Impact of macronutrients and salinity stress on biomass and biochemical constituents in Monoraphidium braunii to enhance biodiesel production

Microalgal lipids are precursors to the production of biodiesel, as well as a source of valuable dietary components in the biotechnological industries. So, this study aimed to assess the effects of nutritional (nitrogen, and phosphorus) starvations and salinity stress (NaCl) on the biomass, lipid content, fatty acids profile, and predicted biodiesel properties of green microalga Monoraphidium braunii. The results showed that biomass, biomass productivity, and photosynthetic pigment contents (Chl. a, b, and carotenoids) of M. braunii were markedly decreased by nitrogen and phosphorus depletion and recorded the maximum values in cultures treated with full of N and P concentrations (control, 100%). These parameters were considerably increased at the low salinity level (up to 150 mM NaCl), while an increasing salinity level (up to 250 mM NaCl) reduces the biomass, its productivity, and pigment contents. Nutritional limitations and salt stress (NaCl) resulted in significantly enhanced accumulation of lipid and productivity of M. braunii, which represented more than twofold of the control. Furthermore, these conditions have enhanced the profile of fatty acid and biodiesel quality-related parameters. The current study exposed strategies to improve M. braunii lipid productivity for biodiesel production on a small scale in vitro in terms of fuel quality under low nutrients and salinity stress.


Microalgal growth under nutrient deficiency and salt stress
The growth rate of green alga Monoraphidium braunii grown in media containing various nitrogen and phosphorus concentrations is shown in Fig. 1A,B.
The growth rate was estimated using optical density (OD 560 ) every two days, the highest growth of M. braunii was detected in cultures accompanied full of nitrogen and phosphorus concentrations (control, 100%), as a result, OD 560 value was (0.232) at the 9th day of cultivation.On the other hand, the depletion of nitrogen and phosphorus concentrations resulted in a significant decrease in the growth rate of Monoraphidium braunii.The highest reduction in the growth rate of M. braunii cultured in medium containing (0.0 N) was 65.09%, whereas 56.90% in medium containing (0.0 P) as compared to the control at the 9th day of cultivation.
Almutairi 15 revealed similar results using Dunaliella salina under nitrogen and phosphorus limitations, reporting that nutrient depletion induces growth failure.Similarly, reduced growth of Chlamydomonas reinhardtii has been found under N deficiency, even after the required phosphate addition 23 .
Regarding salinity stress, the growth rate (OD 560 ) of green microalga M. braunii was improved by increasing the salt concentrations up to 150 mM NaCl, where it increased by 10% over the control (0 NaCl) at 100 mM NaCl, after that, significantly decreased was shown, it decreased by 42.19% at 250 mM NaCl compared to the control at 9th day of cultivation (Fig. 1C).Our results agreed with Pandit et al. 24 revealed that growth rate of Chlorella vulgaris and Acutodesmus obliquus increased by increasing the concentration of NaCl from 0 to 0.1 M and decreased by increasing the dosage from 0.3 to 0.4 M in addition El-Sheekh et al. 25 demonstrated that the addition of NaCl enhanced optical density of S. obliquus when compared to the control.From that perspective, salt stress has been shown to significantly affect growth while interfering with the physiological activities of algal cells.The salt tolerance level and species of microalgae determine their adaptability to saline conditions 26 .On the contrary, Alsull and Omar 27 recorded that lower salinity conditions significantly reduced the optical density and dry weight of Tetraselmis sp. and Nannochloropsis sp.

