Isolation and Screening of Endophytic Fungi Producing Antimicrobial Metabolites from Seaweeds in Southern Thailand

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1 Available online at ST-P-025 Burapha University International Conference 2015 Moving Forward to a Prosperous and Sustainable Community Isolation and Screening of Endophytic Fungi Producing Antimicrobial Metabolites from Seaweeds in Southern Thailand Amina Samae a,c, Sompong O-Thong b,c, Preuttiporn Supapron b and Nugul Intrasungkha b,c* a Graduated student, Program in Biotechnology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand b Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand c Microbial Resource Management Research Unit, Thaksin University, Phatthalung, 93210, Thailand Abstract This study aims to isolate the endophytic fungi from seaweed collected from seacoast of Southern Thailand. It was found that there were 82 fungi isolates consisting of 7 genera which were identified mainly from morphological characteristics, namely, Penicillium sp.1 (25 isolates), Penicillium sp.2 (2 isolates), Aspergillus fumigatus (16 isolates), Aspergillus niger (9 isolates), Aspergillus flavus (3 isolates), Aspergillus sp.1 (1 isolates), Rhizopus sp.1 (1 isolate) and another 25 unidentified isolates. The dual agar diffusion techniques was applied for primary screening of inhibition activity against 7 pathogenic microorganisms such as Bacillus cereus, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella thyphimurium, Bacillus sp. and Candida albicans. however, it was found only 1 isolate (Aspergillus sp.1 or CAS08) which showed the highest inhibition activity among other isolates. Therefore we selected this Aspergillus sp.1 isolate for conducting growth curve studies in conjunction with inhibition activity assays with the same 7 tested human pathogens. The result showed that the most suitable cultural time for the Aspergillus sp.1 was days with Sabouraud, s dextrose broth medium and Staphylococcus aureus ATCC was the most susceptible human pathogen. To optimize the growth of Aspergillus sp.1, we found that glucose and yeast extract were the most suitable carbon and nitrogen sources to obtain highest biomass production and highest antimicrobial activities Published by Burapha University. Keywords : Endophytic fungi; Seaweeds; Southern Thailand; Antibacterial metabolites. * Corresponding author. Tel.: address : nugul@tsu.ac.th 859

2 1. Introduction At present, there are many groups of important alga commonly found in Thailand, such as Red algae group (such as Acanthophora spp. and Gracilaria spp.), Green algae group (such as Enteromorpha spp. Chaetomorpha spp. and Caulerpa racemosa sp.) and Brown algae group (such as Sargassum spp. Padina spp. and Dictyota spp.). These alga can normally be found along Thailand s coastal areas. It is known that many alga are useful for food, cosmetics and medicines. Besides, they contain many bioactive compounds which can be utilized to inhibit various human, plant and animal pathogens, as well as for immunosuppressive and cancer cells prevention. However, the amount of algae has been rapidly decreased by many human activities as well as from the environmental and climate changes. Thus, it is necessary to find new sources of bioactive compounds. Presently, there are many groups of fungi which have symbiotic relationships with plants and do not cause harmful in such plants have been discovered. These fungi are called endophytic fungi and they live both inside and between plant cells. The alga which are the habitats of endophytic fungi will provide nutrients and shelters which are suitable for the endophytic fungi while the endophytic fungi will produce some chemical substances enabling algae to protect them from environmental harms and the development of pathogens, i.e., bacteria, fungi and virus. The bioactive substances which are produced by the endophytic fungi are called secondary metabolites which are very important because, apart from protecting the plants from harm, they can also repel the free radicals (Arunpanichlert et al., 2010) as well as have cytotoxic effects on tumor cells (Sajitha et al., 2013). Therefore, the objective of this study is to search for the algae in order to select the endophytic fungi which can produce new and interesting secondary metabolites. Previously, many studies were conducted mainly in terrestrial plants but so far more researches have been conducted in aquatic plants including algae (Mathan et al., 2013). There is a tendency that such substances can be developed into new medicines for curing many diseases and produce some commercial enzymes for industries and agriculture. 2. Materials and methods 2.1. Collection of sample The endophytic fungi were isolated from fresh and healthy seaweeds samples which were collected from Pattani, Songkhla, Satun and Krabi Provinces. All seaweeds samples were then kept in sterile bottles contained seawater taken from each locations and were transported back to the laboratory for further isolation Isolation of endophytic fungi from seaweeds Surface sterilization technique was generally used to ensure that all isolated were endophytic (Thirunavukkarasu et al., 2011; Suryanarayanan et al., 2010). The sterilized seaweeds were cut into small pieces and transferred onto a Potato dextrose agar (PDA) medium plate amended with the antibiotic (4-5 segments of seaweeds per Petri dish). The medium contained (potato 200 g/l, D-glucose 20 g/l, agar 15g/L and 0.85% NaCl) were sealed using parafilm and incubated at 28 ± 2 ๐ C for 2-3 weeks to observe the emerging of fungal mycelia growth. The fungal hyphae were then removed from the edge of seaweeds portion and transferred into the new PDA medium plate and allowed for growth. After incubation the fungal sample were then sub-cultured and stored for further studies. 860

