Modified Natural Seawater as Growth Medium for Halotolerant Cyanobacterium Aphanothece halophytica to Increase Lipid Content for Biodiesel Production

Modified Natural Seawater as Growth Medium for Halotolerant Cyanobacterium Aphanothece halophytica to Increase Lipid Content for Biodiesel Production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Modified Natural Seawater as Growth Medium for Halotolerant Cyanobacterium Aphanothece halophytica to Increase Lipid Content for Biodiesel Production Sitthichai Thongtha, Kornkanok Aryusuk, Chokchai Kittiwongwattana, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4646793/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Biodiesel derived from cyanobacterial oils becomes attractive as an efficient renewable energy. The present study aims to optimize growth and lipid production of halotolerant unicellular cyanobacterium Aphanothece halophytica cultivated in natural seawater. In this study, A . halophytica was able to grow in natural seawater when supplemented with low concentration of NaNO 3 , whereas no growth occurred without supplementation. The specific growth rate of 0.230 day − 1 and cell concentration of 25.17 x 10 6 cells mL − 1 were achieved in A. halophytica cultivated in natural seawater supplemented with 17.6 mM NaNO 3 and Turk Island salt solution (suitable natural seawater; SNSW) for 14 days. This growth rate was comparable to that of cells grown in normal BG11 plus Turk Island salt solution. The lipid content and fatty acid profiles of A. halophytica varied with changes in NaCl concentrations. The highest lipid content of 50.47% and lipid productivity of 48.33 mg L − 1 day − 1 were obtained in cultures supplemented with 1.89 mmol C-atom L − 1 glucose and 0.75 M NaCl. The optimal medium pH and cultivation temperature for lipid production was 7.5 and 25–35°C, respectively. When cultivating A. halophytica in optimized SNSW with various NaCl concentrations, the highest contents of linoleic and linolenic acids, and the lowest contents of palmitic, stearic, and oleic acids were observed with 0.75 M NaCl. In contrast, cultures grown in optimized SNSW with 0.5 M NaCl showed fatty acid methyl ester profiles rich in monounsaturated fatty acids, which are favorable for high-quality biodiesel production. Growth Lipid production Biodiesel Cyanobacteria Seawater Introduction Global energy demand and consumption have been rising because of the increasing human population (OECD 2011 ). Fossil fuels including coal, natural gas, and petroleum, etc., are presently one of the major sources of energy production. However, fossil fuels are non-renewable, and their production is projected to peak in 2025 and decline thereafter (Mohr et al. 2015 ). Moreover, energy production from fossil fuels results in CO 2 and pollutants emission into the atmosphere. This contributes to and exacerbates the global warming situation, leading to increased severity of climate change (Matthews and Wynes 2022 ). Consequently, research into various kinds of alternative renewable energy has been gaining more attention as means to alleviate the situation. Biodiesel is one of the promising renewable energies that has become more attractive to replace fossil fuels. It is produced by the transesterification or esterification reaction of lipids containing triglycerides and free fatty acids with alcohol (Mandari and Devarai 2022 ). However, there are some factors that limit the use of plant-based biodiesel, including the high requirements of nutrients and land. For these reasons, efforts have been made to utilize biodiesel derived from cyanobacteria which require lower nutritional inputs (Bolatkhan et al. 2020 ) and occupy a smaller land area. Additionally, their high photosynthetic activity results in rapid biomass and lipid accumulation, alleviating the impact of CO 2 emission on global warming (Sadvakasova et al. 2021 ). However, lipid content varies among different cyanobacterial species (Yadav et al. 2021 ). Optimization of growth conditions was previously reported for its positive effects on lipid production by cyanobacteria. These included nutrient compositions, salinity, medium pH and incubation temperature (Cordeiro et al. 2017 ; Miriam et al. 2017 ; Nalley et al. 2018 ; Yalcin 2020 ). Thus, optimization of growth conditions could potentially lead to a reduction in the cost of biodiesel production, facilitating its extensive use in the future. Salinity level is another important factor for lipid production in cyanobacteria. However, its effects may be different, depending upon cyanobacterial species. Growth and lipid content of Hapalosiphon sp. increased as the NaCl concentration rose from 0 to 10 ppt, whereas higher NaCl concentrations only stimulated growth but not lipid yield (Ruangsomboon 2014 ). In contrast, a continuous increase in lipid production was observed in Synechococcus sp. PCC7942 cultivated in medium containing NaCl ranging from 10 to 500 mM (Verma et al. 2019 ). This demonstrates the significance of the investigation of NaCl effect on growth and lipid production in particular cyanobacterial species. The halotolerant unicellular cyanobacterium Aphanothece halophytica , originally isolated from the Solar Lake (Israel), could tolerate high salinity up to 3 M NaCl (Ishitani et al. 1993 ). A previous study showed that A. halophytica provided the highest cell density of 21.6 ± 0.17 x 10 6 cells mL − 1 and accumulated lipid content at 29 ± 0.1% of dry weight, when cultivated in seaweed extract and NPK medium (Miriam et al. 2017 ). Besides lipid production, A. halophytica showed a potential as a H 2 producer when cultivated in nitrogen-deprived BG11 medium supplemented with Turk Island salt solution as well as in natural seawater (Taikhao et al. 2013 ; Taikhao et al. 2015 ). However, the effect of various NaCl concentrations on the lipid production of A. halophytica is not yet known. The present study aimed to investigate the growth and biomass production of A. halophytica cultivated in natural seawater under various NaNO 3 concentrations and in the presence or absence of Turk Island salt solution. Factors affecting lipid production, including carbon sources and concentrations, initial medium pH, and incubation temperature, were investigated. The effect of various NaCl concentrations was also tested and optimized. Additionally, biodiesel properties were determined based on the fatty acid methyl ester profile of lipid extracted from A. halophytica grown under various NaCl concentrations. Materials and methods Cyanobacterial cultivation The unicellular halotolerant cyanobacterium A. halophytica was maintained by cultivation on BG11 agar (pH 7.4) (Rippka et al. 1979 ) supplemented with Turk Island salt solution (Garlick et al. 1977 ) at 30°C under a white-light illumination of 30 µmol photons m − 2 s − 1 . In this experiment, axenic cells of A. halophytica were grown in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with various NaNO 3 concentrations (0.176–17.6 mM). BG11 contains NaNO 3 at a final concentration of 17.6 mM. An initial cell concentration of cyanobacterial culture was adjusted to the optical density of about 0.1 at 730 nm. The culture was shaken at 120 rpm under a white-light intensity of 30 µmol photons m − 2 s − 1 (for 18 h day − 1 ) at 30°C for 14 days. Natural seawater used in this study was collected from Nang Rum Beach, Chonburi Province, Gulf of Thailand, Thailand (12 ° 36.969’ N, 100 ° 55.280’E). The seawater was filtered through a glass microfiber filter GF/F (0.7µm) (Whatman, UK) and adjusted to pH 7.5 with 2 N NaOH prior to sterilization by autoclaving. A. halophytica cultured in BG11 supplemented with Turk Island salt solution was used as a control in all experiments. Growth determination by chlorophyll a content and total cell concentration measurements Growth of A. halophytica was determined by measuring chlorophyll a content and total cell concentration. For chlorophyll a concentration measurement, cells were collected by centrifugation of one mL of cell culture at 7,000×g at 4 0 C for 10 min. Chlorophyll a was extracted from the cell pellet by adding 1 mL of 90% (v/v) methanol and subsequently incubating at room temperature under darkness for 1 h. The chlorophyll a concentration of the extract was determined by measuring the absorbance at 665 nm and calculated according to Mackinney ( 1941 ). Cell concentration was determined based on the direct cell count method using Neubauer hemocytometer (Boeco, Germany) under a light microscope (Olympus CH30RF200, Japan). The specific growth rate (µ) was calculated according to Eq. 1 (Tang et al. 2011 ). Specific growth rate (µ) (day − 1 ) \(=\frac{\text{ln}\left({x}_{1}\right)-\text{ln}\left({x}_{0}\right)}{{t}_{1}-{t}_{0}}\) ……..Eq. 1 x 1 and x 0 are cell numbers at the end and the beginning of the exponential phase, respectively, whereas t 1 and t 0 are durations at the end and the beginning of the exponential phase, respectively. The doubling time required for the population cells to double its cell number was calculated according to Eq. 2 (Guillard 1973 ). Doubling time (day) = \(\frac{\text{ln}2}{\mu }\) ……..Eq. 2 Effect of nitrogen concentration and trace elements in culture medium on growth A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater containing 0, 0.176, 1.76 and 17.6 mM NaNO 3 . Cells were shaken at 120 rpm under a white-light intensity of 30 µmol photons m − 2 s − 1 (for 18 h day − 1 ) at 30°C for 14 days. Chlorophyll a concentration and total cell concentration were measured every 2 days. To increase growth of A. halophytica , Turk Island salt solution containing 0.5 M NaCl, 49 mM MgCl 2 .6H 2 O, 30 mM MgSO 4 .7H 2 O and 8.9 mM KCl (Garlick et al. 1977 ) was added in natural seawater media. Total lipid extraction A. halophytica cells cultivated in various types of media for 14 days were harvested by centrifugation at 7,000×g at 4°C for 10 min. Cells were dried in a hot air oven at 60°C for 24 h. Lipid extraction from dried cyanobacterial cells was performed by the single-step lipid extraction method (Axelsson and Gentili 2014 ). Briefly, 20–30 mg of A. halophytica dry cells were added with 8 mL of chloroform and methanol (2:1, v/v). The mixture was shaken vigorously. Two mL of 0.73% (w/v) NaCl was added in the mixture. The sample was mixed and centrifuged at 7,000×g at room temperature for 5 min. The chloroform layer at the bottom of the tube containing the crude lipid extract was then separated and put into a new tube. Lipid extraction was performed repeatedly five times. The chloroform layer in the lipid crude extract was collected and evaporated using a vacuum evaporator and the crude lipid extract was then weighed. The lipid content was calculated according to Sivaramakrishnan and Incharoensakdi ( 2017 ) and expressed as percentage of lipid per dry cell weight. Lipid productivity was expressed as a unit of extracted lipid weight per culture volume per day. Dry biomass of cyanobacterial cells was obtained from a centrifugation of cell culture at 7,000×g at 4°C for 10 min before drying the cell pellet in a hot air oven at 60°C for 48 h. Effects of carbon source and concentration on lipid content and lipid productivity A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO 3 and Turk Island salt solution. Various carbon sources, i.e., glucose, fructose, sucrose, and lactose, were separately added in the medium at a final concentration of 0.189 mmol C-atom L − 1 which equals to Na 2 CO 3 concentration in BG11. A. halophytica cells were cultivated under previously described conditions for 14 days. Cells were subsequently harvested by centrifugation and total crude lipid was extracted by the previously described protocol. To investigate the optimal concentration of the selected carbon source, different concentrations of the selected carbon source were varied at 0.189, 1.89, 18.9, 189, 379 and 756 mmol C-atom L − 1 . Effects of NaCl concentration on lipid content and lipid productivity A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO 3 and Turk Island salt solution. NaCl was added to the medium to reach the final external concentrations at 0, 0.25, 0.5, 0.75, 1, 2, and 3 M. Cells were grown under previously described conditions. After 14 days of cultivation, cells were harvested by centrifugation and total crude lipid was extracted. Effects of initial pH and temperature on lipid content and lipid productivity A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO 3 , Turk Island salt solution and 0.75 M NaCl. To investigate the effect of initial pH on lipid production, pH of the medium was adjusted to 6, 6.5, 7, 7.5, 8 and 8.5, using HCl and NaOH. Cultivation temperature was tested at 20, 25, 30, 35, and 40°C. After 14 days of cultivation, cells were harvested by centrifugation and total crude lipid was extracted, as described above. Fatty acid profile analysis The composition of fatty acid profile was analyzed at the Lipid Technology Research Laboratory, Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (Bangkhuntien) (Bangkok, Thailand). The crude lipid extracts were transesterified by the protocol modified from Lepage and Roy ( 1984 ). Lipid extract (at least 10 mg) was added with 5% (v/v) HCl in absolute methanol prior to incubation at 85°C for 1 h. Fatty acid methyl esters were analyzed using a gas chromatograph equipped with a flame ionization detector (Agilent 6850, Mulgrave, Australia). Heptadecanoic acid was used as an internal standard. The GC condition was as described in the previous study (Pojjanapornpun et al. 2018 ). The injector and detector temperatures were set at 250°C. One µL of sample was injected in the split mode using a ratio of 500:1. H 2 gas (99.999%) was used as carrier gas at a constant flow rate of 1.5 mL min − 1 . Biodiesel properties of fatty acid methyl ester Several biodiesel properties, i.e., percentage content of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA), degree of unsaturation (DU), saponification value (SV), iodine value (IV), cetane number (CN), long chain saturated factor (LCSF), cold filter plugging point (CFPP), cloud point (CP), pour point (PP), allylic position equivalent (APE), bis-allylic position equivalent (BAPE), oxidation stability (OS), higher heating value (HHV), kinematic viscosity (υ), and density (ρ) were determined based on the fatty acid composition of the obtained methyl ester, by the Biodiesel analyzer software ver. 2.2 (Talebi et al. 2014 ). Statistical analysis All experiments were performed in triplicates. Average values of lipid content and lipid productivity and the standard deviations were presented. The statistical analysis of differences was performed using one-way analysis of variance (ANOVA) with Duncan’s multiple range test at the 95% confidence level. All data were analyzed by a software of IBM SPSS statistics 23 (IBM Corp, USA). Results Growth of A. halophytica cultivated in natural seawater containing various NaNO 3 concentrations with and without supplementation of Turk Island salt solution Growth of A. halophytica based on total cell concentration was observed in natural seawater supplemented with 0.176–17.6 mM NaNO 3 ; however, the highest growth was observed in the control medium followed by that in the seawater supplemented with 17.6 mM NaNO 3 (Fig. S1 A). Supplementation of lower NaNO 3 concentrations decreased the growth, whereas no growth was observed in natural seawater without NaNO 3 supplementation. A similar result was observed with the chlorophyll a content (Fig. S1 B). These results indicated the requirement of NaNO 3 on the cultivation of A. halophytica in natural seawater. Additionally, trace elements available in the Turk Island salt solution further enhanced the growth of A. halophytica cultivated in natural seawater containing various NaNO 3 concentrations (Fig. S2). A. halophytica displayed the highest cell concentration and chlorophyll a content when cultivated in natural seawater supplemented with 17.6 mM NaNO 3 and Turk Island salt solution. The growth under this condition was comparable to that of A. halophytica cells grown in BG11 medium supplemented with Turk Island salt solution. Table 1 shows the cell density at day 14th of cultivation, specific growth rate and doubling time of A. halophytica grown in different media. Supplementation of natural seawater with 17.6 mM NaNO 3 and Turk Island salt solution yielded the highest specific growth rate with 0.230 ± 0.002 day − 1 and the shortest doubling time with 3.01 ± 0.03 days (Table 1 ). They were not significantly different from those of cells grown in enriched BG11 supplemented with Turk Island salt solution (Table 1 ). In contrast, the use of natural seawater alone resulted in the lowest specific growth rate with 0.016 ± 0.001 day − 1 and the longest doubling time with 43.10 ± 0.32 days. The addition of Turk Island salt solution significantly increased the specific growth rate compared to those in the absence of the solution at the same NaNO 3 concentration. Therefore, natural seawater supplemented with 17.6 mM NaNO 3 and Turk Island salt solution was chosen as the suitable medium for cultivation of A. halophytica in natural seawater and named as suitable natural seawater (SNSW). Table 1 Effect of various NaNO 3 concentrations and Turk Island salt solution supplementation in natural seawater on cell density at day14th of cultivation, specific growth rate and doubling time of A. halophytica. BG11 supplemented with Turk Island salt solution was used as a normal control medium. Data were from three independent experiments and expressed as mean ± SD. Type of medium Cell density Specific growth rate (µ) (day − 1 ) Doubling time (×10 6 cells mL − 1 ) at day14th (days) SW + 0 mM NaNO 3 1.25 ± 0.01 f 0.016 ± 0.001 g 43.10 ± 0.32 d SW + 0.176 mM NaNO 3 11.89 ± 1.44 e 0.176 ± 0.009 e 3.94 ± 0.21 b SW + 1.76 mM NaNO 3 13.92 ± 1.10 d 0.188 ± 0.006 d 3.69 ± 0.12 ab SW + 17.6 mM NaNO 3 21.83 ± 0.34 b 0.220 ± 0.001 b 3.15 ± 0.02 ab SW + 0 mM NaNO 3 + Turk 1.63 ± 0.03 f 0.034 ± 0.006 f 20.05 ± 3.81 c SW + 0.176 mM NaNO 3 + Turk 17.08 ± 0.34 c 0.203 ± 0.001 c 3.42 ± 0.02 ab SW + 1.76 mM NaNO 3 + Turk 21.92 ± 0.26 b 0.221 ± 0.001 b 3.14 ± 0.01 ab SW + 17.6 mM NaNO 3 + Turk 25.17 ± 0.65 a 0.230 ± 0.002 a 3.01 ± 0.03 a BG11 + Turk 25.58 ± 0.47 a 0.232 ± 0.001 a 2.99 ± 0.02 a Chemical factors affecting total lipid content and lipid productivity of A. halophytica cultivated in natural seawater Carbon source and concentration Various carbon sources containing 0.189 mmol C-atom L − 1 were tested for their effects on lipid production. A. halophytica grown in SNSW containing glucose gave the highest lipid content with 26.56 ± 0.16% at day 14th and the highest lipid productivity with 8.87 ± 0.07 mg L − 1 day − 1 (Table 2 ). The lipid content and lipid productivity were significantly higher than those of cells grown in sucrose and lactose (Table 2 ). However, SNSW containing glucose and the control medium resulted in statistically comparable lipid content and lipid productivity (Table 2 ). Consequently, glucose was selected as the preferred carbon source for lipid production by A. halophytica. Table 2 Lipid content and productivity of A. halophytica cultivated in natural seawater supplemented with 17.6 mM NaNO 3 and Turk Island salt solution (SNSW) and added with various kinds of carbon sources at final concentration of 0.189 mmol C-atom L − 1 for 14 days. The standard BG11 supplemented with Turk Island salt solution was used as a control medium. Data were from three independent experiments and expressed as mean ± SD. Type of medium Carbon source Lipid content (%) Lipid productivity (mg L − 1 day − 1 ) SNSW SNSW SNSW SNSW SNSW BG11 + Turk - Glucose Fructose Sucrose Lactose - 18.43 ± 1.18 d 26.56 ± 0.16 a 22.33 ± 1.72 b 21.50 ± 1.21 bc 19.94 ± 1.26 cd 24.82 ± 0.20 a 7.12 ± 0.40 d 8.87 ± 0.07 a 8.30 ± 0.13 abc 8.12 ± 0.45 bc 8.00 ± 0.45 c 8.77 ± 0.15 ab The effect of glucose concentration (0 to 756 mmol C-atom L − 1 ) on total lipid content of A. halophytica was investigated. The highest lipid content with 26.50 ± 1.63% and lipid productivity with 10.05 ± 0.63 mg L − 1 day − 1 were obtained in A. halophytica cells cultivated in SNSW containing 1.89 mmol C-atom L − 1 glucose for 14 days (Table 3 ). Too high concentrations of glucose (379 and 756 mmol C-atom L − 1 ) resulted in undetectable lipid content and lipid productivity due to the cell death. In this study, glucose at 1.89 mmol C-atom L − 1 was chosen for further study. Table 3 Lipid content and productivity of A. halophytica cultivated in natural seawater supplemented with 17.6 mM NaNO 3 and Turk Island salt solution (SNSW) and added with various glucose concentrations from 0-756 mmol C-atom L − 1 for 14 days. The standard BG11 supplemented with Turk Island salt solution was used as a control medium. Data were from three independent experiments and expressed as mean ± SD. Type of medium Glucose (mmol C-atom L − 1 ) Lipid content (%) Lipid productivity (mg L − 1 day − 1 ) SNSW - 19.11 ± 1.17 d 7.00 ± 0.46 d SNSW 0.189 23.46 ± 1.77 bc 8.82 ± 0.57 b SNSW 1.89 26.50 ± 1.63 a 10.05 ± 0.63 a SNSW 18.9 22.75 ± 0.63 bc 8.55 ± 0.36 bc SNSW 189 21.48 ± 1.10 c 7.52 ± 0.39 cd SNSW 379 nd nd SNSW 756 nd nd BG11 + Turk - 24.75 ± 0.36 ab 8.88 ± 1.06 b NaCl concentration Under various external NaCl concentrations, the highest lipid content with 50.47 ± 2.46% and lipid productivity with 48.33 ± 0.61 mg L − 1 day − 1 was obtained in A. halophytica cells cultivated in SNSW supplemented with 1.89 mmol C-atom L − 1 glucose and 0.75 M NaCl (Table 4 ). Interestingly, they were approximately 1.3–2.6 folds higher than those of cells cultivated in other NaCl concentrations. The result demonstrated that NaCl concentration played a significant role in lipid production by A. halophytica. Table 4 Lipid content and productivity of A. halophytica cultivated in natural seawater supplemented with 17.6 mM NaNO 3, Turk Island salt solution, and 1.89 mmol C-atom L − 1 glucose and added with various external NaCl concentrations from 0–3.0 M NaCl for 14 days. The standard BG11 supplemented with Turk Island salt solution was used as a control medium. Data were from three independent experiments and expressed as mean ± SD. Type of medium NaCl (M) Lipid content (%) Lipid productivity (mg L − 1 day − 1 ) SNSW + 1.89 mmol C-atom L − 1 glucose - 19.31 ± 1.72 c 6.83 ± 0.42 f SNSW + 1.89 mmol C-atom L − 1 glucose 0.25 28.62 ± 1.14 c 10.27 ± 0.36 e SNSW + 1.89 mmol C-atom L − 1 glucose 0.5 30.51 ± 2.13 c 22.45 ± 0.54 c SNSW + 1.89 mmol C-atom L − 1 glucose 0.75 50.47 ± 2.46 a 48.33 ± 0.61 a SNSW + 1.89 mmol C-atom L − 1 glucose 1 39.89 ± 0.65 b 30.96 ± 1.06 b SNSW + 1.89 mmol C-atom L − 1 glucose 2 28.19 ± 1.90 c 19.92 ± 0.65 d SNSW + 1.89 mmol C-atom L − 1 glucose 3 27.42 ± 1.41 c 2.62 ± 0.29 g BG11 + Turk - 24.15 ± 2.85 c 8.70 ± 0.15 e Physical factors affecting lipid content and lipid productivity of A. halophytica cultivated in natural seawater Effect of Initial medium pH and temperature By variation of initial medium pH from 6.0 to 8.5, A. halophytica cells cultivated in SNSW supplemented with 1.89 mmol C-atom L − 1 glucose and 0.75 M NaCl at initial pH 7.5 gave the highest lipid content with 50.59 ± 4.54% and lipid productivity with 48.52 ± 3.57 mg L − 1 day − 1 . When varying the temperature from 20 to 40°C, cultivation at 30°C gave the highest lipid content with 51.10 ± 1.85% and lipid productivity with 46.24 ± 3.51 mg L − 1 day − 1 Effect of salinity on fatty acid profiles of total lipids The fatty acid composition of total lipids extracted from A. halophytica cells cultivated in SNSW containing 1.89 mmol C-atom L − 1 glucose, supplemented with Turk Island salt solution, under various NaCl concentrations was analyzed by Gas Chromatograph. Qualitatively, ten fatty acids were commonly found among all samples (Table 5 ). These were myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), eicosaenoic acid (C20:1), dihomo-γ-linoleic acid (C20:3) and erucic acid (C22:1). The result showed that fatty acid profiles in A. halophytica depended on the NaCl concentrations. The major types of fatty acid included palmitic acid, oleic acid, linoleic acid, and linolenic acid (Table 5 ). Palmitic acid was mainly found with 24–32% composition in lipid of A. halophytica cells. Interestingly, the highest content of linolenic acid with 32.42 and 31.03% was obtained in A. halophytica cultivated in SNSW plus 1.89 mmol C-atom L − 1 glucose and 0.75 M NaCl, and in BG11 supplemented with Turk Island salt solution, respectively (Table 5 ). In addition, linoleic acid content was the highest with 16.58% at 0.75 M NaCl and the lowest with 6.98% at 0.25 M NaCl. The highest content of linolenic acid with 32.42% was obtained at 0.75 M NaCl (Table 5 ), while the lowest content with 9.89% was observed at 3 M NaCl. Table 5 Effect of salinity on fatty acids profile (% of total fatty acid) of A. halophytica cultivated in natural seawater supplemented with 17.6 mM NaNO 3, Turk Island salt solution, 1.89 mmol C-atom L − 1 glucose and added with various external NaCl concentrations at 0.25, 0.5, 0.75, 1, 2 and 3 M NaCl for 14 days. BG11 supplemented with Turk Island salt solution was used as a control medium. Fatty acid Relative fatty acid composition (%) BG11 NaCl 0.25 M NaCl 0.5 M NaCl 0.75 M NaCl 1 M NaCl 2 M NaCl 3 M Myristic acid (C14:0) 0.91 1.74 1.21 0.40 1.03 0.91 2.04 Palmitic acid (C16:0) 28.20 29.58 32.35 26.48 30.14 24.67 28.43 Palmitoleic acid (C16:1) 0.31 7.85 8.61 4.17 4.44 3.12 1.74 Stearic acid (C18:0) 5.98 9.33 9.38 2.61 7.59 7.80 11.83 Oleic acid (C18:1) 12.15 19.51 20.01 10.23 12.29 14.95 24.91 Linoleic acid (C18:2) 13.44 6.98 7.89 16.58 12.86 14.05 13.88 Linolenic acid (C18:3) 31.03 17.03 17.17 32.42 24.22 20.23 9.89 Eicosaenoic acid (C20:1) 2.93 3.25 1.52 3.17 3.19 5.56 3.14 Dihomo-γ-linoleic acid (C20:3) 2.63 2.39 1.13 2.37 2.42 4.86 2.31 Erucic acid (C22:1) 2.42 2.33 0.73 1.56 1.84 3.86 1.82 Effect of salinity on biodiesel properties Biodiesel properties based on fatty acid composition of A. halophytica grown under different salinities are shown in Table 6 . The result showed that salt stress at 0.75 M NaCl decreased saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) but increased polyunsaturated fatty acid (PUFA) (Table 6 ). On the other hand, an increase in SFA and MUFA and a decrease in PUFA was found in cells cultivated at NaCl concentrations higher or lower than 0.75 M (Table 6 ). In A. halophytica cells grown in SNSW with 1.89 mmol C-atom L⁻¹ glucose and 0.75 M NaCl, SFA, MUFA, and PUFA levels were 29.5, 17.6, and 49.0%, respectively. This was relatively similar to SFA, MUFA and PUFA in enriched BG11 medium with 35.1, 15.4, and 44.5%, respectively. High PUFA content resulted in high degree of unsaturation (Table 6 ). Biodiesel of lipid extracted from A. halophytica under all conditions showed saponification value (SV) with 186.7-203.7 mg g − 1 , iodine value (IV) with 78.9-134.7, cetane number (CN) with 43.6–56.3, cold filter plugging point (CFPP) with − 4.1–11 0 C, cloud point (CP) with 8–12 0 C, pour point (PP) with 1.8–6.2 0 C, allylic position equivalent (APE) with 70.1-108.2, bis-allylic position equivalent (BAPE) with 33.7–81.4, oxidation stability (OS) with 5.0-7.6 h, higher heating value (HHV) with 36.0-38.6 MJ kg − 1 , kinematic viscosity (ν) with 3.2–3.6 mm 2 s − 1 , and density (ρ) with 802–860 kg m − 3 (Table 6 ). Table 6 Biodiesel properties and fatty acid composition of A. halophytica cells cultivated in natural seawater supplemented with 17.6 mM NaNO 3, Turk Island salt solution, 1.89 mmol C-atom L − 1 glucose and added with various external NaCl concentrations at 0.25, 0.5, 0.75, 1, 2 and 3 M NaCl for 14 days. Biodiesel properties were analyzed by BiodieselAnalyzer© version 2.2. Characteristics BG11 NaCl concentration (M) 0.25M 0.5M 0.75M 1M 2M 3M Fatty acid composition (%) SFA 35.1 40.7 42.9 29.5 38.8 33.4 42.3 MUFA 15.4 30.6 30.1 17.6 19.9 23.6 29.8 PUFA 44.5 24.0 25.1 49.0 37.1 34.3 23.8 Biodiesel properties DU 104.3 78.6 80.3 115.6 94.1 92.2 77.3 SV 194.9 197.2 203.7 197.4 197.6 186.7 196.9 IV 122.9 87.3 89.1 134.7 107.7 101.9 78.9 CN 46.7 54.3 53.0 43.6 49.7 52.6 56.3 LCSF 5.8 7.6 7.9 3.9 6.8 6.4 8.8 CFPP 1.8 7.5 8.4 -4.1 4.9 3.5 11.0 CP 9.8 10.6 12.0 8.9 