Volatile Compounds, Fatty Acids Constituents, and Antimicrobial Activity of Cultured Spirulina (Arthrospira fusiformis) Isolated from Lake Mariout in Egypt

In this study, Arthrospira fusiformis previously isolated from Lake Mariout (Alexandria, Egypt) was cultivated in the laboratory using a medium for pharmaceutical grade Arthrospira, named as Amara and Steinbüchel medium. Hot water extract of the Egyptian Spirulina was prepared by autoclaving dried biomass in distilled water at 121°C for 15 min. This algal water extract was analyzed by GC-MS to evaluate its volatile compounds and fatty acids composition. The antimicrobial activity of phycobiliprotein extract from Arthrospira fusiformis using phosphate buffer was evaluated against thirteen microbial strains (two Gram-positive bacteria, eight Gram-negative bacteria, one yeast, and two filamentous fungi). The major components of fatty acids in the hot extract of Egyptian A. fusiformis were hexadecanoic acid (palmitic acid, 55.19%) and octadecanoic acid (stearic acid, 27.14%). The main constituents of its volatile compounds were acetic acid (43.33%) and oxalic acid (47.98%). The most potent antimicrobial effect of phycobiliprotein extract was obtained against two Gram-negative bacteria Salmonella typhi and Proteus vulgaris, filamentous fungus Aspergillus niger, and the pathogenic yeast Candida albicans (all of which showed MIC values of 58.1 μg/ml). Escherichia coli and Salmonella typhimurium come second in their susceptibility to the phycobiliprotein extract from Arthrospira fusiformis and Serratia marcescens and Aspergillus flavus are the least in susceptibility, with MIC values of 116.2 and 232.5 μg/ml, respectively, while phycobiliprotein extract has no antibacterial effect on methicillin-resistant as well as susceptible Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Shigella sonnei. These findings confirmed the nutritional value of Egyptian A. fusiformis isolated from Lake Mariout and suggest the potential use of this strain as an ingredient in the cooking of some foods to increase the level of stearic acid and palmitic acid. Moreover, its effective antibacterial activities against some important and highly resistant to antibiotics bacterial pathogens in addition to its antifungal effects recommend the therapeutic use of its biomass.


