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Effect of Nutrient, Light Intensity and Temperature on the Growth Rates and Metabolism of a Stress-Resistant Bacillariophyta Species – Entomoneis sp.-in Izmir Bay (Aegean Sea)

A unicellular marine microalga, Entomoneis sp. was isolated and studied as had become the dominant species compared to other bacillariophyta species in different environmental fluctuations in Izmir Bay. In effort to better understand the dynamics of this microalga that has achieved unprecedented domination, we conducted a monoculture isolation study. In this study, experiments were planned within the annual range of the Izmir Bay temperature, and the demonstrated behavior of the species in light and nutrient conditions. The stock culture medium was illuminated by approximately 50μmol photons m-2s-1 of illumination with 14/24 daylight. The temperature of the climate chamber was set at the summer (T1 (21±1oC)), spring (T2 (17±1oC)) and winter (T3 (13±1oC)) temperatures of Izmir Bay. Experiments were also conducted with four different light intensities (L1 (50 μmol photons m-2s-1), L2 (25 μmol photons m-2s-1), L3 (5 μmol photons m-2s-1) and L4 (dark)). In this context, nutrient measurements were made on samples of the exponential, stationary and death phase of the culture and nutrient analyses were carried out. The results, which were designed according to ceteris paribus assumptions, were adapted to Michaelis-Menten kinetics. Consequently, considering the lifetime of the diatom at different temperature conditions, T3 was determined as an optimum temperature. Maximum growth rate and process time were observed at this temperature. This explains why these diatoms are abundant available in the winter. Once the light intensity was increased, the growth rate was increased at the T1 and T2 temperatures. However, T3 had a high growth rate at nearly L1 light intensity. Considering the consumption and transformation of different nutrient conditions, different results for both types of microalgae were obtained.


