Growth and photosynthesis of Chlorella strains from polar, temperate and tropical freshwater environments under temperature stress*

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1 Journal of Oeanology and Limnology Vol. 36 No. 4, P , Growth and photosynthesis of Chlorella strains from polar, temperate and tropial freshwater environments under temperature stress* Kok-Keong LEE 1, 2, Phaik-Eem LIM 1, **, Sze-Wan POONG 1, Chiew-Yen WONG 3, 4, Siew-Moi PHANG 1, 5, John BEARDALL 6 1 Institute of Oean and Earth Sienes, University of Malaya, 563 Kuala Lumpur, Malaysia 2 Institute of Graduate Studies, University of Malaya, 563 Kuala Lumpur, Malaysia 3 Shool of Health Sienes, International Medial University, 57 Kuala Lumpur, Malaysia 4 National Antarti Researh Centre, University of Malaya, 563 Kuala Lumpur, Malaysia 5 Institute of Biologial Sienes, University of Malaya, 563 Kuala Lumpur, Malaysia 6 Shool of Biologial Sienes, Monash University, Clayton, Vitoria, 38, Australia Reeived Mar. 28, 217; aepted in priniple May 8, 217; aepted for publiation Jun. 21, 217 Chinese Soiety for Oeanology and Limnology, Siene Press and Springer-Verlag GmbH Germany, part of Springer Nature 218 Abstrat Elevated temperatures as a onsequene of global warming have signifiant impats on the adaptation and survival of miroalgae whih are important primary produers in many eosystems. The impat of temperature on the photosynthesis of miroalgae is of great interest as the primary prodution of algal biomass is strongly dependent on the photosyntheti rates in a dynami environment. Here, we examine the effets of elevated temperature on Chlorella strains originating from different latitudes, namely Antarti, Arti, temperate and tropial regions. Chlorophyll fluoresene was used to assess the photosyntheti responses of the miroalgae. Rapid light urves (RLCs) and maximum quantum yield ( / F m ) were reorded. The results showed that Chlorella originating from different latitudes portrayed different growth trends and photosyntheti performane. The Chlorella genus is eurythermal, with a broad temperature tolerane range, but with strain-speifi harateristis. However, there was a large overlap between the tolerane range of the four strains due to their eurythermal adaptivity. Changes in the photosyntheti parameters indiated temperature stress. The ability of the four strains to reativate photosynthesis after inhibition of photosynthesis under high temperatures was also studied. The Chlorella strains were shown to reover in terms of photosynthesis and growth (measured as Chl a ) when they were returned to their ambient temperatures. Polar strains showed faster reovery in their optimal temperature ompared to that under the ambient temperature from whih they were isolated. Keyword : Antarti; Arti; / F m ; miroalgae; pigments; reovery 1 INTRODUCTION Anthropogeni influenes have led to signifiant hanges in limate. The average global temperature has risen by about.8 C sine 188 and the average global surfae temperature is expeted to inrease by 4 5 C over the next entury (IPCC, 27). In addition to higher average temperatures, global limate hange is also resulting in higher temperature variability, thus inreasing the risks to speies tolerane limits. Temperature is one of the major fators affeting the distribution and produtivity of miroalgae. Understanding the growth and photosyntheti response of miroalgae to the hanges in their thermal environment is ruial to the haraterization of their eophysiology in nature. Temperature effets on the * Supported by the HiCoE Grant (No. IOES-214H) from the Ministry of Higher Eduation (MOHE), Malaysia, the University of Malaya Postgraduate Researh Fund (No. PG A), the Knowledge Management Grant (No. RP1O-13SUS), and the Antarti Flagship Projet (Nos. FP712E12, PV2-215) by the Ministry of Siene, Tehnology & Innovation (MOSTI), Malaysia ** Corresponding author: phaikeem@um.edu.my

2 No.4 LEE et al.: Response of Chlorella spp. to temperature stress 1267 growth rate and photosynthesis of miroalgae is of great interest as the primary prodution of algal biomass is strongly dependent on the prevailing photosyntheti rates in a dynami environment. Photosynthesis is known to be a heat-sensitive proess, and an be inhibited by high temperature before other symptoms of stress are deteted (Camejo et al., 25). Photosystem II (PS II) is reported to be the most thermosensitive omponent of the photosyntheti apparatus ompared with the eletron transport hain, stromal enzymes, PSI ativity and the hloroplasts envelope (Georgieva and Yordanov, 1994; Ralph, 1998). The apaity for photohemial work is influened by the stress levels of ells and any damage to the photohemial apparatus aused by photoinhibition (Eggert et al., 27). Elevated temperature has a signifiant effet on most metaboli proesses. Photoinhibition ours before other ell funtions are damaged (Harrison and Platt, 1986; Davison, 1991). Extreme temperatures indue damage to PSII by limiting eletron transport and arbon fixation of the algae (Levasseur et al., 199; Anning et al., 21). Davison (1991) suggested