J Appl Phycol (2012) 24:319–327 DOI 10.1007/s10811-011-9729-5

Outdoor cultivation of Chlamydomonas reinhardtii for photobiological hydrogen production Stephanie C. Geier & Sabine Huyer & Konstantin Praebst & Moritz Husmann & Christian Walter & Rainer Buchholz

Received: 9 August 2011 / Revised and accepted: 20 September 2011 / Published online: 22 November 2011 # Springer Science+Business Media B.V. 2011

Abstract Photobiological hydrogen production by the unicellular green alga Chlamydomonas reinhardtii has been studied under laboratory conditions to a vast extend but has not been investigated under outdoor conditions yet. Because the hydrogen-producing hydrogenase is very sensitive to oxygen, the production must be performed in a two-stage process: generation of the required algal biomass under oxygenic photosynthesis, followed by hydrogen biosynthesis under anaerobic conditions. In order to design a sustainable process, cultivation and subsequent hydrogen production under cost-free sunlight was investigated in this work for the first time. First, cells were grown in closed photobioreactors under simulated outdoor conditions according to the light intensities of an idealized summer day (up to 2,000 μmol photons m−2 s−1) in order to achieve results independent of varying, and therefore not reproducible, weather conditions. The following outdoor experiments showed comparable growth characteristics and similar cell densities. However, the use of cells grown under outdoor, simulated outdoor, or high light conditions generally resulted in significantly lower hydrogen yields compared to the use of cells cultivated under low and continuous irradiance. In order to lower cultivation costs during the growth phase, the use of 10% CO2 corresponding to the CO2 content of flue gas was investigated. By supplying additional CO2 during growth under the light profile corresponding to an idealized summer day, no significant increase of cell densities could be achieved, but S. C. Geier (*) : S. Huyer : K. Praebst : M. Husmann : C. Walter : R. Buchholz Institute of Bioprocess Engineering, Friedrich‐Alexander Universität Erlangen‐Nürnberg (FAU), Paul-Gordan-Strasse 3, 91052 Erlangen, Germany e-mail: [email protected]

the subsequent hydrogen production increased compared to hydrogen production of cells grown under atmospheric CO2. Keywords Hydrogen . Microalgae . Green algae . Chlamydomonas reinhardtii . Outdoor cultivation . Photobioreactor . Light profiles

Introduction Hydrogen is of major public interest as a clean and environmentally friendly energy source since its combustion only emits water. However, currently, hydrogen is produced by the consumption of fossil fuels or in energydemanding processes like steam reforming. An alternative way of sustainable and non-polluting production of hydrogen could be provided by use of the unicellular green alga Chlamydomonas reinhardtii, which produces hydrogen under anaerobic conditions via photosynthesis and the enzyme hydrogenase. The ability of green algae to produce hydrogen was discovered 70 years ago by Gaffron and Rubin (1942) who detected transiently small amounts of hydrogen after anaerobic incubation of microalgae in the dark. When adapted cells were illuminated, hydrogen evolution increased, suggesting that hydrogen production is linked to photosynthesis. However, hydrogen production under the influence of light lasts only for a short time as the released oxygen inhibits hydrogenase activity (Gaffron and Rubin 1942, Ghirardi et al. 1997). When Melis and coworkers demonstrated that oxygen evolution can be reduced by sulfate deprivation in C. reinhardtii, and thus hydrogen evolution can be prolonged up to 4–5 days at rates of 2 mL h−1 L−1 culture achieving a total hydrogen production of 140 mL L−1 (Melis et al.

320

2000), a further step forward to a practical application of biotechnological hydrogen production was taken. The isolation of the algal hydrogenase (Happe and Naber 1993) as well as many studies on hydrogen pathways (see Posewitz et al. 2009 for a detailed review) led to a deeper understanding of photobiological hydrogen production. All this basic research was performed under controlled laboratory conditions but, in order to fulfill the claim of an environmental friendly, sustainable, and economic process, the application of cost-free sunlight as energy source is inevitable. However, according to the current state of knowledge, only one short report is available on outdoor cultivation of Chlamydomonas sp. in Japan (Akano et al. 1996), but no experimental work on outdoor hydrogen production of C. reinhardtii has been published. Therefore, we investigated in the present study the cultivation of C. reinhardtii under sunlight and its suitability for hydrogen production. Under laboratory conditions, continuous white light with irradiances up to a maximum of 200 μmol photons m−2 s−1 is generally used for growth of C. reinhardtii as well as for the subsequent hydrogen production. In contrast, sunlight reaches in its maximum around midday tenfold of this photon flux density (PFD) compared to laboratory conditions. In addition to this high light stress, the cells are also subject to long dark periods without photosynthetic activity during the night thus losing their productivity for a significant period. In order to be independent of weather-related variations such as clouding, as well as daily and seasonal changes, and in order to achieve reproducible results, for initial experiments, the light course of an idealized summer day was simulated. In a second step, the photobioreactors (PBRs) were placed outside. The cells grown under both of these conditions were subsequently used for photobiological hydrogen production.