Introduction
Microalgae, which are one of the first steps in the food chain of the marine ecosystem, are used to supply the energy and nutrients for aquatic organisms and play an important role in the biogeochemical processes. When the ecosystem is exposed to nutrient, temperature and light fluctuations, the overall efficiency of microalgae can have a dramatic effect on quality growth rates. Having a tremendous population amongst microalgae, diatoms have a significant role on the environmental area such as approximately 20-25% carbon fixation, increasing the marine primary productivity (40%) (Lebeau &Robert, 2003), nutrient cycling in ecosystems (Mendes et al., 2012), and aquaculture (Brindley et al., 2010 ). In addition, various biotechnological applications that consist of renewable energy (Cabello et al., 2015), high value molecules (Brindley et al., 2010), wastewater treatment (Abdelaziz et al., 2014) and ideal feedstock for biofuel production (Gao et al., 2015) can be conducted by mi-croalgae because of their high valuable content, high growth rate and easy cultivation (Cabello et al., 2015;Gao et al., 2015).
An autotrophic diatom Entomoneis sp. usually twist apically, lie in girdle view and appear bilobate, with a fibulate raphe system. It is usually seen as two lobes in the cell and moves in the direction of the lobes perpendicular to the place. It changes direction with the twisting of the cell flaps. During torsion of the cell flaps, the apical axis turns into a fine wave and movement can be observed at various angles. (Dalu et al., 2015;Paillès et al., 2014). According to taxonomic studies, there are 44 known Entomoneis sp in the seas all over the world and this number is increasing (Czarnecki & Reinke, 1982;Poulin & Cardinal, 1983;Osada & Kobayasi, 1985;Paillès et al., 2014;Dalu et al., 2015)). Entomoneis sp. have also been found in a wide variety of seas such as extreme saline waters, Guerrero Negro, Mexico, Marmara Sea sediment and the Adriatic Sea (Clavero & Grimalp, 1999;Paillès et al., 2014;Mucko et al., 2017).
The optimal culture conditions of microalgae such as temperature, salinity, pH, and nutrients can obtain both high photosynthetic efficiency and high growth rate, which is an economically and ecologically important factor. Indeed, it is beneficial for control of the cultivation system in large scale production and biotechnology (Brindley et al., 2010;Cabello et al. 2015). Breuer et al. (2013) submitted that optimal culture conditions could be different under nutrient depletion and repletion provision. Modelling the growth of microalgae using experimental design such as Response Surface Methodology (RSM) design is considered a well-established discipline to determine the nature of the response surface in an experimental region (Keskin Gündoğdu et al., 2016). Providing a large amount of information by using a small number of experiments and being efficient have an advantage for determining the complicated response function (Isleten-Hosoglu et al., 2012;Keskin Gündoğdu et al., 2016). Moreover, scientists have emphasized that experiments are important to identify the response of phytoplankton to predicted future scenarios in order to define the effects on wealth, biomass and resource utilization, detection of ecosystem sensitivity and nutrient carrying capacities (Adalioǧlu et al., 2013;Kutlu & Buyukisik, 2014;Sommer et al., 2012;Stefanidou et al., 2018).
Entomoneis sp., for which there is little information about their growth regimes, have been used for optimizing growth conditions using response surface methodology. The effect of temperature and light regimes has been well established under N and P depletion and repletion growth conditions. Internal nutrient limitation and different light intensities were developed and operated under different temperature conditions (13-17-21 o C) using previously isolated microalgae of Entomoneis sp. in synthetic seawater. Indeed, daily relative growth rates of Entomoneis sp. were calculated by chlorophyll a (chl-a) value.
The aim of this study was to explore the growth regime of Entomoneis sp. and optimization of different culture conditions. Although, Entomoneis sp. was not often observed in mixed culture, in most of the preliminary mesocosm experiments, it became dominant. A few studies have been carried out focused on the taxonomy of the Entomoneis sp., but there is no data itemperature and nutrient depletion/repletion conditions. Investigating behavior of such resistant species in different conditions will increase our understanding of interaction with the ecosystem. In addition, this microalga can be used as a source of biotechnological product because of its various metabolites and pigment content.
In this study, Box-Behnken design (BBD) and one factor design applications were used for optimization of Entomoneis sp. and for understanding their behavior in different culture conditions. In addition, the effect of the collective influence of three factors (temperature (T), light intensity (L) and N/P (nitrate/phosphate) was determined by using BBD and one-factor factorial design. In addition, this is the first study of obtaining the profile of an optimization of Entomoneis sp. culture.

Materials and Methods
The marine water samples were collected seasonally from August 2013 to December 2014 on the İnciraltı coast of Izmir Bay (lat.38,41 o N;long. 27,03 o E). During this period, the determination of resistant species and stock culture propagation studies were performed in mixed samples. Entomoneis sp. emerged as a resistant diatom species in each sample. The schematic diagram of the methodology is shown in Figure 1.

Strain Isolation and Identification
Water samples (minimum 60 L) taken regularly from the coast of İnciraltı were filtered through 50 and 20 μm meshes and the surface of the 20 μm mesh was examined with inverted and stereo microscopes. Entomoneis sp. samples were selected with a microscope, isolated by dilution method, and cultured. Then Entomoneis sp. was obtained in pure form by appropriate mixing and venting technique by feeding in sterile L1 media (Guillard & Hargraves, 1993).

Isolation of Entomoneis sp.
Samples of the mixed marine algal cultures were viewed morphologically in a stereo microscope for initial identification. Monoalgal culture of Entomoneis sp. was isolated by serial dilution technique in the Class II Laminar Flow Biosafety Cabinet with using NIKON SMZ25 that has high resolution and exceptionally bright specification.

