anaerobiosis, which in turn induces the synthesis of hydrogenase with subsequent H
2 production. Moreover, nutrient starvation leads to the accumulation of carbohydrates, important for sustained hydrogen production in the long term [
8,
9]. It also leads to the inhibition of the Calvin–Benson cycle, thereby removing a significant electron sink and thus favoring hydrogen production [
10]. However, cells can only survive for a few days in nutrient-depleted medium and will eventually die. Additionally, it is generally thought that in order to obtain high levels of H
2 production by green algae, light conversion efficiencies will need to be increased. However, the degradation of PSII under nutrient-deprivation leads to a decrease in light conversion efficiencies, especially under high, natural, light conditions.
In the present study we wished to further examine the properties of a strain in which
psbD, encoding the D2 protein of PSII, is expressed under anaerobic conditions [
11,
12]. In order to develop an anaerobically inducible system for
psbD, the nucleus-encoded chloroplast Nac2 protein was used. This protein binds to the 74 nucleotide psbD 5′ UTR and is therefore necessary for processing and stable accumulation of psbD mRNA. In the cy6Nac2.49 construct, the Nac2 coding sequence is fused to the cytochrome c6 (Cyc6) promoter, whose expression is induced by anaerobiosis. Since the background of this strain is nac2-26 (a non-functional allele of nac2, which encodes a protein absolutely required for
psbD messengaer RNA maturation), the only functional Nac2 present is that produced from the construct under anaerobic conditions. In this system PSII synthesis can be regulated in a reversible manner while maintaining all other photosynthetic subunits active in the thylakoid membrane. The advantage of this system is that anaerobiosis can be achieved using cultures grown in nutrient-replete medium. Under these conditions, the cells should in principle remain healthy. This approach thus differs from the classical method in which PSII is inactivated through nutrient depletion, a condition that leads to impairment of cell growth and eventually to cell death. Here, we demonstrate that PSII controllable expression system can improve H
2 production in green algae without the application of nutrient deprivation, therefore avoiding limitations inherent in nutrient deprivation approaches.
3. Discussion
Green algae can grow under photoautotrophic, mixotrophic, photoheterotrophic and heterotrophic conditions [
16,
17]. Essential macro and micronutrient include sulfur, nitrogen, phosphorous, magnesium and trace elements like iron, copper, calcium, zinc, molybdenum, manganese and cobalt among others. In the absence of any of these nutrient, cell division is arrested and cultures stop growing [
18]. In the case of sulfur deprivation, when washed free of sulfur, cultures stop growing within the first 5–20 h after transition into S-deprived medium [
4,
19]. Hydrogen production stops after 4–5 days of sulfur deprivation, and the cells have a spherical morphology with a significant reduction in cell mass [
20]. Under phosphorous deprivation, cultures stop growing after 5 days, whereas H
2 production stops after 10–12 days [
5]. Nitrogen limitation causes cessation of cell growth after 2 days and H
2 evolution stops after approximately 7 days [
6]. In the case of magnesium deprivation, cultures stop growing after 7 days and H
2 production lasts for an additional 7 days [
7]. In all cases production of H
2 eventually stops, despite the continuing presence of energy reserves in the form of starch,
triacyl glycerides (TAGs), and acetate. This could be due to the toxic nature of the accumulated metabolites, or a result of the long-term consequences of nutrient deprivation [
21]. The exact events leading to the termination of hydrogen photo-evolution are not entirely known.
These disadvantages suggest that it will be difficult to develop a practical process for H
2 production in green algae using a nutrient depletion method. However, controllable expression of PSII could be used to reduce oxygen evolution to a rate below respiration, allowing cultures to naturally go anaerobic. The present work used cy6Nac2.49, a genetically modified strain of
C. reinhardtii that activates photosynthesis in a cyclic manner in such a manner that photosynthesis is not active constitutively in the presence of light, but is turned on only in response to a metabolic trigger, anaerobiosis [
11]. In this case the
nac2 gene, which stabilizes the mRNA of
psbD encoding the reaction center polypeptide D2 of PSII, is regulated by an anaerobically induced promoter (Cyc6). Hence when oxygen is absent, expression of the
nac2 gene is enabled and photosynthesis is activated [
11,
12]. Once the oxygen level reaches the threshold for the Cyc6 promoter, transcription ceases and photosynthesis only occurs for a period of time until the PsbD protein is turned over. Once the produced oxygen is consumed by respiration, hydrogenase expression occurs and hydrogenase functions until oxygen reaches a low enough level for the
nac2 gene to be reactivated for another round of photosynthesis [
11,
12].
