ANTIBACTERIAL ACTIVITY OF FIVE SPECIES OF MARINE MICROALGAE

ANTIBACTERIAL ACTIVITY OF FIVE SPECIES OF MARINE MICROALGAE

Teja *, P. Yedukondala Rao, P. Janakiram, D. Sunil Kumar, V. A. Iswarya Deepti and P. Lakshmi Chaya

Department of Marine Living Resources, Andhra University, Visakhapatnam, Andhra Pradesh, India.

ABSTRACT: Antibacterial activity of five marine microalgae Isochrysis galbana, Chaetoceros calcitrans, Tetraselmis suecica, Nannochloropsis oculata, Aphanocapsa sp. was studied from Visakhapatnam. The ethanolic extract of T. suecica, C. calcitrans, and N. oculata s howed inhibitory activity against V. harveyi and S. Aureus with inhibition zone between 11.5 and 20mm. The ethanolic extract of I. galbana showed inhibitory activity against S. aureus only with an inhibition zone of 15mm. Among the four extracts, T. suecica showed maximum inhibitory activity against V. harveyi with an inhibition zone of 20mm. The methanolic extract of T. suecica moderately inhibited the growth of V. harveyi and S. aureus with inhibition zones of 11.5 and 11 mm respectively, whereas the extracts of I. Galbana and C. Calcitrans inhibited only V. harveyi with inhibition zones of 10 and 12 mm respectively, but no activity was found against S. aureus, A. hydrophila and E. coli. So, the studied microalgae could be used as a potential source to extract, bioactive marine natural compounds with antibacterial activity.

Keywords: Marine microalgae, Ethanolic extract, Methanolic extract, Antibacterial activity, Inhibition zone

INTRODUCTION: Natural products from marine organisms have recently acquired importance in the pharmaceutical and pesticide industries. Marine organisms could be a potential source of bioactive secondary metabolites that represent useful leads in the development of new pharmaceutical agents ^{1, 2}. Many chemically distinct marine compounds with various biological activities have been isolated to date, and a number of them are being investigated and/or developed as new pharmaceutical products ^3-5. More than 10,000 compounds have been isolated from marine organisms so far, with hundreds (or) more being discovered every year ^{6, 7}.

Decades of research have demonstrated that marine organisms provide tremendous opportunities for harvesting anti-microbial substances and providing clues for their laboratory synthesis. Microalgae have recently received much attention due to their diverse phytometabolic content with various chemical structures and biological activities ⁸. Algal biomass and its derivatives have many potential applications, ranging from animal feed and aquaculture to human nutrition and health products ^{9, 10}.

Because of their phototropic existence and constant exposure to high oxygen and radical stresses, microalgae also have a high ability to produce a variety of efficient protective chemicals against oxidative and radical stressors ¹¹. With the dawn of molecular biology, the screening of microalgae for antibiotics and other bio-active compounds has received significant attention since Pharma-cological properties were detected in their extracts. Several researchers have investigated a variety of algal-derived substances with bacteriostatic and bactericidal properties ¹². Microalgae have become a prominent source of antibacterial compounds and offer numerous advantages for antimicrobial studies due to their huge biodiversity and rapid growth rate ¹³.

Bioactive substances with antitumor, antileukemia, antibacterial, and antiviral properties have been reported all around the world. The demand for effective and non-toxic antibacterial therapeutics has increased. Antibacterial activity has been reported for a variety of compounds such as fatty acids ¹⁴, terpenoids, carbohydrates ¹⁵, peptides, polysaccharides, and alkaloids ¹⁶.

The production of secondary metabolites from microalgae is usually higher during stressful conditions ¹⁷. In the larval rearing of marine fish, crustaceans, and bivalves, cultured microalgae are commonly used as a live feed.

It has been observed that the addition of microalgae has a positive effect on the larval rearing systems by decreasing the number of opportunistic bacteria ¹⁸. The larvae's enhanced survival rates in tanks supplemented with microalgae were observed in the rearing of many marine fish species ^{19, 20}.

