Light availability modulates the effects of warming in a marine N2 fixer

Abstract. Trichodesmium species, as a group of photosynthetic N2 fixers
(diazotrophs), play an important role in the marine biogeochemical cycles of
nitrogen and carbon, especially in oligotrophic waters. How ongoing ocean
warming may interact with light availability to affect Trichodesmium is not yet clear. We
grew Trichodesmium erythraeum IMS 101 at three temperature levels of 23, 27, and 31∘C under
growth-limiting and growth-saturating light levels of 50 and 160 µmol quanta m−2 s−1, respectively, for at least 10 generations and then
measured physiological performance, including the specific growth rate, N2
fixation rate, and photosynthesis. Light availability significantly modulated
the growth response of Trichodesmium to temperature, with the specific growth rate
peaking at ∼27∘C under the light-saturating
conditions, while growth of light-limited cultures was non-responsive across
the tested temperatures (23, 27, and 31∘C). Short-term thermal
responses for N2 fixation indicated that both high growth temperature
and light intensity increased the optimum temperature (Topt) for
N2 fixation and decreased its susceptibility to supra-optimal
temperatures (deactivation energy – Eh). Simultaneously, all
light-limited cultures with low Topt and high Eh were unable to
sustain N2 fixation during short-term exposure to high temperatures (33–34∘C) that are not lethal for the cells grown under
light-saturating conditions. Our results imply that Trichodesmium spp. growing under low
light levels while distributed deep in the euphotic zone or under cloudy
weather conditions might be less sensitive to long-term temperature changes
that occur on the timescale of multiple generations but are more susceptible to
abrupt (less than one generation time span) temperature changes, such as
those induced by cyclones and heat waves.



Introduction
In vast areas of the oceans, primary production is usually limited by availability of nitrogen (Moore et al., 2013). In addition to recycling within the euphotic zone, biologically available nitrogen sources can be supplied to phytoplankton from upwelling, aerosol deposition and N2 fixation by diazotrophic prokaryotes, supporting new primary production (Dugdale and Goering, 1967). Trichodesmium is one of the major diazotrophic organisms occurring in the pelagic oceans (Zehr, 2011). 30 Trichodesmium is a genus of filamentous cyanobacteria that exists as both single filaments and colonies consisting of tens to hundreds of trichomes, and that is broadly distributed in oligotrophic tropical and subtropical oceans (Capone et al., 1997).
Its contribution to local new production can even be more important than that of nitrate diffusion in some regions LaRoche and Breitbarth, 2005;Mahaffey et al., 2005), and it thus plays a significant role in marine ecosystems and biogeochemical cycles of nitrogen and carbon Zehr, 2011). 35 Trichodesmium has attracted tremendous research interest since its discovery as diazotroph in the early 1960s (Dugdale et al., 1961;Dugdale et al., 1964). Recently, considerable research attention has been focused on evaluating effects of the ongoing ocean climate changes, including sea surface warming associated with global warming, on this keystone organism (Fu et al., 2014;Hutchins and Fu, 2017;Jiang et al., 2018). The IPCC RCP 8.5 scenario predicts that upper ocean temperature will increase by about 3 ℃ on average by the end of the 21st century, and the strongest ocean warming will 40 happen in tropical and subtropical regions (Collins et al., 2013). Because of its important role in marine biogeochemical cycles and marine ecosystems, understanding the responses of Trichodesmium to ocean warming and their underlying mechanisms will be critical to evaluating the potential implications of climate changes on marine primary productivity, food web dynamics and biogeochemical cycles.
