Plastic bags as simple photobioreactors for cyanobacterial hydrogen production outdoors in Moscow region

The strain used was a mutant strain of cyanobacteria Anabaena sp. PCC 7120 (ΔHup), created by disrupting the uptake hydrogenase gene hupL [21], and provided by Prof. Inoue, K., Kanagawa University, Japan.

The inoculum was grown photo-autotrophically in 300-mL Erlenmeyer flasks in Allen–Arnon medium [22] diluted 8 times (AA/8) supplemented with 5 mM TES-KOH buffer (pH 8.2), without adding bound nitrogen but with the addition of streptomycin and spectinomycin, 2 μg mL⁻¹ each [21]. The cultivation was carried out at 27 °C in light (30 μmol photon m⁻² s⁻¹) under air with shaking at 95 rpm.

For experiments, culture (~ 14 L) was grown under the conditions indicated above (except shaking). A set of bottles (1.7 L) were sparged with air + 1% CO₂ through sterile membrane filters with a pore size of 0.2 μm (Pall, Ann Arbor, MI, USA).

To study H₂ production, cyanobacteria were placed in PhBRs—36 cm × 50 cm flexible plastic Be-P bags [20], which have a polyacrylate gas barrier layer. In the first experiment, each PhBR was a flat bag with two ports: for culture and gas sampling [20]. In the second experiment, a rigid internal stainless steel frame was placed inside the bag, increasing the height of the bag to ensure the gas flow from one PhBR to another. This design was equipped with one port for culture sampling and two ports for gas inlet and outlet. Before the start of the experiment, the bags were autoclaved.

The experimental device (Fig. 1) consisted of a water bath to smooth out temperature fluctuations; stabilizing belts to fix the bags, temperature and light sensors connected to the QMbox modular measuring system (RG Tekhnoloji, Russia), and computer to record data. Temperature sensors were submerged in the water bath.

In the Experiment #1, the grown cells were centrifuged (900g, 2 min) and suspended in AA/8-N medium at Chl concentration of 23.3 mg L⁻¹. The volume of the culture in each PhBR was 1 L. The gas phase (3 L) was replaced with Ar supplemented with 5% CO₂, and 3.3% of N₂ was added to the PhBR 1. The cultivation was carried out in Central Russia (Moscow region, 54° 50′ N and 37° 37′ E) from July 20–25, 2016 under natural light and temperature (on the roof of a four-story building).

In the Experiment #2, the culture was not concentrated, but previously adapted to high-light intensities by gradual increase from 30 to 700 μmol photon m⁻² s⁻¹ within 4 days. The outdoor experiments began under reduced light intensity using light filters. The light filter consisted of several layers of netting, which reduced light intensity 2–7 times (the coefficient was determined in laboratory tests). Later, the number of layers varied, providing different light intensities in PhBR 3 and PhBR 4. The volume of culture in the PhBR was 1 L with initial Chl content ~ 4.3 mg L⁻¹, the volume of the gas phase being 3 L. Air supplemented with 0.5–1.0% CO₂ was successively sparged through PhBR 3 and 4 at 20–100 mL min⁻¹. The cultivation was performed outdoors in the same place, August 18–23, 2016 (Fig. 2).

In the Experiment #1, gas phase was analyzed twice a day: at 8:00 and 20:00. H₂ yield and production rate were calculated based on its concentration and the volume of the gas phase of PhBR. Calculations referred to the morning (5:00–8:00), daytime (8:00–20:00), daylight (the whole light period 5:00–20:00) or the whole day (20:00–20:00). Since no H₂ measurements were taken at 5:00, we assumed that at night, there was neither H₂ production nor consumption, therefore, the H₂ concentration obtained at 20:00 the previous day was assumed to be equal to the H₂ concentration at 5:00 the next day.

In the Experiment #2, the rate of H₂ evolution was calculated based on its concentration and the air flow rate, so the H₂ accumulation was further calculated. In this case, we assumed that H₂ concentration at night was zero due to the fast removal of previously formed H₂ with air flow. The current rates of H₂ evolution or averaged rates were calculated during morning, daytime, daylight or the whole day (as indicated in the captions to the figures and tables).

The concentration of H₂ in the gas phase was measured at 22 °C by gas chromatography (LHM80, Moscow, Russia), equipped with a thermoconductivity detector and a 1 m × 3 mm with molecular sieve column (running temperature, 40 °C). Argon was used as the gas carrier [23]. Gas samples (0.5 mL) were withdrawn from PhBR using locked syringes and transferred to chromatograph. Hydrogen concentration was calculated on the basis of calibration using ECOCHROM program.

