Acetate-containing media and low light are non-stressful conditions that elicits H2 production
Hermetically sealed vessels containing 100 ml of low cell density (10 µg chl./ml; ~3 million cells/ml) Chlamydomonas cells cultured in Tris–Acetate–Phosphate (TAP) were placed under four different light conditions (12, 22, 50 and 100 µmol photons m−2 s−1; hereafter, 12 PAR, 22 PAR, 50 PAR, and 100 PAR) and dark. In this work, we will term 12 and 22 PAR as Low Light (LL), 50 PAR as Moderate Light (ML), and 100 PAR as High Light (HL). The headspaces of the culture vessels (40 ml) were not purged, and therefore atmospheric air was present in all vessels at the beginning of the experiments. H2, CO2 and O2 accumulation in the headspace, chlorophyll content, and acetate consumption were measured daily during 10-day experiments (Fig. 1 and Additional file 1: Fig. S1).
Cultures incubated under LL and ML produced H2 after 24 h, at rates of 6.9, 6.0, and 3.7 ml L−1 day−1 for 12, 22, and 50 PAR, respectively (Fig. 1a). After 48 h, little or no H2 production was observed under these light intensities. Interestingly, the H2 accumulation observed under light did not reach a steady-state level, but rather a small decrease was observed after the maximal H2 accumulation was reached. The H2 decrease was most evident under the 12 PAR condition. This observation is likely due to H2 uptake activity. Cultures under 100 PAR resulted in no H2 production. Finally, dark cultures had lower initial H2-production rates relative to 12–50 PAR conditions (average less than 1 ml L−1 day−1), although production was sustained for more than 7 days. Final H2 accumulation in dark cultures, after 8 days, was similar to that obtained under LL after 24–48 h. The atmospheric levels of O2 initially present in the headspaces were totally consumed after 24 h for all conditions except 100 PAR (Fig. 1b). O2 levels in the headspace remained close to zero for 12, 22 PAR and the dark conditions over the 10 days of the experiment. However, under the 50 PAR conditions, O2 accumulation was observed in the headspace after 3 days. At 100 PAR, O2 evolved above the atmospheric level, indicating that PSII activity overtook respiration rates. Acetate uptake also showed light dependence under our experimental conditions (Fig. 1c). Under 22–100 PAR, all the acetic acid contained in the media was essentially consumed after 2–4 days. However, we observed slower acetate uptake kinetics under 12 PAR and dark conditions. After 10 days, there was still acetic acid in the media (5.3 mM and 9.5 mM for 12 PAR and dark, respectively). CO2 quickly accumulated after 24 h in all tested conditions except 100 PAR (Fig. 1d). The media pH remained stable over the 10 days (7.5–7.8). Finally, the chlorophyll concentration increased significantly under 22–100 PAR, whereas there was very little or no increase at 12 PAR and in the dark, respectively (Additional file 1: Fig. S1A).
These data indicate that acetate-containing cultures grown under ≤50 PAR and moderately low cell density can rapidly consume O2 from the headspaces (for a 100:40 medium: headspace v/v ratio), reaching anaerobiosis and producing H2 after 24 h. Maximal H2 production rates were inversely correlated with light intensities. However, dark cultures did not experience a rapid H2-production phase but rather a slow, continued, and sustained level of H2 production. This indicates that light is crucial to induce the rapid H2 production kinetics observed under LL and ML.
Aerations of cultures can double H2 photoproduction
The same experimental conditions described above were also used to monitoring H2 production in aerated vessels. Aeration was performed by opening the caps of the vessels for 5 min, in a sterile atmosphere, every 24 h. H2, CO2 and O2 accumulation in the headspace, acetate consumption, and chlorophyll content were measured daily before aeration during 10-day experiments (Fig. 2; Additional file 1: Fig. S1). H2 and CO2 levels are plotted as total production, whereas O2 levels are plotted as daily measurements. Chlorophyll increased steadily with the intensity of light (Additional file 1: Fig. S1), whereas no changes were observed for pH, which was around 7.6–7.8.