Materials and methods Investigations were carried out with the unicellular green alga C. reinhardtii SAG 83.81 (Experimental Phycology and Culture Collection of Algae of the University of Göttingen, Germany). The alga was cultured using tris-acetatephosphate (TAP) medium (Harris 1989) in PBR screening modules (PSMs) under monoseptic conditions at a temperature of 25°C (Fig. 1). PSMs are sterilizable bubble columns developed for microalgae cultivation with an inner diameter of 49 mm and a working volume of up to 0.9 L. Several PBRs can be used in parallel operation at once, allowing screening as well as optimization investigations by varying different cultivation parameters such as temperature, light

J Appl Phycol (2012) 24:319–327

a exhaust air including foam trap antifoam agent inoculation

sampling

glas tube cooling

supply air

b

Fig. 1 Photobioreactor screening modules: a schematic (modified after Naumann 2009), b set-up for outdoor cultivation of Chlamydomonas reinhardtii

intensity, or media components (König 2007). CO2 (3% or 10%) was added to the process gas (compressed air, 0.45 L min−1) according to the respective experimental requirements. When cultivated in the laboratory, the cylindrical reactor was surrounded by four compact fluorescence lamps (DULUX L 55W/840 2G11 LUMILUX Cool White, Osram, Germany) ensuring a high light yield (surface/volume ratio: 80 m2 m−3). In order to simulate the PFD according to natural sunlight, the irradiance was regulated corresponding to an idealized course of a summer day (Fig. 2) by the use of the

J Appl Phycol (2012) 24:319–327

Fig. 2 Photon flux density (PFD) over a day for Nuremberg, Germany during the summer (○ data from Bavarian State Research Center for Agriculture for the 15th July 2008) and simulated light curve for experiments in photobioreactor screening modules (▼). The dashed line indicates maximum irradiances applied for standard laboratory cultivations

software WinDim (Tridonic, Austria). The required PFD up to 2,000 μmol photons m−2 s−1 could be reached by lining the PSM surrounding chambers with a special reflective foil (Diffuse Light Reflector, DuPont, Luxembourg). Temperature control of the PBR was realized by cooling tubes which were connected to a thermostat. Temperature in the algae suspension was measured with an infrared thermometer (IR-1600A, Voltcraft, Germany). For outdoor cultivation, the PSMs were set up in the courtyard of the institute (Fig. 1b). The PSMs were placed in a holding rack 80 cm above the ground. Sun was available from all sides except in the very early morning when the building caused some shading. In order to avoid high cell-specific irradiances when cell densities are still low, cultures were inoculated in the late afternoon. Irradiance and air temperature were recorded with a LiCor Datalogger Li-1400 in combination with the Quantum Sensor Li-190SA (Li-Cor Biosciences, Germany). Culture density was measured by cell counting with a Neubauer hemacytometer. Lugol’s iodine was used to fix the cells. The optical density of the cultures was determined at 750 nm using an UV–VIS spectrometer. Hydrogen production For standard experimental setup, the cells were grown photoheterotrophically in TAP medium (Harris 1989) under continuous cool-white fluorescence lamps in Erlenmeyer flasks at 90 μmol photons m−2 s−1 in a shaking incubator at 25°C, 100 rpm and 3% CO2. In order to induce hydrogen production, cells were transferred into sulfate-deprived conditions. Therefore, cells were grown to the stationary

