Growth and Lipid Accumulation
3.3 Results and Discussion
3.3.7 Cell viability
83 | P a g e Figure 3.8. Nile Red images showing cellular neutral lipids in T. obliquus KMC24 cells (C:
control cells, –N1 to –N4: one to four days nitrogen starved cells).
84 | P a g e 3.3.8 ROS and MDA content under nutrient starvation
ROS are produced in different cellular compartments of microalgae under unfavorable growth conditions. Microalgal cells trigger several defense systems to scavenge these ROS.
However, under stress conditions, the ROS production rate might exceed the scavenging rate of microalgal cells, thereby causing excess ROS accumulation. This excess ROS causes oxidative injury to the cells by damaging proteins, lipids, and DNA that ultimately causes cell death [19]. The effect of nutrient starvation on ROS fluorescence intensities is shown in Figure 3.9. The ROS fluorescence intensities in A1, A2, A3, B1, B2, B3, -N1, -N2, -N3, -P1, –P2, - P3, -P4 cultures increased significantly (P < 0.034) in comparison to control. This indicated that nutrient starvation was the environmental stress that led to ROS accumulation in the cells of T. obliquus KMC24. Zhang et al. observed similar results, where the ROS level increased significantly (P < 0.0034) in the nitrogen, phosphorus, and sulfur stressed cultures [38].
However, the fluorescence intensities in -N-P cultures were comparatively lower than the control. A gradual decrease in fluorescence intensities was observed with an increase in the duration of -N-P starvation. The dead cells do not possess ROS generating metabolic processes [316]. Therefore, a decrease in cell viability in -N-P cultures as assayed by flow cytometry could be the possible reason for reduced ROS fluorescence intensities. Also, a reduction in fluorescence intensities was observed from the third day of starvation in A, B, -N, -P cultures, probably due to the increase in dead cells with the duration of starvation. In our study, the highest ROS fluorescence intensity was observed in -N2 culture (17051.49 ± 93.15 a.u.).
MDA is a lipid peroxidation end product that is generally released as a stress indicator in microalgae. Hydroxyl radicals are highly reactive species that initiate membrane peroxidation, which results in the release of MDA as an end product [19]. In the present study, except for –N-P cultures, the MDA content was significantly increased (P < 0.0001) under all nutrient-starved conditions compared to the control culture (Fig. 7). The highest MDA content was observed in –N2 culture (3.81 ± 0.02 µM g-1 fresh weight), which was around 2.3-folds higher than the control culture (1.64 ± 0.01 µM g-1 fresh weight). The MDA content was consistent with the ROS level indicating that a linear correlation exists between ROS level and MDA content. Similar to our study, a simultaneous increase in ROS and MDA levels was reported in D. salina under nitrogen-limited conditions [146].
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85 | P a g e Figure 3.9. Effects of nutrient starvation on cell viability, malondialdehyde (MDA) content,
and ROS fluorescence intensity of T. obliquus KMC24. Values are presented as the mean ± standard deviation (n = 3). Values with the different letters represent a significant difference (P < 0.05) between treatments (same letters are not significantly different). The alphabetical
letters are denoted in the ascending order ("a" represents the highest value).
C A1
A2 A3 A4 B1 B2 B3 B4 -N1 -N2
-N3 -N4
-P1 -P2
-P3 -P4
-N-P1 -N-P2
-N-P3 -N-P4
0 6100 12200 18300
C A1
A2 A3 A4 B1 B2 B3 B4 -N1 -N2
-N3 -N4
-P1 -P2
-P3 -P4
-N-P1 -N-P2
-N-P3 -N-P4
0.0 1.3 2.6 3.9
C A1
A2 A3 A4 B1 B2 B3 B4 -N1 -N2
-N3 -N4
-P1 -P2
-P3 -P4
-N-P1 -N-P2
-N-P3 -N-P4
0 33 66 99
p q n o i k f c m a b g l g j h j m e d l
Fluorescence intensity (a.u.)
Treatment
l m k h j d c d ij a b
h f g d ij f g c e i MDA (µM g-1 FW)
-P -N-P
B -N C A
h h h f g bce ab f abde ab cdfg abab f abbc a
Viability (%) a
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86 | P a g e Figure 3.10. Fluorescence images of T. obliquus KMC24 cells stained with 2′, 7′-dichlorodihydrofluoresceine diacetate (DCFH-DA) probe showing the effect of nitrogen starvation in intracellular ROS formation (C: control cells, –N1 to –N4: one to four days nitrogen starved cells).
