Optimal conditions for antimicrobial activity production from two microalgae chlorella marina and Navicula F. Delicatula.
MATERIALS AND METHODS
Isolation and purification of algal isolates
The algal strains (Chlorella marina and Navicula f. delicatula) were isolated from two different locations, namely, El-Agamy (west of Alexandria) and Baltim (East of Alexandria) in the Mediterranean coast of Egypt Figure 2.
Samples were grown in F/2 medium (Guillard and Ryther 1962, Guillard 1975). The algal strains were harvested at their exponential phase of growth which is 12the day for chlorella marina and 14the for Navicula f. delicatula at aerated condition. Harvesting took place by centrifugation at 4000 rpm for 15 min. The isolated strains were identified according to (Tomas C. et al., 1996, Prescott 1968, Cronberg G. et al., 2006).
1. Two gram positive: (Staphylococcus aureus and Micrococus Luteus).
2. Three gram negative: (Serratiam arcescens, Pseudomonas aeruginosa, andE.Coli).
3. The unicellular fungus (Candida albicans).
These test organisms were deposited as culture collection at Microbiology Lab., National Institute of Oceanography and Fisheries Alexandria.
Preparation of the Algal Extracts
The two microalgae were grown in F/2 medium at aerated conditions. We make harvest for growth at stationary face, the culture centrifuged and the pellets were dried in hot air oven (60[degrees]C) till constant weight and used for extraction of antimicrobial agents. 0.5 g of each dried biomass of the two microalgae was extracted in 10 ml each of chloroform, acetone, ethanol, methanol and aqueous. All of the extracts were preserved at -4[degrees] C (Gonzalez Del Val et al., 2001).
Antimicrobial activity test
Screening for antibiotic activity of the tested algal extracts was carried out by the agar diffusion assay according to European Pharmacopoeia (1997). One loop full of each test organism was suspended in 3 ml 0.85% sterile NaCl solution, separately. Nutrient agar (Difeco, UK) was inoculated with this suspension of the respectiveorganism and poured into a sterile Petri dish. According to preliminary test for the most effective dose, 10 1/4 l of dimethyl sulfo-oxide (DMSO) Contained 5 mg of each extract was placed on sterilized paper disc (6 mm diameter). The loaded discs were placed apart from each other on theinoculated agar plate aseptically. Sterilized discs that loaded with DMSO only served as negative control and antibiotic discs (Erythromycin and Ampicillin) served as positive control. A prediffusion for 3h was carried out at 10[degrees]C (Bansemir et al, 2006). Inhibition zones were measured after 24h incubation period at 37[degrees]C for bacteria and at 30[degrees]C after 48h for the fungus species. After incubation, the diameter of the inhibition zone was measured with calipers and the results were recorded in mm (Attaie et al., 1987).
Effect of pH, temperature and light intensity on the production of antimicrobial activity
The F/2 medium (100 ml) was prepared in 250 ml of Erlenmeyer flask. The different growth parameter including pH(5,6,7,8,9,10) ,temperature (20, 25, 30, 35, 40[degrees]C) and light intensity (1000, 2000, 3000 Lux) were optimized independently. Then 10 ml of actively growing log phase inoculum was transferred to the culture flask aseptically and reserved under the fluorescent light for 20 days at aerated condition.
The data were statistically analyzed by applying one-way ANOVA.
