1 INTRODUCTION

Anthropogenic influences have led to significant changes in climate. The average global temperature has risen by about 0.8℃ since 1880 and the average global surface temperature is expected to increase by 4–5℃ over the next century (IPCC, 2007). In addition to higher average temperatures, global climate change is also resulting in higher temperature variability, thus increasing the risks to species' tolerance limits. Temperature is one of the major factors affecting the distribution and productivity of microalgae.

Understanding the growth and photosynthetic response of microalgae to the changes in their thermal environment is crucial to the characterization of their ecophysiology in nature. Temperature effects on the growth rate and photosynthesis of microalgae is of great interest as the primary production of algal biomass is strongly dependent on the prevailing photosynthetic rates in a dynamic environment. Photosynthesis is known to be a heat-sensitive process, and can be inhibited by high temperature before other symptoms of stress are detected (Camejo et al., 2005). Photosystem Ⅱ (PS Ⅱ) is reported to be the most thermosensitive component of the photosynthetic apparatus compared with the electron transport chain, stromal enzymes, PSI activity and the chloroplasts envelope (Georgieva and Yordanov, 1994; Ralph, 1998). The capacity for photochemical work is influenced by the stress levels of cells and any damage to the photochemical apparatus caused by photoinhibition (Eggert et al., 2007).

Elevated temperature has a significant effect on most metabolic processes. Photoinhibition occurs before other cell functions are damaged (Harrison and Platt, 1986; Davison, 1991). Extreme temperatures induce damage to PSⅡ by limiting electron transport and carbon fixation of the algae (Levasseur et al., 1990; Anning et al., 2001). Davison (1991) suggested that elevated temperatures were able to modulate the cellular concentrations of RUBISCO and other Calvin cycle enzymes that in turn decreased the operating quantum efficiency of PSⅡ, ΔF/F_m. Elevated temperatures will thus affect the photosynthesis rate or may induce phenotypic and genotypic changes. Moreover, high temperature stress caused inactivation of PSⅡ reaction centres (Bukhov and Carpentier, 2000; Yamamoto, 2016), causes a shift of the redox equilibrium between the primary acceptor plastoquinone (Q_A) and the secondary acceptor plastoquinone (Q_B) (Pospíši and Tyystjärvi, 1999; Tóth et al., 2007), and induces dissociation of the peripheral antenna complex of PSⅡ from its core complex (Armond et al., 1980; Wen et al., 2005). When exposed to different temperatures above and below their ambient temperature, microalgae show different photosynthetic responses (Salleh and McMinn, 2011). High temperature may cause thylakoid membrane instability, specifically affecting the membrane lipid composition while extreme low temperature causes reduced flexibility of membranes which then become crystalline or freeze (Falkowski and Raven, 2013).

Chlorella species (Chlorophyta), are small, ubiquitous, coccoid phototrophic eukaryotes found in diverse habitats including hot springs and other extreme environments (Phang and Chu, 2004). In nature, these microalgae play a role as primary producers in aquatic ecosystems, but can also serve as a source of nutraceuticals, feedstocks for biofuel, and play a role in wastewater treatment (Chu et al., 2009; Lim et al., 2010). They also show potential for electricity generation (Ng et al., 2014). The biochemical responses of Chlorella to temperature stress have been studied (Teoh et al., 2004, 2013). The protein contents of polar Chlorella were markedly affected by temperature while no clear trends were observed in their lipid and carbohydrate contents (Teoh et al., 2004).

Numerous studies have been done on the various stress factors on algal cells which involve morphological and biochemical changes that are apoptosis-like (Moharikar et al., 2006; Lee and Hsu, 2013). In some cases, cells are not killed even though their photosynthetic activity may have been completely inhibited in the face of moderate to extreme temperature fluctuations. Their performance may decline to a certain extent but when the conditions are favourable, microalgal cells have the ability to fully recover (Lee and Hsu, 2013). Polar Chlorella species have shown eurythermal adaptivity and higher growth rates at temperatures higher than ambient (Teoh et al., 2004; Cao et al., 2016). Therefore, slightly warmer temperatures are predicted to accelerate the recovery of the heat-stressed Chlorella cells, within physiological limits.