that elevated temperatures were able to modulate the ellular onentrations of RUBISCO and other Calvin yle enzymes that in turn dereased the operating quantum effiieny of PSII, Δ F / F m. Elevated temperatures will thus affet the photosynthesis rate or may indue phenotypi and genotypi hanges. Moreover, high temperature stress aused inativation of PSII reation entres (Bukhov and Carpentier, 2; Yamamoto, 216), auses a shift of the redox equilibrium between the primary aeptor plastoquinone ( Q A ) and the seondary aeptor plastoquinone ( Q B ) (Pospíši and Tyystjärvi, 1999; Tóth et al., 27), and indues dissoiation of the peripheral antenna omplex of PSII from its ore omplex (Armond et al., 198; Wen et al., 25). When exposed to different temperatures above and below their ambient temperature, miroalgae show different photosyntheti responses (Salleh and MMinn, 211). High temperature may ause thylakoid membrane instability, speifially affeting the membrane lipid omposition while extreme low temperature auses redued flexibility of membranes whih then beome rystalline or freeze (Falkowski and Raven, 213). Chlorella speies (Chlorophyta), are small, ubiquitous, ooid phototrophi eukaryotes found in diverse habitats inluding hot springs and other extreme environments (Phang and Chu, 24). In nature, these miroalgae play a role as primary produers in aquati eosystems, but an also serve as a soure of nutraeutials, feedstoks for biofuel, and play a role in wastewater treatment (Chu et al., 29; Lim et al., 21). They also show potential for eletriity generation (Ng et al., 214). The biohemial responses of Chlorella to temperature stress have been studied (Teoh et al., 24, 213). The protein ontents of polar Chlorella were markedly affeted by temperature while no lear trends were observed in their lipid and arbohydrate ontents (Teoh et al., 24). Numerous studies have been done on the various stress fators on algal ells whih involve morphologial and biohemial hanges that are apoptosis-like (Moharikar et al., 26; Lee and Hsu, 213). In some ases, ells are not killed even though their photosyntheti ativity may have been ompletely inhibited in the fae of moderate to extreme temperature flutuations. Their performane may deline to a ertain extent but when the onditions are favourable, miroalgal ells have the ability to fully reover (Lee and Hsu, 213). Polar Chlorella speies have shown eurythermal adaptivity and higher growth rates at temperatures higher than ambient (Teoh et al., 24; Cao et al., 216). Therefore, slightly warmer temperatures are predited to aelerate the reovery of the heat-stressed Chlorella ells, within physiologial 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 photosyntheti performane. Their ability to reover from stress aused by exposure to unfavourable temperature was also investigated. 2 MATERIAL AND METHOD 2.1 Algae ulture Four freshwater Chlorella strains were obtained from the University of Malaya Algae Culture Colletion (UMACC), namely UMACC237 ( Chlorella -Ant), UMACC263 ( Chlorella -Ar), UMACC248 ( Chlorella -Temp) and UMACC1 ( Chlorella -Trop). The identifiation of the Chlorella spp. was done based on morphologial and moleular studies (results not shown). Chlorella -Ant was isolated from a soil sample olleted near a wastewater pond at Casey Station, Antartia; Chlorella -Ar was isolated near the Arti Researh Station at Ny- Ålesund, Norway; and Chlorella -Temp (original

3 1268 J. OCEANOL. LIMNOL., 36(4), 218 Vol. 36 ode: LB259) was originally isolated from a freshwater lake in the Netherlands. Chlorella -Trop was isolated from a fish pond at University of Malaya (Phang, 24). The strains were maintained at temperatures lose to those of their original habitats to avoid long-term ultivation effets (Lakeman et al., 29), although some miroalgal strains may show high geneti stability despite long term ultivation (Marija and Dieter, 214). The ultures were maintained in a ontrolled-environment inubator at 4 C for the Arti and Antarti strains, and at 18 C and 28 C for the temperate and tropial strains respetively. All ultures were maintained in Bold s Basal Medium (BBM) (Nihols and Bold, 1965) and illuminated with TLD 18W/ ool white fluoresent lamps (Philips, Amsterdam, the Netherlands) providing ~42 μmol photons/(m 2 s) photosyntheti ative radiation (PAR) on 12 h light: 12 h dark photoperiod. 