RESULTS AND DISCUSSIONS
The antimicrobial activity was evaluated as the diameters of the inhibition zones formed as a result of disc assay method in case of bacteria and fungi. Table 1 showed that the ethanol extracts for chlorella marina showed more activity against Staphylococcus aureus and Serratia marcescens 10.0mm diameter of inhibition zone). On the other hand; the water, chloroform and aceton extract was not active against all tested microorganisms. The aceton extract for Nevicula F. delicatula represented more activity against Staphylococcus aureus, Micrococus Luteus and Pseudomonas aeruginosa. On the other hand; the water, chloroform, and ethanol extract was not active against all tested microorganisms. In the light of the experimental results concerning the antimicrobial activity of the test microorganisms against standard antibiotics showed that when the effects of extracts obtained from marine microalgae were compared with standard antibiotics used in this study, it was found that the effect of standard antibiotics was more than that of extract of chlorella marina and Nevicula f. delicatula. These results go in harmony with those obtained by Ozdemir et al. (2004) and Tuney et al. (2006). With the study of Prakash et al. (2011) on the antimicrobial potential of Oscillatoria sancta and Lyngbya birgei against S. aureus. Scenedesmus exhibited antibacterial activity against S. aureus in methanol and acetone extracts in accordance with Guedes et al. (2011). In addition Ostensvik et al., 1998 who observed that aqueous extracts of Microcystis aeruginosa inhibited B. subtilis, and Rao et al. (2007).
Effect of pH, temperature and light intensity on the production of antimicrobial activity
One optimum PH (8.0) was recorded for antimicrobial agent production from two microalgal genera (Fig.3). No antimicrobial activity could be detected at PH values below 5.0 or above 10.0.The highest growth of chlorella marina was reached at pH 8 and the diameter of inhibition zone recorded (12mm). While in Navicula f. delicatula the diameter of inhibition zone recorded (13mm). It has been well documented by the earlier researcher (Richmond A. 2000, Renaud SM. et al., 1991, Renaud SM. et al., 1995, and Borowizka MA. et al., 1990).
The pH of themedium is very important for growth of microorganisms, for the character of their metabolism and hence for the biosynthesis of antimicrobial products as secondary metabolites. Scytonema ocellatum was found to exhibit maximal scytophycin productivity at pH 8.0-8.5 (Patterson and Boils, 1995).
Temperature is an environmental factor which indirectly affects growth of microalgae and antimicrobial activity (Huang et al., 2008). The results recorded in (Fig. 4) revealed that the highest growth of chlorella marina was reached at temperature 25[degrees]C and the diameter of inhibition zone recorded (12 mm) after 12th days of incubation. While in Nevicula F. delicatula the diameter of inhibition zone recorded (13mm) at temperature 20 after 14th days of incubation. Ame et al. (2003) found that production of higher amounts of the bioactive toxin, microcystin by cyanobacteria was favored at temperature more than 23[degrees]C, although maximum cylindrosperopsinproduction was attained by the cyanobacterium Cylindros permopsis raciborskii at 20[degrees]C (Griffiths and Saker, 2003). Lehtimaki et al. (1997) found that low temperatures (7, 10, 16 C) gave low measurements for nodularin production by cyanobacteria, while the highest production was attained at high temperatures.
The relationship between temperature and growth of microalgae is linear (Takemura et al., 1985). Temperature determines the activity and reaction rates of intracellular enzyme, which will have an influence on algal photosynthesis, respiration intensity, affect the growth of indicated that the light intensities affected the values of the growth and the diameter of inhibition zone Fig. (5), the highest growth of chlorella marina was reached at 3000 lux and the diameter of inhibition zone recorded (11 mm). While in Nevicula F delicatula the diameter of inhibition zone recorded (12mm) at 1000 Lux. On other hand, it was found that the diameter of inhibition zone recorded (7.0 mm) at 3000 Lux for Nevicula F. delicatula but for chlorella marine the diameter of inhibition zone recorded (8.0 mm) at 1000 Lux.
When the light intensity above a certain value, continue increasing in light intensity level will decrease the microalgae growth rate actually, this is called photo inhibition phenomenon. microalgae and to limit its distribution (Tan et al., 2009).
