The effect of various salinities and light intensities on the growth performance of five locally isolated microalgae [Amphidinium carterae, Nephroselmis sp., Tetraselmis sp. (var. red pappas), Aster- omonas gracilis and Dunaliella sp.] in laboratory batch cultures

After a 1.5 year screening survey in the lagoons of Western Greece in order to isolate and culture sturdy species of microalgae for aquaculture or other value added uses, as dictated primarily by a satisfactory potential for their mass-culture, five species emerged and their growth was monitored in laboratory conditions. Amphidinium carterae, Nephroselmis sp., Tetraselmis sp. (var. red pappas), Asteromonas gracilis and Dunaliella sp. were batch cultured using low (20 ppt), sea (40 ppt) and high salinity (50 or 60 or 100 ppt) and in combination with a low (2000 lux) and high (8000 lux) intensity of illumination. The results exhibited that all these species can be grown adequately in all salinities and with best growth in terms of maximum cell density, specific growth rate (SGR) and biomass yield (g dry weight/L) at high illumination (8000 lux). The five species examined exhibited different responses in the salinities used, Amphidinium clearly does best in 20 ppt far better than 40 ppt and even more than 50 ppt. Nephroselmis and Tetraselmis grow almost the same in 20 and 40 ppt and less well in 60 ppt. Asteromonas does best in 100 ppt although it can grow quite well in both 40 and 60 ppt. Dunaliella grows equally well in all salinities (20-40-60 ppt). Concerning productivity as maximum biomass yield at the end of the culture period, first rank is occupied by Nephroselmis with ~3.0 g d.w./L, followed by Tetraselmis (2.0 g/L), Dunaliella (1.58 g/L), Amphidinium (1.19 g/L) and Asteromonas (0.7 g/L) with all values recorded at high light (8000 lux).

From the five species of the present microalgae, three of them (Asteromonas gracilis, Nephroselmis sp. and Amphidinium carterae) has not been studied in a concise manner in laboratory cultures and only fragmented information can be detected on them. So our study aspires to pave the way for future attempts on more elaborate approaches. On the other hand Dunaliella sp. has been extensively studied in every possible way and our study aims to add information for this local strain. The case of Tetraselmis sp. (var. red pappas) is quite peculiar as on the one hand belongs to the family of Tetraselmis, a well studied one, but on the other, its unique feature of coloring red the culture medium suggests that is a novel strain of Tetraselmis with its own potential and worth studying.

