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. I. (2)El-Sayed, A. B. (3) Marwa, M. Reda; (4) Ghazy S. M. A.

(1,4) Environment and Bio-Agriculture Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt

(2) Algal Biotechnology Unit, Fertilization Technology Department, National Research Centre, Giza, Egypt

(3) Center laboratory for environment quality monitoring, National Research Centre, Giza, Egypt

ABSTRACT – Wastes of date pits represent an average of 10% of the total weight of date fruits; therefore, there is a pressing need to identify 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 30 ml.l-1 of waste followed by 40 (0.936 ml. l-

1 waste) compared with control and other waste volumes used (10 and 20ml. l-1). Concerning total chlorophyll, control cultures represented the maximum (5.259 mg. 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% in comparison with 43.7% saturated fatty acids. Algal oil was converted via transesterification with methanol/KOH with sulfuric acid acting 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.

Key words: date wastewater extract, fuel, algal oil, Spirulina platensis, Zarrouk growth medium.

INTRODUCTION

In situ, algal growth cycles are primarily influenced by nutrient availability. Moreover, 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 saline 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).

It is estimated that approximately one-third of the food produced for human consumption is wasted annually. The utilization of food waste as a feedstock for the cultivation of microorganisms offers an effective strategy for recycling organic residues rich in

carbon, nitrogen, and phosphorus compounds (Luque and Clark, 2013). The global production of date palm has increased significantly, rising from 1.8 million tons in 1961 to approximately 7.63 million tons in 2017, with Egypt ranking as the leading producer worldwide (1590414 metric tons) (FAO, 2017). Date palm fruits are particularly rich in monosaccharides, mainly fructose and glucose, along with essential minerals and vitamins, and contain relatively low levels 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). Additionally, it is rich in essential nutrients, including nitrogen, phosphorus, potassium, calcium, and magnesium, and contains high levels of carbohydrates and fats, making it an important source of sugars and dietary fiber (Al-Farsi et al., 2007). Lower-grade (second-grade) dates have been shown to possess sugar contents comparable to those of high-quality dates. Moreover, a substantial quantity of dates exceeding 2 million tons annually (approximately 30% of total production) is discarded due to poor texture and quality (Besbes et al., 2009). These considerable amounts of underutilized dates can be valorized for the production of fructose, ethanol, acetic acid, lactic acid, and other value-added products (Zeinelabdeen et al., 2010).

Spirulina is currently produced by several companies and widely marketed in health food stores worldwide, owing to its high nutritional value. It is considered a rich source of protein (5570%), vitamins, minerals, essential fatty acids, essential amino acids, and pigments such as chlorophyll-a, phycobiliproteins, and carotenoids, making it valuable for applications in the food, chemical, and pharmaceutical industries. In addition, it is recognized as a significant natural source of vitamin B12 and is characterized by its high protein content. Due to the absence of cellulose in its cell wall, Spirulina exhibits high digestibility, with an assimilation rate reaching 8595%. (Kawata et al., 2004, Harriet et al., 2008 and Borowitzka and Borowitzka., 2010). The growth rate of algae is strongly affected by culture conditions, including CO concentration, pH, temperature, nutrient availability (such as nitrogen, phosphorus, hydrogen, potassium, zinc, boron, and magnesium), and the 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 the Algal Biotechnology Unit, National Research Centre (NRC); Egypt. Growth was indoor performed using Zarrouk culture medium (Zarrouk, 1966). Growth culture vessel was created from a completely transparent plexiglas column with initial diameter 7.5cm x 100cm containing 6.0 L of growth medium. Growth conditions were optimized using continuous unilateral illumination suplied by a light bank of five cool white fluorescent lamps (40 watt) and aerated by air-left system from oil-free compressed air was supplied from the lower part of the growth container. The inoculum was prepared in the laboratory, then centrifuged and washed twice using a refrigerated centrifuge (RUNNE HEIDELBERG, RSV-200, Germany)

Date palm waste water extract preparation

Date palm waste was sourced 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 dactylifera) were collected from El-Wahat, Giza Governorate, Egypt. The samples were washed under running tap water for 5 minutes to remove dust and reduce microbial contamination. Following washing, the date stones were separated from the pulp and the material was dried at 55°C for 24 hours in a circulating oven (Baker et al., 2009). The dried samples were then ground using an electric hammer mill and sieved through a 0.9 mm mesh to obtain a fine powder. The prepared samples were stored at 4°C until further 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 selected volumes and concentrations were determined according to the initial total nitrogen levels in the waste compared with that initial 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. Before inoculation, the waste was diluted fourfold with bi-distilled water and subsequently passed through a series of filters with decreasing pore 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 ramped from 50 to 240 °C at a heating rate of 3 °C per minute. Helium served as the carrier gas with a flow rate of 1.1 mL/min. The injector and detector were maintained at 220 °C and 250 °C, respectively. The separated peaks were identified by comparing their profiles with those of standard 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 three moles of alcohol are required to esterify one mole of oil, 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 until neutrality was achieved, then dried over anhydrous sodium sulfate. The remaining alcohol was removed using a rotary evaporator. The progress of the esterification reaction was monitored by thin-layer chromatography through periodic sampling at defined time intervals until completion.