2.2 Experimental design Bath mode ulturing was onduted at 4 to 33 C for the polar strains ( Chlorella -Ant and Chlorella - Ar), 18 to 38 C for Chlorella -Temp, and 18 to 43 C for Chlorella -Trop. The ultures inubated at 4 C for the Chlorella -Ant and Chlorella -Ar, and at 18 C and 28 C for the Chlorella -Temp and Chlorella -Trop strains respetively, were set as ontrol in this experiment. The inoulum was 1% (v/v) from the exponential phase ultures standardized at an optial density of at 62 nm (OD 62 ). Tripliate ultures of eah strain were grown in 1 L Erlenmeyer flasks with 5 ml BBM and maintained in ontrolled environment shaking inubators at 8 r/min (Hoteh Model 718, Taiwan, China) at the range of temperatures mentioned earlier. Irradiane of ~42 μmol photons/(m 2 s) PAR was provided diretly from above the flasks with a 12 h light:12 h dark yle and was measured with a Li-Cor Light Meter (Model L1-25A). Cultures were allowed to grow until stationary phase (day 1). Growth was monitored by measuring absorbane at OD 62 using a UV-18 UV-VIS spetrophotometer (Shimadzu, Japan). Cell density was estimated by ell ounting using a haemoytometer (improved Double- Neubaur, Assistent, Germany). Speifi growth rate ( μ, /d) was alulated using the following formula: μ (/d)=(ln N 2 ln N 1 )/( t 2 t 1 ), where N 1 and N 2 represent the ell onentrations at times t 1 and t 2, respetively, within the whole logarithmi phase. Chlorophyll- a (Chl a ) ontent was analyzed spetrophotometrially after extration of the filtered samples (Whatman GF/C, 5 μm) in aetone (Strikland and Parsons, 1972). The absorbane of the pigment extrat was measured at 665 nm (OD 665 ), 645 nm (OD 645 ) and 63 nm (OD 63 ). The onentration of Chl a was alulated using the following formula: Chl a (mg/m 3 )=( C a V a )/ V, where, C a =11.6OD OD OD 63 ; V a =volume of the aetone (ml) used for extration; V = volume of ulture (L); Chl a (mg/l)=chl a (mg/m 3 )/1. The extration and quantifiation of total arotenoids followed the method desribed by Vonshak and Borowitzka (1991). The absorbane of the pigment extrat was measured at 452 nm (OD 452 ). The total arotenoid ontent (μg/ml) was alulated using the following formula: Total arotenoids (μg/ml)=(od V e )/ V, where, V e =volume of the aetone (ml) used for extration; V =volume of ulture (ml). 2.3 Measurement of photosynthesis performane Non-invasive fluoresene measurements were obtained using a high-resolution Water-Pulse Amplitude Modulated (PAM) fluorometer (Walz GmbH, Effeltrih, Germany). The ultures were dark adapted for a period of 15 min before the generation of rapid light urves (RLCs). A weak measuring light (.15 μmol photons/(m 2 s)) from the PAM fluorometer was used to measure the fluoresene yield without induing photosynthesis, termed as minimum fluoresene ( F o, open PSII reation enters). A saturating pulse (>3 μmol photons/(m 2 s) for.8 s) was used to determine maximum fluoresene ( F m in the dark) when all PSII reation entres were losed. PSII quantum yield, also known as the maximal effiieny of PSII, was determined as followed aording to Shreiber et al. (1995): ϕ PSII max = / F m =( F m F o )/ F m, where F m and F o are the maximum and the minimum fluoresene yields in the dark-adapted state, respetively. RLCs were generated for all samples using the Water PAM software (WinControl, Walz). Light-emitting diodes (LEDs) provided the eightstepwise inrement of atini light used in the generation of RLCs. The atini light levels used were, 48, 15, 158, 233, 358, 53, 812 and μmol photons/(m 2 s), eah lasting for 1 s. While we reognise that a short exposure time, espeially in dark-adapted ultures, will not provide

4 No.4 LEE et al.: Response of Chlorella spp. to temperature stress 1269 Speifi growth rate, μ (/d).1 Chlorella-Trop Chlorella-Ant Chlorella-Ar Chlorella-Temp Temperature ( C) Fig.1 Speifi growth rates, μ (/d) of the four strains at various temperatures Data are mean±sd of tripliate samples. aurate measures of steady state eletron transport, all samples were proessed in the same way so the hanges between treatments provide a reliable measure of the relative alterations to ell performane. Photosyntheti parameters suh as photosyntheti effiieny ( α ), maximum rate of relative eletron transport (retr max ) and photoadaptive index ( E k ) were determined to ompare the RLCs quantitatively (Ralph and Gademann, 25). The relative eletron transport rate (retr) at a given irradiane was alulated by multiplying the effetive photohemial effiieny and the irradiane (Genty et al., 1989). RLCs were onstruted by plotting retr against PAR data and subsequently fitted mathematially to a single exponential funtion (Platt et al., 198), using a Marquardt-Levenberg regression algorithm: Ed - Pm P P m 1 e, where P is the retr at a given irradiane, P m is the maximum potential retr (retr max ), α is the initial slope of the fitted RLC before the onset of saturation (light-limiting ondition effiieny) and E d is the inident irradiane (4 7 nm). The funtion beomes an asymptoti maximum retr value by assuming that there is no photoinhibition (Jassby and Platt, 1976). The interept of the α value with retr max gave the saturation irradiane for eletron transport ( E k ) whih is defined as: E k =retr max / α. Non-Photohemial Quenhing (NPQ) is a measurement of the photoprotetive xanthophyll yle whereby exessive light is dissipated as heat to avoid negative impats to the photosyntheti eletron transport hain. NPQ was alulated using the formula of Shreiber (24): NPQ=( F m F m ')/ F m ', where F m is the maximal fluoresene after a dark adaptation period and F m ' is the maximum fluoresene in the steady state light-adapted state (attained after 5 1 min). NPQ was alulated and quantified by measuring the hange in F m to the final value F m (Δ F %). The relative inhibition of α