The positive effect of microalgae introduced to larval rearing tanks has been attributed to the stabilization of the nutritional value of live food organisms added to the tanks ²¹, as well as to non-nutritional aspects such as the stimulation of the larvae’s digestive system and immune system and the effect of the bacterial communities associated with the microalgae ^22-24. In light of the above, the present investigation focused on the antibacterial activity of five marine microalgal species viz. Isochrysis galbana, Chaetoceros calcitrans, Tetraselmis suecica, Nannochloropsis oculata and Aphanocapsa sp. (Cyanobacteria) against selected bacterial pathogens Staphylococcus aureus (Gram+ve), Vibrio harveyi, Aeromonas hydrophila and Escherichia coli (Gram -ve), which cause diseases in human and aquaculture species. This study was undertaken in the dept. of Marine Living Resources, Andhra University, Visakhapatnam, India during 2017-18.

MATERIALS AND METHODS:

Microalgal Culture: The stocks of five marine microalgal species I. galbana, C. calcitrans, T. suecica, N. oculata and Aphanocapsa sp. were used in this study procured from the regional center of Central Marine Fisheries Research Institute (CMFRI), Visakhapatnam, Andhra Pradesh, India. The microalgae are cultured in Conway ²⁵ medium at salinity 34 ppt and at 23±2 °C temperature under 12:12 light-dark cycle with a light intensity of 80 µmol photons. m^-2.s^-1.

Biomass Harvest: At the end of the exponential phase, the algal culture was kept at 1-4° C, where the water could be in a minimum cooling point without freezing for 2-5 days until all the cells settled down. The period was varied according to cell size and density, i.e. larger and high-density cells settled early. Then the upper clear culture medium was discarded, and the biomass was washed twice with distilled water and dried in a hot air oven at 45-50° C for 48h.

Preparation of Crude Extract: Individually dried microalgal samples were powdered finely and soaked for 48 h in two solvents, i.e. ethanol and methanol, separately in a ratio of 1:8 (w/v). The solvent was decanted and then concentrated using a rotary evaporator (Heidolph G3). The final concentration was adjusted to 100mg/ml.

Antibacterial Activity: The concentrated crude extract thus obtained was tested against four pathogenic bacteria of the shrimp, fish, and human viz. Vibrio harveyi (MTCC: 3438), Staphylococcus aureus (ATCC: 11632), Aeromonas hydrophila (MTCC: 1739), and Escherichia coli (MTCC: 1678). Antibacterial activities of the extracts were analyzed using the agar well diffusion technique followed by ²⁶.

The isolates viz. S. Aureus (gram +ve), V. harveyi (-ve), A. hydrophila (-ve) and E. coli (-ve) were inoculated individually into sterile nutrient broth taken in four different test tubes and incubated at 37° C for 18 h. Young cultures aforementioned were swabbed onto the surface of 3.8% Muller’s Hinton Agar (MHA) plates separately. In each of these plates, wells of 6 mm diameter were made using a sterile cork borer. Exactly 50 µl of each crude extract was filled in respective wells and allowed to diffuse at room temperature for 2 h. The respective solvents were used as controls. Then plates were inverted and incubated at 37°C for 24 h. Antibacterial activity was expressed in terms of zone of inhibition diameter (mm) using the Kirby-Bauer scale ²⁷.

Sensitivity of Bacterial Pathogens to Commercially Available Antibiotics: Simul-taneously the inhibitory activity of six commercially available antibiotics viz. Chloramphenicol, Gentamycin, Tetracycline, Erythromycin, Furozolidone and Streptomycin were tested against S. aureus, V. harveyi, A. hydrophila and E. coli in question for comparative study by using agar disc diffusion method.

The antibiotic discs (6mm) were placed on the MHA plates. Antibacterial activity was expressed in terms of zone of inhibition diameter (mm) using the Kirby-Bauer scale ²⁷.