Biomass yield and biomass productivity under nutrient deficiency and salt stress
The cellular dry weight (CDW) and biomass productivity were measured on the last day of culture (9th day).Because they indicate how quickly different microalgal strains grow, biomass yield and productivity are essential factors in the production of biofuel 28 .The most important species for significant biomass production are those with faster growth rates and higher biomass productivity 29 .Biomass yield and the productivity of M. braunii significantly reduced (at p ≤ 0.05) as the nitrogen concentration was decreased (Fig. 2A).The highest decrease in biomass yield and its productivity of 0.245 g/L and 0.030 g/L/d, respectively was observed in 100% N deficiency culture as compared to the control (0.582 g/L and 0.072 g/L/d, respectively), with a reduction of 42% and 41%, respectively lower than control.In line with these results, the nitrogen deficiency in the synthetic media caused considerable reductions in dry weight and biomass productivity 30 .Nitrogen plays an important role in photosynthesis and contributes to the creation of numerous structural and functional molecules, including proteins, enzymes, and nucleic acids, which can be attributed to the high biomass yield 31 .
Dry weight and biomass productivity of Monoraphidium braunii in the late exponential phase markedly decreased under phosphorus depletion, especially in 100% of phosphorus deficiency culture, with a reduction of 58% and 62% compared to the control (100% P), respectively (Fig. 2A).In this respect, Xin et al. 32 showed that phosphorus starvation reduced the total biomass of Scenedesmus sp.These findings revealed that nitrogen depletion had a greater effect on M. braunii growth than phosphorus depletion.On the other hand, the deficiency of nitrogen and phosphorus caused the fixing of carbon to lipids or carbohydrates synthesis.As a result, N and P stress conditions are regarded as the most critical for lipid metabolism 33 .
Concerning salt stress, dry weight and biomass productivity of M. braunii were considerably increased up to 150 mM NaCl.In culture treated with 100 mM NaCl, the highest dry weight and biomass productivity were observed (0.62 g L -1 and 0.078 g L -1 d -1 , respectively ), with an increase of 20% and 19%, respectively when compared to the control followed by 50 mM NaCl (0.588 g L -1 and 0.074 g L -1 d -1 , respectively) corresponding (0.517 and 0.065 g L -1 d -1 , respectively) in the control.While increasing salinity levels reduces the biomass and its productivity, the highest percentage reduction in biomass and biomass productivity was 48.74 and 46.15%, respectively less than the corresponding control after 9 days of incubation at 250 mM NaCl (Fig. 2B).Low concentrations of NaCl are beneficial for some metabolic processes and promote microalgae growth.On the other hand, high levels of NaCl concentrations may cause inhibition of growth and death of cells 34 , these findings were consistent with our research.In contrast, El-Sheekh et al. 2 reported that 300mM NaCl exhibited the greatest growth of Microchloropsis salina as compared to other salinity concentrations.Normal salt stress affects phytoplankton through (1) mechanisms of ion homeostasis (2) alterations in cellular ionic ratios caused by selective ion permeability in the membrane, (3) Osmolytes are classified into types: osmolytes and osmoprotectants 35 .Microalgae have unique mechanisms for adjust with salinity stress.Mechanisms may involve accumulating osmo-protective solutes, producing antioxidant enzymes, regulating ion exchange processes, and shifting from active cell division to energy storage through lipids.As a result, microalgae can tolerant salinity stress and grow more efficiently 36 .

Photosynthetic pigments under nutrient deficiency and salt stress
During this study, the pigments, chlorophyll (Chl.)a, b, and carotenoids were assessed every two days which could be used as a biomass criterion and an indicator for the growth of microalgae.The results in Fig. 3 revealed that exposure of M. braunii cultures to the reduction in phosphorus concentration was accompanied by a significant decrease in photosynthetic pigment contents (Chl.a, b, and carotenoids).
100% nitrogen deficiency caused Chl.a, b, and carotenoids decrease by 46.74, 47.04, and 62.35%, respectively contrasted with control (100% P).A similar trend was observed in the results of phosphorus reduction cultures (Fig. 4), where the Chl.a, b, and carotenoid contents decreased by 47.64, 52.27, 75.44%, respectively, after 9 days of cultivation.Phosphorus is a vital abiotic macronutrient for the development of cellular structure, nucleic acid synthesis, algal growth, and protein synthesis.Low protein content and chlorophyll destruction are both consequences of phosphorus deficiency 37 .According to Breuer et al 38 , nitrogen deficiency causes a reduction in protein, which results in a decrease in chlorophyll content in microalgae cells.This apparent reduction in chlorophyll content could be explained by nitrogen restriction on the photosynthetic system within algal cells 30 .In this regard, Xin et al. 32 stated that phosphorus starvation reduced the chlorophyll content of Scenedesmus sp.
Salt toxicity limits the rate net of CO 2 assimilation, which lowers the chlorophyll (Chl) and growth 39 .The content of photosynthetic pigments, Chl a, Chl b and carotenoids of M. braunii were measured every two days are revealed here (Fig. 5).Chl.a, Chl.b, and carotenoids of M. braunii were considerably increased with increasing salinity up to 100 mM NaCl (23, 24 and 22% over the control, respectively).The maximum percentage reduction in Chl.a, Chl.b, and carotenoids was recorded 40%, 36% and 24%, respectively with 250 mM NaCl compared to the control on the 9th day of cultivation (Fig. 5).These results were consistent with those of Fal et al. 40 , who found that at 0 and 200 mM NaCl, Chl a, and Chl b levels of C. reinhardtii were significantly decreased from 7.15 mg L -1 and 1.67 mg L -1 to 5.28 mg L -1 and 1.24 mg L -1 , respectively.Pandit et al. 24 discovered similar results using Chlorella vulgaris under salinity stress, reporting that low salinity dose increased photosynthetic pigments and decreased at higher concentrations.On the contrary, Pandit et al. 24 , using Acutodesmus obliquus under salinity stress, found that low salinity dose reduced photosynthetic pigments.Higher salinities cause lower chlorophyll contents due to osmotic and toxic ionic stress, which reduces the photosynthesis rate and thus lowers chlorophyll and protein content 29 .Chl a deficiency may be a sign of oxidative stress brought on by increased chlorophyllase activity, which promotes the degradation of Chl 41 .Also, it could be related to a decrease in Rubisco activity as a result of the low CO 2 uptake 39 .

