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Research Journal of Microbiology

Year: 2007  |  Volume: 2  |  Issue: 8  |  Page No.: 606 - 618

Niromycin A: An Antialgal Substance Produced by Streptomyces endus N40

S.A. El-Shirbiny, M.F. Ghaly, Y.M. El-Ayoty and N.S. Fleafil

Abstract

The antialgal activity of 107 Actinomycete isolates were isolated from different localities in El-Sharkia, El-Dakahliya and El-Meniya governorates. Egypt. Isolate No. 40 (isolated from Ekhtab. Aga, Dakahliya) was selected and identified as Streptomyces endus N40. The optimum culture conditions for the production of the antialgal metabolite were studied. It has been found that the optimum incubation temperature for the production of the antialgal metabolite from Streptomyces endus N40 was 28°C after incubation period (8 days) at pH 6.5. The most favorable carbon, nitrogen, phosphorus, microelement and vitamin sources were maltose (6 g L-1), asparagine (1.486 g L-1), K2HPO4 (I g L-1), FeSO4.7H2O (0.01 g L-1) and (inositol) (0.01 mg L-1), respectively. The maximum antialgal activity was obtained against Anabaena sp. Anabaena flos-aqueae, Nostoc sp. and Anacystis nidulans. The antialgal substance had extracted using xylene solvent Formulation and identification of the antialgal substance that produced by Streptomyces endus N40 was carrying out using IR, Mass, NMR spectra and elementary analysis and these results were emphasized that the molecular weight equal 279.33 KDa with chemical formula (C15H21NO4) and identified as Niromycin A.

Table 1 and Fig. 1 revealed that the highly sensitive algal species to the block agar born actinomycete was detected against Anabaena sp. which gave a clear zone equal (26 mm) followed by Nostoc sp. which gave a clear zone equal (24 mm). While a clear zone of Anabaena flos-aaueae indicated 22 mm in a similar manner results in Table 2 showed chlorosis and complete lysis of Anacystis nidualans cells when treated with 1 mL Streptomyces endus N40 suspension.


Table 1: Inhibition zone (mm) of Streptomyces endus N40 against tested cyanobacteria

Fig. 1: Inhibition zone (mm) of Streptomyces endus N40 against tested cyanobacteria

The maximum drop in algal growth is indicated after 10 days.

Optimization of the Environmental Conditions and Nutritional Requirements for Antialgal Substance Production by Streptomyces endus N40
The data recorded in Table 5-12 revealed that the highest biomass yield and antialgal substance production by Streptomyces endus N40 was achieved after incubation for 8 days at 28°C and pH 6.5 under shaking condition. Optimization for the maximum production of lytic agent from Streptomyces endus N40 was carried out using different carbon, nitrogen, phosphorus, microelement and vitamin sources. The best medium has (g L-1): maltose 6 g; asparagine 1.486 g; K2HPO4 1 g; NaCI 0.5 g; MgSO4.7H2O 0.5 g; CaCO3 3 g; FeSO4.5H2O 0.01 g; vitamin Bio inositol), 0.01 mg and distilled water up to 1000 mL. These are agreement with the results obtained by (Naki et al., 2000; Ramadan, 2000; Toshio et al., 2000; Yutaka el al., 2001; Gupte and Kalkani, 2002; Ammar et al., 2003; Hammad, 2004; Ghaly et al., 2005).


Table 2: Antialgal activity induced by Streptomyces endus N40 in liquid media (inhibition expressed as a reduction in optical density at 665 nm)
Untreated alga = Anacystis alga; Treated alga = Anacystis nidulans alga + Streptomyces endus N40

Table 3: The activity of different levels of Niromycin A on Anacysfis nidulans

Table 4: A comparative study of the characteristic properties of the tested substance in relation to reference substance (Niromycin A)

Table 5: Effect of the different incubation periods on the production of antialgal substance by Streptomyces endus N40

Table 6: Effect of different pH values on the production of antialgal substance by Streptomyces endus N40

Table 7: Effect of different incubation temperature on the production of antialgal substance by Streptomyces endus N40

Table 8: Effect of different carbon sources on the production of antialgal substance by Streptomyces endus N40

Table 9: Effect of different nitrogen sources on the production of antialgal substance by Streptomyces endus N40