10.8 8.0 10.0 PP 3.9 4.7 6.2 2.9 5.0 1.8 4.00 APE 101.1 67.5 70.1 108.2 86.5 83.5 72.4 BAPE 75.5 41.0 42.2 81.4 61.3 54.5 33.7 OS 5.2 7.5 7.3 5.0 5.8 6.0 7.6 HHV 37.3 37.5 38.6 37.7 37.6 36.0 37.8 υ 3.3 3.5 3.6 3.2 3.4 3.3 3.6 ρ 837 835 860 848 842 802 839 SFA: Saturated fatty acid (%), MUFA: Monounsaturated fatty acid (%), PUFA: Polyunsaturated fatty acid (%), DU: Degree of unsaturation (% wt.), SV: Saponification value (mg g − 1 ), IV: Iodine value (g I 2 100 g − 1 ), CN: Cetane number, LCSF: Long-chain saturated factor, CFPP: Cold filter plugging point (°C), CP: Cloud point (°C), PP: Pour point (°C), APE: Allylic position equivalent, BAPE: Bis-allylic position equivalent, OS: Oxidation stability (h), HHV: Higher heating value (MJ kg − 1 ), υ: Kinematic viscosity (mm 2 s − 1 ), ρ: Density (kg m − 3 ). Discussion BG11 medium contains various macro- and micronutrients that facilitate the growth of cyanobacteria and is commonly utilized for cyanobacterial cultivation (Prihantini et al. 2019 ). Turk Island salt solution contains several salts including NaCl, which is essential for growth of halophilic microorganisms. Nevertheless, in marine cyanobacterial cultivation for biodiesel production, the utilization of BG11 supplemented with Turk Island salt solution proves to be economically impractical. In this study, biomass and lipid production yields from A. halophytica comparable to those obtained with BG11 were achieved by optimization of natural seawater and growth parameters. NaNO 3 was found to be essential for promoting the growth of A. halophytica in natural seawater, as its absence inhibited growth (Fig. S1 ). The optimal NaNO 3 concentration was 17.6 mM. In addition, Turk Island salt solution in natural seawater further enhanced chlorophyll a content and total cell concentration (Fig. S2). This was likely due to the presence of high Mg 2+ concentration which is the core constituent of chlorophyll molecules and a cofactor of many important enzymes involved in energy metabolisms (Cowan 2002 ; Scholnick and Keren 2006 ). Carbon is the major constituent of the lipid molecules. Among the tested carbon sources, glucose was the preferred carbon source for lipid production by A. halophytica as indicated by the elevated levels of lipid content and lipid productivity, compared to other carbon sources. This agreed with the previous report that glucose supplementation into the N-limited medium of Chlorella protothecoides resulted in an approximately 4 folds increase of the lipid content (Miao and Wu 2004 ). Chlorella sorokiniana and Chlorella vulgaris ESP-31 grown in medium containing 5 and 10 g L − 1 glucose, respectively, gave higher lipid content than those without glucose addition (Wan et al. 2011 ; Yeh and Chang 2012 ). However, different results were observed in other microalgal species. The addition of 2 and 0.5-5 g L − 1 glucose did not show an increase of lipid content in Chlorella sp. and Chlorella pyrenoidosa , respectively (Cheirsilp and Torpee 2012 ; Zhang et al. 2014 ). In Scenedesmus obliquus , the enhancement of lipid production by glucose supplementation was due to the increased biomass (Mandal and Mallick 2009 ). In this study, the optimal glucose concentration for lipid production was 1.89 mmol C-atom L − 1 glucose (Table 3 ), providing about 1.5 folds lipid content and lipid productivity higher than those without glucose addition. This corresponded with the previous study that S. obliquus grown in medium supplemented with 1.5% (w/v) or 0.089 mmol C-atom L − 1 glucose gave the highest lipid content which was approximately 10 folds higher than that of cells grown without glucose addition (Mandal and Mallick 2009 ). By comparison of lipid content with other cyanobacteria, A. halophytica cultivated in SNSW supplemented with 1.89 mmol C-atom L − 1 glucose and 0.75 M NaCl gave very high lipid content at 50.47 ± 2.46% by weight (Table 7 ). Other filamentous and unicellular cyanobacteria cultivated in different types of media show 4–32% lipid content. Based on lipid productivity, A. halophytica exhibited the second highest lipid productivity (48.33 ± 0.61 mg L − 1 day − 1 after Anabaena sp. ( g24 ) which showed lipid productivity at 79.10 ± 4.34 mg L − 1 day − 1 (Table 7 ). This was likely due to the higher biomass productivity of Anabaena sp. (g24) (489.66 ± 12.77 mg L − 1 day − 1 ) as opposed to a much lower biomass productivity of A. halophytica (95.76 ± 0.25 mg L − 1 day − 1 ). Despite its lower lipid productivity, A. halophytica maintained a significant advantage as a biodiesel production source because of its ability to grow and accumulate lipids in natural seawater, unlike freshwater Anabaena sp. ( g24 ). This advantage presented an economically viable strategy for practical application. Table 7 Comparison of biomass productivity, lipid content and lipid productivity of cyanobacteria Cyanobacteria Type of medium Biomass productivity (mg L − 1 day − 1 ) Lipid content (%) Lipid productivity (mg L − 1 day − 1 ) References Anabaena cycadeae BG11 0 131.67 ± 2.46 9.75 ± 0.25 12.84 ± 0.58 Nagappan et al., 2020 Anabaena cylindrica BG11 42.63 ± 0.20 4.79 ± 0.03 2.04 ± 0.01 Patel et al., 2018 Anabaena cylindrica BG11 0 303.06 ± 41.60 6.95 ± 0.20 21.02 ± 2.30 Nagappan et al., 2020 Anabaena doliolum BG11 0 183.95 ± 5.52 9.02 ± 0.52 16.58 ± 0.46 Nagappan et al., 2020 Anabaena f ertillissima BG11 0 31.76 ± 1.96 7.60 ± 2.26 2.44 ± 0.87 Nagappan et al., 2020 Anabaena sp. ( g24 ) BG11 0 489.66 ± 12.77 16.15 ± 0.47 79.10 ± 4.34 Nagappan et al., 2020 Aphanothece halophytica Natural seawater + Turk + 0.75 M NaCl 95.76 ± 0.25 50.47 ± 2.46 48.33 ± 0.61 This study Leptolyngbya foveolarum HNBGU001 BG11 + 300 mg L − 1 S + 45.9 mgL − 1 Carbonate + 10 mgL − 1 P + 375 mgL − 1 N 154.80 ± 3.60 32.10 ± 0.40 49.60 ± 0.70 Singh and Kumar, 2021 Leptolyngbya sp. ISTCY101 BG11 61.21 ± 0.09 19.00 ± 0.01 11.63 ± 0.02 Singh and Thakur, 2015 Leptolyngbya sp. ISTCY101 Wastewater 85.00 ± 0.28 25.00 ± 0.18 21.25 ± 0.05 Singh and Thakur, 2015 Leptolyngbya sp. ISTCY101 BG11 + 50 mM NaHCO 3 79.80 ± 0.15 20.00 ± 0.11 15.96 ± 0.02 Singh and Thakur, 2015 Lyngbya sp. BG11 33.79 ± 0.49 10.30 ± 0.03 3.48 ± 0.05 Patel et al., 2018 Nostoc muscorum BG11 40.47 ± 0.43 7.49 ± 0.08 3.03 ± 0.03 Patel et al., 2018 Nostoc muscorum BG11 0 91.03 ± 1.70 8.45 ± 1.21 7.68 ± 0.96 Nagappan et al., 2020 Nostoc sp. MCC41 BG11 0 539.96 ± 39.44 15.69 ± 2.33 84.27 ± 6.42 Nagappan et al., 2020 Oscillatoria sp. BG11 40.00 ± 0.14 8.49 ± 0.01 3.40 ± 0.01 Patel et al., 2018 Oscillatoria PBGA3 BBM 84.17 ± 4.88 12.23 ± 1.36 10.29 ± 1.15 Thangavel et al., 2018 Phormidium sp. BG11 56.32 ± 0.53 8.39 ± 0.09 4.73 ± 0.05 Patel et al., 2018 Synechococcus 7942 BG11 57.37 ± 0.18 11.01 ± 0.31 6.32 ± 0.02 Patel et al., 2018 Synechocystis 6803 BG11 62.43 ± 0.21 13.10 ± 0.12 8.18 ± 0.03 Patel et al., 2018 Synechocystis sp. BG11 423.52 ± 6.62 3.61 ± 0.48 15.27 ± 1.78 Nagappan et al., 2020 Tolypothrix sp. BG11 0 212.83 ± 11.6 7.74 ± 1.99 16.59 ± 5.12 Nagappan et al., 2020 Tolypothrix sp. PBGA1 BBM 102.71 ± 4.72 10.58 ± 2.73 10.87 ± 2.80 Thangavel et al., 2018 Westiellopsis sp. BG11 0 262.50 ± 12.73 9.3 ± 0.66 24.45 ± 2.90 Nagappan et al., 2020 BBM: Bold’s Basal Medium, BG11: Blue green 11 medium, BG11 0 : nitrogen-deficient Blue green 11 medium At various medium pH levels and temperatures, A. halophytica cultivated in SNSW at pH 7.5 and 30°C exhibited the highest lipid content and lipid productivity. This result was consistent with previous studies. pH 7 and 7.5 were ideal pH for lipid production in microalgae Tetraselmis suecica and Chlorella sp. (Moheimani 2013 ) whereas in marine microalga Nannochloropsis salina , the highest lipid accumulation with 24.75% by mass was obtained at pH 8 treatment (Bartley et al. 2014 ). The actual mechanism of the pH change affecting lipid production is not yet known. Since pH 7.5 is relatively neutral, the increase in lipid accumulation was unlikely a stress response. Another study showed that Scenedesmus acutus provided the highest fatty acid productivity at 42.10 mg L − 1 day − 1 , when grown at 30°C (El-Sheekh et al. 2017 ). Since the growth conditions at pH 7.5 and 30°C were relatively mild, the elevation of lipid accumulation in A. halophytica was unlikely a stress response. Physiologically, lipid accumulation is one of physiological acclimations to enhance environmental stress tolerance in cyanobacteria. It was previously investigated for its role in the detoxification of reactive oxygen species induced by Na + (Yang et al. 2024 ). Thus, the application of salinity stress to increase biodiesel production was gaining more interest (Yang et al. 2024 ). In this study, fluctuation of lipid production by A. halophytica was observed at different NaCl concentrations. Generally, natural seawater contains high salinity of about 0.5 M or 3.0-3.5% (w/v) NaCl (Xie et al. 1997 ). This concentration is too high to cultivate freshwater cyanobacteria due to the salinity stress by ion homeostasis mechanism and the change of the cellular ionic ratios from the membrane selectivity permeability (Pandit et al. 2017 ). However, A. halophytica could grow in medium containing 0.25-3.0 M NaCl (Waditee et al. 2002 ). The optimal concentration of NaCl for lipid production depended on the type and characteristics of cyanobacterial or microalgal species and cultivation times. The optimal NaCl concentration for lipid accumulation by A. halophytica was 0.75 M which provided the highest lipid content and lipid productivity (Table 4 ). In contrast, freshwater green algae Chlorella vulgaris and Acutodesmus obliquus showed a maximum lipid content of 49.5 and 43.4%, respectively, when cultivated in modified BG11 medium containing 0.4 M NaCl for 15 days (Pandit et al. 2017 ). The marine microalga Nannochloropsis oculata CS179 gave the highest lipid content with 32.1% at NaCl concentration of 25% (w/v) or 4.3 M (Gu et al. 2012 ). Additionally, NaCl at 20 g L − 1 or 0.34 M could enhance lipid content with 32.4% in freshwater microalga Desmodesmus abundans (Xia et al. 2014 ). This emphasizes the significance of optimizing NaCl concentration for biodiesel production in microalgal species. Cyanobacteria responded to the salinity stress by changing various kinds of physiological mechanisms such as uptake, efflux, and changing in sodium ion inside and outside of the cell and cellular membrane fluidity. These led to the metabolic changes of fatty acid profiles. In this study, fatty acid profiles of A. halophytica were different under various NaCl concentrations. The most common fatty acids in A. halophytica contained 16–18 C-atoms which are the main components of biodiesel (Miao and Wu, 2007 ). A previous study showed that A. halophytica cultivated in SNPK (Seaweed extract + NPK) medium displayed the highest relative percentage of fatty acid methyl esters of 11-octadecenoic acid (C18:1) and 13-docosenoic acid (C22:1) at 32.39 and 55.88%, respectively (Miriam et al. 2017 ). This was different from the present study. A. halophytica cultivated in SNSW supplemented with 0.75 M NaCl displayed 10.23% oleic acid (C18:1) and 1.56% erucic acid (C22:1). Additionally, a decrease in SFA and MUFA but an increase in PUFA under salinity stress at 0.75 M NaCl were observed (Table 6 ). This contrasted with a previous study where 0.4 M NaCl increased SFA and MUFA but decreased PUFA in Acutodesmus obliquus and Chlorella vulgaris (Pandit et al. 2017 ). Thus, fatty acid profiles may be influenced by not only the microalgal species, but also the cultivation conditions. Biodiesel properties depended on the fatty acid profiles, especially the content of SFA. Fatty acid methyl esters with long chain and unsaturated fatty acids provide the high quality of biodiesel (Shekh et al. 2016 ). It has been reported that MUFA is more advantageous than SFA and PUFA with respect to oxidative stability, cold flow, and combustion properties (Knothe 2009 ). Because optimized SNSW and BG11 media yielded relatively similar composition of SFA, MUFA and PUFA, this suggested optimized SNSW was suitable for biodiesel production. High cetane number (CN) indicated a high quality for good ignition, less knocking and low emission of nitrous oxide (Arias-Penarands et al. 2013 ). The present study revealed that the highest CN of 56.3 was achieved with cultivation of A. halophytica at 3 M NaCl concentration, whereas a CN of 43.6 was observed in the biodiesel derived from the cultivation at 0.75 M NaCl. Thus, the latter did not meet the specified CN standard given by ASTM D6751-08, EN 14214 and IS 15607 (Mandotra et al. 2014 ). According to the standard, maximum iodine value (IV) is determined as 120 g I 2 100 g − 1 . The IV above the standard at 134.7 g I 2 100 g − 1 was observed in biodiesel derived from the culture at 0.75 M NaCl, because of the high percentage of PUFA. From the above results, fatty acids of A. halophytica cells cultivated at 0.75 M NaCl were suggested to be suitable source for PUFA production rather than biodiesel production. Table 6 shows that all kinematic viscosity (3.2 to 3.6 mm 2 s − 1 ) met the standard limit of ASTM D6751-08 (1.9-6.0 mm 2 s − 1 ); however kinematic viscosity at 3.6 mm 2 s − 1 of the biodiesel derived from the cultivation at 0.5 and 3 M NaCl met the standard of EN 14214 (3.5-5.0 mm 2 s − 1 ). It is worth noting that only the density of biodiesel derived from A. halophytica cultivated at 0.5 M NaCl with 860 kg m − 3 was within the range of standard (860–900 kg m − 3 ). Therefore, fatty acids of A. halophytica cells cultivated at 0.5 M NaCl that exhibited the good biodiesel properties were suitable as a source of biodiesel production. In conclusion, natural seawater containing 17.6 mM NaNO 3 and Turk Island salt solution (SNSW) provided a comparable growth of A. halophytica with the enriched BG11 medium supplemented with Turk Island salt solution. The optimal conditions for lipid production by A. halophytica were cultivation in SNSW supplemented with 1.89 mmol C-atom L − 1 glucose and 0.75 M NaCl at pH 7.5 and 25–35 ℃. Adjustment of NaCl concentration is the key factor for maximizing lipid production by A. halophytica. The highest lipid content with 50.47 ± 2.46% and lipid productivity with 48.33 ± 0.61 mg L − 1 day − 1 were obtained at 0.75 M NaCl. From this maximum lipid production yield, the high content of PUFA linoleic acid at 32.42% and linolenic acid at 16.58% were observed. However, the biodiesel properties based on the fatty acids composition under these conditions were relatively poorer than those obtained from higher or lower NaCl concentrations than 0.75 M. Thus, for biodiesel production by A. halophytica , it was suggested to cultivate cells at 0.5 M NaCl. Declarations Funding information This study was financially supported by a research grant from the School of Science, King Mongkut’s Institute of Technology Ladkrabang. ST thanks School of Science, King Mongkut’s Institute of Technology Ladkrabang for his scholarship (RA/TA-2562-D-011). Author Contribution ST performed experiments, collected data and drafted the manuscript. KA helped to analyse fatty acid compositions. CK and AI provided revisions to scientific content of the manuscript. SP conceived the ideas of the study, analysed and interpreted data and wrote the main manuscript text. All authors reviewed the manuscript. 