TAG and starch accumulation
For both the wild-type (wt) S. obliquus (UTEX 393) and a starchless mutant (slm1) of S. obliquus[28], duplicate nitrogen run-out experiments were performed to investigate the difference in carbon partitioning between the wt and the slm1 under nitrogen depleted conditions (Figure 1). Reactors were inoculated at 50 mg DW/l and cultivated at an incident light intensity of 100 μmol m-2 s-1 until the biomass concentration was 0.3-1 g DW/l, after which the incident light intensity was increased to 500 μmol m-2 s-1. The moment of inoculation is considered as t = 0.
Nitrogen was depleted from the culture medium at a biomass concentration of approximately 1.5-2 g/l and occurred 70 to 100 h after inoculation (Figure 1). After nitrogen was depleted, carbon assimilation and biomass formation continued, mainly as a result of accumulation of TAG (both the wt and the slm1) and starch (the wt only), which is consistent with previous observations [7, 28, 29]. The wt increased more rapidly in biomass concentration than the slm1 during initial nitrogen starvation and also achieved a higher maximum biomass concentration (Figure 1A,B).
In the wt cultivation, starch and TAG were accumulated simultaneously after nitrogen was depleted. Initially starch was produced at a much higher rate than TAG (Figure 1C), but when nitrogen starvation progressed starch synthesis stopped. Starch reached a maximum content of 38 ± 2% (average of duplicate cultivations ± deviation of duplicates from average) of dry weight after 168 ± 2 h and a maximum concentration of 3.6 ± 0.2 g/l after 223 ± 10 h. Subsequently starch was degraded. The starch concentration at the end of the cultivation decreased to 0.5 g/l (6% of dry weight). During this period TAG synthesis continued, and the TAG content reached a maximum of 45 ± 1% of dry weight (4.5 ± 0.1 g/l) (Figure 1C; Figure 2A). The simultaneous degradation of starch and production of TAG in the wt could indicate that degradation products of starch are used for the synthesis of TAG. This interconversion has also been suggested previously for Pseudochlorococcum sp. [11], C. reinhardtii[19], Coccomyxa sp. [19], and Chlorella zofingiensis (also known as Chromochloris zofingiensis) [13], as well as for conversion of chrysolaminarin into TAG in the diatom Cyclotella cryptica[30].
In the slm1, the production of starch is negligible. As a result, the TAG content increases more rapidly in the slm1 than in the wt during initial nitrogen starvation; the TAG content in the slm1 reached a maximum of 57 ± 0.2% of dry weight (5.2 ± 0.2 g/l) after 433 ± 70 h (Figure 1C,D; Figure 2A).
In neither the wt nor the slm1 can the combined accumulation of starch and TAG completely account for the increase in dry weight after nitrogen depletion. This difference between the measured biomass constituents and dry weight concentration is relatively constant and accounts for approximately 20 to 30% of dry weight during the entire cultivation for both the wt and the slm1. Proteins are most likely not part of this residual biomass as no nitrogen source is available for protein synthesis; also protein synthesis out of non-protein nitrogen present in the biomass can only contribute very little, because this fraction of non-protein nitrogen in the biomass is very small [31]. It is likely that the cell wall will account for a substantial part of this residual biomass. Although little is known about the cell wall composition of S. obliquus and other microalgae, it is hypothesized that this residual biomass consists largely of carbohydrates (other than starch) such as cellulose, which is known to be a major constituent of the cell wall of S. obliquus and other microalgae [32, 33].
Yields, productivity, and implications for large-scale production
Using the measured TAG concentration at each time point (Figure 2A) and the amount of light supplied specific to the reactor volume (calculated as the incident light intensity multiplied by the area-to-volume ratio of the reactor), the time-averaged yield of TAG on photons was calculated for each time point (the yield of TAG on light achieved over the period between inoculation and each time point) (Figure 2B). Because almost no TAG is produced during nitrogen replete conditions, this yield of TAG on light is very low during the initial part of the cultivation. After nitrogen depletion, the time-averaged yield increases to a maximum of 0.217 ± 0.011 and 0.144 ± 0.004 g TAG/mol photon for the slm1 and wt, respectively (Figure 2B). This illustrates that the slm1 can achieve a 51% higher time-averaged yield of TAG on light than the wt. Similarly, the maximum volumetric productivity, calculated between inoculation and each time point, was enhanced in the slm1 by 35% compared to the wt and increased from a maximum of 0.265 ± 0.004 in the wt to a maximum of 0.359 ± 0.008 g TAG l-1 day-1 in the slm1 (Figure 2C). During the period that these maxima in yield and volumetric productivity were maintained, the TAG content increased to over 40% of dry weight for both the wt and the slm1 (Figure 1C,D; Figure 2B,C).
