Bioactive Compounds from Microalgae Spirulina platensis as Antibacterial Candidates Against Pathogen Bacteria

Microbial infection by bacteria has caused severe health problems worldwide. Treatment with antibiotics as the current solution has several drawbacks and triggers the phenomenon of bacterial resistance. Therefore, there is an urgency to look for a natural antimicrobial that is safer and has fewer side effects. One of the most promising antibacterial agents is Spirulina platensis. This research was conducted to evaluate the antibacterial activity of microalgae S. platensis against Propionibacterium acne, Staphylococcus epidermidis, and Enterobacter aerogenes and identify compounds from the active fraction of microalgae. Biomass was extracted with ethanol 96% using the reflux method then partitioned with immiscible solvents such as hexane, ethyl acetate, and water. Partial purification was carried out by chromatography techniques such as thin-layer chromatography and column chromatography. The compounds of active fractions were identified by GC-MS analysis. The result showed that ethyl acetate extract had vigorous antibacterial activity against all tested bacteria. The highest activity (14.4 ± 0.63 mm and 16.9 ± 1.48 mm) was achieved against P. acne; followed by S. epidermidis (13.05± 0.14 mm and 13.15 ± 0.0 mm), and E. aerogenes (11.7 ± 2.05 mm and 12.6 ± 1.90 mm), at concentrations 20,000 ppm and 30,000 ppm, respectively. The results indicated that the extract is more sensitive to Gram-positive bacteria (P. acne and S. epidermidis) than Gram-negative bacteria (E. aerogenes). Purification of the extract resulted in fraction 2 and fraction 6 as the most potential fractions for further analysis and identification. Based on the antibacterial activity, inhibition zones of fractions are wider than extracts. It could be assumed that the purification process enhances the activity of a sample. GC-MS analysis revealed that the dominant compounds of fractions 2 and 6 were bis (2-ethylhexyl) phthalate (67.76%) and 1,2-Benzendicarboxilic acid, bis (2-ethylhexyl) ester (50,88%), respectively. This result indicated that the ethyl acetate fraction of the microalgae S. platensis has the potential as a natural antibacterial.


Introduction
Microbial infections have become one of the significant causes of health problems and mortalities globally [1]. Among microbes, most infectious diseases are caused by bacteria. A common skin disease called acne vulgaris can be caused by several skin bacteria, i.e., Staphylococcus epidermidis [2] and Propionibacterium acne [3]. Acne vulgaris, otherwise known as acne, is a common and chronic inflammatory disorder that affects the pilosebaceous unit, associated with hair follicles [4]. This disease usually affects teenagers and young adults [5], and the causes are associated with increased sebum production, hyperkeratinization, bacterial colonization, and inflammation. The occurrence of skin tissue damage is triggered by enzymes secreted by bacteria P. acne [6]. Bacteria Enterobacter aerogenes has been reported as

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Check for updates important opportunistic and multiresistant bacterial pathogens for humans. This species is categorized as nosocomial pathogens, which contribute to several infections, such as endocarditis, bacteremia, skin infection, respiratory, urinary, and gastrointestinal tract [7].
The efforts to overcome bacterial infections have been carried out using antibiotics. However, antibiotics have several drawbacks related to toxicity, cost, and the indiscriminate and excessive use of antibiotics may lead to microbial resistance [1]. Therefore, it is necessary to search for natural sources of safer drugs with limited side effects than synthetic ones. Hopefully, this can reduce and replace the synthetic compounds used daily. In the last decades, researchers have been interested in searching for antimicrobial candidates from plants and microalgae such as cyanobacteria [8,9]. One of the most interesting species from cyanobacteria is Spirulina platensis, which produces many metabolites, making them a potential source of medicine [10]. Spirulina biomass is rich in metabolites, composed of 60-70% proteins, carbohydrates, fatty acids, chlorophyll a, phycocyanin, carotenes, vitamins especially provitamin A, vitamin C, vitamin E, and minerals such as iron, calcium, magnesium, potassium, sodium, zinc, phosphorus [11].
A previous study by Bellahcen (2019) [12] showed that the essential oil of Spirulina inhibits the growth B. antharcis, S. epidermidis, and E. coli with the higher inhibition zone of 28 mm. Methanol extract of S. platensis also showed inhibition activity against S. aureus , P. acne, Streptococcus pyogenes, S. epidermidis, Bacillus cereus, Proteus mirabilis, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa, E. coli, and Vibrio cholerae [13,14]. Therefore, the present study was conducted to evaluate the antibacterial activity of microalgae S. platensis against bacteria Propionibacterium acne, Staphylococcus epidermidis, and Enterobacter aerogenes and identify compounds from the active fraction of microalgae.

