Growth Performance, Oil Profile and Fuel Properties of Spirulina Platensis Grown in Organic Carbon Waste Extract

Growth Performance, Oil Profile and Fuel Properties of Spirulina Platensis Grown in Organic Carbon Waste Extract

(1)El-Awady, R. M.; (2)El-Sayed, A. B.; (3)Marwa, M. Reda; and (4)Ghazy, S. M.

1,4Environment and Bio-Agriculture Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt 2 Algal Biotechnology Unit, Fertilization Technology Department, National Research Centre, Giza, Egypt 3Center laboratory of environment quality monitoring, National Research Centre, Giza, Egypt

Abstract – Wastes of date pits represent an average of 10% of date fruits. Thus, there is an urgent need to find practical and economical use applications for this waste fraction. This study was achieved aimed to evaluate whether date waste water extract (40ml. l-

1) as an alternative carbon source affects Spirulina platensis growth in concern cell metabolites (dry weight, chlorophyll, carotenes, and oils). Furthermore, the extracted oil was then evaluated as a new fuel source.

In the laboratory, Spirulina was grown under different concentrations of date palm waste Extract (DPWE) within a vertical tubular photobioreactor (15 units x 14 L). In situ, the photobioreactor was made from fully transparent Plexi-Glass. Scaling up was then carried out within open ponds (75m3)

Zarrouk growth medium presented the control one, while the formulated growth medium containing organic carbon was compared during in, and out-door growth.

Data revealed the enhancing effect of date waste on all examined concentrations in concern dry weight, and total carotenes, while the opposite manner was observed with chlorophyll content under the higher concentrations of date waste.

The maximum dry weight (0.983g. l-1) was obtained from cultures received 30ml.l-1 of waste followed by 40 (0.936ml. l-1 waste) compared with control and other waste volumes used (10 and 20ml. l-1). Concerning total chlorophyll, control cultures represented the maximum (5.259mg.l-1), however 20 ml.l-1 of waste resulted in the maximum increase. Increments ratio and other evaluated parameters exhibited the same pattern. Carotenes mostly represented the dry weight response.

The produced oil was richer in unsaturated fatty acids being 56.3% compared to 43.7% saturated fatty acids. Algal oil was then trans-esterified with methanol/KOH in presence of sulfuric acid as a catalyst. Prediction of fuel priorities showed the superior effect of waste on the produced oil as compared with those of Zarrouk produced oil.

INTRODUCTION

In natural habitats, algal growth cycles depend mainly on the nutrients supply. In addition, there are more than 22 countries invested in Spirulina mass production and the total industrial production of Spirulina is almost 3000 tons per year (Shimamatsu, 2004). Spirulina flourishes in alkaline brackish water and its growth medium should provide it by all essentials nutrients (Raoof et al., 2006). The nutritional inputs mainly carbon and nitrogen limiting the successful investment on this sector. Carbon represented about 50% of algal cell composition and a100 tons algae biomass can fix183 tons CO2 (Chisti, 2007). Artificially, 16.8g. l-1 NaHCO3 and 2.5g.l-1 of NaNO3 were recommended for the proper growth of Spirulina (Zarrouk, 1966). The use of organic carbon sources instead of sodium bicarbonate could reduce the production coast and decreasing of liberated sodium ions and bicarbonate oxidation. Such technique was performed mainly using the organic food wastes including corn steam liquor (El-Sayed et al., 2015); citrate waste (El-Sayed, 2010 and El-Sayed et al., 2012); okara waste (Sheraz et al., 2017); cheese whey (Asmaa et al., 2023 a, b), carboxymethyl cellulose (Reem et al., 2022); bagasse wastes (El-Sayed et al., 2020, 2022 and Nashwa et al., 2022), and date palm (El-Awady et al ., 2020). As for nitrogen, urea was used instead nitrate (K or Na salt) which also contains a little amount of carbon (El-Shafey et al., 1999; El-Sayed, 1999 and El-Sayed et al., 2001).

The wasted human consumption yearly is accounted by about one third of the food produced. The think of food waste application as feedstock in micro-organisms cultivation allows the recycling of waste matters consisting of carbon, nitrogen and phosphorous compounds (Luque and Clark, 2013). Date palm has increased from 1.8 million tons in 1961 to 7.627624 million tons in 2017; Egypt is the top palm date producer in the world (1590414 metric tons) (FAO, 2017). Date palm fruit is very rich in monosaccharides (fructose and glucose), minerals, vitamins with small amount of sucrose (Hasnaoui et al., 2010). Fructose and glucose constitute over 75% of the dry weight of pitted dates. Fructose is the sweetest natural sugar; 30% sweeter than sucrose,

while glucose has only 70% of the sweetness of sucrose (Mostafazadeh et al., 2011). It is also rich in several nutrients such as nitrogen, phosphor, potassium, calcium, magnesium, etc. and has a high carbohydrate and fat content and is a vital source of sugar and dietary fiber (Al-Farsi et al., 2007). A second-grade (or low-grade) dates showed the same sugar content as dates of high quality. Lost dates, which account for more than 2,000,000 tones every year (30% of production), are discarded due to their inadequate texture (Besbes et al., 2009). These huge amounts of unused dates could be utilized for the production of fructose, ethanol, acetic acid, lactic acid and other valuable products (Zeinelabdeen et al., 2010).

Spirulina is now produced by several companies and sold in many health food stores around the world because it serves as a rich source of protein (55-70%), vitamins, minerals, essential fatty acids, essential amino acids and pigments such as chlorophyll- a, phycobiliproteins and carotenoids for food, chemical and pharmaceutical industry. It is the major known source of vitamin B12, having high protein content. Due to lack of cellulose in its cell wall, 8595% is assimilated by the organism (Kawata et al., 2004, Harriet et al., 2008 and Borowitzka and Borowitzka., 2010). Growth conditions can affect the growth rate of algae according to its level in algal growth media, including CO2 levels, pH, temperature, nutrients availability (nitrogen, phosphorus, hydrogen, potassium, zinc, boron and magnesium) and presence or absence of other organisms (Juneja et al., 2013).