C -N1 -N2 -N3 -N4
C -N1 -N2 -N3 -N4
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87 | P a g e 3.3.9 Correlation between lipid content and ROS level under nutrient starvation
The correlation between lipid content and ROS level under various nutrient-starved conditions was revealed using Pearson's correlation analysis (Figure 3.10). The ROS fluorescence intensity for all the nutrient stress conditions is represented on the x-axis, while the lipid content (%) is represented on the y-axis. The Pearson correlation coefficient was 0.908, indicating a high correlation between lipid content and ROS. In addition, a high correlation coefficient under individual nutrient stress conditions was obtained by plotting individual fluorescence intensities versus corresponding lipid content (Figure 3.11). These results further confirmed that the ROS levels were positively correlated with the lipid contents for all the nutrient-starved conditions. It has been reported that microalgal strains with high tolerance to oxidative stress are more efficient for producing biodiesel as compared to non- tolerant strains [317]. In the present study, the highest ROS fluorescent intensity and lipid content was observed in -N2 cultures. This indicated that T. obliquus KMC24 cells under two days of nitrogen starvation are highly oxidative stress tolerant with potential for biodiesel production. In a similar study, a positive linear correlation between ROS and lipid accumulation under different culture conditions was observed in Chlorella pyrenoidosa [38].
It has been reported that enhanced lipid production might be mediated by oxidative stress [146].
Recent evidence also indicated that ROS might act as a secondary messenger for various stress factors, which controls cellular responses to extracellular stress [318]. Some of the hypotheses on the mechanisms of ROS-mediated lipid accumulation are as follows:
Lipids are highly reduced molecular entities. Therefore, NL overproduction necessitates significant amounts of NADPH, which is primarily obtained through the oxidative pentose phosphate (OPP) pathway. Oxidative stress causes the carbon metabolic flux to change from glycolysis to the OPP pathway through post-translational modification of glycolytic enzymes [318]. Hence, this could be a possible mechanism through which oxidative stress enhances NL accumulation.
The stored NL bodies are accumulated within lipid droplets, and studies have reported that endoplasmic reticulum (ER) stress activates the production of lipid droplets [319]. As ROS stimulates ER stress, this could be another possible way through which ROS increases lipid droplets formation [318].
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88 | P a g e
ROS might function as a mediator for enhanced lipid accumulation through autophagy under unfavorable conditions. Suzuki et al. [320] reported that ROS accumulation triggers autophagy in eukaryotic cells, while Scott et al. [321] reported that ROS stimulates autophagy, thereby causing the cells to degrade macromolecules and shift large amounts of carbon to energy storage compounds, such as lipids. Zhao et al. reported that salinity stress induces ROS and activates cellular autophagy, which further regulates lipid synthesis [322]. Couso et al. suggested that autophagy is required for the synthesis of TAG in nitrogen or phosphate-starved cells of Chlamydomonas [323].
Furthermore, ROS are also demonstrated to modulate cellular responses at the level of signal transduction pathways [324].
Figure 3.11.The correlation between lipid content and ROS level of T. obliquus KMC24 under various nutrient-starved conditions. Lines are linear fit with Pearson correlation
coefficient (r).
15 20 25 30 35 40 45
4000 6000 8000 10000 12000 14000 16000 18000
Lipid content (% DCW)
ROS fluorescence intensity (a.u.)
r=0.908, p<0.01
C A B -N -P -N-P
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89 | P a g e Figure 3.12. Relationship between the reactive oxygen species and corresponding lipid content (%) of T. obliquus KMC24 under individual culture condition. Lines are linear fit
with Pearson correlation coefficient (r).
