In the present study, we observed that the carbon source and its concentration exerted an important influence on cell growth, which in the case of bicarbonate was in a concentration-dependent manner (Fig. 1a). The improved microalgal biomass yield in cultures supplied with rising levels of NaHCO3 has been previously reported and associated with the induction of carbon concentration mechanisms (CCM) (Vadlamani et al. 2019). These CCM serve as a mean to increase the flux of inorganic carbon from the extracellular medium into the chloroplast (Giordano et al. 2005). Part of the CCM relies on the expression of carbonic anhydrases that interconvert CO2 and NaHCO3 and whose Km values are reported to be 10–20 mM (Mirjafari et al. 2007). Under standard cultivation conditions (i.e., low turbulence regimes and atmospheric CO2 levels), the carbon fixation rate and the underlying biomass production can be kinetically limited by substrate concentration at the carbon fixation site for RuBisCO, since the Km (CO2) has been shown to be close to 30 µM for green algae (Tcherkez et al. 2006). Therefore, high external carbon doses would help to compensate the low carbon availability at the fixation sites, particularly in the form of NaHCO3, which remains dissolved much longer than CO2. This in turn leads to a higher carbon fixation capacity, and the subsequent improvement of biomass productivity and yields.
NaHCO3 is commonly used as a carbon source in the growth media of the cyanobacterium Spirulina; nonetheless, it has not been extensively explored in microalgae with most research conducted using CO2 (Arun et al. 2020; Umetani et al. 2021); however, NaHCO3 has been recently considered as an alternative carbon feedstock for microalgae that improves biomass yield and also has proven to induce the accumulation of metabolites such as TAGs and carotenoids (Gardner et al. 2013). Moreover, Srinivasan et al. (2018) suggested that bicarbonate ions could also promote reactive oxygen species scavenging and biomass formation in algal cells even upon nutrient limitation conditions.
Remarkably, our results showed that B. braunii LB 572 not only can grow faster in the presence of high NaHCO3 levels (up to 2%) but also, such conditions promoted sustained TAG accumulation (see “Lipidome remodeling of B. braunii cells grown under high carbon dosage” sections) without the need of a second stage of nutrient deprivation that is traditionally used for lipid accumulation in algal systems.
N- and P-deficiency conditions tested in this study were detrimental for B. braunii growth when compared with the control (Fig. 1a). N is a major constituent of biomass, forming part of a wide range of biomolecules such as nucleic acids, proteins, and cofactors. Moreover, N assimilation represents an important sink for reducing power, especially when the main N source supplied is an oxidized form such as NO3− (Grund et al. 2019). In that sense, low N availability implies a lower capacity of biomass formation (Bolton 2006). For instance, Zhila et al. (2005) found that B. braunii IPPAS H-252 cultures produced two times less biomass in a N-deprived media as compared to the controls. Similar outcomes have also been documented by other authors with different B. braunii strains (Lupi et al. 1994; Choi et al. 2011; Barajas-Solano et al., 2016) . On the other hand, P deprivation also limited the biomass production in B. braunii cultures (Fig. 1a), since it constitutes another vital nutrient that is required to synthesize several P-containing molecules such as nucleic acids, GPLs, inositol triphosphate, NADP(H), ATP, among others. Furthermore, P plays a crucial role in the regulation of energy production, amino acids, nucleic acids and CHOs metabolism, and several reactions related to the progression of the cell cycle are activated by phosphorylation (Cui et al. 2017). Consequently, P deficiency is likely to hinder to some extent the biomass yield as reported elsewhere (Kamalanathan et al. 2016; Li et al. 2016).
Effect of nutritional schemes on differential biomass composition
N deprivation is expected to trigger lipid accumulation in algae cells (Kumari et al. 2014), as observed in this study (Fig. 1b), and in previous reports (Breuer et al. 2012; Pancha et al. 2014; Jerez et al. 2016). However, overall lipid productivity could be a more relevant indicator of culture performance. Here we show that lipid productivity was significantly enhanced in the cells incubated with the highest NaHCO3 level when compared with the controls and nutrient-starved samples (Fig. 1c). This is due to the significantly greater biomass and therefore lipid productivity reached with the highest carbon dosages, even though their lipid content was somehow lower than the value measured for ND cells (Fig. 1b).