Introduction
Te flamentous blue-green microalga Arthrospira fusiformis (commercially known as Spirulina platensis) was proved to have various nutritional and medicinal properties and was considered a "miracle from the sea" or "superfood" by scientifc communities. Tis edible, microscopic, and alkalophilic cyanobacterium belongs to the microalgae class Cyanophyta [1]. It contains higher amounts of vitamins, protein, minerals, and the like than any other single-cell protein [2,3]. It has photoautotrophic as well as auxotrophic growth since it has the capability to utilize organic compounds in dark and grow autotrophically in light [4,5]. Interestingly, there is no need to cook or specially treat Spirulina to increase its protein availability, which makes its production simple and preserves its valuable constituents including polyunsaturated fatty acids and vitamins [6].
Due to the nutritional value and pharmaceutical activities of Arthrospira fusiformis, it has been used for human and animal nutrition for centuries. Tis microalga is marketed worldwide in many stores of healthy foods [7][8][9]. It represents a good supply of bioactive ingredients for the diet including essential amino acids and fatty acids, high protein content (60-70%), pigments of photosynthesis, minerals, carotenoids, and vitamins [10,11]. Several studies have reported the use of Arthrospira fusiform as a food ingredient (ingredient in pasta and enriched bread) or food additive (added to freeze-dried yogurts) [12][13][14].
Five solvents have been commonly used to extract the bioactive ingredients (with antioxidant and antimicrobial activities) of Arthrospira fusiformis including ethyl acetate, dichloromethane, methanol, hexane, and petroleum ether [24,34]. Abedin and Taha [35] tested supernatant, hexane, and methanolic extracts of Tolypothrix ceytonica, Anabaena oryza, and Spirulina platensis for antifungal activity against various phytopathogenic fungi (Aspergillus niger, Fusarium verticillioides, Aspergillus favus, Alternaria brassicae, and Helminthosporium sp.) and fungus causing human disease (Candida albicans). Tey found that Spirulina platensis extracts have the highest antifungal efect on tested fungi. Kaushik and Chauhan [36] reported that methanol, ethyl acetate, hexane, and dichloromethane extracts of Spirulina platensis could inhibit the growth of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi. Tey showed that methanolic extract has the most potent antibacterial activity, while all extracts have no antibacterial efect on Klebsiella pneumoniae [36]. In 2010, Sharaf et al. [20] isolated and authenticated a new Egyptian Arthrospira fusiformis strain from the brackish Lake Mariout at the southwest of Alexandria city. Te phosphate bufer and hot water extracts of this strain were confrmed to have antiherpetic activity (could inhibit the multiplication of herpesvirus before and during virus infection of host cells) [20,21].
Najdenski et al. [37] confrmed that ethanol extracts and fatty acids from Arthrospira fusiformis have in vitro antibacterial activity against Staphylococcus aureus and Streptococcus pyogenes. Additionally, phycobiliproteins isolated from Arthrospira fusiformis were proved to have antimicrobial activity [37]. Ould Bellahcen et al. [38] isolated essential oil by hydrodistillation from Spirulina platensis collected from Lake Foum Elouad, Laayoune (South of Morocco) and cultivated in the laboratory using Zarrouk's medium. Te essential oil was analyzed by GC/ MS and GC/FID. Tey reported that the major components of essential oils in the Moroccan Spirulina were heptadecane (41.7%) and tetradecane (25.3%) [38]. Tey also reported for the frst time in the Moroccan Spirulina, the presence of geosmin; a key component of odor in cyanobacteria [38].
A new medium was formulated by Amara and Steinbüchel [39] for pharmaceutical grade Arthrospira. Te formulated medium was derived from a combination of George's and Zarrouk's media. Amara and Steinbüchel (A-St) medium at concentrations of 1.5-2x (high saline medium) has the ability to inhibit the growth of diferent forms of cyanobacteria and microalgae including Chlorella, whereas only Arthrospira could survive in this salinity.
Te current work aimed to identify volatile compounds and fatty acids composition extracted by hot water from Egyptian Arthrospira fusiformis that was previously isolated from Lake Mariout and authenticated by Sharaf et al. [20]. Te hot water extract was analyzed by GC-MS to predict its volatile compounds and fatty acids composition if Arthrospira is consumed as an ingredient in cooking or as a hot extract. Additionally, the in vitro antimicrobial activities of crude phycobiliprotein sodium phosphate extract of Egyptian Arthrospira fusiformis were evaluated against thirteen microbial strains.

Growth Medium. Te composition of Amara and
Steinbüchel (A-St) medium 1.5x is as follows: 13.82 g/l NaHCO 3 , 10.71 g/l NaCO 3 , and 0.75 g/l K 2 HPO 4 (part A); 2.25 g/l NaNO 3 , 0.85 g/l K 2 SO 4 , 1.5 g/l NaCl, 0.22 g/l MgSO 4 .7H 2 O, 0.011 g/l CaCl 2 .2H 2 O, 0.012 g/l FeSO 4 .2H 2 O, and 0.1 g/l EDTA-Na 2 .2H 2 O (part B); 0.02 g/l ferric citrate (part C); and 0.1 g/l peptone, and 0.01 g/l yeast extract (part D) [39]. All parts of this medium except for part D were flter sterilized separately through a syringe flter of 0.22 μm pore size from TPP (St. Louis, MO), while part D was autoclaved. Tis medium at 1.5x concentration was used for cultivation of the investigated Arthrospira fusiformis strain to inhibit the growth of Chlorella which was contaminating the seed culture of Arthrospira.

Growth Conditions. Arthrospira fusiformis was cultivated in Amara and Steinbüchel medium in 20 l bottles.
Tese bottles were aerated and agitated through a sterilized plastic tube using an air pump (150 bubbles/min) [40]. Bottles were incubated at 25°C and light from a forescent lamp/sunlight for 9 days at pH 9.3.

Collection of Microalga.
Te microalga was collected from 20 l cultivation bottles, washed ten times with double distilled water and dispersed in a mesh bottom frame, and dried at 40°C in an oven until the moisture content reached approximately 13%. Te samples were then homogenized using a grinder prior to the extraction method.