Table 10: Effect of different phosphorus sources on the production of antialgal substance by Streptomyces endus N40

Table 11: Effect of different microelements on the production of antialgal substance by Streptomyces endus N40

Table 12: Effect of different vitamins on the production of antialgal substance by Streptomyces endus N40

Isolation and Purification of the Lytic Agent
Streptomyces endus N40 cultivated in 250 mL Erlenmeyer flask containing the optimized medium (100 mL) in each. After autoclaving, suspension of Streptomyces endus N40 was inoculated to the different flasks under aseptic condition. All the flasks were incubated at 28°C for 8 days. After the elapse of incubation period, the fermented flasks were collected and the spores of actinomycete as well as its hyphae were separated. The aliquot of fermented media were concentrated to 500 mL by dialysis method (cellulose bag), extracted by xylene at pH 7.0 (Ahmed et al., 2002) and the organic layer was collected and concentrated under vacuum by using rotary evaporator to dryness. The obtained residual fraction was purified by using thin layer chromatography, which manifested through ultraviolet lamp one spot at Rf = 0.7. The spots were collected by its elution and the physicochemical characteristics (IR, Mass spectrum, NMR spectrum and elementary analysis). (Williams and Fleming, 1987; Hayakawa et al., 1994; Stefani and Agodi, 2000) indicated that this substance is known as Niromycin A (Fig. 2-4). These microanalyses are carried in microanalysis center in Cairo University.

Identification of the Antialgal Substances Produced by Streptomyces endus N40
Taking into consideration the elementary analysis, IR, mass and NMR spectra and also on the basis of the recommended keys for the identification of antibiotics and in view of the comparative study of the recorded properties of the antibiotics, it could be stated that the substance extracted from Streptomyces endus N40 is named as Niromycin A (Table 4). The identification is carried out according to (Berdy, 1980a-c; Umezawa, 1977).

Elementary Analysis
The extracted substance from Streptomyces endus N40 was found to contain carbon (C = 64.5%), hydrogen (H = 7.58%), nitrogen (N = 5.01%) and oxygen (O = 22.91%).


Fig. 2: Infra-red of Streptomyces endus N40 metabolite

Fig. 3: Mass spectrum of Streptomyces endus N40 metabolite

Fig. 4: NMR spectrum of Streptomyces endus N40 metabolite

Infra-Red Spectrum (IR)
Besides the elementary analysis the identification of the compound was also confirmed by spectroscopic measurements. The infra-red spectrum of this compound showed an isopropanol band in the range of 1360 and 1385 cm-1; aromatic ring in 1270,1122. 1006, 958. 784 cm-1; CH-aliphatic in 2924. 2855 cm-1; CH-aromatic in 3050 cm-1; amidic carbonyl group in 1702 cm-1; NH group in 3424 cm-1; free OH group in 3651 cm-1 (Fig. 3).

Molecular Weight
The FD-MS spectrum showed the molecular peak at m/z 279.33 (Fig. 4).

Molecular Formula
The molecular formula was determined on the basis of the results obtainedfrom the mass spectrometric and elementary analysis of the extracted compound as C15H21NO4.

NMR Spectrum of the Antibiotic
The 1H NMR of the antibiotics produced by Streptomyces endus N40 was investigated by Nuclear Magnetic Resonance (NMR). It could be deduced that the substance molecule is characterized by the following:

Presence of two methyl groups (singlet at 1, 3).
Presence of four protons of the sugar molecules (multiplete at 2.26-2.40).
Presence of the carbon (C2-H) (singlet at 5.04).
Presence of the phenyl ring (multiplete at 7.48-7.64).
Presence of the NH proton (singlet at 10.03).
Presence of the free OH proton (singlet at 14.40).

Taking into consideration the elementary analysis, IR, mass and NMR spectra, the structure of the present substance could be suggested as follows:

All these results were applied according to spectroscopic methods in organic chemistry.