In the present study, four Chlorella strains originating from different latitudes were exposed to a range of temperatures to investigate their responses based on growth and photosynthetic performance. Their ability to recover from stress caused by exposure to unfavourable temperature was also investigated.

2 MATERIAL AND METHOD 2.1 Algae culture

Four freshwater Chlorella strains were obtained from the University of Malaya Algae Culture Collection (UMACC), namely UMACC237 (Chlorella-Ant), UMACC263 (Chlorella-Arc), UMACC248 (Chlorella-Temp) and UMACC001 (Chlorella-Trop). The identification of the Chlorella spp. was done based on morphological and molecular studies (results not shown). Chlorella-Ant was isolated from a soil sample collected near a wastewater pond at Casey Station, Antarctica; Chlorella-Arc was isolated near the Arctic Research Station at Ny-Ålesund, Norway; and Chlorella-Temp (original code: LB259) was originally isolated from a freshwater lake in the Netherlands. Chlorella-Trop was isolated from a fish pond at University of Malaya (Phang, 2004). The strains were maintained at temperatures close to those of their original habitats to avoid long-term cultivation effects (Lakeman et al., 2009), although some microalgal strains may show high genetic stability despite long term cultivation (Marija and Dieter, 2014). The cultures were maintained in a controlled-environment incubator at 4℃ for the Arctic and Antarctic strains, and at 18℃ and 28℃ for the temperate and tropical strains respectively. All cultures were maintained in Bold's Basal Medium (BBM) (Nichols and Bold, 1965) and illuminated with TLD 18W/54-765 cool white fluorescent lamps (Philips, Amsterdam, the Netherlands) providing ~42 μmol photons/(m²·s) photosynthetic active radiation (PAR) on 12 h light: 12 h dark photoperiod.

2.2 Experimental design

Batch mode culturing was conducted at 4 to 33℃ for the polar strains (Chlorella-Ant and Chlorella Arc), 18 to 38℃ for Chlorella-Temp, and 18 to 43℃ for Chlorella-Trop. The cultures incubated at 4℃ for the Chlorella-Ant and Chlorella-Arc, and at 18℃ and 28℃ for the Chlorella-Temp and Chlorella-Trop strains respectively, were set as control in this experiment. The inoculum was 10% (v/v) from the exponential phase cultures standardized at an optical density of 0.2 at 620 nm (OD₆₂₀). Triplicate cultures of each strain were grown in 1 L Erlenmeyer flasks with 500 mL BBM and maintained in controlled environment shaking incubators at 80 r/min (Hotech Model 718, Taiwan, China) at the range of temperatures mentioned earlier. Irradiance of ~42 μmol photons/(m²·s) PAR was provided directly from above the flasks with a 12 h light:12 h dark cycle and was measured with a Li-Cor Light Meter (Model L1-250A). Cultures were allowed to grow until stationary phase (day 10).

Growth was monitored by measuring absorbance at OD₆₂₀ using a UV-1800 UV-VIS spectrophotometer (Shimadzu, Japan). Cell density was estimated by cell counting using a haemocytometer (improved Double Neubaur, Assistent, Germany). Specific growth rate (μ, /d) was calculated using the following formula:

where N₁ and N₂ represent the cell concentrations at times t₁ and t₂, respectively, within the whole logarithmic phase.

