4.3 Antimicrobial activity test
4.3.1 Antimicrobial activity of microalgae against Aeromonas hydrophila…
Antimicrobial activity of methanol extracts of Tetraselmis and Chlorella was demonstrated at variable extents against Aeromonas hydrophila in the current study (Figure 4.5).Larger inhibition zone appeared around the disc loading of extract of Tetraselmis with a diameter of 6.65mm (p<0.01).
Page | 41 Figure 4.5Antimicrobial activity of microalgae against Aeromonas hydrophila. a) Agar plate indicates clear zone of bacterial growth surrounding the antibiotic (A), Tetraselmis (T) and Chlorella (C) with no zone of inhibition around control disc (N).
b) Both Tetraselmis and Chlorella showed significantly higher zone of inhibition with Tetraselmis the highest. Statistical tests done by ANOVA, error bars represent standard error of means, p**<0.01.
4.3.2 Antimicrobial activity of microalgae against Lysinibacillus sphaericus
The antimicrobial activity of methanol extracts of Tetraselmis and Chlorella was observed against Lysinibacillus sphaericus(Figure 4.6). Both the microalgae had little effect on the bacteria though the results were not statistically significant (p>0.05).
Figure 4.6Antimicrobial activity of microalgae against Lysinibacillus. a) Agar plate indicates clear zone of bacterial growth surrounding the antibiotic (A), Tetraselmis (T), and Chlorella (C) with smaller inhibition zone and no zone around control disc (N). b) Statistical analysis of microalgae with control showed no significant difference in antibacterial activities. Statistical tests done by ANOVA, error bars represent standard error of means, ns: not significant.
4.3.3 Antimicrobial activity of microalgae against Staphylococcus
Against Staphylococcus saprophyticus, Chlorella showed greater activity compared to Tetraselmis with a diameter of 6.57mm (p<0.01) (Figure 4.7). Larger inhibition zone
Page | 42 appeared around the disc loading of extract of Chlorella with a diameter of 6.57mm (p<0.01).
Figure 4.7Antimicrobial activity of microalgae against Staphylococcus. a) Agar plate indicates clear zone of bacterial growth surrounding the antibiotic (A)Tetraselmis (T) and Chlorella (C) with no zone around the control disc (N). b) Both the microalgae showed significantly higher antibacterial activity. Statistical tests done by ANOVA, error bars represent standard error of means, p**<0.01.
4.4 Antimicrobial index of microalgae extracts
Antimicrobial activity of microalgae extracts was calculated using the formula which was mentioned earlier in the Materials and Methods chapter. The relative percentage (%) inhibition zones of Tetraselmis extract have presented the highest against Aeromonas hydrophila, followed by Staphylococcus and the lowest against Lysinibacillus sphaericus. While Chlorella exposed relative percentage the highest against Staphylococcus followed by Aeromonas hydrophilic and Lysinibacillus sphaericus.
Table 4.1 Relative percentage inhibition zones of Tetraselmis and Chlorella against Aeromonas hydrophila, Lysinibacillus sphaericus and Staphylococcus
Selected bacteria Relative percentage (%) inhibition zones Tetraselmis sp. Chlorella sp.
Aeromonas hydrophila 1.99 1.168
Lysinibacillus sphaericus 0.879 1.077
Staphylococcus 1.156 1.262
Page | 43 4.5 Minimal inhibitory concentration (MIC)
The MIC of the extracts from Chlorella and Tetraselmis grown under different conditions were determined against Aeromonas hydrophila and Staphylococcus. Due to insufficient amount, it was not possible to determine MIC value of these microalgae against Lysinibacillus sphaericus. The MIC value was determined as the minimum concentration of microalgae capable to prevent bacterial growth. It was observed that Tetraselmis has strong antimicrobial activity against Aeromonas hydrophila, however, Chlorella has against Staphylococcus (Table 4.2).