Moreover, the fact that the increased lipid productivity remained constant between D6 and D12 might result convenient from a process design perspective, since it would facilitate the operation of a continuous process for lipid-rich biomass production and its subsequent transformation, rather than designing a two-stage system consisting of a cell growth phase, followed by a nutritional stress and lipid accumulation stage (Biller et al. 2015). Furthermore, it might help to improve systems such as the in situ milking proposed by some researchers (Hejazi and Wijffels 2004; Kleinert and Griehl 2020), where the basic concept relies on the continuous solvent-based lipid extraction, omitting the cellular disruption step.
Botryococcus braunii is also known for its capacity to produce and excrete polysaccharides originated from the internal fibrillar layer that play a crucial role as an adhesive matrix for colony formation and protection (Giraldo Calderón et al., 2018). These polysaccharides have been investigated for their wide range of applications in material science and microbiology (Navarro Gallón et al. 2019). In this study, we observed that ND and PD cultures had more similar amounts of in-CHOs compared to those in 1 and 2% NaHCO3 supplemented cultures, which might imply that this specific strain is prone to re-route the assimilated carbon towards more reduced and energetically dense molecules such as the lipids under certain nutritional scenarios. Some authors have suggested that CHOs production and excretion are associated with growth, while the highest yields are obtained at the final stages of the active growth, but also during the stationary and decline phases due to nutrient depletion (Blifernez-Klassen et al. 2018), as in the case of the control and PD cultures of our study (Fig. 1e). However, it is important to consider that the metabolic phenotypes are highly variable across B. braunii races, even for the same strain (Eroglu et al. 2011; Jin et al. 2016).
Some of the cultures underwent a substantial decrease in the Chl a content among the two sampling points (Fig. 1f). In the case of the cultures amended with NaHCO3, this situation may have occurred due to a lower prevalence of light harvesting complexes associated with reduced requirements for passive non-photochemical energy dissipation upon high carbon availability (Pessarakli 2016). The results revealed that the YNO was lower (P < 0.05) (Fig. 2c) and the YNPQ higher (P < 0.05) (Fig. 2e) for 2% NaHCO3 cells compared to the controls at most irradiance values, and even more compared to the culture with 0.1% NaHCO3. Similarly, the N-starved cultures displayed a significant decrease in Chl a content between D6 and D12 (Fig. 1f) following a common trend found in the literature (Zhila et al. 2005; Jerez et al. 2016). This reduction could be related to the fact that the Chl a is a N-rich molecule that could be degraded under N scarcity for recycling purposes, and its biosynthesis is also prone to be downregulated under such conditions (Hockin et al. 2012). In this regard, a proteomic analysis of Thalassiosira pseudonana (CCMP 1335) revealed that N deficiency conditions resulted in a 1.5- to 2.5-fold decrease of the expression levels of five proteins involved in chlorophyll biosynthesis (Hockin et al. 2012).
The P-starved cultures were the less sensitive in terms of chlorophyll production (Fig. 1f) since the Chl a content remained unaltered in PD cultures throughout the course of the experiment. A similar result was found by (Kamalanathan et al. 2016) with Chlamydomonas reinhardtii.
Fv/Fm is considered as an indicator parameter of the level of photosynthetic stress and photoinhibition (Malapascua et al. 2014). During the first days of incubation, Fv/Fm remained similar across most of the tested conditions with values close to 0.7 (Fig. 1i); this indicates that B. braunii was not undergoing a metabolic pressure that significantly affected its capacity to perform photosynthesis during the first stages of growth. Nevertheless, the lower Fv/Fm value measured for the 0.1% NaHCO3 culture indicates that a limited carbon availability impairs the metabolic fitness. This may be attributed to the overproduction of reductive power that cannot be rapidly channeled to carbon fixation due to the low intracellular CO2 level. This overreduction could be detrimental for the reaction centers at the PSII and could provoke a decline in its photochemical quantum yield (Pessarakli 2016). In contrast, at D12, Fv/Fm remained high for the controls, while the values for the remainder of the conditions were diminished (0.487–0.585) (P < 0.001), probably due to the reduction in the cellular chlorophyll content (Fig. 1f), adding to the nutrient shortage effect in ND and PD cultures (Jerez et al. 2016).
