3.2. Pigment Profile During Phototrophic Growth
The characteristics of the chromatographic and mass spectrometric data for the different pigments analyzed in the present study are shown in
Table S1.
Scenedesmus exhibits the typical carotenoid profile of the Chlorophyta taxon, which mainly contains lutein, β-carotene, and relative amounts of minor xanthophylls, such as violaxanthin and neoxanthin [
47]. The chlorophyll fraction has been generally described as comprised by chlorophyll
a and chlorophyll
b, a feature of this taxonomic group of green algae. However, our detailed analysis reveals the presence of intermediary chlorophyll metabolites within the chlorophyll profile.
Figure 4 displays the structures of the chlorophyll derivatives present in the profile of
Scenedesmus obliquus.
It was observed that the total amount of carotenoids (
Table 2) increased with the radiation time until the microalgae reached the stationary phase (144 h), to subsequently present a steady state until the end of the phase. In
Scenedesmus, this behavior is due to the response of the main carotenoids, lutein and β-carotene, to the continuous illumination. As it has been stated [
39], the same carotenoid kind may develop different roles in the cell depending on its location. According to the observed data (
Table 2), lutein and β-carotene behave as primary photosynthetic pigments in
Scenedesmus, although β-carotene could perform secondary activities in other chlorophytes, and even transported into oil droplets where they accumulate under stress conditions [
17]. Regardless, lutein and β-carotene are photoprotective pigments, minimizing the photoinhibition through additional roles as quenchers or scavengers [
39]. However, the minor xanthophylls display a different behavior under continuous radiation in
Scenedesmus cells. Neoxanthin, violaxanthin, luteoxanthin, and antheraxanthin increased their concentrations in the microalgae culture even after the stationary growth phase. Specifically, violaxanthin and antheraxanthin are involved in the so-called xanthophyll cycle, intimately related with the ability to dissipate the excess of absorbed light. During high light irradiance conditions, the de-epoxidation reaction of violaxanthin to produce antheraxanthin reduces the light-harvesting efficiency in the antenna [
48]. Finally, although neoxanthin could be considered as a light harvesting pigment, it also develops a role as photoprotective compound, reacting towards reactive oxygen species and preventing cell damage [
49].
In relation to the response of the chlorophyll fraction to the continuous irradiance (
Table 3), it was observed that light exposure initially induces chlorophyll synthesis. Although this result was anticipated, prolonged irradiance times (which means an excess of light) result in a net degradation of the chlorophyll fraction. The detailed analysis of the chlorophyll profile during the phototrophic growth of
Scenedesmus shows chlorophyll
a and
b as the main pigments, but the accumulation of the intermediary metabolites pheophytin and pheophorbide
a was also concomitant. Pheophytin
a (
Figure 4b) is produced by the substitution of the central Mg
2+ ion by hydrogens, while pheophorbide
a (
Figure 4a) involves an additional dephytylation step at the C17
3 position. However, the outstanding results are the production of a heterogeneous profile of oxidized chlorophylls. Among them, the 13
2-hydroxy-compounds stand out, which result from the oxidation at the C13
2 carbon atom (R
2 is OH in
Figure 4) in chlorophyll of the
a and
b series, and in pheophorbide
a. Furthermore, the formation of a lactone functional group is considered a further step in the oxidative level of the original chlorophyll structure [
31]. In this sense, it was very surprising to find 15
1-hydroxy-lactone chlorophyll
b (
Figure 4c) in the chlorophyll profile of
Scenedesmus under radiation conditions.
