**2. Materials and methods**

#### **2.1 Chlorella culture and growing conditions**

*Chlorella* sp. DT (DT) was discovered on the surface of power transmission cables near a mountain in central Taiwan, and *Chlorella pyrenoidosa* 211-8b (8b) was acquired from the Algal Collection Center at the University of Gottingen, Germany [26, 27]. Stock cultures were maintained at an initial concentration of 4 μg Chl mL<sup>−</sup><sup>1</sup> in 200 mL *Chlorella* medium in a 6 × 50 cm column at 32 ± 1°C in a water bath with continuous irradiance of 120 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> and bubbling of 4% CO2. In this study, six different temperatures and two different irradiance levels were used, but the growth conditions were similar.

#### **2.2 Chlorophyll (Chl) determination**

The growth of algal cultures was monitored by measuring the total Chl (Chl *a* + Chl *b*) content according to the method of Hoffman and Werner [28].

**173**

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase…*

An algal culture of 5 mL was centrifuged for 5 min at 2000 × g (Sigma, MK-201, Germany). After the supernatant was removed, the algal cell pellet was collected, 5 mL of 100% methanol was added, and the mixture was heated at 60°C for 3 min. After centrifugation at 2000 × g for 10 min to remove any cell debris, the Chl extract was obtained. To determine the total Chl and Chl *a*/*b* ratios, the concentrations of Chl *a* and Chl *b* were measured spectrophotometrically according to

Chl *a* (μg mL−1) = 16.5 × A665nm − 8.3 × A650nm; (1)

Chl *b* (μg mL−1) = 33.8 × A650nm − 12.5 × A665nm; (2)

Total Chl (μg mL−1) = 4 × A650nm + 25.5 × A665nm. (3)

ln [Chl]*<sup>t</sup>*<sup>2</sup> <sup>−</sup> ln [Chl]*<sup>t</sup>*<sup>1</sup> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *<sup>t</sup>*<sup>2</sup> <sup>−</sup>*t*<sup>1</sup>

where [Chl]*t*1 and [Chl]*t*2 are the initial and final concentrations, at time t1 and

Chlorophyll fluorescence was measured using a modulated chlorophyll fluorometer (Hansatech Instruments Ltd., Norfolk, UK). Algal samples were collected at the

a period of 10 min at room temperature before measurement. The fiber-optic probe of the fluorometer was then placed into the chamber with 2 mL of the algal samples. The minimum fluorescence (F0) with all open PSII reaction centers was determined

cence (Fm) with all closed PSII reaction centers was induced by a saturating pulse of

ratio of the variable to maximum fluorescence (Fv/Fm) represented the maximal

Algal cells from the log phase were collected by centrifugation. The cell pellet was washed three times and resuspended with extraction buffer (100 mM K2PO4 and 5 mM EDTA, pH 7.0). Algal cells were then broken down by sonication (V500, SONIC, USA) and centrifuged at 10,000 × g (MK-201, Sigma, Germany) for 15 min. The supernatant obtained was the algal crude extract. Protein concentrations of algal crude extracts were determined by the method of Lowry et al. [31] using BSA as the

The activity of the SODs was determined by measuring the inhibited reduction of cytochrome c (cyt c) because the SOD competed with cyt c for superoxide radicals, thus inhibiting cyt c reduction [32]. The reduction of cyt c was measured by monitoring the change in absorbance at 549 nm (cyt c absorption) when xanthine oxidase was added to a reaction mixture of K2PO4 (pH 7.8), xanthine, and

) was derived from the difference in

s<sup>−</sup><sup>1</sup>

). The variable fluorescence (Fv) was calculated as Fm-Fo, and the

) and measured after applying the actinic light

(4)

, and dark adapted for

). The maximum fluores-

*DOI: http://dx.doi.org/10.5772/intechopen.89024*

Hoffman and Werner's equations:

The cell specific growth rate (μ, day<sup>−</sup><sup>1</sup>

μ =

**2.3 Measurement of chlorophyll fluorescence parameters**

indicated times, adjusted to a concentration of 4 μg Chl mL<sup>−</sup><sup>1</sup>

s<sup>−</sup><sup>1</sup>

standard. The algal crude extracts were then subjected to a SOD assay.

by a weak non-actinic modulated light (<0.1 μmol m<sup>−</sup><sup>2</sup>

**2.4 Spectrophotometrical assay of SOD activity**

white light (1 s, 13,000 μmol m<sup>−</sup><sup>2</sup>

s<sup>−</sup><sup>1</sup>

photochemical efficiency [29, 30].

cellular Chl content over time as follows:

t2, respectively.

(300 μmol m<sup>−</sup><sup>2</sup>

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase… DOI: http://dx.doi.org/10.5772/intechopen.89024*

An algal culture of 5 mL was centrifuged for 5 min at 2000 × g (Sigma, MK-201, Germany). After the supernatant was removed, the algal cell pellet was collected, 5 mL of 100% methanol was added, and the mixture was heated at 60°C for 3 min. After centrifugation at 2000 × g for 10 min to remove any cell debris, the Chl extract was obtained. To determine the total Chl and Chl *a*/*b* ratios, the concentrations of Chl *a* and Chl *b* were measured spectrophotometrically according to Hoffman and Werner's equations:

$$\text{Chl } a \text{ } (\text{\(\mu g mL\)}^{-1}) = \text{16.5} \times \text{A}\_{665\text{nm}} - \text{8.3} \times \text{A}\_{650\text{nm}} \text{\textdegree} \tag{1}$$

$$\mathrm{Chl}\,b\,\left(\mathrm{\mu g\,mL^{-1}}\right) = \mathrm{33.8} \times \mathrm{A}\_{650\,\mathrm{nm}} - \mathrm{12.5} \times \mathrm{A}\_{66\,\mathrm{5mm}}\,\mathrm{\,\,\,}\tag{2}$$

$$\text{Total Chl } \left(\text{\(\mu\text{g mL}^{-1}\)} = \text{4} \times \text{A}\_{650\text{nm}} + \text{25.5} \times \text{A}\_{665\text{nm}}.\tag{3}$$

The cell specific growth rate (μ, day<sup>−</sup><sup>1</sup> ) was derived from the difference in cellular Chl content over time as follows:

$$\begin{array}{l} \text{s} \\ \text{s} \text{follows} \end{array} \begin{array}{l} \\ \text{s} \\ \\ \text{ln} = \frac{\text{ln} \left[ \text{Chl} \right]\_{t\_1} - \text{ln} \left[ \text{Chl} \right]\_{t\_1}}{t\_2 - t\_1} \end{array} \tag{4}$$

where [Chl]*t*1 and [Chl]*t*2 are the initial and final concentrations, at time t1 and t2, respectively.