Preparation of Water Extract from Microalga.
Distilled water (100 ml) was added to 1 g of the dried samples, and the samples were autoclaved at 121°C for 15 min to remove microbes [41,42]. Te autoclaved samples were centrifuged at 2220 ×g for 10 min, and then the supernatant was collected as the water extract solution and lyophilized [43]. We used hot water extraction only to analyze the components consumed by people if they use Spirulina as a hot extract or food ingredient in cooking. Algal hot water extract was scanned by GC-MS (Scion GC-456, Netherlands) in Central Laboratory for Scientifc Services and Environmental Assessment of SRTA-City (Alexandria, Egypt) to evaluate its volatile compounds and fatty acids composition.
Te concentration of C-phycocyanin was calculated as previously reported by Siegelman and Kycia [45] as follows: where A 615 is the absorbance measured at 615 nm and A 652 is the absorbance measured at 652 nm.

Test Microorganisms for Antimicrobial Activity.
Te antimicrobial activity of phycobiliprotein extract from Arthrospira fusiformis was tested against thirteen microbial strains. Previously identifed methicillin-resistantStaphylococcus aureus (MRSA) clinical isolate obtained from the blood of a patient at Almery University Hospital (Alexandria, Egypt) was used in this study [46]. Candida albicans ATCC 10231 and Staphylococcus aureus ATCC 25923 strains were obtained from Becton Dickinson (France). Salmonella typhi ATCC 19430, Escherichia coli ATCC 25922, Salmonella typhimurium LT2, and Shigella sonnei ATCC 25931 were purchased from an American-type culture collection (ATCC, USA). Aspergillus niger, Aspergillus favus, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratia marcescens, and Proteus vulgaris were collected from Mycology Center of Al-Azhar University (Cairo, Egypt), and Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Assiut Branch (Egypt). A culture aliquot (100 μl) of each strain of bacteria was added to Luria Bertani (LB) broth, incubated overnight at 37°C, and then stored in 20% glycerol at −80°C to be used as seeds stock [47]. Stock cultures of C. albicans, A. niger, and A. favus were maintained on potato dextrose agar (PDA) overnight at 30°C for C. albicans and at 25°C for 5 days for A. niger and A. favus [47]. To evaluate antibacterial activity, cationadjusted Mueller-Hinton (CAMH) broth and Mueller-Hinton agar were used, while antifungal activity was evaluated using potato dextrose broth and potato dextrose agar.

Antimicrobial Activities of Phycobiliprotein Extract from
Arthrospira fusiformis. Susceptibility screening of test microorganisms to phycobiliprotein extract from Arthrospira fusiformis and diferent antibacterial and antifungal standards was performed using the agar well difusion technique on Mueller-Hinton agar and Potato dextrose agar for bacteria and fungi, respectively [47]. Plates were overlaid with 100 μl of an overnight culture of each bacterial pathogen at a cell density of 10 6 CFU/ ml, and 100 μl of an overnight culture of C. albicans at a cell density of 10 4 cells/ml based on McFarland turbidity standard [47,48]. Potato dextrose agar plates were overlaid with 1 ml fungal suspension (6 mm disc of the fungal growth suspended in sterile water) of 5 days old culture of A. niger and A. favus. Ten, wells were cut in the agar media by using a sterile 6 mm cork borer and flled with 100 μl of phycobiliprotein extract at a concentration of 0.93 mg/ml. Fusidic acid standard disc obtained from Mast Diagnostics (Merseyside, UK), chloramphenicol from Bioshop (Ontario, Canada), or amphotericin-B purchased from HyClone (Logan, Utah, USA) were used as a positive control, while sterile water was used as a negative control. Te culture plates were kept at 4°C for 2 h to allow proper difusion of tested antimicrobials through the inoculated media before being incubated at 30°C and 37°C for 24 h in the case of C. albicans and bacterial cultures, respectively, and for 5 days at 25°C in case of fungal cultures. Te presence of the inhibition zones was examined and recorded in (mm) of three replicates.