Previous studies demonstrate that without resource limitation the growth versus temperature curve is unimodal in 45 Trichodesmium with lower and upper tolerance limits separately at 18-20 ℃ and 32-34 ℃ and optimum temperature at 26-28 ℃ (Breitbarth et al., 2007;Chappell and Webb, 2010;Fu et al., 2014). Based on these findings and the spatial heterogeneity of present temperatures and projected warming of Trichodesmium's habitat (Capone et al., 1997;Collins et al., 2013), the effects of ocean warming on Trichodesmium can be spatially diverse, generally benefiting those occurring in relatively high latitude but being harmful to those occurring near to the equator (Breitbarth et al., 2007;Fu et al., 201450 Thomas et al., 2012. However, this pattern can be distorted and complicated by resource limitations. For example, it is shown that iron limitation, which is commonly experienced by Trichodesmium in nature (Hutchins and Boyd, 2016;, substantially increases the optimum temperature in Trichodesmium (Jiang et al., 2018). Similar to iron availability, light is also among the key environmental drivers for Trichodesmium (Cai and Gao, 2015;Cai et al., 2015;Breitbarth et al., 2008). Trichodesmium can be distributed from the sea surface down to 150 m depth where light 55 intensity at noon ranges from > 2000 μmol quanta m -2 s -1 to < 10 μmol quanta m -2 s -1 (Davis and McGillicuddy, 2006;Olson et al., 2015). Moreover, Trichodesmium is known to be able to partially regulate its vertical position in water column by buoyancy adjustment (Villareal et al. 2003). Currently, ocean warming effects on Trichodesmium have been widely examined under single, saturating light conditions, providing important knowledge on this diazotroph's physiological responses to temperature changes (Breitbarth et al., 2007;Fu et al., 2014;Jiang et al., 2018;Levitan et al., 2010). However, 60 how it responds to warming under both light-limiting and saturating conditions is also of general significance, but has been rarely studied (Boatman et al., 2017).
The growth of phytoplankton is a holistic result of many biochemical and physiological activities, so its responses to environmental changes are dependent on species-specific physiology. Normally, warming increases enzyme activities, accelerating biochemical reactions (Gillooly et al., 2001). For phytoplankton, reduced growth at high temperature might be 65 the result of less carbon availability due to the higher thermal sensitivity of respiration compared to that of photosynthesis (Padfield et al., 2015). This negative effect of high temperature might be exacerbated by directly reduction of photosynthesis under light limitation. In the diazotroph Trichodesmium, besides photosynthesis and respiration, N2 fixation process might also play a critical role in its growth response to environmental changes. Little has been documented on this aspect (Jiang et al., 2018). 70 In this study, we explored the combined effects of temperature and light in Trichodesmium erythraeum IMS 101. We measured the specific growth rate, photosystem functions and N2 fixation rate in Trichodesmium cultures acclimated to two light levels and three temperatures. Moreover, we measured their short-term thermal responses for N2 fixation. In this paper, "acclimation" and "acclimated" indicate that cultures were given enough time (several weeks) to respond to the environmental changes so that balanced growth was achieved, and "short-term" refers to acute processes and changes 75 occurring within hours. Although adaptation (over several hundreds of generations) has been demonstrated to be critical in evaluating the responses in phytoplankton to environmental changes (Hutchins et al., 2015;Li et al., 2017;Padfield et al., 2015;Schaum et al., 2018;Tong et al., 2018), it is beyond the scope of this study.

Culture conditions 80
Triplicate cultures of Trichodesmium erythraeum (strain IMS101, originally isolated from the North Atlantic Ocean by (Prufert-Bebout et al. 1993)) were established under six different culture conditions. These included factorial combinations of three temperatures (23±1, 27±1 and 31±1 ℃) and two light intensities (saturating light, 160 ± 20 μmol quanta m -2 s -1 and limiting light, 50 ± 6 μmol quanta m -2 s -1 ). These three growth temperatures are representatives of present and future temperatures of Trichodesmium habitats (Breitbarth et al., 2007;Fu et al., 2014). The limiting and saturating light levels 85 were established based on a pilot experiment (Supplementary Fig. S1(a)) and previous studies (Cai et al., 2015;Breitbarth et al., 2008;Garcia et al., 2011). All the cultures were run semi-continuously by continual dilutions (every 2-4 days) in the artificial seawater medium YBCII without combined nitrogen (Chen et al., 1996) in 1-L glass flasks maintained in plant growth chambers (HP300G-C, Ruihua, China). Light was provided by LED tubes (FSL, China) with a 12:12 Light:Dark cycle. Different levels of light intensity were achieved using neutral density filters. Cultures were continuously bubbled with 90 air (outdoor) so that the cyanobacteria floated as single filaments. The cells were allowed to acclimate to each condition for at least 10 generations. Acclimation was confirmed by balanced growth with stable specific growth rate. Then, we started the sampling and data collection.