Preparation of the Synthetic Seawater Medium and Stock Culture
The isolated culture from Izmir Bay was maintained in filtered and sterilized L1 medium which was suggested by Guillard & Hargraves (1993) with some modification of nitrogen and phosphate concentrations. Bioassay conditions were prepared under monoculture conditions and stock cultures of microalgae were inoculated into 300mL medium with 33±1 psu of artificial salinity in 500mL Erlenmeyer flasks. Then the microalgae stock cultures were incubated under a controlled temperature of 21±1 o C and illumination of 50μmol photons m -2 s -1 on the surface of the flasks and agitated by hand in a 10:14 dark:light regime. Sparging of the glass bottles was bubbled by filter-sterilized air (0.2 µm filter, Sartorius, Germany).

Experimental Design and Optimization
The experiments were carried out in two steps. In the first step, Entomoneis sp. was isolated and cultivated in different culture conditions. The operation parameters and growth of Entomoneis sp. were analyzed in this step. The second step included study of the roles of the Entomoneis sp. community in different nutrient concentrations and environmental factors in a lab-scale batch experiment. The parameters, including light intensity, nutrient removal and temperature were monitored in this experiment.
Design Expert software (version 7.0.0, Stat-Ease Inc., Minneapolis, MN) utilizes a 3-factor, 3-level design which is useful for investigating quadratic response surfaces and constructing second-order polynomial models. Important factors for achieving valid responses are selected using different designs. The process is usually followed by statistical analysis. One-factor design, which is made for each combination of the levels of each factor, is used for 1 numerical factor using 3 and 5 levels for a linear and quadratic model, respectively. In addition, it is a more powerful design due to reducing the error variance. Box-Behnken design (BBD) requires that factors are varied over three levels. Requiring fewer total runs may decrease the cost of experiments (Isleten-Hosoglu et al., 2012;Keskin Gündoğdu et al., 2016).
The second-order polynomial function is used for correlating the independent variables and foreseeing the optimum point and response. The Equation is made for three factors: Y=β 0 + β 1 X 1 + β 2 X 2 + β 3 X 3 + β 12 X 1 X 2 + β 13 X 1 X 3 + β 23 X 2 X 3 + β 11 X 1 2 + β 22 X 2 2 + β 33 X 3 2 where Y expresses the response variable, and X 1 , X 2 and X 3 are the coded levels of the independent variables. Also, b 0 is the model constant, b 1 , b 2 and b 3 are linear coefficients, b 12 , b 13 and b 23 are interaction effect coefficients, and b 11 , b 22 and b 33 are quadratic coefficients (interaction and quadratic terms). After the optimization experiments, the data were analyzed by one-way analysis of variance (ANOVA) using response surface methodology. Then Duncan's Multiple Range Test (DMRT) was used for a post-hoc test to measure specific differences between pairs of means with SPSS statistical software (SPSS for Windows ver.18.0). In the statistical analyses, results based on a confidence level of p ≤ 0.05 and 95% were evaluated as being statistically significant.

Preparation of the culture conditions
Stock cultures of Entomoneis sp. for inoculation were performed from an exponential growth phase (approx. 35,000 cells/mL). 125mL of this culture was diluted with sterilized artificial seawater to a final volume of 1000mL. Initial pH of batch cultivations was adjusted to 8.1-8.2 and was not controlled during the process. Changing of the illumination (L1, L2, L3, L4), temperature and nutrient concentration of the culture conditions is presented in Table 1.

Determination of the growth rates
Experiments were conducted to evaluate the effects of sectional changes of T, L and different N / P ratios on the growth rates of Entomoneis sp. in a synthetic seawater medium. During the process, Entomoneis sp. cultures were sustained in Climatic Room Laboratory conditions. Growth rates were drawn as different nutrient concentrations with in vivo chl-a analysis using a Turner Design Trilogy® Laboratory Fluorometer.
Daily growth rates µ (1/d) were calculated according to equation (1) The relationship between substrate, biomass and growth rate is typically defined by equations (2)