Chlorophyll-a (Chl a) content was analyzed spectrophotometrically after extraction of the filtered samples (Whatman GF/C, 0.45 μm) in acetone (Strickland and Parsons, 1972). The absorbance of the pigment extract was measured at 665 nm (OD₆₆₅), 645 nm (OD₆₄₅) and 630 nm (OD₆₃₀). The concentration of Chl a was calculated using the following formula:

where, C_a=11.6OD₆₆₅–1.31OD₆₄₅–0.14OD₆₃₀; V_a=volume of the acetone (mL) used for extraction; V_c= volume of culture (L); Chl a (mg/L)=Chl a (mg/m³)/1 000.

The extraction and quantification of total carotenoids followed the method described by Vonshak and Borowitzka (1991). The absorbance of the pigment extract was measured at 452 nm (OD₄₅₂). The total carotenoid content (μg/mL) was calculated using the following formula:

where, V_e=volume of the acetone (mL) used for extraction; V_c=volume of culture (mL).

2.3 Measurement of photosynthesis performance

Non-invasive fluorescence measurements were obtained using a high-resolution Water-Pulse Amplitude Modulated (PAM) fluorometer (Walz GmbH, Effeltrich, Germany). The cultures were dark adapted for a period of 15 min before the generation of rapid light curves (RLCs). A weak measuring light (0.15 μmol photons/(m²·s)) from the PAM fluorometer was used to measure the fluorescence yield without inducing photosynthesis, termed as minimum fluorescence (F_o, open PSⅡ reaction centers). A saturating pulse (> 3000 μmol photons/(m²·s) for 0.8 s) was used to determine maximum fluorescence (F_m in the dark) when all PSⅡ reaction centres were closed. PSⅡ quantum yield, also known as the maximal efficiency of PSⅡ, was determined as followed according to Schreiber et al. (1995):

where F_m and F_o are the maximum and the minimum fluorescence yields in the dark-adapted state, respectively. RLCs were generated for all samples using the Water PAM software (WinControl, Walz). Light-emitting diodes (LEDs) provided the eight stepwise increment of actinic light used in the generation of RLCs. The actinic light levels used were 0, 48, 105, 158, 233, 358, 530, 812 and 1 216 μmol photons/(m²·s), each lasting for 10 s. While we recognise that a short exposure time, especially in dark-adapted cultures, will not provide accurate measures of steady state electron transport, all samples were processed in the same way so the changes between treatments provide a reliable measure of the relative alterations to cell performance. Photosynthetic parameters such as photosynthetic efficiency (α), maximum rate of relative electron transport (rETR_max) and photoadaptive index (E_k) were determined to compare the RLCs quantitatively (Ralph and Gademann, 2005). The relative electron transport rate (rETR) at a given irradiance was calculated by multiplying the effective photochemical efficiency and the irradiance (Genty et al., 1989). RLCs were constructed by plotting rETR against PAR data and subsequently fitted mathematically to a single exponential function (Platt et al., 1980), using a Marquardt-Levenberg regression algorithm:

where P is the rETR at a given irradiance, P_mis the maximum potential rETR (rETR_max), α is the initial slope of the fitted RLC before the onset of saturation (light-limiting condition efficiency) and E_dis the incident irradiance (400–700 nm). The function becomes an asymptotic maximum rETR value by assuming that there is no photoinhibition (Jassby and Platt, 1976). The intercept of the α value with rETR_max gave the saturation irradiance for electron transport (E_k) which is defined as:

Non-Photochemical Quenching (NPQ) is a measurement of the photoprotective xanthophyll cycle whereby excessive light is dissipated as heat to avoid negative impacts to the photosynthetic electron transport chain. NPQ was calculated using the formula of Schreiber (2004):

where F_m is the maximal fluorescence after a dark adaptation period and F_m' is the maximum fluorescence in the steady state light-adapted state (attained after 5–10 min). NPQ was calculated and quantified by measuring the change in F_m to the final value F_m (ΔF%). The relative inhibition of α and rETR_max in relation to the control was calculated as:

where X are the values of α or rETR_max.