#### **2.3 Measurement of chlorophyll fluorescence parameters**

Chlorophyll fluorescence was measured using a modulated chlorophyll fluorometer (Hansatech Instruments Ltd., Norfolk, UK). Algal samples were collected at the indicated times, adjusted to a concentration of 4 μg Chl mL<sup>−</sup><sup>1</sup> , and dark adapted for a period of 10 min at room temperature before measurement. The fiber-optic probe of the fluorometer was then placed into the chamber with 2 mL of the algal samples. The minimum fluorescence (F0) with all open PSII reaction centers was determined by a weak non-actinic modulated light (<0.1 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> ). The maximum fluorescence (Fm) with all closed PSII reaction centers was induced by a saturating pulse of white light (1 s, 13,000 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> ) and measured after applying the actinic light (300 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> ). The variable fluorescence (Fv) was calculated as Fm-Fo, and the ratio of the variable to maximum fluorescence (Fv/Fm) represented the maximal photochemical efficiency [29, 30].

#### **2.4 Spectrophotometrical assay of SOD activity**

Algal cells from the log phase were collected by centrifugation. The cell pellet was washed three times and resuspended with extraction buffer (100 mM K2PO4 and 5 mM EDTA, pH 7.0). Algal cells were then broken down by sonication (V500, SONIC, USA) and centrifuged at 10,000 × g (MK-201, Sigma, Germany) for 15 min. The supernatant obtained was the algal crude extract. Protein concentrations of algal crude extracts were determined by the method of Lowry et al. [31] using BSA as the standard. The algal crude extracts were then subjected to a SOD assay.

The activity of the SODs was determined by measuring the inhibited reduction of cytochrome c (cyt c) because the SOD competed with cyt c for superoxide radicals, thus inhibiting cyt c reduction [32]. The reduction of cyt c was measured by monitoring the change in absorbance at 549 nm (cyt c absorption) when xanthine oxidase was added to a reaction mixture of K2PO4 (pH 7.8), xanthine, and

*Microalgae - From Physiology to Application*

it catalyzes the dismutation of O2·

ing temperatures [21–23].

**2. Materials and methods**

**2.1 Chlorella culture and growing conditions**

continuous irradiance of 120 μmol photons m<sup>−</sup><sup>2</sup>

the growth conditions were similar.

**2.2 Chlorophyll (Chl) determination**

and regulatory systems to avoid this "energy excess" [6–9].

oxygen species (ROS), which can damage the photosynthetic apparatus and further decrease photosynthetic efficiency [5]. Therefore, in response to wide daily and seasonal fluctuations in temperature and light, algae must possess some protective

Upon initial exposure to low temperature or high irradiation, excessive excitation pressure may be induced between the rate of energy absorbed via the photosynthetic antenna and energy utilization [4, 5, 10–12]. One of the protection mechanisms that algae and higher plants employ to avoid receiving too much light energy is to adjust their chlorophyll (Chl) a/b ratios and the structure of the photosystem I and II (PSI and PSII) antenna complexes in response to different combinations of light intensity and temperature [2, 13–15]. Light-harvesting complexes (LHCs) with modified Chl composition have the ability to absorb different levels of light energy depending on the environmental conditions [16–18]. Another protective mechanism of algae and plants after receiving too much light energy is to adjust the antioxidant response of the scavenging system such that any

excess excitation pressure is transferred to the superoxide radical (O2·

and other derived reactive oxygen species (ROS) [19]. Superoxide dismutase (SOD, EC 1.15.1.1) is known as the first line of cellular defense against oxidative stress, and

SOD classified on the basis of their metal cofactors: the copper/zinc (CuZnSOD), iron (FeSOD), and manganese (MnSOD) isoenzymes [20]. SOD activity increases in cells in response to diverse environmental stresses including high light and chill-

and temperature in algal cultures that may inform optimization of production system in manufacturing [24, 25]. The two warm-climate green algae, *Chlorella* sp. DT (DT) and *Chlorella pyrenoidosa* 211-8b (8b), were compared in their photosynthetic activity and antioxidant enzymatic responses under relatively high irradiance and various chilling temperatures [26, 27]. To determine the capacity of these algae to absorb light, their Chl contents and Chl a/b ratios were measured. Photochemical efficiency and the extent of photodamage were assessed by quantifying the chlorophyll fluorescence emission of PSII [28, 29]. The responses of SOD antioxidant enzymes to chilling and high-light acclimation were also examined because they

enabled correlation with cell growth and photosynthetic activity [30].

*Chlorella* sp. DT (DT) was discovered on the surface of power transmission cables near a mountain in central Taiwan, and *Chlorella pyrenoidosa* 211-8b (8b) was acquired from the Algal Collection Center at the University of Gottingen, Germany [26, 27]. Stock cultures were maintained at an initial concentration of 4 μg Chl mL<sup>−</sup><sup>1</sup> in 200 mL *Chlorella* medium in a 6 × 50 cm column at 32 ± 1°C in a water bath with

study, six different temperatures and two different irradiance levels were used, but

The growth of algal cultures was monitored by measuring the total Chl (Chl *a* + Chl *b*) content according to the method of Hoffman and Werner [28].

s<sup>−</sup><sup>1</sup>

and bubbling of 4% CO2. In this

The primary objective of this present work was to explore combinations of light

<sup>−</sup> to H2O2 and O2. There are three distinct types of

<sup>−</sup>) pathway

**172**

cyt c (oxidized) at room temperature. After a few seconds, the algal crude extract was added to the reaction mixture. SOD activity was then calculated as 50% of the inhibited reduction rate of cyt c. An extinction coefficient of ε549nm = 21 mM<sup>−</sup><sup>1</sup> cm<sup>−</sup><sup>1</sup> for cyt c was used.