Chemical Analysis
In vivo chl-a was measured daily using a Turner Design Trilogy® Laboratory Fluorometer. The method is based on the measurement of chlorophyll fluorescence in living cells (Holm-Hansen et al., 1965). However, there was extractive chl-a analysis (in vitro) in each life phase according to standard chl-a methodology (Strickland & Parsons, 1972). Experiments were ended when the algal population had decreased to approximately 200 µg/L of in vivo chl-a. The cultures were checked for contamination with inverted and stereo microscopes.
Samples were taken for the dissolved inorganic nutrient in every phase of the bioassay. Samples were filtered through pre-combusted Whatman GF/F filters. All the different conditions of the experiments were collected in 100mL polyethylene bottles. Then the samples were kept in a deepfreeze (-20°C) until the chemical analysis. The colorimetric methods adopted were performed by T80 Plus UV/VIS Spectrophotometer.

Results
In the set of experiments, different methods of classification were applied with different temperature, light intensity and nutrient content. Various kinds of varieties and classification methods were made separately for the comparisons and installation reliability.
Within the scope of this study, microbial dynamics of Entomoneis sp. culture related to different nutrient and light intensity levels were investigated. Nitrogen and phosphorus are macro elements that can be consumed by Entomoneis sp. cultures or form compounds in the oxidation stages from chemical routes. In addition, these nutrients are necessary for living and for multiplying living cells in different oxidation levels.
Environmental factors such as nutrient concentration, light intensity, pH, temperature, and concentration of inoculation are parameters affecting nutrient uptake of microalgae. The inoculation values for the interaction of these parameters and for the optimization of environmental conditions of the Entomoneis sp. cultures were kept constant at about 35x10 3 cells/mL.
In vitro chl-a samples were extruded within 24 hours. Concentration values afterwards were determined by Parsons et al., (1984) using the T80 Plus UV/VIS Spectrophotometer. In the coming years, one way to predict the effect of these changes in environmental conditions on Entomoneis sp. culture structure, such as changes in chlorophyll, is to model development processes under the changing environmental conditions using the ecophysiological differences. Temperature and light intensity are some of the potential ecophysiological properties to be used in such a statistical model. These properties can relate growth rate, nutrient consumption and harvesting processes to each other and can help to observe biogeochemical effects (Aysel & Aysel, 2002;Sabancı & Koray, 2001).