Methodology
This research used microalgae S. platensis and was conducted in several steps, including cultivation of microalgae, extraction of bioactive compounds, evaluation of the antibacterial activity, partial purification with some chromatography techniques until the identification of compounds with Gas Chromatography-Mass Spectrometry (GC-MS).

Extraction of Bioactive Compounds
Sonication (40 Hz for 15 min) was conducted before extraction to break the microalgae cell and optimize the extraction process. Dried biomass was extracted with ethanol 96% (Merck) at 70℃ using the reflux method [15]. The extract was collected and concentrated using a rotary evaporator (Janke & Kunkel RV 05-ST) at 40℃ and stored for further use. Ethanolic extract was subjected to a partition process using immiscible solvents [16], such as hexane (Merck), distilled water, and ethyl acetate (Merck). The yield of each extract was calculated.

Antibacterial Assay
Antibacterial activity of extracts was evaluated against several skin bacteria, i.e., P. acne, S. epidermidis, and E. aerogenes, using the paper-disk diffusion method [8] in a double-layer medium. As much as 50 µL of each bacteria suspension 10 7 cell/ml was added to the semisolid Nutrient Agar (Merck) medium. Extract concentrations were 20,000 ppm and 30,000 ppm. Chloramphenicol 20 ppm was used as a positive control, while each solvent was a negative control. All treatments were incubated at 37℃ for 18-24 hours. Inhibition zone was measured and expressed in mm.

Phytochemical Analysis
Phytochemical content, including alkaloid, flavonoid, saponin, tannin, triterpenoid, and sterol, were analyzed according to Farnsworth & Harborne [17,18]. Alkaloid was analyzed using Mayer's, Wagner's, and Dragendorff's reagents. Flavonoid was identified according to the cyanidin reaction of Willstatter, which would detect the y-benzopyrone nucleus in the compound. Saponin was detected with the froth test, by the production of honeycomb froth after vigorous shaking of the solution. Tannin was identified by adding ferric chloride solution to the extract and should result in a dark blue or blackish-green color. In comparison, terpenoid compounds were identified with anisaldehyde sulfuric acid.

GC-MS Analysis
The active fraction was diluted in 1 ml ethanol (Merck), then 2 µl was injected into GC-MS 5973 N (Agilent Technologies). This instrument was equipped with DB 5 column (60 m in length and 0.25 mm in diameter) at injection temperature 70℃ and final temperature 290℃ and was using helium as carrier gas. The mass spectra of the component were analyzed based on Wiley 10 N.14 Library.