321

phase (about 1.2×107 cells mL−1). In case of the cultures under simulated light profiles or cultivated outdoors, cells were harvested 4–7 h after light onset on the third day by centrifugation (2,000×g, 15 min). The cells were resuspended in 30 mL medium, transferred into 50 mL reaction tubes and centrifuged (2,000×g, 7 min) again. The now firm pellet was resuspended in 40 mL sulfate-free TAP medium per tube. In the sulfate-free TAP medium, all sulfates were replaced with chloride salts at the same molar concentrations and the Hutner’s trace elements solution was omitted. The pH was adjusted to 7.7 according to Kosourov et al. (2003). The concentrated algae suspensions of all tubes were combined and the exact cell density of this cell concentrate was determined. To accomplish the required final cell density in the PBR for hydrogen production (H2PBR) of 7–9×106 cells mL−1, the necessary volume of the sulfate-deprived cell concentrate was calculated and transferred to the H2-PBR, which was prefilled with sulfate-free TAP medium. The H2-PBR consisted of commercially available screw neck glass bottles with an inner diameter of 52 mm (designed for a volume of use of 100 mL) and 82 mm (designed for a volume of use of 500 mL). In order to minimize oxygen containing headspace to a few mL, the H2-PBRs were filled with algal suspension to a maximum of 126 or 593 mL, respectively. Final cell densities in the H2-PBR were determined again and corrected when necessary. With this method, sulfate concentrations below the detection threshold of 1 mg L−1 were achieved. Measurements of sulfate concentrations were done according to DIN 38405-D5 and DIN EN ISO10304-2-D20. The H2-PBRs were then sealed with a custom-build polyoxymethylen plug with a Viton o-ring, in which a stainless steel capillary as an off-gas port was pressed through. A perfluoralkoxy tube (Chromatographie Handel Müller, Germany) with an inner diameter of 1/16 inch was attached to this port and placed into an upside down cylinder filled with water. The H2-PBRs were kept for up to 180 h under continuous illumination from one side at 200 μmol photons m−2 s−1 and 21°C room temperature. Determination of the concentrations of produced hydrogen Gas evolved by the algae in the H2-PBRs was conducted via PFA tubing, attached to the gas ports, leading into an upside down graduated glass cylinder filled with water. During the experiment, the volume of the produced gas was measured directly by the volume of the displaced water in the graduated cylinders. Quantification of produced hydrogen in the off-gas was performed at the end of the hydrogen production phase by a BCP-H2 sensor (BlueSens, Germany). Before each set of measurements, the sensor was purged with N2, set to zero and checked with test gas (Linde, Germany).

322

Results Cultivation under simulated outdoor light conditions In order to achieve reproducible results, algal growth was first investigated in the laboratory under the simulation of light profiles according to an idealized summer day in mid Europe (Fig. 3). TAP medium was used as growth medium in all experiments, because only photoheterotrophically cultivated cells reach relevant hydrogen yields (Kosourov et al. 2007). The temperature was kept at 25°C. In these experiments, the growth pattern of C. reinhardtii (Fig. 3) differed significantly from the logistic growth behavior of cells grown under continuous illumination up to 1,000 μmol photons m−2 s−1 (Geier 2011). In contrast to cultures under continuous light, the cultures grown under the simulated summer day showed an increase of cell density mainly during the dark periods. During the day, cell counts stayed constant with few changes, but in the second and third nights, cell densities increased rapidly. The cell densities increased from cell counts of 8×105 cells mL−1 at the beginning of the respective second night periods to cell densities in the range of 3×106 cells mL−1 at their ends. The second rapid increase led to a fourfold increase from these cell densities to cell counts in the range of 1.2×107 cells mL−1 within 10 h in the respective third night periods. The cell densities decreased marginally with the onset of the fourth light period. Because CO2 is essential for photosynthesis and can be provided in the form of abundantly available flue gases, the use of 10% CO2 corresponding to the average CO2 content

Fig. 3 Growth of Chlamydomonas reinhardtii in photobioreactor screening modules under controlled laboratory conditions and simulation of the light profile of an idealized summer day. Culture ‘simulation 1’ (○ single batch) and ‘simulation 2’ (□ single batch) were bubbled with air. ‘Simulation 3’ (▼) was bubbled with 10% CO2 and run as triplicates