Lipid content and lipid productivity of M. braunii under nutrient deficiency and salt stress
Lipid content of M. braunii increased significantly during cultivation under nitrogen and phosphorus depletion, reaching a maximum of 229.63 and 212.09 mg g -1 at 75% nitrogen and phosphorus deficiency, respectively, which represented more than twofold of the control (Fig. 6).The increase in lipid content in algal cells in response to nitrogen deficiency is thought to be a defense mechanism to prevent protein breakdown and a decline in growth dry weight 42 .The reduction of N and P was effective for the lipid productivity of M. braunii as shown in Fig. 6.A 75% reduction in nitrogen and phosphorus was the most effective condition, where lipid productivity increased by more than threefold and twofold (up to 209 and179%, respectively) of the control grown under 100% nitrogen (N-NO 3 -) and phosphorus (P-PO 4 3-), followed by sever nitrogen (100% deficiency) induced lipid productivity up to 2.2 fold of the control for M. braunii.These findings revealed that decreasing nitrogen concentration had a greater effect on M. braunii lipid accumulation and productivity than phosphorus concentration reduction.These results are in agreement with other studies that investigated the effects of the reduction in nitrogen and phosphorus concentrations on lipid production 15,43 .In general, nitrogen concentration has a significant effect on the biochemical compositions and growth of microalgae; conversely, nitrogen depletion in culture medium results in a reduction in growth with concurrent enhanced lipid productivity 44 .Nitrogen starvation is an effective method for increasing lipid production in microalgal cells.Lipids produced in the case of nitrogen deficiency are two-fold those produced in the case of nitrogen-sufficient medium 45 .Phosphorus plays a critical role in the transfer of energy.In the deficiency, it increases the lipid accumulation of algae.Nutrient deficiency (i.e., nitrogen and phosphorus limited) for algal cells is not unusual in the natural environment, where nutrient limitation shifts the organism's metabolic pathway.For instance, the metabolism of lipids changes from neutral lipid storage to membrane lipid synthesis when nitrogen and phosphorus are deficient, as a result, the total lipid content of green algae increase 46 .
Concerning saline stress, the lipid content and lipid productivity of M. braunii increased significantly as the NaCl concentration increased from 50 to 250 mM, increasing more than threefold of the control at 250 mM NaCl (Fig. 6).
Numerous studies have found that unfavorable conditions, such as nutrient deficiency or high salinity, promote higher lipid levels 40,47 .These results are in agreement with another study in which the lipid content in Botryococcus braunii grown in 0.50 M NaCl was higher than those without NaCl 48 .Hang et al. 49 found that a high NaCl concentration (200 mM) significantly increased lipid content (41.1%) in C. reinhardtii compared to the control (20.8%) after 3 days of application.To increase the production of lipids and value-added products, microalgae's stress tolerance and environmental stress tolerance are frequently altered 50 .
According to Fal et al. 40 , the content of lipids also increased significantly, rising from 9.35% and 27.18% under 0 and 200 mM NaCl conditions, respectively.This lipid accumulation, specifically neutral lipids, reduces the osmotic pressure and fluidity of cell membranes, which helps to maintain the membrane integrity in response to salt stress 41 .According to Dahmen et al. 51 , a high salinity growth medium increased microalga cellular content by enhancing TAG formation.The most effective microalgae for producing biodiesel are essentially determined by their lipid accumulation and lipid productivity both of which are influenced by biomass production 31.www.nature.com/scientificreports/