Chlorophyll-a (Chl-a) concentration and specific growth rate
Chl-a concentration was spectrophotometrically quantified by gently filtering the cells onto glass-fiber filters (GF/F, 95 Whatman), followed by extraction in pure methanol at 4 ℃ for 24 h and centrifugation at 6000g for 10 minutes. The absorbance spectrum of the supernatant was determined from 400 nm to 700 nm using a spectrophotometer (DU800, Beckman, USA). Chl-a concentration was calculated as: [Chl-a] (μg mL -1 ) =12.9447*(A665-A750), where A665 and A750 were respectively the absorbances at 665 and 750 nm (Ritchie, 2006). Because Chl-a concentration is a good proxy for biomass in Trichodesmium (Breitbarth et al. 2007), Chl-a concentrations 100 measured at different days were analysed using Eq. (1) to obtain the specific growth rate: In(Chl-a (t)) = μ * t + b (1) where Chl-a(t) is the Chl-a concentration at time t (d), μ is the specific growth rate (d -1 ), and b is interpreted as the natural logarithm of Chl-a concentration at day 0. Because the cultures were semi-continuously maintained, Chl-a concentrations at each time point was corrected by the dilution ratios with the assumption of no dilutions. 105

Short-term thermal response for N2 fixation
N2 fixation rates were determined using the acetylene reduction assay assuming a ratio of 4:1 to convert ethylene production to N2 fixation (Capone, 1993). To examine the responses of N2 fixation in the cells grown at different temperatures and light levels to short-term temperature changes, we simultaneously measured N2 fixation at five temperatures ranging from 19 to 35 ℃. For each replicate, a 25ml aliquot of the culture was taken and dispensed into five vials. Five vials each of which 110 contained 5ml culture were separately placed in five different zones of two multi-zone culture chambers (HP100-2 and HP100-3, Ruihua, China) and allowed to equilibrate to different target temperatures for 30 minutes. Pilot experiments had showed that 30 minutes was enough for temperature equilibrium. After temperature equilibration, each vial was spiked with 1 ml (12.5% of headspace volume) pure acetylene and incubated for another 30 minutes under the growth light level. The quantity of ethylene produced was determined using a gas chromatograph with flame ionization detector (Clarus 580, 115 PerkinElmer, USA).
Typically, the short-term thermal response curves for N2 fixation were unimodal and negatively skewed, which could be accommodated to a modified version (Padfield et al., 2015;Schaume et al. 2018) of the Sharpe-Schoolfield model (Schoolfield et al., 1981;Sharpe and DeMichele, 1977): where N(T) is the N2 fixation rate (μmol N2 mg Chl-a -1 h -1 ) at temperature T (Kelvin, K), Ea is the activation energy (electron volt, eV) for N2 fixation, being indicative of the steepness of the slope leading to a thermal optimum, Eh is the deactivation energy (electron volt, eV) characterizing high temperature induced inactivation above deactivation temperature Th (K), and N(Tc) is the N2 fixation rate at an arbitrary reference temperature Tc (here, Tc = 25 ℃) used for normalization. According to Eq. (2), the optimum temperature (Topt, K) corresponding to the maximal N2 fixation rate (Nmax, μmol N2 mg 125 Chl-a -1 h -1 ) is: Additionally, we also obtained the N2 fixation rate at growth temperature (Ngrowth) by bring corresponding growth temperatures to Eq. (2).

Chl-a fluorometry 130
Photosystem II (PSII) effective quantum yield (ΦPSII) and photosynthetic relative electron transport rate (rETR) were measured by using the Multiple Excitation Wavelength Chlorophyll Fluorescence Analyzer (MULTI-COLOR-PAM, Walz, German) equipped with the US-T temperature control unit (Walz, Germany). Aliquots of 1.5 ml of the culture were taken to determine effective quantum yield (ΦPSII) under actinic light levels that were the same as those of the growth conditions. Then, ΦPSII(E) values were successively measured at seven levels of light intensity (E) ranging from 0 to 1064 μmol quanta 135 m -2 s -1 . Samples were allowed to acclimate to each light level for 3 minutes before ΦPSII(E) measurements (Suggett et a., 2007). Relative electron transport rate (rETR) at each light level was calculated as: rETR = E * ΦPSII(E) (Ralph and Gademann, 2005). The light response curve of rETR was analysed according to the model of (Eilers and Peeters, 1988): Photosynthetic parameters including photosynthetic light harvesting efficiency (α), rETR maximum (rETRmax) and light 140 saturation point (Ek) can be calculated as: During the measurements, sample temperature was maintained at the corresponding growth temperatures using the US-T 145 temperature control unit (Walz, Germany).