2.4 Stress and recovery

The stress-inducing temperatures for each strain were selected based on the initial experiments with a range of increasing temperatures. Temperatures of 34℃ were used for both Chlorella-Ant and Chlorella Arc, 40℃ for Chlorella-Temp and 44℃ for Chlorella Trop. These temperatures represented the condition when the F_v/F_m decreased sharply during the growth cycle (see results), indicating a highly-stressed condition of the culture. These selected temperatures may lead to culture death with prolong exposure. The Chlorella strains were allowed to recover by returning them to their ambient temperatures after the high temperature treatments. Two threshold levels were selected; F_v/F_m≈0.4 (assigned as point 'A') and F_v/F_m≈0.2 (assigned as point 'B'). These points were selected on the basis that at F_v/F_m≈0.4, most of the culture was still able to grow while at F_v/F_m≈0.2, no net increase of biomass was observed. Biomass (Chl a) was estimated at F_v/F_m≈0.4 and F_v/F_m≈0.2. The relative rate of recovery is defined as the time taken for the culture to recover from F_v/F_m≈0.4 and F_v/F_m≈0.2, to its original F_v/F_m.

2.5 Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) followed by a post-hoc test using Tukey's HSD test. Statistical analyses were carried out using Statistica 10 (StatSoft Inc., USA).

3 RESULT 3.1 Microalgal growth and pigment contents

The Chlorella strains used in this study tolerated a wide range of temperatures. Chlorella-Trop displayed a higher specific growth rate (μ, /d) than the rest of the strains under various temperature treatments (Fig. 1) with the highest μ (0.579/d) attained at 28℃, but μdecreased markedly with increasing temperature above the optimum. Chlorella-Ant and Chlorella-Arc showed similar growth trends across the temperature treatments. Both polar strains could grow from 4℃ to the upper temperature limit of 33℃. The upper temperature limit is defined as the highest temperature that a culture can tolerate. The μ of polar strains increased with increasing temperatures, whereby the highest μ values for Chlorella-Ant and Chlorella-Arc were recorded as 0.405/dat 20℃ and 0.356/dat 10℃, respectively. However, there was no consistent growth trend observed for Chlorella-Temp but its highest μ was recorded as 0.469/d at 18℃. The lethal temperature (temperature at which the culture could not survive) was the lowest for Chlorella-Ant and Chlorella-Arc. Temperatures at and above 33℃, 38℃ and 43℃ were fatal to the polar, temperate and tropical Chlorella respectively.

The Chl a:carotenoids ratio (μg:μg) was also used as an indicator of the stress condition of the culture (Table 1). The Chl a:car ratio (μg:μg) were significantly lower in Chlorella-Ant, Chlorella-Arc and Chlorella-Trop at temperatures above 25, 30, and 33℃, respectively. Chlorella-Temp grown at 18℃ showed the lowest Chl a:car ratio amongst the strain grown at different temperatures. The highest Chl a: car ratio in Chlorella-Ant and Chlorella-Arc were observed at 15℃ and 20℃, respectively. It was found that under the influence of cultivated temperatures, Chlorella-Ant, Chlorella-Arc and Chlorella-Trop showed a strong correlation between their specific growth rate and Chl a:car ratio (Table S1) with R²=0.933, 0.864 and 0.900 respectively (Pearson correlation, P < 0.01).