Broth Microdilution Susceptibility
Assay. Te minimum inhibitory concentrations (MICs) of phycobiliprotein extract from Arthrospira fusiformis against test pathogens were determined by broth microdilution technique [49]. Two 96well microtiter plates (Greiner, Frickenhausen, Germany) were inoculated with test microorganisms that were susceptible to the phycobiliprotein extract in the agar well Te Scientifc World Journal difusion assay, and then 100 μl of CAMH broth or potato dextrose broth containing phycobiliprotein extract in twofold serial dilutions were added. Te concentrations of phycobiliprotein extract ranged from 58.12 μg/ml to 0.93 mg/ml. Te plate inoculated with bacteria and C. albicans was incubated at 30°C for 24 h, whereas the plate inoculated with fungi was incubated at 25°C for 5 days. Te MICs were determined by measuring the absorbance at 600 nm to test bacterial pathogens and C. albicans and calculating fungal sporulation using a hemocytometer. All MIC determinations were performed in duplicate. Te MIC was defned as the lowest concentration at which growth was completely inhibited. Bacteria in CAMH broth and fungi in potato dextrose broth were used as control.

Statistical Analysis.
Te susceptibility assay of test microorganisms to phycobiliprotein extract from Arthrospira fusiformis was carried out in triplicate and the obtained results were demonstrated as mean ± SD of triplicate. Data analysis was done by using Student's t-test and McNemar's test. A P value of less than 0.05 was regarded as statistically signifcant.

GC-MS Analysis of Algal Hot Water Extract.
Te percentage composition of the volatile compounds and fatty acids identifed in the hot water extract of Arthrospira fusiformis isolated from Lake Mariout and cultivated in Amara and Steinbüchel medium is presented in Table 1. A total of two fatty acids, one fatty acid methyl ester, and three volatile compounds were identifed. Te major components of A. fusiformis fatty acids were hexadecanoic acid (palmitic acid, 55.19%) and octadecanoic acid (stearic acid, 27.14%). Te main constituents of its volatile compounds were acetic acid (43.33%) and oxalic acid (47.98%). GC-MS chromatograms of fatty acids and volatile compounds composition of algal hot water extract are presented in Figures S1 and S2. GC-MS chromatogram of algal hot water extract revealed the presence of a secondary metabolite or bioactive (2H-Pyran, tetrahydro-2-(12-pentadecynyloxy), and monoterpenoid (3,7-dimethyl-1,6-octadiene).

Antimicrobial Activities of Phycobiliprotein Extract from
Arthrospira fusiformis. Te test microorganisms difered in their susceptibility to phycobiliprotein extract from Arthrospira fusiformis. Te Gram-positive bacteria MRSA and S. aureus in addition to the Gram-negative bacteria P. aeruginosa, K. pneumonia, and S. sonnei were resistant (no inhibition zones) to the phycobiliprotein extract. Te most potent antimicrobial efect of phycobiliprotein extract was obtained against two Gram-negative bacteria Salmonella typhi and Proteus vulgaris, flamentous fungus Aspergillus niger, and the yeast Candida albicans (with the largest inhibition zone of 20 mm) as recorded in Table 2. Test microorganisms that showed susceptibility (inhibition zones) to phycobiliprotein extract from Arthrospira fusiformis are demonstrated in Figure S3.