The Growth of Microalgae S. platensis
The increase in optical density indicates the growth of microalgae. The higher the optical density value, the more cells produced. Based on Fig. 1, the culture did not experience a lag phase (Figure 1). This cultivation was initiated with a high optical density at the logarithmic phase, so the culture grew faster with the suitable medium for growth. A previous study [21] revealed that high cell density at initial cultivation shortens the lag phase and significantly increases the logarithmic phase. Besides that, the biomass produced was also higher. Another factor that influences the absence of the log or adaptation phase is the Zarrouk medium, which is known as the maintenance medium for Spirulina. The adaptation phase occurs as a response of cells to the new growth medium. The more different the growth medium, the longer the adaptation phase [22]. The logarithmic phase occurred from day 1 to day 6, characterized by the addition of cells due to cells dividing rapidly. Day 7 to day 10 reached a stationary phase characterized by a balance between the rate of cell growth and cell death. In the late cultivation period, the OD starts to decrease as an indication of the death phase. Cell dividing almost does not occur while the death cell occurs massively [23]. The culture was harvested on day 7 at the stationary phase to get maximum compounds. From the experiment, dry biomass yield gained 1.3 ± 0,1 g.L -1 . This result was relatively low as compared to the many research that reported the maximum biomass dry weight of Spirulina in Zarrouk medium ranging from 0.69 to 6.90 g.L -1 [11]. This can be explained because, in the stationary phase, the culture is more focused on metabolites rather than growth productivity.
Harvesting S. platensis is relatively easy and straightforward because of its enormous size, around 200-300 µm in length and 5-70 µm in width [24]. Therefore filtration method was used in harvesting.

Extraction of bioactive compounds
The reflux method was used in this process, which involves heat to accelerate and maximize the extraction process. The first extraction used ethanol and produced 6.95 g of extract (yield 27.8%). The next step is partitioning with hexane, ethyl acetate, and water, producing an extract of 8.48%, 2.24%, and 7.92%, respectively (Table 1). The yield of extract indicated that ethanol is quite effective in extracting biomass. Wang (2010) [25] explained that ethanol has an excellent ability to penetrate the cellular membrane to extract intracellular materials. In addition, the yield of the extract also depends on the type of solvents, temperature, extraction time, and the chemical composition of the sample [26].
Each solvent only dissolves compounds with the same/ similar polarity, as the general principle in solvent extraction is "like dissolve like" [26]. In the partition process, non-polar compounds contained in the ethanol extract, such as alkanes, fatty acids, pigments, terpenoids, and alkaloids, will be extracted into hexane solvent. The semi-polar compounds such as alkaloids and flavonoids are extracted into ethyl acetate solvent. In contrast, polar compounds such as flavonoid glycosides, tannins, and alkaloids will be extracted into water solvents [27].

Antibacterial activity of the extract
Antibacterial activity of the extract was shown by inhibition zone formation. The diameter of the inhibition zone depends on the type of solvent used and the tested antibacterial activity [10]. The result showed that ethyl acetate extract has antibacterial activity against all tested bacteria and showed the highest activity (14.4 ± 0.63 mm and 16.9 ± 1.48 mm against P. acne; 13.05± 0.14 mm and 13.15 ± 0.0 mm against S. epidermidis; 11.7 ± 2.05 mm and 12.6 ± 1.90 mm against E. aerogenes, at concentrations 20,000 ppm and 30,000 ppm, respectively), followed by ethanol extract in the second place (9.05 ± 1.27 mm and 10.5 ± 0.49 mm against P. acne; 10.15 ± 0.0 mm and 9.7 ± 0.63 mm against S. epidermidis). Hexane and aqueous extracts showed no activity against all tested bacteria ( a medium category. Davis & Stout (1971) [28] stated that extracts with an inhibitory diameter of more than 20 mm were included in the very strong category, the inhibition diameter ranged from 10-20 mm was included in the strong category, the inhibition diameter ranged from 5-10 mm was included in the medium category. The inhibition diameter was less than 5 mm was included in the weak category. Negative control did not show activity, while positive control (chloramphenicol) showed activity against all tested bacteria.
Compared to the positive control, the antibacterial activity of the extract is lower. Nevertheless, the activity showed the potential of the extract as a natural antibacterial. It is indicated that optimizing processes such as extraction methods, solvent selection, and other strategies still need to be evaluated to maximize the activity. Chloramphenicol as positive control is a broadspectrum antibiotic that can inhibit Gram-positive and Gram-negative bacteria [29].  The results indicated that the extract is more sensitive to Gram-positive bacteria (P. acne and S. epidermidis) than Gram-negative bacteria (E. aerogenes). Gram-positive bacteria are generally more easily inhibited or killed than Gram-negative bacteria. It is due to differences in cell wall structure between Gram-positive and Gram-negative bacteria. Gram-positive bacteria have a thick cell wall and only have a single layer (monolayer). In contrast, the cell wall of Gram-negative bacteria is thin and has 3 layers (multilayer), making it challenging to absorb antibacterial compounds [30]. Gram-positive bacteria have a hydrophilic side, i.e., carboxyl, amino acids, and hydroxyl so that bacteria Gram-positive are more sensitive to antibacterial compounds. The formation of the inhibition zone is highly dependent on the amount of antibacterial agent that is dropped onto the disc, the solubility of the antibacterial agent in the medium, the diffusion coefficient, and the effectiveness of the antibacterial agent [31].