Table 4.2 MIC value of microalgae against Aeromonas and Staphylococcus
Microalgae MIC value (mg/ml)
Aeromonas hydrophila Staphylococcus
Chlorella 40 20
Tetraselmis 10 20
Page | 44 CHAPTER 5
DISCUSSION
Natural pigments such as carotenoids, chlorophylls, phenolics, hycocyanin, phycoerythrin and phycobiliproteins are produced by microalgae are well known.
They are used in nutraceutical, pharmaceutical and in food applications (Guerin et al., 2003).The antibiotic content of algal species may vary depending on many ecological and biogeographically factors. Various strains of microalgae are known to produce intracellular and extracellular metabolites with diverse biological activities such as anti algal, antibacterial, antifungal, and antiviral activity.
The present study was aimed at the screening of antimicrobial activities of three microalgae belonging to the genus Tetraselmis, Chlorella and Nanochloropsis against one Gram-negative Aeromonas hydrophila and two Gram-positive bacteria Lysinibacillus sphaericus and Staphylococcus. All the samples were collected from Bata (Labeo bata) and Tilapia (Oreochromis niloticus) fish were positive for Aeromonas hydrophila, Lysinibacillus sphaericus and Staphylococcus. Furthermore, Tetraselmis and Chlorella had variable degrees of suppressive activities against all three bacteria. Marine microalgae present many bioactive compounds such as fatty acid, acrylic acid, halogenated aliphatic compounds, terpenes, sulphur-containing hetero cyclic compounds, carbohydrates and phenols that have antimicrobial effects recognized for long time (Kannan et al.,2010).In this study, methanolic extracts of the marine microalgae were used to determine the antimicrobial activities. Previous study reported that, extraction of algal samples of Chlorella and Nannochloropsis was performed using methanol (Qusem et al., 2016).
In the current study, bacterial samples were collected from gill and intestine of Bata (Labeo bata) and Tilapia (Oreochromis niloticus) fish and were positive for Aeromonas hydrophila, Lysinibacillus sphaericus and Staphylococcus. Previous study reported that, bacterial samples collected from eye, stomach, kidneys, gills and intestine of Tilapia (Oreochromis niloticus) were positive for Lactobacillus sp., Bacillus firmus, Aeromonas hydrophila, Pseudomonas anguilliseptica, and Pseudomonas fluorescenses (Austin et al., 2012; Cruickshank et al., 1975). Due to
Page | 45 limitations of budget, we aimed only three bacterial species and could possibly get more if explored extensively.
Present study demonstrated that Chlorella is effective to prevent growth of Gram- positive bacteria Staphylococcus than Gram-negative bacteria Aeromonas. The findings are in agreement with a previous study where antibacterial activity of Chlorella vulgaris extract was reported against Bacillus subtilis, Staphylococcus aureus and Klebsiella pneumonia (El-deen et al., 2014). Mokbel and his co-workers (2005) tested the effect of butanedioic acid against some Gram-negative and positive bacteria and found that butanedioic acid has antibacterial activity against S. aureus, B.
cereus, S. enteritidis and E. coli. Chlorella contains several compounds that might have inhibitory effects on the growth of pathogenic bacteria. Myristic acid, palmitic acid and butanedioic acid have good antimicrobial activity that is contained by Chlorella. Myristic acid, palmitic acid and fatty acids the bactericidal properties of fatty acids are well known (Kabara et al., 1972; Mokbel et al., 2005). On the contrary, Tetraselmis showed better antibacterial activities to Gram-negative bacteria Aeromonas followed by Gram-positive bacteria Staphylococcus in this study.
Tetraselmis contains high amounts of oleic acid, fatty acids, linoleic acid and palmaitic acid which might have antimicrobial activity against the bacterial species (Maadane et al., 2017). So, it can be considered that Tetraselmis sp. and Chlorella sp.
have present antibacterial activity against Gram-positive and Gram-negative bacteria.
An earlier study demonstrated that Nannochloropsis oculata had moderate antibacterial activity against Bacillus subtilis (Qusem et al., 2016). However, the current study opposes this statement as no effect of Nannochloropsis was observed against any of the three-bacterial species studied.