Aiming to assess the influence of the different conditions over the irradiance tolerance and adaptation capacity, RLC were constructed to relate a series of irradiance levels and the derived rETR (Fig. 1j, k). At D6, the highest rETR were recorded for 2% NaHCO3, which were also the cells with the fastest growth rate. Higher carbon availability could lead to increased electron consumption rates due to the prevalence of carboxylation reaction over oxygenation performed by RuBisCO, decreasing the energy losses by non-photochemical energy dissipation and favoring the photochemical conversion (Pessarakli 2016). A similar outcome with a green algae was reported by Vadlamani et al. (2019), as these authors found that elevated NaHCO3 concentrations benefit the biomass productivity and the photosynthetic performance of Chlorella sorokiniana. Furthermore, it has been reported that the response to bicarbonate is strain-specific, and the enhancement of the photosynthetic metabolism occurs only under certain concentration thresholds (Yeh et al. 2010; White et al. 2012).
Interestingly, the cultures exposed to nutrient limitation (i.e., ND and PD) exhibited relatively high photosynthesis production values either at D6 and D12, and ND cultures displayed only a slight decay at D12, although it remained even higher than the rETR of the control (Fig. 1j, k). The ND cultures also suffered a drastic decline of the Chl a content (Fig. 1f), which might lead to smaller light-harvesting complexes (LHCs) and consequently reduced cellular light absorption capacity (Zhao et al. 2017; Giraldo et al. 2021). Accordingly, we hypothesize that this reduction in the light harvested per cell may diminish the inhibitory effects of high irradiance and alleviate at some extent the pressure over the reaction centers at the PSII (Maxwell et al. 1995; Neidhardt et al. 1998). This was supported by the decrease in the Y(NPQ) (Fig. 2e) at the expense of an increase in the Y(II) (Fig. 2a) that resulted in higher rETR values (Zhao et al. 2017). However, from a different perspective, the energy partition factor between the PSII and PSI would have suffered changes due to possible alterations in the LHCs structure, triggered by the reduction in Chl a content (Baker 2008; Hogewoning et al. 2012). In such scenario and taking into account the changes in the cellular light absorption properties, the electron transfer rate values should be more accurately expressed as the absolute ETR, which is estimated based on the absorptance and partition factor measurements (Malapascua et al. 2014); however, the determination of these parameters was beyond the scope of the present study.
In the case of PD cultures, the photosynthetic production was not significantly affected either at D6 or D12 (Fig. 1j, k). This outcome, together with the unaltered Chl a content (Fig. 1f), could be explained by the fact that P is not as essential as N for chlorophyll biosynthesis (Kamalanathan et al. 2015). Additionally, scarcity of extracellular P is not detrimental for the photosynthetic apparatus (Kamalanathan et al. 2016), probably due to the rapid accumulation of intracellular reservoirs of polyphosphate bodies prior the transfer of the cells to the P-free medium (Powell et al. 2009). Furthermore, the elevated lipid accumulation in PD cultures compared to the control would have stimulated the reductive power demand from the electron transport chain, causing the augmented Y(II) that accounted for the higher rETR values (Fig. 1j–k).
Despite the differences found in the rETR values, which reflect the photosynthetic productivity, the overall response in terms of irradiance tolerance and inhibition was similar among all the tested conditions. The saturation point ranged between 200 and 300 µmol photons m−2 s−1, and above 400 µmol photons m−2 s−1 the decline in the rETR values denoted the photoinhibition region (Fig. 1j, k). Interestingly, Yoshimura et al. (2013) found a relationship between the irradiance and the temperature that affects the specific growth rate in B. braunii var Showa. In that case, the authors found that at 20 °C, the growth is somewhat inhibited with irradiance levels above 350 µmol photons m−2 s−1. Conversely, in a different study, the photosynthesis in B. braunii became saturated at 1000 µmol photons m−2 s−1 of red light (Sakamoto et al. 2012). Nonetheless, the different wavelengths used in each study can partially account the discrepancies.
The quenching analysis revealed that the different nutritional contexts evaluated for B. braunii cells had a significant impact on the dissipation and use of light energy. The cellular response (i.e., high YII and low YNO; Fig. 2a, c, respectively) under the highest NaHCO3 concentration could be a consequence of a combined effect between an enhanced capacity to use the reducing power through the carbon fixation pathway, pulling electrons from the electron transport chain, and a concomitant maintenance of a stable intra-thylakoidal pH gradient that keeps the YNPQ (Fig. 2e) similar among the groups (Malapascua et al. 2014). At D12 in contrast, the YNO (Fig. 2c) surpassed significantly the YII (Fig. 2a) and YNPQ (Fig. 2e) in the culture with the lowest NaHCO3 concentration, showing the photosynthetic impairment derived from the unavailability of carbon as the main electron sink.
