Syazana ANUWAR, Ming-Li TEOH,,3*, Wei-Hsum YAP, Fong-Lee NG & Siew-Moi PHANG,4

1 School of Biosciences, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Selangor Darul Ehsan, Malaysia;

2 Institute of Ocean and Earth Sciences (IOES), University of Malaya, 50603 Kuala Lumpur, Malaysia;

3 National Antarctic Research Centre, Institute of Graduate Studies, 50603 Kuala Lumpur, Malaysia;

4 Faculty of Applied Sciences, UCSI University, 56000 Kuala Lumpur, Malaysia

Abstract Global warming has been the subject of concern in today’s world with elevating temperature causing the melting of polar ice and increasing sea level. The aim of this study was to investigate the physiological and photosynthetic performance of two polar Chlorella, namely Chlorella UMACC 250 and Chlorella UMACC 234 to elevating temperatures as might be experienced under future warming scenarios. The cultures were exposed to three different temperatures of 4 ℃,8 ℃ and 12 ℃. The growth and photosynthetic activity were determined every 2 d for a period of 10 d. At the end of the experiment, the cultures were harvested and analysed for biochemical composition. Both Chlorella strains were able to tolerate higher temperatures than their ambient temperature. The final pigments content showed an increasing trend with increased temperatures for both strains. The photosynthetic activities were measured using pulse-amplitude modulation (PAM) fluorometer. The photosynthetic parameters including maximum quantum efficiency (Fv/Fm), maximum relative electron transport rate (rETRmax), light harvesting efficiency (α) and photoadaptive index (Ek) were derived from the rapid light curves (RLCs). Both Chlorella strains showed a slight decline in growth and photosynthetic activities at the initial part of the experiment. However, they showed the ability to recuperate with Chlorella UMACC 250 recovers better compared to Chlorella UMACC 234. Both Chlorella strains showed similar trend in their carbohydrate content at 12 ℃ , while the protein content of Chlorella UMACC 234 decreased when exposed to increasing temperatures. The results indicated that polar Chlorella are able to survive at increased temperatures throughout the experiment.

Keywords polar, Chlorella, photosynthesis, pulse-amplitude modulation (PAM) fluorometry

1 Intr oduction*

Microalgae consists of both prokaryotic and eukaryotic organisms that rapidly grow in aquatic environment including freshwater, marine water and interestingly, waste water environment (Ravindran et al., 2016). They are unicellular organisms similar to higher plants, capable to undergo photosynthesis to convert light energy to chemical energy. Microalgae is said to be contributing to approximately half of the global primary productivity since they have a more rapid growth and turnover rates compared to higher plants (Gao et al., 2012). Besides being the primary producer of the aquatic ecosystem, microalgae are recognised as living-cell factories. Their biochemical composition such as proteins, carbohydrates and lipids are important for the production of biofuels, and wastewater treatment (Li et al., 2007; Priyadarshani and Rath, 2012). Interestingly, microalgae are also extensively used for feedstock and various food supplements that are beneficial for human’s health (Chu, 2012; Kokou et al., 2012; Chew et al., 2017).

The growth of microalgae is regulated by many abiotic factors such as temperature, pH and light intensity (Mendes et al., 2012). Changing environment temperature will initiate changes in microalgae’s physiological, chemical and molecular activities (Falkowski and Oliver, 2007; Li et al., 2011). This alterations and changes are a necessary response for them in an attempt of long-term survival. This process is known as acclimation (Valledor et al., 2013). The temperature that ranges between optimal and lethal is varied among different species. Some species might have a narrow range indicating their sensitivity to temperatures, while other species have wider range of temperatures that indicates they are able to survive by adaptation or acclimation (Ras et al., 2013).

Chlorellasp. are known to have optimum growth rates over a broad range of temperatures (Ras et al., 2013; Teoh et al., 2013) and has been highlighted as non-fastidious since it is able to colonize different types of natural environments successfully. A study by Kessler (1985) reported that the optimum growth rates of 17 different tropicalChlorellastrains were between 26℃ and 36℃. However, the polar microalgae,Chlorella saccharophilawas able to grow in temperature range of 5℃ to 15℃ for over two weeks (Vona et al., 2004). The microalgaeChlorellasp. has also been used traditionally by the Japanese as a food supplement as it contains a remarkably high protein content (41%-58%) along with some other species of microalgae such asSpirulina maximaandSynechoccussp. (de Morais et al., 2015; Ravindran et al., 2016).