3.2 Photosynthetic performance

Effects of temperature on photosynthetic parameters varied amongst strains with regards to their latitudinal origin. During cultivation, Chlorella Ant, Chlorella-Arc and Chlorella-Trop were able to maintain a relatively stable F_v/F_m (=ϕPSⅡ_max) at their ambient temperatures and at temperatures near T_opt (Fig. 2). However, ϕPSⅡ_max of Chlorella-Trop presented a downward trend from day 6 at 38℃ and 40℃ (Fig. 2a). For both Chlorella-Ant and Chlorella Arc, ϕPSⅡ_max values were at maximum values during incubation at temperatures near 10℃, but declined as temperature rose to above 20℃. ϕPSⅡ_max in both polar Chlorella were somewhat stable, albeit with comparatively lower values after day 6 at 28℃ and 30℃ (Fig. 2b, c). ϕPSⅡ_max of Chlorella-Ant could still be detected on day 8 while ϕPSⅡ_max of Chlorella-Arc was already zero on day 6 at 33℃. A sharp decrease in ϕPSⅡ_max was observed at 33℃ for both polar Chlorella and at 43℃ for Chlorella-Trop. The variation in ϕPSⅡ_max for Chlorella-Temp was temperature-independent, whereby ϕPSⅡ_max fluctuated around 0.5 from 18℃ to 38℃ (Fig. 2d).

The four Chlorella strains grown at various temperatures showed different trends in the inhibition of both rETR_max and light harvesting efficiency (α) (Fig. 3). Generally, the inhibition of rETR_max and α was increased with increasing temperature. For Chlorella-Trop, increasing temperature up to 38℃ did not inhibit rETR_max, however, temperatures beyond 38℃ lowered the rETR_max (Fig. 3a). Chlorella Ant did not show inhibition of rETR_max from 10 to 25℃, but the percentage of inhibition increased significantly (P < 0.05) with further increase in temperature (Fig. 3b). Although the inhibition of rETR_max in Chlorella-Arc was relatively higher than Chlorella-Ant, the Antarctic strain (R²=0.916, P < 0.01) displayed better correlation (Table S2) between temperature and inhibition of rETR_max than Chlorella-Arc (R²=0.775, P < 0.01) and Chlorella Trop (R²=0.604, P < 0.01). Chlorella-Arc showed 26.8%, 7.1%, 58.7%, 82.7% and complete inhibition of rETR_max at 20℃, 25℃, 28℃, 30℃ and 33℃, respectively (Fig. 3c). Inhibition of α was essentially temperature dependent especially at temperatures beyond the T_opt for all strains studied. The values of α consistently declined in both polar Chlorella and Chlorella-Trop with increasing temperature. The effect of temperature on α was the most strongly correlated for Chlorella-Trop (R²=0.669, P < 0.01) followed by Chlorella-Arc (R²=0.636, P < 0.05) and Chlorella-Ant (R²=0.598, P < 0.01). Significant inhibition (P < 0.05) of rETR_max was only observed in Chlorella-Temp at 38℃ with 30.8% inhibition but α was generally suppressed across 25℃ to 38℃ (Fig. 3d).

Generally, at 1 216 μmol photons/(m²·s) Chlorella Temp presented the lowest NPQ values ranging from 0.147 to 0.245 across the experimented temperatures (Fig. 4). NPQ in both polar Chlorella strains was activated in response to temperature stress. NPQ at 1 216 μmol photons/(m²·s) peaked at 4℃ with 0.535 followed by 0.382 at 33℃ for Chlorella-Ant. For Chlorella-Arc, NPQ at 1 216 μmol photons/(m²·s) peaked at 4℃ with 0.543 followed by 0.440 at 30℃. Chlorella-Trop recorded a largely downward trend in NPQ with increasing temperature.

All the Chlorella strains were able to recover from exposure to high temperature after being returned to their ambient or optimal temperature (Fig. 5, Table 2). The recovery rate of ϕPSⅡ_max depended on the degree of damage. Recovery from F_v/F_m≈0.2 was generally faster than from F_v/F_m≈0.4 (Table 2). Only Chlorella Trop showed the inverse trend. Comparatively, Chlorella-Trop had the highest relative rate of F_v/F_m recovery, followed by Chlorella-Temp. Meanwhile, Chlorella-Ant and Chlorella-Arc showed faster relative recovery rates at their optimal temperature of 20℃ (denoted by Aꞌ and Bꞌ) than in their ambient condition (4℃) (Fig. 5b, c).