Isolation and purification
After many monthly collections of water samples from various lagoons of W. Greece such as Messolonghi and its saltworks (prefecture of Etoloakarnania), the lagoon of Kalogria and Pappas (pref. of Achaia) and the lagoon of Kotyhi (pref. of Ilia), the samples were transported to the laboratory and 200 mL of each sample were put in glass 1-L conical Erlenmeyer flasks containing 800 mL of 40 ppt sterilized water enriched with Walne's nutrient formula [13]. The so called maintenance flasks were left for one week to mature, supplied through a 1-mL pipette with filtered air (~ 0.5 flask volume/min) fed by a central blower, exposed to continuous light of 3000 lux emitted by white light LED tubes, in a airconditioned room of 20-22 o C temperature. The maintenance flasks were left 2-3 weeks to develop microalgae population evident by coloration of the water and microscopic examination. Those with no sign of coloration after 3 weeks were discarded. By this practice we deliberately focused on algae species that can either solely or in companion with other species be fully adapted to seawater salinity and dominate the culture. After the confirmation of the well establishment of one or more species in the maintenance flasks, serial dilutions were performed in successive steps using 20-mL glass Erlenmeyer flasks filled with sterilized and fertilized as above water of the same salinity (40 ppt). The inoculated flasks were left to mature for 20 days in a special thermo-regulated chamber at 19 o C in low ambient continuous illumination of 1300 lux and daily mildly hand agitated. After 20 days they were examined microscopically and if a monoculture was observed the content of this flask was transferred in 500-mL flasks prepared with new fertilized medium and left to mature (20-22 o   The cultures lasted 15 days and from the 8 th day the drop of the growth rate and the "entry" in the static (stationary) phase of each culture became visible in all conditions (although in a different way in each). At the salinity of 20 ppt ( Figure 3-left) the evolution of the culture showed a much more intense increase in high light (8000 lux) compared to low light (2000 lux). In both lights there was a very short (2 days) initial phase of adaptation (lag phase) after which, especially in high light, the increase became strongly exponential (log phase). In the high light culture the final cell density (~6.5 x 10 6 cells/mL) was almost triple that of the low light (~2.3 x 10 6 cells/mL). The pH fluctuated in the alkaline region with values 8.3 -9.3 and from the beginning it showed higher values in the high light (a sign of more intense photosynthesis) and then (from the 11 th day), an abrupt synchronized drop (sign of aging) in both lights. At salinities of 40 and 50 ppt (Figure 3-middle & right) the increase of the growth curves and the final densities attained after an initial adaptation phase of 2 and 3 days respectively, were much lower compared to 20 ppt and the stationary phase was reached on the 10 th day (on 14 th day at 20 ppt). At the salinity of 40 ppt the final density in high light was ~3.1 x 10 6 cells/mL almost triple that of low light (~1.4 x 10 6 cells/mL). At the salinity of 50 ppt even lower values of final densities were recorded in both high (~2.0 x 10 6 cells/mL) and low light (~0.65 x 10 6 cells/mL). The pH at these salinities followed the same fluctuation pattern observed in 20 ppt with alkaline higher values (>9.0) in high light as compared to low light (<9.0) and near the end of the culture period a synchronized drop to almost identical lower values (8.0 -8.5). For the calculation of the specific growth rate (SGR) and the doubling or generation time (tg) the interval 3 -8 th day was chosen as it showed similarity in the upward trend of the growth curve in all salinities. There was a clearly higher growth rate in high light (0.295 -0.232 -0.302) for salinities 20 -40 -50 ppt respectively, compared to the values (0.189 -0.167 -0.143) for low light at the corresponding same salinities (Table 1). Statistically the values differed from each other except those of 20 and 50 ppt which in high light were statistically equal. As a reflection of the above growth rates, the generation times (tg) were shorter in the high light condition (2.35 -2.98 -2.29 days for the salinities of 20 -40 -50 ppt respectively) compared to the values (3.67 -4. 16 -4.85) for low light at the corresponding same salinities. • The period for the calculation of SGR and tg was from the 3 rd to the 8 th day.
• The different superscripts indicate a statistically significant difference at the 0.05 level of confidence (statistical processing with ANOVA and then pair-wise comparison with Tukey's test). Where there is a second superscript it means statistically equal to the value of the condition of the corresponding letter.     • The period for the calculation of SGR and tg was from the 4 th to the 8 th day.
• The different superscripts indicate a statistically significant difference at the 0.05 level of confidence (statistical processing with ANOVA and then pair-wise comparison with Tukey's test). Where there is a second superscript it means statistically equal to the value of the condition of the corresponding letter.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 November 2021
For the yield of the cultures (Figure 8) in biomass as dry weight per liter of culture (g/L), the values were calculated on the 17 th day. It is clearly shown and in agreement with the increase of the curves in Figure 7 that the salinities of 20 and 40 ppt resulted in much higher yield (2.88 and 2.99 g/L respectively) compared to the salinity of 60 ppt (1.61 g/L) at high light (8000 lux). In all salinities at low light the yields were much lower around 1 g/L and statistically equal.   Figure 11) and in all treatments after an initial delay-adaptation period of 2-4 days an intense and continuous exponential growth, much more pronounced in the high light, was observed. The only culture that entered the stationary phase was that of 60 ppt-high light which resulted in substantially lower final densities in both high and low light (5.8 x 10 6 and 2.2 x 10 6 cells/mL respectively) compared to 20 ppt (10 x 10 6 and 4.3 x 10 6 cells/mL respectively) and 40 ppt (9.6 x 10 6 and 3.3 x 10 6 cells/mL respectively).
Nevertheless the specific growth rate (SGR) calculated for the period from the 3 nd till the 9 th day for all treatments, did not exhibit a huge difference between all salinities at high light (Table 3)    • The period for the calculation of SGR and tg was from the 3 rd to the 9 th day.
• Number of measurements = 18.   (Figure 13). The case of the chlorophyte Asteromonas gracilis is very special as its natural habitat is exclusively ultra-salty areas in many parts of the Earth. It is a species that can, prefers, and grows in salinities much higher than seawater. In our study we isolated it from the saltern basins of the Messolonghi lagoon and it is easily maintained at a salinity of about 100 ppt [11]. From the literature [12] and from preliminary tests it was found that this species does not grow at all in salinities around 20 ppt, so our cultures were formed at 40, 60 and 100 ppt.  The specific growth rate (SGR) calculated for the period from the 3 nd till the 9 th day based on the findings of the growth curves of Figure 15 in order to obtain values containing on the one hand a part of the initial lag phase (3 rd day) and on the other a representative day of the exponentially (log) phase around its middle period (9 th day). The highest growth rate of 0.280 doublings/day was recorded in 100 ppt-XL and in 60 ppt-XL (0.273) which were statistically equal ( Table   4)   • The period for the calculation of SGR and tg was from the 3 rd to the 9 th day.
• The different superscripts indicate a statistically significant difference at the 0.05 level of confidence (statistical processing with ANOVA and then pair-wise comparison with Tukey's test). Where there is a second superscript it means statistically equal to the value of the condition of the corresponding letter.
The yield in g dry weight/L was measured at the final day of the culture period (17 th day) and from the very first glance becomes evident that the high light condition resulted in much more production of biomass compared to the low light counterpart in each salinity. The maximum value (0.70 g/L) was recorded in the treatment of 100 ppt-XL and the lowest     In all treatments ( Figure 19) the lag phase was very short (2-3 days) after which a sharp elevation of the growth curve characterized the log phase especially in the high light of the 40 and 60 ppt salinities in contrast to the low light curves where the transition to the log phase was long and smooth.  • The period for the calculation of SGR and tg was from the 3 rd to the 8 th day.
• The different superscripts indicate a statistically significant difference at the 0.05 level of confidence (statistical processing with ANOVA and then pair-wise comparison with Tukey's test). Where there is a second superscript it means statistically equal to the value of the condition of the corresponding letter.
The specific growth rate (SGR) calculated for the period from the 3 nd till the 8 th day was based on the uniformity of the shape of the growth curves of all treatments in Figure 19. The highest growth rate of 0.405 doublings/day was recorded in 60 ppt-XL and in 40 ppt-XL (0.387) which were statistically equal ( Table 5)

