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.
INTRODUCTION
Presently, the direct application of chemicals to control algal blooms harm and/or eventually disrupt the aquatic ecosystem by killing off beneficial plankton and even fish (McGuire et al., 1984; Reynolds, 1984; Sevrin-Reyssac and Pletikosic, 1990). Consistent with the panecological and environmental approaches to lake water conservation, many countries are currently seeking to develop suitable biological control agents (Imai et al., 1993; Kim et al., 2003; Manage et al., 2000; Boudjella et al., 2006; Volka and Furkert, 2006).
In particular, blooms of the cyanobacterium Microcystis aeruginosa Lemmermann f. Aeruginosa are widespread in eutrophic lakes and reservoirs throughout the world (Carmichael, 1992; Han et al., 2002; Hong et al., 2002; Choi et al., 2005) and may lead to the production of microcystin, a hepatotoxin that affects fish, birds, wild animals, livestock and humans. It is associated with allergies, irritation reactions, gastroenteritis, liver diseases and tumors (An and Carmichael, 1994; Bell and Codd, 1994; Dawson, 1998). Historically, these cyanobacterial (algal) blooms have also caused other problems, such as foul odors, decreased aesthetic value, deterioration of water quality and deoxygenation of water (Sigee et al., 1999). Also, Oscillatoria rubescens and O. agardii have been reported to produce hepatotoxins (Carpenter and Carmichael, 1995) and may be responsible for dermatitis or skin irritation when people are exposed to polluted water (Gorham and Carmichael, 1988). In addition, they produce unpleasant odors (Jüttner, 1976; Slater and Blok, 1983; Tsuchiya et al., 1992).
The search for new antibiotics continues to be of utmost importance in research programs around the world because of the increase of resistant pathogens and toxicity of some used antibiotics. Among microorganisms, actinomycetes are one of the most investigated groups particularly members of the genus Streptomyces from which, a large number of antibiotics was obtained and studied (Okami and Hotta, 1988). The vast majority of actinomycetes have originated from soil (Davies and Williams, 1970) and their isolation method deal almost exclusively with those suitable for Streptomyces species which grow rapidly on soil dilution plates. However, in recent years, the rate of discovery of new antibiotics in the genus Streptomyces was declining and isolation of other actinomycete genera, appeared to be necessary to assess the health hazard and to find novel strains producing commercially valuable antibiotics.
The genus Streptomyces has received considerable attention especially for its importance as a source of several secondary metabolites particularly antibiotics (Sanglier et al., 1993a; Young, 1993; Lazzarini et al., 2000; Habib et al., 2001; Kokare et al., 2004; Choi et al., 2005; Boudjella et al., 2006; Volk and Furkert, 2006). Some antibiotics have been found to use in therapy of plant and human diseases which are caused by bacteria, virus, amoeba and other fungi. This wide range of usefulness makes the substance producing actinomycetes are most important (Brooks et al., 1998; Castillo et al., 2003; Hammad, 2004; Sujatha et al., 2004). Fogg et al. (1973) reported that the metabolites of Streptomycetes have antimicrobial activities against cyanobacteria as Anabaena cylindrical and Tolypothrix tenuis and shriveling in the vegetative cells of A. cylindrical causes discoloration and complete lysis. The lytic activity explained the basis of the interaction; between the antibiotics and cell wall functional and or structural components which led to lytic appearance (Whyte et al., 1985). Gunnison and Alexander (1974) reported that the activity of cellulase or polygalacturonase enzymes producing Streptomycete (G4) against the cell wall of Chlamydomonas reinhardtii reached to 89 or 98% and Ulothrix fimbrata reached to 64 or 84%, respectively and attributed the lysis of Cylindrospermum sp.; to Streptomycete's lysozyme. In this study, we report the discovery of an antialgal actinomycetes active against Anabaena sp., Anabaena flos-aqueae, Nostoc sp. and Anacystis nidulans and also reported the antialgal features of this actinomycetes in regards to its activity at various algal and actinomycetal growth phases. We report also on the purification of lytic agent and the effect of the purified antibiotic on the tested alga.
MATERIALS AND METHODS
Strains and Media
Strain No. 40 was isolated on starch-nitrate agar medium from soil cultivated
with pepper according to Waksman (1959). Experimental organism (Isolate No.
40) was classified by using ISP (International Streptomyces Project) according
to Shirling and Gottlieb (1966) and Bergey's Mannual (1989) and identified as
Streptomyces endus N40. Anabaena sp., Anabaena flos-aqueae,
Nostoc sp. and Anacystis nidulans were taken from the algal lab.
Faculty of Science, Zagazig University and cultivated on watanabe medium (Watanabe,
1951) and BG11 medium (Stanier et al., 1971). This study takes place
in the Phycology and Water Research Lab, Faculty of Science, Zagazig University,
during January 2005.
Assessment of Algal Lytic Actinomycete
Streptomyces endus N40 was streaked on agar plates and incubated
for 8 days at 28°C. After detectable, the colonies of Streptomyces endus
N40, the solid medium was cut into discs (5 mm) by using cork borer and
placed on watanabe medium cultivated with cyanobacteria (Anabaena sp.,
Anabaena flos-aqueae and Nostoc sp.) according to Yamamoto (1978).
After the elapse of incubation periods, the clear inhibition zones induced by
Streptomyces endus N40 discs were measured by millimeter. With respect
to the growth conditions of Anacystis nidulans, 0.1 mL of Streptomyces
endus N40 suspension was seeded to the nutritive liquid medium of BGII,
the initial optical density of algal medium at zero time was calculated at 665
nm. Then all the flasks were incubated for 2 weeks under normal growth condition.
The optical density (growth rate) was calculated every 2 days Uchida et al.
(1998).
Separation of Lytic Agent Against Cyanobacteria
Optimization for the maximum production of lytic agent from Streptomyces
endus N40 was carded out using different carbon, nitrogen, phosphorus, microelement
and vitamin sources and take in consideration the best; pH value, incubation
period and incubation temperature as previously mentioned. The best medium having
(g L-1); maltose 6 g; asparagine 1.486 g; K2HPO4
1 g; NaCl 0.5 g; MgS04.7H2O, 0.5 g; CaCO3 3
g; FeSO4.5H2O, 0.01 g; vitamin Bio inositol), 0.01 mg
and distilled water up to 1000 mL. After growing Streptomyces endus N40
on the optimum medium, the cultured broth (5 L) was centrifuged at 2000 rpm.
And the resulting supernatant was dialyzed (cellulose bag method) against hypertonic
sweet solution. Subsequently, the resulting residue was mixed with xylene (3
times). The organic layers were combined and concentrated under vacuum to about
50 mL using a rotary evaporator. To the concentrated layer, a non-polar organic
solvent (petroleum ether 40-60) was added drop by drop untill yellowish crystalline
substance appeared. The solid fraction was washed with ether and then dried
in air. Continuous purification of substance using TLC (silica gel G54) was
carried out. The major spots appeared at Rf = 0.7 was gathered and eluted with
petroleum ether (40-60). The partially purified fractions were microanalyzed
using IR, Mass and NMR spectra and elementary analysis for complete identification
of substance.
RESULTS
Antagonistic Activity of Stereptomyces isolate on Tested Cyanobacteria
Clear inhibition zones represented in 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.