It is noteworthy to highlight the different behavior of chlorophyll derivatives from
a series (
Figure 4a, CH
3 at C7) from that observed for the
b series (
Figure 4a, CHO at C7). The chlorophyll compounds from
a series, except pheophytin
a, were biosynthesized until the maximum growth stage was reached (144 h), and subsequently a progressive degradation initiated. However, metabolites from chlorophyll
b series (chlorophyll
b, 13
2-hydroxy-chlorophyll
b and 15
1-hydroxy-lactone chlorophyll
b) showed their maximum concentrations between 48 and 72 h of illumination, around half of the period required to reach the residence time. After the apex peak, the metabolites of the chlorophyll
b initiated a net degradation. The interconversion of chlorophyll
a and
b, through the denominated chlorophyll cycle (
Figure 1 [
50]), is an essential mechanism in photosynthetic organisms, as they can adapt their photosynthetic apparatus to the irradiance level. At high levels of illumination, the organism reduces the antenna complexes to avoid excess of photons, so that the production of reactive oxygen species (ROS) is minimized. As antenna complexes are rich in chlorophyll b compounds, at high irradiances the relative amounts of chlorophyll
b decreased. On the contrary, at low irradiance (shadow) conditions, the organism rises the antenna complexes to capture as many photons as possible, which results in an increase of the chlorophyll compounds of the
b series. Consequently, microalgae modify the ratio of chlorophyll
a/b according to the irradiance levels [
51]. As it can be observed in
Table 4, at the initial 72 h of growth the ratio of
a/b series decreased in
Scenedesmus, as the biosynthesis rate of chlorophyll
b was higher than that for the chlorophyll
a. However, when the quantity of light was excessive for the culture (after 72 h of continuous illumination), the antenna complexes decreased, the concentration of chlorophyll
b diminished and, consequently, the
a/b ratio increased. Similar changes in the
a/b ratio have been observed for
Chlorella and
Dunalliela [
52]. At high irradiances, the energy received by chlorophyll
a molecule is higher than its capacity to transfer it towards the photosynthetic electron transport chain, and chlorophyll
a switches to the triplet excited stage [
39]. Next, overexcited chlorophyll
a molecule is quenched by molecular oxygen yielding ROS. As we can observe in
Table 3 and
Table 4, the interconversion between chlorophyll
a and
b contents is the preferred mechanism of
Scenedesmus cells to avoid the formation of ROS at high irradiances.
Nevertheless, once the maximum concentrations for chlorophyll
a (144 h,
Table 3) and
b (72 h in
Table 4) were reached, the net degradation of chlorophyll compounds was not exhaustive. Otherwise,
Scenedesmus cells reached a steady state for the chlorophyll content (around 6200 mg/kg dw. for chlorophyll
a, 1900 mg/kg dw. for chlorophyll
b) until the end of the controlled period. It seems that once the top biosynthetic capabilities were accomplished, the microalgae found an ‘ideal’ chlorophyll content, which allows an equilibrated photosynthetic performance, that is, a productive one but not harmful, at least at the irradiance assayed for
Scenedesmus. As it has been previously stated, photoacclimation is complete only when a balanced growth condition is achieved [
53]. However, this is accurate when the chlorophyll content is determined as a whole value. As we have shown, a detailed study of the complete chlorophyll profile allows to observe different biosynthetic capabilities with some chlorophyll metabolites reaching steady state earlier, precisely to fit with the photoacclimation at 144 h.
Pheophorbide
a and pheophytin
a are currently considered the metabolites of the chlorophyll degradation pathway (
Figure 1). In fact, pheophorbide, pheophytin, and pyropheophorbide have been associated with the chlorophyll degradation in cyanobacteria in sedimentary surfaces [
54] and chlorophyll senescence in marine environments [
34]. Recently, the gene responsible of the formation of pheophytin (SGR) a has been identified in
Chlamidomonas reinharditii [
24]. However, while the kinetics of production and degradation of pheophorbide
a is parallel to the chlorophyll
a, the profile of metabolism of pheophytin
a seems to progress in a different fashion and not correlated with the metabolism of chlorophyll
a. The maximum concentration of pheophytin
a was observed at 24 h, while its progressive decay through the continuous illumination period made the interpretation of the results in base to its implication in the chlorophyll degradation pathway challenging.