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90 | P a g e 3.3.10 Responses of cellular antioxidants under nutrient starvation
Microalgal cells stimulate powerful intrinsic antioxidant systems such as enzymatic (CAT, APX) and non-enzymatic (polyphenols) metabolites to counter ROS toxicity. CAT is a heme-containing enzyme that allows the cell to remove H2O2 in an energy-efficient way by catalyzing the conversion of H2O2 into O2 and H2O [146]. CAT is absent in chloroplasts, so H2O2 is degraded in the chloroplast by APX. APX is an ascorbate-based antioxidative defense system that scavenges H2O2 [19]. The effect of nutrient starvation on the antioxidative defense system is illustrated in Figure 3.12. Except for –N-P cultures, the CAT and APX activity was significantly increased (P < 0.0001) under all nutrient-starved conditions, indicating a correlation with the corresponding H2O2 contents. The highest CAT and APX activity of 4.21
± 0.05 U mg-1 protein and 2.79 ± 0.04 U mg-1 protein respectively was obtained in -N2 cultures, which were around 1.26- and 1.93-folds higher than those of control cultures (3.32 ± 0.07 U mg-1 protein and 1.44 ± 0.09 U mg-1 protein, respectively). The increase in the activities of CAT and APX strongly suggests that oxidative stress is induced under nutrient-starved conditions in T. obliquus KMC24 cells.
Polyphenols function as a substrate for the hydrogen peroxide-scavenging enzyme peroxidase and inhibit ROS dissemination by modifying peroxidation kinetics and reducing cell membrane fluidity [19]. However, limited studies are available on the impact of nutrient starvation on the polyphenol content of microalgae. In the present study, except for -N-P, A4, B4, and -N4 cultures, the total polyphenol content was enhanced under all nutrient-starved conditions compared to the control culture. The highest total polyphenol content was observed in -N2 culture (159.39 ± 2.82 µg g-1 FW), which was around 2.7-folds higher than the control culture (58.37 ± 0.69 µg g-1 FW). However, on increasing the duration of nutrient starvation (3 and 4 days), a decrease in both enzymatic and non-enzymatic activities was observed.
Moreover, the responses of cellular antioxidants were significantly lower (P < 0.0001) than the control in –N-P cultures. A decrease in ROS accumulation could be the possible reason for the reduced activity of MDA, CAT, APX, and polyphenols in these cultures. Thus, it was observed that activities of CAT, APX, and polyphenols were consistent with lipid content suggesting a strong connection between oxidative stress and lipid accumulation.
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91 | P a g e Figure 3.13. Activities of the enzymatic and non-enzymatic antioxidants of T. obliquus
KMC24 under various nutrient-starved conditions. Values are presented as the mean ± standard deviation (n = 3). Values with the different letters represent a significant difference (P < 0.05) between treatments (same letters are not significantly different). The alphabetical
letters are denoted in the ascending order ("a" represents the highest value).
C A1
A2 A3 A4 B1 B2 B3 B4 -N1 -N2
-N3 -N4
-P1 -P2
-P3 -P4
-N-P1 -N-P2
-N-P3 -N-P4
0.0 1.6 3.2 4.8
C A1
A2 A3 A4 B1 B2 B3 B4 -N1 -N2
-N3 -N4
-P1 -P2
-P3 -P4
-N-P1 -N-P2
-N-P3 -N-P4
0.00 0.95 1.90 2.85
C A1
A2 A3 A4 B1 B2 B3 B4 -N1 -N2
-N3 -N4
-P1 -P2
-P3 -P4
-N-P1 -N-P2
-N-P3 -N-P4
0 45 90 135 180
mnn l lm ghgh c ef jk a ab ij de gh cdab fg hi de b k
CAT (U mg-1 protein)
Treatments
n o m mn fg gh de def
b
l a l ef hi f d j gh f c
k
APX (U mg-1 protein)
C A B -N -P -N-P
m m i kl e c de fg kl b a
de j kl ef i m h d gh
Polyphenol (µg g-1 FW)
jk
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92 | P a g e 3.3.11 Influence of nutrient starvation on fatty acid composition
The fatty acid compositions of T. obliquus KMC24 for all culture conditions are presented in Table 3.2. The major fatty acid components were palmitic acid (C16:0), palmitoleic acid (C16:1), oleic acid (C18:1n9c), elaidic acid (C18:1n9t), and arachidic acid (C20:0), accounting for about 97% of total FAME content for almost all nutrient stress conditions. A shift in monounsaturated fatty acid (MUFA) biosynthesis towards saturated fatty acid (SFA) production was observed when the cells were cultivated in A and B medium, where the SFA C16:0 and C20:0 were increased.