Experimental Design and Optimization
Response surface methodology is an important statistical and mathematical techniques that is useful for developing, improving and optimizing a process (Keskin Gündoğdu et al., 2016). In this study, the Box-Behnken model for three variables (temperature, light intensity and N/P ratio), along with their minimum and maximum levels, was used as the experimental design model for optimization of growth of Entomoneis sp. In the experimental design model, temperature (13-21 o C), light intensity (0-50 μmol photons m -2 s -1 ) and N/P ratio (0-110) were taken as input variables. The statistical analysis of the chl-a model is shown in Table 2. Temperature, light intensity and N/P ratio were accomplished by means of analysis of variance (ANOVA). As shown in Table 2, the model with the F-value of 23.09 was significant (p<0.05). In terms of the ANOVA test, there was a significant interaction between temperature and light intensity on chl-a concentration of Entomoneis sp. (p<0.05).
The interaction effects of the light intensity and temperature on the chl-a concentration are shown with the 3-dimensional plot in Fig. 2. Light intensity is directly proportional to the chlorophyll concentration while N/P ratio is fixed at 11. The maximum chlorophyll a concentration was seen when light intensity (43.15 μmol photons m -2 s -1 ) and temperature value (13.7 o C) were at the maximum and minimum level in that order, while the N/P ratio was kept at the central level of 11. Optimization results also showed that the growth of Entomoneis sp. was inhibited by increasing temperature value. As shown in Fig. 2, when the intensity of light decreased and the temperature increased, the chlorophyll levels decreased in the culture.
Significant studies have been carried out on diatomic metabolism, mostly under the N restriction. Our trial results also showed that chlorophyll a concentration might be associated with seven different nitrate concentrations  as 0-1.1-4.4-11-27-110-247 N/P range applied to Entomoneis sp. (Collos et al., 1980;Geider et al., 1993;Lomas & Glibert, 2000). The effect of the proportions of inorganic nutrients has been studied in both a laboratory culture and an outdoor mesocosms. Most work on the subject focuses on changes in the inorganic N/P ratio. Growth rate optimization is shown in Fig. 3. Light intensity and temperature directly affected the growth rate and when the temperature was below and above 17 degrees, the growth rate was decreased. The maximum growth rate was seen when light intensity was up to 25 μmol photons m -2 s -1 and temperature value was approximately 17 o C, while the N/P ratio was kept at the low level of 4.4 and chl-a value was approximately 1002 µg/L. Optimization results also showed that the growth of Entomoneis sp. was not inhibited by high light intensity. In this study, N/P ratio was kept at 4.4 for the statistical experimental design, because some microalgae species have been found to be more adaptive to growing in nitrogen-limited cultures (Isleten-Hosoglu et al., 2012). This optimization results of μ is 0.41 day -1 at 14.6 o C and 40 μmol photons m -2 s -1 . If we increase the desirability of optimization the values are changed as seen in Fig. 4.
The interaction effects of the light intensity and temperature on the growth rate are shown with the 3-dimensional plot in Fig. 4. The maximum chlorophyll a concentration was seen when light intensity (43 μmol photons m -2 s -1 ) and temperature value (13.7 o C) were at the maximum and minimum level in that order, while the N/P ratio was kept at the central level of 27. Optimization result is clear where the growth rate is 0.396 day -1 and chl-a value is 1108 µg/L. The optimization results also showed that the growth of Entomoneis sp. was inhibited by increasing temperature value. As shown in Fig. 4, when the intensity of light increased, and the temperature decreased, the growth rate levels increased in the culture.
The interaction effects of the all the factors with two groups are determined and discussed for Entomoneis sp.
culture. As shown in Table 2 and post hoc test to measure specific differences between pairs of means, light intensity vs. temperature had a significant difference for both chlorophyll and growth rate responses. However, differences of N/P ratio vs. temperature and N/P ratio vs. light intensity had fewer mean differences than the corresponding Rp, which is the critical value for comparing differences in Duncan's Multiple Range Test. This means that the differences of N/P ratio were not significant with changing of temperature and light intensity, separately.