After these maxima in yield and volumetric productivity were achieved, the difference in performance of the wt and the slm1 became smaller when the cultivation progressed. This can be explained by the degradation of starch in the wt and possible interconversion into TAG. This could enhance the TAG contents in the wt at the end of the cultivation, resulting in a smaller difference between the slm1 and wt at the end of the cultivation.
Due to the different behavior of the wt and slm1, there is a difference in the biomass concentration in the wt and slm1 cultivation (Figure 1). This did not result in a difference in light absorption rates between the wt and slm1, because nearly all light was absorbed in all cultures; therefore, a difference in the biomass concentration or pigmentation will only result in a difference in the light gradient in the photobioreactor. Furthermore, because all cultures were provided with the same amount of NO3, the amount of light absorbed per N-mol and per amount of catalytic biomass (assuming that the amount of catalytic biomass is proportional to the amount of nitrogen) is exactly the same.
When algae are cultivated using sunlight, the amount of light that can be provided to the photobioreactor is limited to the insolation to that area. The maximum areal productivity is therefore directly proportional to the yield on light that can be achieved. Maximizing this yield of TAG on light can therefore contribute to improving the areal productivity of microalgal TAG production. The time point where the highest time-averaged yield of TAG on light is achieved is therefore proposed as the optimum time point to harvest the culture. Previously it has been shown that this yield can be enhanced by improving the photobioreactor design [2, 34] as well as optimizing cultivation conditions [29]. In this work it is shown that this yield on light can be improved by 51% by using a starchless mutant, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. It should be noted that at the moment this maximum was reached, the TAG content was over 40% of the dry weight.
In this work, all cultivations were performed using continuous illumination. However, during day-night cycles starch contents in microalgae oscillate, and starch can likely provide energy for nocturnal respiration [35, 36]. This might complicate cultivation of starchless mutants in day-night cycles. In higher plants such as Arabidopsis thaliana it is indeed reported that starchless mutants show decreased growth rates and decreased net photosynthesis rates when grown under day-night cycles, whereas these are indistinguishable from their wild types during continuous illumination [37, 38]. The slm1 mutant, however, does not show decreased growth under day-night cycles under nitrogen replete conditions, and possibly the role of starch can be taken over by other storage metabolites [28]. Further investigation of the behavior of slm1 under day-night cycles and nitrogen depleted conditions would be of future interest.
Photosynthetic energy distribution in the wt compared to the slm1
The biomass productivity was lower in the slm1 than in the wt during the initial period of nitrogen starvation. Because exactly the same amount of light was supplied, this might at first suggest a reduced photosynthetic efficiency in the slm1. However, lipids (for example, TAG) are much more energy dense than carbohydrates (37.6 kJ/g for lipids compared to 15.7 kJ/g for carbohydrates [39]). The difference in metabolic costs required to produce TAG and starch can completely explain the observed difference in biomass productivity. To illustrate this, we compare the photosynthetic requirement for the observed biomass production after nitrogen depletion in the wt and the slm1. To calculate this photosynthetic requirement, it is assumed that after nitrogen depletion only TAG, starch, and other carbohydrates (such as cell wall cellulose) are produced. The TAG and starch concentration are measured at each time point (Figure 1), and it is assumed that the remaining newly produced biomass consists of other carbohydrates (calculated as the amount of dry weight produced minus the amounts of TAG and starch produced). The photosynthetic requirement to produce the biomass that is made between nitrogen depletion and time point t can then be calculated by summing the quotients of the measured concentration of each biomass constituent at time point t and the photosynthetic yield of that biomass constituent (Eq. 1):
(1)
In Eq. 1, Ci(t) represents the concentration of component i (g/l) at time point t and Yi,light represents the photosynthetic yield of component i (g product/mol photon). These photosynthetic yields are estimated to be 1.02 g TAG/mol photon, 3.24 g starch/mol photon, and 3.24 g carbohydrate/mol photon (see Appendix A).