J Appl Phycol (2012) 24:319–327

of flue gas (Negoro et al. 1992; Doucha et al. 2005) was investigated. The cultures bubbled with 10% CO2 showed the same growth pattern as the cultures under the simulated summer day and reached marginally higher maximum cell densities (1.7×107 cells mL−1; Fig. 3) as compared to the maximum achieved cell densities in air-bubbled cultures (1.4×107 cells mL−1; Fig. 3, simulation 2). Outdoor cultivation In contrast to the simulated experiments under laboratory conditions, temperature and irradiance can vary significantly during the course of a day in the case of real outdoor cultivation. Therefore, PBRs were placed outdoors in the subsequent step. In order to investigate the influence of varying temperature besides the influence of fluctuating irradiance during the cultivation, two PBR were operated at 25°C and one was not temperature-controlled (Fig. 4a). Because additional CO2 in the process gas had only a minor impact on cell densities (Geier 2011), the cultures were bubbled with air. During the second half of the first day, the cultures operated at 25°C showed marginally higher cell counts than the culture without temperature control, which heated up to 44°C (temperature in the algal suspension) at midday of the first day. Due to insufficient cooling capacities, the cultures under temperature control also heated up but reached only a maximum of 32°C in the algal suspension at midday. From the second day (t=38°C) onward, the cell density of the culture without temperature control did not differ from the cultures operated at 25°C, although the temperature of the former culture heated up to 40°C at midday on the second day and 37°C at midday on the third day. Cultures under temperature control only reached 32°C at midday on the second day and 30°C at midday on the third day. Cooling of the air temperature to 12°C in the night (Fig. 4b) did not have any obvious influence on growth in the case of the cultures without temperature control. During the second day, the cell density of all cultures decreased. After a rapid increase in cell counts during the third night, all cultures achieved similar maximum cell densities of about 1.2×107 cells mL−1 at the fourth day (t=60 h). In order to investigate a potential daily harvest of the biomass, one culture, with a cell density of 1.3×107 cells mL−1, was depleted after 60 h to 30% of the filling volume and was refilled with fresh medium to the filling volume before subharvest (repeated batch). Within 20 h, the cell density reached 76% of the value measured before the subharvest, with 1.0×107 cells mL−1. The reference batch operated at 25°C, which was not harvested, reached at t=60 h a maximum cell density of 1.2×107 cells mL−1, the batch without temperature control achieved an insignificantly lower cell count with 1.1×107 cells mL−1.

J Appl Phycol (2012) 24:319–327

a

b

323

hydrogen production, cultures showed four distinct phases: an aerobic phase with evolution of photosynthetic oxygen, a lag phase without any gas evolution, during which oxygen is consumed, and a hydrogen production phase followed by a final dying of the cells. Cells routinely grown in Erlenmeyer flasks at 90 μmol photons m−2 s−1 and transferred into H2-PBR with fresh sulfate-free media for hydrogen production followed the gas production pattern of these characteristic phases (Fig. 5; reference): within the first 20 h after transfer into sulfate-free media, the cells showed a first gas evolution due to photosynthesis activity. After several hours, a second phase of gas evolution began in which a total of 86 mL L−1 hydrogen gas was produced. In contrast to this, gas and related hydrogen production of cells obtained from cultures bubbled with air and grown under simulated summer light profiles as well as cells grown outdoors repeatedly resulted in very low volumes, which were not sufficient for hydrogen determination (data not shown). In order to investigate this phenomenon, in a first step hydrogen production was performed with cells grown at 3% CO2 under different irradiances. In a second experiment, the influence of CO2 supply during the growth phase on hydrogen production was investigated using cells grown bubbled with 10%CO2.

Fig. 4 Outdoor cultivation of Chlamydomonas reinhardtii in photobioreactor screening modules: a growth of cultures under temperature control (○ ‘outdoor 1,’ □ ‘outdoor 2’) and of culture without temperature control (▼ ‘outdoor 3’). Culture ‘outdoor 2’ (□) was harvested at t=60 h and refilled with fresh medium (all cultures were run as single batches). b air temperature (○) and photon flux density (PFD, ●) during outdoor cultivation

The cultures under outdoor conditions (Fig. 4a) showed similar growth to that of the cultures grown under simulated light profiles under controlled laboratory conditions (Fig. 3). During the first day, the cells of all cultures hardly propagated. Over the second and third nights, the cell densities increased rapidly. The cell densities of the outdoor cultures and the lab cultures grown under simulated light profiles decreased during the second light period. During the light phases, the outdoor cultures reached higher cell counts compared to the cultures grown under simulated light profiles. Hydrogen production In order to induce hydrogen production, harvested cells were transferred from growth medium into sulfate-free medium. During the adaption to sulfate deprivation and the subsequent