RESULTS:

Ethanol Extract: The ethanolic extract of T. suecica highly inhibited the growth of V. harveyi and S. aureus with inhibition zones of 20 and 13 mm respectively, but no activity was found against A. hydrophila and E. coli, whereas the extracts of C. calcitrans and N. oculata also inhibited V. harveyi and S. aureus with inhibition zones of 13 and 12 mm & 13 and 11.5 mm respectively, but showed no inhibitory activity against A. hydrophila and E. coli.

The extract of I. galbana highly inhibited the growth of S. aureus only with an inhibition zone of 15 mm. The extract of Aphanocapsa sp. showed no inhibitory activity against all four tested pathogenic bacteria Table 1 Fig. 1 and 2.

Methanol Extract: The methanolic extract of T. suecica moderately inhibited the growth of V. harveyi and S. aureus with inhibition zones of 11.5 and 11 mm respectively, but no activity was found against A. hydrophila and E. coli, whereas the extracts of I. galbana and C. calcitrans inhibited only V. harveyi with inhibition zones of 10 and 12 mm respectively, but no activity was found against S. aureus, A. hydrophila and E. coli. Extracts of Aphanocapsa sp. and N. oculata showed no inhibitory activity against all four tested pathogenic bacterial strains Table 2, Fig. 3 and 4.

Sensitivity of Bacterial Pathogens with Commercially Available Antibiotics: Six selected antibiotics were tested against four bacterial pathogens, but results presented here were only against two bacteria i.e. V. harveyi and S. aureus, as there was no inhibitory activity with microalgal extracts on A. hydrophila and E. coli.

Hence, the comparison has been confined to V. harveyi and S. aureus only. Out of six antibiotics, four showed sensitivity on V. harveyi and S. aureus.

Furazolidone and streptomycin showed activity against V. harveyi but no activity against S. aureus. Among six antibiotics tested, tetracycline showed the highest sensitivity of 22 mm inhibition zone against V. harveyi and S. aureus Table 3, Fig. 5.

