Growth, lipid, and pigment properties of locally isolated (Kastamonu,

Chlorella has become one of the most studied and produced microalgae, with Spirulina among the hundreds of species since the beginning of microalgal biotechnology. The growth performance of microalgae and the biochemical composition of the biomass may also vary significantly by strain. Therefore, it is thought that searching for new strains from aquatic environments is important in providing the most suitable microalgae for production. An isolated strain from Daday Stream was cultured in the laboratory at Kastamonu University. BG-11 was used as a medium, and CO2 from the air was used as a carbon source in the experiments. The initial cell number was arranged to 1.0×106 cells mL-1, and the highest cell number was found on the 17th day as 40.52×106 cells mL-1. Chlorophyll a and carotenoids were determined at the end of the experiment and were found as 3.48±0.08 µg mL-1 and 1.16±0.02 µg mL-1, respectively. Total lipid amount and fatty acid composition analysis were also conducted at the end of the study. According to the analyses, the lipid content of Chlorella sp. was found to be 15.37±0.00% (w/w). ∑SFA (saturated fatty acid), ∑MUFA (monounsaturated fatty acid), and ∑PUFA (polyunsaturated fatty acid) ratios were calculated to be 31.30±1.21%, 4.99±0.34% and 63.71±2.65%, respectively.


Introduction
Natural sources have decreased considerably since the increase in human population and industrial production.
Water resources provide an excellent habitat for microalgae growth and proliferation.However, studies on the taxonomy and diversity of these group of microorganisms are still limited.
Microalgae stand out with their large-scale producibility and usability in different industrial fields such as aquaculture, nutrition, pharmaceuticals, and even energy (Shah et al., 2018;Durmaz et al., 2020;Mehariya et al., 2021).
Chlorella has become one of the most studied and produced microalgae, with Spirulina among the hundreds of species since the beginning of microalgal biotechnology (Sugiharto, 2020).
Alteration of the biochemical composition of microalgae by culture conditions is already known for decades (Renaud et al., 1991;Ram et al., 2019).New studies are carried out every day for this purpose.Searching for more efficient production of microalgae to obtain target metabolites at higher concentrations causes these efforts.However, this is not the only way to achieve the desired production biochemical composition.The growth performance of microalgae and the biochemical composition of the biomass may also vary significantly by strain.Therefore, it is thought that searching for new strains from aquatic environments is crucial in providing the most suitable microalgae for production.This study was aimed to examine isolate and biochemical composition of new strain from the Daday Stream.

Material and Methods
The water sample was collected from the Daday Stream (41°27'18.4"N 33°42'15.2"E) in Kastamonu, Türkiye.The Autoclaved (121°C, 15 mins) falcon tubes were used for that purpose.First, the samples were inoculated into petri dishes on the same day.After several repeats of the inoculation process, an isolated microalgae strain was obtained.The strain was inoculated to the tubes and flasks to secure appropriate stock.
The culture growth performance and lipid content of the isolated strain were investigated and, for this purpose BG-11 was used as a medium, and cultures were mixed with air.0.2 µm syringe filters were used in aeration to avoid contamination.
The temperature was 21°C during the experiment period.

Growth
The experiment and the analysis were completed in triplicate.Cell numbers were counted under the light microscope daily.The specific growth rate (µ) was calculated according to the formula given in the Eq. ( 1): (X: cell number;

Pigments
Chlorophyll a and carotenoids were determined spectrophotometrically. 5 mL samples were centrifuged, and supernatants were discarded.After adding 5 mL methanol, samples were mixed by vortex and placed in ultrasound for 15 mins.Lastly, samples were centrifuged again, and supernatants were used to determine absorbance values.Pigment amounts were calculated using the formulas below (1-2).

Results
After the isolation process, the obtained strain was examined under the microscope.The size of the cells varied between 4.42-8.32µm, and the average cell size was calculated as 5.90±1.00µm.Cells were single and non-motile.Also, singlecell formation was observed without flagella.According to the visual identification, the strain was determined to be Chlorella sp.(Vuuren et al., 2006;Bellinger & Sigee, 2010).

Growth
Cell numbers were counted daily for 18 days of the experiment period.The initial cell number was arranged to 1.0×10 6 cells mL -1 ; the highest cell number was found on the 17th day as 40.52×10 6 cells mL -1 (Figure 2).The specific growth rate reached the highest point at the beginning of the experiment (4 th day) and was calculated as 1.08±0.06.When specific growth was turned negative at the 18 th day, the experiment was terminated.Dry weight analysis was conducted on the 18 th day of the experiment and found 2.47±0.15g L -1 .
According to the cell number of the day, cellular weight was calculated as 60.15±3.83pg cell -1 .

Pigments & Lipid
Chlorophyll a and carotenoids were determined at the end of the experiment and were found as 3.48±0.08µg mL -1 and 1.16±0.02µg mL -1 , respectively.Total lipid amount and fatty acid composition analysis were also made at the end of the study.According to the results, the lipid content of Chlorella sp.

Discussion
Chlorella is one of the most common species found in freshwater.As stated above, the current strain isolated from the Daday Stream was cultured under laboratory conditions.2.47±0.15g L -1 dry weight was obtained in this study with 60.15±3.83pg cell -1 cellular weight.The cell weight of Chlorella vulgaris is given as 6-18 pg cell -1 in the study on the effect of different culture media on growth (Chia et al., 2013).Even if it is stated that cellular weight is alterable by culture nutrient composition, Chlorella sp.(Daday Stream) cell weight is higher than that.In another study, Chlorella vulgaris culture with modified BG-11 provided 0.9±0.001g L -1 biomass after 12 days (Wong et al., 2017).Results of the study on 5 different Chlorella strains and their biomass production with 4 different culture media show that both the strain and the culture medium are effective in growth performance (Sharma et al., 2016).Since the dry weights varied between 0.9-1.7 g L -1 in the mentioned studies, it can be stated that the Chlorella sp.(Daday Stream) strain exhibited higher growth performance.
The lipid ratio and fatty acid composition of microalgae are also alterable by culture medium and strain.For instance, it is indicated that Chlorella sp. 1 strain's lipid accumulation varied between 14-16% depending on the medium.Also, lipid accumulation of another strain (Chlorella sp. 2) was given between 8-10% in the same study (Sharma et al., 2016).
Chlorella sp.T4 can accumulate 25.87% lipid, another strain from South Africa.This result is higher than the lipid ratio of Chlorella sp.(Daday sample).However, the T4 strain with 0.77 g L -1 biomass production is three times lower than Chlorella sp.(Daday sample), which leads to lower lipid production (Gumbi et al., 2022).

Conclusion
According to the result of this study, the locally isolated Chlorella sp.strain showed promising growth performance.
Lipid and pigment accumulation ratios were also acceptable if it was considered that any optimization or stress conditions were not applied.Further investigations may help to improve the growth and desired biochemical composition of the Chlorella sp.strain.

Compliance With Ethical Standards
also measured.Cell size measurement was conducted with ImageJ (National Institutes of Health, USA) software by using digital images of microalgae samples on a Neubauer hemocytometer (Figure1).

Figure 1 .
Figure 1.Chlorella sp.(Daday Stream) cell size on Neubauer hemocytometer Dry weights were determined at the end of the experiment.0.45 µ Whatman filters were weighed after drying at 105°C for 2 h.Then, 5 mL samples from each culture were filtered and dried at the same temperature until the weights became constant and were weighted.