Gas chromatography (GC) analysis of fatty acids
Characteristics of fatty acid profiles determine whether a lipid is suitable for biodiesel production even if lipid content and productivity are important factors in determining the best conditions for biofuel production 52 .As a result, the current study included a complete analysis of fatty acid composition.The fatty acid profile of Monoraphidium braunii showed an increase in saturated fatty acids (SFAs) C14:0, C16:0, and C18:0 and monounsaturated fatty acids (MUFAs) C16:1 and C18:1 while exhibiting a decrease in in in polyunsaturated fatty acids (PUFAs) C18:2 and C18:3 under reduced nitrogen and phosphorus concentrations, as well as salinity stress when compared to the control conditions (Table 1 and Fig. 7).Saturated fatty acids (SFAs) exhibited the maximum proportion, which varied from 60.93 to 76.98% of total fatty acids (TFAs), followed by polyunsaturated fatty acids (PUFAs) (12.29 to 29.45%) and monounsaturated fatty acids (MUFAs) (7.62% to 18.46%) in Monoraphidium braunii.The biomass produced the highest percentage of SFAs (76.98%) under 250 mM NaCl followed by 75% of nitrogen deficiency (74.5%), in excess of 20% of biomass produced in the control.The saturated fatty acids, palmitic acid (C16:0) and stearic acid (C18:0) were the most abundant fatty acids in the M. braunii oil biodiesel under different conditions.Monounsaturated fatty acids (MUFAs) in the M. braunii were represented by oleic acid (C18:1) and palmitoleic acid (C16:1), oleic acid was the major monounsaturated fatty acid that was found in M. braunii oil biodiesel where the highest value 15.85% of total fatty acids at 75% of phosphorus deficiency.Polyunsaturated fatty acids in the M. braunii showed the highest value (29.45%) at the control condition (100% N and P) and the highest decrease by 41% of the control at 250 mM NaCl.These data agree with Sukkrom 53 reported that the C 16 and C 18 fatty acid groups accounted for more than 80% of the total fatty acids in microalgae cultured in all media.Also, Widianingsih et al. 54 stated that the most common fatty acids found in biodiesel were palmitic, stearic, oleic, and linolenic acids.These results were also consistent with Dahmen et al. 51 who observed that salinity stress increased total SFAs and MUFAs while decreasing PUFAs in microgreen algae.Variations in the fatty acid profile in the medium as salinity levels rise are required to keep the membrane fluid intact and prevent its destruction 35 .
The current study discovered that the FA profile changed with different conditions, resulting in fatty acid content variation.These findings were in line with those of Srinuanpan et al. 55 , which exposed that variation in the fatty acid profile caused by nutrient manipulation that was considered favorable for biodiesel quality.
The proportions of SFA, MUFA, and PUFA in the FAs compositions varied significantly under various circumstances (p ≤ 0.05) (Fig. 7).This might be a result of variation in lipid production and composition of fatty acid in response to various stressors 56 .Algal lipids had higher SFA levels than biodiesel's traditional soybean and canola oil feedstock 57 .Furthermore, microalgae with low growth rates in nitrogen and phosphorus-deficient conditions accumulate more saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) into neutral lipids.Contrarily, higher growth causes more accumulation of polyunsaturated fatty acids (PUFA) 58 .As a result, we can conclude that Monoraphidium braunii is a suitable producer of saturated fatty acids that are easily convertible to biodiesel under nitrogen and phosphorus deficiency as well as salinity stress.