In addition, the HPLC-ESI/APCI-hrTOF-MS analyses of the chlorophyll fraction revealed the existence of a specific chlorophyll oxidative metabolism (
Table 2 and
Table 3) during the
Scenedesmus phototrophic cultivation. As stated before (
Figure 2), hydroxylation at C13
2 is the first step in the oxidative pathway of chlorophylls. Hence, 13
2-hydroxy-chlorophyll
a and
b increased their concentrations in the cell with the continuous illumination for the initial 72 h period, and afterwards a progressive degradation was observed. In any case, it is important to highlight that the maximum of 13
2-hydroxy-chlorophyll
a cellular content was not concurrent with the maximum concentration of chlorophyll
a, which pointed towards a specific linking reaction between both compounds instead of an unspecific process. Noteworthy, 13
2-hydroxy-pheophorbide
a was also produced around 3 days of illumination, once pheophorbide
a is biosynthesized in the microalgae. A further oxidative process is the generation of the lactone rearrangement at the C15
1 position (
Figure 4). During the phototrophic growth of
Scenedesmus a progressive accumulation of 15
1-hydroxy-lactone chlorophyll
b is observed, reaching the maximum value after 72 h (
Table 3). In our experimental conditions it seems 72 h is the timeframe for
Scenedesmus to reach the ‘buffer capacity’ (from the point of view of chlorophylls) and manage both the excess of energy and, consequently, the potential accumulation of ROS. Afterwards, profound physiological changes are required to avoid oxidative stress, as the commented restructuration of antenna complexes.
Moreover, no 15
1-hydroxy-lactone chlorophyll
a formation was detected in any moment of the phototrophic growth, although chlorophyll
a is the main chlorophyll pigment in the chlorophyll profile of
Scenedesmus. In fact, although a chlorophyll metabolite with this functional group is not easy to distinguish [
1], it is the 15
1-hydroxy-lactone chlorophyll
a catabolite observed (if any) in photosynthetic organisms, but not the 15
1-hydroxy-lactone chlorophyll
b catabolic product [
30]. Indeed, both proportionally and in absolute concentration, the total biosynthesized chlorophyll oxidative compounds of the
b series overcame those of the
a series. To the best of our knowledge, this is the first time to describe such phenomenon. The biochemical origin of the oxidized chlorophyll metabolites is still under discussion. In higher plants, different enzymatic systems have been assumed as responsible for such oxidation (lipoxygenase and/or peroxidase) [
29,
30,
31]. However, although different oxidative mechanisms have been observed in microalgae (peroxidase, superoxide dismutase, polyphenol oxidase, glutathione peroxidase, etc.) [
55,
56], none of them have been correlated with the chlorophyll metabolism so far. Two possible hypotheses can explain the higher rate of oxidation of chlorophyll
b catabolism. Thus, the preferential accumulation of chlorophyll
b catabolites could be due to an unknown chlorophyll
b affinity by the pool of oxidative enzymes pool, or this singularity could be caused by the different localization of both chlorophyll series in the photosynthetic apparatus. Further research is required to unravel the exact mechanism.
3.3. Pigment Evolution During Heterotrophic Growth
As it can be seen in
Table 5, heterotrophy means carotenoid degradation for
Scenedesmus obliquus in our experimental conditions, although at very different rates depending on the carotenoid sort. The initial 24 h in darkness produces a significant carotenoid degradation except for neoxanthin, while the concentration of β-carotene and violaxanthin decreased by half. This decrease was extended in a lower degree for lutein. From 24 to 48 h of growth in the darkness, carotenoids were highly stable, the next 24 h interlude (72 h) only being a significant stage for the stability of neoxanthin and violaxanthin. Extending the heterotrophic culture of
Scenedesmus obliquus far from 96 h implied a carotenoid degradation of at least 85%. In fact, carotenoid production in heterotrophic cultivation requires additional oxidative stress: high salt concentration, high light, etc. [
13]. In any case, it is important to highlight the different stability of carotenoids in heterotrophic conditions, to face the future biotechnological strategies aimed to enhance the production of carotenoids.