#### **2.5 Native PAGE analysis of SOD**

About 5–20 μg of protein from algal cell crude extracts suspended in sample buffer comprising 12.5 mM Tris–HCl (pH 6.8), 0.02% (w/v) bromophenol blue, and 4% (v/v) glycerol was loaded into each well. SODs in the algal extract were separated by native polyacrylamide gel electrophoresis (native PAGE) (10%). After electrophoresis the gels were washed with 100 mM K2PO4 buffer (pH 7.8) for 10 min and incubated in staining buffer composed of 20 mM K2PO4 buffer (pH 7.0), 0.05 mM riboflavin, 0.1 mM nitroblue tetrazolium (NBT), and 0.2% (w/v) TEMED in the dark at room temperature for 30 min [33]. Then gels were washed twice with 100 mM K2PO4 butter (pH 7.8) and exposed to light until the development of colorless bands. The colorless bands on the purple-stained gel indicated the existence of SOD because the free radicals produced by riboflavin are removed by the SOD, and as a consequence the colorless oxidized NBT in the SOD band is not converted into its purple reduced form. The reaction was then stopped by immersing the gels in deionized water. The SOD isoenzymes were identified on the basis of their sensitivity to KCN (5 mM) or H2O2 (10 mM), which were added into the staining buffer when required. MnSODs are resistant to both inhibitors, Cu/ZnSODs are sensitive to both inhibitors, and FeSODs are resistant to KCN but sensitive to H2O2.

### **3. Results**

### **3.1 Enhanced inhibition of cell growth by a combined stress of doubled irradiance and chilling**

The Chl content was monitored during acclimation because it represented not only the level of cell growth but also the capacity for light absorption [34]. Under a moderate irradiance of 120 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> at 32°C (**Figure 1A**), the Chl content of DT algal culture increased with acclimation time. The DT culture exhibited a maximum specific growth rate of 2.2 μg Chl mL<sup>−</sup><sup>1</sup> day<sup>−</sup><sup>1</sup> and reached a stationary phase on Day 3 at a content of 92 μg Chl mL<sup>−</sup><sup>1</sup> . At 20°C, DT growth was inhibited on Day 1, but growth resumed at a slower rate than the control on Day 2. When the temperature was lowered to 15, 10, or 7°C, DT stopped growing and even showed negative growth rates; 15°C seemed to be a critical temperature at which no net growth was observed. Under a doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> at 32°C (**Figure 1B**), DT cells exhibited a maximum specific growth rate of 2.7 μg Chl mL<sup>−</sup><sup>1</sup> day<sup>−</sup><sup>1</sup> and reached the stationary phase on Day 1 at a content of 70 μg Chl mL<sup>−</sup><sup>1</sup> . At 20°C, DT cells initially ceased growth under this doubled irradiance but resumed growth on Day 3. Transferring cultures to temperatures below 20°C promoted cell death, while the critical temperature for avoiding the negative growth rate now rose to 17°C, two degrees higher than for moderate irradiation.

A similar response was observed in 8b cells during acclimation at the various temperatures. The 8b exhibited maximum specific growth rates of 2.1 and 2.7 μg Chl mL<sup>−</sup><sup>1</sup> day<sup>−</sup><sup>1</sup> at 32°C on Day 1 under irradiance of 120 and 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> , respectively, and reached the stationary phase at about 98 and 69 μg Chl mL<sup>−</sup><sup>1</sup> on Day 3 (**Figure 2A, B**). However, once moved to temperatures below 20°C

**175**

photon m<sup>−</sup><sup>2</sup>

*than the symbol).*

**Figure 1.**

*m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup>*

irradiance.

under 240 μmol photons m<sup>−</sup><sup>2</sup>

s<sup>−</sup><sup>1</sup>

s<sup>−</sup><sup>1</sup>

Chl content than DT after Day 3. For 8b, the critical temperatures below which no net cell growth occurred and cell death was observed under both 120 and 240 μmol

*Changes in the Chl content and photosynthetic activity of DT under irradiance of 120 or 240 μmol photons* 

*Each point represents the mean ± SD (n = 4) from duplicate cultures (where not visible, error bars are smaller* 

 *during cultivation between 32 and 7°C. the total Chl content (A, B), the Chl* a*/*b *ratio (C, D), and the Fv/Fm ratio (E, F) of DT were measured each day. The initial cultivation concentration was 4 μg Chl mL<sup>−</sup><sup>1</sup>*

 irradiance were 15°C and above 17°C, respectively. Therefore, although a slightly higher maximum specific growth rate was observed initially at 32°C, lower temperatures induced enhanced inhibition of cell growth in both DT and 8b, and this was further inhibited under doubled

, the 8b culture after Day 3 produced slightly lower

*.* 

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase…*

*DOI: http://dx.doi.org/10.5772/intechopen.89024*

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase… DOI: http://dx.doi.org/10.5772/intechopen.89024*

#### **Figure 1.**

*Microalgae - From Physiology to Application*

**2.5 Native PAGE analysis of SOD**

for cyt c was used.

**3. Results**

Chl mL<sup>−</sup><sup>1</sup>

Chl mL<sup>−</sup><sup>1</sup>

mL<sup>−</sup><sup>1</sup>

day<sup>−</sup><sup>1</sup>

day<sup>−</sup><sup>1</sup>

**irradiance and chilling**

cyt c (oxidized) at room temperature. After a few seconds, the algal crude extract was added to the reaction mixture. SOD activity was then calculated as 50% of the inhibited reduction rate of cyt c. An extinction coefficient of ε549nm = 21 mM<sup>−</sup><sup>1</sup>

About 5–20 μg of protein from algal cell crude extracts suspended in sample buffer comprising 12.5 mM Tris–HCl (pH 6.8), 0.02% (w/v) bromophenol blue, and 4% (v/v) glycerol was loaded into each well. SODs in the algal extract were separated by native polyacrylamide gel electrophoresis (native PAGE) (10%). After electrophoresis the gels were washed with 100 mM K2PO4 buffer (pH 7.8) for 10 min and incubated in staining buffer composed of 20 mM K2PO4 buffer (pH 7.0), 0.05 mM riboflavin, 0.1 mM nitroblue tetrazolium (NBT), and 0.2% (w/v) TEMED in the dark at room temperature for 30 min [33]. Then gels were washed twice with 100 mM K2PO4 butter (pH 7.8) and exposed to light until the development of colorless bands. The colorless bands on the purple-stained gel indicated the existence of SOD because the free radicals produced by riboflavin are removed by the SOD, and as a consequence the colorless oxidized NBT in the SOD band is not converted into its purple reduced form. The reaction was then stopped by immersing the gels in deionized water. The SOD isoenzymes were identified on the basis of their sensitivity to KCN (5 mM) or H2O2 (10 mM), which were added into the staining buffer when required. MnSODs are resistant to both inhibitors, Cu/ZnSODs are sensitive

to both inhibitors, and FeSODs are resistant to KCN but sensitive to H2O2.