4 DISCUSSION

Despite the use of a low number of strains from each latitude, which hampers our ability to draw hard conclusions about a general relationship between temperature response and latitude, the results of this study are in broad agreement with other work on various Chlorella strains from different latitudes (Teoh et al., 2013; Cao et al., 2016) and the responses of growth and photosynthesis were similar to those found in psychrophilic and temperate species of other green algae such as Chlamydomonas (Lukeš et al., 2014). The four Chlorella strains were found to be able to grow and photosynthesize at temperatures one and a half to six times higher than their ambient growth temperatures. Although the exposure time in the present study was short, at a maximum of ten days, even at the extremes of growth-permissive temperatures, the cultures were able to undergo at least one cell division over this time. For example, the μ of Chlorella-Trop under stress conditions was 0.167/d which implies that there was a turnover of at least one generation within the ten days.

4.1 The eurythermal adaptivity of polar Chlorell a strains

This study extrapolated the possible interactions between the elevated temperatures associated with global climate change and the sensitivity of Chlorella from different latitudes, in terms of their growth and photosynthetic responses. Microalgae are able to acclimatize to changes in temperature within a short time span. The ranges of temperature used in this study varied according to the latitudinal origin of the strains and were designed to exceed their respective tolerated upper temperature limit. When exposed to a new temperature, microalgae would respond and adapt by altering their biochemical composition, pigment contents, and metabolites via regulation of gene expression (Morgan-Kiss et al., 2006; Song et al., 2014), to maintain their normal functions and prevent mortality from extreme stress. Beyond the optimal temperature range, algae would function less efficiently, and may die if the stress level is too extreme. The results showed that the polar Chlorella were able to grow at much higher temperatures, with faster growth rates than that achieved at the ambient temperature. According to Vincent (2007), the growth rates of polar species at low temperatures are less impressive compared to growth at higher temperatures. Generally, the growth rates of Chlorella increased until the respective optimum temperatures (T_opt) were reached. Incubation of the Chlorella strains at supra-optimal conditions significantly affected their growth. Chlorella-Ant and Chlorella-Arc survived temperatures up to 34℃, although their optimum growth temperatures were around 20℃. Cold-adapted microorganisms include both psychrotolerant species and psychrophiles (Morgan-Kiss et al., 2006). Both polar Chlorella strains in this study are psychrotolerant as shown by the wide range of temperatures that they tolerated. As for the tropical Chlorella, an increase in temperature resulted in decreased μ and photosynthetic performance, indicating that the strain was already living at its upper limit of growth temperature. High temperature-induced stress was clearly evident when the strains were incubated at temperatures above their respective optimal temperature.

Elevated temperature had an adverse effect on the Chl a content per unit biomass, thus the photosynthetic rate per cell may decrease. Generally, Chl a decreased under temperature stress but carotenoids often remain constant or high. A reduction in Chl a might imply degradation of PSⅡ and PSI reaction centres (Luder et al., 2002). Carotenoids not only serve as accessory light-harvesting pigments but also protect photosynthetic systems as non-enzymatic antioxidant compounds against reactive oxygen species (ROS) generated during stressful conditions (Phillips et al., 1995). Photosynthetic pigments are susceptible to attack by ROS and this could lead to a decrease in the growth rate of Chlorella spp. (Takahashi et al., 2004). Chlorella cells in this study appeared to have adjusted their photoprotective carotenoids to attenuate high temperature-induced stress. The strong correlation between Chl a:car ratio and specific growth rate suggested that this ratio can be used as an indicator of the physiological status of the Chlorella species.