The aim of the current work is to use palm waste as a novel alga growth medium which in turn reduces the production costs via the utilization of initial organic carbon as well as minerals and other organic components.

MATERIALS AND METHODS

Alga and growth conditions

Microalgae Spirulina platensis belonging to Cyanophyta was obtained from Algal Biotechnology Unit, National Research Centre (NRC); Egypt. Growth was indoor performed using Zarrouk growth medium (Zarrouk, 1966). Growth container was created from fully transparent Plexi Glass column with initial diameter 7.5cm x 100cm containing 6.0 L of growth medium. Growth conditions were optimized by continuous light illumination which provided from one side light bank with five white cool fluorescent lamps (40 watt) and aerated by air-left system from free oil compressed air from the lower hold of growth container. Inoculum was laboratory prepared after centrifugation and washing two times using laboratory cooling centrifuge (RUNNE HEIDELBERG, RSV-200, Germany).

Date palm waste water extract preparation

Date palm waste was obtained from Food Technology Research Institute, Soybean Processing Center, Agriculture Research Center, Giza, Egypt. It was freshly collected and freeze till use. Samples of waste dates "Phoenix dactyliferaquot; were collected from El-Wahat, Giza Governorate, Egypt. The samples were washed under running tap water for five min to remove dust and reduce the number of contaminating microorganisms. After washing, the date stones were separated from flesh then dried at 55°C for 24 hrs. (s) using circulated oven (Baker et al., 2009). The dried fruits were powdered using an electric hammer mill and passed through a 0.9 mm sieve to obtain a fine powder. Samples were stored at 4oC until use.

Chemical analysis of Date palm waste

Protein was determined as total nitrogen (T.N) x 6.25, where T.N was determined based on Micro-Kjeldahl method (Ma and Zauzag, 1942); carbohydrate by phenol sulfuric method (Smith et al., 1956); oils by weight differences of Soxhelt extracted samples using n-hexane : isopropanol solvent mixture (3:2, v:v) according to A.O.A.C (2000) and fibers (Holst, 1982). The sample was collected and subjected to elemental analysis after ashing (500oC / muffle furnace) and dissolved in 1.0 N HCl. The filtered digest was then measured for P, K, Ca and Na (Chapman and Pratt, 1974). Total carbon, organic carbon and organic matter were determined (Shimadzu, 2012 and Baird et al., 2017).

Date palm waste water-extract treatments

The used volumes and concentrations were selected based on their initial concentration of total nitrogen in waste compared with those original growth medium (Zarrouk). Thus, algal growth was achieved under enriching growth medium by 0.0, 10, 20, 30 and 40 ml.l-1 of waste extract. Prior inoculation, waste was diluted by 4 fold of bi-distilled water and then filtered through sequences filtered sizes.

Alga growth parameters

During the whole growth period, the investigated growth parameters were dry weight, total chlorophyll and total carotenes. Dry weight (g.l-1) was determined by filtering a define volume over pre-weighted filter membrane (0.45µm). After drying, weight difference expressing the accumulated dry weight within a define growth period. Total chlorophyll (mg.l-1) extracted by 95% DMSO (Burnison, 1980); measured at 666nm and calculated according to (Seely et al., 1972). After saponification, total carotenes (mg.l-1) were extracted by 95% DMSO and their absorbance was 468 nm (Boussiba et al., 1992). Parallel, growth analysis was performed according to the adopted equations of Pirt (1973).

Oil extraction

The wet Spirulina biomass was freeze and then soaked with n-hexane solvent (Gouvein and Oliveira, 2009) to extract oils. After centrifugation, the precipitate was re-extracted and all of the supernatant was collected and water was eliminated using separation funnel. Based on the pre dry weight estimated, oil content was determined as weight differences.

Fatty acids methyl ester

An oil sample was first methylated following the procedures of Ludde et al. (1960) and it was then analyzed for its fatty composition by Gas – liquid chromatography.

GC analysis was performed by using a Perkin Elmer Auto System XL model equipped with flame ionization detector (FID). A fused silica capillary column DB5 (60m x 0.32mm i.d.) was used. The oven temperature was programmed from 50 to 240°C at a rate of 3°C/min. Helium was used as the carrier gas, at flow rate 1.1 ml/min. The injector and detector temperatures were 220 and 250°C, respectively. The isolated peaks were identified by comparison with those of authentic fatty acids (Supelco TM 37 component FAME mix).

Biodiesel from algae was prepared by trans-esterification of Spirulina oil with methanol in presence of potassium hydroxide and then neutralized by 2% of sulfuric acid which precipitate the liberated potassium ions into potassium sulfate. Although the stoichiometric number of moles of alcohol needed to esterify one mole of oil is three, the reaction was made using six moles alcohol rather than three. The alcohol was used in such excess of alcohol as to force the reaction which is reversible in the forward direction so that all oil will be completely esterified. The reaction was carried at the boiling point of the alcohol. At the end of the reaction, it was left to cool and then carefully transferred to a suitable separating funnel where it was separated into two clear layers. The upper layer which contained esters, traces of alcohol and acid catalyst was separated from the lower one which contained glycerol, alcohol and acid catalyst. Then, the upper layer was washed with distilled water till neutralization, dried over anhydrous sodium sulfate and the residual alcohol was evaporated using rotary evaporator. It should be notified that the reaction progress was followed up during the esterification process by thin layer chromatographic analysis of samples drawn over definite time intervals until completed.

Fuel proprieties estimation based on FAMEs profile

The cetane number (CN), saponification value (SV), iodine value (IV), The degree of unsaturation (DU), long chain saturated factor (LCSF) and cold filter plugging point (CFPP) were determined by experimental equations according to Francisco et al. (2010); Nascimento et al. (2013); Knothe (2007); Sarin et al. (2009); Krisnangkura (1986) and Agarwal et al. (2010). International biodiesel standards such as European (EN 14214), American (ASTM D6751), and Indian (IC15607) provide biodiesel fuel specifications (Hoekman et al. 2012).