and retr max in relation to the ontrol was alulated as: Inhibition(%)=1 (1 X sample / X ontrol ), where X are the values of α or retr max. 2.4 Stress and reovery The stress-induing temperatures for eah strain were seleted based on the initial experiments with a range of inreasing temperatures. Temperatures of 34 C were used for both Chlorella -Ant and Chlorella - Ar, 4 C for Chlorella -Temp and 44 C for Chlorella - Trop. These temperatures represented the ondition when the / F m dereased sharply during the growth yle (see results), indiating a highly-stressed ondition of the ulture. These seleted temperatures may lead to ulture death with prolong exposure. The Chlorella strains were allowed to reover by returning them to their ambient temperatures after the high temperature treatments. Two threshold levels were seleted; / F m (assigned as point A ) and / F m (assigned as point B ). These points were seleted on the basis that at / F m, most of the ulture was still able to grow while at / F m, no net inrease of biomass was observed. Biomass (Chl a ) was estimated at / F m and / F m. The relative rate of reovery is defined as the time taken for the ulture to reover from / F m and / F m, to its original / F m. 2.5 Statistial analysis Data were analyzed by one-way analysis of variane (ANOVA) followed by a post-ho test using Tukey s HSD test. Statistial analyses were arried out using Statistia 1 (StatSoft In., USA). 3 RESULT 3.1 Miroalgal growth and pigment ontents The Chlorella strains used in this study tolerated a wide range of temperatures. Chlorella -Trop displayed a higher speifi growth rate ( μ, /d) than the rest of the strains under various temperature treatments (Fig.1) with the highest μ (79/d) attained at 28 C, but μ

5 127 J. OCEANOL. LIMNOL., 36(4), 218 Vol. 36 Table 1 Comparison of growth parameters and pigment ontents of Chlorella -Trop, Chlorella -Ant, Chlorella -Ar and Chlorella -Temp ultivated at a range of temperatures Speies T ( C) Cell density (1 6 ells/ml) Chl a (μg/ml) Chl a (μg/1 6 ells) Total arotenoids (μg/ml) Total Carotenoids (μg/1 6 ells) Chl a / arotenoid ratio (μg:μg) ±2 b 3.36±9 b 9±.3 b, 1.51±2 a,b.13±.2 a,b, 2.23±.1 a ± b 4.16±.16 a 6±.1 a 1.76±.4 a.15±. a 2.36±.4 a ±1 a 3.6± b 4±.3,d 1.32±.7 b.1± ±.3 a Chlorella -Trop ± 3.21±.5 b 5±.1 a,b 1.41±.3 b.15±. a 2.28±.2 a ±9 e 1.42±.12 3±.1,d ±.5.12±.1 b, 2.2±.4 b ±.4 d 1.6±.16 2±.2,d.96±.9.13±.1 a,b, 1.68± ±.3 f.1±.3 d.18±.4 d.8±.2 d.14±.2 a,b 1.26±.13 d ±.4 e 1.16±.2 e ±. b 6±. e.16±. b 2.53±.4 a ±8 1.99±.3 9±.2 b 7±.3.15±.1 b 2.59±.4 a ±3 b 2.58±.18 b 7±.1 b.98±.8 b.14±. b 2.63±.3 a Chlorella -Ant ±.18 a 2.86±.1 a 7±.2 b 1.1±.4 a.14±.1 b 2.59±.1 a ±3 b 1.86±.6 8±.2 2±.1.1± ±.5 a ±.5 d 1.4±.5 d 4±.1 b ±.2 d.14±. b 2.36±.1 b ±.1 f 1.6±.8 e 6±.4 a 5±.4 e ±.2 a 2.34±.5 b 33 3±.6 g.14±.1 f.19±.3 d.6±.1 f.9± ± ±.2 d,e 1.49±.9,d 6±.2 a ±.5.15±.1 a,b 2.48±.6 a,b 1 6.2±2 2.2±.7 b 4±. a,b 8±.2 b.13±. a,b 2.58±.2 a,b ±7 b 2.6±.1 a 1±.2 a,b 1.±.4 a.12±.1 b, 2.61±.6 a,b Chlorella -Ar ±2 a 2.75±.9 a 8±.1 b 1.2±.3 a.1±.,d 2.69±.2 a ±2 b 1.6±.2 ±.1 2±..8±. e 2.59±.3 a,b ±.2 d 1.41±.1,d ±.2 b 8±.4.12±.1 b, 2.43±.1 b 3 3.7± e 1.32±.4 d 6±.3 a 6±.2.15±.2 a 2.35±.5 b 33 4±.2 f.9±.2 e ±.3.5±.1 d.1±.1 d,e 1.91± ±5 b 2.89±.16,d 4±.3 b, 1.31±.1 b, ±.1 b 2.21±.6 d ±3 a 3.46±6 b, 4±.4 b, 1.52±.9 a,b.19±.2 b 2.28±.5,d ±9 a 4.18±1 a 8±.8 b, 1.69±.17 a ±.3 b 2.47±.2 a,b Chlorella -Temp ±7 a 4.32±3 a 3±.2 b, 1.73±.9 a 1±.1 b 2.49±.2 a,b ±.19 b 3.52±.18 b 6±.5 b 1.4±.5 b 2±.1 b 2.52±.5 a ± b 2.6±.9 d 2±.3 1.1±.6.18±.1 b 2.36±.7 b, 38.92±.8 7±.1 e 3±.5 a ±. d 3±.3 a 2.22±.4 d Data are represented as mean±sd from tripliate samples. Different letter aross a olumn indiates signifiant differene ( P <.5, n =3) between growth temperatures. dereased markedly with inreasing temperature above the optimum. Chlorella -Ant and Chlorella -Ar showed similar growth trends aross the temperature treatments. Both polar strains ould grow from 4 C to the upper temperature limit of 33 C. The upper temperature limit is defined as the highest temperature that a ulture an tolerate. The μ of polar strains inreased with inreasing temperatures, whereby the highest μ values for Chlorella -Ant and Chlorella -Ar were reorded as 5/d at 2 C and 56/d at 1 C, respetively. However, there was no onsistent growth trend observed for Chlorella -Temp but its highest μ was reorded as 69/d at 18 C. The lethal temperature (temperature at whih the ulture ould not survive) was the lowest for Chlorella -Ant and Chlorella -Ar. Temperatures at and above 33 C, 38 C and 43 C were fatal to the polar, temperate and tropial Chlorella respetively. The Chl a :arotenoids ratio (μg:μg) was also used as an indiator of the stress ondition of the ulture (Table 1). The Chl a:ar ratio (μg:μg) were signifiantly lower in Chlorella -Ant, Chlorella -Ar