Fig. 5 Gas evolution depending on cultivation conditions during growth phase. Cells routinely cultivated at 90 μmol photons m−2 s−1 and 3% CO2 in shaker flasks (■ single batch, ● run in duplicates) were used as a reference. When cultures were grown at 200 μmol photons m−2 s−1 and 3% CO2 in photobioreactor screening modules (○ duplicates), gas production was significantly less. Gas production and hydrogen yield (see also Table 1) could be increased by bubbling with 10% CO2 during previous growth (□ duplicates). Cells grown under the light profile of a summer day bubbled with air or 3% CO2, respectively, generally yielded in only small off-gas volumes, but when bubbled with 10% CO2 (▼ single batch), collected volume of off-gas increased. Previous experiments showed that the standard error between multiple reference batches independently run from each other account for 12% (Geier 2011)

324

Fig. 6 Change of cell size (● determined by cell-specific scattering) during cultivation under the light profile of a summer day. During the day, cells size increased until the early afternoon. After cell division is completed in the beginning of the night, cell size significantly declined again. For better comparison, growth of ‘simulation 2’ is given (□)

Hydrogen production of the cells previously grown under the reference conditions (Erlenmeyer flasks at 90 μmol photons m−2 s−1 and 3% CO2) started at 15 h and 24 h, respectively, after a short period of photosynthetic gas evolution (Fig. 6). Cells grown in PSMs at 200 μmol photons m−2 s−1 and 3% CO2 did not produce hydrogen until 43 h, i.e. double the adaption time of the cells grown under low light. Cells grown at 10% CO2 and 200 μmol photons m−2 s−1 required up to thrice the adaption time (64 h) as compared to the cells grown under reference conditions in flasks, but produced hydrogen at a higher initial rate and for a longer time period than the cells grown at 3% CO2 and 200 μmol photons m−2 s−1. When cells for hydrogen production were grown under a simulated light profile of a summer day but with 10% CO2, a very small gas volume was produced. An overview of the produced amounts of hydrogen is given in Table 1. Hydrogen production of cells grown at 200 μmol photons m−2 s−1 could be more than doubled from 20 mL L−1 to 48 mL L−1 hydrogen when cells were grown at 10% CO2 compared to growth at 3% CO2. However, when cells were cultivated under the light profile of a summer day at 10% CO2, produced gas volumes were significantly lower and contained only 8.5 mL L−1 hydrogen.

Discussion The growth patterns of cells cultivated under a simulated summer light profile and under outdoor conditions (Figs. 3 and 4a) differ significantly from the logistic growth behavior of cells grown under continuous illumination

J Appl Phycol (2012) 24:319–327

(data not shown, see Geier 2011). This growth pattern is characteristic for Chlamydomonas underlying light–dark cycles. At the beginning of the dark period, several cell divisions are induced within hours, but daughter cells are kept in the mother cell until all divisions are completed. After a few hours of the dark period, all cells are released simultaneously (Harris 1989), which explains the rapid increase in cell counts, in particular, during the second and the third nights. The associated increase in cell size during the light period and the rapid decrease before cell division is shown in Fig. 6. Under optimal growth conditions in the laboratory, cells under dark–light cycles divide three times so that one mother cell results in eight daughter cells (Harris 2009). In the outdoor experiments as well as in the cultivations under simulated light profiles of a summer day, cell densities increased approximately by the fourfold, indicating that these conditions only allowed the division of a mother cell into four daughter cells. Cell densities of the outdoor cultivations, as well as the cultures under simulated light profile of a summer day, showed a minor decline in cell counts during the second half of the light period due to the high irradiances of up to 2,000 μmol photons m−2 s−1 during midday which were reached in the experiments under simulated light profiles as well as in the outdoor cultures. When light adsorption exceeds the photosynthetic capacity, electrons are directly transferred to oxygen because NADP+ is not sufficiently present in the oxidized form. The resulting reactive oxygen species (ROS) can cause further ROS in the cell and eventually lead to cell death (Niyogi 2009), which is indicated by the decreasing cell counts during the second half of the day. Here, differences are identifiable between the cultivation sets: the outdoor cultures reach higher cell densities during the day compared to the cultures under simulated light profile, which can be attributed to the higher photosynthetic activity of the cells under the higher temperatures outdoors (Richmond 1984). With increase of temperature toward the optimum, cells are less exposed to photodamage by unused photons and electrons due to the higher rate of utilized photons. In addition to the beneficial effect of temperature, the outdoor cultures were not continuously exposed to irradiance peaks due to clouding as was the case during the second halves of the second and third day of cultivation (Fig. 4b), hence reaching marginally higher cell densities. However, suboptimal temperatures (≥40°C, depending on species and growth conditions) accelerate the damage of PSII due to a decreased rate of photosynthesis (Lawlor 1990). This negative influence of high temperature on growth is obvious in the outdoor cultures without temperature control during the first day, as the cell density is still very low (Fig. 4a) and the algal suspension reached up to 42°C and the high cell specific radiation was a higher impact.

