**Table 1.**

*Theoretical excessive excitation pressure.*

*Microalgae - From Physiology to Application*

**4.1 Imbalance in excitation pressure**

**4. Discussion**

photon m<sup>−</sup><sup>2</sup>

s<sup>−</sup><sup>1</sup>

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

(**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.

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

irradiance data are dispersed from the 240 μmol photon m<sup>−</sup><sup>2</sup>

doubled irradiance data. Obviously, doubling the irradiance did not simply double

(T, temperatures below 32°C) so that the theoretical excessive excitation pressure of

*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* 

 *irradiance. The dotted line curves represent theoretical specific growth rates at 120 (+) and 240 (×)* 

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

the effect of the temperature reduction on the specific growth rate.

s<sup>−</sup><sup>1</sup>

s<sup>−</sup><sup>1</sup>

(32*<sup>o</sup>* C−T2)/10

of 10°C (**Figure 7A**) and the doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup>

**182**

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

**Figure 8.**

*μmol photon m<sup>−</sup><sup>2</sup>*

 *s<sup>−</sup><sup>1</sup>*

 *irradiance.*

2.3-fold at 20°C, of 3.3-fold at 15°C, and so on were calculated relative to the control (onefold at 32°C) (**Table 1**). The diminished activities caused by theoretical excessive excitation pressure were plotted as a function of acclimation temperature (**Figure 8**). Subsequently, another plot was obtained for the doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> , assuming that the excessive excitation pressure was twice the value under 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance. However, the experimental curves did not follow the theoretical ones, implying that regulation of the response to the combined light and temperature stresses was more complicated than expected.

In our experiments, under a moderate irradiance of 120 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> , DT and 8b showed no significant differences in growth rates and photochemical efficiency when subjected to various low temperatures. However, under a doubled irradiance of 240 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> , DT had a slightly higher growth rate than 8b at temperatures below 20°C. This suggests that DT might possess a more efficient energy dissipation system against the combined stress of low temperatures and high irradiation than 8b. These results are in agreement with reports that the impact from photoinhibition due to low temperature and high light varies greatly across different green algal species [41, 43–45]. Although a greater specific growth rate was obtained under 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance compared to 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance, neither DT nor 8b favored high irradiance because a smaller Chl content was found during the stationary phase, that is, less biomass was generated.

In order to control light energy absorption and transfer, the LHC must modify the pigment composition of the Chl *a*/*b* ratio, and this is related to alterations in the photosynthetic apparatus under various conditions [16–18]. In the present study, decreases in both the Chl content and the Chl *a*/*b* ratio under low temperatures and high lights occurred simultaneously, suggesting a degradation of Chl molecules or the rearrangement of the LHCII complex [12]. A Chl *a/b* ratio of about 2.5 was obtained in both DT and 8b, which was similar to the green alga *Dunaliella salina* (2.3) [16], smaller than in *Chlorella vulgaris* (7.2) [2], and larger than in *Bryopsis maxima* (1.5) [38]. The lowering of Chl *a*/*b* ratios in DT and 8b is likely a mechanism to avoid absorbing too much light during acclimation [17]. The restoration of the Chl *a*/*b* ratio to 2.6 during 20°C acclimation might derive from the bleaching of Chl *b*, which is expected to absorb higher light excitation energy.

Despite the apparent decrease in the Fv/Fm ratios in our 10 and 7°C acclimation experiments, an initial increase and then a quenching of Fo was observed (data not shown). This phenomenon has been found in *C. vulgaris* and is suggested as being due to a rise in the xanthophyll cycle for dissipating excessive energy [43]. The reduction in both Fm and Fo implied changes in antenna size, thereby minimizing the absorbance of incident light [43]. Because Fo originates from the Chl *a* of the PSII-associated antenna, an increase in Fo is indicative of decreased energy transfer from LHCII to PSII. A large reduction in Fo has generally been regarded as a symptom of serious damage to the photosynthetic apparatus.