Besides, MIC was carried to study the antimicrobial activity of Tetraselmis sp. against Aeromonas hydrophila due to the largest inhibition zone of 1.99mm compared to Chlorella sp. and other test bacteria. Here Chlorella sp. antibacterial activity against Staphylococcus due to the largest inhibition zone of 1.262mm compared to Tetraselmis sp. and other test bacteria. MIC value of Tetraselmis sp. was resulted at the concentration of 10mg per ml to inhibit Aeromonas hydrophilic and Chlorella sp.
showed the 20mg per ml to inhibit Staphylococcus (1.5 x 108CFU/ml or 0.5 McFarland standards).So these extracts can control well the growth of the fish
Page | 46 pathogenic bacteria. Microalgal cells present different types of bioactive compounds such as bactericidal or bacteriostatic. These bioactive compounds action modes have been exposed for growth inhibition or killing of bacteria by some of the functional molecules. The effective of microalgae extracts used were sensitive to Gram-positive and Gram-negative bacteria. This is because of the difference cell wall structures of the Gram-positive and Gram-negative bacteria. The penetration, binding and antimicrobial activity of the compound was determined by the difference cell wall structures of bacteria. The cell wall structures of Gram-positive have thick peptidoglycan layer, lipid and polysaccharides that can dissolve in water that assist as a positive ion transport and carboxyl, amino acid and hydroxyl consist at a hydrophilic side of the bacteria which increase the effectiveness of antibacterial compounds to the bacteria. While for the Gram-negative bacteria, it has complex cell wall structures which are lipoprotein as outer layer, lipopolysaccharide as middle layer and the third layer of peptidoglycan. Phytochemical comprising microalgal substance may act by inducing cellular membrane perturbation, interference with certain microbial metabolic processes and modulation of signal transduction or gene expression. Free fatty acids could initiate peroxidative processes and preclude the synthesis of bacterial fatty acids. Besides free fatty acids might interact with cellular membranes of microbial cells causing leakage of molecules from these cells reduction of their nutrient uptake or inhibition of their respiration (Suresh et al., 2014)
In the present study, the antimicrobial activities of Tetraselmis and Chlorella extracts were attributed to their contents of fatty acids, carotenoids and polyphenols as discussed above. These substances probably act together, either in an independent or synergistic manner. Resistance to antibiotics by bacteria is a global threat. The resistance of microbes is due to indiscriminate utilization of commercial antimicrobials. Many researchers investigated antimicrobial resistance and concluded to explore newer antimicrobial substances from several medicinal plants and algae (Algesboopathi et al., 2012). In this regard, microalgae can be used as antimicrobial drug because there is no side effect and low cost.
Page | 47 CHAPTER 6
CONCLUSION
The current study demonstrated antibacterial activity of three-species of marine microalgae against three-species of bacteria isolated from gill and intestinal samples of Bata (Labeo bata) and Tilapia (Oreochromis niloticus) fish. It was observed that Chlorella has the greatest propensity to suppress Staphylococcus; however, Tetraselmis was very suppressive against Aeromonas. Nannochloropsis showed no sensitivity against Aeromonas, Staphylococcus and Lysinibacillus.
Page | 48 CHAPTER 7
RECOMMENDATIONS AND FUTURE PERSPECTIVES
According to this research work, the following recommendations may be done:
Antibiotics have high market price with side effects. Marine microalgae contain various bioactive compounds which inhibit the bacterial pathogens. The bioactive compounds may be suitably used for the other bacterial pathogens. Farmers may get good result when using of marine microalgae particularly, Tetraselmis and Chlorella sp. When fish are attacked by bacterial diseases then they can use marine microalgae because it is low cost, no side effect and high antimicrobial properties. Further studies are directed to identify the specific components of microalgae that are responsible for the sensitivities against bacterial infections.
Page | 49 LIMITATIONS
Isolation and identification of microalgae directly from the marine sources was not possible in the current study due to time limitation. We used the microalgae that we already have in our laboratory stored. A larger bacterial sample size could increase the sensitivity and specificity of the results. We also unable to grow enough microalgae for the antimicrobial sensitivity testing and determination of MIC values.
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