Fuel proprieties estimation based on FAMEs profile

The cetane number (CN), saponification value (SV), iodine value (IV), degree of unsaturation (DU), long chain saturated factor (LCSF) and cold filter plugging point (CFPP) were calculated using empirical equations based on established methods 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 was carried out using SPSS (2011), IBM SPSS Statistics for Windows (Version 20.0). Significant differences among variables were assessed using Duncans multiple range test (Duncan, 1955). All experiments were conducted in triplicate, and the results were presented as the mean of three replicates. One-way analysis of variance (ANOVA) was applied to evaluate statistical significance at a probability level of P 0.05.

RESULTS AND DISCUSSION

Date palm water extracts 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

Biochemical constituent
Protein	CHO	Oil	Fiber	Ash	TC	OC	OM
%
3.86	72.15	2.77	8.98	6.76	1.67	1.28	2.21

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

	C	N	P	K	Ca	Mg	Na
%				Ppm
*Date Palm	1.67	0.06	0.03	360	4.15	45.5	50.5
Zarrouk	0.24	0.042	0.009	67.0	11.0	21	5680

*Values of the original extract

On this context, the used digested effluent by Soni et al. (2017) serves as an inexpensive nutrient source, rich in C:N:P at a ratio of 24:0.14:1, and effectively supports the growth of Spirulina.. This ratio enhanced the biomass composition, raising the proportions of protein, carbohydrates, and lipids to 68%, 23%, and 11%, respectively. On the other hand, a latex serum enriched with 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) enhanced dry weight accumulation in all S. platensis cultures compared to the control, which relied solely on nutrients from the standard growth medium. Here, the resulting net 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 standard growth medium, respectively (Table 3). Thus, 30 ml.l-1 of PDWE concentration seems to be the most superior one that reached the highest 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). A significant increase in biomass production of the examined strain was observed with increasing PDWE concentrations, and a positive correlation (r² = 0.0827) was recorded between the dry biomass produced by the tested S. platensis and PDWE levels. (Fig. 1A).

The increase in dry weight during growth may be attributed to the high initial concentrations 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

1

0.8

0.6

0.4

0.2

0

Z T1 T2 T3 T4

6

DW (g.l-1)

T.Ch. (mg.l-1)

5

4

3

2

1

0

Z T1 T2 T3 T4

Treatment

c

S E P

1

T.Car. (mg.l-1)

.8

.6

.4

.2

0

Z T1 T2 T3 T4

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 a marked rise in chlorophyll content of the tested alga was observed as PDWE concentrations increased, accompanied by a 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 A significant increase in carotenoid content was observed compared to the control, which received only the standard treatment. 0.0 ml.l-1 of PDWE. A very slight decrease was observed at PDWE concentrations of 10, 20, and 40 mL·L¹, which was not statistically significant at P 0.05. The highest carotenoid content was recorded at 30 mL·L¹, followed by 40 mL·L¹ and then 20 mL·L¹ (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) followed by 30 ml. l-1 of PDWE (0.068 g. l-1/d) 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). A decline in chlorophyll content was observed at concentrations of 10 and 20 mL·L¹, which corresponded with a negative growth rate, along with an increased doubling time and a lower percentage increase (Table 3).

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

Parameter	Waste concentration (%)
	Control	1.0	2.0	3.0	4.0
	Dry weight
DW (g.l-1)	0.802ab	0.477b	0.816a	0.983a	0.936a

GR (g.l-1 d-1)	0.087	0.030	0.03	0.068	0.061
DT (hrs.)	7.95	35.17	39.93	10.98	13.73
DM	1.76	0.60	0.65	1.36	1.22
PI%	70.51	32.00	32.83	60.04	54.99
	Total chlorophyll
Chlorophyll (mg l-1)	5.259a	2.437ab	2.898ab	5.011a	4.910a
GR (mg l-1 d-1)	0.1592	0.0281	0.0350	0.1109	0.0842
DT (hrs.)	42.808	21.611	20.751	65.639	65.332
DM	3.2126	0.5664	0.7068	2.2377	1.6996
PI%	89.1506	10.2078	33.9604	78.612	65.6147
	Total carotenes
Total carotenes	0.450b	0.368b	0.297b	0.862a	0.780b
GR (mg l-1 d-1)	0.171	0.088	0.057	0.117	0.092
DT (hrs.)	4.235	8.867	0.589	7.078	7.937
DM	10.326	5.331	3.444	7.099	5.549
I%	89.715	68.341	7.365	76.489	71.188

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

A positive correlation (r² = 0.3267) was observed in the chlorophyll content of the tested 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. Carotenoid levels decreased at concentrations of 10 ml·L¹ and 20 ml·L¹, leading to a negative growth rate, prolonged doubling time, and a reduced 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.