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Bioresour Technol 105:120–127 Zhang W, Zhang P, Sun H, Chen M, Lu S, Li P (2014) Effects of various organic carbon sources on the growth and biochemical composition of Chlorella pyrenoidosa . Bioresour Technol 173:52–58 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Sep, 2024 Reviews received at journal 27 Sep, 2024 Reviewers agreed at journal 22 Aug, 2024 Reviewers invited by journal 08 Jul, 2024 Editor assigned by journal 28 Jun, 2024 Submission checks completed at journal 28 Jun, 2024 First submitted to journal 27 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4646793","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329830237,"identity":"25c1c93d-f433-4482-948a-bae54d70754a","order_by":0,"name":"Sitthichai Thongtha","email":"","orcid":"","institution":"King Mongkut’s Institute of Technology Ladkrabang","correspondingAuthor":false,"prefix":"","firstName":"Sitthichai","middleName":"","lastName":"Thongtha","suffix":""},{"id":329830238,"identity":"5b817536-b7a5-42f5-a359-1a546fbf357a","order_by":1,"name":"Kornkanok Aryusuk","email":"","orcid":"","institution":"King Mongkut’s University of Technology Thonburi (KMUTT)","correspondingAuthor":false,"prefix":"","firstName":"Kornkanok","middleName":"","lastName":"Aryusuk","suffix":""},{"id":329830239,"identity":"b57ffda3-7588-4052-8757-e4bf2c77f3dd","order_by":2,"name":"Chokchai Kittiwongwattana","email":"","orcid":"","institution":"King Mongkut’s Institute of Technology Ladkrabang","correspondingAuthor":false,"prefix":"","firstName":"Chokchai","middleName":"","lastName":"Kittiwongwattana","suffix":""},{"id":329830240,"identity":"355b3c44-183e-4b16-bec7-3760ae22ebd9","order_by":3,"name":"Aran Incharoensakdi","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Aran","middleName":"","lastName":"Incharoensakdi","suffix":""},{"id":329830241,"identity":"1eff0c93-8806-427e-876c-6b908738671e","order_by":4,"name":"Saranya Phunpruch","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYFADCebDDAwGDDJgDmMDUVrYkoFaDHjAnIPEaeExBpJEaJFvP/vwc2GbHYO5dM9noxsFf3gY2A8/YP64A7cWgzPpxtIz25IZLOec3ZycA3IYT5oBw8EzeLQwpDFI87YxMxjcyN18GKyFIQfosDY8Dut/xvybt60eqCXnMUQL/xv8WhhupLEBbTkM0sIMcZgEAVsMbjxjs+Y5d5zHckaasXGOgTEPm8QzgwNn8Tosjfk2T1m1nLlE8mPpnD9ycvz8yQ8fVOJzGAgwsjHwGMA4bEB8gIAGIPgDCrpRMApGwSgYBTgAAGQTR/6fV6x0AAAAAElFTkSuQmCC","orcid":"","institution":"King Mongkut’s Institute of Technology Ladkrabang","correspondingAuthor":true,"prefix":"","firstName":"Saranya","middleName":"","lastName":"Phunpruch","suffix":""}],"badges":[],"createdAt":"2024-06-27 08:10:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4646793/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4646793/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60787126,"identity":"735aa75d-7e2f-40ba-a67f-fd7d03a7b114","added_by":"auto","created_at":"2024-07-22 06:03:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1325612,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4646793/v1/fe8e1d64-19dd-40e8-889f-71abd720899a.pdf"},{"id":60786748,"identity":"a688d6ef-59e4-4b74-84ca-254a2fbc857f","added_by":"auto","created_at":"2024-07-22 05:55:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":131049,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4646793/v1/e251ffdb998f6d0a277f38f8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modified Natural Seawater as Growth Medium for Halotolerant Cyanobacterium Aphanothece halophytica to Increase Lipid Content for Biodiesel Production","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobal energy demand and consumption have been rising because of the increasing human population (OECD \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Fossil fuels including coal, natural gas, and petroleum, etc., are presently one of the major sources of energy production. However, fossil fuels are non-renewable, and their production is projected to peak in 2025 and decline thereafter (Mohr et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, energy production from fossil fuels results in CO\u003csub\u003e2\u003c/sub\u003e and pollutants emission into the atmosphere. This contributes to and exacerbates the global warming situation, leading to increased severity of climate change (Matthews and Wynes \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, research into various kinds of alternative renewable energy has been gaining more attention as means to alleviate the situation.\u003c/p\u003e \u003cp\u003eBiodiesel is one of the promising renewable energies that has become more attractive to replace fossil fuels. It is produced by the transesterification or esterification reaction of lipids containing triglycerides and free fatty acids with alcohol (Mandari and Devarai \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, there are some factors that limit the use of plant-based biodiesel, including the high requirements of nutrients and land. For these reasons, efforts have been made to utilize biodiesel derived from cyanobacteria which require lower nutritional inputs (Bolatkhan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and occupy a smaller land area. Additionally, their high photosynthetic activity results in rapid biomass and lipid accumulation, alleviating the impact of CO\u003csub\u003e2\u003c/sub\u003e emission on global warming (Sadvakasova et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, lipid content varies among different cyanobacterial species (Yadav et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Optimization of growth conditions was previously reported for its positive effects on lipid production by cyanobacteria. These included nutrient compositions, salinity, medium pH and incubation temperature (Cordeiro et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Miriam et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nalley et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yalcin \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, optimization of growth conditions could potentially lead to a reduction in the cost of biodiesel production, facilitating its extensive use in the future.\u003c/p\u003e \u003cp\u003eSalinity level is another important factor for lipid production in cyanobacteria. However, its effects may be different, depending upon cyanobacterial species. Growth and lipid content of \u003cem\u003eHapalosiphon\u003c/em\u003e sp. increased as the NaCl concentration rose from 0 to 10 ppt, whereas higher NaCl concentrations only stimulated growth but not lipid yield (Ruangsomboon \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In contrast, a continuous increase in lipid production was observed in \u003cem\u003eSynechococcus\u003c/em\u003e sp. PCC7942 cultivated in medium containing NaCl ranging from 10 to 500 mM (Verma et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This demonstrates the significance of the investigation of NaCl effect on growth and lipid production in particular cyanobacterial species. The halotolerant unicellular cyanobacterium \u003cem\u003eAphanothece halophytica\u003c/em\u003e, originally isolated from the Solar Lake (Israel), could tolerate high salinity up to 3 M NaCl (Ishitani et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). A previous study showed that \u003cem\u003eA. halophytica\u003c/em\u003e provided the highest cell density of 21.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 x 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and accumulated lipid content at 29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% of dry weight, when cultivated in seaweed extract and NPK medium (Miriam et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Besides lipid production, \u003cem\u003eA. halophytica\u003c/em\u003e showed a potential as a H\u003csub\u003e2\u003c/sub\u003e producer when cultivated in nitrogen-deprived BG11 medium supplemented with Turk Island salt solution as well as in natural seawater (Taikhao et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Taikhao et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the effect of various NaCl concentrations on the lipid production of \u003cem\u003eA. halophytica\u003c/em\u003e is not yet known.\u003c/p\u003e \u003cp\u003eThe present study aimed to investigate the growth and biomass production of \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in natural seawater under various NaNO\u003csub\u003e3\u003c/sub\u003e concentrations and in the presence or absence of Turk Island salt solution. Factors affecting lipid production, including carbon sources and concentrations, initial medium pH, and incubation temperature, were investigated. The effect of various NaCl concentrations was also tested and optimized. Additionally, biodiesel properties were determined based on the fatty acid methyl ester profile of lipid extracted from \u003cem\u003eA. halophytica\u003c/em\u003e grown under various NaCl concentrations.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCyanobacterial cultivation\u003c/h2\u003e \u003cp\u003eThe unicellular halotolerant cyanobacterium \u003cem\u003eA. halophytica\u003c/em\u003e was maintained by cultivation on BG11 agar (pH 7.4) (Rippka et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1979\u003c/span\u003e) supplemented with Turk Island salt solution (Garlick et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) at 30\u0026deg;C under a white-light illumination of 30 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In this experiment, axenic cells of \u003cem\u003eA. halophytica\u003c/em\u003e were grown in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with various NaNO\u003csub\u003e3\u003c/sub\u003e concentrations (0.176\u0026ndash;17.6 mM). BG11 contains NaNO\u003csub\u003e3\u003c/sub\u003e at a final concentration of 17.6 mM. An initial cell concentration of cyanobacterial culture was adjusted to the optical density of about 0.1 at 730 nm. The culture was shaken at 120 rpm under a white-light intensity of 30 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for 18 h day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 30\u0026deg;C for 14 days. Natural seawater used in this study was collected from Nang Rum Beach, Chonburi Province, Gulf of Thailand, Thailand (12\u003csup\u003e\u0026deg;\u003c/sup\u003e 36.969\u0026rsquo; N, 100\u003csup\u003e\u0026deg;\u003c/sup\u003e 55.280\u0026rsquo;E). The seawater was filtered through a glass microfiber filter GF/F (0.7\u0026micro;m) (Whatman, UK) and adjusted to pH 7.5 with 2 N NaOH prior to sterilization by autoclaving. \u003cem\u003eA. halophytica\u003c/em\u003e cultured in BG11 supplemented with Turk Island salt solution was used as a control in all experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth determination by chlorophyll\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003econtent and total cell concentration measurements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGrowth of \u003cem\u003eA. halophytica\u003c/em\u003e was determined by measuring chlorophyll \u003cem\u003ea\u003c/em\u003e content and total cell concentration. For chlorophyll \u003cem\u003ea\u003c/em\u003e concentration measurement, cells were collected by centrifugation of one mL of cell culture at 7,000\u0026times;g at 4 \u003csup\u003e0\u003c/sup\u003eC for 10 min. Chlorophyll \u003cem\u003ea\u003c/em\u003e was extracted from the cell pellet by adding 1 mL of 90% (v/v) methanol and subsequently incubating at room temperature under darkness for 1 h. The chlorophyll \u003cem\u003ea\u003c/em\u003e concentration of the extract was determined by measuring the absorbance at 665 nm and calculated according to Mackinney (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1941\u003c/span\u003e). Cell concentration was determined based on the direct cell count method using Neubauer hemocytometer (Boeco, Germany) under a light microscope (Olympus CH30RF200, Japan). The specific growth rate (\u0026micro;) was calculated according to Eq.\u0026nbsp;1 (Tang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpecific growth rate (\u0026micro;) (day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(=\\frac{\\text{ln}\\left({x}_{1}\\right)-\\text{ln}\\left({x}_{0}\\right)}{{t}_{1}-{t}_{0}}\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;..Eq.\u0026nbsp;1\u003c/p\u003e \u003cp\u003ex\u003csub\u003e1\u003c/sub\u003e and x\u003csub\u003e0\u003c/sub\u003e are cell numbers at the end and the beginning of the exponential phase, respectively, whereas t\u003csub\u003e1\u003c/sub\u003e and t\u003csub\u003e0\u003c/sub\u003e are durations at the end and the beginning of the exponential phase, respectively. The doubling time required for the population cells to double its cell number was calculated according to Eq.\u0026nbsp;2 (Guillard \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDoubling time (day) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{\\text{ln}2}{\\mu }\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;..Eq.