The Activity of Different Levels of Purified Antibiotic
The effect of different concentrations of the substance produced from Streptomyces endus N40 against Anacystis nidulans cyanobacterium is determined. The results in Table 3 showed the growth of algal species with addition of Niromycin A at different concentrations (10, 20, 30 or 40 μg mL-1) to 50 mL liquid algal media. This growth calculated by colourimetric method at 665 nm after 14 and 16 days. Niromycin A inhibit the algal growth recording 43.5, 54.3, 56.5 or 58.7%, 14 days after incubation, respectively but the absorbance recorded 71.3, 73.8, 75 or 76.3%, respectively after 16 days incubation. The maximum inhibition of Anacystis nidulans recorded 76.3% with Niromycin A at a rate of 40 μ mL after 16 days.

DISCUSSION

Bacteria with antialgal activity against algae have been previously reported and the list includes Pseudomonas sp. (Kodani et al., 2002), Alcaligenes denitrificans (Manage et al., 2000) and Streptomyces phaeofaciens (Yamamoto et al., 1998). Indeed, several members of the genus Streptomyces have been previously reported as having cyanobacteria-killing activity (Safferman and Morris, 1962; Yamamoto et al., 1998). However, there is no report demonstrating the in situ application of antialgal bacteria against a cyanobacterial bloom. One of the major reasons is probably the unpredictability of the antialgal effects of bacteria on other members of the freshwater ecosystem (EPA, 2002).

Before application of an antialgal agent to freshwater systems, there should be information on (1) the antialgal activity against the target alga, (2) the effects on the other organisms in the freshwater ecosystem and (3) a forecast of the algal dynamics after the removal of the target alga (Choi et al., 2005). Here, the Streptomyces endus N40 release an algicidal substance and its concentration was time dependent. The maximum inhibitory effect was obtained after 10 days that equal 87% compared with its corresponding control. These results are in agreement with that of Safferman and Morris (1962) who found 213 cultures of soil isolated actinomycetes (out of 403 isolates) had inhibitory effects against the cyanobacteria (including species of Anacystis, Fremyella, Lyngbya. Nostoc. Phormidium and Plectonema). Also, Gromov et al. (1972) indicated that the Flexibacter flexilis var. algavorum lyses the cells of filamentous blue-green algae belonging to the genera Anabaena, Phormidium and Nostoc. On the other hand, Al-Tai (1982) found that the extra-cellular products of an actinomycete (AN6) were able to lyse cyanobacteria, fungi, bacteria and green algae. Thereafter, Whyte et al. (1985) indicated that the pure cultures of Streptomyces achromogenes were shown to lyse Anabaena cylindrica and Tolypothrix lenuis.

Yamamoto and Suzuki (1990) attributed that the lytic activity of Streprtomycetes under investigation secrete some lysozyme and protease which causing cell lysis of the toxic strain of Microcystis aeruginosa. At the same time, Yamamoto et al. (1998) showed that Streptomyces phaeofaciens produced compounds causing extensive lysis of Microcystis cells. However, Sigee et al. (1999) found that formation of the lytic agent by Streptomyces exfoliatus occurs independently with the presence of cyanobacteria, the ability to destroy these organisms is probably due to the antagonist of actinomycetes. Hence, Choi et al. (2005) study the effects of the antialgal bacterium S. neyagawaensis on several dominant algae in the Paltang, Juam, Daechung Reservoir and Naktong River and found that S. neyagawaensis had an effect on A. Xos-aquae and A. cylindrica but not A. macrospora and A. aynis. It also affected strains within a species differently. For example, the antialgal activity was 38.8% on M. aeruginosa NIES-44 and 70.2% on M. aeruginosa NIES-298.

Also (Choi et al., 2005) reported that, there are two possible explanations: (1) the culture conditions utilized in this experiment were not suitable for bacterial growth and (2) M. aeruginosa exudates may suppress bacterial growth. The first explanation is based on the divergence between optimum culture conditions for the antialgal bacterium and the cyanobacterium. The bacterium did not grow well at pH 9 and 25°C. The second explanation is that the toxicity of microcystin, from M. aeruginosa, is known to inhibit growth of organisms such as cladocerans, copepods and mosquito larvae (Sathiyamoorthy and Shanmugasundaram, 1996; Singh et al., 2003).

In this study the purified antialgal substance was identified using the elementary analysis, IR, mass and NMR spectra and also on the basis of the recommended keys for the identification of antibiotics as Niromycin A. Niromycin A have an inhibitory effect on the algal growth and the maximum inhibition of Anacystis nidulans recorded 76.3% with Niromycin A at a rate of 40 μ mL after 16 days.

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