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International Journal of and Bioengineering Research. ISSN 2231-1238, Volume 4, Number 3 (2013), pp. 275-282 © Research India Publications http://www.ripublication.com/ ijbbr.htm

Marine Fungi as a Source of Secondary Metabolites of

K. Manimegalai1, N.K. Asha Devi2 and S. Padmavathy3

Department of Zoology and , Thiagarajar College (Autonomous), Theppakulam, Madurai- 625009, TamilNadu, India.

Abstract

Marine fungi have been shown to be tremendous sources for new and biologically active secondary metabolites which are reflected by the increasing number of published literature dealing with compounds from this group of fungi. As a result to these efforts, more than a hundred secondary metabolites from marine fungi have been described. The mycobiota of the coastal water were collected from five different localities in and around Mahabalipuram beach. The filamentous fungi were identified and assigned to eight genera. Greater populations as well as a wider spectrum range of fungal genera and were obtained in Mahabalipuram beach while other locations were the poorest one. The genera of highest incidence and their respective numbers of species were: Cephalosporium (37.6%, 8 spp.) (23.72%, 6 spp.) and (21.28%, 16 spp.). The species which showed the highest incidence in all cases was P. chrysogenum, followed by P. citrinum, A. niger, A. flavus, A.fumigatus Cephalosporium acremonium and sp. Several other genera and species were detected at quite low occurrence. The investigation of the content of marine fungal strains of Cephalosporium acremonium and P. citrinum showed broad spectrum activities and the partial chemical structures of the compounds were identified using IR and NMR studies. Secondary metabolites isolated during this study represented different structural classes of natural product. This indicates that marine fungi have a wide range of biosynthetic capabilities for the production of novel structures.

Keywords: Filamentous fungi; Secondary metabolites.

276 K. Manimegalai et al

1. Introduction Marine-derived fungi are considered to be the potential sources for new and biologically active secondary metabolites (Liberra and Lindequist, 1995; Konig and Wright, 1996; Pietra, 1997; Biabini and Laatsch 1998) which is reflected by the increasing number of published literature dealing with compounds from this group of fungi. Filamentous fungi are a profilic source of biologically active secondary metabolites (Namikoshi et al., 2002). are provided with immense treasure, thus open many areas of multidisciplinary work. The novelty of marine derived compounds and their wide spectrum of applications may find way in getting solution to many dreadful diseases like AIDS, Cancer, Alzheimer’s disease and arthritis which are difficult to be cure today. The present work deals with the study of marine filamentous fungi. The secondary metabolites from marine fungi are of special interest due to the scarce information about it. Hence the aim of the present study was to isolate the marine fungi from the waters of Bay of Bengal and screening for their antagonistic activity against bacterial and fungal human .

2. Methodology Water samples collected from five different localities (Kovalam, Mudaliarkuppam, Thiru-Vidanthai, Sadras and Mahabalipuram) in and around Mahabalipuram, Tamilnadu, India. The water samples were collected in clean, sanitized and autoclaved bottles and transported to the laboratory for further analysis. Potato dextrose agar (Hi Media) using sea water was supplemented with streptomycin (20 mg/l) to inhibit the growth of bacteria and actinomycetes used to culturing the fungal colonies, which were incubated at 37o C for 24-48 hours. The isolated fungal colonies were identified based on the colony morphology and microscopic characteristics (Cappuccino, and Sherman, 1999; Holt et al., 1994). A total of 39 fungal strains isolated (Marine Agar, Himedia) from the coastal water samples were screened for the production of antibacterial substance. In primary screening, antimicrobial activity was assessed against the target pathogenic microorganisms. From the pure cultures, marine bacterial strains were spotted on target organisms swabbed in Muller Hinton Agar (MHA) plates. Based on the zone of inhibition, each bacterial strain was selected for secondary screening and further analysis. Secondary screening was done by agar well diffusion assay for testing the antagonistic activities of marine fungal isolates. Selected isolates of marine fungal samples isolated were cultured in 300 ml in Potato dextrose broth for the production of secondary metabolites in 500 ml Erlenmeyer flasks. Flasks were incubated on a rotary shaker at 220 rev/min. After 7 days of cultivation, the culture broth was centrifuged at 10000 rpm for 20 minutes to remove the cell and filtered through Millipore filter (0.45µm) to get cell free extract. Antibacterial activities were assayed in duplicate, using 50µl of marine fungal samples in well diffusion assay (Schillinger and Lucke,1989) and the test organisms were (NCIM 2079), Micrococcus luteus (NCIM 2871), Streptococcus mutans (MTCC 890) Escherichia