4.2 Photosynthetic response to temperature variations

PAM-fluorometry offers a rapid and non-invasive way to measure high temperature induced photophysiological stress in the four Chlorella strains, with respect to geographical habitat differences. The present study showed significant differences (P < 0.05) in the parameters of photosynthetic performance following exposure to various temperatures. The maximum quantum yield (F_v/F_m) of PSⅡ was found to vary significantly under incubation at different temperatures. The values of F_v/F_m observed under the ambient condition were 0.64–0.71, 0.65–0.71, 0.67– 0.71, and 0.50–0.63 for Chlorella-Ant, Chlorella Arc, Chlorella-Trop and Chlorella-Temp respectively, indicating healthily photosynthesizing cells. However, these values were significantly decreased at higher temperatures and with longer exposure periods, indicating the increase of physiological stress. The decrease in F_v/F_m suggested functional disorder of the photosynthetic apparatus and damage to PSⅡ, and cells subsequently increased their NPQ (Ralph and Gademann, 2005; Campbell et al., 2006).

Apart from regulating light utilization for photosynthesis, Chlorella cells also control the amount of light absorbed. The value of α quantifies the light harvesting efficiency (Ralph and Gademann, 2005). Values of α in all strains showed different degree of inhibition under the ranges of temperature tested. In contrast to the observation by Cao (2016) and co-workers, the current finding indicated down regulation of light capture efficiency under high temperature stress. Changes in α can be attributed to the variation in pigment composition of light harvesting complexes (LHCs) and the efficiency of energy transfer from the light-harvesting antenna to PSⅡ reactions centres (Ralph and Gademann, 2005; Serôdio et al., 2006). Damage to the photosynthetic apparatus (as reflected in the decline in F_v/F_m) arising from excessively high temperature may also contribute to the drop in α.

Excessively high temperature causes a decrease in the electron transport rate, as was observed in this study. The rETR_max provides an approximation of the rate of electron flow via PSⅡ into the photosynthetic electron transport chain and is related to the overall photosynthetic capacity of microalgae (Juneau et al., 2005). The percentage inhibition of rETR_max was higher for the cultures of Chlorella-Ant, Chlorella Arc and Chlorella-Trop, but not for Chlorella-Temp, exposed to higher temperature, indicating stress to photosynthesis which intensified with increasing temperature. The rETR_maxvaluesof Chlorella-Ant and Chlorella-Arc were decreased above 25℃ and 20℃, respectively. This implied that the electron transport rates of the microalgae were higher at temperatures below their respective optimum temperatures. It has been suggested that the enzymes of the dark reaction system might operate more rapidly and thus be able to consume NADPH₂ and ATP at a faster rate (Kirk, 1994) as temperatures increase until it reaches the optimum temperature. Defew et al. (2004) observed a similar pattern in a temperate microalgal community, whereby rETR_max increased with temperature until the optimal temperature (20℃) was reached and then declined as the enzymes that control the RUBISCO activity, for example RUBISCO activase and others involved in carbon fixation, were deactivated (MacIntyre et al., 1997).

It has been demonstrated that the effect of high temperature and high irradiance can be minimized if NPQ is activated (Salleh et al., 2010). NPQ helps to regulate and protect PSⅡ against photoinhibition. Normally, NPQ is associated with the xanthophyll cycle. The xanthophyll cycle in Chlorophyta consists of the pH-dependent conversion from violaxanthin to an intermediate, antheraxanthin, and subsequently to zeaxanthin (Müller et al., 2001). When the absorption of light energy exceeds the capacity for light utilization, NPQ is activated to dissipate light energy in the form of heat (Müller et al., 2001). Enhanced NPQ activities were observed in Chlorella-Ant and Chlorella-Arc at 4℃ and at a higher temperature of 33℃ for Chlorella-Ant and 30℃ for Chlorella-Arc. The marked decline in NPQ observed in Chlorella Trop as temperature increased above 28℃ suggest the diminishing ability of this strain to dissipate excess irradiance as heat. Adverse temperatures are believed to disrupt the NPQ process in cells. With regards to this, Chlorella strains are likely to show species-specific xanthophyll cycle parameters such as rate constants and the extent and kinetics of depoxidation (Lavaud et al., 2004).