Determination of the growth rates
At the beginning of the experiment, the diversity of the experiment was achieved by considering the inorganic N/P ratio in the medium calculated on molar basis (Table 1).
Growth rates depending on the light intensity, temperature and N/P ratios are shown in Fig.5. All experiments were started to measure the growth rate under the light cycle of 14:10 L:D. In order to observe nutrient changes in the experiments, a blank medium was also prepared in which no nutrients were added. Also, a different concentration of dissolved inorganic nitrogen or phosphate was added on the first day. The effect of different temperatures, N/P ratio and light intensity on phases was observed in the growth curve formation in which we examined the variation of chlorophyll a in vivo with time. The growth curve shown in Fig. 5 gives information on how many days the culture will complete the lag phase in, and on what days it will be in the logarithmic phase and will pass to the death phase. According to the study, the increase of the in vivo chl-a approached saturation on the 25th-30th days.
When considering the amount of chl-a production seen as a control of cell viability and activity of different  nutrient conditions, Entomoneis sp. reached the maximum amount of average chlorophyll a concentration (1200±200 µg/L chl-a) at the temperature of 13 o C (T3) degrees and at the light intensity of L2. At the other T1 and T2 degrees, 700±50 and 800±100 µg/L chl-a were reached in L3 and L2 light intensity, respectively (Fig. 5).
A similar phenomenon was observed in N/P ratio of up to 11 experiments where the maximum chlorophyll was observed at L2 light intensity. A concentration of 1400 µg/L chl-a was obtained with 27.5 of N/P ratio at temperature T3 where the maximum amount of chlorophyll was obtained in L2 light intensity in both sets of N and P variations. This situation proves that these species are found more frequently in the winter months and that production increases due to temperature. At all temperatures, chl-a concentration of L3 light intensity is lower than other light intensities and the maximum amount of chl-a is achieved for longer periods. L4 light intensity did not make a significant difference for each temperature and gave the lowest amount of 250 µg/L chl-a and growth rate of 0.25 day -1 .
At the T3 temperature, Entomoneis sp. grew rapidly and reached its maximum growth rate. The growth rate of Entomoneis sp. was higher in the experiments where the temperature was T3 and the light intensity was L2. However, the longest exponential phases were determined at light intensity of L3. The L3 growth curves of Entomoneis sp. were similar under varying N/P ratios (Fig.5). There were no significant differences in growth rates of Entomoneis sp. cultures grown on combinations of nitrate and ammonia.
In the monod equation, in which all N/P values set at temperature T1 are considered, the maximum growth rate reached (µ max ) was calculated as 0.3 day -1 at L1 and L3, and 0.25 day -1 at L2 and L4 (Fig. 6). However, in experiments in which N/P values were greater than 11, half-saturation constant (K s ) ranged from 0.03 to 0.59, while in experiments in which N/P values were below 11, they ranged between 0.02 and 0.68. The K s sequence showed similarity in almost all experimental sets, decreasing as light intensity increased (K s_L2 <K s_L1 <K s_L3 <K s_L4 ).
At temperature T2, the µ max reached (0.5 day -1 ) was greater compared with the experimental sets at T1. At this temperature, growth rates were in the order of µ max_L1 >µ-max_L2 > µ max_L3 >µ max_L4 . In experiments having N/P values of 0-11, the K s sequence was calculated as K s_L1 <K s_ L2 <K s_L3 <K s_L4 . In cases where values were higher than  11, no significant difference in K s values was observed.
In the experiments conducted, at temperature T3, Entomoneis sp. reached the death phase at a later time by remaining at the stationary phase for longer. Particularly at L3 light intensity, cells remained in the exponential phase for a long period and did not progress to the death phase. In the monod equation, in experiments where N/P values were greater than 11, the highest µ max value that was determined at L3 light intensity was calculated as 0.72 day -1 , while K s reached 0.88 units. In the same experimental set, while no significant sequence was observed in µ max or K s values according to light intensity, the µ max value was observed in the L1 and L2 experimental sets when N/P values were 27.5 and in L3 when N/P value was 18.3. In all experiments, a decrease in growth rate was observed when N/P value was 11. In experiments where N/P values were lower than 11, the sequence of µ max values was µ max_ L3 >µ max_L2 > µ max_L1 >µ max_L4 . In the L1, L2 and L3 experimental sets, K s was calculated as 0.036, 0.0023 and 0.76 respectively. µ max values were around 0.3; 0.4-0.5 and 0.6-0.7 day -1 at T1, T2 and T3 temperatures respectively.
According to the experimental sets and the optimization studies made for these experimental sets, the values found when chl-a values were maximized matched the results of the experiments. While the maximum growth rate determined was 0.72 d -1 , the mean maximum growth rate for all experimental sets was calculated as 0.37 d -1 . In the study conducted by Sarthou et al. (2005) , in a light-filled environment and under nutrient-sufficient conditions, the maximum specific growth values obtained were calculated between 0.2 d -1 and 3.3 d -1 . In this optimization study, it was found that the determined growth rates also ranged between these values. In another similar study, variations in growth rate of Entomoneis sp. resulting from addition of bird droppings to its growth environment were examined (Jauffrais et al., 2015). Growth rate obtained with enrichment was measured as 0.9 day -1 . In this study, in which optimization was carried out according to seasonal variations in its own ecosystem, the growth rate of 0.72 day -1 is close to those data.