Statistical analyses
Statistical analyses were performed with the R language (version 3.5.3). [Chl-a] versus time in each of the triplicate cultures for each treatment was fitted to Eq.(1) using function "lm" in package "stats" to get the specific growth rate (μ). The significance of differences in specific growth rate (μ) between treatments was tested using two-way analyses of variance 150 (ANOVA) (function "aov" in package "stats"), followed by Tukey's test for pairwise comparison (function "TukeyHSD" in package 'stats'). The homogeneity of variance assumption and the residuals normality assumption were separately checked by Levene's test (function "leveneTest" in package "stats") and Shapiro-Wilk test (function "shapiro.test" in package "stats").
The significance level was set to 0.05.
To test whether parameters N(Tc), Ea, Eh, Th and Topt differ between different treatments, we fitted short-term thermal 155 responses for N2 fixation to Eq.(2) using nonlinear mixed effects models ("nlme" package). Models included random effects on each of the parameters of Eq.(2) by replicate. The structure of the fixed effects of the nonlinear mixed effects model was determined by trying all possible models (625 models) and selecting the one with the lowest small sample-size corrected Akaike information criterion (AICc) (see Supplementary Table S3 for all tested models' AICc). AICc was calculated using function "AICc" in package "MuMIn". 160 Light curve of rETR in each of the triplicate cultures for each treatment was fitted to Eq. (4) using function "nls" in package "stats". The significance of differences in photosynthetic light harvesting efficiency (α), rETR maximum (rETRmax), light saturation point (Ek) and effective quantum yield (ΦPSII) between treatments was tested using the same statistical methods as those for μ. The significance level was set to 0.05.
The value of deactivation energy (Eh) for N2 fixation, reflecting the thermal susceptibility to supra-optimal temperatures, was affected by both light availability and growth temperature, but not by their interaction (Table 3). Eh tended to be lower in 220 Trichodesmium cultures grown under high temperature and high light intensity. With the highest Topt (30.0 ± 0.3 ℃ (±S.E.M)) and the lowest Eh (1.47 ± 0.14 eV(±S.E.M)) among all treatments, light-saturated cultures acclimated to 31℃ were the only cultures that were able to maintained considerable N2 fixation rates at assay temperatures as high as 34 ℃ (Fig. 3). In addition, both light availability and growth temperature affected the deactivation temperature (Th) for N2 fixation in Trichodesmium and no interaction between these two drivers was found on Th (Table 3). Th in light-saturated cultures 225 tended to be higher than that in light-limited cultures regardless of the growth temperature. Th was lower in cultures grown at 31 ℃ compared to that in cultures grown at 23 or 27 ℃ under both light levels. The activation energy (Ea) for N2 fixation was affected by growth temperature but not by growth light intensity ( Table 3)

Discussion
In this study, light availability not only affected growth rate and N2 fixation directly, but also modulated their responses to temperature changes in Trichodesmium IMS 101. Reduced energy supply due to light limitation leads to lowered nitrogen fixation and thus reduced growth in the diazotroph. The specific growth rate were maximal at 27 ℃ for saturating lightgrown cells, but were virtually insensitive to temperature changes across the tested temperature (23, 27 and 31 ℃) for light-235 limited cultures.
The interactions between temperature and light on Trichodesmium demonstrated in this work are relevant to natural light and temperature variations and to Trichodesmium global change physiology and biogeography. Light supplies energy for photosynthesis, growth and other key activities, such as N2 fixation in cyanobacterial diazotrophs. The observed phenomenon that the growth rate becomes less sensitive to temperature changes (Fig 1(a) and Fig. 4) in Trichodesmium IMS 240 101 under limiting light levels can be attributed to insufficient energy supply for the cells to respond to temperature changes.