6 No.4 LEE et al.: Response of Chlorella spp. to temperature stress C 23 C 28 C 33 C 38 C.1 4 C 43 C a Time (d) 4 C 1 C 15 C 2 C 25 C 28 C.1 4 C 1 C 15 C 2 C 25 C 28 C 3 C 33 C Time (d).1 3 C.1 35 C 33 C 38 C d Time (d) Time (d) Fig.2 Maximum quantum yield ( / F m ) of (a) Chlorella-Trop, (b) Chlorella-Ant, () Chlorella-Ar and (d) Chlorella-Temp grown in different temperatures Data are mean±sd of tripliate samples. 18 C 25 C 28 C 3 C 33 C b and Chlorella -Trop at temperatures above 25, 3, and 33 C, respetively. Chlorella -Temp grown at 18 C showed the lowest Chl a:ar ratio amongst the strain grown at different temperatures. The highest Chl a: ar ratio in Chlorella -Ant and Chlorella -Ar were observed at 15 C and 2 C, respetively. It was found that under the influene of ultivated temperatures, Chlorella -Ant, Chlorella -Ar and Chlorella -Trop showed a strong orrelation between their speifi growth rate and Chl a:ar ratio (Table S1) with R 2 =.933,.864 and.9 respetively (Pearson orrelation, P <.1). 3.2 Photosyntheti performane Effets of temperature on photosyntheti parameters varied amongst strains with regards to their latitudinal origin. During ultivation, Chlorella - Ant, Chlorella -Ar and Chlorella -Trop were able to maintain a relatively stable / F m (= ϕ PSII max ) at their ambient temperatures and at temperatures near T opt (Fig.2). However, ϕ PSII max of Chlorella -Trop presented a downward trend from day 6 at 38 C and 4 C (Fig.2a). For both Chlorella -Ant and Chlorella - Ar, ϕ PSII max values were at maximum values during inubation at temperatures near 1 C, but delined as temperature rose to above 2 C. ϕ PSII max in both polar Chlorella were somewhat stable, albeit with omparatively lower values after day 6 at 28 C and 3 C (Fig.2b, ). ϕ PSII max of Chlorella -Ant ould still be deteted on day 8 while ϕ PSII max of Chlorella -Ar was already zero on day 6 at 33 C. A sharp derease in ϕ PSII max was observed at 33 C for both polar Chlorella and at 43 C for Chlorella -Trop. The variation in ϕ PSII max for Chlorella -Temp was temperature-independent, whereby ϕ PSII max flutuated around from 18 C to 38 C (Fig.2d). The four Chlorella strains grown at various temperatures showed different trends in the inhibition of both retr max and light harvesting effiieny ( α ) (Fig.3). Generally, the inhibition of retr max and α was inreased with inreasing temperature. For Chlorella -Trop, inreasing temperature up to 38 C did not inhibit retr max, however, temperatures beyond 38 C lowered the retr max (Fig.3a). Chlorella - Ant did not show inhibition of retr max from 1 to 25 C, but the perentage of inhibition inreased

7 1272 J. OCEANOL. LIMNOL., 36(4), 218 Vol. 36 Inhibition (%) a ' retr max α,d Temperature ( C) d b a a' Inhibition (%) b e ',d' d' d d e' d e' Temperature ( C) ' b a a' Inhibition (%) d e ' d e d' d' d' Temperature ( C) ' b a a' Inhibition (%) d b,',' Temperature ( C) Fig.3 Perentage inhibition of light harvesting effiieny (α) and maximal relative eletron transport rate (retr max ) of (a) Chlorella-Trop, (b) Chlorella-Ant, () Chlorella-Ar and (d) Chlorella-Temp grown at different temperatures Data were normalized against the relevant ambient temperature for eah strain. Vertial bars denote standard deviations from tripliate samples and negative values represent a stimulation of the parameters. Different letters indiate signifiant (P<.5) differenes. ' a a' signifiantly ( P <.5) with further inrease in temperature (Fig.3b). Although the inhibition of retr max in Chlorella -Ar was relatively higher than Chlorella -Ant, the Antarti strain ( R 2 =.916, P <.1) displayed better orrelation (Table S2) between temperature and inhibition of retr max than Chlorella -Ar ( R 2 =75, P <.1) and Chlorella - Trop ( R 2 =4, P <.1). Chlorella -Ar showed 26.8%, 7.1%, 58.7%, 82.7% and omplete inhibition of retr max at 2 C, 25 C, 28 C, 3 C and 33 C, respetively (Fig.3). Inhibition of α was essentially temperature dependent espeially at temperatures beyond the T opt for all strains studied. The values of α onsistently delined in both polar Chlorella and Chlorella -Trop with inreasing temperature. The effet of temperature on α was the most strongly orrelated for Chlorella -Trop ( R 2 =69, P <.1) followed by Chlorella -Ar ( R 2 =36, P <.5) and Chlorella -Ant ( R 2 =98, P <.1). Signifiant inhibition ( P <.5) of retr max was only observed in Chlorella -Temp at 38 C with 3.8% inhibition but α was generally suppressed aross 25 C to 38 C (Fig.3d). NPQ.1 Chlorella-Trop Chlorella-Ant Chlorella-Ar Chlorella-Temp Temperature ( C) Fig.4 The NPQ of Chlorella -Trop, Chlorella -Ant, Chlorella - Ar and Chlorella -Temp grown at different temperatures The NPQ value is obtained at the end of eah rapid light urve with the atini irradiane of μmol photons/(m 2 s). Generally, at μmol photons/(m 2 s) Chlorella - Temp presented the lowest NPQ values ranging from.147 to 45 aross the experimented temperatures (Fig.4). NPQ in both polar Chlorella strains was ativated in response to temperature stress. NPQ at μmol photons/(m 2 s) peaked at 4 C with 35