The H2 production in aerated cultures under LL was 2.4 times higher than in non-aerated cultures under the same light conditions (Figs. 2a vs 1a). For cultures under 50 PAR and dark, the aeration either slightly increased or reduced H2 production, respectively, relative to non-aerated cultures. Under LL, unlike the non-aerated vessels, H2 production was sustained for 3 days. Afterwards, very little H2 production was observed. For LL grown cultures, atmospheric levels of O2 entering in the vessels daily were completely consumed during the first 3 days (Fig. 2b). By the 4th day, increased levels of O2 were detected in the headspace indicating that the O2 consumption rates were progressively decreasing. Similar results were observed for cultures under 50 PAR, although appearance of O2 in the headspaces was detected earlier as the light intensity was increased. For all light intensities, except darkness, headspace O2 accumulation exceeded the initial O2 atmospheric levels, indicating net O2 photoproduction (Fig. 2b). Unlike the non-aerated cultures, all the acetate initially contained in the media was eventually consumed in the aerated cultures under all the conditions tested (Fig. 2c). More specifically, in the light, acetic acid was consumed after 2–3 days. We observed a strong correlation among the H2 production, the level of O2 in the headspace, and the consumption of acetate, indicating that these three processes are closely interconnected. H2 was produced as long as acetate was present in the media. Once acetate was consumed, O2 was accumulated and H2 production ceased. The higher the light intensity, the faster the acetate was consumed, the faster O2 accumulated in the headspace, and the faster H2 production ceased. Notably, increasing the light intensity not only reduced the length of the H2-production phase but also reduced the daily H2-production rates (e.g., 5 ml L−1 day−1 for 12PAR vs 2.6 ml L−1 day−1 for 50 PAR). However, in the dark, H2 production dropped drastically reinforcing the idea that light is crucial for the H2 production rates observed in the light. CO2 production was significantly higher for all the conditions tested when comparing to non-aerated cultures (Figs. 2d vs 1d).
Release of H2 partial pressure highly promotes H2 production in mixotrophic LL cultures
Two possibilities might explain the higher H2 accumulation observed in aerated cultures relative to non-aerated cultures: (1) the increased acetic acid uptake observed in aerated cultures may result in higher H2 production, or (2) release of the H2 partial pressure from the headspace may favor the equilibrium displacement of the reversible reaction catalyzed by hydrogenases toward H2 production and preventing H2 uptake [27]. When the headspaces of 12 PAR grown cultures were N2-purged daily, H2-production rates during the first 3 days were similar to those observed in aerated cultures (Fig. 3a). However, N2-purged cultures kept producing H2 for 10 days, while aerated cultures stopped producing H2 by the 4th day (Fig. 3b). Total H2 production over the 10 days in the purged cultures was 1.4 times higher than in aerated cultures. Interestingly, acetate uptake in purged cultures was severely impaired compared to aerated cultures (Fig. 3b). Purged cultures consumed only around 7 mM of acetic acid but produced more H2 than aerated cultures, which consumed all the acetate initially present in the media (17.4 mM). These data indicate that enhanced H2 production under aeration is mainly due to partial H2 pressure release and not to the increased acetate uptake rates.
To further test which of the two initial hypotheses were true, we added to non-aerated cultures pure O2 daily to the headspaces at atmospheric levels using a syringe (Additional file 2: Fig. S2). Cultures remained hermetically sealed for 9 days without releasing the H2 partial pressure. The results showed high acetate uptake rates, but this did not improve H2 production, which was similar to that obtained for non-aerated cultures. O2 from the headspaces was consumed for 4 days; after 5 days, O2 started to accumulate. This indicates that despite the fact that cultures reached anoxia for 4 days and that acetate was totally consumed, the H2 partial pressure in the headspaces blocked further H2 production.
Acetic acid and O2 supplementation leads to sustained H2 production under LL conditions
Cultures were subjected to identical experimental conditions described for aerated cultures for 4 days. After 4 days, cultures consumed most of the initial acetate (17.4 mM) contained in the media and did not significantly produce H2 afterwards (Fig. 2a). The addition of extra acetic acid (8.7 mM final concentration) on the 4th day promoted a second boost of H2 production under all light conditions except on cultures kept in the dark (Fig. 4a). After acetic acid supplementation, H2 production was sustained for another one (50 PAR) or two (12 and 22 PAR) days before ceasing again. Addition of a second dose of acetic acid on the 7th day (once H2 production stopped and acetate was consumed again) promoted a 3rd H2-production boost. Supplementation with acetic acid did not significantly change the pH of the media over the 10 days of the experiment, which remains about 7.5–7.8. In agreement with our previous data, LL grown cultures produced more H2 than 50PAR cultures. At 12 PAR, the maximal H2 production rate was 21.2 ml per day, and net total accumulation was 14.9 and 4.0 times higher compared to non-aerated and aerated without supplementation cultures, respectively. Similar to aerated cultures, we observed a good correlation between H2 production, acetate uptake, and O2 consumption in the headspace (Fig. 4a–c). Addition of extra acetic acid to the medium increased CO2 production under all conditions tested, compared to non-supplemented cultures (Figs. 4d vs 2d). Supplementation with acetic acid without performing aeration resulted in cell death (data not shown). This is likely due to the low acetic acid uptake observed in the absence of O2 (see below section), which in turn results in acidification of the media (pHs of the dead cultures were of 5.6). Overall, the data showed that H2 production can be sustained in fed-batch type bioreactors under LL with both aeration and acetic acid supplementation.