The N deprivation showed to have an important impact not only on the carbon distribution into the different biomass components but also on the energy partitioning. At D6, the prevalence of YII (Fig. 2b) over YNPQ (Fig. 2f) in ND cultures compared to the controls suggests that such nutritional context favors the photochemical conversion of the absorbed energy and reduces the need for active photoprotective mechanisms. This outcome led us to hypothesize that a higher reductive power demand in the form of NADPH to sustain the elevated rates of lipid biosynthesis (Fig. 1b) may be pulling the absorbed energy into the electron transport chain, rather than dissipating the exitonic energy as heat or fluorescence. Alternatively, Figueroa et al. (2021) reported that the green alga Ulva rigida, exhibited higher ETR values under low nitrogen (5 µM nitrate) and carbon (380 ppm CO2) conditions compared to low nitrogen and high carbon (700 ppm CO2) conditions. This was explained due to the disturbance in the dynamics of the NADPH and ATP production for carbon assimilation and the changes in the C:N ratio derived from the N reallocation inside the cells. At D12, the drastic reduction of Chl a level, along with the prolonged N starvation, led to the stimulation of active photoprotection mechanisms expressed as YNPQ (Fig. 2f) that resulted in the decline of the contribution of the YII (Fig. 2b).
On the other hand, P starvation showed a substantial impact on the distribution among the passive and active energy dissipation mechanisms. During the first days of incubation, the contribution of the active photoprotective mechanisms (i.e., xanthophyll cycle) was more active than the passive dissipation of the energy surplus as heat (i.e., YNO) (Fig. 2d). Accordingly, P-deprived B. braunii cells could preferentially activate the energy-mediated photoprotection systems (Cui et al. 2017). The increase in YNPQ (Fig. 2f) could serve as a mechanism to cope with the light-induced stress and the accumulated proton motive force derived from the substrate-dependent limitation in the phosphorylation reactions and the limited carbon fixation rate, as similarly reported by (Wykoff et al. 1998). A comparative proteomic analysis performed with Karlodinium veneficum revealed a significant metabolic remodeling response under P starvation (Cui et al. 2017). These authors indicated that some of the most P-consuming processes (e.g., cell proliferation) were downregulated, while some energy-producing/consuming metabolic pathways remained active or were even upregulated such as glycolysis, tricarboxylic acid cycle, and lipids metabolism. Additionally, the level of expression of several LHC-related proteins was increased upon P-deprivation, which explains the conservation of the photosynthetic activity observed under such conditions. Even though our results are consistent with those shown in the cited reports, the specific mechanism governing the energy distribution and its relationship with the lipid metabolism is a complex process which remains largely at the level of speculation for B. braunii and is open to be further experimentally explored.
Effects of nitrogen deprivation in B. braunii lipidome
N-deprivation of microalgae triggers membrane lipid turnover and breakdown (Li et al. 2012; Simionato et al. 2013; Martin et al. 2014; Han et al. 2017; Kokabi et al. 2020) and de novo synthesis of fatty acids (FA) (Li et al. 2012; Kokabi et al. 2020), to supply precursors for TAG assembly. Lipid turnover and degradation under N deprivation is central to keep membrane homeostasis under stress, especially in chloroplasts, thus explaining the increase of DGDG to MGDG ratio, which corresponds to the major chloroplast lipids (Kobayashi 2016). This is in agreement with our observations of a major abundance of DGDG compared to MGDG species (Fig. 4b, c), and with results published by other authors (Li et al. 2014; Han et al. 2017; White et al. 2019; Kokabi et al. 2020).
MGDG reduction under nutrient limitation is consistent with its role as the major thylakoid constituent and its function in the photosynthetic pigment-protein complexes (White et al. 2019). Therefore, the loss of cellular viability following N-deprivation could be avoided by blocking photosynthetic electron transport or redirecting the exitonic energy through non-photochemical dissipation routes (Fig. 2f), relieving a detrimental overreduction of the photosynthetic electron transport chain (Li et al. 2012). Hence, membrane lipids recycling into TAGs may represent a protective mechanism to minimize the destruction of the thylakoid and chloroplast envelope membranes (Han et al. 2017). DGDG increase, on the other hand, suggests a physiological adaptive response to nutrient stress for the functional and structural stabilization of the chloroplast (Han et al. 2017; White et al. 2019).