Biodiesel properties
Biodiesel properties are primarily determined by the fatty acids profile of the feedstock used.The most suitable fatty acid methyl esters (FAMEs) for high-quality biodiesel are those with longer chain lengths and lower levels of carbon chain unsaturation 59 .Biodiesel of Monoraphidium braunii which is cultivated in nitrogen and phosphorus medium starvation as well as salt stress had high saturated and low unsaturated fatty acid content, which is preferable for high-quality biodiesel because saturated fatty acids promote transesterification 60 .The values of unsaturation degree (Du), kinematic viscosity (KV), specific gravity (SG), cloud point (CP), cetane number (CN), iodine value (IV), saponification value (SV), higher heating value (HHV), Long-chain saturation factor (LCSF) and cold filter plugging point (CFPP) were estimated in present study (Table 2), were found to be in line www.nature.com/scientificreports/with LCSF and CFPP in the international standards (ASTM D6751 and EN 14,214).The degree of unsaturation (DU) was calculated by multiplying double bonds C=C and the mass fraction of each individual fatty acid, and it mainly indicates the number of unsaturated FAs.The degree of unsaturation (DU) was decreased with nitrogen and phosphorus starvation as well as salt stress to reach to lowest value of 0.49% in the case of 200 mM NaCl.The high DU value of the biofuel indicated that it was more difficult to form deposits and had better lubricity 34 .The cetane number (CN) increases with increasing saturation level of fatty acid and represents the ignition quality of the biodiesel and it 61 .The results showed an increase in cetane number under different stresses where the highest value is 59.58 at 200 mM of NaCl compared to 58.98 at the control, followed by CN value 58.99 and 58.98 at 50% phosphorus and nitrogen deficiency, respectively compared to 57.44 in the control biodiesel which is due to the high content of palmitic acid (C16:0) and stearic acid (C18:0).These values confirm that the microalgae biodiesel has a high ignition quality and meets the lowest CN values prescribed by international EN-14214, ASTM D675140 biofuel standards, the Australian standard, and the Brazilian National Petroleum Agency.It was noted that the cetane number varies greatly depending on engine speed, the lower the engine speed, the lower the cetane number (as low as 20) of fuel it can use 62 .The high value of cetane number is often related to enhanced performance and cleaner fuel combustion.A high CN value is preferred for biodiesel production because it is proportional to oxidative stability and ignition quality and can be achieved at lower unsaturation levels 63 .There is a strong positive correlation between low NOx emissions and a high cetane number, which is a desirable characteristic in the production of clean fuel 64 .Considering that fossil diesel has a cetane number of 49.6 65 .Iodine value (IV) means the amount of iodine absorbed in 100 g of the fuel sample by double bonds of the FAME molecules.A higher iodine value indicates higher fats and oils, lower value of IV is favorable for biodiesel quality 17 , the results showed IV value was decreased with nitrogen and phosphorus starvation as well as NaCl to reach the lowest value of 49.51 (g I 2 .100g -1 oil) in case of 200 mM of NaCl and IV found to be 56.02 and 56.14 (g I 2 .100g -1 oil) at the depletion of phosphorus and nitrogen by 50%, respectively that proportionated to the USFAs 60 .The IV value of M. braunii with and without stress was lower than the maximum IV (120 g I2/100 g) of biodiesel prescribed by EN 14,214  The Saponification value (SV) is the amount of KOH (mg) required to saponify one gram of oil.The molecular weights of triglycerides are defined as SV in the oil.It is in reverse to the average molecular weight or the chain length of the fatty acids 66 .Thus, long-chain fatty acids are present in the lipids indicating a low SV.The limit of saponification value was not specified in biodiesel standards such as ASTM D6751, EN 14,214, and IS 15,607.The SV of M. braunii under nutrient deficiency and salt stress were 209.24-210.59mg KOH/g oil, which is close to those of M. reisseri SIT04 (208.02-214.51mg KOH/g oil) and S. obliquus SIT06 (210.95-216.73mg KOH/g oil) 67 .
Low-temperature properties such as cold filter plugging point (CFPP) and cloud point (CP) of biodiesel were determined based on FA composition.Most of the treated cultures FAMEs showed an increase in CFPP and high values more than biodiesel standard EN 14,214 (− 13 to − 5 °C) and ASTM D6751(≤ 5/ ≤ − 20 °C) prescribed due to the decrease of polyunsaturated fatty acids (PUFAs) and high level of SFA, mainly the palmitic acid (C16:0) and stearic acid (C18:0), thus the higher proportion of these fatty acids will affect the low-temperature properties 68 .To confirm that a suitable fuel supply reaches injectors at various operating temperatures, biodiesel must have an appropriate kinematic viscosity (υ) 69 .Kinematic viscosity (υ), which has an inverse relationship with temperature, affects the engine's CFPP performance at low temperatures.According to ASTM6751-02 and en14214, the kinematic viscosity limits are 1.9-6.0mm 2 s -1 and 3.5-5.0mm 2 s -1 , respectively.For M. braunii strains, the overall kinematic viscosity of the biodiesel fuel is within the range.A specific cloud point (CP) value has not been specified by the biodiesel standards (ASTMD6751 and EN1424) because of the wide seasonal and temperature.In the present study, the results revealed that the determination cloud point for algal culture with different concentrations of nitrogen and phosphorus, as well as NaCl, increased under deficiency conditions where it ranged from 9.11 °C at the control to 12.82 °C at 50% P.This is in line with the previous studies that CP value of −2.47 to 9.71 °C70 .A higher heating value (HHV) of biodiesel is an important property that indicates the amount of heat released by the fuel combustion of a unit quantity of fuel.The present results showed a lower HHV value under cultivation stress conditions except for 100 and 250 mM of NaCl exerts HHV more than control, where it ranged from 39.40 to 39.97 Mj kg −1 .These values were acceptable according to the previous studies 56 , and they agree with earlier findings for Anabaena variabilis (39.87 Mj kg −1 ) 48 .Accordingly, its biodiesel properties produced by growing in the nitrogen and phosphorus starvation as well as salinity stress in this study can be applied to modern engines.In general, the results shown in Table 2 were maintained by the study, which asserted that most of the properties of biodiesel came from the species of microalga that were studied and met the limit values set by ASTM D6751 and EN 14,214:2012 + A2:2019 biodiesel standards 71 .www.nature.com/scientificreports/It is worth noting that growing microalga Monoraphidium braunii in nutrient-restricted environments and saline stress has advantages for both the economy and the environment and is the recommended best approach for producing a lot of algal lipid content, productivity, and biodiesel.Furthermore, it was exposed that the properties of biodiesel of Monoraphidium braunii grown under these conditions are highly aligned with global biodiesel standards (ASTM D6751 and EN 14,214), assembly it is a strong contender for large-scale biodiesel production.
According to the findings of this study, the native strain M. braunii could be considered a promising feedstock for sustainable energy production.