Statistical analysis

Statistical analysis systems were carried out using SPSS (2011), IBM SPSS Statistics for Windows, Version 20.0. Variables having significant differences were compared using Duncans multiple rang tests (Duncan, 1955). All experiments were replicated three times where data were presented as means of three replicates. Data obtained were analyzed statistically to determine the degree of significance using one-way analysis of variance (ANOVA) at probability level P 0.05.

RESULTS AND DISCUSSION

Date palm water extract chemical composition

As present in Table (1); the percentage of the total protein, fat, crude fiber, total ash and total carbohydrates in dry date palm fruit wastes were 3.86, 2.77, 8.98, 6.76, and 72.15, respectively. Such biochemical composition seems to be more promising in alga nutrition due to the high initial load of carbon as sugars (72.15%); where carbon nutrition of algae in general represented the maximum figure in production costs which accounted by 70% (Zaborsky, 1985). Spirulina carbon nutrition seems to be a special case, where the main carbon source received from NaHCO3 (16.8g. l-1). Utilization of such concentration led to liberation of high sodium ions (27.39% of salt molecular weight); which not utilized by alga and liberated to the growth medium resulted in salt stress. Thus, organic carbon seems to be more efficient (El-Sayed and Almutirii, 2024).

Table 1. Biochemical profile of Date Palm waste

CHO = carbohydrates; TC= total carbon; OC= organic carbon and OM = organic matter

In addition, the percentage of the organic carbon (Table 2), total carbon, organic matter, N and P of date palm wastes extract were 1.28, 1.67, 2.21, 0.06, 0.03 %, respectively and K, Ca, Mg and Na were 360, 41.5, 45.5, 50.5 ppm, respectively; in which support the growth medium by all mineral nutrition requirements.

Table 2. Macro and secondary-nutrients content in Palm Date and Zarrouk growth medium

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*Values of the original extract

On this context, the used digested effluent by Soni et al. (2017) is a source of low-cost nutrients rich in C: N: P with a ratio of 24: 0.14: 1, supports the growth of Spirulina. This ratio increased the proportion of protein, carbohydrates, and fats in biomass to be 68, 23, and 11%, respectively. On the other hand, latex serum containing mineral salts and organic compounds with Mg:P:N:C medium in a ratio of 0.2:0.3:3:1 gave 0.350 g.l-1 carotenoids of Spirulina biomass, whereas the medium was 0.407 g.l-1 for two months. Expectedly, the initial content of macronutrients (NPK) is able to fulfill the proper growth of Spirulina alga.

Comparing the initial content of date waste extract with those of the recommended medium in concern macro (CNPK) and secondary-nutrients (Ca-Mg-Na) showing that the sum of carbon ions received from 16.8g. l-1 NaHCO3 supporting growth medium by 2.4 g carbon. On the other hand, the given concentrations of date wastes (1.0 4.0%) only support growth medium by 0.0084, 0.0168, 0.0252 and 0.0336 g.l-1 of carbon ions. However these concentrations are very low comparing with the original medium, their effect surpass the original due to the role of bicarbonate in Spirulina growth medium in open air cultures.

The loss of carbon (CO or carbonate ions) from bicarbonate salts is a well-documented process that occurs through different mechanisms, depending on environmental and chemical conditions of algal growth medium. For instance, thermal decomposition took place under warm conditions producing CO2 gas and carbonate ions (Na2CO3). In addition, Lowering media reaction (pH) during growth increase bicarbonate release carbon dioxide (Lindsay, 1979); low activity of cell metabolism in concern carbonic anhydrase enzyme (Tripathi et al., 2007); direct liberation of carbon dioxide to the outer media especially in open and aerated cultures, where exposure to sunlight can accelerate CO2 degassing from bicarbonate-rich waters (Drever, 1997) and evaporation concentrates bicarbonates, leading to precipitation of carbonate salts and CO release (Stumm and Morgan, 1996).

Effect of palm date wastes extract concentrations on S. platensis cell metabolites Dry weight

The palm date waste extract (PDWE) increases the dry weight accumulation of all S. platensis grown cultures as compared with control cultures that received only nutrients from recommended growth medium. Here, the net obtained biomass was 0.566, 0.146, 0.323, 0.594 and 0.515 g. l-1 with 0.0, 10, 20, 30 and 40ml/l of PDWE enriched S. platensis grown culture, respectively (Table 3). Thus, 30 ml.l-1 of PDWE concentration seems to be the most superior one that reached the maximum growth rate (0.098 g. l-1.d-1), followed by 40 ml.l-1 of PDWE (0.0936 g. l-1.d-1); then 20 ml.l-1 of PDWE (0.0816 g. l-1.d-1). The significant increase in biomass production by the examined strain was observed with increasing PDWE concentrations and the positive correlation (r2= 0.0827) was recorded among dry biomass produced by the examined S. platensis (Fig. 1A).

The growth dry weight enhancement could be attributed to the high initial load of minerals and organic carbon from waste as growth was proceeded in association with the waste volumes increments. 10 and 20 ml.l-1 of waste recorded a negative results comparing with control one in which might attributed to the insufficient nutrients supporting or alga need more long time to adapt such limitation. More than 30.0 ml.l-1 of waste represented a slight decrease comparing with control or 30.0 ml.l-1 wastes, which might goes back the acidic reaction by high sugar content.

a b c

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T.Ch. (mg.l-1)

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Fig. 1. a) Dry weight (g.l-1); b) total chlorophyll (mg.l-1) and c) total carotenes (mg.l-1) of Spirulina platensis as affected by different Date Palm waste concentrations. Z=Zarrouk; T1=1.0, T2=2.0; T3=3.0 and T4=4.0 PDWE

When growth data was subjected to the evaluation as chlorophyll content, data revealed that the highest chlorophyll content was recorded by control grown cultures (5.259 mg.l-1) followed by concentration of 30ml l-1 (5.011 mg l-1) comparing with other treatments (Table 4); however significant increase in chlorophyll production by the examined alga was observed with increasing PDWE concentrations and the positive correlation (r2= 0.1103) was recorded among chlorophyll produced by S. platensis (Fig.1B).