As mentioned earlier, the total lipid content of ND B. braunii cells was higher than in the control samples (Fig. 1b). After examining the lipidome, we noticed an augmented abundance ratio in most DAGs (Fig. 3c, d). The increased DAG accumulation may take place through several mechanisms reported in microalgae subjected to nutrient limitation: (i) acylation of de novo synthesized FAs (Li et al. 2012; Martin et al. 2014), (ii) or acylation of FAs recycled from membrane lipids which are liberated through the activity of acyl hydrolases (Sung Ho Cho and Thompson 1986; Li et al. 2012; Martin et al. 2014), into the glycerol backbone to produce glycerolipids such as DAG; or (iii) glycolipid- (Li et al. 2012) and GPL-derived DAG (Martin et al. 2014), that results from the removal of the polar head of these membrane lipids. The lipidome analysis also showed that around 93% and 87% of the TAGs identified were accumulated in D6 and D12 samples, respectively (Fig. 4d). TAG species with low degree of unsaturation (e.g., TAG-48:0/-48:2/-50:0/-50:1/-52:1/-52:2/-54:2/-56:2/-58:1) are proposed to be the result of de novo synthesis pathways (Simionato et al. 2013; Li et al. 2014); nevertheless, they could also result from FA recycling (Simionato et al. 2013; Li et al. 2014). The increased abundance of highly unsaturated TAGs (e.g., TAG-50:4/-50:5/-50:6/-52:4/52:5/-52:6/-52:7/-52:8/-52:9) leads us to hypothesize that they may result from membrane-derived polyunsaturated FA, in agreement with the reduction in glycolipid abundance (Fig. 4b, c), which may be indicative of membrane derived-FA recycling into TAGs (Li et al. 2012, 2014; Kokabi et al. 2020).
With respect to membrane lipids, we identified several GPLs in B. braunii ND cells, namely PC, PG, and PS (Fig. 5a, b), detected mostly in D12 samples. The PG and PS abundance ratio was reduced, while the opposite was encountered for PC. These results share some similarities and differences with those published by other authors. For instance, Martin et al. (2014) reported a decrease in total GPLs (e.g., PE, PC, PS, PI) content of around 70% and 33% for Chlorella sp., and Nannochloropsis sp., respectively, with the exception of PG that experienced an increased in both microalgae. Li et al., (2014) conducted a study with Nannochloropsis sp., reporting the depletion of PC, PI, and PG, concurrent with the accumulation of TAG. White et al., (2019) in a study with Chlorella sp., recorded a reduction for several GPL classes (e.g., PE, PC, and PG). From our results, it is not possible to explain the preferential accumulation of PCs over the other GPLs identified, such as the non-nitrogen containing PG. In P-deprived microalgae cells, there are reports of PC and DGDG increase, which may suggest the re-assembly of DGDG from PC, hypothesizing that PC may transiently increase under P deprivation for its reuse in DGDG biosynthesis (Jouhet et al. 2003; Kokabi et al. 2020). In ND B. braunii cells at D12, we found increased abundance for 71% and 73% of the identified DGTSs (Fig. 4a) and DGDGs (Fig. 4b) respectively, as well as the global increase of neutral lipids (DAG, TAG; Figs. 3d and 4d, respectively). However, the occurrence of a similar mechanism that may explain the concomitant PC, DGTS, DGDG, and neutral lipids accumulation that may point towards a possible redirection of PC-derived intermediates to structural and neutral lipids biosynthesis, requires further studies to be confirmed. In respect to the structural lipid DGTS, we found an increase taking place for a relevant number of species not only in PD at D6 but also in ND cells at D6 and D12 (Fig. 4a), contrary to reports of overall DGTS reduction in nutrient-limited microalgae cultures (Han et al. 2017; White et al. 2019). In addition, given the increased abundance of PC species found under N deprivation (Fig. 5b), we did not find a clear pattern of PC substitution with DGTS under the conditions evaluated in this study, as reported by other authors (Sato 1992; Harwood 2006).