J Appl Phycol (2012) 24:319–327

325

Table 1 Hydrogen production depending on cultivation parameters during growth (see also Fig. 5) Hydrogen production (continuous light, 200 μmol photons m−2 s−1)

Conditions during growth PBR

Light (μmol photons m−2 s−1)

CO2 (vol.%)

Hydrogen (mL L−1)

Flask PSM PSM PSM PSM

90 200 200 ≤2,000 ≤2,000

3% 3% 10% Air 3%

86.0 (±0.03%) 86 1.00 19.8 (±32%) 86 0.23 48.0 (±17%) 132 0.36 H2 could not be determined due to small off-gas volumes H2 could not be determined due to small off-gas volumes

PSM

≤2,000

10%

8.5

Process time (h)

87

Space-time yield (mL L−1 h−1)

0.09

All presented batches were run as duplicates except of the hydrogen production with cells grown with 10% CO2; standard error is given in %. PBR Photobioreactor, PSM photobioreactor screening module

Most laboratory strains of C. reinhardtii adapt to irradiances of up to 1,000 μmol photons m−2 s−1, but bleach at higher irradiances and eventually die (Niyogi 2009). However, the simulation of light intensities according to a summer day and the outdoor cultivation with peak values of 2,000 μmol photons m−2 s−1 demonstrates that C. reinhardtii can endure this photo stress and thus can be cultivated under high irradiances. The course of growth under outdoor conditions correlates closely with growth under simulated light conditions, demonstrating that the simulation of light profiles in PSMs is a practical tool in order to investigate outdoor cultivation independent of seasons and field work. Under both simulated and real outdoor conditions, a cell density of 1.2×107 cells mL−1 was reached, which is sufficient for hydrogen production. Furthermore, it could be shown that a repeated batch with a daily harvest of 70% of the reactor volume is feasible. Hydrogen production Cells grown under simulated light profiles of a summer day or cultivated outdoors produced only very little gas volumes in the subsequent experiments for hydrogen production compared to cells grown at continuous illumination and lower irradiances. The relatively slight increase of light intensity from 90 μmol photons m−2 s−1 to 200 μmol photons m−2 s−1 during growth resulted in a decline of hydrogen production by 76% (Table 1). In addition, with increasing maximum irradiances during growth, the onset of hydrogen production is delayed. Tsygankov and coworkers (2002) observed a similar effect of adaption to the irradiances during the growth phase. Cells cultivated under higher irradiances (120 μmol photons m−2 s−1) during the growth stage than during the stage of hydrogen production, showed no hydrogen production when cells were illuminated with 20 μmol photons m−2 s−1 during sulfate deprivation or produced significantly less hydrogen when cells were

illuminated with 110 μmol photons m−2 s−1. Highest hydrogen production was reached when cells grown at 25 μmol photons m −2 s −1 were subject to sulfate deprivation at 110 μmol photons m−2 s−1 (Tsygankov et al. 2006). These authors also reported that anaerobic conditions developed later, when cells were grown under similar or higher light intensities compared to light intensities during hydrogen production (Tsygankov et al. 2006). Because photodamage of D1, which is an essential protein of the water-splitting reaction at PSII, increases at linear correlation with increasing light (Niyogi 2009), and the rate of D1 repair is reduced due to sulfate deprivation (Wykoff et al. 1998), it can be assumed that cells, which are adapted to lower light during growth, experience an additional photoinhibition of D1 due to higher light intensities under sulfate-deprived conditions than during growth (Tsygankov et al. 2006). Chlamydomonas reinhardtii, like other algae, adjusts its photosynthesis apparatus according to the ambient light condition. However, in contrast to other green algae, such as Chlorella or Dunaliella, C. reinhardtii mainly increases the number of PSII with increasing light, leading to a doubling of the ratio of PSII:PSI at 400 μmol photons m−2 s−1 compared to 47 μmol photons m−2 s−1 (Neale and Melis 1986). Because of this effect of light adaption during growth, adapted cells might produce more oxygen or over a longer period due to the higher quantity of PSII. Consequently, the rate of oxygen evolution exceeds the rate of respiration which is not affected by sulfate deprivation (Melis et al. 2000; Cao et al. 2001), so that anaerobic conditions are established later. Assuming a steady reduced rate of the water-splitting reaction, a higher overall oxygen concentration might be present in the reactor due to a larger proportion of PSII and the consequently higher oxygen release in the case of the high light-adapted cells. As a result, the oxygen sensitive hydrogenase does not achieve its full activity because its activity is already reduced to 50% below 1% molecular oxygen (Ghirardi et al. 1997).

