4. Results
4.2 Cellular composition of E. huxleyi determined via FTIR
4.3.1 Pigment composition in different growth phases
In this result section, the general pigment content and differences between both life-cycle stages at each respective time point will be described first. In a second step, the differences between growth phases will be addressed.
It is important to note that pigment results are presented in relative abundance per cell, as quantification via external standard calibration curves was not possible due to limited concentration of many commercially available standards. This is a common issue for marine microalgae, as they contain many uncommon pigments (Egeland, 2011). As analytes can give variable responses in UV-vis-spectrometers, it is possible that the abundance of one pigment is not necessarily comparable to another pigment (Jeffrey, 1997; Smith, 2004). For example, chlorophyll a is known to give a smaller response compared to fucoxanthin, at the same injected weight (Jeffrey, 1997). Studies have reported that fucoxanthin derivatives give comparable responses, the same is true for chlorophyll c derivatives (Egeland, 2011; Garrido, 2016). This does not influence conclusions about relative changes between life-cycle stages and growth phases.
Ten pigments could be detected in both life-cycle stages and all growth phases (Figure 13).
They were identified as chlorophyll a, chlorophyll c2, chlorophyll c2-MGDG, chlorophyll c3, diadinoxanthin, diatoxanthin, fucoxanthin, h4k-fucoxanthin, hex-fucoxanthin and β-carotene, based on comparison of retention times and absorption spectra from commercially available standards (Supplementary Table 2).
Figure 13: Relative pigment abundances per cell for E. huxleyi life-cycle stages harvested in different growth phases. Diploid (2N, dark grey) and haploid (1N, light grey) stages are shown. Cells harvested in (a) early-exponential phase (t1), (b) late-exponential phase (t2), (c) stationary phase (t3). Note different scale of y-axis. Statistical differences between both life-cycle stages are indicated by asterisks, as determined via t-test (* indicates p-value ≤0.05, ** ≤0.01). Mean values and SD of two biological and two technical replicates are shown. Chl c2-MGDG, chlorophyll c2-monogalactosyldiacylglycerol.
chloro phyll a chloro
phyll c2 chl c2-MG
DG chloro
phyll c3 diad
inoxanthin diato
xanthin fucoxanthin
h4k-fucoxanthin hex-fucoxanthin
β-carotene
0 200 400 600 800
Stationary growth phase Late-exponential growth phase
c b
Pigments [rel. ab. cell-1 ]
2N t1 1N t1
a
chloro phyll a chloro
phyll c2 chl c2-MG
DG chloro
phyll c3 diad
ino xanthin
diato xanthin
fucoxanthin h4k-fucoxanthin
hex-fucoxanthin β-carotene
0 30 60 90 120
Pigments [rel. ab. cell-1 ]
2N t2 1N t2
Early-exponential growth phase
chloro phyll a chloro
phyll c2 chl c2-MG
DG chloro
phyll c3 diad
ino xanthin
diato xanthin
fucoxanthin h4k-fucoxanthin
hex-fucoxanthin β-carotene
0 30 60 90 120
Pigments [rel. ab. cell-1 ]
2N t3 1N t3
The haploid stage generally showed a higher variability between biological replicates, as indicated by the higher standard deviation (SD). Comparison of both life-cycle stages in the individual growth phases revealed a similar pigment composition in the early-exponential growth phase, where not significant differences were observed (Figure 13 a). However, life-cycle specific differences were observed between both stages in the late-exponential and stationary phase, as the diploid stage contained significantly more h4k-fucoxanthin than the haploid stage in both later growth phases, as well as more diatoxanthin in the stationary phase (Figure 13 a, b).
Hex-fucoxanthin was a highly abundant pigment in both life-cycle stages. It was found in significantly higher abundance (p≤0.05) compared to chlorophyll a at all time points for the diploid stage, as well as in late-exponential phase for the haploid stage. However, as stated above, chlorophyll a gives a smaller detection response compared to fucoxanthin, which is why hex-fucoxanthin is not necessarily present in higher abundance than chlorophyll a. Direct comparison of fucoxanthin derivatives is possible due to high structural similarity. Hex-fucoxanthin comprised 76-87 % of total Hex-fucoxanthins in both life-cycle stages and all growth phases. In comparison, fucoxanthin (5-15 %) and h4k-fucoxanthin (4-15 %) proportions were minor.
The chlorophyll c derivatives are also comparable with regard to detection response.
Chlorophyll c2 and chlorophyll c3 were the most abundant chlorophyll c pigments in both life-cycle stages, together making up 80-86 % of total chlorophyll c. In contrast, the proportion of chlorophyll c2-MGDG was comparably small, amounting to 15-20 % of total chlorophyll c. The proportion of chlorophyll c2 and c3 varied slightly between growth phases and life-cycle stages.
In the diploid stage, both chlorophyll c2 and c3 were similarly abundant in the early-exponential (p=0.06) and stationary phase (p=0.09), whereas the chlorophyll c3 abundance was significantly higher in the late-exponential phase (p=0.02, 1.1-fold). This was not the case for the haploid stage, as no significant differences between chlorophyll c2 and chlorophyll c3 were observed.
To further examine growth phase-dependent changes in pigment composition in the individual life-cycle stages, the fold-change of cellular pigments was calculated, comparing pigment abundance of harvesting time points to the first harvesting time point (Figure 14).