3 2.3. Identification by morphological characteristics of endophytic fungi The fungi isolates were inoculated on to PDA medium plate and incubated for 2-3 weeks at 28±2 ๐ C to observe the colonies, morphology and measured their diameter of colonies. A square piece of culture medium was cut together with mycelia, put on a sterile glass slide, added a drop of Lactophenol Cotton Blue stain, and covered with a coverslip. The morphology of the conidia chains and spore were observed under microscope for giving codes using culture characteristic such as colony surface, texture, pigmentation etc Preliminary screening of antimicrobial activity To select the suitable growth medium for fungi isolates, all pure cultures were grown in different culture media such as Potato dextrose agar (PDA), Malt extract agar (MEA), Sabouraud s dextrose agar (SDA) and Corn meal agar (CMA) for primary screening antimicrobial activity using Dual agar diffusion technique with some modification. There were 7 pathogenic microorganisms used in this study, namely, Bacillus cereus, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella thyphimurium, Bacillus sp. and Candida albicans. The start cultures were incubated in Nutrient broth for 24 hour at 37 ๐ C. The microbial at diluted 0.5 McF with 0.85% NaCl and then were swabbed onto a sterile Nutrient agar (NA) for bacteria and Yeast malt agar (YMA) for yeast. The antimicrobial activity assay was compared by measuring zone of inhibition produced by endophytic fungi against pathogenic bacteria and yeast Optimization of culture media Selection of suitable culture media (Mathan et al., 2013) To select the suitable growth medium, the isolate CAS08 or Aspergillus sp.1 was grown in different culture media such as Potato dextrose broth (PDB), Czapek dox broth (CDB) Malt extract broth (MEB), Sabouraud s dextrose broth (SDB), Corn meal broth (CMB) and Nutrient broth (NB). For biomass production and bioactive metabolites production, the medium in which the isolate exhibited maximum antibiotic production expressed in terms of zone of inhibition was then used as the optimized medium for further study Effect of incubation period on biomass and bioactive metabolite productions Sabouraud dextrose broth medium was used as a basal medium. 100 milliliters of the medium dispersed in 250 millilitres conical flask and sterilized, the fungal were inoculated with 5 pieces, inoculum obtained from 7 days old spore culture of Aspergillus sp.1 and using different incubation period ranges incubated at 28±2 ๐ C. After that incubation the growth of the isolate was determined as dry mycelia weight. The mycelia were harvested by centrifuged at 8,000 rpm for 10 min, washed by distilled water and then allowed to dry at70 ๐ C and expressed as dry weight mycelia. The production of bioactive metabolites was expressed by measuring the diameter of inhibition zone against pathogenic microorganisms such as Bacillus cereus, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella thyphimurium, Bacillus sp. and Candida albicans. 861