Discussion
Few studies are found in the literature obtaining the profile of a growth rate of Entomoneis sp. and its optimization study. What is actually found in the studies of many research groups are the analyses of their chemical content in freshwater microalgae and physical properties and focus on the marine mixed culture studies. The analyses were carried out by the inhibition of growth and the evaluation of changes in the photosynthetic pigments (Dudkowiak et al., 2011;Jauffrais et al., 2015;Satoh et al., 2005;Soares et al., 2012). Jauffrais et al. (2015) conducted a study on the Atlantic French coast in the Gulf of Burgas at the same time as our experiment. This study shows that coastal bird feces can significantly affect growth of diatom and biochemical composition by addition of dissolved matter. In media fortified with bird feces, the maximum growth rate reached at least 35% higher compared to our study.
The current results show that the growth rate of Entomoneis sp. is affected by temperature. Jauffrais et al. (2017) investigated the effect of bacteria on growth, biomass, elemental (C & N) and biochemical composition, and extracellular polymeric substances (EPS) excretion by two marine benthic diatoms, Halamphora coffeaeformis and Entomoneis paludosa. In the study, in which a growth curve for diatoms in environments containing and not containing antibiotics was created, batch cultures of Entomoneis sp. reached a maximum growth rate of 1.80 ± 0.10 day -1 at a temperature of 17°C, a salinity of 35 and a light:dark cycle of 14:10 in an antibiotic-free environment. Under the same conditions but in an environment in which antibiotics were applied, however, a maximum growth rate of 1.50 ± 0.10 day -1 was determined (Jauffrais et al., 2017). In untreated cultures, high bacterial abundance promoted cell division, increased growth rate and cellular abundance of E. paludosa. The 0.72 day -1 growth rate determined in the present study was found to be low when also compared with the data in the literature in terms of the likelihood of bacteria being present. Since the possibility of presence of bacteria was not focused on in the study, experiments were conducted via an Entomoneis sp. culture environment realized as a monoculture. According to these results, to ensure an increase in growth rate, ideas can be created for future studies on suitable bacterial combinations for Entomoneis sp. suited to interdependent life.
Furthermore, the effect of suitable temperature and light intensity on growth rate was observed. The minimum and maximum temperature and light intensity ranges created for the Entomoneis sp. monoculture isolated on the İnciraltı coast in the Aegean Region by considering the seasonal temperatures of the sea were applied for optimization specific to that region.
For algae to display their best performance, the correct ecosystem environment is needed and only in such a situation will growth rates be high. In this study, the optimum temperature was found for development of Entomoneis sp. and a laboratory model was created to determine how the seasonal transition temperature in Izmir Bay will affect the development of the diatom Entomoneis sp. diatom in the euphotic zone depending on the changing light intensity over depths.

Conclusions
Examining the different environmental conditions for the Entomoneis sp. culture isolated in İnciraltı, Izmir, the environmental optimization and research into the effect of the temperature changes and differences in light intensity in Izmir Bay on the growth, behavior and metabolic production of Entomoneis sp. have been carried out for the first time in this region. The direct effect of these independent variables was thus determined and subsequently used to make predictions about the response as growth rate and chl-a for given levels of each factor. This essay has focused on three factors as nutrient, light and temperature affecting the chl-a and growth rate of Entomoneis sp. on the İnciraltı coast of Izmir Bay. This is the first regional optimization of Entomoneis sp. for Izmir Bay. The major outcome of our experiments was the fact that Entomoneis sp., which is one of the most resistant to environmental stress among diatoms, showed a significant sensitivity to temperature. This study provides sufficient insights into achieving an increase in process growth rate, which could make the approach appealing to various biomass and pigment production and to the understanding of regional conditional differences for the monoculture study and optimization of condition for Entomoneis sp.