A final aspect that worth highlighting is related to the preservation of membrane homeostasis. The increase of the DGDG/MGDG ratio is a common observation made in several studies of microalgae subjected to conditions were N deprivation/limitation is the stressor agent (Lu et al. 2013; Abida et al. 2015); however, there is also evidence that other lipid remodeling responses can take place in microalgae coping with the same stressor. The studies performed with N-deprived Ettlia oleoabundans (Matich et al. 2018) and C. reinhardtii (Siaut et al. 2011), showed that a global reduction of chloroplast lipids (MGDG, DGDG, and SQDG), besides PG can occur concomitantly with TAGs accumulation. In the study performed with Chlorella sp., and Nannochloropsis sp. (Martin et al. 2014), a reduction of MGDGs and DGDGs was also reported, but SQDGs underwent a significant reduction in Chlorella sp., while in Nannochloropsis sp., the decrease was less drastic, and in both cases the PG content increased. Similar results with Nannochloropsis sp. were obtained by Li et al. (2014) in respect to MGDG, DGDG, and SQDG reduction, alongside the drop of PG content. Finally, in Chlamydomonas nivalis (Lu et al. 2013), the increased DGDG/MGDG ratio was accompanied by a reduction of SQDG and a concomitant increase of PG. The above examples show that it is not possible to make a generalization regarding the remodeling responses of membrane lipids exhibited by microalgal cells under stress conditions. In our study with B. braunii, we observed a reduced abundance for MGDGs at the early and late stages of growth (Fig. 4c), with a concomitant increase in the abundance of over 60% of the identified DGDGs (Fig. 4b), which is in agreement with some of the studies aforementioned. However, the proper development of the chloroplasts and the functioning of the photosynthetic machinery requires an adequate balance among several lipid classes (Wada and Mizusawa 2009; Martin et al. 2014). Hence, the lipid remodeling responses under stress conditions not only involve galactolipids, but also other lipid classes, such as PG and sulfolipids, that despite not being comparatively as abundant as galactolipids, their importance should not be overlooked. From our data, it is not possible to infer all the mechanisms deployed by N-deprived B. braunii cells to maintain membrane homeostasis, especially in chloroplasts, given the observed PG and MGDG reduction. There is evidence that PG deficiency may be at least partially compensated by the anionic lipid SQDG, (Abida et al. 2015; White et al. 2019), which is not the case in ND B. braunii cells (Fig. 5b), or by the not charged lipid DGDG (Abida et al. 2015), that appears as the most likely possibility according to the abundance ratio calculations (Fig. 4b). The differences in the results of membrane lipid remodeling in this and other published studies may suggest a species-specific response to stress and may be indicative that in B. braunii the shunting of carbon flux from the membrane lipids to TAG biosynthesis occurred mainly by glycolipids (i.e., MGDG) and to a lesser extent by betaine lipids rather than GPLs degradation. Further research is necessary to unveil the mechanism of lipid membrane homeostasis in B. braunii under stress conditions.
In addition to the mentioned mechanisms for boosting glycerolipids accumulation, other existing macromolecules could also be degraded (i.e., CHOs and proteins) into lipid biosynthesis precursors (Li et al. 2010, 2011). In our study, the significant reduction of in- and ex-CHOs in ND cells (Fig. 1d, e), especially at D12, might point towards a shift of carbon partitioning into neutral lipids as primary carbon and energy storage product, at the expense of CHOs.
Effects of phosphate deprivation in B. braunii lipidome
P is a critical nutrient in all domains of life (Dyhrman 2016). The effects of P deprivation have been widely studied in microalgae and cyanobacteria (Martin et al. 2011; Cañavate et al. 2017; Matich et al. 2018; Kokabi et al. 2020). The effect of P deprivation in boosting cellular lipid accumulation has rendered contradictory results in microalgae. There are reports of minor changes (Cañavate et al. 2017; Matich et al. 2018) or even reduction (Cañavate et al. 2017), while there are other cases where an increased lipid content is observed (Kamalanathan et al. 2015; Cañavate et al. 2017). In our study, we found that P deprivation triggers B. braunii lipid accumulation only at early stages of growth, since a longer exposure to this stress condition has the opposite effect, causing a significant reduction of the total lipid content (Fig. 1b). Several remodeling events took place in the lipidome of PD B. braunii cells compared to the control samples. The abundance/reduction ratios of DAG (Fig. 3c, d), DGTS (Fig. 4a), and DGDG (Fig. 4b) species were not just influenced by the absence of P in the medium, but also by the time of exposure to this stress, since the abundance pattern changed drastically in the two time points evaluated. On the contrary, the response was more consistent for the structural glycolipid, MGDG (Fig. 4c), since we noticed a global reduction of MGDG at early (D6) and late (D12) stages of culture.