Light is an essential key for growth of microalgae. Microalgae uses light to process the photosynthetic, but the light energy cannot be stored by microalgae, so the light should be supplied sustainably. The microalgae cannot use all the supplied light because microalgae cannot absorb all the photons, and too much light will cause light inhibition for the surface layer of microalgae. Through the photosynthetic process, for autotrophic microalgae to convert carbon dioxide in the air into organic compounds, visible light is the main source of energy (Carvalho et al., 2011) since the chlorophylls, phycobilins and carotenoids in microalgae can be absorbed in the visible light range. At stationary phase, the data
The ethanol extract for chlorella marina showed more activity against Staphylococcus aureus and Serratiam arcescens 10 mm diameter of inhibition zone with pH 8.0 in 3 5psu of salinity at 25[degrees]C and light intensity 3000 lux during 12th day of incubation at aerated condition in F/2 medium On the other hand the aceton extract for Nevicula F. delicatula more activity against Staphylococcus aureus, Micrococus Luteus and Pseudomonas aeruginosa. with pH 8.0 in 35psu of salinity at 20C and light intensity 1000 lux during 14th day of incubation at aerated condition in F/2 medium.
My sincere thanks are also extended to all the staff members of Phycological Lab., Botany Department, Faculty of Science, and University of Beni-Suef Egypt. Thanks are also extended to all members of marine Biotechnology Laboratory, National Institute of Oceanography and Fisheries -Alexandria.
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R. Elkomy  *, I.B.M. Ibraheem , M. Shreadah , R. Mohammed 
 Biotechnology Laboratory-National Institute of Oceanography and Fisheries-Alexandria, Egypt.
 Botany and microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt.
 Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Egypt.
(Received: 09 October 2015; accepted: 03 November 2015)
* To whom all correspondence should be addressed. E-mail: email@example.com
Caption: Fig. 1. Locations for isolated microalgae I-Navicula f. delicatula (El Agamy and II-Chlorella marina (Baltim)
Caption: Fig. 2. Effect of different pH on antimicrobial activity production of chlorella marina & Navicula f. delicatula.
Caption: Fig. 3. Effect of different temperature on antimicrobial activity production of chlorella marina& Navicula f. delicatula
Caption: Fig. 4. Effect of different light intensity on antimicrobial activity production of Chlorella marina& Navicula f. delicatula
Table 1. Antibacterial and antifungal activity of the investigated chloroform, acetone, ethanol, methanol and water extracts of two microalgal genera using the agar plate by diffusion assay method. Standard antibiotics Diameter of inhibition zone(mm) Gram (+V) S.aureus M.luteus Erythromycin 20[micro]g/disc 10.5 14 Ampicillin 10[micro]g/disc 11.5 15 Microalgal sp. Solvent extracts chlorella chloroform -- -- marina acetone -- -- ethanol 10 methanol 7 -- water -- -- Navicula f. chloroform -- -- delicatula acetone 10 9 ethanol -- -- methanol 7 7 water -- -- Standard antibiotics Diameter of inhibition zone(mm) bacteriaGram (-V) bacteria P.aerug E.coli S.marcescens Erythromycin 20[micro]g/disc 10.5 11.0 15.5 Ampicillin 10[micro]g/disc 10.5 11.5 11 Microalgal sp. Solvent extracts chlorella chloroform -- -- -- marina acetone -- -- -- ethanol 9 10 methanol -- -- 5 water -- -- -- Navicula f. chloroform -- -- -- delicatula acetone 10 -- 11 ethanol -- -- -- methanol 8 -- 8 water -- -- -- Standard antibiotics Diameter of inhibition zone(mm) Fungal sp. C.albicans Erythromycin 20[micro]g/disc -- Ampicillin 10[micro]g/disc -- Microalgal sp. Solvent extracts chlorella chloroform -- marina acetone -- ethanol methanol 6 water -- Navicula f. chloroform -- delicatula acetone 8 ethanol -- methanol 5 water -- -- = No inhibitory effect; width 1 to 8 mm = week activity; width 8 to mm = moderate activ es; width > 10 mm = strong activity.
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|Author:||Elkomy, R.; Ibraheem, I.B.M.; Shreadah, M.; Mohammed, R.|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Dec 1, 2015|
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