Studies have shown that the protein content decrease in some microalgae when exposed to elevated temperature (Borbély et al., 1985; Teoh et al., 2004; de Castro Araujo and Garcia, 2005; Carvalho et al., 2009). Carbohydrates are regard as microalgae’s cellular fuel and possess a vital function as structural support of the membranes. The decrease in this compound can result in affected growth and metabolism of microalgae’s cells (de Castro Araujo and Garcia, 2005). Interestingly, there are a few contradicting results reported associated with carbohydrates concentration in microalgae. Increasing temperature normally leads to increasing of carbohydrates contents of some microalgae (Ogbonda et al., 2007) but some microalgae has higher carbohydrates content at lower temperature (Teoh et al., 2004; de Castro Araujo and Garcia, 2005).

Photosynthetic activities of microalgae can be measured using a pulse amplitude modulated (PAM) fluorometer. PAM fluorometry is a frequently used method in the determination of photosynthetic activities in various marine life including macro and microalgae in both the laboratory andin situ(Beer and Axelsson, 2004). This technique has been used to measure a wide range of processes associated with photosynthesis where the wellbeing of the algae studied can be assessed since photosynthesis is crucial in photosynthesizing organisms (Sjollema et al., 2014). PAM fluorometry is a non-invasive and non-destructive as well as rapid method that is able to provide direct and immediate information on the photosynthetic activity of the algae compared to the growth inhibition test that requires at least 72 h to produce results. With this more convenient technique, photosynthetic light response curves are easier to be obtained (Ritchie, 2008).

The world is facing global warming at an alarming rate. Water bodies experienced warming at a faster rate than air temperature where ice cover has been depleting, suggesting enhanced radiative warming (Larsen et al., 2014). Anthropogenic emissions since the beginning of Industrial Revolution have led to 1 ℃ of global warming. It is reported that the probability of the warming to reach 1.5 ℃ between the year 2030 and 2052 is high if these emissions persist (IPCC, 2014; Guilyardi et al., 2018). The weather events and the extreme climate change has been of major concern since they have led to disruptions to modern and past societies (Coumou and Rahmstorf, 2012; Cook et al., 2014). However, global warming does not happen overnight. The warming of the earth was observed since the mid-20thcentury (IPCC, 2011) and recent climate changes are starting to show effects on many natural and human systems (IPCC, 2007). Thus, it is important for us to fully understand the effects of elevated temperatures on organisms, particularly on microalgae. It is beneficial to have the information regarding the physiological response of microalgae to increasing temperature to predict future distributions of microalgae. This knowledge can aid in the findings of the mechanism of survival of microalgae, therefore can be used in future generation of biofuel generation.

46.Self-same bundle of rags: Since her commitment was been broken, all of the riches and luxuries she received have been revoked102. She is returned to the same state in which she began the story.Return to place in story.

The aim of this study was to investigate the response of twoChlorellastrains originating from the Antarctic to elevated temperatures. The growth, photosynthetic activities, pigments and biochemical composition in these microalgae were determined.

2 Materials and methods

2.1 Algal cultures

The microalgae used in this study, namelyChlorellaUMACC 250 andChlorellaUMACC 234 were obtained from the University of Malaya Algae Culture Collection (UMACC) (Table 1). TheChlorellaUMACC 250 was isolated from samples collected around Marion Island, Sub-Antarctic (Chu et al., 2002), while UMACC 234 was collected from snow originated from Beal Island, Antarctic. The cultures were maintained in a controlled-environment incubator at 4 ℃, illuminated with cool white fluorescent lamps (42 μmol·m-2·s-1) on 12 h∶12 h light-dark cycle. All cultures were grown in Bold’s Basal Medium (BBM) (Nichols and Bold, 1965).

2.2 Experimental design

The microalgal cultures were grown in 2 L conical flasks and placed in light- and temperature-controlled incubators set at different temperatures of 4℃ (ambient temperature), 8℃ (projected future warming scenarios) and 12℃ (projected extreme future warming scenarios). Illumination was provided by cool fluorescent lamps (42 μmol·m-2·s-1) on 12 h∶12 h light-dark cycle. The experiment was carried out in triplicate with the total volume of 1600 mL of each flask. The inoculum (10%) used was from exponential phase cultures standardised at an optical density at 750 nm (OD750nm) of 0.2.