Microalgae and growth conditions
The green alga Monoraphidium braunii was isolated for the first time from a water sample from South Valley University, south Egypt and grown in Erlenmeyer flasks of 500 ml in Beijerinck media 72 under continuous illumination fluorescent light of 100 µmol m −2 s −1 at 25 °C and aerated with an air pump.The algal cultures were subjected to different deficiencies of nitrogen, and phosphorus (25%, 50%, 75%, and 100%, Table 3).Beijerinck media with 100% nitrogen and phosphorus concentrations were considered the standard treatment (control).Salinity stress was investigated using various concentrations of NaCl (50, 100, 150, 200, and 250 mM) added to the culture media, where the culture without NaCl was used as a control.Algal cells were added to the media to give a concentration of 5%.After 9 days of cultivation, the microalgal strains were harvested to determine and compare the biochemical analysis, fatty acids profile, and biodiesel properties.

Optical density
To estimate the growth of each algal culture, optical density at 560 nm (OD560) was determined using a spectrophotometer every two days.This wavelength was recommended for green algae by Wetherel 73 .Plot the growth curve of the respective algae cultures by using the absorbance obtained.www.nature.com/scientificreports/

Biomass yield and biomass productivity
Algal growth was measured also by determining the cellular dry weight (CDW) of algal species and biomass productivity.To measure the biomass productivity of M. braunii, the algal suspension (30 mL) was centrifuged at 4000 rpm on the last day of culture for 15 min, followed by several thorough washes with distilled water to remove trace of the growth medium, then dried overnight at 60 °C until constant weight.Biomass productivity was calculated using Eq.(1), as discussed by Abomohra et al. 74 .
where CDW E and CDW L represent the cellular dry weight (CDW) at the early exponential phase (t E ) and at the time of the late exponential phase (t L ) as g/L, respectively.