**3.1 Enhanced inhibition of cell growth by a combined stress of doubled** 

Under a moderate irradiance of 120 μmol photons m<sup>−</sup><sup>2</sup>

stationary phase on Day 3 at a content of 92 μg Chl mL<sup>−</sup><sup>1</sup>

exhibited a maximum specific growth rate of 2.2 μg Chl mL<sup>−</sup><sup>1</sup>

The Chl content was monitored during acclimation because it represented not only the level of cell growth but also the capacity for light absorption [34].

Chl content of DT algal culture increased with acclimation time. The DT culture

inhibited on Day 1, but growth resumed at a slower rate than the control on Day 2. When the temperature was lowered to 15, 10, or 7°C, DT stopped growing and even showed negative growth rates; 15°C seemed to be a critical temperature at which no net growth was observed. Under a doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup>

at 32°C (**Figure 1B**), DT cells exhibited a maximum specific growth rate of 2.7 μg

rate now rose to 17°C, two degrees higher than for moderate irradiation.

. At 20°C, DT cells initially ceased growth under this doubled irradiance but resumed growth on Day 3. Transferring cultures to temperatures below 20°C promoted cell death, while the critical temperature for avoiding the negative growth

A similar response was observed in 8b cells during acclimation at the various temperatures. The 8b exhibited maximum specific growth rates of 2.1 and 2.7 μg

and reached the stationary phase on Day 1 at a content of 70 μg Chl

at 32°C on Day 1 under irradiance of 120 and 240 μmol photons

, respectively, and reached the stationary phase at about 98 and 69 μg Chl

on Day 3 (**Figure 2A, B**). However, once moved to temperatures below 20°C

s<sup>−</sup><sup>1</sup>

at 32°C (**Figure 1A**), the

. At 20°C, DT growth was

and reached a

s<sup>−</sup><sup>1</sup>

day<sup>−</sup><sup>1</sup>

cm<sup>−</sup><sup>1</sup>

**174**

m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup>

mL<sup>−</sup><sup>1</sup>

*Changes in the Chl content and photosynthetic activity of DT under irradiance of 120 or 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> during cultivation between 32 and 7°C. the total Chl content (A, B), the Chl* a*/*b *ratio (C, D), and the Fv/Fm ratio (E, F) of DT were measured each day. The initial cultivation concentration was 4 μg Chl mL<sup>−</sup><sup>1</sup> . Each point represents the mean ± SD (n = 4) from duplicate cultures (where not visible, error bars are smaller than the symbol).*

under 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> , the 8b culture after Day 3 produced slightly lower Chl content than DT after Day 3. For 8b, the critical temperatures below which no net cell growth occurred and cell death was observed under both 120 and 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance were 15°C and above 17°C, respectively.

Therefore, although a slightly higher maximum specific growth rate was observed initially at 32°C, lower temperatures induced enhanced inhibition of cell growth in both DT and 8b, and this was further inhibited under doubled irradiance.

#### **Figure 2.**

*Changes in the Chl content and photosynthetic activity of 8b under irradiance of 120 or 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> during cultivation between 32 and 7°C. the total Chl content (A, B), the Chl* a*/*b *ratio (C, D), and the Fv/Fm ratio (E, F) of 8b were measured each day. The initial cultivation concentration was 4 μg Chl mL<sup>−</sup><sup>1</sup> . Each point represents the mean ± SD (n = 4) from duplicate cultures (where not visible, error bars are smaller than the symbol).*

#### **3.2 The readjustment of Chl a/b ratios**

In order to understand the influence of Chl composition on excitation energy transfer, the Chl *a*/*b* ratio was analyzed. Under 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance, the variation in the Chl *a*/*b* ratios of DT (**Figure 1C**) and 8b (**Figure 2C**) had similar trends at various temperatures. At 32°C, during acclimation, the Chl *a*/*b* ratios of both strains decreased slightly with time but remained between 2.4 and 2.1. When the cultures were moved to lower temperatures, the Chl *a*/*b* ratios changed dramatically. At 20°C, the Chl *a*/*b* ratios of both strains decreased to 1.7 by Day-1 but climbed back to about 2.1 by Day 2. At 15°C, the Chl *a*/*b* ratios of both strains were reduced to 1.0 on Day 1 and then remained at this value until the end of the experimental period. Under the lower temperatures of 10 and 7°C, the Chl *a*/*b* ratios of both strains rapidly declined to 0.4.

**177**

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase…*

and 8b (**Figure 2D**) acclimated to 32°C were similar to the ratios observed under

time, while it returned to a value of 2.6 on Day-3 and was higher than the value of

To assess the photochemical efficiency of PSII, the ratio of the variable to maximum fluorescence (Fv/Fm) was measured [28, 29]. In both algal cultures, the Fv/Fm ratios of the DT and 8b controls stayed initially in the 0.83–0.85 range at 32°C

by the end of the acclimation period (**Figures 1E, 2E**). The Fv/Fm ratios of both *Chlorella* strains were higher than those of most green algae but close to those of healthy green leaves of higher plants [28, 35–37]. This may be due to the antenna sizes of *Chlorella* PSII being different from those of other algae but similar to higher plants because the measured Chl fluorescence is assumed to originate from PSII [29, 38]. Algal cells grown at 20°C exhibited almost constant Fv/Fm ratios, which were similar to those at 32°C, although the cell growth rates were slower than those at 32°C. Once the cultures were transferred to 15°C, a significant decrease in the Fv/Fm ratios was observed, first falling to 0.40 for DT and 0.42 for 8b on Day 1 but by Day 2 recovering to 0.57 and 0.60 and staying at this value throughout the rest of the cultivation period. When the cultures were transferred to lower temperatures, the Fv/Fm ratios of DT and 8b fell rapidly to 0.08 and 0.10 at 10°C and 0.04 and 0.03 at 7°C, respectively, on Day 1 and continued to decrease to nearly zero by the end of

s<sup>−</sup><sup>1</sup>

and 8b strains also remained in 0.79–0.80 range (**Figures 1F, 2F**). However, the Fv/Fm ratios changed dramatically with lower temperatures. At 20°C, the Fv/Fm ratio of DT decreased to 0.20 on Day 2 but returned to 0.70 on Day 3, while in 8b it decreased to 0.40 but returned to 0.65 on Day 3. At 10 or 7°C, the Fv/Fm ratios of both strains declined to zero on Day 1, indicating that photosynthetic activity was immediately and completely inhibited. The Day 2 Fv/Fm ratios of both 8b and DT at 17°C and 7°C (**Figures 1E, F, 2E**, **F**) showed peaks that were probably due to

**3.4 Shielding effects of high algal cell concentrations on light absorption**

DT and 8b cultures that started at concentrations of 4 and 6 μg Chl mL<sup>−</sup><sup>1</sup>

recovered and by Day-3 were 0.67 for DT and 0.63 for 8b (**Figure 3E, F**).