\u0026nbsp;2\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of nitrogen concentration and trace elements in culture medium on growth\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. halophytica\u003c/em\u003e was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater containing 0, 0.176, 1.76 and 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e. Cells were shaken at 120 rpm under a white-light intensity of 30 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for 18 h day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 30\u0026deg;C for 14 days. Chlorophyll \u003cem\u003ea\u003c/em\u003e concentration and total cell concentration were measured every 2 days. To increase growth of \u003cem\u003eA. halophytica\u003c/em\u003e, Turk Island salt solution containing 0.5 M NaCl, 49 mM MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, 30 mM MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO and 8.9 mM KCl (Garlick et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) was added in natural seawater media.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal lipid extraction\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated in various types of media for 14 days were harvested by centrifugation at 7,000\u0026times;g at 4\u0026deg;C for 10 min. Cells were dried in a hot air oven at 60\u0026deg;C for 24 h. Lipid extraction from dried cyanobacterial cells was performed by the single-step lipid extraction method (Axelsson and Gentili \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Briefly, 20\u0026ndash;30 mg of \u003cem\u003eA. halophytica\u003c/em\u003e dry cells were added with 8 mL of chloroform and methanol (2:1, v/v). The mixture was shaken vigorously. Two mL of 0.73% (w/v) NaCl was added in the mixture. The sample was mixed and centrifuged at 7,000\u0026times;g at room temperature for 5 min. The chloroform layer at the bottom of the tube containing the crude lipid extract was then separated and put into a new tube. Lipid extraction was performed repeatedly five times. The chloroform layer in the lipid crude extract was collected and evaporated using a vacuum evaporator and the crude lipid extract was then weighed. The lipid content was calculated according to Sivaramakrishnan and Incharoensakdi (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and expressed as percentage of lipid per dry cell weight. Lipid productivity was expressed as a unit of extracted lipid weight per culture volume per day. Dry biomass of cyanobacterial cells was obtained from a centrifugation of cell culture at 7,000\u0026times;g at 4\u0026deg;C for 10 min before drying the cell pellet in a hot air oven at 60\u0026deg;C for 48 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of carbon source and concentration on lipid content and lipid productivity\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. halophytica\u003c/em\u003e was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution. Various carbon sources, i.e., glucose, fructose, sucrose, and lactose, were separately added in the medium at a final concentration of 0.189 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which equals to Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e concentration in BG11. \u003cem\u003eA. halophytica\u003c/em\u003e cells were cultivated under previously described conditions for 14 days. Cells were subsequently harvested by centrifugation and total crude lipid was extracted by the previously described protocol. To investigate the optimal concentration of the selected carbon source, different concentrations of the selected carbon source were varied at 0.189, 1.89, 18.9, 189, 379 and 756 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of NaCl concentration on lipid content and lipid productivity\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. halophytica\u003c/em\u003e was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution. NaCl was added to the medium to reach the final external concentrations at 0, 0.25, 0.5, 0.75, 1, 2, and 3 M. Cells were grown under previously described conditions. After 14 days of cultivation, cells were harvested by centrifugation and total crude lipid was extracted.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of initial pH and temperature on lipid content and lipid productivity\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. halophytica\u003c/em\u003e was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e, Turk Island salt solution and 0.75 M NaCl. To investigate the effect of initial pH on lipid production, pH of the medium was adjusted to 6, 6.5, 7, 7.5, 8 and 8.5, using HCl and NaOH. Cultivation temperature was tested at 20, 25, 30, 35, and 40\u0026deg;C. After 14 days of cultivation, cells were harvested by centrifugation and total crude lipid was extracted, as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFatty acid profile analysis\u003c/h2\u003e \u003cp\u003eThe composition of fatty acid profile was analyzed at the Lipid Technology Research Laboratory, Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut\u0026rsquo;s University of Technology Thonburi (Bangkhuntien) (Bangkok, Thailand). The crude lipid extracts were transesterified by the protocol modified from Lepage and Roy (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Lipid extract (at least 10 mg) was added with 5% (v/v) HCl in absolute methanol prior to incubation at 85\u0026deg;C for 1 h. Fatty acid methyl esters were analyzed using a gas chromatograph equipped with a flame ionization detector (Agilent 6850, Mulgrave, Australia). Heptadecanoic acid was used as an internal standard. The GC condition was as described in the previous study (Pojjanapornpun et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The injector and detector temperatures were set at 250\u0026deg;C. One \u0026micro;L of sample was injected in the split mode using a ratio of 500:1. H\u003csub\u003e2\u003c/sub\u003e gas (99.999%) was used as carrier gas at a constant flow rate of 1.5 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBiodiesel properties of fatty acid methyl ester\u003c/h2\u003e \u003cp\u003eSeveral biodiesel properties, i.e., percentage content of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA), degree of unsaturation (DU), saponification value (SV), iodine value (IV), cetane number (CN), long chain saturated factor (LCSF), cold filter plugging point (CFPP), cloud point (CP), pour point (PP), allylic position equivalent (APE), bis-allylic position equivalent (BAPE), oxidation stability (OS), higher heating value (HHV), kinematic viscosity (υ), and density (ρ) were determined based on the fatty acid composition of the obtained methyl ester, by the Biodiesel analyzer software ver. 2.2 (Talebi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicates. Average values of lipid content and lipid productivity and the standard deviations were presented. The statistical analysis of differences was performed using one-way analysis of variance (ANOVA) with Duncan\u0026rsquo;s multiple range test at the 95% confidence level. All data were analyzed by a software of IBM SPSS statistics 23 (IBM Corp, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGrowth of\u003c/b\u003e \u003cb\u003eA. halophytica\u003c/b\u003e \u003cb\u003ecultivated in natural seawater containing various NaNO\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e \u003cb\u003econcentrations with and without supplementation of Turk Island salt solution\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGrowth of \u003cem\u003eA. halophytica\u003c/em\u003e based on total cell concentration was observed in natural seawater supplemented with 0.176\u0026ndash;17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e; however, the highest growth was observed in the control medium followed by that in the seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Supplementation of lower NaNO\u003csub\u003e3\u003c/sub\u003e concentrations decreased the growth, whereas no growth was observed in natural seawater without NaNO\u003csub\u003e3\u003c/sub\u003e supplementation. A similar result was observed with the chlorophyll \u003cem\u003ea\u003c/em\u003e content (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). These results indicated the requirement of NaNO\u003csub\u003e3\u003c/sub\u003e on the cultivation of \u003cem\u003eA. halophytica\u003c/em\u003e in natural seawater. Additionally, trace elements available in the Turk Island salt solution further enhanced the growth of \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in natural seawater containing various NaNO\u003csub\u003e3\u003c/sub\u003e concentrations (Fig. S2). \u003cem\u003eA. halophytica\u003c/em\u003e displayed the highest cell concentration and chlorophyll \u003cem\u003ea\u003c/em\u003e content when cultivated in natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution. The growth under this condition was comparable to that of \u003cem\u003eA. halophytica\u003c/em\u003e cells grown in BG11 medium supplemented with Turk Island salt solution.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the cell density at day 14th of cultivation, specific growth rate and doubling time of \u003cem\u003eA. halophytica\u003c/em\u003e grown in different media. Supplementation of natural seawater with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution yielded the highest specific growth rate with 0.230\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the shortest doubling time with 3.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 days (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). They were not significantly different from those of cells grown in enriched BG11 supplemented with Turk Island salt solution (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the use of natural seawater alone resulted in the lowest specific growth rate with 0.016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the longest doubling time with 43.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 days. The addition of Turk Island salt solution significantly increased the specific growth rate compared to those in the absence of the solution at the same NaNO\u003csub\u003e3\u003c/sub\u003e concentration. Therefore, natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution was chosen as the suitable medium for cultivation of \u003cem\u003eA. halophytica\u003c/em\u003e in natural seawater and named as suitable natural seawater (SNSW).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of various NaNO\u003csub\u003e3\u003c/sub\u003e concentrations and Turk Island salt solution supplementation in natural seawater on cell density at day14th of cultivation, specific growth rate and doubling time of \u003cem\u003eA. halophytica.\u003c/em\u003e BG11 supplemented with Turk Island salt solution was used as a normal control medium. Data were from three independent experiments and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of medium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCell density\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpecific growth rate\u003c/p\u003e \u003cp\u003e(\u0026micro;) (day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eDoubling time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e(\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003eat day14th\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e(days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;0 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e43.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;0.176 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e11.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.176\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;1.76 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e13.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.188\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e21.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.220\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;0 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e20.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;0.176 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e17.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.203\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;1.76 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e21.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.221\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW\u0026thinsp;+\u0026thinsp;17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e25.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.230\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBG11\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e25.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.232\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChemical factors affecting total lipid content and lipid productivity of\u003c/b\u003e \u003cb\u003eA. halophytica\u003c/b\u003e \u003cb\u003ecultivated in natural seawater\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCarbon source and concentration\u003c/h2\u003e \u003cp\u003eVarious carbon sources containing 0.189 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were tested for their effects on lipid production. \u003cem\u003eA. halophytica\u003c/em\u003e grown in SNSW containing glucose gave the highest lipid content with 26.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16% at day 14th and the highest lipid productivity with 8.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The lipid content and lipid productivity were significantly higher than those of cells grown in sucrose and lactose (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, SNSW containing glucose and the control medium resulted in statistically comparable lipid content and lipid productivity (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consequently, glucose was selected as the preferred carbon source for lipid production by \u003cem\u003eA. halophytica.