8 No.4 LEE et al.: Response of Chlorella spp. to temperature stress a Time (h) Control A B.8 b Time (h) Control A B A' B' Time (h) Control A B A' B'.8.1 d Time (h) Control A B Fig.5 (a) Chlorella-Trop, (b) Chlorella-Ant, () Chlorella-Ar and (d) Chlorella-Temp grown at 44 C, 34 C, 34 C and 38 C respetively until the / F m dereased reahing the thresholds of A and B The ultures were then subjeted to reovery under ambient onditions. For Chlorella-Ant and Chlorella-Ar, the reovery was additionally onduted at 2 C (dotted line). Table 2 Relative reovery rate and hanges in biomass before and after reovery (Chl a ) Strain A B A' B' UMACC 1 Chlorella -Trop UMACC 237 Chlorella -Ant UMACC 263 Chlorella -Ar UMACC 248 Chlorella -Temp Relative rate of reovery (h) 8.88E-3±1.19E E-3±2.57E ±.6 72±.14 Chl a (μg/ml) 1.11±.7 87±.6 Relative rate of reovery (h) 1.72E-3±6.61E E-3±1.86E E-3±1.53E E-3±2.7E-4 46±.3 39±.7 46±.3 39±.7 Chl a (μg/ml) 47±.7 83±.5 1.3±.4 13±.12 Relative rate of reovery (h) 1.44E-3±4.97E-5 2.2E-3±2.27E E-3±1.7E E-3±3.E-5 79±.1 65±.1 79±.1 65±.1 Chl a (μg/ml).978±.4 64±.3.881± ±.15 Relative rate of reovery (h) 2.12E-3±1.97E E-3±2.27E ±.7 98±.7 Chl a (μg/ml) 76±.17 58±.7 A: relative reovery rate at ambient temperature from / F m ; B: relative reovery rate at ambient temperature from / F m ; A': relative reovery rate at optimal temperature from / F m ; B': relative reovery rate at optimal temperature from / F m. followed by 82 at 33 C for Chlorella -Ant. For Chlorella -Ar, NPQ at μmol photons/(m 2 s) peaked at 4 C with 43 followed by 4 at 3 C. Chlorella -Trop reorded a largely downward trend in NPQ with inreasing temperature. All the Chlorella strains were able to reover from exposure to high temperature after being returned to their ambient or optimal temperature (Fig.5, Table 2).

9 1274 J. OCEANOL. LIMNOL., 36(4), 218 Vol. 36 The reovery rate of ϕ PSII max depended on the degree of damage. Reovery from / F m was generally faster than from / F m (Table 2). Only Chlorella - Trop showed the inverse trend. Comparatively, Chlorella -Trop had the highest relative rate of / F m reovery, followed by Chlorella -Temp. Meanwhile, Chlorella -Ant and Chlorella -Ar showed faster relative reovery rates at their optimal temperature of 2 C (denoted by Aꞌ and Bꞌ) than in their ambient ondition (4 C) (Fig.5b, ). 4 DISCUSSION Despite the use of a low number of strains from eah latitude, whih hampers our ability to draw hard onlusions 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., 213; Cao et al., 216) and the responses of growth and photosynthesis were similar to those found in psyhrophili and temperate speies of other green algae suh as Chlamydomonas (Lukeš et al., 214). 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 ultures were able to undergo at least one ell division over this time. For example, the μ of Chlorella -Trop under stress onditions was.167/d whih 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 interations between the elevated temperatures assoiated with global limate hange and the sensitivity of Chlorella from different latitudes, in terms of their growth and photosyntheti responses. Miroalgae are able to alimatize to hanges in temperature within a short time span. The ranges of temperature used in this study varied aording to the latitudinal origin of the strains and were designed to exeed their respetive tolerated upper temperature limit. When exposed to a new temperature, miroalgae would respond and adapt by altering their biohemial omposition, pigment ontents, and metabolites via regulation of gene expression (Morgan-Kiss et al., 26; Song et al., 214), to maintain their normal funtions and prevent mortality from extreme stress. Beyond the optimal temperature range, algae would funtion less effiiently, and may die if the stress level is too extreme. The results showed that the polar Chlorella were able to grow at muh higher temperatures, with faster growth rates than that ahieved at the ambient temperature. Aording to Vinent (27), the growth rates of polar speies at low temperatures are less impressive ompared to growth at higher temperatures. Generally, the growth rates of Chlorella inreased until the respetive optimum temperatures ( T opt ) were reahed. Inubation of the Chlorella strains at supra-optimal onditions signifiantly affeted their growth. Chlorella -Ant and Chlorella -Ar survived temperatures up to 34 C, although their optimum growth temperatures were around 2 C. Cold-adapted miroorganisms inlude both psyhrotolerant speies and psyhrophiles (Morgan-Kiss et al., 26). Both polar Chlorella strains in this study are psyhrotolerant as shown by the wide range of temperatures that they tolerated. As for the tropial Chlorella, an inrease in temperature resulted in dereased μ and photosyntheti performane, indiating that the strain was already living at its upper limit of growth temperature. High temperature-indued stress was learly evident when the strains were inubated at temperatures above their respetive optimal temperature. Elevated temperature had an adverse effet on the Chl a ontent per unit biomass, thus the photosyntheti rate per ell may derease. Generally, Chl a dereased under temperature stress but arotenoids often remain onstant or high. A redution in Chl a might imply degradation of PSII and PSI reation entres (Luder et al., 22). Carotenoids not only serve as aessory light-harvesting pigments but also protet photosyntheti systems as non-enzymati antioxidant ompounds against reative oxygen speies (ROS) generated during stressful onditions (Phillips et al., 1995). Photosyntheti pigments are suseptible to attak by ROS and this ould lead to a derease in the growth rate of Chlorella spp. (Takahashi et al., 24). Chlorella ells in this study appeared to have adjusted their photoprotetive arotenoids to attenuate high temperature-indued stress. The strong orrelation between Chl a:ar ratio and speifi growth rate suggested that this ratio an be used as an indiator of the physiologial status of the Chlorella speies.