Marine Fungi as a Source of Secondary Metabolites of Antibiotics 277 coli (MTCC 46),(NCIM2200), Salmonella typhi (MTCC 734, Vibrio cholerae(MTCC 1738) and Klebsiella pneumoniae (NCIM 2957). NEXUS – 672 and 8400S Shimadzu MODEL Fourier Transform Infra Red Spectroscopy (Japan) were used for the analysis of the culture supernatants. The spectrum was taken in the mid IR region of 400 – 4000 cm-1. Bruker (300 MHz) Nuclear Magnetic Resonance (NMR) Spectroscopy was used for the analysis of twelve culture supernatants. The samples were dissolved by using deutrated chloroform (CDCl3) as solvent.

3. Results After primary screening, among 39 marine fungal isolates 9 were selected for further studies on the basis of antagonistic activity and growth characteristics. Based on morphological and microscopical characteristics, they were identified as Aspergillus niger (MF-16), (MF-31), (MF-5), Penicillium chrysogenum (MF-2), Penicillium citrinum (MF-27), Cephalosporium acremonium (MF-29), Cladosporium sp. (MF-36), Geotrichum sp. (MF-33) and sp. (MF-24). Selected 9 fungal filtrated samples were tested against selected human pathogens. Cephalosporium acremonium and Penicillium citrinum revealed the efficiency of broad spectrum antibacterial activity. Hence, the strain Cephalosporium acremonium and Penicillium citrinum (Figure 1 & 2) isolated from the coastal region of Mahabalipuram have been selected for further studies significant antibacterial activity. Except the gram negative bacteria of Klebsiella pneumoniae, other three gram negative bacteria were sensitive to Penicillium chrysogenum. Cladosporium sp. also showed strong antibacterial activity against gram negative bacteria but not against Salmonella typhi. Of the 9 marine fungal samples, 2 species of Aspergillus niger and Alternaria sp. also showed antibacterial activity against gram positive bacteria of Staphylococcus aureus. (Table - 1; Figure.2). Chemical characterization using FTIR analysis and NMR analysis for the experimental samples of Cephalosporium acremonium and Penicillium citrinum probably indicate the presence of aliphatic amines and imidazole rings by the fungal supernatants concluded that they probably belongs to antimicrobial compounds, since the presence of this type of structures are the characteristic feature of antimicrobials (Table-2).

Table 1: Secondary screening for antibacterial activities by marine fungi.

S. Target organisms Zone of inhibition in mm No MF MF MF MF MF MF MF MF MF 2 5 16 24 27 29 31 33 36 1 Escherichia coli (MTCC 46) 12 - - - 18 18 14 - 14 2 Klebsiella pneumoniae - - - - 16 16 - - 14 (NCIM 2957)

278 K. Manimegalai et al

3 Micrococcus luteus (NCIM 14 - 11 11 20 20 10 10 - 2871) 4 Pseudomonas aeruginosa 12 - - - 14 14 - - 12 (NCIM2200) 5 Salmonella typhi (MTCC 16 - - - 16 16 - - - 734) 6 Staphylococcus aureus 16 - 15 14 14 16 - - - (NCIM 2079) 7 Streptococcus mutans 12 - - - 16 16 - - - (MTCC 890) 8 Vibrio cholerae (MTCC 12 - - - 16 18 - - 12 1738) MF-2: Penicillium chrysogenum, MF-5: Aspergillus fumigatus, MF-16: Aspergillus niger, MF-24: Alternaria sp., MF-27: Penicillium citrinum MF-29:Cephalosporium acremonium, MF-31: Aspergillus flavus, MF-33: Geotrichum sp., MF-36: Cladosporium sp. * Culture supernatant concentration is 100µl. Each data point is the mean of three experiments

Table 2: IR & NMR Spectroscopic data of the selected culture cell free supernatant samples.