#### **4.2 Differential SOD response**

Since SOD is the first line of cellular defense against oxidative stress to remove O2· <sup>−</sup>, monitoring how SOD responds to photoinhibition during acclimation may provide more information about photoprotection [20]. It is known that SOD activity increases in cells in response to diverse environmental stresses including high light intensities and low temperatures and that SOD isoforms are expressed differently to protect against a subset of oxidative stresses under various environmental conditions [46, 47]. In particular, each of the SOD isoforms is independently regulated according to the degree of oxidative stress experienced in the respective subcellular compartments [48].

**185**

O2·

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

<sup>−</sup> substrate [20–23].

**Acknowledgements**

**Abbreviations**

PS photosystem Chl chlorophyll

Fm maximum fluorescence Fv variable fluorescence LHC light-harvesting complex SOD superoxide dismutase ROS reactive oxygen species

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

**5. Conclusion**

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

point where the specific growth rate of the algal cells was zero, DT possessed higher SOD activities and more isoforms than 8b. To clarify further which SOD isoform responded to light or temperature, SOD activities were measured under the lower

The results showed that the original SOD isoforms, which are likely sensitive to low temperature, were amplified by at 10°C and the newly induced SOD isoforms, which

The green algae *Chlorella* species DT (DT) and *Chlorella pyrenoidosa* 211-8b (8b) were very alike in their cell growth rate (total Chl), light energy absorption regulation (Chl *a*/*b* ratio), and photochemical efficiency (Fv/Fm) under optimal

s<sup>−</sup><sup>1</sup>

more new SOD isoforms for removing free radicals than 8b.

article is dedicated in memory of Professor Pao-Chung Chen.

7°C. Upon exposure of the cultures to a doubled irradiance of 240 μmol photons

The authors acknowledge that this work was partly supported by grants to Lee-Feng Chien from the National Science Council (now Ministry of Science and Technology) of Taiwan (NSC89-2312-B-005-007 and NSC93-2311-B-005-017). This

, DT exhibited higher cell growth rates than 8b at chilling temperatures of 20°C and 15°C. It was also found that under the combined stresses of chilling temperature and relatively high irradiance, DT possessed higher SOD activity and

are likely sensitive to light, appeared under the doubled irradiance treatment. Our data also suggested that the regulation of the antioxidant response to chilling was different from the response to irradiation. This raises the interesting question of why the regulation of antioxidant defenses is so highly complex and varied under a range of oxidative stresses even though they are targeting the same

s<sup>−</sup><sup>1</sup>

irradiation, which was the

s<sup>−</sup><sup>1</sup>

irradiance (**Figure 7A**)

(**Figure 7B**).

s<sup>−</sup><sup>1</sup>

and as temperatures decreased from 32 to

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

At 15°C acclimation and 120 μmol photon m<sup>−</sup><sup>2</sup>

temperature conditions of 10°C and 120 μmol photon m<sup>−</sup><sup>2</sup>

and at 15°C under the doubled irradiance of 240 μmol photon 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*

At 15°C acclimation and 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiation, which was the point where the specific growth rate of the algal cells was zero, DT possessed higher SOD activities and more isoforms than 8b. To clarify further which SOD isoform responded to light or temperature, SOD activities were measured under the lower temperature conditions of 10°C and 120 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> irradiance (**Figure 7A**) and at 15°C under the doubled irradiance of 240 μmol photon m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> (**Figure 7B**). The results showed that the original SOD isoforms, which are likely sensitive to low temperature, were amplified by at 10°C and the newly induced SOD isoforms, which are likely sensitive to light, appeared under the doubled irradiance treatment.

Our data also suggested that the regulation of the antioxidant response to chilling was different from the response to irradiation. This raises the interesting question of why the regulation of antioxidant defenses is so highly complex and varied under a range of oxidative stresses even though they are targeting the same O2· <sup>−</sup> substrate [20–23].