In order to understand whether the initial concentration of algal cells affected light absorption during chilling acclimation, cell growth was measured at different

By Day 3 following the initial cessation of growth at 20°C (**Figure 3A, B**), the

ratios of DT decreased to 1.2, 1.7, and 2.0 with respect to initial concentrations of

Day 3 (**Figure 3C**). The Chl *a*/*b* ratios of 8b showed similar variations with concen-

DT and 8b initially decreased to 0.58 and 0.60 on Day-1; however, the ratios soon

At 15°C, the cell growth of DT and 8b gradually declined with time regardless of the initial Chl concentrations. Nevertheless, at initial concentrations of 4 and 6 μg

by Day 1, but then they increased close to control values by

s<sup>−</sup><sup>1</sup>

irradiance, the Chl *a*/*b* ratios of DT (**Figure 1D**)

, but decreased slightly to 0.74–0.75

irradiance, at 32°C the Fv/Fm ratios of the DT

under the doubled irradiance of

(**Figure 3A, B**). The Chl *a*/*b*

(**Figure 3D**). The Fv/Fm ratios of

were

. At lower temperatures, the Chl *a*/*b* ratios decreased with

s<sup>−</sup><sup>1</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.89024*

s<sup>−</sup><sup>1</sup>

**3.3 Change in PSII photochemical efficiency**

with an irradiance of 120 μmol photons m<sup>−</sup><sup>2</sup>

Under 240 μmol photon m<sup>−</sup><sup>2</sup>

120 μmol photons m<sup>−</sup><sup>2</sup>

the acclimation period.

experimental variations.

240 μmol photons m<sup>−</sup><sup>2</sup>

2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup>

Under 240 μmol photon m<sup>−</sup><sup>2</sup>

initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup>

 s<sup>−</sup><sup>1</sup> .

quicker to resume growth than those at 2 μg Chl mL<sup>−</sup><sup>1</sup>

tration to DT, with the exception of 2 μg Chl mL<sup>−</sup><sup>1</sup>

2.4 recorded for 8b.

Under 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance, the Chl *a*/*b* ratios of DT (**Figure 1D**) and 8b (**Figure 2D**) acclimated to 32°C were similar to the ratios observed under 120 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> . At lower temperatures, the Chl *a*/*b* ratios decreased with time, while it returned to a value of 2.6 on Day-3 and was higher than the value of 2.4 recorded for 8b.

### **3.3 Change in PSII photochemical efficiency**

*Microalgae - From Physiology to Application*

**3.2 The readjustment of Chl a/b ratios**

both strains rapidly declined to 0.4.

In order to understand the influence of Chl composition on excitation energy

*Changes in the Chl content and photosynthetic activity of 8b under irradiance of 120 or 240 μmol photons* 

*Fv/Fm ratio (E, F) of 8b were measured each day. The initial cultivation concentration was 4 μg Chl mL<sup>−</sup><sup>1</sup>*

*Each point represents the mean ± SD (n = 4) from duplicate cultures (where not visible, error bars are smaller* 

 *during cultivation between 32 and 7°C. the total Chl content (A, B), the Chl* a*/*b *ratio (C, D), and the* 

the variation in the Chl *a*/*b* ratios of DT (**Figure 1C**) and 8b (**Figure 2C**) had similar trends at various temperatures. At 32°C, during acclimation, the Chl *a*/*b* ratios of both strains decreased slightly with time but remained between 2.4 and 2.1. When the cultures were moved to lower temperatures, the Chl *a*/*b* ratios changed dramatically. At 20°C, the Chl *a*/*b* ratios of both strains decreased to 1.7 by Day-1 but climbed back to about 2.1 by Day 2. At 15°C, the Chl *a*/*b* ratios of both strains were reduced to 1.0 on Day 1 and then remained at this value until the end of the experimental period. Under the lower temperatures of 10 and 7°C, the Chl *a*/*b* ratios of

s<sup>−</sup><sup>1</sup>

irradiance,

*.* 

transfer, the Chl *a*/*b* ratio was analyzed. Under 120 μmol photon m<sup>−</sup><sup>2</sup>

**176**

**Figure 2.**

*than the symbol).*

*m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup>*

To assess the photochemical efficiency of PSII, the ratio of the variable to maximum fluorescence (Fv/Fm) was measured [28, 29]. In both algal cultures, the Fv/Fm ratios of the DT and 8b controls stayed initially in the 0.83–0.85 range at 32°C with an irradiance of 120 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> , but decreased slightly to 0.74–0.75 by the end of the acclimation period (**Figures 1E, 2E**). The Fv/Fm ratios of both *Chlorella* strains were higher than those of most green algae but close to those of healthy green leaves of higher plants [28, 35–37]. This may be due to the antenna sizes of *Chlorella* PSII being different from those of other algae but similar to higher plants because the measured Chl fluorescence is assumed to originate from PSII [29, 38]. Algal cells grown at 20°C exhibited almost constant Fv/Fm ratios, which were similar to those at 32°C, although the cell growth rates were slower than those at 32°C. Once the cultures were transferred to 15°C, a significant decrease in the Fv/Fm ratios was observed, first falling to 0.40 for DT and 0.42 for 8b on Day 1 but by Day 2 recovering to 0.57 and 0.60 and staying at this value throughout the rest of the cultivation period. When the cultures were transferred to lower temperatures, the Fv/Fm ratios of DT and 8b fell rapidly to 0.08 and 0.10 at 10°C and 0.04 and 0.03 at 7°C, respectively, on Day 1 and continued to decrease to nearly zero by the end of the acclimation period.

Under 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance, at 32°C the Fv/Fm ratios of the DT and 8b strains also remained in 0.79–0.80 range (**Figures 1F, 2F**). However, the Fv/Fm ratios changed dramatically with lower temperatures. At 20°C, the Fv/Fm ratio of DT decreased to 0.20 on Day 2 but returned to 0.70 on Day 3, while in 8b it decreased to 0.40 but returned to 0.65 on Day 3. At 10 or 7°C, the Fv/Fm ratios of both strains declined to zero on Day 1, indicating that photosynthetic activity was immediately and completely inhibited. The Day 2 Fv/Fm ratios of both 8b and DT at 17°C and 7°C (**Figures 1E, F, 2E**, **F**) showed peaks that were probably due to experimental variations.