TAG accumulation under P stress has been reported in a number of studies (Rodolfi et al. 2009; Abida et al. 2015). In B. braunii cells, TAGs were predominantly built up at the early stage of growth (Fig. 4d) as reported for other microalgae upon short-term exposure to P deprivation (Mühlroth et al., 2017). However, after prolonged PD stress, TAG accumulation seemed to slow down, with a significant number of TAGs being degraded. Considering the abundance ratio trend, we determined that at D6, the higher abundance of DAGs and TAGs, and to some extent several polar lipids (i.e., DGDG and DGTS), were responsible for the increase in total lipid content. At D12, the reduction of a number of GPLs, galactolipids, DGTS, and TAGs, may account for the reduced total lipid content.
Meanwhile, our results of GPLs and SQDG reduction upon exposure of cells to prolonged P deprivation (Fig. 5a, b), differed to some extent from the work published by other authors. In cyanobacteria and eukaryotic algae, it is reported that the P-rich PG can be substituted by other non-P-containing lipids (i.e., SQDG, DGDG), while the extraplastidial PC can be replaced by taxonomically specific betaine lipids (Abida et al. 2015; Mühlroth et al. 2017). In our case, we noticed increase in DGTS at the early stage of growth (D6) (Fig. 4a); while at later stages, both SQDG (Fig. 5b) and DGTS species were reduced. These findings are also partially opposed to schemes of GPLs recycling proposed for other microalgae, where GPLs are broken down to glycerol-3-phophate, FAs, and DAG, and the products are used to synthesize mainly DGTS and SQDG in exponential growth and, later, for the synthesis of TAGs and galactolipids in stationary growth (Mühlroth et al. 2017). In our study, we could hypothesize that in B. braunii cells, GPLs degradation products may be preferentially directed to TAG biosynthesis, but only at late stages of growth (Fig. 5b), since GPLs degradation was negligible at D6 (Fig. 5a). This might coincide with the need to mobilize GPL-bounded P, possibly acting as secondary P storage upon cell exposure to prolonged P deprivation. On the other hand, the overall trend of MGDG degradation found in B. braunii samples (Fig. 4c) leads us to suggest that this galactolipid class may be recycled into neutral lipids (i.e., DAG and TAG) at D6 (Figs. 3c and 4d) and D12 (Fig. 4d), and may act as a precursor for other structural lipids (i.e., DGTS, DGDG) at early stages of growth (Fig. 4a, b).
The above findings indicate that at early stages of growth, B. braunii probably had availability of intracellular P that might have been stored before the cells were transferred to the P-deprived medium (Casadevall et al. 1985), and was sufficient to sustain cellular metabolism and growth at a similar rate to B. braunii cells under P-replete conditions (Fig. 1a). However, upon exposure to prolonged P-deprivation, the P levels may become critically low with a subsequent arrest of growth and a reduced lipid content. It is well documented that algae are capable of absorbing P in amounts largely exceeding cell requirements, and such P surplus is stored as pholyphosphate granules (Dyhrman 2016), that constitute a reservoir to sustain cell growth in a P-free medium. These results indicate that B. braunii may be capable of storing P surplus, which nevertheless was not sufficient to sustain growth and lipid production under conditions of prolonged P deprivation.
The GPLs decrease following P depletion found across microalgae and observed in the present study is compatible with a role for GPLs as P storage compounds (Dyhrman 2016; Cañavate et al. 2017), and constitutes one of several mechanisms exhibited by microalgae for maintaining P homeostasis. However, the magnitude of this role seems to be highly variable as it depends on the total microalgal GPL content, which is susceptible to turnover under the influence of P availability (Cañavate et al. 2017). In species with an intrinsically low GPL content, it is very unlikely that GPLs are involved in P stock regulation, as may be the case of B. braunii, giving the low number of GPLs detected compared to other structural lipids. This finding provides evidence that a true physiological exchange between betaine lipids and GPL cannot always be assumed (Cañavate et al. 2017). It is suggested that betaine lipids might play a more diversified role than GPL in microalgal lipid metabolism and that betaine lipids replacing GPLs in P-scarce environments cannot be regarded as a common phenomenon (Cañavate et al. 2017).