On the contrary, it was remarkable to observe the behavior of the chlorophyll fraction at heterotrophic culture conditions. During the initial 48 h of growth, the total amount of chlorophylls was constant and after that time interval, the chlorophyll profile initiated a phase of net degradation with increased rate at the end of the controlled period. Such modification in the chlorophyll metabolism is coincident with an increase of biomass. The initial steady state of the chlorophyll content means that the biosynthetic and the degradative reactions are evolving at the same rate. Although the exact quantity is unknown, the half-life of a chlorophyll molecule is estimated around several hours [
57]. This fact implies that during the steady state of chlorophylls in the initial 48 h of heterotrophic culture, biosynthetic and degradative reactions are running in
Scenedesmus cells. Regarding the biosynthetic metabolism, as stated before green algae can synthesize chlorophylls in dark conditions. Consequently, during 48 h of heterotrophic cultivation of
Scenedesmus, a continuous synthesis of chlorophylls took place, although at the same rate as the degradative reactions. The first assumption to consider is that under heterotrophic conditions, the cell does not invest energy in chlorophyll synthesis but focuses on the cell division and growth process with the available resources. In fact, it has been argued that glucose can inhibit the chlorophyll biosynthesis, by means of an inhibitory activity towards the precursor coprophorphyrin III [
58]. On the contrary, some reports have shown a certain degree of chlorophyll retention during heterotrophic growth [
59], as we have found for
Scenedesmus. The exact physiological meaning of such energetic investment is unknown to date, although our results are an important starting point for future biotechnological applications aimed to enhance the chlorophyll production.
In addition, the detailed analysis of the chlorophyll profile during the heterotrophic growth of
Scenedesmus shows accumulation of chlorophyll metabolites produced during the chlorophyll degradation, that mirror the masked reactions that were under progress. Pheophorbide, chlorophyllide, and pheophytin are intermediary catabolites during the chlorophyll degradation pathway.
Table 5 shows a significant increment of pheophorbide and chlorophyllide
a at the end of the controlled period, concomitant with the main degradation of chlorophylls. However, pheophytin levels continuously decreased through the cycle, showing no parallelism with the chlorophyll breakdown. The results suggest that the operating pathways during the heterotrophic cultivation of
Scenedesmus are better related with the chlorophyllase (CHL) pathway (
Figure 1) than with pheophytinase one (PPH). Homologous PPH proteins have been found through BLASTP (Basic Local Alignment Search Tool for Proteins) searches in green algae but not in cyanobacteria, and it has been proposed that PPHs are also likely to be operative in the green algae [
60], although no functional analysis has been developed so far. Although such data are not available, PPH seems to not be responsible for the chlorophyll degradation during heterotrophic conditions, at least during the culture conditions assayed in
Scenedesmus.
To the best of our knowledge, accumulation of 13
2-hydroxy-chlorophylls is described for the first time in this study during the heterotrophic culture of green microalga, although no 15
1-hydroxy-lactone derivatives were detected. Interestingly, the heterotrophic strategy only induced oxidation in chlorophyll
a molecules and no oxidized chlorophyll
b compounds were detected in any moment of the cycle. 13
2-hydroxy-chlorophyll
a production, observed during the initial 48 h of growth in the darkness could involve a role during the chlorophyll turnover, although the main synthesis is accomplished with the net degradation of chlorophylls at the end of the cultivation period. As stated before, the exact role of oxidized chlorophylls in phytoplankton is unclear, but associated with defense, grazing, senescence, or even death cell [
33,
34,
35,
36,
37,
38]. Our results show both production and degradation kinetics during the heterotrophic culture of
Scenedesmus, with more than a plausible role during the chlorophyll degradation. Consequently, the results obtained in
Table 5 open a door for future research, with a focus on the biochemical mechanisms involved in the chlorophyll oxidative metabolism during the heterotrophic cultivation of green microalgae.