The inhibitory effect of PDWE on chlorophyll accumulation with increasing of the used volume might goes back to the high initial organic carbon and sugars content which blocked the physiological function of chlorophyll in concern fixation the atmospheric carbon dioxide. Thus, alga obligated to grow in heterotrophic mode reducing inert chlorophyll content on the expense of carotenoids.

Another claim on the inhibitory effect of high PDWE concentrations on chlorophyll accumulation could be attributed to their effect on growth medium salinity and acidity. It is well known that organic carbon increments on algal growth medium especially in the case of nitrogen deficiency, high light irradiation and/or salinity, chlorophyll tended to markedly decreased and disappeared in extern conditions (El-Shafey et al., 1999).

Concerning, carotenoids content of S. platensis, it was shown that medium with 30 ml.l-1 PDWE was recorded a significant increase in carotenoids compared to control one that received 0.0 ml.l-1 of PDWE. Avery slight decrease could be observed with 10, 20 and 40 PDWE which is statistically non-significant at P 0.05. Highest carotenoid content was recorded for 30 ml.l-1 followed by 40 ml.l-1 and then 30 ml.l-1 (control) of PDWE medium (0.862, 0.780 and 0.450 mg l-1 with increases 0.671, 0.537 and 401 mg l-1, respectively).

Growth analysis confirmed such hypothesis, where the maximum growth rate for S. platensis was obtained with control (0.087 g. l-1. d-1 ) followed by 30 ml. l-1 of PDWE (0.068 g. l-1. d-1) with the lowest generation time (doubling time) was recorded as 7.95 and 10.98 hours as compared with other concentrations used (Table 3).

Spirulina platensis growth rate as total chlorophyll was maximized with the control cultures which reached 0.1592 mg. l-1.d-1 followed by 30 ml.l-1 (0.1109 mg. l-1. d-1) and 40 ml.l-1 ( 0.0842 mg. l-1. d-1). Chlorophyll decline was observed with concentrations of 10 ml.l-1 and 20 ml.l-1 which resulted in a negative growth rate corresponding with high doubling time and lower percentage increase (Table 3).

Table 3. Growth analysis of cell metabolites of S. platensis as affected by PDWE enriched growth medium.

0.65

PDWE= Palm date water extract; GR= growth rate; DT= doubling time; DM= degree of multiplication and PI= increase percentage.

Positive correlation (r2= 0.3267) was recorded among chlorophyll produced by the examined S. platensis (Fig.1C). As mentioned in chlorophyll case, organic carbon and salinity increase the carotenoids content due to the blocking of photosynthesis via carbon dioxide fixation and cells tended to increase their dry weight through the high synthesis and condensation of fatty acids through carotenoids. Thus, the increasing of fatty acids is closely associated by the hyper accumulation of non-green pigments (carotenes and carotenoids). Thus, it may be concluded that different concentrations of date palm waste used are most effective on carotenoids accumulation rather than dry weight or/and chlorophyll- accumulation. Furthermore, date palm waste saves the proper growth with cell metabolites (dry weight: chlorophyll: carotenes) balance as visually observed of green colors of Spirulina cultures.

Spirulina platensis growth rate was maximized with the superior carotenoid concentration control cultures to reach 0.171 mg. l-1. d-1, followed by 30 ml.l-1of PDWE cultures 0.117 mg.l-1.d-1 and 40 ml.l-1of PDWE cultures 0.092 mg. l-1.d-1. Carotenoids decline was observed with concentrations of 10 ml.l-1and 20 ml/l which resulted in a negative growth rate corresponding with high doubling time and lower percentage increase.

Oil content and fatty acids methyl esters

Oil content of the grown Spirulina under different concentrations of date palm waste represented variable results which might attribute to the differences in carbon source and the initial concentration. It was early reported that increasing of organic carbon in algal growth medium triggered the bioaccumulation of oils and carotenoids. However the used concentration of bicarbonate seems to be more in carbon, the stability of low organic carbon in growth medium increases the efficiency use of such concentrations. Date Palm waste suggested more than one benefit in algal growth medium including waste utilization, minimizing cost, avoid carbon gasses use and emission, increasing biomass content rich in both chlorophylls (protein) and carotenoids (oils).

Among the different waste concentrations used, 3.0 and 4.0% of waste surpass other treatments even control one. Table (6) represented the oil content of Spirulina platensis dried biomass grown under different concentrations of palm date water extract. In general, oil content of Spirulina platensis is almost low comparing with different algae species (49% on dry weight basis); however some reported the rise in oil content up to 1113% under stress conditions (Becker, 2007).

Table 4. Oil content (%) of Spirulina platensis grown under different PDWE concentrations.

Here, cultures which grown under the recommended growth medium (Zarrouk) resulted in 5.92% oil content. Stressed cultures of Spirulina due to waste addition represented different pattern, where lower concentrations (1.0 and 2.0%) of waste led to slight decreases in oil content (5.09 and 5.21%). On the other hand, moderate and high waste concentrations of waste (3.0 and 4.0%) led to marked increases of oil content as compared with controls one (7.06 and 7.81%).

The enhancing effect of organic wastes on algal oil might goes back to the supplying algae by carbon and nutrients which stimulate mixotrophic growth especially in the case of growth limitation due to salinity or nitrogen deficiency.

Fatty acids methyl ester profile (Table 5) showed that addition of date palm waste as a source of organic carbon for Spirulina growth led to obvious increase in saturation index due to the rise of saturated fatty acids (1.76) comparing with those grown under recommended growth medium which reached 0.96. Initially, unsaturated fatty acids still the highest percent. Also, long chain fatty acids represented the high content

Table 5. Fatty acids composition (%) of Spirulina oil

Fatty acid profile of Spirulina platensis as presented in Table (5) showed that palmatic C16:0, oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) were the most prevalent and total saturated fatty acid of Spirulina represented 44.7% of the total fatty acids compared to unsaturated fatty acids (56.3 %).

Fatty acids methyl esters with 4 and more double bonds are susceptible to oxidation and this reduces their acceptability to use in biodiesel. Results indicated that fatty acids with 4 or more double bonds (C 20:4) reaching 2.04 and 2.87%; while C20:5 represented 3.17 and 2.02% for Zarrouk grown Spirulina and waste, respectively. The European biodiesel standards limit the contents of FAMEs with four and more double bonds to a maximum of 1.0 %.