Treatment	Cont.	1.0	2.0	3.0	4.0
Oil %	5.92	5.09	5.21	7.06	7.81

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

F.A	M.W	C/D	FAME	Cont.	PDWE
Caprylic acid	144.216	C8:0	158.243	2.16	3.47
Capric acid	172.270	C10:0	186.297	1.05	2.49
Lauric acid	200.324	C12:0	214.351	0.56	1.89
Myristic acid	228.378	C14:0	242.405	1.17	2.14
Palmitic acid	256.432	C16:0	270.459	25.12	29.48
Stearic acid	284.486	C18:0	298.513	8.42	12.57
Behenic acid	340.594	C 22:0	354.621	9.62	11.09
SFA				48.1	63.13
Palmitoleic acid	254.432	C16:1	268.459	2.08	1.89
Oleic acid	282.486	C18:1	296.513	14.03	10.52
MUSFA				16.11	12.41
Linoleic acid	280.486	C18:2	294.513	12.61	9.54
Linolenic acid	278.486	C18:3	292.513	16.09	9.07
Arachidonic acid	304.540	C 20:4	318.567	2.04	2.87
Eicosapentaenoic acid	302.540	C 20:5	316.567	3.17	2.02
PUSFA				33.91	23.5
FA (TS)				48.1	63.13
USFA (TU)				50.02	35.91
Saturation index (TS/TU)				0.96	1.76

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 acid methyl esters containing four or more double bonds are highly prone to oxidation, which limits their suitability for biodiesel applications. The results showed that fatty acids with four 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. European biodiesel standards limits on the allowable content of FAMEs with four and more double bonds to a maximum of 1.0 %.

Microalgae exhibit a favorable fatty acid composition that is well suited for biodiesel production with enhanced oxidative stability. Moreover, the physical and chemical properties of biodiesel derived from microalgal oil such as density, viscosity, acid value, and heating value are largely comparable to those of conventional diesel fuel. (Miao and Wu, 2006). The European standard EN 14214 restricts the proportion of linolenic acid methyl esters in biodiesel intended for automotive use to a maximum of 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 rich in oleic acid are known to offer a well-balanced set of fuel properties, including ignition quality, heat of combustion, cold filter plugging point, oxidative stability, viscosity, and lubricity, all of which are influenced by the structural characteristics of their constituent 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). An appropriate balance between saturated and unsaturated fatty acids is crucial for the effective use of microalgal 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 biodiesel would exhibit a very low tendency toward oxidation.. 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 consumes 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 biodiesel is inversely related to the average molecular weight of its 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 effect 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

Parameter	DU	SV	IV	CN	LCSF	CFPP	OS	PP	CP	FP
Zarrouk	83.93	156.4	7.46	44.97	21.15	5.0	4.55	2.1	9.35	155.99
PDWE	59.41	157.9	68.14	46.64	25.87	6.48	5.49	4.59	10.51	169.9

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 rangedbetween -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 a critical parameter affecting fuel handling, storage, and its flow behavior within engine fuel lines. (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).

The flash point (FP) is defined as the lowest temperature at which a substance emits sufficient vapor to ignite briefly.. It 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 be attributed to the lowest methanol content, residual carbon or/and long chain fatty acids.

Alternatively, the carbon residue from the combustion of the fuel blend is 11.1% which is surpassing 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 enhances the safety of biodiesel fuel during handling and storage compared to conventional diesel. 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 safer. In addition, the cetane number of the solar/spirulina biodiesel blend is 38. This value is significantly below the minimum requirement for 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 derived from Spirulina offers three key advantages over conventional diesel: reduced corrosion of injection components caused by acidic sulfur oxides, decreased engine wear from ash deposits, and lower 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 study was conducted as part of the activities of the Algal Biotechnology Unit at the National Research Centre, Dokki, Cairo, Egypt, under the supervision of Prof. Dr. Abo El-Khair B. El-Sayed. The authors gratefully acknowledge the contributions of 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 writing original draft preparation and writing-review

Saad M. A. Ghazy; Analysis, conceptualization, writing-review and editing.

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

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

Funding: This study did not receive any external funding.

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

The authors declare that they have no financial or personal conflicts of interest that could have influenced the research presented in this paper.

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