4 Effect of carbon sources on biomass and bioactive metabolite production To study the effect of 5 different carbon sources, namely, glucose, sucrose, starch, mannitol and lactose were compared. 1% of each carbon sources were added to the basal medium individually. Each flask containing different carbon sources were inoculated with a 8 millimeters mycelia disc of 7 days old fungal cultures and incubated at 28±2 ๐ C for 10 days. After the incubation period mycelia dry weight and the production of bioactive metabolites were examined and recorded Effect of nitrogen sources on biomass and bioactive metabolite production To study the effect of different carbon sources, peptone, beef extract, yeast extract, ammonium chloride and ammonium sulphate were used. 1% of each nitrogen sources were added to the basal medium individually. Each flask containing different carbon sources were inoculated with a 8 millimeters mycelia disc of 7 days old fungal cultures and incubated at 28±2 ๐ C for 10 days. After the incubation period mycelia dry weight and the production of bioactive metabolites were examined and recorded. 3. Results and Discussions 3.1. Survey of seaweeds and endophytic fungi We surveyed and collected seaweeds from only 4 southern provinces in Thailand (Pattani, Songkhla, Satun and Krabi Provinces). It was found 8 types of seaweed groups consisting of Caulerpa racemosa, Chaetomorpha antennina, Chaetomorpha crassa, Enteromorpha clarthrata, Enteromorpha intestinalis, Enteromorpha prolitera, Gracilaria fisheri and Sargassum sp. (Fig.1). From these seaweeds, we obtained 82 endophytic fungi isolates, of which were found the most from Enteromorpha spp. (26 isolates), followed by Chaetomorpha spp. (16 isolates), Sargassum sp. (15 isolates) and Caulerpa racemosa (14 isolates), respectively (Fig.2). The more commonly group of algae found were Gracilaria spp., Enteromorpha spp. and Caulerpa racemosa, especially Enteromorpha spp. which were found in the highest number and had special characteristics, i.e., fast growth, resistance to a wide range of salinities, tolerance to water shortage and ability to grow in the areas of brackish water (Thanatthawongcharoen, 2012). As a result, we found the highest amount and diversity of endophytic fungi living in this type of seaweed Identification of endophytic fungi Based on morphological characteristics, we identified endophytic fungi isolated from all seaweeds, could be divided into 7 genera, namely, Penicillum sp.1 for 25 isolates (30.5%), Penicillum sp.2 for 2 isolates (2.44%), Aspergillus fumigatus for 16 isolates (19.5%), Aspergillus niger for 9 isolates (11.0 %), Aspergilus flavus for 3 isolates (3.7%), Aspergillus sp.1 for 1 isolates (1.2%), Rhizopus sp. for 1 isolate (1.2%) and also found 25 unidentified isolates (30.5%) (Fig.2). However, the fungi isolates which contained only hypha without spore production still be classified into the morphotype group which considered from thier colony characteristics. As for the endophytic fungi with spore production, they were identified into the Colletotrichum, Nigrospora and Phoma genera and the Xylariaceae family. From the results it was found some similarities to other researcher finding, for example Thirunavukkarasu et al., (2011) also reported the isolation of Penicillium sp.1, Aspergillus sp., Aspergillus niger and other endophytic fungi from Caulerpa racemosa, Sargassum sp. and Gracilaria edulis samples. These endophytic fungi can produce secondary 862

5 metabolites showing some biological activities such as inhibiting pathogens in humans and fish (Mathan, et al., 2013), antiprotozoal and cytotoxicity (Zhu, et al., 2009), L-asparaginase production (Thirunavukkarasu, et al., 2011) and L-glutaminase enzymes production (Sajitha, et al., 2013) etc. A B C D E F G Fig.1 Seaweeds found in this area Pattani, Songkhla, Satun and Krabi Provinces include (A) Cualerpa racemosa; (B) Chaetomorpha antennina; (C) Chaetomorpha crassa; (D) Enteromorpha clarthrata; (E) Enteromorpha intestinalis; (F) Enteromorpha prolitera; (G) Gracilaria fisheri and (H) Sargassum sp. H 863