326

Hydrogen production in C. reinhardtii depends not only on the anaerobic expression of hydrogenase but also on the supply of sufficient amounts of substrate in the form of electrons and protons. One source of the required protons and electrons is the remaining water-splitting activity of PSII, which passes electrons via the electron transport chain to PSI and eventually to the hydrogenase. The other source of substrate is the degradation of endogenous starch, which induces electrons into the photosynthetic electron transport chain via chlororespiration (Gfeller and Gibbs 1984; Gibbs et al. 1986). In a previous study under continuous light of 200 μmol photons m−2 s−1, the cell-specific accumulation of starch significantly increased tenfold, when CO2 was increased from atmospheric levels to 3% CO2 (data not shown). In order to increase hydrogen production by promoting starch accumulation, the CO2 content of the process gas during growth at 200 μmol photons m−2 s−1 was increased from 3 to 10% CO2, which led to more than double the amount of hydrogen (Table 1). As cultures under strong light require higher amounts of inorganic carbon in order to build up a relevant quantity of starch (Tsygankov et al. 2006), it can be assumed that the increased CO2 availability to the cells during growth led to higher starch accumulation and thus increased the rate of gas evolution and prolonged hydrogen production. However, when cells are grown under the light profile of a summer day at 10% CO2, the subsequently produced gas volumes were significantly lower and contained only small amounts of hydrogen which is probably due to the combined impact of starch consumption during the dark phase and adaption to high light during growth. However, bearing in mind that hydrogen production of cells from cultures bubbled with air grown under simulated light profiles of an idealized summer day as well as grown outdoors repeatedly resulted in very low volumes, the achievement of detectable hydrogen amounts of cells grown at 10% CO2 could be considered as a vital improvement. Tsygankov et al. (2002) showed that accumulation of starch varies during growth under light–dark cycles and that cells harvested after 4 h of light showed the highest hydrogen production due to a high starch accumulation. The later the cells were transferred from growth under light to hydrogen production, the less hydrogen was produced. In the present study, cells were harvested 4–7 h after light onset. As the transfer method to sulfate-free medium further required 1–2 h, it is possible that the time of harvest or the starch content at start of the hydrogen production, respectively, was not optimal. By determining the maximum starch content during outdoor growth and investigating the impact of bubbling with high CO2 concentrations on starch contents, time of harvest could potentially be optimized further and thus improve hydrogen production. Besides the influence of CO2 and light intensity, also heat stress during

J Appl Phycol (2012) 24:319–327

growth might influence the accumulation of starch and metabolism, in general, and therefore have an impact on the ability to produce hydrogen. In conclusion, the outdoor cultivation of C. reinhardtii could be demonstrated for the first time. In the subsequently performed hydrogen production under controlled laboratory conditions, only a maximum of 10% of hydrogen amounts produced by cells grown under laboratory conditions was reached. Further research will be required to investigate whether this is due to the high irradiances and high temperature at midday or due to unfavorable starch contents or a combination of all. Because hydrogen production is inhibited under irradiances above 300 μmol photons m−2 s−1 (Kim et al. 2006; Giannelli et al. 2009; Geier 2011), an additional future challenge lies within the design of a specific PBR or process for outdoor hydrogen production under irradiances up to 2,000 μmol photons m−2 s−1 in order to overcome this hurdle and produce hydrogen under economic and sustainable aspects. Acknowledgement The financial support of this research was provided by the Bavarian State Ministry of the Environment and Public Health (StMUG).