Discussion
A considerable bulk of data exists in the literature that concern the effect of various manipulation of physicochemical parameters on the growth and biochemical output of microalgae cultures. Two major drawbacks are recognizable on reviewing this issue in the literature. First, the immense variation in the tools (e.g. vessels) and techniques (e.g. batch culture or continuous, nutrients, light, temperature, etc) used. Second, the fragmented information that the results of each work transport to the reader. An example of this is the calculation of specific growth rate (SGR) which in the majority of papers is presented with no reference of the exact culture time on which was calculated upon. This can ensue in great discrepancies of the calculated values and comparisons are greatly hindered. In our study we calculated SGR from around the end of lag (stationary) phase till the middle of log (exponential) phase. Have it be otherwise (i.e. from the well advanced log phase) our values would have been much higher. This has the meaning of a more realistic approach each particular tested species presents in reality. It makes little sense to record SGR only during the short log phase whilst a long lag phase has preceded it, e.g. [17].
It is logical and essential to rank the priorities on the path to investigate and know well every species needs and potentials in culture terms. With no doubt the first priority is to investigate the maximum possible biomass that can be produced under economically feasible conditions. For this purpose a kind of default set of parameters should be implemented in order to be considered as a starting point. In the present paper an ordinary indoor temperature of 20-21 o C, a classic nutrient medium (Walne), a sufficient range of illumination (2000-8000 lux), bubble aeration with no addition of CO2, not too small not too big vessels of 1-2 L and non manipulated pH were chosen for our purpose which was to investigate the evolution of the growth curve, the growth rate and the dry weight yield of each species in salinities of brackish, sea, and hypersaline waters. Only after gathering results from experimentation with similar to the above mentioned conditions the procedures for further manipulations in order to achieve maximum biochemical products (e.g. pigments, lipids, etc) can have meaning. It is of little usefulness to find that a certain species produces a lot of a valuable substances in conditions that otherwise result in minimum biomass from a very small growth rate and a very long culture period.
Of the 5 species of microalgae cultured in the present paper only one in terms of Genus (Dunaliella sp.) is represented quite well in the relevant literature followed by Tetraselmis. Amphidinium has only limitedly been studied and even less Nephroselmis. Asteromonas gracilis is almost neglected at all although its importance as feed for the rotifer Brachionus plicatilis is well documented [18,19] and presents also some other advantageous features [15].