10 No.4 LEE et al.: Response of Chlorella spp. to temperature stress Photosyntheti response to temperature variations PAM-fluorometry offers a rapid and non-invasive way to measure high temperature indued photophysiologial stress in the four Chlorella strains, with respet to geographial habitat differenes. The present study showed signifiant differenes ( P <.5) in the parameters of photosyntheti performane following exposure to various temperatures. The maximum quantum yield ( / F m ) of PSII was found to vary signifiantly under inubation at different temperatures. The values of / F m observed under the ambient ondition were 4 1, 5 1, 7 1, and 3 for Chlorella -Ant, Chlorella - Ar, Chlorella -Trop and Chlorella -Temp respetively, indiating healthily photosynthesizing ells. However, these values were signifiantly dereased at higher temperatures and with longer exposure periods, indiating the inrease of physiologial stress. The derease in / F m suggested funtional disorder of the photosyntheti apparatus and damage to PSII, and ells subsequently inreased their NPQ (Ralph and Gademann, 25; Campbell et al., 26). Apart from regulating light utilization for photosynthesis, Chlorella ells also ontrol the amount of light absorbed. The value of α quantifies the light harvesting effiieny (Ralph and Gademann, 25). Values of α in all strains showed different degree of inhibition under the ranges of temperature tested. In ontrast to the observation by Cao (216) and o-workers, the urrent finding indiated downregulation of light apture effiieny under high temperature stress. Changes in α an be attributed to the variation in pigment omposition of light harvesting omplexes (LHCs) and the effiieny of energy transfer from the light-harvesting antenna to PSII reations entres (Ralph and Gademann, 25; Serôdio et al., 26). Damage to the photosyntheti apparatus (as refleted in the deline in / F m ) arising from exessively high temperature may also ontribute to the drop in α. Exessively high temperature auses a derease in the eletron transport rate, as was observed in this study. The retr max provides an approximation of the rate of eletron flow via PSII into the photosyntheti eletron transport hain and is related to the overall photosyntheti apaity of miroalgae (Juneau et al., 25). The perentage inhibition of retr max was higher for the ultures of Chlorella -Ant, Chlorella - Ar and Chlorella -Trop, but not for Chlorella -Temp, exposed to higher temperature, indiating stress to photosynthesis whih intensified with inreasing temperature. The retr max values of Chlorella -Ant and Chlorella -Ar were dereased above 25 C and 2 C, respetively. This implied that the eletron transport rates of the miroalgae were higher at temperatures below their respetive optimum temperatures. It has been suggested that the enzymes of the dark reation system might operate more rapidly and thus be able to onsume NADPH 2 and ATP at a faster rate (Kirk, 1994) as temperatures inrease until it reahes the optimum temperature. Defew et al. (24) observed a similar pattern in a temperate miroalgal ommunity, whereby retr max inreased with temperature until the optimal temperature (2 C) was reahed and then delined as the enzymes that ontrol the RUBISCO ativity, for example RUBISCO ativase and others involved in arbon fixation, were deativated (MaIntyre et al., 1997). It has been demonstrated that the effet of high temperature and high irradiane an be minimized if NPQ is ativated (Salleh et al., 21). NPQ helps to regulate and protet PSII against photoinhibition. Normally, NPQ is assoiated with the xanthophyll yle. The xanthophyll yle in Chlorophyta onsists of the ph-dependent onversion from violaxanthin to an intermediate, antheraxanthin, and subsequently to zeaxanthin (Müller et al., 21). When the absorption of light energy exeeds the apaity for light utilization, NPQ is ativated to dissipate light energy in the form of heat (Müller et al., 21). Enhaned NPQ ativities were observed in Chlorella -Ant and Chlorella -Ar at 4 C and at a higher temperature of 33 C for Chlorella -Ant and 3 C for Chlorella -Ar. The marked deline in NPQ observed in Chlorella - Trop as temperature inreased above 28 C suggest the diminishing ability of this strain to dissipate exess irradiane as heat. Adverse temperatures are believed to disrupt the NPQ proess in ells. With regards to this, Chlorella strains are likely to show speies-speifi xanthophyll yle parameters suh as rate onstants and the extent and kinetis of depoxidation (Lavaud et al., 24). 4.3 Reovery from high temperature stress The inhibition of / F m of the Chlorella speies was temperature and latitude dependent. Here, / F m was used as an indiator of the physiologial health of the miroalgae. As the seleted strains in normal onditions showed variation in / F m with values flutuating between and, thus this range was