While thermal biological responses are mainly based on enzymatic performance, light limitation suppresses syntheses of enzymes (Raven and Geider, 1988), and thus subsequently limits thermal responses. Although light-limited phytoplankton cells typically allocate more resources to light-harvesting systems to compensate for light shortages, at very low irradiances this compensation cannot prevent light harvesting capacity from being a limiting factor for enzyme synthesis and growth 245 (Raven and Geider, 1988). Field investigations show that vertical distributions of Trichodesmium can reach to depths greater than 100 m, where light is absolutely limiting and temperature is lower compared to surface temperature (Olson et al., 2015;Rouco et al., 2016). According to the typical values of surface solar irradiances and vertical extinction coefficient in tropical and subtropical oceans (Olson et al., 2015), the daily light dose received by the light-limited cultures in our study corresponds to that received by Trichodesmium at a depth of 50-60 m. The contribution of biomass and N2 fixation by 250 Trichodesmium at depths greater than 50 m might range from 7% to 28% (Davis and McGillicuddy, 2006;Olson et al., 2015). Therefore, the evaluation of potential warming effects on Trichodesmium should not be constrained to the populations inhabiting light-saturated environments (upper tens of meters) (Breitbarth et al., 2007;Jiang et al., 2018), making 3-Dimensional models indispensable. In existing 3-Dimensional model studies involving Trichodesmium (Boyd and Doney, 2002; J. K. Moore et al., 2001), the combined effects of temperature and light on Trichodesmium biological activities are 255 simply assumed to be additive, which is proven to be inappropriate in this work. While the absolute values of N2 fixation rate under light limiting and saturating levels cannot be directly compared on the basis of Chl-a content, since lower light level resulted in more cellular Chl-a content ( Supplementary Fig. S1(b)), comparison of the thermal response patterns can generate useful information for improving model predictions of diazotrophic responses to ocean climate changes.
Thermal responses for organisms are known to be useful in evaluating thermal acclimation potential and probing low and 260 high temperature tolerances (Gunderson et al, 2010;Somero, 2010;Way and Yamori, 2014). In this work, the shape of the short-term thermal response curves for N2 fixation is normalization-independent because cells were exposed to different assay temperatures for only one hour, hardly changing the elemental stoichiometry or cellular pigments component. When exposed to abrupt temperature gradients, the light-saturated cells acclimated to higher temperature exhibited higher Topt values (Table 3) and lower thermal susceptibility to supra-optimal temperatures (Eh; Table 3). This indicates an increased 265 capability for the diazotroph to tolerate short-term warming impacts. However, light limitation made the cells more susceptible to warming due to decreased Topt and increased Eh for N2 fixation (Table 3). Moreover, with light limitation, acclimation to high temperature did not help Trichodesmium cells tolerate short-term supral-optimal temperature. On the other hand, Chl-a fluorescence data shown that the PSII in light-limited cultures was as healthy as that in cells grown under saturating light (Fig. 1(c), 2), and it has been shown that damage to PSII usually occurs at temperatures above 45 ℃ (Yamori 270 et al., 2014). Therefore, the collapse of N2 fixation at high temperature was not likely caused by the dysfunction of the photosystems, but might be caused by the uncoupling of adenosine triphosphate (ATP) synthesis to electron transport, since proton leakiness of the thylakoid membrane has been frequently proposed as a problem at high temperature . This is consistent with the observation that supra-optimal temperature inhibition of N2 fixation was aggravated by light limitation (Fig. 3). In addition, damage to nitrogenase at high temperatures might also be one of the reasons responsible 275 for the faster drop of N2 fixation at high temperature in light-limited cultures (Gallon et a., 1993). This is because the extra investment of resources in repair of damaged nitrogenase could not be supported under light-limiting conditions (Fig. 3(b)).
Therefore, light availability exerts critical control on the acclimation potential of N2 fixation in Trichodesmium to warming.
Acclimation to different temperatures also affected the activation energy (Ea) for N2 fixation in Trichodesmium IMS 101 (Table 3). For Trichodesmium species, N2 fixation can be controlled by supply of ATP/reducing equivalents, mainly coming 280 from photosynthesis, and the inherent catalytic capacity of the nitrogenase. These two processes may exhibit different temperature dependence, i.e. different Ea. The Ea of the controlling process determines the N2 fixation Ea (Hikosaka, et al,, 2006;Staal et al., 2003). Therefore, the differences in N2 fixation Ea between cultures grown at different temperatures may reflect that N2 fixation was primarily controlled by different processes in cultures acclimated to different temperatures.