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLipid content and productivity of \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution (SNSW) and added with various kinds of carbon sources at final concentration of 0.189 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 14 days. The standard BG11 supplemented with Turk Island salt solution was used as a control medium. Data were from three independent experiments and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of medium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarbon source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLipid content\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLipid productivity\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eday\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003cp\u003eBG11\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003cp\u003eFructose\u003c/p\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003cp\u003eLactose\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e26.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e22.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e21.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e19.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e24.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e8.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e8.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e8.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e8.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe effect of glucose concentration (0 to 756 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on total lipid content of \u003cem\u003eA. halophytica\u003c/em\u003e was investigated. The highest lipid content with 26.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63% and lipid productivity with 10.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were obtained in \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated in SNSW containing 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose for 14 days (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Too high concentrations of glucose (379 and 756 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) resulted in undetectable lipid content and lipid productivity due to the cell death. In this study, glucose at 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was chosen for further study.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLipid content and productivity of \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution (SNSW) and added with various glucose concentrations from 0-756 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 14 days. The standard BG11 supplemented with Turk Island salt solution was used as a control medium. Data were from three independent experiments and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of medium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003cp\u003e(mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLipid content\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLipid productivity\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eday\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e8.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e10.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e8.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e7.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBG11\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e8.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eNaCl concentration\u003c/h2\u003e \u003cp\u003eUnder various external NaCl concentrations, the highest lipid content with 50.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46% and lipid productivity with 48.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was obtained in \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated in SNSW supplemented with 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and 0.75 M NaCl (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, they were approximately 1.3\u0026ndash;2.6 folds higher than those of cells cultivated in other NaCl concentrations. The result demonstrated that NaCl concentration played a significant role in lipid production by \u003cem\u003eA. halophytica.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLipid content and productivity of \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3,\u003c/sub\u003e Turk Island salt solution, and 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and added with various external NaCl concentrations from 0\u0026ndash;3.0 M NaCl for 14 days. The standard BG11 supplemented with Turk Island salt solution was used as a control medium. Data were from three independent experiments and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of medium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003cp\u003e(M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLipid content\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eLipid productivity\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eday\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u0026thinsp;+\u0026thinsp;1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e19.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u0026thinsp;+\u0026thinsp;1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e28.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u0026thinsp;+\u0026thinsp;1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e30.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u0026thinsp;+\u0026thinsp;1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e50.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u0026thinsp;+\u0026thinsp;1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e39.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u0026thinsp;+\u0026thinsp;1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e28.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNSW\u0026thinsp;+\u0026thinsp;1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e27.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBG11\u0026thinsp;+\u0026thinsp;Turk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e24.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhysical factors affecting lipid content and lipid productivity of\u003c/b\u003e \u003cb\u003eA. halophytica\u003c/b\u003e \u003cb\u003ecultivated in natural seawater\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eEffect of Initial medium pH and temperature\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eBy variation of initial medium pH from 6.0 to 8.5, \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated in SNSW supplemented with 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and 0.75 M NaCl at initial pH 7.5 gave the highest lipid content with 50.59\u0026thinsp;\u0026plusmn;\u0026thinsp;4.54% and lipid productivity with 48.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. When varying the temperature from 20 to 40\u0026deg;C, cultivation at 30\u0026deg;C gave the highest lipid content with 51.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85% and lipid productivity with 46.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of salinity on fatty acid profiles of total lipids\u003c/h2\u003e \u003cp\u003eThe fatty acid composition of total lipids extracted from \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated in SNSW containing 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose, supplemented with Turk Island salt solution, under various NaCl concentrations was analyzed by Gas Chromatograph. Qualitatively, ten fatty acids were commonly found among all samples (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These were myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), eicosaenoic acid (C20:1), dihomo-γ-linoleic acid (C20:3) and erucic acid (C22:1). The result showed that fatty acid profiles in \u003cem\u003eA. halophytica\u003c/em\u003e depended on the NaCl concentrations. The major types of fatty acid included palmitic acid, oleic acid, linoleic acid, and linolenic acid (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Palmitic acid was mainly found with 24\u0026ndash;32% composition in lipid of \u003cem\u003eA. halophytica\u003c/em\u003e cells. Interestingly, the highest content of linolenic acid with 32.42 and 31.03% was obtained in \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in SNSW plus 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and 0.75 M NaCl, and in BG11 supplemented with Turk Island salt solution, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In addition, linoleic acid content was the highest with 16.58% at 0.75 M NaCl and the lowest with 6.98% at 0.25 M NaCl. The highest content of linolenic acid with 32.42% was obtained at 0.75 M NaCl (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), while the lowest content with 9.89% was observed at 3 M NaCl.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of salinity on fatty acids profile (% of total fatty acid) of \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3,\u003c/sub\u003e Turk Island salt solution, 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and added with various external NaCl concentrations at 0.25, 0.5, 0.75, 1, 2 and 3 M NaCl for 14 days. BG11 supplemented with Turk Island salt solution was used as a control medium.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFatty acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003eRelative fatty acid composition (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003cp\u003e0.25 M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003cp\u003e0.5 M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003cp\u003e0.75 M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003cp\u003e1 M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003cp\u003e2 M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003cp\u003e3 M\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyristic acid (C14:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePalmitic acid (C16:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e28.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePalmitoleic acid (C16:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStearic acid (C18:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOleic acid (C18:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinoleic acid (C18:2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinolenic acid (C18:3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEicosaenoic acid (C20:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDihomo-γ-linoleic acid (C20:3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErucic acid (C22:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of salinity on biodiesel properties\u003c/h2\u003e \u003cp\u003eBiodiesel properties based on fatty acid composition of \u003cem\u003eA. halophytica\u003c/em\u003e grown under different salinities are shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The result showed that salt stress at 0.75 M NaCl decreased saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) but increased polyunsaturated fatty acid (PUFA) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). On the other hand, an increase in SFA and MUFA and a decrease in PUFA was found in cells cultivated at NaCl concentrations higher or lower than 0.75 M (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In \u003cem\u003eA. halophytica\u003c/em\u003e cells grown in SNSW with 1.89 mmol C-atom L⁻\u0026sup1; glucose and 0.75 M NaCl, SFA, MUFA, and PUFA levels were 29.5, 17.6, and 49.0%, respectively. This was relatively similar to SFA, MUFA and PUFA in enriched BG11 medium with 35.1, 15.4, and 44.5%, respectively. High PUFA content resulted in high degree of unsaturation (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Biodiesel of lipid extracted from \u003cem\u003eA. halophytica\u003c/em\u003e under all conditions showed saponification value (SV) with 186.7-203.7 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, iodine value (IV) with 78.9-134.7, cetane number (CN) with 43.6\u0026ndash;56.3, cold filter plugging point (CFPP) with \u0026minus;\u0026thinsp;4.1\u0026ndash;11 \u003csup\u003e0\u003c/sup\u003eC, cloud point (CP) with 8\u0026ndash;12 \u003csup\u003e0\u003c/sup\u003eC, pour point (PP) with 1.8\u0026ndash;6.2 \u003csup\u003e0\u003c/sup\u003eC, allylic position equivalent (APE) with 70.1-108.2, bis-allylic position equivalent (BAPE) with 33.7\u0026ndash;81.4, oxidation stability (OS) with 5.0-7.6 h, higher heating value (HHV) with 36.0-38.6 MJ kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, kinematic viscosity (ν) with 3.2\u0026ndash;3.6 mm\u003csup\u003e2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and density (ρ) with 802\u0026ndash;860 kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBiodiesel properties and fatty acid composition of \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated in natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3,\u003c/sub\u003e Turk Island salt solution, 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and added with various external NaCl concentrations at 0.25, 0.5, 0.75, 1, 2 and 3 M NaCl for 14 days. Biodiesel properties were analyzed by BiodieselAnalyzer\u0026copy; version 2.2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e \u003cp\u003eNaCl concentration (M)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.75M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eFatty acid composition\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e38.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e33.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e42.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMUFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e23.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e29.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePUFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e34.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e23.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"13\" rowspan=\"14\"\u003e \u003cp\u003eBiodiesel properties\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e104.