Figure 14: Heat map of the fold-changes of pigments per cell in E. huxleyi life-cycle stages, comparing growth phases. Diploid (2N) and haploid (1N) stages are shown. (a, d) Early-exponential growth phase (t1) compared to early-exponential growth phase (t1). (b, e) Late-exponential growth phase (t2) compared to early-exponential growth phase (t1). (c, f) Stationary growth phase (t3) compared to early-exponential growth phase (t1). Asterisks indicate p-value of t-test (* indicates ≤0.05, ** indicates ≤0.01). Fold-changes were calculated from mean values of two biological and two technical measurements.
In the diploid life-cycle stage (2N), nine of ten pigment abundances decreased in the late-exponential phase compared to the early-late-exponential phase (Figure 14 b). Eight of them were significantly decreased, including all chlorophylls, diadinoxanthin, fucoxanthin, hex-fucoxanthin and β-carotene (2.7-6.3-fold). Diatoxanthin increased non-significantly (1.5-fold).
In the stationary phase, significant decreases were even more prominent for the same eight pigments (3.6-13.7-fold), compared to the early-exponential phase (Figure 14 c). Diatoxanthin was significantly increased (2.8-fold).
In the haploid-life cycle stage, the decreasing pattern of pigment abundances was similar, although the change was not significant, due to a higher SD. All pigment abundances non-significantly decreased (2.6-8.7-fold) from early to late-exponential phase (Figure 14 e). The decrease was more pronounced for all pigment abundances in the stationary phase compared to the early-exponential phase (6.0-21.3-fold), however again statistically non-significant (Figure 14 f).
Pigment abundances are often additionally normalized to chlorophyll a (Stolte, 2000;
Zapata, 2004; Llewellyn, 2007; Lefebvre, 2010), which allows even more detailed interpretations of a cell’s physiological status. The resulting pigment ratios are shown in Figure 15. Also, a de-epoxidation ratio can be calculated (Llewellyn, 2007; Ragni, 2008), which describes the activity of the xanthophyll cycle (photoprotection via non-photochemical quenching), in which diatoxanthin (dtx) and diadinoxanthin (ddx) are involved:
de − epoxidation ratio = ( dtx
(dtx+ddx)) Eq. 6
Figure 15: Heat map of the fold-changes of pigment to chlorophyll a ratios in E. huxleyi life-cycle stages, comparing growth phases. Diploid (2N) and haploid (1N) E. huxleyi life-cycle stages. (a, d) Late-exponential growth phase (t2) compared to early-exponential growth phase (t1). (b, e) Stationary phase (t3) compared to early-exponential growth phase (t1). (c, f) Stationary phase (t3) compared to late-exponential growth phase (t2). Asterisks indicate p-value of t-test (* indicates ≤0.05, ** indicates ≤0.01). Fold-changes were calculated from mean values of two biological and two technical measurements. Chl, chlorophyll; caro, carotenoids;
MGDG, monogalactosylgdiacylglycerol; fuco, fucoxanthin; ddx, diadinoxanthin; dtx, diatoxanthin; β-caro, β-carotene.
As mentioned above, the abundance of chlorophyll a per cell decreased during growth in both life-cycle stages. Trends for pigment to chlorophyll a ratios were similar for both life-cycle stages, although most changes were non-significant for the haploid stage. Only significant changes will be mentioned hereafter, fold-changes in parenthesis are depicted for the late-exponential- and stationary phase, respectively.
Regarding the general pigment distribution, the ratio increased in the diploid stage for total chlorophyll c to chlorophyll a (both 1.3-fold) and carotenoids to chlorophyll a (1.2- and 1.5-fold) in the late-exponential and stationary phase, compared to the early exponential growth phase. Taking a more detailed look into the individual chlorophyll c pigments, the increase was especially caused by chlorophyll c3 (1.7 and 1.9-fold). Furthermore, fucoxanthin abundance decreased in the diploid stage compared to chlorophyll a (1.4- and 2.0-fold), whereas both h4k-fucoxanthin (2.0-fold in stationary phase) and hex-h4k-fucoxanthin per chlorophyll a increased (1.4- and 1.5-fold).
Regarding the xanthophyll cycle carotenoids, the ratio of diatoxanthin to chlorophyll a especially increased in the diploid stage (6.6- and 19.3-fold), similar to the results on a cellular basis (Figure 14). Consequently, the calculated de-epoxidation ratio increased in the diploid stage in the late-exponential (3.7-fold) and even more in the stationary phase (7.9-fold), whereas it only increased in the late-exponential phase in haploid stage (2.1-fold in late-exponential phase). Absolute values for the de-epoxidation ratio are shown in Table 6. As mentioned above, increased de-epoxidation ratios indicate increased xanthophyll cycling and therefore a more intense photoprotection via non-photochemical quenching.
Table 6: The de-epoxidation ratio of xanthophyll cycle pigments for diploid (2N) and haploid (1N) E. huxleyi life-cycle stages harvested in three different growth phases. Mean value and SD of two biological and three technical measurements. Asterisks indicate significant difference of values at later growth phases compared to the early-exponential growth phase (p-value of t-test, * indicates ≤0.05, ** indicates ≤0.01).
De-epoxidation ratio [-]
Growth phase 2N 1N
early-exponential 0.08±0.003 0.18±0.08 late-exponential 0.29±0.10* 0.37±0.09*
stationary 0.62±0.03** 0.20±0.10