4.3 Recovery from high temperature stress

The inhibition of F_v/F_m of the Chlorella species was temperature and latitude dependent. Here, F_v/F_m was used as an indicator of the physiological health of the microalgae. As the selected strains in normal conditions showed variation in F_v/F_m with values fluctuating between 0.5 and 0.7, thus this range was selected to represent a "healthy" photosynthetic state of these strains. In addition, it was observed that by exposing different stresses the F_v/F_m value began to decline below this range. Therefore, the value of 0.5 was chosen as a threshold defining the onset of stress along with the values 0.4 and 0.2 considered different levels of stress. Also as noted by Reeves et al., (2011), F_v/F_m values of ~0.1 are usually considered representative of dead cells. We noticed that at F_v/F_m 0.4, all of the strains were still able to increase biomass (Chl a), whereas most of the strains (excluding Chlorella-Trop) showed no net increase of biomass at F_v /F_m 0.2. Although the recovery process took several days, the Chlorella strains in this study were able to restore their photosynthetic fitness from two different levels of stress (F_v/F_m≈0.4 and F_v/F_m≈0.2) even at high temperatures of 34℃ for polar Chlorella, 38℃ for Chlorella-Temp and 44℃ for Chlorella-Trop. Chlorella-Trop had the highest relative rate of recovery amongst the four strains when returned to its optimum temperature. Polar strains showed higher relative rate of recovery at 20℃ than 4℃, which suggested that the protein and enzymes for repair processes are at their optimal condition at 20℃. A number of studies (Rautenberger et al., 2015; Wong et al., 2015; Rivas et al., 2016) stated that repair processes such as the turnover and re-synthesis of D1 protein are enzymatically driven, thus the repair processes are strongly dependent on temperature (within physiological limits).

Long term exposure to high temperatures above the ambient condition may result in conformation changes and denaturation of proteins (Jensen and Knutsen, 1993). Extreme temperatures decrease the repair rate of D1 protein and thus can have damaging effects on the operation of the photosynthetic apparatus. Long et al. (1994) demonstrated that photoinhibition is associated with the loss of D1 protein, and this blocks the recovery of the PSⅡ. It was predicted that longer exposure time to excessively high temperature will cause irreversible damage to the photosynthesis apparatus of the cells and may eventually be fatal if no net recovery process takes place.

5 CONCLUSION

The growth and photosynthetic performance of Chlorella spp. incubated at different temperatures were characterized. The findings showed how Chlorella from different geographical regions responded differently to temperature stress with changes in the photosynthetic parameters and pigment contents. Different Chlorella species are adapted to grow under different thermal habitats. Chlorella-Ant and Chlorella-Arc were found to exhibit eurythermal adaptability with a wide temperature tolerance. They are presumed to have evolved from the temperate regions and possessed different mechanisms to cope with abiotic stresses. While Chlorella-Temp was rather insensitive to the range of tested temperatures, Chlorella-Trop was already living at its upper growth temperature limit. Hence further increase in temperature negatively affected the growth of this strain. The stable temperature in the tropical freshwater environment may be the reason for Chlorella-Trop's lack of ability to tolerate large temperature fluctuations. Although variations in temperature may alter the primary productivity of Chlorella spp. to a certain extent, the rise in temperature per se is unlikely to cause negative impacts on the polar Chlorella as they were able to survive at rather high temperatures. Future work should include identifying the genes and biological pathways involved in the response to heat stress. A fundamental understanding of the thermal tolerance of Chlorella from different latitudes is crucial to predict how these microalgae will fare in the future climate scenario of rising global temperatures. However, work on additional strains from each latitudinal region is required to make definitive conclusions about the relationship between latitude and temperature response.

6 DATA AVAILABILITY STATEMENT

All data generated or analysed during this study are included in this published article and its supplementary information files.

Electronic supplementary material

Supplementary material (Supplementary Tables S1–S2) is available in the online version of this article at https://doi.org/10.1007/s00343-018-7093-x.