### **3.4 Shielding effects of high algal cell concentrations on light absorption**

In order to understand whether the initial concentration of algal cells affected light absorption during chilling acclimation, cell growth was measured at different initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup> under the doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> .

By Day 3 following the initial cessation of growth at 20°C (**Figure 3A, B**), the DT and 8b cultures that started at concentrations of 4 and 6 μg Chl mL<sup>−</sup><sup>1</sup> were quicker to resume growth than those at 2 μg Chl mL<sup>−</sup><sup>1</sup> (**Figure 3A, B**). The Chl *a*/*b* ratios of DT decreased to 1.2, 1.7, and 2.0 with respect to initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup> by Day 1, but then they increased close to control values by Day 3 (**Figure 3C**). The Chl *a*/*b* ratios of 8b showed similar variations with concentration to DT, with the exception of 2 μg Chl mL<sup>−</sup><sup>1</sup> (**Figure 3D**). The Fv/Fm ratios of DT and 8b initially decreased to 0.58 and 0.60 on Day-1; however, the ratios soon recovered and by Day-3 were 0.67 for DT and 0.63 for 8b (**Figure 3E, F**).

At 15°C, the cell growth of DT and 8b gradually declined with time regardless of the initial Chl concentrations. Nevertheless, at initial concentrations of 4 and 6 μg

#### **Figure 3.**

*Effect of initial cultivation concentration on photosynthesis under 20°C and 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance. The total Chl content (A, B), the Chl a/b ratio (C, D), and the Fv/Fm ratio (E, F) of* Chlorella *DT and 8b were measured in cultures with initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup> at 20°C with irradiation of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> . Each point represents the average of two measurements from duplicate cultures (where not visible, error bars are smaller than the symbol).*

Chl mL<sup>−</sup><sup>1</sup> , both strains were slower to die than cultures starting out at 2 μg Chl mL<sup>−</sup><sup>1</sup> (**Figure 4A, B**). Neither DT nor 8b at 2 μg Chl mL<sup>−</sup><sup>1</sup> resumed growth at 15°C, and no significant difference was recorded between the two strains. The Chl *a*/*b* ratios of DT and 8b (**Figure 4C, D**) rapidly decreased on Day 1 from 2.32 to 0.50, 1.03, and 1.65 with respect to the initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup> , and no increases were observed for the duration of the acclimation period. The Fv/Fm ratios of DT and 8b fell dramatically to near zero on Day 1 regardless of the initial concentrations, and no significant recovery was seen (**Figure 4E, F**).

The results suggested that the initial concentration (2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup> ) of algal cells did affect light absorption, but temperature was the major factor determining cell growth (**Figures 3**, **4**). DT had a slightly greater tolerance at 20°C than 8b because its Chl a/b ratios attained levels higher than the control (Day 0), even though the Chl a/b ratios of 8b also returned to slightly above the control level.

**179**

protein sec<sup>−</sup><sup>1</sup>

**Figure 4.**

*than the symbol).*

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase…*

However, neither DT nor 8b could overcome the stress of low temperatures of 15°C

*irradiance. The total Chl content (A, B), the Chl a/b ratio (C, D), and the Fv/Fm ratio (E, F) of* Chlorella

*represents the average of two measurements from duplicate cultures (where not visible, error bars are smaller* 

tion, the specific growth rate of algal cells remained zero, implying that the energy input and output seemed to reach a critical point. To understand the contribution of antioxidants in scavenging ROS produced during chilling acclimation, the SOD activities were assayed with a spectrophotometrical method. It was found that DT had an approximately twofold higher rate of SOD activity (0.46 μmol mg<sup>−</sup><sup>1</sup>

sion of SOD isoforms was examined after activity staining on native PAGE, three

protein sec<sup>−</sup><sup>1</sup>

 s<sup>−</sup><sup>1</sup> .

*. Each point* 

 *s<sup>−</sup><sup>1</sup>*

irradia-

s<sup>−</sup><sup>1</sup>

). Moreover, when the expres-

and below combined with the doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup>

*Effect of initial cultivation concentration on photosynthesis under 15°C and 240 μmol photon m<sup>−</sup><sup>2</sup>*

*DT and 8b were measured in the cultures with initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup>*

For the duration of the 15°C acclimation with 120 μmol photon m<sup>−</sup><sup>2</sup>

**3.5 Differential induction of multiple SOD isoforms**

) than 8b (0.21 μmol mg<sup>−</sup><sup>1</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.89024*

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase… DOI: http://dx.doi.org/10.5772/intechopen.89024*

#### **Figure 4.**

*Microalgae - From Physiology to Application*

**178**

Chl mL<sup>−</sup><sup>1</sup>

*irradiation of 240 μmol photons m<sup>−</sup><sup>2</sup>*

**Figure 3.**

, both strains were slower to die than cultures starting out at 2 μg Chl mL<sup>−</sup><sup>1</sup>

no significant difference was recorded between the two strains. The Chl *a*/*b* ratios of DT and 8b (**Figure 4C, D**) rapidly decreased on Day 1 from 2.32 to 0.50, 1.03, and 1.65 with respect to the initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup>

*Effect of initial cultivation concentration on photosynthesis under 20°C and 240 μmol photon m<sup>−</sup><sup>2</sup>*

*DT and 8b were measured in cultures with initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup>*

*irradiance. The total Chl content (A, B), the Chl a/b ratio (C, D), and the Fv/Fm ratio (E, F) of* Chlorella

increases were observed for the duration of the acclimation period. The Fv/Fm ratios of DT and 8b fell dramatically to near zero on Day 1 regardless of the initial concen-

The results suggested that the initial concentration (2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup>

algal cells did affect light absorption, but temperature was the major factor determining cell growth (**Figures 3**, **4**). DT had a slightly greater tolerance at 20°C than 8b because its Chl a/b ratios attained levels higher than the control (Day 0), even though the Chl a/b ratios of 8b also returned to slightly above the control level.

resumed growth at 15°C, and

*. Each point represents the average of two measurements from duplicate* 

, and no

 *s<sup>−</sup><sup>1</sup>*

 *at 20°C with* 

) of

(**Figure 4A, B**). Neither DT nor 8b at 2 μg Chl mL<sup>−</sup><sup>1</sup>

*cultures (where not visible, error bars are smaller than the symbol).*

 *s<sup>−</sup><sup>1</sup>*

trations, and no significant recovery was seen (**Figure 4E, F**).