Table 1 Details of the microalgae selected for this study

Growth was monitored every 2 d based on at least two parameters: OD750nmand cell number or OD750nmand chlorophyll-a(chl-a).Chl-awas determined by spectrophotometry after extraction of the filtered samples using glass-fibre filters, 0.47 μm in acetone (Strickland and Parsons, 1968). Chl-awas determined using the following formula:

where,Ca= 11.6 (OD665nm) - 1.31 (OD645nm) - 0.14 (OD630nm),Va= Volume of acetone (mL) used for extraction,

Carotenoid content was determined using the following formula:

Carotenoid (μg·mL-1)∶(OD452nm× 3.86 ×Va) / (Vc) , where,Va= Volume of acetone (mL) used for extraction,

As OD750nmwas measured for all samples, specific growth rate (μ, d-1) based on this parameter was calculated using the following formula:μ(d-1) = (LnN2- LnN1)/(t2-t1) whereN2is OD750nmatt2,N1is OD750nmatt1, andt2andt1are times within the exponential phase (Guillard, 1973). Photosynthetic activity was also determined every 2 d using pulse-amplitude modulation (PAM) fluorometer. The samples were harvested at the end of the experiment (stationary phase, day 10) by filtration and used for determination of dry weight and biochemical analysis (protein and carbohydrate).

2.3 Dry weight determination

Blank glass-fibre filters (Fioroni, 0.47 μm) were dried in a forced-air oven at 100℃ for 24 h and weighed. A known volume of the algal culture was filtered on a pre-weighed filter and was let dry for 24 h at 100℃. The algal dry weight (DW) was determined using the following equation (APHA, 1989):

2.4 Biochemical analysis

The protein concentration of cells was determined using the dye-binding method after extraction in 0.5 mol·L-1NaOH (Bradford, 1976). Carbohydrates contents were extracted in 2 mol·L-1HCl and the concentration were determined using the phenol-sulphuric acid method (Kochert et al., 1978).

2.5 Photosynthetic activity

The photosynthetic activities of microalgae were measured every 2 d using a Diving-PAM fluorometer (Walz, Germany) (McMinn et al., 2010; Keng et al., 2013). Rapid light curve (RLC) was obtained using software control (Wincontrol, Walz). All cultures were dark-adapted for 15 min prior to exposing them to different light level. The maximum quantum efficiency (Fv/Fm) was used to indicate the physiological condition of phytoplankton where the health status of the cells can be estimated. The equation used:Fv/Fm= (Fm-F0)/FmwhereFmis the maximum fluorescence andF0is the minimum fluorescence resulting in the variable fluorescenceFv. The maximum photosynthetic efficiency (α) was determined from the initial slope of the RLC. The quantum yield and irradiance obtained from the end of each light interval was used to calculate the relative electron transport rate (rETR). The RLC is a condition where it consists of eight consecutive ten-second intervals of actinic light with increasing intensity. The photoadaptive index (Ek) was obtained from the curve fitting model (Platt et al., 1980).Ekis the interception point of theαvalue with the maximum relative electron transport rate (rETRmax) whereEk=rETRmax/α.

2.6 Da ta analysis

The data such as specific growth rate, photosynthetic activities and biochemical composition were analysed by one-way ANOVA followed by comparison of means using Neuman-Keuls Test (Statistica software, Version 5). The differences were considered significant atp＜ 0.05.

3 Results

3.1 Gr owth trends

Based on specific growth rate measurement (μ, d-1),ChlorellaUMACC 234 grew best at its ambient temperature (0.35 ± 0.02 d-1), and specific growth rate decreased dramatically with further increase in temperature (Figure 1a) (p＜ 0.05). Meanwhile, the growth ofChlorellaUMACC 250 was at the highest temperature exposure of 12 ℃ (0.24 ± 0.01 d-1).

BothChlorellasp. from the polar region showed similar trends in their chl-acontent (Figure 1b). There was a marked increase of chl-acontent when the cultures were exposed to increasing temperatures, 459.20 ± 0.01 mg·m-3for UMACC 250 and 748.76 ± 0.03 mg·m-3for UMACC 234 at 12℃. Same trend was observed in their carotenoid concentration (Figure 1c) where both strains has the highest reading at the highest temperature exposure 0.23 ± 0.01 μg·mL-1forChlorellaUMACC 250 and 0.41 ± 0.03 μg·mL-1forChlorellaUMACC 234.

Of the two Antarctic microalgae, theChlorellaUMACC 250 strain appeared to be more tolerant to temperature stress whileChlorellaUMACC 234 was sensitive to temperatures higher than their ambient. In general, polar microalgae are able to survive at a higher temperature range from their ambient temperature.