6 Fig.2 Number of endophytic fungi isolates obtained from seaweeds Preliminary screening antimicrobial activity We found only one strain, Aspergillus sp.1 (or islolate CAS08) showed the best antimicrobial activities by inhibiting the growth of 5 pathogenic microorganisms up (> 10 millimeters of inhibition zone), including of Bacillus cereus, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella thyphimurium and Bacillus sp. It was found that Pseudomonas aeruginosa ATCC showed the inhibitory effect similar to Bacillus sp., and Bacillus cereus, when cultured in Sabouraud s dextrose agar and Potato dextrose agar. This fungus also showed the most inhibitory efficiency against Bacillus sp. (with 20.7 millimeters of inhibition zone) in Sabouraud s dextrose agar medium (Table1 and Fig.3). Bacillus cereus and Bacillus sp. are Gram-positive bacteria, facultatively anaerobic, endospore forming and large rod which can cause diseases in human. These bacteria can cause two different sickness types, first type, after contaminated food is eaten the bacteria make a toxic substance within small intestine leading to diarrhoea. The second type occurs if Bacillus cereus. and Bacillus sp. produce different kind of toxins in contaminated food. These toxins are most commonly affects rice and other starchy foods (Food and Drug Administration, 2012). Therefore, the Aspergillus sp.1 endophytic fungi were then selected for further studying of optimal cultural time of secondary metabolite production. 864

7 Table 1 Antimicrobial activities assay from Aspergillus sp.1 grown on 4 culture media. Aspergillus sp. 1 media Zone of inhibition against microbial pathogens (mm.)* PDA 16.4± ± ± ± ± ± SDA 13.7± ± ± ± ± ± MEA 16.0± ± ± ± ± CMA 7.7± ± ± ± *Tested pathogenic microorganisms : 1) Bacillus cereus, 2) Staphylococcus aureus ATCC 25923, 3) Pseudomonas aeruginosa ATCC 27853, 4) Escherichia coli ATCC 25922, 5) Salmonella thyphimurium, 6) Bacillus sp. and 7) Candida albicans ** (-) no inhibition. Fig.3 Antimicrobial activity against two microbial pathogen species (A) Pseudomonas aeruginosa ATCC 2785, (B) Bacillus sp. by using Aspergillus sp.1 growth on the culture medium : (1) PDA, (2) SDA (3) MEA and (4) CMA 3.4. Optimization of cultural media for Aspergillus sp Basal and selection of the culture media To select the suitable of growth media and produce high amount of bioactive compounds, we compared 6 culture media to support the high growth and antimicrobial activities of Aspergillus sp.1. Fig 4 revealed the effect of different growth on biomass and bioactive metabolite production. Among the tested media, maximum mycelia dry weight g/l was obtained in Sabouraud s dextrose broth, followed by Potato dextrose broth (0.971 g/l), Corn meal broth (0.961 g/l), Nutrient broth (0.947 g/l), Czapek s dox broth (0.926 g/l) and Malt extract broth (0.909 g/l), respectively. whereas maximum bioactive metabolites were obtained in 865