Phytochemical Analysis
Phytochemical analysis showed the presence of flavonoid and tannin in ethyl acetate extract (Table 3). These compounds have polar properties to be found in semi-polar ethyl acetate extract. Flavonoids show the antibacterial effect through 3 mechanisms, i.e., inhibiting the cell membrane function [32], inhibiting nucleic acid synthesis, and inhibiting the energy metabolism [33]. Meanwhile, tannin is also able to inhibit microorganisms [34].

Isolation and Partial Purification of Compounds
Thin-layer chromatography (TLC) was the first step in purifying compounds. Solvent with the best separation will be used for column chromatography. The result showed that hexane, ethyl acetate, and ethanol with a ratio 7.5:1.5:1 gave the best separation and the greatest number of spots (Figure 2). Rf value of the spots were 0.78; 0.68; 0.52; 0.4; 0.36; 0.32; 0.28; 0.24; 0.2; 0.16. A good eluent can separate compounds in large quantities marked by the appearance of spots. The spots formed are tailless, and the distance between the spots is clear [18].  (Table 4). These fractions were subjected to further analysis.

Antibacterial activity of fractions
The result showed that the inhibition zones of fractions are wider than extracts (Table 5). It could be assumed that the purification process enhances the activity of the sample. The strongest fractions activity was achieved against S. epidermidis (14.25 ± 0.98 mm and 17.65 ± 2.12 mm for fractions 2 and 6, respectively). Fractions 2 and 6 were subjected for identification with GC-MS according to the activity against all tested bacteria. Both fractions had strong activity because of the inhibition zone at the range of 10-20 mm, except fraction 6 against P. acne, which was categorized as a medium category [28]. Fraction 5, even though it had higher activity than fraction 6, also had a high deviation standard. That is why fraction 6 was picked up for the following analysis. Fractions 7 and 8 were not used for further analysis because they had higher concentrations than the others (20,000 ppm).

Conclusion
Several drawbacks related to antibiotics to treat bacterial infections have prompted researchers to look for safer natural antimicrobials. S. platensis is a promising microalga due to its metabolites content and bioactivity. Extract of ethyl acetate was successfully obtained from S. platensis and showed strong antibacterial activity against all tested bacteria, i.e., P. acne, S. epidermidis, E. aerogenes. Based on the result, the extract was more sensitive to Gram-positive bacteria than Gram-negative bacteria, influenced by the structure and component of the bacterial cell wall. Partial purification with the chromatography method showed fraction 2 and fraction 6 as the potential antibacterial fraction for further analysis and identification. The purification process is assumed to enhance the activity of a sample, indicated by the wider inhibition zones formed. GC-MS analysis revealed that the dominant compounds of fractions 2 and 6 were bis (2-ethylhexyl) phthalate (67.76%) and 1.2-Benzendicarboxilic acid, bis (2-ethylhexyl) ester (50.88%), respectively. Further purification is needed to gain a pure compound responsible for the antibacterial activity. Overall, optimizing processes such as extraction methods, solvent selection, and other strategies still need to be evaluated to maximize the activity.