Acetate uptake depends on O2 availability. The inhibitory effect of DCMU on H2 production under LL is linked to O2 availability and impairment of acetate uptake
To determine the role of PSII activity in the observed H2 production under our experimental conditions, DCMU was added to the cultures.
The addition of DCMU to the non-aerated cultures incubated under 12–50 PAR produced a substantial reduction of H2 accumulation compared to the cultures containing no DCMU (78.8–69.5 % reduction at 24 h) (Fig. 1e vs a). We also observed an impairment in acetate uptake in non-aerated cultures treated with DCMU under all light intensities; the acetate concentration initially dropped but no significant acetate uptake was observed after 24–48 h (Fig. 1g). Interestingly, in aerated cultures, this reduction in H2 production when using DCMU (Fig. 2e) was significantly smaller than the one obtained for non-aerated cultures (e.g., 19.2 and 40.1 % at 12 and 22 PAR, respectively). Moreover, addition of DCMU to aerated cultures caused a much lower inhibition of the acetate uptake than in non-aerated cultures with DCMU (Figs. 2g vs 1g). Indeed, acetate was totally consumed in aerated cultures with DCMU after 4–5 days, while in non-aerated cultures, about 10 mM of acetate remained in the media after 10 days. When daily purged cultures under 12 PAR were supplemented with DCMU, both acetate uptake and H2 production dropped severely (Fig. 3). DCMU caused a 64 % inhibition of H2 production, and acetic acid uptake was practically inexistent. The effects of DCMU on H2 production and acetate uptake in purged cultures resemble the observed in non-aerated cultures. Finally, simultaneous addition of both acetic acid and DCMU on the 4th and 7th days in aerated cultures resulted in partial inhibition of H2 production (e.g., 21.5 % reduction for 12 PAR) relative to cultures supplemented with acetic acid but without DCMU (Fig. 4e vs a). However, acetate uptake was not significantly affected in these cultures (Fig. 4g vs c). These data are very similar to those obtained with aerated cultures without extra acetic acid supplementation.
Two main conclusions can be obtained from these data. First, we show that acetate uptake depends on the availability of O2. In the presence of DCMU, daily aeration of the cultures can supply the O2 needed to consume all the acetate from the media (Fig. 2g), whereas in non-aerated or purged vessels with DCMU, the lack of O2 inhibits acetate uptake (Fig. 1g). The initial drop in the acetate levels observed in non-aerated cultures containing DCMU after 24–48 h is likely due to the initial presence of O2 in the non-purged headspace; as O2 level decreased, no more acetate was consumed (Fig. 1g). Moreover, acetate uptake does not depend on the PSII activity per se. However, indirectly, the PSII activity contributes providing O2. This can be observed in non-aerated and aerated cultures without DCMU (Figs. 1c, 2c): daily aeration of the cultures can supply the O2 needed to consume all the acetate from the media, whereas in non-aerated vessels the PSII activity is the only source of O2 that enables acetate uptake. The light dependence of acetate uptake observed in both non-aerated and aerated cultures without DCMU can be explained by the relative activity of the PSII and the availability of the photo-evolved O2. Previous observations describing acetate consumption in the light and termed as photoassimilation of acetate (or acetate photometabolism) [21, 28] must be linked, at least partially, to the photogeneration of O2 but not to the ATP availability.