H
2 production in regular TAP medium by the cy6Nac2.49 strain was examined under light/dark regime 10-h:14-h using two different light intensities, 10 and 50 W·m
−2. The maximal rate of H
2 production by the cy6Nac2.49 cultures was obtained at a light intensity of 10 W·m
−2 and was equal to ~0.9 mmol·L
−1, which is about 4.5 times higher than that obtained with wild-type cultures
Figure 3A. However, the level of H
2 in the cy6Nac2.49 strain at 50 W·m
−2 was about ~0.3 mmol·L
−1, slightly higher than what was produced by wild-type cultures
Figure 3A. The high levels of H
2 production in the cy6Nac2.49 strain indicate that it is able to sustain hydrogenase activity much longer than the wild-type, suggesting that driving
psbD expression by an anaerobically induced promoter keeps oxygen production below the compensation point [
11,
12].
Previous work has demonstrated that the inhibitory effect of high light intensity on H
2 photo-production is related to the enhanced O
2 evolution activity of PSII, with the fast build-up of the O
2 gas inactivating hydrogenase, stopping H
2 production [
22]. The optimal average light intensity for H
2 production was shown to be 30–40 µE·m
−2·s
−1, which is equal to about ~10 W·m
−2. Measurement of photosynthetic O
2 evolution by the cy6Nac2.49 strain and wild-type with a Clark type electrode demonstrated a higher rate of O
2 evolution in the cy6Nac2.49 strain at 50 W·m
−2 compared to that at 10 W·m
−2 (
Table 1)
. The level of photosynthetic O
2 evolution of the cy6Nac2.49 strain was about 10 times lower than that of the wild-type, indicating an effective turn-off of PSII activity in the cy6Nac2.49 strain by the Cyc6 promoter under nutrient-replete conditions (
Table 1)
. Additionally, the low photosynthetic yield of cy6Nac2.49 cultures, measured as the maximum fluorescence, also suggests turn-off of PSII activity
Figure 3B,C. As was mentioned above, maximum H
2 production in cy6Nac2.49 cultures, ~0.9 mmol·L
−1, was obtained in nutrient-replete conditions under a light/dark regime. For comparison, the hydrogen production of wild-type
C.reinhardtii under
S-deprivation is about ~3.27 mmol·L
−1 [
19]; under Mg-deprivation, ~6 mmol·L
−1 [
7]; under
P-deprivation, ~2.45 mmol·L
−1 [
5]; under
N-deprivation, ~1.5 mmol·L
−1 [
6]. Despite the lower level of H
2 production demonstrated in the cy6Nac2.49 strain cultures under nutrient-replete conditions in comparison with the amounts of H
2 obtained under nutrient-deprived conditions with wild-type cultures, application of controllable expression of PSII eliminates limitations associated with nutrient deprivation approaches. More importantly, under a light/dark regime H
2 production by the cy6Nac2.49 cultures increased in every subsequent light period, and by the end of the last light period was 4.5-fold higher than the wild-type cultures. From this perspective, the application of the cy6Nac2.49 strain in long-term experiments for H
2 production in fed-batch cultures is of future interest.
Further improvements of this system could come about by taking into account other factors besides the turn-off of PSII activity that could affect H2 production in Chlamydomonas. These could include considerations of starch accumulation, which serves as an additional source of electrons for hydrogen production, and the competition of hydrogen production with other electron sinks such as the Calvin–Benson cycle and cyclic electron flow. In principle genetic engineering could be applied to circumvent these limitations.