Lipidome remodeling of B. braunii cells grown under high carbon dosage
B. braunii cells cultivated under different NaHCO3 regimes displayed a group of lipids whose abundance was increased or decreased concomitantly with the NaHCO3 concentration (Figs. 3c, d and 4a–d). The accumulation of structural lipids (i.e., DGTS, DGDG, MGDG) was particularly enhanced at early stages of growth (Fig. 4a–c), particularly for 1 and 2% NaHCO3 samples, in contrast to the condition-dependent reductions observed under nutrient (i.e., N and P) deprivation. In studies performed with N-deprived C. reinhardtii, it was suggested that DGTS remodeling occurs in a temporal manner in response to overall cell density and environmental conditions (Yang et al. 2015). We observed an increased abundance of the structural lipids in particular for 1 and 2% NaHCO3 cells, which corresponded to the conditions where the highest biomass productivities were reached (Fig. 1a). Thus, it is expected that cells with the highest division rates require larger amounts of structural lipids, whose synthesis was enhanced, especially at the earliest time point. It was also remarkable to note the differential profile of nutrient consumption from the medium, where we observed that in cells grown on medium with 0.1% NaHCO3, the depletion of P from the medium occurred faster (Fig. 1h), which may trigger a similar response to P-deprived cells, as we also noticed a similar pattern of DGDG and MGDG degradation at D6 and D12 (Fig. 4b, c), as well as DGTS reduction at D12 (Fig. 4a).
The total lipid content was increased compared to the control samples, and despite it was slightly lower than the N-deprived treatment, we did not observe a substantial reduction of TAGs at the later stage of growth (Fig. 4d). It is interesting to note that when high carbon dosages are supplied to B. braunii in the form of NaHCO3, the neutral lipids fraction (i.e., DAG, and TAG) is enriched compared to the control samples; and unlike the N-starved cells, accumulation of TAGs occurred at later stages of growth with minimal degradation (Fig. 4d). DAG species were preferentially accumulated at the early stage of growth (D6) (Fig. 3c), since samples taken at D12 showed a reduction of the abundance ratios for a substantial number of DAGs (Fig. 3d). Whether this may be the result of a reduced rate of DAGs synthesis or a higher flux towards the reactions of fatty acyl assembly to produce glycerolipids, or both, requires further research to be determined. Another question that remains open is the origin of the precursors for TAG formation at later stages of growth, since DAG accumulation seems to slow-down compared to the control samples, while a concomitant reduction was also appreciated for the structural lipids, DGTS, and galactolipids, which may point to a mechanism of lipid recycling into TAGs. However, further research is necessary to elucidate the contribution of the de novo synthesis and lipid remodeling routes to neutral lipid accumulation in B. braunii cells cultivated under a high carbon dosage regime. The effect of a larger TAG accumulation was more evident in the medium with 1% and 2% NaHCO3, which underline the importance of keeping a proper C/N ratio to sustain high biomass and lipid yield.
After taking a close look at the TAGs that were accumulated at the early (D6) and late stages of growth (D12), we did not identify a pattern that might be indicative of the preferential accumulation of a specific group of TAGs, since over 70% of the accumulated species were common to all samples. On the other hand, after comparing the TAGs distributions of samples taken at the same sampling points, of cells grown on NaHCO3 and the N-deprived cultures, we identified a group of TAGs whose abundance ratios were increased exclusively at the early and the late stage of growth, which corresponded mostly to polyunsaturated species (Fig. 4d). The question remains as to what can be attributed this common feature, since the extracellular medium of cells cultivated with NaHCO3 still contained considerable amounts of nutrients (i.e., P and N), especially in 1 and 2% NaHCO3 samples (Fig. 1g, h). Thus, a nutrient deficiency response at least for these two elements does not seem to be a likely explanation. On the other hand, the increase of these TAGs occurred on samples taken at specific days, and therefore the possibility remains that the change in the profile of these TAGs may be related to the stage of growth of the culture and to some extent may also be associated with lipid remodeling events, given the condition-specific reductions observed for some polyunsaturated species of chloroplast lipids, as well as GPLs, betaine lipids, and DAGs. Further research will be required to have an in-depth understanding of the physiological mechanisms behind these changes.
The use of NaHCO3 has been evaluated in different algae, with interesting results in terms of larger biomass and/or lipid accumulation (Pimolrat et al. 2010; Gardner et al. 2013). In B. braunii, there are but a few reports about the use of NaHCO3 as carbon source and its effect on growth and total lipid content (Tanoi et al. 2011; Wei-Bao Kong 2012; Salbitani et al. 2019), and a detailed analysis about the lipid composition and the impact of this carbon source on other determinants of growth and productivity was lacking.