Second, we demonstrate that DCMU can affect differently H2 production in LL cultures. The addition of DCMU to non-aerated and purged cultures grown under 12 PAR caused very similar effects on inhibition of H2 production (72.4 vs 64 %, respectively) (Figs. 1e, 3a) and on acetate uptake (essentially blocked) (Figs. 1g, 3b). The effect of DCMU, however, is clearly different in aerated cultures cultivated under 12 PAR (with or without supplementation with acetic and DCMU) where H2 production was only partially inhibited (nearly 20 %) (Figs. 2e, 4e) and acetate uptake was not severely impaired (Figs. 2g, 4g). Note that the degree of inhibition of the H2 production caused by DCMU correlates with the O2 availability and the rates of acetate uptake. The higher H2 inhibition was obtained when O2 availability and acetate uptake were very low.
Finally, addition of DCMU to non-aerated cultures grown under HL elicited a small H2 production (Fig. 1e). H2 production was only possible under HL when DCMU was present in the medium. The presence of this inhibitor was crucial to reach anaerobiosis under HL (Fig. 1f), unlike in the 12–50 PAR grown cultures. Similar results have been found in autotrophic sulfur-depleted cultures [29].
Mobilization of starch reserves are not linked to H2 production in acetate-containing media under LL
Starch mobilization has been proposed to contribute to H2 production in mixotrophic nutrient-replete cultures via the PSII-independent pathway [16, 17, 21]. It has been suggested that under aerobic conditions acetate would first stimulate starch accumulation, which would be later degraded under anoxic conditions and provide the PQ pool with reductive equivalents. Hence, either the starch reserves present prior the beginning of the experiments or those accumulated during the experiments might potentially contribute to H2 production under LL.
We analyzed the starch accumulation patterns in the LL cultures (Fig. 5). In non-aerated cultures, starch accumulated progressively doubling its initial concentration at the end of the 8-day experiment (Fig. 5a). Similarly, in daily purged cultures under LL, starch initially decreased after 24 h but then accumulated during 6 days reaching a steady level afterwards (Fig. 5a). In aerated cultures, an initial starch accumulation phase was observed followed by a degradation phase. We observed a good synchronization between the disappearance of acetate from the media (Fig. 2c) and the starch degradation phase in aerated cultures under LL (Fig. 5a). The addition of DCMU to non-aerated cultures clearly impaired starch accumulation (Fig. 5b), likely reflecting the poor acetate uptake measured in these cultures (Fig. 1g). The addition of DCMU to aerated cultures had only a minor effect on the starch accumulation pattern (Fig. 5b), which is in agreement with the poor inhibition of acetate uptake observed in these cultures (Fig. 2g), and demonstrate that starch accumulation does not require PSII activity but acetate uptake.
Overall, we observed no correlation between starch accumulation/degradation patterns and H2 production because the starch accumulation phases correlated with the maximal H2-production rates in all cases, and there was no further H2 production when starch was degraded (e.g., aerated cultures, see Figs. 5 vs 2 and 2e).
Comparing H2 production in LL mixotrophic nutrient-replete and S-depleted cultures
H2 production under LL mixotrophic S-depleted conditions was tested using the previously described experimental conditions.
Non-aerated, S-depleted cultures at 12PAR (without purged head space) showed an initial H2-production phase resembling that obtained with S-replete cultures. After 7 days, a second H2-production boost was observed (Supplemental Fig. 3a). While the first H2-production boost might share similar physiological processes with nutrient-replete cultures, the second H2 boost could be more specific to S-deprivation physiology. Interestingly, non-aerated S-depleted cultures presented higher tolerance to the inhibitory effect of the H2 partial pressure than nutrient-replete cultures. Maximal H2 accumulation in non-aerated S-depleted cultures (13.7 ml L−1) was 2 times higher than in non-aerated S-replete cultures (6.9 ml L−1), but similar to the production obtained in aerated S-replete cultures (16.4 ml L−1) (Supplemental Fig. 3b). S-depleted aerated cultures produce no H2 (Additional file 3: Fig. S3B) because cells were unable to consume the O2 that daily entered in the headspaces and anaerobiosis never occurred (data not shown) revealing that O2 consumption rates in S-depleted cultures are lower than in S-replete cultures. S-depleted daily purged cultures showed H2 production rates quite similar to those of S-replete purged cultures (Additional file 3: Fig. S3C).
Overall, the results showed that although S starvation can improve H2 production in non-aerated cultures, similar levels of H2 can be obtained in S-replete, aerated cultures in a shorter period of time. Moreover, S-depleted, purged cultures did not show any significant advantage over S-replete purged cultures. Hence, under our experimental conditions, removal of S from the cultures does not contribute a significant advantage over nutrient-replete media under LL.