Broth Microdilution Susceptibility Assay.
Results of broth microdilution susceptibility assay revealed that S. typhi, P. vulgaris, C. albicans, and A. niger were the most susceptible test organisms with minimal inhibition concentration of 58.1 μg/ml, while the Gram-negative bacterium S. marcescens and the flamentous fungus A. favus were the least susceptible ones (MIC of 232.5 μg/ml) ( Table 3).
In 2010, Sharaf et al. [20] isolated a new Arthrospira fusiformis strain from the brackish Lake Mariout at the southwest of Alexandria city in Egypt. Te strain was proved to have unique tolerance for salinity, and its phosphate bufer and hot water extracts could directly inactivate herpes simplex viral particles before infection of host cells. Tey recommended applying Spirulina crude extracts as a treatment for recurrent herpetic infection [20].
In this study, we identifed volatile compounds and fatty acids composition extracted by hot water from Arthrospira fusiformis isolated by Sharaf et al. [20] and tested the antimicrobial activities of its phycobiliprotein extract using sodium phosphate bufer. Te obtained results revealed that two fatty acids, one fatty acid methyl ester, and three volatile compounds were identifed in the hot water extract of Arthrospira fusiformis. Te major components of its fatty acids were palmitic acid (55.19%), and stearic acid (27.14%). Our results support and agree with a previous report by Abd El-Hameed et al. [63], which demonstrated that adding Spirulina platensis (Arthrospira fusiformis) to raw and cooked spaghetti leads to an increase of fve fatty acids including palmitic acid, and stearic acid. Tey also observed that the fatty acid profle of spaghetti prepared with the incorporation of Spirulina platensis has a higher resistance to the thermal treatment applied during the cooking [63]. Te main constituents of volatile compounds in hot extract were acetic acid (43.33%), and oxalic acid (47.98%). GC-MS chromatogram of algal hot water extract also revealed the presence of a secondary metabolite, 2H-Pyran, tetrahydro-2-(12-pentadecynyloxy, with a percentage of 3.7. Tis secondary metabolite has potential application as an inhibitor to non-small cell lung cancer as well as antibacterial and antioxidant activities [64,65].
In the present study, results obtained from antimicrobial activities assays of the phycobiliprotein extract of Arthrospira fusiformis revealed that eight of the tested bacterial and fungal pathogens were susceptible to the phycobiliprotein extract but the most potent antimicrobial efects were observed against Salmonella typhi, Proteus vulgaris, Aspergillus niger, and Candida albicans. No antibacterial efect was found on methicillin-resistant as well as susceptible Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Shigella sonnei. Tese results agree in part with previous reports by Murugan [66], and Zamani et al. [67], which showed that C-phycocyanin of Arthrospira fusiformis has antifungal activities against Aspergillus niger, Aspergillus favus, and Candida albicans. Tese results also are in agreement with previous studies by Sun et al. [22], Sadeghi et al. [23], and Abdel-Moneim et al. [24], which  Te results obtained from this work suggest the potential use of biomass of A. fusiformis isolated from Lake Mariout as a food additive. Our fndings combined with the results obtained by Sharaf et al. [20,21] also recommend that A. fusiformis isolated from Lake Mariout is a potential antimicrobial against some bacterial and fungal pathogens and antiviral therapeutic.

Conclusion
Tere is a growing interest in the use of microalga Arthrospira fusiformis not only for food applications but also as safe pharmaceutical products with antimicrobial and antioxidant activities as well as corrective properties  Mean of three assays; C-chloramphenicol and FC-fusidic acid antibacterial standards at concentrations of 50 and 10 μg/ml, respectively; AMP-amphotericin-B antifungal standard at a concentration of 100 μg/ml; R-resistant (no inhibition zone); NT-not tested. Te Scientifc World Journal against tumor growth, anemia, and malnutrition. Terefore, we cultivated a previously isolated Egyptian Arthrospira fusiformis in the laboratory and analyzed its volatile compounds and fatty acids composition in the hot extract. We used hot water extraction only to analyze the components (volatile compounds and fatty acids) consumed by people if they use Spirulina as a hot extract or food ingredient in cooking. Te antimicrobial activity of its phycobiliprotein extract was also evaluated against thirteen pathogens, eight of these pathogens were susceptible to the phycobiliprotein extract. Te major fatty acids found were palmitic acid and stearic acid, which suggests the promising application of Egyptian A. fusiformis biomass as an ingredient in the cooking of some foods such as pasta or bread to increase the level of the two most commonly consumed saturated fatty acids; stearic acid and palmitic acid. Overall, our fndings revealed that A. fusiformis isolated from Lake Mariout has nutritional as well as efective antimicrobial properties and suggest the potential therapeutic use of its biomass.

Data Availability
Data are contained within the article and supplementary materials.

Ethical Approval
Not applicable.

Consent
Not applicable.

Conflicts of Interest
Te authors declare no conficts of interest.  Figure S1: GC-MS chromatogram of fatty acids composition of algal hot water extract. Figure S2: GC-MS chromatogram of volatile compounds composition of algal hot water extract. Figure S3: Test microorganisms that showed susceptibility to phycobiliprotein extract from Arthrospira fusiformis. (Supplementary Materials)