Amphidinium carterae
The interest for culture of this species and in general of the dinoflagellates is based on their capacity to produce bioactive metabolites (amphidinolides and amphidinols) and possibly biofuels although there are concerns about their ability to withstand shear stress caused by turbulence in photobioreactors [20][21][22][23]. The chlorophyte Nephroselmis sp. has drawn interest due to its potential for production of lipids, carotenoids and various antioxidants [27][28][29] and as an excellent feed for Artemia [30]. Its documented mixotrophic ability [29] is an advantage for its mass culture as it can overcome occasionally shortage of nutrients and even benefit from the bacterial load usually present in old cultures. Because of its special ability for mixotrophy the cultures of Nephroselmis almost never collapse the viable range for microalgae [31], it is logical to expect even higher yields if we culture Nephroselmis using higher temperatures (e.g. 25-30 o C) than 20-21.5 o C used in the present study. A lot of future research is needed for the culture of Nephroselmis concerning the effect of many factors on its growth characteristics for establishing a reliable and trustworthy culture protocol.

Tetraselmis sp. (var. red pappas)
This strain of the species Tetraselmis sp. was given the arbitrary name "var. red pappas" due to its originating place of the lagoon pappas in W. Greece. It is a chlorophyte that differs from other species of genus Tetraselmis in its peculiar and astonishing characteristic of imparting a dark-red coloration to its culture medium ( Figure 10) in conditions of illumination of 8000 lux when it reaches the stationary phase, as happened in our culture. This unique phenomenon has never been reported in the literature and we cannot give a plausible explanation other than it should be due to the accumulation of extracellular substances excreted by the senescent cells. Definitely cannot be attributed to carotenoids as the absorbance spectrum of the devoid of cells supernatant of centrifuged culture samples did not present any peaks corresponding to any kind of pigments, being almost absolutely zero-valued and flat along the range of 350-750 nm. So we assume that a naturally excretion of organic matter by its intact or lysed cells can be the cause for this phenomenon as such excretions are known to occur in microalgae in various intensities depending on species, conditions, and phase of the culture [32][33][34][35][36][37]. As the red coloration occurs only late in the stationary phase it is probable to be due to high molecular substances (carbohydrates or humic species from decomposition of cells) [36,38,39] than to low molecular ones (peptides or small proteins) that are known to be excreted during the exponential phase of the culture [32,38,39].
In the literature there are various studies on culture of several species of the genus Tetraselmis (Tetraselmis sp., T. suecica, T. chui) but they vary extremely in their conditions used and the output data. What seems from all of them to be in accordance with our data, is that Tetraselmis is growing best at salinities of 35 ppt or lower and that the high illumination is more productive [40][41][42][43][44][45][46][47]. This was confirmed in our study as the maximum density of Tetraselmis reached the level of ~10 x 10 6 cells/mL in the salinities of 20 and 40 ppt and high illumination (8000 lux), far higher than the corresponding one of 60 ppt (~6 x 10 6 cells/mL) and even higher from their counterparts of low (2000 lux) illumination (~4.1 x 10 6 , 3.2 x 10 6 and 2.1 x 10 6 cells/mL at salinities 20, 40 and 60 ppt respectively). Based on our experience of many years in culturing Tetraselmis suecica and now the present Tetraselmis sp. (var. red pappas), we never experienced densities over 10 x 10 6 cells/mL. In this respect the value of 35 x 10 6 cells/mL in [45] using low intensity light of 1588 lux is in our opinion questionable. We consider also as questionable the notation of [43] that their culture of T. suecica entered the stationary phase on the 4 th day and presented a growth rate of 0.8 yielding biomass dry weight of 0.57 g/L as in our cultures, Tetraselmis for 17 days kept its exponential phase at salinities of 20 and 40 ppt and entered the stationary phase on day 14 and only at salinity of 60 ppt. Our higher yield in biomass of about 2.0 g d.w./L is supporting the overall published merits of genus Tetraselmis as an ideal species for aquaculture feed [48], candidate for biodiesel production [49] and easy to manipulate due mainly to its high tolerance to extreme salinities [50,51].