S. Supernata Characteristic band in IR Characteristic peak in NMR No nt samples spectrum (Wave number (cm-1) data (Chemical shift ppm)

1 Penicillium 1025.2, 1217 – Aliphatic amines, 2-7, 8-11 – Alkyl - NH(2) – citrinum 3367.82 – N-H- 1º, 2º Amines Amines, RCONH (MF27) 2 Cephalospo 1620.88 – N-H, 3403.74 – 1-2.5 – Allylic amides, rium 1º, 2º Amines (Pyrroles) alkenes, 3.2-8 – Amines acremoniu m (MF29)

Figure 1

Penicillium citrinum Cephalosporium acremonium Figure 2: Antibacterial activity of MF-27: Penicillium citrinum MF-9: Cephalosporium acremonium and MF-2: Penicillium chrysogenum against Gram-negative Salmonella typhi organisms.

Marine Fungi as a Source of Secondary Metabolites of Antibiotics 279

against Gram- Salmonella typhi

MF-27

MF-2 MF-29

4. Discussion Fungi growing in the sea can be grouped into obligate and facultative marine fungi. According to a definition made by Kohlmeyer (1974), "obligate marine fungi are those that grow and sporulate exclusively in a marine or estuarine (brackish water) habitat; facultative marine are fungi from freshwater or terrestrial areas able to grow also in the natural marine environment". In 1991 Kohlmeyer & Volkmann-Kohlmeyer listed 321 filamentous higher marine fungi (Kohlmeyer & Kohlmeyer, 1979). In the present study two of our isolates from the coastal regions of Mahabalipuram (Tamil Nadu, India) were identified as Cephalosporium acremonium and Penicillium citrinum. A number of antibiotics have been obtained from culture broths of filamentous fungi to date. Studies on marine filamentous fungi are useful and sustainable source of bioactive marine natural products (Kobayshi et al., 1997 and Namikoshi et al., 2000; 2001; Khudyakova et al., 2004). The present study reveals that among 39 selected fungal isolates, Penicillium chrysogenum and Cephalosporium acremonium showed potent antibacterial activity against all the tested organisms. Cephalosporin C is the first classical example of β-lactam isolated from a marine derived fungi Cephalosporium acremonium (Blunden, 2001). Chemical characterization using FTIR analysis for the experimental samples probably showed the presence of aliphatic and aromatic structure possessed by them. Based on the NMR spectrum analysis the presence of aliphatic amines and imidazole rings by the fungal supernatants concluded that they probably belongs to antimicrobial compounds, since the presence of this type of structures are the characteristic feature of antimicrobials. The world of fungi provides a fascinating and almost endless source of biological diversity, which is a rich source for exploitation (Manoharachary et al., 2005). The structural similarity between some of the metabolites in this study and previously reported compounds from marine fungi indicates that the marine fungal origin of these compounds must be considered.

280 K. Manimegalai et al

5. Conclusion The present investigation reported marine fungal isolate may play an important role in the eradication of dreadful diseases. Further characterization of the isolate and the purified compound will explore them as a new entity in the discovery of natural products from marine microbes. The need for new, safe and more effective antimicrobials are a major challenge to the pharmaceutical industry today especially with the increase in opportunistic infections in the immunocomprimised host. In conclusion, the marine biotechnology industry faces a unique challenge for the millennium: Inventing a new generation of tools and processes that will enable a greater understanding of the and its resources and lead to the discovery of new bioproducts for the future, and designing methods for the sustainable development of these unique bioproducts.

6. Acknowledgements Authors thank Department of Zoology and Microbiology, Thiagarajar College (Autonomous), Madurai -9 for providing all facilities.

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