*Effect of initial cultivation concentration on photosynthesis under 15°C and 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance. The total Chl content (A, B), the Chl a/b ratio (C, D), and the Fv/Fm ratio (E, F) of* Chlorella *DT and 8b were measured in the cultures with initial concentrations of 2, 4, and 6 μg Chl mL<sup>−</sup><sup>1</sup> . Each point represents the average of two measurements from duplicate cultures (where not visible, error bars are smaller than the symbol).*

However, neither DT nor 8b could overcome the stress of low temperatures of 15°C and below combined with the doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> .

#### **3.5 Differential induction of multiple SOD isoforms**

For the duration of the 15°C acclimation with 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiation, the specific growth rate of algal cells remained zero, implying that the energy input and output seemed to reach a critical point. To understand the contribution of antioxidants in scavenging ROS produced during chilling acclimation, the SOD activities were assayed with a spectrophotometrical method. It was found that DT had an approximately twofold higher rate of SOD activity (0.46 μmol mg<sup>−</sup><sup>1</sup> protein sec<sup>−</sup><sup>1</sup> ) than 8b (0.21 μmol mg<sup>−</sup><sup>1</sup> protein sec<sup>−</sup><sup>1</sup> ). Moreover, when the expression of SOD isoforms was examined after activity staining on native PAGE, three

distinct colorless bands were observed in DT (**Figure 5**) while only two bands were observed in 8b (**Figure 6**). The SOD activities of both strains were generally amplified with time and decreasing temperature. At the same time, some new SODs were induced, and some were diminished.

As shown in **Figure 5**, the DT control contained two DTMnSODs and three DTFeSODs, which were verified with inhibitors of H2O2 and KCN. Once the culture was moved to 15°C, the SOD activities of the DT increased greatly on Day-1 and reached a maximum on Day 2. By Day 4, at least 10 SOD isoforms were observed in DT including two new DTMnSODs and three new DTFeSODs. However, by Day 8, SOD activities declined, and some isoforms disappeared, leaving only three DTFeSODs and two DTMnSODs present. Similarly, as shown in **Figure 6**, the 8b control contained two 8bMnSODs and two 8bFeSODs. The SOD activities of 8b were amplified on Day 2 and reached a maximum on Day 4, while one new 8bMnSOD and two new 8bFeSODs were induced. At the end of acclimation, the SOD activity declined, and only two 8bFeSODs and two 8bMnSODs were present.

#### **Figure 5.**

*Native PAGE analysis of SOD from a crude extract of DT grown at 15°C under 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance. In each well 5 μg of crude extract proteins was loaded. In comparison to the control (A), SOD isoforms were recognized by adding the inhibitors H2O2 (5 mM) (B) and 2 mM KCN (2 mM) (C). In total, nine SODs were induced differentially in DT with regard to six FeSODs and three MnSODs. The numbers represent the order of discovery.*

#### **Figure 6.**

*Native PAGE analysis of SODs from crude extracts of 8b grown at 15°C under 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance. In each well 5 μg of crude extract proteins was loaded. In comparison to the control (A), SOD isoforms were recognized by adding the inhibitors H2O2 (5 mM) (B) and 2 mM KCN (2 mM) (C). Seven SODs were induced differentially in DT with regard to original four FeSODs and three MnSODs in 8b. The numbers represent the order of discovery.*

**181**

**Figure 7.**

*irradiance (A) or 15°C under 240 μmol photon m<sup>−</sup><sup>2</sup>*

*loaded. The numbers represent the order of discovery.*

m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup>

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase…*

These results suggest that DT and 8b utilize different strategies for scavenging O2·

For further identification of which SOD isoforms responded to light stress and to temperature stress, the SODs were analyzed under lower temperature or doubled irradiance. In the DT culture, the original SOD isoforms of DTFeSOD1, DTFeSOD2, DTMnSOD1, and DTMnSOD2 were amplified in response to both the lower temperature of 10°C (**Figure 7A**) and to a doubled irradiance of 240 μmol photons

 (**Figure 7B**). DTFeSOD3 disappeared on Day 1, probably because it was sensitive to both higher light and lower temperature. A newly induced DTFeSOD4 appeared on Day 1 in response to doubled irradiance, but it was not detected until

*Native PAGE analysis of SOD from crude extract of DT and 8b grown at 10°C under 120 μmol photon m<sup>−</sup><sup>2</sup>*

 *s<sup>−</sup><sup>1</sup>*

*was loaded in each well; in (B), 10 μg of crude extract proteins (except 1 μg proteins of DT on Day-2) was* 

We found that MnSOD1 and FeSOD1 were the most abundant isoforms in both *Chlorella* strains, accounting for about 60–70% of the estimated total SOD activity. The other 30% is made up of other isoforms. The main FeSOD in both strains was particularly responsive to temperature [39]. Although there are three distinct types of SOD isoenzymes, only FeSOD and MnSOD were found in both *Chlorella* stains. Our observation of no CuZnSOD in either strain agrees with Asada et al. [40].

−.

 *s<sup>−</sup><sup>1</sup>*

 *irradiance (B). In (A), 15 μg of crude extract proteins* 

*DOI: http://dx.doi.org/10.5772/intechopen.89024*

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase… DOI: http://dx.doi.org/10.5772/intechopen.89024*

These results suggest that DT and 8b utilize different strategies for scavenging O2· −. We found that MnSOD1 and FeSOD1 were the most abundant isoforms in both *Chlorella* strains, accounting for about 60–70% of the estimated total SOD activity. The other 30% is made up of other isoforms. The main FeSOD in both strains was particularly responsive to temperature [39]. Although there are three distinct types of SOD isoenzymes, only FeSOD and MnSOD were found in both *Chlorella* stains. Our observation of no CuZnSOD in either strain agrees with Asada et al. [40].