3.2 Biochemical content

ChlorellaUMACC 250 showed an increased value of carbohydrates at 12 ℃ (24.37% ± 5.15%, DW) after a marked decrease at 8℃ (11.84% ± 4.12%, DW). Similar finding was observed inChlorellaUMACC 234 where the value of carbohydrates increased at 12 ℃ (19.55% ± 6.70%, DW). Carbohydrate was produced more at highest temperature (Figure 2a). The opposite result was obtained for the protein content (Figure 2b) whichChlorellaUMACC 234 shows a dramatic decrease (p＜ 0.05) of protein content when temperature reaches 12℃ (16.08% ± 1.64% DW). However,ChlorellaUMACC 250 shows increased protein content at 12℃ (19.4%, DW) although experiencing a noted decrease to 13.6% DW at 8 °C from 29.2% DW at 4℃. Based on the data, more protein was produced byChlorellaUMACC 250 in order to survive in elevated temperature.

3.3 Photosynthetic activity

The maximum quantum efficiency (Fv/Fm) (Figure 3a) ofChlorellaUMACC 250 showed consistent reading from its ambient temperature to the highest temperature exposure with a significant increase (p＜ 0.05) from 0.55 ± 0.02 at 4 °C to 0.60 ± 0.01 at 12 °C.ChlorellaUMACC 234 shows a significant increase (p＜ 0.05) from 0.37 ± 0.04 at its ambient temperature to 0.47 ± 0.01 at 8 °C after 10 d of exposure. TheFv/Fmindicates the health of the microalgae being studied andChlorellaUMACC 234 showed potentially compromised health when temperature reached 12 °C. The light harvesting efficiency (α） (Figure 3b) forChlorellaUMACC 250 were reported highest at the highest temperature (0.40 ± 0.05).ChlorellaUMACC 234 shows similar trend withαthe highest at 12℃ (0.26 ± 0.00). The maximum relative electron transport rate,rETRmax(μmol·m-2·s-1, electrons) (Figure 3c) forChlorellaUMACC 250 showed a decreasing trend as temperature increased. In contrast,ChlorellaUMACC 234 shows highrETRmaxreading at highest temperature (276.67 ± 90.05 μmol·m-2·s-1, electrons). There was no consistent trend for the photoadaptive index,Ek(Figure 3d) of both strains whereChlorellaUMACC 250 shows consistent decreased inEkreading as temperature elevated whileChlorellaUMACC 234 has highestEkreading at 8 °C (1273.38 ± 492.93 μmol·m-2·s-1, photons) and started to decrease as temperature reached 12 °C (1096.06 ± 450.51 μmol·m-2·s-1, photons).

Figure 2 Carbohydrate content (a) and protein content(b) of UMACC 250 and UMACC 234. Vertical bars denote standard deviations from triplicate samples. Different alphabets above the bar charts indicate significant differences (p ＜ 0.05) between temperatures for each species.

4 Discussion

Figure 3 a, Maximum quantum efficiency (Fv/Fm); b, Light harvesting efficiency (α）; c, Maximum relative electron transport rate, rETRmax; d, Photoadaptive index, Ek of both UMACC 250 and UMACC 234. Vertical bars denote standard deviations from triplicate samples. Different alphabets above the bar charts indicate significant differences (p ＜ 0.05) between temperatures for each species.

Based on the results, both polarChlorellaUMACC 250 andChlorellaUMACC 234 strains have distinct growth trends in accordance to temperature (Figure 1). It can be observed that the polar UMACC 250 is eurythermal where it can tolerate a wider range of temperature (Lee et al., 2018) from 4℃ to 12℃ and can adapt a temperature higher than its ambient temperature, it has the highest specific growth rate (μ) at highest temperature exposure. This proves that polar microalgae are psychrotrophic since they are cold-tolerant microorganism that possesses the ability to grow at low temperatures but have optimal growth temperature at above 20℃(Shukla et al., 2011). Similar finding was reported by Teoh et al. (2004) where the polar microalgae were able to survive even at 30.℃ In current study, the findings indicated thatChlorellaUMACC 234 strain is more sensitive to temperature elevation when compared toChlorellaUMACC 250 since its specific growth rate (μ) decreases markedly as temperature increases. It can be said thatChlorellaUMACC 234 has reached its optimum growth temperature close to its ambient temperature. At temperature below the optimum level, growth will increase with increasing temperature but will decrease significantly at temperature above the optimum level (Teoh et al., 2010). Temperature elevations are commonly link to increasing growth of microalgae as proven in a study by Shukla et al. (2013) where increased temperature of up to 23℃stimulated the growth of polarChlorella mirabilis.