Using this calculation, it appears that although the slm1 has a lower biomass productivity, the minimum photosynthetic requirement to produce that biomass is similar (Figure 3). This indicates that the slm1 does not have a reduced photosynthetic efficiency, but only seems to differ from the wt in terms of carbon partitioning.
In the wt, the calculated photosynthetic requirement also increases at the end of the cultivation, where no substantial increase in dry weight concentration is observed. This can be explained by an increase in energy density of the biomass due to a change in biomass composition (increase in TAG and decrease in starch content). This requires additional energy, which is provided by photosynthesis.
In these calculations it was assumed that the residual biomass (difference between the produced dry weight and the measured amounts of TAG and starch) consists in large part of cell wall material that is made of carbohydrates such as cellulose [33]. If this were a different biomass constituent with a different photosynthetic yield than carbohydrates, it would affect the calculated photosynthetic requirement. However, the estimated amount of this remaining fraction is similar in the wt and the slm1. Therefore, this will not result in a biased comparison.
It is observed in the wt that starch is first produced and subsequently degraded (Figure 1). This turnover is not taken into account in these calculations. However, the turnover of starch (synthesis of starch out of GAP and subsequent degradation of starch into GAP) only costs 1 ATP per glucose monomer and would only result in a minor change in the calculated photosynthetic requirement.
Photosynthesis
The pigmentation of the cell determines the amount of light that can be absorbed and ultimately be used for photosynthesis. The absorbance cross section of the biomass was measured and used as a proxy for the pigmentation. An up to eightfold decrease in the biomass specific absorbance cross section (m2/g DW) was observed at the end of the cultivation compared to the point before nitrogen depletion in both the wt and the slm1 (Figure 4B). A decrease in pigmentation during nitrogen starvation is commonly observed in microalgae [40]. The volumetric absorbance cross section (m2/l), however, remained more or less constant throughout the entire experiment. This suggests that the decrease in biomass specific absorbance cross section is mainly a result of dilution of pigments over newly formed biomass and is likely caused to a lesser extent by net degradation of pigments.
In addition to a change in absorbance cross section, the absorbance spectrum, and thus the pigment class composition, changed drastically (Figure 4C). Photoprotective pigments (carotenoids) can be produced in response to physiological stress to prevent photo-oxidative damage [40, 41]. The ratio of chlorophyll over carotenoids decreased during nitrogen starvation as is apparent from the increase in absorbance at 483 nm (the observed absorbance maximum of carotenoids) relative to the absorbance at 680 nm (the observed absorbance maximum of chlorophyll) (Figure 4D). The decrease in absorbance cross section between the slm1 and wt was similar, but the slm1 showed a higher ratio of absorbance at 483 nm/680 nm as the nitrogen starvation progressed. This suggests that the slm1 has relatively more carotenoids than the wt. This difference between the slm1 and wt became more apparent when nitrogen starvation progressed (Figure 4D). These observations suggest that a progressively smaller fraction of the absorbed light is available for photosynthesis when nitrogen starvation progresses due to an increased carotenoid/chlorophyll ratio.
The variable fluorescence/maximum fluorescence ratio (Fv/Fm) was measured and can be used as a proxy for the intrinsic (or maximum) PSII quantum yield [10, 23]. Although it does not directly reflect the photosynthetic efficiency achieved in the photobioreactor, it is often used as a diagnostic value for the photosynthetic performance [42]. Immediately after inoculation, the Fv/Fm ratio started substantially below the maximum value that was observed (Figure 4A). This could possibly be due to a shock in biomass concentration and light intensity as a result of inoculation. During the nitrogen replete growth phase, the Fv/Fm ratio increased gradually to a maximum of 0.78, which is consistent with maximum values observed in other studies [23]. Once nitrogen was depleted, the Fv/Fm ratio gradually decreased, as is commonly observed [10, 23]. This could be an indication of increased damage to the photosystems. Fv/Fm ratios in the slm1 are comparable to or even slightly higher than the Fv/Fm ratios in the wt. This is consistent with the observation presented in Figure 3, that the photosynthetic performance is not negatively affected in the slm1 and that the main difference with the wt is an improved carbon partitioning towards TAG in the slm1.