11 1276 J. OCEANOL. LIMNOL., 36(4), 218 Vol. 36 seleted to represent a healthy photosyntheti state of these strains. In addition, it was observed that by exposing different stresses the / F m value began to deline below this range. Therefore, the value of was hosen as a threshold defining the onset of stress along with the values and onsidered different levels of stress. Also as noted by Reeves et al., (211), / F m values of ~.1 are usually onsidered representative of dead ells. We notied that at / F m, all of the strains were still able to inrease biomass (Chl a ), whereas most of the strains (exluding Chlorella -Trop) showed no net inrease of biomass at / F m. Although the reovery proess took several days, the Chlorella strains in this study were able to restore their photosyntheti fitness from two different levels of stress ( / F m and / F m ) even at high temperatures of 34 C for polar Chlorella, 38 C for Chlorella -Temp and 44 C for Chlorella -Trop. Chlorella -Trop had the highest relative rate of reovery amongst the four strains when returned to its optimum temperature. Polar strains showed higher relative rate of reovery at 2 C than 4 C, whih suggested that the protein and enzymes for repair proesses are at their optimal ondition at 2 C. A number of studies (Rautenberger et al., 215; Wong et al., 215; Rivas et al., 216) stated that repair proesses suh as the turnover and re-synthesis of D1 protein are enzymatially driven, thus the repair proesses are strongly dependent on temperature (within physiologial limits). Long term exposure to high temperatures above the ambient ondition may result in onformation hanges and denaturation of proteins (Jensen and Knutsen, 1993). Extreme temperatures derease the repair rate of D1 protein and thus an have damaging effets on the operation of the photosyntheti apparatus. Long et al. (1994) demonstrated that photoinhibition is assoiated with the loss of D1 protein, and this bloks the reovery of the PSII. It was predited that longer exposure time to exessively high temperature will ause irreversible damage to the photosynthesis apparatus of the ells and may eventually be fatal if no net reovery proess takes plae. 5 CONCLUSION The growth and photosyntheti performane of Chlorella spp. inubated at different temperatures were haraterized. The findings showed how Chlorella from different geographial regions responded differently to temperature stress with hanges in the photosyntheti parameters and pigment ontents. Different Chlorella speies are adapted to grow under different thermal habitats. Chlorella -Ant and Chlorella -Ar were found to exhibit eurythermal adaptability with a wide temperature tolerane. They are presumed to have evolved from the temperate regions and possessed different mehanisms to ope with abioti stresses. While Chlorella -Temp was rather insensitive to the range of tested temperatures, Chlorella -Trop was already living at its upper growth temperature limit. Hene further inrease in temperature negatively affeted the growth of this strain. The stable temperature in the tropial freshwater environment may be the reason for Chlorella -Trop s lak of ability to tolerate large temperature flutuations. Although variations in temperature may alter the primary produtivity of Chlorella spp. to a ertain extent, the rise in temperature per se is unlikely to ause negative impats on the polar Chlorella as they were able to survive at rather high temperatures. Future work should inlude identifying the genes and biologial pathways involved in the response to heat stress. A fundamental understanding of the thermal tolerane of Chlorella from different latitudes is ruial to predit how these miroalgae will fare in the future limate senario of rising global temperatures. However, work on additional strains from eah latitudinal region is required to make definitive onlusions about the relationship between latitude and temperature response. 6 DATA AVAILABILITY STATEMENT All data generated or analysed during this study are inluded in this published artile and its supplementary information files. Referenes Anning T, Harris G, Geider R J. 21. Thermal alimation in the marine diatom Chaetoeros alitrans (Baillariophyeae). Eur. J. Phyol., 36 (3): , Armond P A, Björkman O, Staehelin L A Dissoiation of supramoleular omplexes in hloroplast membranes A manifestation of heat damage to the photosyntheti apparatus. Biohim. Biophys. Ata, 61 : Bukhov N G, Carpentier R. 2. Heterogeneity of photosystem II reation enters as influened by heat treatment of barley leaves. Physiol. Plantarum, 11 (2): , Camejo D, Rodríguez P, Morales MA, Dell Amio J M,

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