Photosynthetic pigment
The pigment contents (Chl.a,Chl.b, and carotenoids) were assessed using a spectrophotometer every two days in agreement with the method of Metzner et al. 75 .Pigment fractions were calculated by µg/ ml algal suspension.

Determination of total lipids and productivity
To extract total lipids from algae, El-Sheekh et al. 76 modified protocol was used.Cells were homogenized in a 2:1 mixture of chloroform and methanol.After an orbital shaker the mixture was stirred at room temperature for 20 min.0.2 ml of 0.9% sodium chloride solution was used to wash the solvent.The homogenate was separated into two phases using centrifugation.The lipid extracts were dried in 30 min at 80 °C, an argon stream, in pre-weighted glass vials, cooled in a desiccator, and then weighed.Andarad and Costa 77 (Eq.2) were used to determine lipid productivity, which was modified by Abomohra et al. 74 .
where L 0 and L D indicate the total lipids (mg L -1 ) at the first day of cultivation (T 0 ) and the desired days phase (T D ), respectively.

Fatty acids methyl ester (FAMEs) determination
The lipid extracts were trans-esterified prior to gas chromatography analysis to fatty acid methyl esters (FAMEs) 78 .
A DB-WAX column (30 m × 250 m internal diameter and 0.25 m film thickness) was installed in the GC.The constituents were identified by comparing the spectrum stored in the Wiley and NIST Mass Spectral Library data to the spectrum fragmentation pattern of the algal extracts.
where C = refers to the number of C=C double bonds in fatty acid, while Mf represents mass fraction of each individual fatty acid.
In the previous equations, D is referred to the number of double bonds in the fatty ester, M is the molecular weight of the fatty ester, and N is the percentage of the certain fatty ester in the oil sample.In the above Eq.( 11), C16, C 18 , C , C 22 , and C 24 refer to mass fraction of saturated fatty acids containing C16, C18, C20, C22, and C24, respectively.

Statistical analysis
The experiments were carried out in triplicate and the results are shown as means standard deviation (± SD).Statistical analyses were carried out by using SPSS (version 23).One-way analysis (ANOVA) was used to compare mean values and to determine whether the results were statistically significant at probability levels ≤ 0.05, the differences were significant the post-hoc Dunn's multiple range test was used.

Figure 1 .
Figure 1.The growth curve of green microalga M. braunii grown in nitrogen (A) and phosphorus (B) deficiency and salt stress (C).Data are means of replicates with standard deviation (SD).

Figure 2 .
Figure 2. Biomass and biomass productivity of green microalga M. braunii grown in nutrients deficiency (A) and salt stress (B).Data are means of replicates with standard deviation.Means followed by different letters are significantly different according to Duncan's multiple range comparison (DMRTs), and the same letters are not significantly different.

Figure 3 .
Figure 3. Chl.a, (A) Chl.b (B) and carotenoid (C) contents of green microalga Monoraphidium braunii grown under nitrogen deficiency.Data are means of replicates with standard deviation.

Figure 4 .
Figure 4. Chl.a, (A) Chl.b (B) and carotenoid (C) contents of green microalga Monoraphidium braunii grown under phosphorus deficiency.Data are means of replicates with standard deviation.

Figure 5 .
Figure 5. Chl.a, (A) Chl.b (B) and carotenoid (C) contents of green microalga Monoraphidium braunii grown under different concentrations of salt stress (NaCl).Data are means of replicates with standard deviation.

Figure 6 .
Figure 6.Lipid content and lipid productivity of green microalga M. braunii grown in nutrients deficiency (A) and salinity stress (B).Data are means of replicates with standard deviation.Means followed by different letters are significantly different according to Duncan's multiple range comparison (DMRTs), and the same letters are not significantly different.

Table 2 .
Biodiesel characteristics based on the fatty acids profile of M. braunii cultivated in nutrient deficiency and salt stress in comparison with the international standards.DU: Degree of unsaturation, KV: kinematic viscosity, SG: Specific gravity, CP: Cloud point, CN: Cetane number, IV: Iodine value, SV: Saponification value, HHV: Higher heating value, LCSF: Long-chain saturation factor, CFPP: Cold filter plugging point.ASTM International (formerly American Society for Testing and Materials)-EN 14,214, The European biodiesel specification.

Table 3 .
The concentrations of the different nutritional compositions of Beijerinck media (mg L −1 ).