Interestingly, the accumulation of C16:0 increased with the duration of nitrogen starvation. The SFA content increased from 22.35% in control to 81.83% in –N4 cultures, whereas the MUFA content decreased from 75.36% in control to 18.83%. Nitrogen starvation for four days increased the SFA content by almost fourfold. However, the exact mechanism involved in the increase in SFA is still unknown. Fernandes et al. suggested that under nutrient deficiency, the ratio of carbon to mineral substrates (microalgal growth nutrients) increases, thereby increasing CO2 availability, which may further improve SFA accumulation over unsaturated fatty acid (UFA) [325]. High content of SFA produces biodiesel with higher CN and superior oxidative stability [38]. High SFA content also mitigates the auto-oxidation of biodiesel, thereby improving its shelf life. Similar results were observed by Chandra et al., where nitrogen depletion and carbon supplementation, respectively, increased the degree of saturation of the total fatty acid pool [326]. PUFA are specifically sensitive to oxidation; thus, the reduction of UFA upon nitrogen starvation indicated oxidative damage [327,328]. Except for –P4 cultures, under all nutrient stress conditions, the value of C18:3 was found to be ⩽12%, which is the permissible limit according to European standard EN 14214.
The production of polyunsaturated fatty acid (PUFA) was found to increase with the duration of phosphorus starvation. The PUFA content increased from 2.29% in control to 46.65% in –P4 cultures. However,the increase in PUFA production was coupled with a decline in SFA and MUFA content. SFA and MUFA are generated in the chloroplast, which serves as substrates for PUFA synthesis. As the duration of phosphorus starvation increased, a lower ratio of (SFA + MUFA)/PUFA was witnessed, which indicated a shift in SFA and MUFA biosynthesis towards PUFAs production. It has been reported that microalgae accumulate PUFA in mitochondrial membranes to protect the cell membranes from oxidative stress [329].
Combined nitrogen and phosphorus starvation significantly increased (P < 0.05) the MUFA content. The proportions of C16:1, C18:1n9c, C18:1n9t was found to increase gradually in -N- TH-2569_156151002
93 | P a g e P medium, while C16:0 content declined compared to the control. The MUFA content increased from 75.36% in control to 91.36% when the cells were starved for four days in -N-P medium.
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94 | P a g e Table 3.2. Fatty acid methyl esters (FAME) composition of T. obliquus KMC24 under various nutrient-starved conditions.
Treatments Fatty acid (%) SFA
(%)
MUFA (%)
PUFA C16:0 C16:1 C18:1n9c C18:1n9t C18:2 C18:3 C20:0 C22:0 C22:1 C24:0 (%)
C 13.69 5.45 36.31 33.44 - 2.29 1.86 6.63 0.16 0.17 22.35 75.36 2.29
A1 16.74 6.95 32.10 28.43 - 0.08 - 10.02 2.72 2.96 29.72 70.2 0.08
A2 20.01 4.48 28.94 31.62 - 0.26 6.07 7.10 1.47 0.05 33.23 66.51 0.26
A3 24.60 12.50 50.40 - - 1.1 6.64 2.16 2.34 0.26 33.66 65.24 1.1
A4 32.04 10.01 1.52 37.05 - 0.37 8.39 2.01 3.68 4.93 47.37 52.26 0.37
B1 24.67 5.35 22.40 15.94 - 2.03 22.06 0.78 4.04 2.73 50.24 47.73 2.03
B2 30.64 2.43 14.40 13.12 - 1.51 24.24 0.53 12.65 0.48 55.89 42.6 1.51
B3 33.47 2.56 11.19 11.49 - 0.84 29.08 0.35 6.25 4.77 67.67 31.49 0.84
B4 38.96 2.60 11.23 11.15 - 0.81 21.42 0.36 6.03 7.44 68.18 31.01 0.81
-N1 25.32 2.74 11.40 11.44 - 0.52 31.33 - 5.50 11.75 68.4 31.08 0.52
-N2 38.20 2.39 10.37 10.53 - 0.48 21.21 - 5.62 11.20 70.61 28.91 0.48
-N3 47.50 0.66 12.62 9.47 - 1.96 20.63 - 0.19 6.97 75.1 22.94 1.96
-N4 49.30 1.08 6.94 5.37 - 0.13 22.48 - 4.65 10.05 81.83 18.04 0.13
-P1 0.50 3.92 15.63 13.92 11.38 1.29 - 34.41 8.83 10.12 45.03 42.3 12.67