FIG. 1: INHIBITORY ACTIVITY OF THE CRUDE MICROALGAL ETHANOLIC EXTRACTS ON VIBRIO HARVEYI

FIG. 2: INHIBITORY ACTIVITY OF THE CRUDE MICROALGAL ETHANOLIC EXTRACTS ON STAPHYLOCOCCUS AUREUS

FIG. 3: INHIBITORY ACTIVITY OF THE CRUDE MICROALGAL METHANOLIC EXTRACTS ON VIBRIO HARVEYI

FIG. 4: INHIBITORY ACTIVITY OF THE CRUDE MICROALGAL METHANOLIC EXTRACTS ON STAPHYLOCOCCUS AUREUS

FIG. 5: ANTIBIOTIC SENSITIVITY ON PATHOGENIC BACTERIA

TABLE 1: ANTI-BACTERIAL ACTIVITY OF ETHANOLIC EXTRACTS OF FIVE MARINE MICROALGAE

NA-No activity

TABLE 2: ANTIBACTERIAL ACTIVITY OF METHANOLIC EXTRACTS OF FIVE MARINE MICROALGAE

NA-No activity

TABLE 3: BACTERIAL SENSITIVITY WITH COMMERCIALLY AVAILABLE ANTIBIOTICS

NA-No activity

DISCUSSION: The chrysophyte Isochrysis galbana has been reported to have a wide range and degree of inhibitory activity against pathogens such as P. aeruginosa, K. Pneumonia, Proteus vulgaris, P. fluorescens, S. typhiand B. subtilis ^{15, 28-32}. In the present study ethanolic extract of I. galbana also showed high inhibition (IZ-15mm) against S. aureus, whereas methanolic extract showed moderate inhibition (IZ-10mm) against V. harveyi. Chaetoceros launderi, tested against fungi and significant activity was observed against all the dermatophytes ³³. Selvendran & Michael Babu ³⁴ reported Chaetoceros calcitrans is best controller of all the shrimp bacterial pathogens. George et al. ³⁵ reported methanolic extract of Chaetoceros showed moderate activity against V. harveyi & S. aureus, but no activity with ethanolic extract. In the present study, C. calcitrans showed only moderate activity against shrimp bacterial pathogens like V. harveyi & S. aureus. The present study showed that the microalgal extracts obtained from Tetraselmis suecica claimed to be the best inhibitor against the growth of V. harveyi and S. aureus. This study is in concurrence with the one where T. suecica showed activity against a number of pathogenic Vibrio species including V. alginolyticus, V. parahaemolyticus and V. vulnificus ³⁶. Similarly, Tetraselmis showed inhibitory activity against soil bacteria ¹⁵, V. harveyi ³⁷ and E. coli & S. aureus ^{28, 30}. Over 132 marine microalgae when screened against six strains of bacteria revealed that methanolic and hexane extracts were more effective against S. aureus and Streptococcus faecalis and less effective with regard to Bacillus subtilis ³⁸. Ethanolic extract of Noctiluca scintillans showed antibacterial activity against E. coli while extract in acetone inhibited the growth of S. faecalis ³⁹. In the present study ethanolic extracts of I. galbana, C. calcitrans, T. suecica and N. Oculata showed the best results which are in concurrence with the report that the growth of bacterial pathogens i.e. Psuedomonas vulgaris (26.9%), Shigella sp. (26.3%) and Salmonella typhi (22.6%) ³².

Ochromonas sp., Prymnesium parvum, and a number of blue-green algae produce toxins that may have potential pharmaceutical applications ¹⁶and Oscillatoria sp. showed activity against S. aureus & E. coli ⁴⁰, but in the present study, both ethanolic & methanolic extracts of Aphanocapsa sp. showed no activity against all four bacterial pathogens. The temperature in incubation, pH of the culture medium, incubation period, medium constituents, and light intensity are the important factors influencing antimicrobial agent production ⁴¹.

In the present study, streptomycin showed no inhibition activity against S. aureus, whereas ethanolic extract of N. oculata has shown moderate to high inhibition against both S. aureus and V. harveyi. Surendhiran et al. ⁴² tested N. oculata FAME against different microbial strains; among them E. coli was found to be more sensitive with an inhibition zone of 27 mm than other microorganisms such as B. subtilis (16 mm) and S. aureus (17 mm). Hassi et al. ³⁰ also mentioned ethanolic extract of N. gaditana showed positive against E. coli. But in the present study N. oculata were did not show any activity against E. coli. The strains of V. harveyi were sensitive to the antibiotics tested except Ampicillin ⁴³. He also reported that chloramphenicol showed the highest zone of inhibitions i.e., 31, 25, 26 mm, against V. harveyi strains of PSU 2015, AAHRC1, and AAHRC2, respectively. In the present study, all the tested antibiotics showed inhibition against V. harveyi and S. aureus, but furazolidone and streptomycin showed no activity against S. aureus. Tetracycline showed the highest zones of inhibition (22 mm) against V. harveyi and S. aureus. In the present study, almost all microalgal extracts have shown greater or equal inhibitory activity against V. harveyi and S. aureus.

CONCLUSION: Since, marine microalgae seem to be a potential source for antibacterial compounds and especially show inhibitory activity against the bacterial pathogens of fish and shrimp more like commercial antibiotics. Further explorations towards this venture are needed for healthy and sustainable aquaculture.

ACKNOWLEDGMENTS: The authors are thankful to Dr. K. Umadevi, CSIR-NMITLI laboratories, Department of Marine Living Resources, Andhra University, for providing the algal culture lab facility.

Authors' Agreement to Authorship and Submission: All the authors agreed to the authorship and submission of this manuscript to the International Journal of Pharmaceutical Sciences and Research for peer review.

Funding: This research has not received any grant from funding agencies in the public, commercial, or not-for-profit sectors.

Statement of Informed Consent, Human/Animal Rights: No conflicts, informed consent, or human or animal rights are applicable to this study.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Teja G, Rao PY, Janakiram P, Kumar DS, Deepti VAI and Chaya PL: Antibacterial activity of five species of marine microalgae. Int J Pharm Sci & Res 2022; 13(11): 4763-70. doi: 10.13040/IJPSR.0975-8232.13(11).4763-70.

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