For further identification of which SOD isoforms responded to light stress and to temperature stress, the SODs were analyzed under lower temperature or doubled irradiance. In the DT culture, the original SOD isoforms of DTFeSOD1, DTFeSOD2, DTMnSOD1, and DTMnSOD2 were amplified in response to both the lower temperature of 10°C (**Figure 7A**) and to a doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> (**Figure 7B**). DTFeSOD3 disappeared on Day 1, probably because it was sensitive to both higher light and lower temperature. A newly induced DTFeSOD4 appeared on Day 1 in response to doubled irradiance, but it was not detected until

#### **Figure 7.**

*Microalgae - From Physiology to Application*

induced, and some were diminished.

distinct colorless bands were observed in DT (**Figure 5**) while only two bands were observed in 8b (**Figure 6**). The SOD activities of both strains were generally amplified with time and decreasing temperature. At the same time, some new SODs were

As shown in **Figure 5**, the DT control contained two DTMnSODs and three DTFeSODs, which were verified with inhibitors of H2O2 and KCN. Once the culture was moved to 15°C, the SOD activities of the DT increased greatly on Day-1 and reached a maximum on Day 2. By Day 4, at least 10 SOD isoforms were observed in DT including two new DTMnSODs and three new DTFeSODs. However, by Day 8, SOD activities declined, and some isoforms disappeared, leaving only three DTFeSODs and two DTMnSODs present. Similarly, as shown in **Figure 6**, the 8b control contained two 8bMnSODs and two 8bFeSODs. The SOD activities of 8b were amplified on Day 2 and reached a maximum on Day 4, while one new 8bMnSOD and two new 8bFeSODs were induced. At the end of acclimation, the SOD activity declined, and only two 8bFeSODs and two 8bMnSODs were present.

*Native PAGE analysis of SODs from crude extracts of 8b grown at 15°C under 120 μmol photon m<sup>−</sup><sup>2</sup>*

*Native PAGE analysis of SOD from a crude extract of DT grown at 15°C under 120 μmol photon m<sup>−</sup><sup>2</sup>*

*irradiance. In each well 5 μg of crude extract proteins was loaded. In comparison to the control (A), SOD isoforms were recognized by adding the inhibitors H2O2 (5 mM) (B) and 2 mM KCN (2 mM) (C). In total, nine SODs were induced differentially in DT with regard to six FeSODs and three MnSODs. The numbers* 

*irradiance. In each well 5 μg of crude extract proteins was loaded. In comparison to the control (A), SOD isoforms were recognized by adding the inhibitors H2O2 (5 mM) (B) and 2 mM KCN (2 mM) (C). Seven SODs were induced differentially in DT with regard to original four FeSODs and three MnSODs in 8b. The* 

 *s<sup>−</sup><sup>1</sup>*

 *s<sup>−</sup><sup>1</sup>*

**180**

**Figure 6.**

**Figure 5.**

*represent the order of discovery.*

*numbers represent the order of discovery.*

*Native PAGE analysis of SOD from crude extract of DT and 8b grown at 10°C under 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance (A) or 15°C under 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance (B). In (A), 15 μg of crude extract proteins was loaded in each well; in (B), 10 μg of crude extract proteins (except 1 μg proteins of DT on Day-2) was loaded. The numbers represent the order of discovery.*

Day 1 under moderate irradiance, implying that DTFeSOD4 was probably more sensitive to light than to low temperatures. In 8b culture, in addition to the original SOD isoforms of 8bFeSOD1, 8bFeSOD2, 8bMnSOD1, and 8bMnSOD2, some new isoforms were induced. They were amplified in response to the lower temperature of 10°C (**Figure 7A**) and the doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> (**Figure 7B**) on Day 1. However, 8bMnSOD1 declined on Day 2. In spite of new SOD isoforms being amplified and in spite of the expectation that the SODs would prevent cell death, under the two combined stresses, the algal cells were still dying.

### **4. Discussion**

#### **4.1 Imbalance in excitation pressure**

The specific growth rates on Day 1 from DT and 8b were plotted as a function of the cultivation temperatures (**Figure 8**). This showed that the specific growth rates decreased exponentially with decreasing temperatures from 32 to 10°C. Our results did not follow the previous observation of Sandnes et al. [41] where the specific growth rate of the green alga *Nannochloropsis oceanica* increased linearly with increasing low irradiance in the 17–26°C range. The curves fitted for the 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance data are dispersed from the 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> doubled irradiance data. Obviously, doubling the irradiance did not simply double the effect of the temperature reduction on the specific growth rate.

Furthermore, the relationship of specific growth rates versus cultivation temperatures was theoretically simulated in accordance with the excessive excitation pressure. The temperature coefficient (Q10) represents the factor by which the speed of a biochemical reaction approximately doubles for every 10°C rise. Although some evidence indicated that Q10 in plants is temperature dependent [42], a Q10 of 2 was used here to theoretically estimate excessive excitation pressure. Therefore, the excessive excitation pressure due to the reduction in biochemical processes was calculated as 2 (32*<sup>o</sup>* C−T2)/10 (T, temperatures below 32°C) so that the theoretical excessive excitation pressure of

#### **Figure 8.**

*Plots of measured and theoretical specific growth rates versus temperatures in DT and 8b. The solid line curves represent the measured specific growth rates at 120 (●, DT; ▲, 8b) and 240 (○, DT; Δ, 8b) μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance. The dotted line curves represent theoretical specific growth rates at 120 (+) and 240 (×) μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance.*

**183**

**Irradiation at 120 μmol photons m−2 s−1**

**Temperature**

33°C 20°C 15°C 10°C

7°C **Table 1.**

*Theoretical excessive excitation pressure.*

1-fold 2.5-fold 3.9-fold 4.9-fold 6.1-fold

**Theoretical excessive excitation pressure (fold)**

**DT cell specific growth rate (μ) on Day-1** 

**8b cell specific growth** 

**Temperature**

**Theoretical excessive** 

**DT cell specific growth** 

**8b cell specific** 

**growth rate (μ) on** 

**Day-1 (μg Chl day−1**

2.71

0.16

−0.36

−0.35

**)**

**rate (μ) on Day-1** 

**(μg Chl day−1**

2.66 0.13 −0.14 −0.36

**)**

**excitation pressure** 

**(fold)**

2-fold

**rate (μ) on Day-1** 

**(μg Chl day−1**

2.07 0.27 0.01 −0.04 −0.11

*Excessive excitation pressure was calculated upon the assumptions of temperature factor Q10 equaling to 2 for biochemical processes and light pressure factor equaling to 2 for double irradiance.*

15°C

7.0-fold

17°C

6.1-fold

20°C

4.9-fold

33°C

**)**

**(μg Chl day−1**

2.15 0.25 −0.01 −0.13 −0.69

**)**

*Changes in Photochemical Efficiency and Differential Induction of Superoxide Dismutase…*

*DOI: http://dx.doi.org/10.5772/intechopen.89024*

**Irradiation at 240 μmol photons m−2 s−1**