-P2 0.96 8.17 30.49 20.20 5.51 18.39 16.28 - - 35.63 38.66 25.71
-P3 1.08 8.44 33.66 30.07 24.09 2.66 - - - - 1.08 72.17 26.75
-P4 1.84 10.09 39.08 - 26.52 20.13 2.34 - - - 4.18 49.17 46.65
-N-P1 1.43 10.98 39.17 30.98 - 0.15 15.17 1.98 0.14 - 18.58 81.27 0.15
-N-P2 0.55 6.57 41.75 39.31 - 0.08 9.69 1.87 0.16 0.02 12.13 87.79 0.08
-N-P3 - 8.59 43.26 38.79 6.30 1.12 - 1.81 0.08 0.05 1.86 90.72 7.42
-N-P4 - 10.15 42.47 38.64 - 0.88 6.19 1.23 0.10 0.34 7.76 91.36 0.88
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95 | P a g e 3.3.12 Influence of nutrient starvation on biodiesel properties
Based on the fatty acid composition, the biodiesel quality was assessed for all the growth conditions using the well-defined empirical equations (Table 3.3). Biodiesel with a high CN indicates improved ignition delay time and quality of combustion. The CN is positively related to the SFA content [38]. A high content of SFA under A, B, and -N growth conditions resulted in a higher CN. Owing to the lower SFA content under phosphorus starvation, comparatively a lower CN was obtained. Except for -P4, the CN for all other nutrient stress conditions was over 47, which is the permissible limit according to ASTM D6751. The DU and IV describes the oxidative stability of fuel, where a lower DU and IV favors better oxidation stability. As compared to all the nutrient stress conditions, T. obliquus KMC24 under nitrogen starved conditions possessed lower DU and IV. The viscosity defines the flow of fuel in an engine. Generally, lower viscosity results in good flow properties with high atomization quality and high biofuel penetration ability. Under all growth conditions, the kinematic viscosity was maintained in the range of 3.48 mm2 s-1 to 4.46 mm2 s-1. CFPP value predicts the flow performance of biofuel at low temperatures. Under all growth conditions, the CFPP value was comparatively lower than the ASTM D6751 and EN 14214 standards, thereby indicating excellent low temperature performance of biodiesel. Thus, it was observed that T. obliquus KMC24 under nitrogen starved conditions have high lipid content, combustion quality, low temperature performance and ignition delay time, thereby making T. obliquus KMC24 cells a potential factory for the biosynthesis of biodiesel.
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96 | P a g e Table 3.3. Biodiesel properties of T. obliquus KMC24 under various nutrient-starved conditions.
Treatments
Quality parameters of biodiesel
CN ɳ
(mm2 s-1)
SV (mg KOH g-1)
IV (g I2 100 g−1 oil)
HV (MJ Kg-1)
DU CFPP (°C)
C 57.46 3.86 196.30 73.96 40.20 79.94 -11.80
A1 60.23 3.97 193.79 63.17 40.13 70.36 -10.76
A2 60.64 3.93 195.37 60.43 40.19 67.03 -9.64
A3 58.19 3.83 197.93 61.69 40.27 67.44 -8.07
A4 63.54 3.96 195.02 47.77 40.19 53 -5.53
B1 64.04 4.04 191.61 47.74 40.19 51.79 -8.05
B2 66.13 4.11 189.16 40.09 40.19 45.62 -6.01
B3 68.61 4.18 188.54 29.51 40.19 33.17 -5.04
B4 68.53 4.15 189.78 29.04 40.19 32.63 -3.16
-N1 69.55 4.32 184.08 28.45 40.08 32.12 -7.83
-N2 69.38 4.22 188.13 26.35 40.08 29.87 -3.42
-N3 68.77 4.07 193.16 25.73 40.08 26.86 -0.25
-N4 71.53 4.20 189.63 15.78 40.08 18.3 0.37
-P1 64.06 4.46 175.08 59.64 40.08 53.72 -16.31
-P2 56.71 4.01 185.69 84.35 39.92 90.08 -16.15
-P3 48.39 3.63 197.10 113.78 39.54 95.6 -16.11
-P4 42.09 3.48 195.33 142.89 39.71 142.47 -15.85
-N-P1 57.71 3.91 194.59 73.96 40.03 50.59 -15.98
-N-P2 56.34 3.89 195.56 79.43 40.19 48.64 -16.29
-N-P3 52.35 3.74 197.77 95.75 40.15 66.77 -16.48
-N-P4 54.92 3.82 196.88 84.91 40.28 54.48 -16.48
Standard ASTM D6751/EN
14214
≥47 1.9–6.0 ≤ 370 ≤ 120 40 - ≤ +5
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