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e115.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e94.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e92.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e77.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e197.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e203.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e197.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e197.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e186.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e196.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e122.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e134.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e107.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e101.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e78.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e49.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e52.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e56.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCFPP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e108.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e86.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e83.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e72.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBAPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e81.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e54.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e33.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHHV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e36.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e37.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eυ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eρ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e848\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e839\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eSFA: Saturated fatty acid (%), MUFA: Monounsaturated fatty acid (%), PUFA: Polyunsaturated fatty acid (%), DU: Degree of unsaturation (% wt.), SV: Saponification value (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), IV: Iodine value (g I\u003csub\u003e2\u003c/sub\u003e 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CN: Cetane number, LCSF: Long-chain saturated factor, CFPP: Cold filter plugging point (\u0026deg;C), CP: Cloud point (\u0026deg;C), PP: Pour point (\u0026deg;C), APE: Allylic position equivalent, BAPE: Bis-allylic position equivalent, OS: Oxidation stability (h), HHV: Higher heating value (MJ kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), υ: Kinematic viscosity (mm\u003csup\u003e2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), ρ: Density (kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBG11 medium contains various macro- and micronutrients that facilitate the growth of cyanobacteria and is commonly utilized for cyanobacterial cultivation (Prihantini et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Turk Island salt solution contains several salts including NaCl, which is essential for growth of halophilic microorganisms. Nevertheless, in marine cyanobacterial cultivation for biodiesel production, the utilization of BG11 supplemented with Turk Island salt solution proves to be economically impractical. In this study, biomass and lipid production yields from \u003cem\u003eA. halophytica\u003c/em\u003e comparable to those obtained with BG11 were achieved by optimization of natural seawater and growth parameters. NaNO\u003csub\u003e3\u003c/sub\u003e was found to be essential for promoting the growth of \u003cem\u003eA. halophytica\u003c/em\u003e in natural seawater, as its absence inhibited growth (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The optimal NaNO\u003csub\u003e3\u003c/sub\u003e concentration was 17.6 mM. In addition, Turk Island salt solution in natural seawater further enhanced chlorophyll \u003cem\u003ea\u003c/em\u003e content and total cell concentration (Fig. S2). This was likely due to the presence of high Mg\u003csup\u003e2+\u003c/sup\u003e concentration which is the core constituent of chlorophyll molecules and a cofactor of many important enzymes involved in energy metabolisms (Cowan \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Scholnick and Keren \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCarbon is the major constituent of the lipid molecules. Among the tested carbon sources, glucose was the preferred carbon source for lipid production by \u003cem\u003eA. halophytica\u003c/em\u003e as indicated by the elevated levels of lipid content and lipid productivity, compared to other carbon sources. This agreed with the previous report that glucose supplementation into the N-limited medium of \u003cem\u003eChlorella protothecoides\u003c/em\u003e resulted in an approximately 4 folds increase of the lipid content (Miao and Wu \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cem\u003eChlorella sorokiniana\u003c/em\u003e and \u003cem\u003eChlorella vulgaris\u003c/em\u003e ESP-31 grown in medium containing 5 and 10 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose, respectively, gave higher lipid content than those without glucose addition (Wan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yeh and Chang \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, different results were observed in other microalgal species. The addition of 2 and 0.5-5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose did not show an increase of lipid content in \u003cem\u003eChlorella\u003c/em\u003e sp. and \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e, respectively (Cheirsilp and Torpee \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In \u003cem\u003eScenedesmus obliquus\u003c/em\u003e, the enhancement of lipid production by glucose supplementation was due to the increased biomass (Mandal and Mallick \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this study, the optimal glucose concentration for lipid production was 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), providing about 1.5 folds lipid content and lipid productivity higher than those without glucose addition. This corresponded with the previous study that \u003cem\u003eS. obliquus\u003c/em\u003e grown in medium supplemented with 1.5% (w/v) or 0.089 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose gave the highest lipid content which was approximately 10 folds higher than that of cells grown without glucose addition (Mandal and Mallick \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBy comparison of lipid content with other cyanobacteria, \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in SNSW supplemented with 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and 0.75 M NaCl gave very high lipid content at 50.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46% by weight (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Other filamentous and unicellular cyanobacteria cultivated in different types of media show 4\u0026ndash;32% lipid content. Based on lipid productivity, \u003cem\u003eA. halophytica\u003c/em\u003e exhibited the second highest lipid productivity (48.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after \u003cem\u003eAnabaena\u003c/em\u003e sp. (\u003cem\u003eg24\u003c/em\u003e) which showed lipid productivity at 79.10\u0026thinsp;\u0026plusmn;\u0026thinsp;4.34 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This was likely due to the higher biomass productivity of \u003cem\u003eAnabaena\u003c/em\u003e sp. (g24) (489.66\u0026thinsp;\u0026plusmn;\u0026thinsp;12.77 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as opposed to a much lower biomass productivity of \u003cem\u003eA. halophytica\u003c/em\u003e (95.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Despite its lower lipid productivity, \u003cem\u003eA. halophytica\u003c/em\u003e maintained a significant advantage as a biodiesel production source because of its ability to grow and accumulate lipids in natural seawater, unlike freshwater \u003cem\u003eAnabaena\u003c/em\u003e sp. (\u003cem\u003eg24\u003c/em\u003e). This advantage presented an economically viable strategy for practical application.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of biomass productivity, lipid content and lipid productivity of cyanobacteria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyanobacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eType of medium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiomass productivity\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eday\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLipid content\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLipid productivity\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eday\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnabaena cycadeae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnabaena cylindrica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnabaena cylindrica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e303.06\u0026thinsp;\u0026plusmn;\u0026thinsp;41.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnabaena doliolum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e183.95\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnabaena\u003c/em\u003e f\u003cem\u003eertillissima\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnabaena\u003c/em\u003e sp. (\u003cem\u003eg24\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e489.66\u0026thinsp;\u0026plusmn;\u0026thinsp;12.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79.10\u0026thinsp;\u0026plusmn;\u0026thinsp;4.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAphanothece halophytica\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNatural seawater\u0026thinsp;+\u0026thinsp;Turk\u0026thinsp;+\u0026thinsp;0.75 M NaCl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e95.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e50.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e48.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eThis study\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeptolyngbya foveolarum\u003c/em\u003e HNBGU001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u0026thinsp;+\u0026thinsp;300 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e S\u0026thinsp;+\u0026thinsp;45.9 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Carbonate\u0026thinsp;+\u0026thinsp;10 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e P\u0026thinsp;+\u0026thinsp;375 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e154.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSingh and Kumar, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeptolyngbya\u003c/em\u003e sp. ISTCY101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSingh and Thakur, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeptolyngbya\u003c/em\u003e sp. ISTCY101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWastewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSingh and Thakur, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeptolyngbya\u003c/em\u003e sp. ISTCY101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u0026thinsp;+\u0026thinsp;50 mM NaHCO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSingh and Thakur, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLyngbya\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNostoc muscorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNostoc muscorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNostoc\u003c/em\u003e sp. MCC41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e539.96\u0026thinsp;\u0026plusmn;\u0026thinsp;39.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84.27\u0026thinsp;\u0026plusmn;\u0026thinsp;6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eOscillatoria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eOscillatoria\u003c/em\u003e PBGA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThangavel et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhormidium\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSynechococcus\u003c/em\u003e 7942\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSynechocystis\u003c/em\u003e 6803\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSynechocystis\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e423.52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTolypothrix\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e212.83\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.59\u0026thinsp;\u0026plusmn;\u0026thinsp;5.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTolypothrix\u003c/em\u003e sp. PBGA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThangavel et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWestiellopsis\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBG11\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e262.50\u0026thinsp;\u0026plusmn;\u0026thinsp;12.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNagappan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eBBM: Bold\u0026rsquo;s Basal Medium, BG11: Blue green 11 medium, BG11\u003csub\u003e0\u003c/sub\u003e: nitrogen-deficient Blue green 11 medium\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAt various medium pH levels and temperatures, \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in SNSW at pH 7.5 and 30\u0026deg;C exhibited the highest lipid content and lipid productivity. This result was consistent with previous studies. pH 7 and 7.5 were ideal pH for lipid production in microalgae \u003cem\u003eTetraselmis suecica\u003c/em\u003e and \u003cem\u003eChlorella\u003c/em\u003e sp. (Moheimani \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) whereas in marine microalga \u003cem\u003eNannochloropsis salina\u003c/em\u003e, the highest lipid accumulation with 24.75% by mass was obtained at pH 8 treatment (Bartley et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The actual mechanism of the pH change affecting lipid production is not yet known. Since pH 7.5 is relatively neutral, the increase in lipid accumulation was unlikely a stress response. Another study showed that \u003cem\u003eScenedesmus acutus\u003c/em\u003e provided the highest fatty acid productivity at 42.10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, when grown at 30\u0026deg;C (El-Sheekh et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Since the growth conditions at pH 7.5 and 30\u0026deg;C were relatively mild, the elevation of lipid accumulation in \u003cem\u003eA. halophytica\u003c/em\u003e was unlikely a stress response.