The microalgae possess a favorable fatty acids profile that can be utilized for biodiesel production with high oxidation stability. Physical and chemical properties such as density, viscosity, acid value, heating value etc. of biodiesel from microalgae oil are very much comparable to those of fuel diesel (Miao and Wu, 2006). The European standard (En 14214) limits linolenic acid methyl ester content in biodiesel for vehicle use to 12%, and must meet other criteria relating to the exit of total unsaturation of the oil which is indicated by its iodine value. Standard of the iodine value of biodiesel not exceed 120-130 g iodine.100g-1 biodiesel. Accordingly the concentration of C 18:3, in the present study needs to be decreased.

Highest amount of oleic acid (C18:1) (10.52 14.03 %) was detected in both Spirulina cultures. Oils with high oleic acid content have been reported to have a reasonable balance of fuel, including their ignition quality, combustion heat, cold filter

plugging point, Oxidation stability, viscosity and lubricity which are determined by the structure of their component fatty acids (Knothe, 2008 and Rashid et al., 2008). The highest oleic content of Spirulina is making it most suitable for the production of good quality biodiesel. The addition of methyl oleic has been suggested to improve the oxidation stability (Knothe, 2008). The proper percentage of saturated and unsaturated fatty acids is very important to the use of microalgae feedstock (Deng et al., 2009). (Schenk et al., 2008) indicated that the ideal mixture of fatty acids has been suggested to be C16:1, C18:1, and C14:0 in the ratio of 5:4:1. Such a biodiesel would have the properties of very low oxidative potential. Accordingly in the present study the concentration of C16:1 and 14:0 needs to be increased. (Gouveia and Oliveira, 2009) suggested the addition of other oil to the microalgae oil to improve its quality.

Fuel proprieties

Fuel proprieties of Spirulina platensis grown under Zarrouk medium and PDWE represented some variation due to the saturation index and carbon chain. The degree of unsaturation (DU) of biodiesel indexes the double bonds capacity of FAMEs which affects the other fuel proprieties. For instance, higher DU led to decrease the cetane number and lowering pour and cold- point. Here, moderate values were obtained (59.41 -83.93); which are lowest than the moderate one (120 Rapeseed and 130Canola) and nearly to palm oil (50-60).

Saponification value (SV); referring to the sum of fatty acids (carboxyl groups) corresponding to fatty acid chain length, where short chain consume more alkaline moles and resulted in high SV. Most of Spirulina fatty acids chain are up to 16 C (ca; 94%) 1 in which reduces the SV values in both grown cultures and slightly equal (156.4 -157.6 mgKOH.g-1 oil). Data was found in the common average is ranged between 175 and 205 mg KOH/g. The saponification value (SV) of the biodiesel has an inverse relationship to the average molecular weight of the fatty acids (Knoth, 2002). The SV of the esters is higher than that of oil due to the fact that, the process of transesterification removes the heavier glycerol molecules from the oil, consequently reducing the average molecular weight of the methyl esters (Wagutu et al., 2009).

Iodine value (IV); expresses the degree of unsaturation of FAMEs, where the double ponds saturated by iodine (gI2.100g-1 biodiesel). Its affect fuel properties, where the cetane number is lower with its higher IV values. European biodiesel standard (EN 14214) showed the normal of such value must be 120 g I2.100 g-1. The profile of other sources ranged from 10-15 (Coconut oil) to 120-140 (Soybean biodiesel). The obtained results on the present case are 74.6 with Zarrouk grown alga and 68.14 with PDWE grown Spirulina.

Table 6. Some fuel properties of Spirulina platensis grown under 4.0% of PDWE

DU (degree of unsaturation); SV (saponification value); IV (iodine value); CN (cetane number); LCSF (long- chain saturated factor); CFPP (cold filter plugging point); OS (oxidative stability); PP (pour point); CP (cloud point) and FP (flash point).

Cetane number (CN) which measure the time delay between injection and ignition commonly ranged between 47 (ASTM D6751) and 51(EN 14214). Higher DU minimized CN values, while long chain FAMEs raised it. The present case represented 44.97-46.64 with Zarrouk and PDWE grown Spirulina, respectively.

The saturation based index defined as Long Chain Saturation Factor (LCSF) predict some properties of biodiesel, such as the Cold Filter Plugging Point (CFPP) and represented the contribution of saturated fatty acids ( C:16) in the produced biodiesel. Their values are closely related to old Filter Plugging Point (CFPP).

Cold Filter Plugging Point (CFPP) was defined as the lowest temperature at which a fuel will still pass through a standardized filter within a specified time. In practice it estimates the temperature at which wax crystals or solidified FAMEs plug fuel filters, causing engine starvation or failure in cold conditions. It was ranged between -3oC (the better) to 15.

The pour point (PP) of biodiesel varies with feedstock and the pour point of Spirulina is 2.1- 4.9 0C markedly varied, but both of them are in the acceptable limits. The pour point is of great significance in connection with handling, storage and flow in fuel lines of engines (3 °C to 5 °C for vegetable oil and 10 to 15 °C).

High pour point means the possibility of having some troubles using this fuel blend especially during cool seasons.

The Cloud Point (CP) is the temperature degree allow crystallization of wax crystals due to saturated FAMEs) in cool weather. Maximum cloud point was observed with palm oil (13-15oC); while the minimum was obtained by Canola oil (-3oC).

Spirulina biofuel was found in the middle area in this concern (9.35 and 10.51oC) for either grown alga as compared with palm oil (13-15 oC) or animal fat (12-16oC).

Flash point (FP) was defined as thelowest temperature permits the enough emission of vapor to ignite momentarily. It is usually lies between 120 and 170 °C which also not expressed the quality of biodiesel due to the presence of free or excess- methanol during transesterification processes. However, ASTM D6751 covered FP by 93 °C and 120 °C for EN 14214. In Spirulina case, such value reached 155.99oC with Zarrouk grown alga and 169.9 oC with those grown with PDWE. This might attributed to the lowest methanol content, residual carbon or/and long chain fatty acids.