References Akano T, Miura Y, Fukatsu K, Miysaka H, Ikuta Y, Matsumoto H, Hamasaki A, Shioji N, Mizoguchi T, Yagi K, Maeda L (1996) Hydrogen production by photosynthetic microorganisms. Appl Biochem Biotechnol 57–58:677–688 Cao H, Zhang L, Melis A (2001) Bioenergetic and metabolic processes for the survival of sulfur-deprived Dunaliella salina (Chlorophyta). J Appl Phycol 13:25–34 Doucha J, Straka F, Lívanský K (2005) Utilization of flue gas for cultivation of microalgae Chlorella sp. in an outdoor open thinlayer photobioreactor. J Appl Phycol 17:403–412 Gaffron H, Rubin J (1942) Fermentative and photochemical production and hydrogen on algae. J Gen Physiol 26:219–240 Geier SC (2011) Photobiologische Wasserstoffproduktion mit der einzelligen Grünalge Chlamydomonas reinhardtii unter verfahrenstechnischen Aspekten. Dissertation, University of Erlangen-Nuremberg Gfeller RP, Gibbs M (1984) Fermentative metabolism of Chlamydomonas reinhardtii: I. Analysis of fermentative products from starch in dark and light. Plant Physiol 75:212–218 Ghirardi ML, Togasaki RK, Seibert M (1997) Oxygen sensitivity of algal H2-production. Appl Biochem Biotechnol 63–65:141– 152 Giannelli L, Scoma A, Torzillo G (2009) Interplay between light intensity, chlorophyll concentration and culture mixing on the hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors. Biotechnol Bioeng 104:76–90 Gibbs M, Gfeller RP, Chen C (1986) Fermentative metabolism of Chlamydomonas reinhardtii: III. Photoassimilation of acetate. Plant Physiol 82:160–166 Happe T, Naber JD (1993) Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii. Eur J Biochem 214:475–481

J Appl Phycol (2012) 24:319–327 Harris EH (ed) (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic, San Diego Harris EH (2009) Cell division. In: Harris EH (ed) The Chlamydomonas sourcebook: Volume1. Introduction to Chlamydomonas and its laboratory use, 2nd edn. Academic, San Diego, pp 65–88 Kim J, Kang CD, Park TH, Kim MS, Sim SJ (2006) Enhanced hydrogen production by controlling light intensity in sulfurdeprived Chlamydomonas reinhardtii culture. Int J Hydrogen Energy 31:1585–1590 König T (2007) Gewinnung und Charakterisierung antiviraler Wirkstoffe aus aquatischen Mikroorganismen. Dissertation, University of Erlangen-Nuremberg Kosourov S, Seibert M, Ghirardi ML (2003) Effects of extracellular pH on the metabolic pathways in sulphur-deprived, H2-producing Chlamydomonas reinhardtii cultures. Plant Cell Physiol 44:146– 155 Kosourov S, Patrusheva E, Ghirardi ML, Seibert M, Tsygankov A (2007) A comparison of hydrogen photoproduction by sulphurdeprived Chlamydomonas reinhardtii under different growth conditions. J Biotechnol 128:776–787 Lawlor DW (1990) Photosynthese: Stoffwechsel-Kontrolle-Physiologie. Georg Thieme Verlag, Stuttgart Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M (2000) Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol 122:127–135 Naumann I (2009) Sulfoquinovosyldiacylglyceride—antiviral aktive Substanzen. Dissertation, University of Erlangen-Nuremberg

327 Neale PJ, Melis A (1986) Algal photosynthetic membrane complexes and the photosynthetic-irradiance curve: a comparison of light-adaption responses in Chlamydomonas reinhardtii (Chlorophyta). J Phycol 22:531–538 Negoro M, Shioji N, Ikuta Y, Makita T, Uchiumi M (1992) Growth characteristics of microalgae in high-concentration CO2 gas, effects of culture medium trace components, and impurities thereon. Appl Biochem Biotechnol 34–35:681–692 Niyogi KK (2009) Photoprotection and high light response. In: Stern DB (ed) The Chlamydomonas sourcebook: organellar and metabolic processes, 2nd edn. Academic, San Diego, pp 847–870 Posewitz MC, Dubini A, Meuser JE, Seibert M, Ghirardi ML (2009) Hydrogenases, hydrogen production, and anoxia. In: Stern DB (ed) The Chlamydomonas sourcebook: organellar and metabolic processes, 2nd edn. Academic, San Diego, pp 217–255 Richmond A (1984) Cell response to environmental factors. In: Richmond A (ed) Handbook of microalgal mass cultivation. CRC Press, Boca Raton, pp 69–99 Tsygankov A, Kosourov S, Seibert M, Ghirardi ML (2002) Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures. Int J Hydrogen Energy 27:1239–1244 Tsygankov A, Kosourov S, Tolstygina IV, Ghirardi ML, Seibert M (2006) Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int J Hydrogen Energy 31:1574–1584 Wykoff DD, Davies JP, Melis A, Grossman AR (1998) The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii. Plant Physiol 117:129–139