Chl a concentration was measured spectrophotometrically at 664 nm (U–VIS 1240 Mini, Shimadzu, Japan) after extraction with 85% methanol. The light intensity (photosynthetically active radiation, 400–700 nm) was determined with a quantum meter (Quantum Meter, model: QMSW-SS, USA). To check pH value, 5 mL of culture was taken through the culture port using a sterile syringe. If necessary, pH was adjusted to 8.2 with 0.5 M NaHCO₃. In both experiments, the culture was mixed manually by gently shaking the bag twice a day during sampling. The axenity of cyanobacteria culture was checked by light microscopy (LOMO MIKMED-2, Russia).

Statistical data processing was performed in the SigmaPlot 11.0 program. The average values of the results ± 95% confidence interval are presented.

Continuous monitoring of light intensity and temperature during the two experiments allowed us to characterize weather conditions. During Experiment #1 (Fig. 3a), the average daily temperature was 21.7 ± 0.4, maximal 31.4, and minimal 16.1 °C. The average daily illumination was 290 ± 40, maximal 1520, and minimal < 10 μmol photon m⁻² s⁻¹. During Experiment #2 (Fig. 3b), the average daily temperature was 20.6 ± 0.4, maximal 30.5, and minimal 14.9 °C. The average daily illumination was 340 ± 42, maximal 1650, and minimal < 10 μmol photon m⁻² s⁻¹. Hence, in Experiment #1, the illumination was lower, and in Experiment #2, the temperature was lower.

As indicated above, both the PhBRs were initially filled with Ar + CO₂, but 3.3% of N₂ was added to the gas phase of PhBR 1. The patterns of H₂ accumulation were similar in both PhBRs (Fig. 4a). The maximum rates of H₂ production were at midday, but they varied over the observation period (Fig. 4b). The decrease in the maximum rate at 40th and 64th h correlated with the low daytime temperature and illumination (Fig. 4a).

The average rate of H₂ production in the daytime in PhBRs 1 and 2 increased with the average light intensity (Fig. 5a). Similar dependence was shown in other outdoor experiments [16]. In more detail, the influence of light intensity was studied in Experiment #2.

A decrease in the average rate of H₂ evolution in the morning was observed with a decrease in temperature in the morning hours (Fig. 5b) or during the preceding night (not shown). Earlier, a low daily temperature was supposed to be the main limiting factor in obtaining H₂ outdoors [16]. A decrease in H₂ production activity with decreasing temperature is a matter of common knowledge [24, 25]. However, the effect should not be as dramatic as shown in Fig. 5b: about 10 times decrease was seen in H₂ production rate with temperature decrease by only 3 °C. We would like to attribute it to the mutual effect of low temperature and high-light intensity. Earlier in short-term laboratory experiments, we showed that at 16 °C, the photosynthetic activity of this strain was inhibited at light intensity above 300 μmol photon m⁻² s⁻¹, while at higher temperatures no inhibition was observed even at 2800 μmol photon m⁻² s⁻¹ [25]. Although the average light intensity in the morning was only about 100 μmol photon m⁻² s⁻¹ and the average morning temperature was 15.1–16.6 °C, the duration of the impact was much longer. Therefore, the photoinhibition may be one of the explanations for this dependence.

Torzillo and Vonshak [26] demonstrated that photoinhibition of photosynthetic activity was much higher at temperatures below and above the optimum, in the open ponds or closed systems, correspondingly. Outdoor experiments with Synechocystis PCC 6803 in 50 L tubular PhBR showed that photoinhibition strongly affected maximum quantum yield of PSII at low biomass concentration [17].

The presence of N₂ in the Ar gas phase might be helpful for prolonged H₂ production during 20 days as was shown earlier [27]. However, in our rather short experiments, it significantly affected neither the current H₂ production rates, nor its accumulation (Fig. 4a, b).

It should be noted that the final N₂ concentration in PhBR 2 (without N₂ addition) did not exceed 0.3%, which proves that these bags have negligible permeability to at least N₂. We cannot judge the permeability to H₂ based on our data, but the previous investigation shows that it was very low [20]. The final oxygen concentration was ~ 10% (Fig. 4c), i.e., lower than that in the air and due evidently to the photosynthetic activity of cyanobacteria. The maximum increase in O₂ concentration was observed in the first 2 days of the experiment, later the O₂ concentration did not increase (Fig. 4c). This could be due to a decrease in photosynthetic activity, therefore 40 mL of 0.5 M NaHCO₃ was added at 113th h (not shown). However, O₂ evolution did not resume, and H₂ evolution ceased. Perhaps this was due to inadequate mixing, because control pH measurement (7.2) turned out to be incorrect—actual pH was 10.2, which was the reason for experiment termination. Mixing technique in PhBR of such type should be thought out. It is well-known that appropriate stirring strongly affected the biomass output of cyanobacteria [28]. However, for a large-scale cultivation in the future, mixing seems to be improbable to execute.