On the other hand, the carbon residue from the combustion of the fuel blend is 11.1% which is higher than that recommended of diesel fuel. If this property is high, it would result in excessive deposits of carbon in the combustion chamber of the engine, intake valves and injector tip (injector coking).Injector coking results in poor fuel atomization and hence a lot of ignition troubles. This would greatly reduce the efficiency of the combustion process and, hence the engine performance. The flash point of the Spirulina biodiesel / solar blend (table 6) was higher than the diesel fuel 96 oC and 55 oC respectively. This makes the biodiesel fuel safer during handling and storage than diesel fuel. While it was 134 for biodiesel from Nannochloropsis oculata (Almarales et al., 2012); and 145 for biodiesel from C. vulgaris (Ahmed, et al.,2013) and accordingly the biodiesel from C. vulgaris is the most safer. In addition, the cetane number of the solar/spirulina biodiesel blend is 38 which is much lower than the minimum requirement of a diesel fuel (55). On the other hand, cetane number of C.vulguris biodiesel was 53(Ahmed, et al, 2013), while that of Scenedesmus obliquus was 51.3 (Mandal and Mallick, 2012). Thus, it is expected that the knocking noise of the engine using this fuel blend of Spirulina will be the highest. The results proved that the biodiesel was nearly free from sulfur and non- burnable matters. It could be concluded that this biodiesel of Spirulina have three major advantages over regular diesel oil-less corrosion of injection parts by acidic sulfur oxides-less wear of engine itself by ash deposits and less environmental pollution.

CONCLUSION

A part of Spirulina production challenge in concern mineral and carbon nutrition could solve using organic wastes including date palm waste which contains sufficient amounts of nutrients as well as organic carbon. Such hypothesis reduces the production costs due to minimizing bicarbonate addition and reduces the injury effect of high sodium ions liberated from carbonate utilization.

The oil content of Spirulina platensis is relatively low (49% of dry weight) compared with many other microalgae species such as Nannochloropsis, Botryococcus, and Chlorella, which can accumulate 2070% lipids under appropriate conditions. Therefore, Spirulina is valued more for its protein and pigment production rather than as a lipid-rich biofuel source.

ACKNOWLEDGMENT

This research work has been carried out as a part of activities of Algal Biotechnology Unit, National Research Centre, Dokki, Cairo, Egypt, (Prof. Dr. Abo El-Khair B. El-Sayed). The authors express thanks to all other staff members.

Author contributions:

Abo El-Khair B. El-Sayed; Conceptualization, biostimulator resources, methodology writing-original draft preparation, writing- review and editing.

Marwa, M. Reda; Conceptualization, supervision, software, validation, formal analysis writingoriginal draft preparation and writing-review

Sara S. A. El-Sawy; Analysis, conceptualization, writing-review and editing.

Reda, M. El-Awady; Analysis, conceptualization, writing-review and editing.

All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding

Email Address of the Corresponding Author in the manuscript: (Reda El-awady) elawady185@gmail.com

"The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper".

REFERENCES

1. A.O.A.C. (2000). Association of Official Analytical Chemists. Official method of analysis. 17th Ed. Vol. 11. Washington U.S.

2. Abo El-Khair B. El-Sayed, Marwa, M. Reda, Adel W. Almutairi and Charalampos Mavromatis (2023). Productivity and biochemical constituents of

   Chlorella vulgaris grown in Net-House Photobioreactor. Journal of Taibah University for Science. 17(1); 2194843.

3. Agarwal, M., Singh, K. and Chaurasia, S. P. (2010). Prediction of Biodiesel Properties from Fatty Acid Composition using Linear Regression and ANN Techniques. Indian Chemical Engineer, 52(4), 347-361.

4. Ahmed, F.; Amin, U.K. and Yasar, A. (2013). Transesterification of oil extracted from different species of algae for biodiesel production. Afric. J. of Environ. Sci. and Tech. 7(6), 358-364.

5. El-Sayed, A. B. (2010). Immobilized-microalga Scenedesmus sp. for biological desalination of Red Sea Water: II. Effect on macronutrients removal. Journal of American Science, 6(9):637-643.

6. Al-Farsi M.; Alasalvar, C.; Al-Abid, M.; Al-Shoaily, K.; Al-Amry, M. and Al-Rawahy, F. (2007). Compositional and functional characteristics of dates, syrups, and their by-products. Food Chemistry, 104(3): 943-947.

7. Almarales, A.; Chenard, G.; Abdala, R.; Gomes, D. A.; Reyes, Y. and Tapanes, N. O. (2012). Hydroesterification of Nannochloropsis oculata

   microalgas biomass to biodiesel on AlO supported NbO catalyst. Natural Science, 4(4), 204-210

8. Asmaa Salah, Reham M. El-Bahbohy, Abo El-Khair B. El-Sayed, Ayman Amin and Hoda Sany. (2023a). The impact of inoculum preparation media on pollutant removal through phycoremediation of agricultural drainage water by Desmodesmus sp. Python-International Journal of Experimental Botany. 92(10)2875-2890.

9. Asmaa Salah. Hoda Sany. Abo El-Khair B. El-Sayed. Reham M. El-Bahbohy. Heba I. Mohamed and Ayman Amin. (2023b). Growth performance and biochemical composition of Desmodesmus sp. green alga grown on agricultural industries waste (Cheese Whey) Water Air Soil Pollut. 234:770.

10. American Standard for testing of materials (ASTM) D- 86, D- 455, D- 93, D- 224, D- 97, D- 976, D- 1298, D- 4294, D- 189, D- 482, D- 874.

11. Baird, R.B; Eaton, A.D.; Rice, E.W. Standard Methods for the Examination of Water and Wastewater, 23rd ed.; Washington, D.C.: American Public Health Association, American Water Works Association, Water Environmental Federation, pp. 5-32.

12. Baker, H.G.; Baker, I. and Hodges, S.A. (2009). Sugar composition of nectars and fruits consumed by birds and bats in the Tropics and Subtropics. Biotrop., 30: 559 -586.