Preliminary evidence supporting this hypothesis came from the various effects of assay light intensity on the values of Ea for 285 N2 fixation between light-limited cultures grown at 23 ℃ and 27 ℃ (Supplementary Table S1, Fig. S2). For Trichodesmium grown under limiting light level, the lower Ea values in cultures acclimated to 23 ℃ was significantly elevated by the increased assay light intensity which can provide more ATP/reducing equivalents (Supplementary Table S1; Fig. S2(a)).
This suggests the constraint should be the supply of ATP/reducing equivalents. The higher Ea values in cultures acclimated to 27 ℃ were insensitive to the assay light intensity changes, suggesting N2 fixation should not be controlled by the supply 290 of ATP/reducing equivalents at this optimal temperature, but may possibly be controlled by inherent catalytic capacity of the nitrogenase (Supplementary Table S1; Fig. S2(b)).
The short-term thermal responses for N2 fixation mirror thermal shock responses. If cells are exposed to the thermal changes for longer time, acclimation will definitely change the thermal responses for N2 fixation in Trichodesmium (Breitbarth et al., 2007;Fu et al., 2014;Staal et al., 2003). To compare the short-term and acclimated thermal responses for 295 N2 fixation, we calculated the corresponding values of Ea, Eh and Topt, being respectively 0.93 ± 0.64 eV(±S.E.M), 1.86 ± 1.19 eV(±S.E.M) and 27.1 ± 1.0 ℃(±S.E.M), for fully-acclimated N2 fixation within the range of 20-34 ℃ growth temperatures in Trichodesmium IMS 101 (Breitbarth et al., 2007). These values of Ea and Eh are comparable to those derived from short-term thermal response for N2 fixation in the same strain grown under light-saturating condition and 31 ℃ in our study (Table 3), but the Topt value is lower than that from short-term thermal response. On the other hand, we have tried to 300 derive values of Ea, Eh and Topt for acclimated N2 fixation rates in another three Trichodesmium erythraeum strains (strains RLI, KO4-20 and 21-75) (Fu et al., 2014), but the model fitting failed to converge. Instead of been negatively skewed, the thermal response curves of acclimated N2 fixation in these three Trichodesmium strains are nearly symmetrical. These comparisons show that thermal response for N2 fixation in Trichodesmium are strains-specific, and are affected on the time scale of acclimation process. 305 In the oceans, Trichodesmium and other pelagic phytoplankton are often exposed to abrupt temperature changes due to strongly disturbed weather conditions, such as tropical cyclones, and marine heat waves. Global warming has been predicted to increase both tropical cyclone intensities, and the frequency of the most intense tropical cyclones (Elsner et al. 2008;Knutson et al., 2010;Wehner et al., 2018). Upper ocean temperature declines prior and during cyclone event, and then increases abruptly afterwards (Li et al., 2009), accompanied by strong variations of surface solar radiation and stratification 310 (Sriver and Huber, 2007). These abrupt temperature changes occurring in nature are not as acute as those in our experiment.
For example, temperature changes caused by cyclone and heat waves are on the scale of 0.5 -1 ℃ per day (Babin et al., 2004;Beca-Carretero et al., 2018). Nonetheless, these temperature changes occur within one generation of Trichodesmium because of its low growth rate, leaving not enough time for full acclimation. Therefore, the values of Ea, Eh, Th and Topt provided in this study can likely serve as proxies for some types of abrupt natural temperature increases. 315

Code/Data availability
All data obtained in this study are in Supplement. Table 1 Results of two-way ANOVA for specific growth rate, N2 fixation rate, effective quantum yield and parameters derived from rETR light curve with interactions between "Temperature" and "Light". See Supplementary Table S2 Table 2 The light harvesting efficiency (α), relative election transport rate maximum (rETRmax) and light saturation point (Ek), derived from the light curves (Fig. 2)  Model parameters of thermal responses for N2 fixation. The structure of the fixed effect is: N(Tc) ~ Temperature * Light; Ea ~ Temperature; Eh ~ Light + Temperature; Th ~ Light + Temperature. "+" and "*" represent additive and 495 interactive effects, respectively.   conditions; values represent the means ± standard deviations of biological replicates(n=3); Solid lines illustrate the best fit to Eq. (4) with 95% confidence intervals as dashed lines.

Figure 4
The combined effects of temperature and light intensity on the specific growth rate in Trichodesmium IMS 101; data from published literatures involving at least two growth temperatures and this study. 525