\u003c/p\u003e \u003cp\u003ePhysiologically, lipid accumulation is one of physiological acclimations to enhance environmental stress tolerance in cyanobacteria. It was previously investigated for its role in the detoxification of reactive oxygen species induced by Na\u003csup\u003e+\u003c/sup\u003e (Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, the application of salinity stress to increase biodiesel production was gaining more interest (Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, fluctuation of lipid production by \u003cem\u003eA. halophytica\u003c/em\u003e was observed at different NaCl concentrations. Generally, natural seawater contains high salinity of about 0.5 M or 3.0-3.5% (w/v) NaCl (Xie et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). This concentration is too high to cultivate freshwater cyanobacteria due to the salinity stress by ion homeostasis mechanism and the change of the cellular ionic ratios from the membrane selectivity permeability (Pandit et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, \u003cem\u003eA. halophytica\u003c/em\u003e could grow in medium containing 0.25-3.0 M NaCl (Waditee et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The optimal concentration of NaCl for lipid production depended on the type and characteristics of cyanobacterial or microalgal species and cultivation times. The optimal NaCl concentration for lipid accumulation by \u003cem\u003eA. halophytica\u003c/em\u003e was 0.75 M which provided the highest lipid content and lipid productivity (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, freshwater green algae \u003cem\u003eChlorella vulgaris\u003c/em\u003e and \u003cem\u003eAcutodesmus obliquus\u003c/em\u003e showed a maximum lipid content of 49.5 and 43.4%, respectively, when cultivated in modified BG11 medium containing 0.4 M NaCl for 15 days (Pandit et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The marine microalga \u003cem\u003eNannochloropsis oculata\u003c/em\u003e CS179 gave the highest lipid content with 32.1% at NaCl concentration of 25% (w/v) or 4.3 M (Gu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally, NaCl at 20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e or 0.34 M could enhance lipid content with 32.4% in freshwater microalga \u003cem\u003eDesmodesmus abundans\u003c/em\u003e (Xia et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This emphasizes the significance of optimizing NaCl concentration for biodiesel production in microalgal species.\u003c/p\u003e \u003cp\u003eCyanobacteria responded to the salinity stress by changing various kinds of physiological mechanisms such as uptake, efflux, and changing in sodium ion inside and outside of the cell and cellular membrane fluidity. These led to the metabolic changes of fatty acid profiles. In this study, fatty acid profiles of \u003cem\u003eA. halophytica\u003c/em\u003e were different under various NaCl concentrations. The most common fatty acids in \u003cem\u003eA. halophytica\u003c/em\u003e contained 16\u0026ndash;18 C-atoms which are the main components of biodiesel (Miao and Wu, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). A previous study showed that \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in SNPK (Seaweed extract\u0026thinsp;+\u0026thinsp;NPK) medium displayed the highest relative percentage of fatty acid methyl esters of 11-octadecenoic acid (C18:1) and 13-docosenoic acid (C22:1) at 32.39 and 55.88%, respectively (Miriam et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This was different from the present study. \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in SNSW supplemented with 0.75 M NaCl displayed 10.23% oleic acid (C18:1) and 1.56% erucic acid (C22:1). Additionally, a decrease in SFA and MUFA but an increase in PUFA under salinity stress at 0.75 M NaCl were observed (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This contrasted with a previous study where 0.4 M NaCl increased SFA and MUFA but decreased PUFA in \u003cem\u003eAcutodesmus obliquus\u003c/em\u003e and \u003cem\u003eChlorella vulgaris\u003c/em\u003e (Pandit et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, fatty acid profiles may be influenced by not only the microalgal species, but also the cultivation conditions.\u003c/p\u003e \u003cp\u003eBiodiesel properties depended on the fatty acid profiles, especially the content of SFA. Fatty acid methyl esters with long chain and unsaturated fatty acids provide the high quality of biodiesel (Shekh et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It has been reported that MUFA is more advantageous than SFA and PUFA with respect to oxidative stability, cold flow, and combustion properties (Knothe \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Because optimized SNSW and BG11 media yielded relatively similar composition of SFA, MUFA and PUFA, this suggested optimized SNSW was suitable for biodiesel production.\u003c/p\u003e \u003cp\u003eHigh cetane number (CN) indicated a high quality for good ignition, less knocking and low emission of nitrous oxide (Arias-Penarands et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The present study revealed that the highest CN of 56.3 was achieved with cultivation of \u003cem\u003eA. halophytica\u003c/em\u003e at 3 M NaCl concentration, whereas a CN of 43.6 was observed in the biodiesel derived from the cultivation at 0.75 M NaCl. Thus, the latter did not meet the specified CN standard given by ASTM D6751-08, EN 14214 and IS 15607 (Mandotra et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). According to the standard, maximum iodine value (IV) is determined as 120 g I\u003csub\u003e2\u003c/sub\u003e 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The IV above the standard at 134.7 g I\u003csub\u003e2\u003c/sub\u003e 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was observed in biodiesel derived from the culture at 0.75 M NaCl, because of the high percentage of PUFA. From the above results, fatty acids of \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated at 0.75 M NaCl were suggested to be suitable source for PUFA production rather than biodiesel production. Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows that all kinematic viscosity (3.2 to 3.6 mm\u003csup\u003e2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) met the standard limit of ASTM D6751-08 (1.9-6.0 mm\u003csup\u003e2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); however kinematic viscosity at 3.6 mm\u003csup\u003e2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the biodiesel derived from the cultivation at 0.5 and 3 M NaCl met the standard of EN 14214 (3.5-5.0 mm\u003csup\u003e2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). It is worth noting that only the density of biodiesel derived from \u003cem\u003eA. halophytica\u003c/em\u003e cultivated at 0.5 M NaCl with 860 kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e was within the range of standard (860\u0026ndash;900 kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e). Therefore, fatty acids of \u003cem\u003eA. halophytica\u003c/em\u003e cells cultivated at 0.5 M NaCl that exhibited the good biodiesel properties were suitable as a source of biodiesel production.\u003c/p\u003e \u003cp\u003eIn conclusion, natural seawater containing 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution (SNSW) provided a comparable growth of \u003cem\u003eA. halophytica\u003c/em\u003e with the enriched BG11 medium supplemented with Turk Island salt solution. The optimal conditions for lipid production by \u003cem\u003eA. halophytica\u003c/em\u003e were cultivation in SNSW supplemented with 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and 0.75 M NaCl at pH 7.5 and 25\u0026ndash;35 ℃. Adjustment of NaCl concentration is the key factor for maximizing lipid production by \u003cem\u003eA. halophytica.\u003c/em\u003e The highest lipid content with 50.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46% and lipid productivity with 48.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were obtained at 0.75 M NaCl. From this maximum lipid production yield, the high content of PUFA linoleic acid at 32.42% and linolenic acid at 16.58% were observed. However, the biodiesel properties based on the fatty acids composition under these conditions were relatively poorer than those obtained from higher or lower NaCl concentrations than 0.75 M. Thus, for biodiesel production by \u003cem\u003eA. halophytica\u003c/em\u003e, it was suggested to cultivate cells at 0.5 M NaCl.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding information\u003c/h2\u003e \u003cp\u003eThis study was financially supported by a research grant from the School of Science, King Mongkut\u0026rsquo;s Institute of Technology Ladkrabang. ST thanks School of Science, King Mongkut\u0026rsquo;s Institute of Technology Ladkrabang for his scholarship (RA/TA-2562-D-011).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eST performed experiments, collected data and drafted the manuscript. KA helped to analyse fatty acid compositions. CK and AI provided revisions to scientific content of the manuscript. SP conceived the ideas of the study, analysed and interpreted data and wrote the main manuscript text. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArias-Penarands MT, Cristiani-Urbina E, Montes-Horcasitas CM, Esparza-Garcia F, Torzillo G, Canizares-Villanueva RO (2013) \u003cem\u003eScenedesmus incrassatulus\u003c/em\u003e CLHE-Si01: a potential source of renewable lipid for high quality biodiesel production. Bioresour Technol 140:158\u0026ndash;164\u003c/li\u003e\n \u003cli\u003eAxelsson M, Gentili F (2014) A single-step method for rapid extraction of total lipids from green microalgae. PLoS One 9:e89643\u003c/li\u003e\n \u003cli\u003eBartley ML, Boeing WJ, Dungan BN, Holguin FO, Schaub T (2014) pH effects on growth and lipid accumulation of the biofuel microalgae \u003cem\u003eNannochloropsis salina\u003c/em\u003e and invading organisms. 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Bioresour Technol 173:52\u0026ndash;58\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Growth, Lipid production, Biodiesel, Cyanobacteria, Seawater","lastPublishedDoi":"10.21203/rs.3.rs-4646793/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4646793/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiodiesel derived from cyanobacterial oils becomes attractive as an efficient renewable energy. The present study aims to optimize growth and lipid production of halotolerant unicellular cyanobacterium \u003cem\u003eAphanothece halophytica\u003c/em\u003e cultivated in natural seawater. In this study, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehalophytica\u003c/em\u003e was able to grow in natural seawater when supplemented with low concentration of NaNO\u003csub\u003e3\u003c/sub\u003e, whereas no growth occurred without supplementation. The specific growth rate of 0.230 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and cell concentration of 25.17 x 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were achieved in \u003cem\u003eA. halophytica\u003c/em\u003e cultivated in natural seawater supplemented with 17.6 mM NaNO\u003csub\u003e3\u003c/sub\u003e and Turk Island salt solution (suitable natural seawater; SNSW) for 14 days. This growth rate was comparable to that of cells grown in normal BG11 plus Turk Island salt solution. The lipid content and fatty acid profiles of \u003cem\u003eA. halophytica\u003c/em\u003e varied with changes in NaCl concentrations. The highest lipid content of 50.47% and lipid productivity of 48.33 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were obtained in cultures supplemented with 1.89 mmol C-atom L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and 0.75 M NaCl. The optimal medium pH and cultivation temperature for lipid production was 7.5 and 25\u0026ndash;35\u0026deg;C, respectively. When cultivating \u003cem\u003eA. halophytica\u003c/em\u003e in optimized SNSW with various NaCl concentrations, the highest contents of linoleic and linolenic acids, and the lowest contents of palmitic, stearic, and oleic acids were observed with 0.75 M NaCl. In contrast, cultures grown in optimized SNSW with 0.5 M NaCl showed fatty acid methyl ester profiles rich in monounsaturated fatty acids, which are favorable for high-quality biodiesel production.\u003c/p\u003e","manuscriptTitle":"Modified Natural Seawater as Growth Medium for Halotolerant Cyanobacterium Aphanothece halophytica to Increase Lipid Content for Biodiesel Production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 05:55:34","doi":"10.21203/rs.3.rs-4646793/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-27T10:02:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-27T09:53:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213005417850616910216200082882520154848","date":"2024-08-22T15:56:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-08T08:25:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-28T08:12:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-28T05:34:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2024-06-27T08:09:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c923a9bb-23e7-48cf-83c2-3f4f18edf1d3","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-18T00:53:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 05:55:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4646793","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4646793","identity":"rs-4646793","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}