Asteromonas gracilis
This extremely halotolerant (in respect to higher than seawater salinities) chlorophyte [15,16,52] which has been proven as a suitable live food for rotifers in marine fish hatcheries [18,19] and a candidate for biofuel production [53] has drawn little attention for mass culture. To the best of our knowledge our study is the first one focused on the basics of its growth in batch culture in order to be considered as a starting point for future more elaborated studies. Compared to other cultured microalgae, it is the biggest in cell size (18-22 μm) and because of this its maximum density is around 6.5 x 10 5 cells/mL (Figure 15-right) which was attained at the highest (100 ppt) of salinities tested and in high illumination (8000 lux). The salinity of 100 ppt in high light presented also the highest yield of 0.7 g/L as compared to 40 and 60 ppt (both ~0.5 g/L). All these values are higher from the only reported in the literature [53] of ~0.4 g/L in the culture of which he conducted the experiment in higher temperature (25 o C, ours 20-21.5 o C) but with weaker illumination (2500 lux, ours 8000 lux for the above mentioned values) and recorded the end of exponential (log) phase on day 9 while in our culture even on day 17 the exponential (log) phase had not been ended. We feel that our results apart from corroborating (at least in part) the findings of [53], must be considered as a more realistic approach due to the meticulous planning of our experimentation that had the sole purpose to study the parameters of growth of A. gracilis. Definitely we can conclude that this species grows best at salinities over 60 ppt and apart from its highest cell density and yield attained, an indirect proof of it is the shortest lag phase in 100 ppt (3 days) as compared to 5 and 8 days recorded in 60 and 40 ppt respectively.
From all the above however, the opinion that A. gracilis cannot be cultured in lower than 100 ppt salinities must be discarded because as it is shown in Figure 15 (left and middle) grows well also in both 40 and 60 ppt.

Dunaliella sp.
Species of the genus Dunaliella have been the subject of numerous studies over the years as this chlorophyte is notorious for its high salt tolerance by producing excessive amounts of glycerol and carotenoids [54,55] and from long ago has found a positive response by aquaculturists aiming to the production of value added products [56,57]. Being so, it comes of no surprise to meet in the literature highly variable data concerning its growth performance in terms of density of culture (cells/mL), growth rate and biomass yield as a consequence of the various volumes and conditions used in each particular experimentation. In the present study our batch culture was an experiment using medium volumes (2L), average easily attainable temperature (20-21.

Conclusions
In all the five species cultured, two things were common to all and deserve special attention. First, that growth was far