Since in Experiment #1, both PhBRs showed similar results, the influence of light intensity (four different values) was studied in parallel in two PhBRs (two values for each PhBR). The light intensity was changed using filters as indicated in Materials and Methods.

Both the maximum and average (per daylight) H₂ production rates were the lowest in the unshadowed culture at 1400 µmol photon m⁻² s⁻¹ (Table 1), which suggested the possibility of photoinhibition. It is well-known that the effect of light intensity may be different in various strains of cyanobacteria [10]. For example, low-light intensity was recommended for laboratory H₂ production by some filamentous cyanobacteria [29, 30]. Besides, this effect may also depend on other factors: the cell concentration, duration of illumination and range of the light intensities. Data of Experiment #2 (Table 1) differ from those of Experiment #1 (Fig. 5a), where no light inhibition was observed. We can ascribe it to lower-light intensities, higher Chl concentration (~ 20 mg Chl L⁻¹) as well as lower O₂ concentration (due to Ar gas phase) in Experiment #1. Our data (Fig. 5a) referred to prolonged H₂ production. When cell suspension of the same culture in short-term (2 min) experiments was tested, we observed no inhibition even at 2800 µmol photon m⁻² s⁻¹ [25]. In short-term experiments, the Hup⁻ mutant of A. variabilis PK84 demonstrated only a slight sensitivity to O₂ concentration [31]. In our Experiment #2 at various light intensities, there were no essential differences in O₂ concentrations measured against the background of air flow (not shown). However, since the air flow was delivered above the surface, the intimate conditions in culture may be different and pO₂ may be elevated. Generally speaking, the light delivery to the culture was inhomogeneous due to absence of continuous mixing [28]. In a well-mixed outdoor culture A. variabilis PK84, the H₂ rate did not decrease but conversion efficiency of light/H₂ energy decreased at high daily irradiance [16].

Experiments #1 and #2 were carried out with different initial Chl concentrations (Table 2). However, an increase in Chl concentration in the course of the experiment was not observed, there was even a slight decrease. This could be explained by a decrease in the specific content of Chl due to high natural light intensity, which was noted earlier [32]. Indeed, OD (another growth indicator), in contrast to Chl concentration, increased by approximately 50% during Experiment #2 (PhBRs 3, 4). This can be due to accumulation of saccharides. On the other hand, an increase in OD with a decrease in Chl content may also indicate bacteriological contamination of the culture. Such a change in parameters in Experiment #2 (PhBR 4) was actually accompanied by contamination, which was confirmed by microscopy after 110 h (not shown). At the same time, pO₂ also decreased, and the H₂ production ceased, which were subsequently not restored (therefore, the experiment has been stopped).

The average rate of H₂ production did not exceed 7.2–7.4 mL L⁻¹ day⁻¹ during 5 days, whereas the maximal volumetric H₂ production rate amounted to 18.5–20.6 mL L⁻¹ day⁻¹ (Table 2), but it took place only for a few hours (2–4 h). The maximal rate per 1 mg Chl amounted to 3.8 mL day⁻¹ in Experiment #2 with lower Chl content. Hydrogen accumulation reached 33.2 mL L⁻¹. Compared to the best outdoor experiments [14, 16], the rather short duration and quite low values of H₂ production in our study can be due to several reasons: the simple PhBR design, poor mixing and culture heterogeneity, and uncontrolled pH. Natural conditions were not optimal as well. Contamination was still a problem even if cyanobacteria were cultivated with antibiotics. As a disadvantage, we should mention poor reliability of plastic bags when using without mechanical protection. Actually, some bags were rejected after the start of experiments due to damage.

We would like to emphasize that during almost 2 decades, there were only a few attempts of outdoor H₂ production using filamentous or unicellular cyanobacteria [14, 16, 17]. Despite the successful laboratory experiments and theoretical considerations, actual outdoor H₂ production is still a challenge. It seems that practical devices need to be more complicated and cost consuming to provide reliable hydrogen. Besides, weather conditions (geographic region) should be chosen carefully. In general, our very first attempt in outdoor cultivation proves that photoautotrophic H₂ production by cyanobacteria in Moscow region is feasible, however, there is still great room for improvement.