BothChlorellastrains show increasing trend of chl-aand carotenoid content as temperature increases. Past studies have reported that adaptation to low temperature imitates the acclimation to condition of high irradiance (El-Sabaawi and Harrison, 2006), thus lower chlorophyll content is considered as a microalgae’s normal response to high irradiance. An increased in pigment composition under high temperature indicates that higher energy is supplied by light harvesting process (Geider et al., 1997). Furthermore, pigment synthesis might speed up at elevated temperature (Cao et al., 2016). However, different environmental conditions exposures affect chlorophyll contents in a species-specific manner (da Silva Ferreira and Sant’Anna, 2017). High temperature can lead to stressful condition upon microalgae that eventually speeds up the production of excess free radicals. In order to mitigate the harmful effects of these radicals, algae cells will produce molecules with anti-oxidant properties, hence the increasing content of carotenoids (Ras et al., 2013).

In general, the biochemical content for each polar strain varies. Carbohydrate content for bothChlorellastrains is abundant at highest temperature exposure withChlorellaUMACC 250 being the highest. However, inconsistency of carbohydrates accumulation with temperature have been observed in previous studies. For example, a study conducted on the microalgaePavlova lutherishows that carbohydrate content increases at temperature of 18℃, and slightly decrease afterwards at higher temperature of 22 ℃ (Carvalho et al., 2009).

In current study, protein content in both strains are much lower in increasing temperature compared to their ambient temperature withChlorellaUMACC 234 shows a consistent dramatic decrease as temperature elevates. This result is similar to the findings where polarChlorellaaccumulated protein and lipids under lower temperatures and as temperature elevates; its metabolism tends to synthesis soluble sugar (Cao et al., 2016). Typically, the biochemical content of a fast-growing photosynthetic microalgal cell is generally associated with a low carbohydrate and high protein content. However, under environmental stress conditions where cells might have reached their stationary growth phase,more photoassimilated carbon is link to carbohydrates or lipids (Zhu et al., 1997). In spite of that, there is no consistent trend in biochemical composition in response to temperature. It was found to be species specific (Teoh et al., 2010).

Under varying environmental conditions, photosynthetic organisms will strive to maintain an equilibrium between energy supply through electron transport and energy utilized via carbon fixation (Maxwell et al., 1994). Photosynthetic performance measured by using fluorescence can be associated with its (relative) electron transport rate and followed by growth rate, thus giving a faster estimates of productivity (Baker, 2008; Malapascua et al., 2014). The reading ofFv/Fmindicates the ‘health’ of the microalgae being studied where value ＞ 0.5 is considered as healthy.ChlorellaUMACC 250 shows value of ＞ 0.5 until day 10 for all temperature exposures indicating healthy microalgae. In contrast,Fv/Fmvalue forChlorellaUMACC 234 shows ＜ 0.5 in all temperature exposures suggesting the depletion of nutrient in experimented cultures and possible temperature stress due to the transportation of flasks from one temperature to another.

Theαis a function for light absorption efficiency. It is regard as the measure of efficiency of light harvesting activity of microalgae. Theαreading for both strains are highest at 12. ℃As temperature increases, the demand for available carbon will also increases, as well as a possible enhancement of RuBisCo activity (Fu et al., 2007). Therefore, bothChlorellastrains were able to change to greater light-harvesting efficiency.ChlorellaUMACC 250 has the highest value of 0.4 at 12℃, suggesting that it harvests light more efficiently compared to UMACC 234. This can be seen as one of the reasons whyChlorellaUMACC 250 shows a higherμat elevated temperature.

TherETRis an estimation of the electron flow rate through the photosynthetic chain (Malapascua et al., 2014; Gomes et al., 2017). Interestingly, despite being the more tolerant strain,ChlorellaUMACC 250 showed significant decrease inrETRmaxfrom values above 300 m-2·s-1at 4 ℃ to values below 300 m-2·s-1at 12. It has been concluded ℃ that strains originated from bright environments tend to have higher values than those that grow in low light environment. The same trend was observed in the photoadaptive index,EkwhereChlorellaUMACC 250 had decreasing value as temperature increased. The finding was explained in a study where the rise inαduring exposure to increased temperature led to a lower light level for saturation ofEk. This shows that the efficiency of light harvesting had increased (Fu et al., 2007).

In conclusion, both polarChlorellastrains studied are able to grow at temperature higher than their ambient temperature. They tend to decrease their protein content and increase their carbohydrates content in order to adapt to elevated culture temperatures. Further research involving proteomic studies to investigate the types of proteins expressed during the period of high temperature exposure of the polar microalgae will be carried out.

AcknowledgmentsThis study was funded by a research grant from the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme (Grant no. FRGS/1/2017/STG05/TAYLOR/02/2). The authors would like to thank University of Malaya (UM) and Institute of Ocean and Earth Sciences (IOES) for the collaboration and laboratory access. Gratitude is also given to Taylors’s University (TU) for providing laboratory access. We appreciate very much two anonymous reviewers, and guest editor for their helpful and constructive comments on the manuscript of this paper.