4. Materials and Methods
4.1. Cell Growth
Liquid cultures of C. reinhardtii strain cy6Nac2.49, kindly provided by Solarvest Bioenergy Inc., and its parental wild-type were cultivated photoheterotrophicaly, photomixotrophically, and autotrophicaly using, as appropriate, regular TAP and TP medium (Tris (2.42 g/L), phosphate solution (1 mL/L of K2HPO4 (288 g/L), KH2PO4 (144 g/L)), 1 mL/L of Hutner's trace elements). For autotrophic and photomixotrophic growth, the carbon dioxide concentration in the gas phase was adjusted, up to ~40%, at the beginning of the experiment by injecting pure carbon dioxide into the sealed 165 mL cylindrical vials which contained air. Vials were filled with 100 mL of culture and the pH was adjusted to 7.2, and the pressure in vials was equilibrated to atmospheric. As needed during cultivation, the pH was adjusted manually to 7.2. All cultures were shaken continuously on an orbital shaker (100 rpm) under continuous white light from LED lamps with an intensity of about 10 W·m−2 (approximately 48 μmol·m−2·s−1) or under a 10-h:14-h light/dark regime at two different light intensities, 10 and 50 W·m−2 (approximately 240 μmol·m−2·s−1). Growth was measured as an increase in cell density as monitored spectrophotometrically at 600 nm, a minimum in in vivo chlorophyll absorbance, and by measuring cellular chlorophyll (Chl) content. All of the experiments were carried out in triplicate and results are given as the mean + standard deviation.
4.2. H2 Production under a Light/Dark Regime
Liquid cultures of C. reinhardtii strain cy6Nac2.49 and the parental wild-type, grown as described above with TAP medium, were inoculated by injecting the pre-inoculum into fresh TAP medium and dilution to a specific Chl concentration. Cultures were incubated in sealed 165 mL cylindrical vials under an air atmosphere at the start of the experiment, each vial contained 100 mL of culture. After an initial dark period (14 h), cultures were exposed to continuous white light (LED) for 10 h, followed again with the periods of dark (14 h) and light (10 h), with a total experimental duration of 100 h. Two light intensities, 10 W·m−2 (48 μmol m−2·s−1 and 50 W·m−2 (240 μmol·m−2·s−1, were used. The gas phase in the vials during the light period was analyzed by gas chromatography using a Shimadzu GC-8A (Shimadzu, Nakagyo-ku, Kyoto, Japan) equipped with 1 m × 0.3715 cm column packed with molecular sieve 5A using argon as carrier gas; the temperature of the injector and column were 110 °C and 60 °C, respectively and the current was 70 mA. Carbon dioxide production was measured in the same gas chromatograph equipped with 80/100 Porapak Q (RestekTM, Fishersci, Ottawa, ON, Canada) column using helium as a carrier gas, the temperature of injector and column were 100 °C and 40 °C, respectively, and the current was 100 mA.
During the light/dark periods, the quantum fluorescence yield of the cy6Nac2.49 and wild-type strains was measured as well as growth, estimated as total Chl. Photosynthetic yields were analyzed using a Handy PEA (Hansatech Instruments version 1.06 connected to a sensor of the same manufacture, King’s Lynn, Norfolk, UK) during the light/dark regime. The measurements were made through the glass at the bottom of the sealed bottles. A saturating pulse (1500 µmol photons m
−2·s
−1) of light (duration of 1 s) was applied to record Fv/Fm, the maximum fluorescence yield in the dark periods, while δFv/Fm’ represents the maximum fluorescence yield recorded during periods of illumination [
23,
24].
4.3. Oxygen Evolution and Consumption
Maximal O2 evolution in the light and respiration in the dark were measured with a Clark-type O2 electrode (Hansatech Instruments, King’s Lynn, Norfolk, UK). Measurements were conducted at 25 °C on vigorously stirred samples of the wild-type and cy6Nac2.49 strains in TAP medium. O2 evolution was determined when cultures were illuminated at light intensities of 10 and 50 W·m−2. Respiration rates were determined in the dark prior to measurements of photosynthetic oxygen evolution in illuminated cultures.
4.4. Other Analytical Procedures
The chlorophyll concentrations of 95% ethanol cell extracts were measured spectrophotometrically by the method of Spreitzer [
25]. Samples were taken directly from vials with a sterile syringe and pelleted by centrifugation at 13000 rpm (MiniSpin, Eppendorf, NY, USA) for 3 min for starch, Chl and acetate measurements. The pellets and supernatants were separated and stored frozen at −20 °C until all samples were ready for processing. The amount of starch accumulated inside the cells was determined in the pellet according to the method developed by Gfeller and Gibbs (1984) [
26], except that ethanol was used instead of methanol for cell disruption and pigment extraction. Acetate concentrations in the supernatants were determined using a colorimetric assay kit (BioVision Inc., Milpitas, CA, USA).