13. Becker, E.W. (2007). Micro-algae as a source of protein. Biotechnology Advances, 25(2), 207210.

14. Besbes, S.; Drira, L.; Blecker, C.; Deroanne, C. and Attia, H. (2009). Adding value to hard date (Phoenixdactylifera L.): Compositional, functional and sensory characteristics of date jam. Food Chemistry, 112(2), 406-411.

15. Borowitzka, M.A. and L.J. Borowitzka (2010). Microalgae biotechnology. Cambridge Univ. press, New York., pp: 57-58.

16. Boussiba, S.; Fan, L. and Vonshak, A. (1992). Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. Methods Enzymol., 213: 386-371.

17. Burnison, K. (1980). Modified dimethyl sulfoxide (DMSO) extraction for chlorophyll analysis of phytoplankton. Can. J.Fish. Aquat. Sci., 37: 729-733.

18. Chisti, Y. (2007). Biodiesel from microalgae.Biotechnol Adv25:294306.

19. Chpman, H.D. and Pratt, P.F. 1974. Methods for Soils, Plants and Waters. Agricultural Division Sciences, California Univ., Berkeley, USA.

20. Deng X., Li Y., and Fei X. (2009), Microalgae: A Prom- ising feedstock for biodiesel. Afr. J. Microbiol. Res. 3, 1008-1014.

21. Drever, J.I. (1997). The Geochemistry of Natural Waters. Prentice Hall.

22. Duncan, D.B. (1955). Multiple ranges and multiple F-test. Biomet., 11: 1-42.

23. El-Awady, R. M.; A. B. El-Sayed, K. M. El-Zabalawy, and M. A. El-Mohandes. (2020). Bio-mass Production of Chlorella vulgaris grown on date wastes under different stress conditions. Al-Azhar Journal of Agricultural Research, 45 (2); 62-75.

24. El-Sayed A. B. (2010). Carotenoids accumulation in the green alga Scenedesmus sp. incubated with industrial citrate waste and different induction stresses, Nat. Sci., 8(10): 3440. (ISSN: 1545-0740).

25. El-Sayed, A. B.; Nashwa, A. Fetyan, Fatma. M. Ibrahim and Sadik, M.W. (2022). Biomass Fatty acids profile and fuel properties prediction of bagasse wastes grown Nanochloropsis oculata. Agriculture, 12, 1201.

26. El-Sayed, A.B. (1999). Some physiological studies on green algae. Ph.D. Thesis, Plant Physiology, Fac. Agric. Cairo Univ., Egypt.

27. El-Sayed, A.B. and Almutairi. A.W. (2024). Nutrient balance for enhanced recovery of stressed Spirulina platensis. Environmental Science and Pollution Research, 31(45):56685-56696.

28. El-Sayed, A.B.; Nashwa A.H. Fetyan, Fatma Ibrahim, Sayed A. Fayed and M.W. Sadik. (2020). Application of bagasse extract in economic

    Nannochloropsis oculata mass production. Egyptian Journal of Chemistry,63(12); 5183 5192.

29. El-Sayed, A.B.; Abdalla, F. E. and Abdel-Maguid, A. A. (2001). Use of Some Commercial Fertilizer Compounds for Scenedesmus Cultivation. Egyptian J. of Phycology, 2, 9-16.

30. El-Sayed, A.B.; Battah M.G. and El-Sayed, E.W. (2015). Utilization efficiency of artificial carbon dioxide and corn steam liquor by Chlorella vulgaris. Biolife, 32, 391-403.

31. El-Sayed, A.B.; Hoballah, E.M. and Khalafallah, M.A. (2012). Utilization of citrate wastes by Scenedesmus sp. I- enhancement of vegetative growth. Journal of Applied Sciences Research, 8(2): 739-745.

32. El-Shafey, Y.H.; El-Fouly, M.M.; Khalil, M.M.; Abdalla, F.E. and El-Sayed, A.B. (1999). Secondary carotenoid accumulation in some green algae species. The First Congress on the Recent Technologies in Agriculture. 27-29 Nov., 1999, Fac. of Agric., Cairo Univ. Cairo, Egypt.

33. FAO stat. (2017). Faostat.fao.org/default.aspx. Available at: http://faostat3.fao.org/

34. Francisco, E.C.; Neves, D.B.; Lopes, E.J. and Franco, T.T. (2010). Microalgae as feedstock for biodiesel production: carbon dioxide sequestration, lipid production and biofuel quality, J. Chem. Technol. Biotechnol. 85: 395-403.

35. Gouveia, a, L. and Otiveira, A.G. (2009): Miroalgae as a raw material for biofuels production J. Ind. Mircobial Biotechnology. 36:269-274.

36. Harriet V.; Ulisses I.; Oliveira J.L.B. and Santanna, E.S. (2008). Cultivation of Arthrospira (Spirulina) platensis in desalinator wastewater and salinated synthetic medium: protein content and amino-acid profile. Braz. J. Microbiol., 39: 98-101.

37. Hasnaoui A.; Elhoumaizi, M.A.; Asehraou, A.; Simdic, M.; Deroanne, C. and Hakko, A. (2010). Chemical composition and microbial quality of dates grown in Figuig oasis of Morocco, Int. J. of Agric. and Biol., 12(2):311- 314.

38. Hoekman, S. K.; Broch, A.; Robbins, C.; Ceniceros, E. and Natarajan, M. (2012). Review of biodiesel composition, properties, and specifications.

    Renewable and Sustainable Energy Reviews, 16(1), 143-169.

39. Holst, D. O. (1982). In: J. Assoc. Off. Anal. Chem.; 65 (2); 265-269.

40. Juneja, A.; Ceballos, R.M. and Murthy, G.S. (2013). Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review. Energies, 6:46074638. doi:10.3390/en60 94607.

41. Kawata, Y.; Yano, K.S.H. and Toyomizu. M. (2004). Transformation of Spirulina platensis strain C1 (Arthrospira sp.) with Tn5 Transposase – Transposon DNA Cation Liposome Complex. Marine Biotechnology, 6: 355-363.

42. Knothe, G. H. (2008). Designer biodiesel optimizing fatty ester composition to improve fuel properties Energy fuel 22, 1358-1364.

43. Knothe, G. (2002). Structure indices in FA chemistry. How relevant is the iodine value? Journal of the American Oil Society. JAOCS; 79(9), 847-854.

44. Krisnangkura, K.A. (1986). Simple method for estimation of cetane index of vegetable oil methyl esters. J. Am. Oil Chem. Soc. 63: 552-553.

45. Lindsay, W.L. (1979). Chemical Equilibria in Soils. Wiley-Interscience.

46. Ludde, F.E.; R.A. Barvord and R.W.Reimenschnider (1960). Direct conversion of lipid components to their fatty acid methyl ester, J. Am. Oil chem. Soc. 37,447-451.

47. Luque, R. and Clark, J.H. (2013). Valorisation of food residues: waste to wealth using green chemical technologies. Sustainable Chemical Processes, 1186/2043-7129, pp. 1-10.

48. Ma, T.S. and Zauzag, C. (1942). Microkjeldahl determination of nitrogen. A new indicator and an improved rapid method. Ind. Eng. Chem. Ed., 14, 280.

49. Mandal, S. and Mallick, N. (2012). Biodiesel production by green microalgae Scenedesmus obliquus in a recirculatory Aquaculture System.Applied and Environ. Microalgae, 78, 5929-5934.

50. Miao,X and Wu,Q (2006). Biodiesel production from hetrtrophic microalgal oil.Bio-resour Technology,97:841-846

51. Mostafazadeh, K.; Sarshar, M.; Javadian, Sh.; Zarefard, M.R. and Z.A. Haghighi (2011). "Separation of fructose and glucose from date syrup using resin chromatographic method: experimental data and mathematical modeling," Sep. Purif. Technol., 79(1): 72-78.

52. Nashwa A. H. Fetyan, Abo El-Khair B. El-Sayed, Fatma M. Ibrahim, Yasser A. Attia and Mahmoud W. Sadik. (2022). Bioethanol production from defatted biomass of Nannochloropsis oculata microalgae grown under mixotrophic conditions. Environmental Science and Pollution Research, 29: 2588 2597.

53. Pirt, S. J. (1973). Principle of Microbe and Cell Cultivation. Blackwell Scientific Publication, 4-7.

54. Raoof, B.; Kaushik. B. D. and Prasanna, R. (2006). Formulation of a lowcost medium for mass production of Spirulina. Biomass and Bioenergy, 30(6):537- 542.

55. Rashid, U., Anwar, F., Moser B. R., and Knothe G. (2008). Moringa oleifera oil: A Possible source of biodiesel. Bioresour. Technol. 99, 8175-8179.

56. Reem H.I. Hassan; Nadia A.M. El-Said and A. B. El-Sayed. (2022). Effect of algae extracts on growth, yield, and volatile oil on fennel Foeniculum vulgare Mill plant. Scientific Journal of Flowers and Ornamintal Plants. 9(4); 363-372.

57. Sarin, A.; Arora, R.; Singh, N.P. and Malhotra, R.K. (2009). Influence of metal contaminants on oxidation stabiity of Jatropha biodiesel. Energy, 34(9), 1271-1275.

58. Schenk, P. M.; Thomas-Hall, S. R.; Stephens, E.; Marx, U. C.; Mussgnug, J. H.; Posten, C.; Kruse, O.; Hankamer, B. (2008). Second generation biofuels: High-efficiency microalgae for biodiesel production. Bioenergy Research, 1 (1); 20-43.

59. Seely, G.R.; Voncan, M.J. and Widaver, W.E. (1972). Preparative and analytical extraction of pigments from brown algae with dimethyl sulfoxide. Mar. Biol., 12: 175-179.

60. Selim, K.; Abd El-Bary, M. and Ismaeel, O. (2012). Effect of irradiation and heat treatments on the quality characteristics of Siwi date fruit (Phoenix dactylifera L.).xd Agro. Life Sci. J., 1: 103-111.

61. Sheraz, M. Kamal; El-Sayed, A.B.; Manar, T.; Amal A. and Hoida, A.M. El-Shazly (2017). Use of okara waste for algae nutrition. Arab Univ. J. Agric. Sci.; 25(2); 271-279.

62. Shimadzu (2012). TOC in Daily Practice. SCA-130-517,

63. Shimamatsu, H. (2004). Mass production of Spirulina, an edible microalga, Hydrobiologia; 512:3944.

64. Smith, F.; Gilles, M.A.; Haithon, J.K. and Goees, P.A. (1956). Colorimetric method for determination of sugar related substances. Annl. Chem., 28: 350- 356.

65. Soni, R.A.; Sudhakar, K.; Rana, R.S. SpirulinaFrom growth to nutritional product: A review. Trends Food Sci. Technol. 2017, 69, 157171.

66. SPSS (2011), IBM SPSS Statistics for Windows, Version 20.0.

67. Stumm, W., & Morgan, J.J. (1996). Aquatic Chemistry. Wiley.

68. Tripathi, R.D. et al. (2007). Soil processes and microbial interactions under saline and alkaline conditions. Environmental Reviews, 15(4), 328358.

69. Wagutu, A. W., Chhabra, S. C., Thoruwa, C. L., Thoruwa, T. F., & Mahunnah, R. I. A. (2009). Indigenous Oil Crops as a Source for Production of Biodiesel in Kenya. Bulletin of the Chemical Society of Ethiopia, 23(3), 359-370.

70. Zaborsky, O. R. (1985). Feeds from Spirulina: Process Engineering and genetic engineering analysis of co-products. (OMEC International, Inc. Washington D.C).

71. Zarrouk, C. (1966). Contribution a letude dune cyanobacterie: influence de divers facteurs physiques et chimiques sur la croissance et la photosynthese de

    Spirulina maxima (Setchell et Gardner) Geitler. PhD thesis, University of Paris, France.

72. Zeinelabdeen, M.A.; Abasaeed, A.E.; Gaily, M.H.; Sulieman, A.K. and Putra, M.D. (2013). Coproduction of Fructose and Ethanol from Dates by S. cerevisiae ATCC 36859.International Journal of Chemical, Molecular, Nuclear, Materials and Metallurgical Engineering, 7(10).