8 Potato dextrose broth (21.2 and 20.3 mm against Escherichia coli ATCC and Staphylococcus aureus ATCC 25923) followed by Nutrient broth (19.2 mm against Staphylococcus aureus ATCC 25923), Malt extract broth, (19.0 mm against Escherichia coli ATCC 25922) and minimum production of bioactive metabolite was observed in Czapek s dox broth (7.7 mm against Staphylococcus aureus ATCC 25923). In similar to other studies, Mathan et al., (2013) also reported the potato dextrose broth medium was the most suitable medium for the maximum on both biomass and bioactive metabolite production. Fig.4 Effect of different growth media on biomass and antibacterial activity against of Aspergillus sp.1 endophytic fungi, microbial pathogen : Bacillus cereus (Ba); Staphylococcus aureus ATCC (St); Escherichia coli ATCC (Ec); Salmonella thyphimurium (Sa) and Bacillus sp. (B.sp.) Effect of incubation period on biomass and bioactive metabolite production From growth curve studies, it was found the most suitable period for bioactive metabolite production that showing the best antimicrobial activities was days, and inhibited the most tested pathogenic microorganisms up to 5 types, including Bacillus cereus, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella thyphimurium and Bacillus sp., obtained in Sabouraud s dextrose broth. This isolate was found the most efficiency to inhibit Staphylococcus aureus ATCC with the inhibition zone diameter of 17.2 millimeters (Fig.5). The preliminary examination revealed that there are 19 types could inhibit pathogenic microorganisms in human and fish with high efficiency by measuring from inhibition zone at > 10 mm. From this finding, we selected the fungi Aspergillus sp.1 for future study aiming to seek the possibility of a future antibiotics production which can inhibit pathogenic microorganisms (Mathan, et al., 2013) 866

9 Fig. 5 Effect of incubation period on biomass growth in Sabouraud dextrose broth and antibacterial activity Against of Aspergillus sp.1 endophytic fungi, microbial pathogen : Bacillus cereus (Ba); Staphylococcus aureus ATCC (St); Escherichia coli ATCC (Ec); Salmonella thyphimurium (Sa) and Bacillus sp. (B.sp.) Effect of carbon sources on biomass and bioactive metabolite production Among the various carbon sources tested, glucose was found the best carbon source for biomass production (0.971g/l) whereas the maximum bioactive metabolite was obtained in lactose source (18.23 mm against Staphylococcus aureus ATCC 25923) and the minimum bioactive metabolite was produced in sucrose source (3.67 mm against Salmonella thyphimurium) (Fig.6). Merlin et al., (2013) reported the higher biomass (3.37 mg/ml) and bioactive metabolite production µg/ml obtained by culturing Fusarium solani LCPNCF01 in dextrose amended medium. Fig. 6 Effect of carbon sources on biomass growth in Sabouraud s dextrose broth and antibacterial activity against of Aspergillus sp.1 endophytic fungi, against pathogenic microorganisms: Bacillus cereus (Ba); Staphylococcus aureus ATCC25923 (St); Escherichia coli ATCC (Ec); Salmonella thyphimurium (Sa) and Bacillus sp. 867

10 Effect of nitrogen sources on biomass and bioactive metabolite production The nitrogen sources on biomass and bioactive metabolite production by Aspergillus sp.1 endophytic fungi. Maximum biomass production (0.921g/l) was observed in culture filtrate supplemented with yeast extract and Maximum antibacterial activity (29.7 mm and 28.9 mm, zone of inhibition against Escherichia coli ATCC and Staphylococcus aureus ATCC respectively. Fig.5). Whereas peptone was the least utilized nitrogen compound by the isolate and even the bioactive production was very low. The results are in good agreement with Mathan et al., (2013). Similar results were obtained in the present study where maximum biomass and bioactive metabolite production (56 mg/25ml), (21 mm zone of of inhibition against Escherichia coli and Vibrio parahaemolyticus). These results similar to other studies, Merlin et al., (2013) reported the higher biomass and bioactive metabolite production obtained by culturing Fusarium solani LCPNCF01 in yeast extract amended medium. Fig. 7 Effect of nitrogen sources on biomass growth in Sabouraud s dextrose broth and antibacterial activity against of Aspergillus sp.1 endophytic fungi, against pathogenic microorganisms: Bacillus cereus (Ba); Staphylococcus aureus ATCC25923(St); Escherichia coli ATCC (Ec); Salmonella thyphimurium (Sa) and Bacillus sp. 4. Conclusion In the present study, we obtained 82 endophytic fungi isolates from seaweeds collected from the southern Thailand which consisting of 7 genera by identifying mainly from morphological characteristics. These fungi showed various extent of the antimicrobial activities against many pathogenic microorganisms. Among all isolates obtained, we found only one strain which could not be identified by molecular characteristics, Aspergillus sp.1 or CAS08 isolate showing the highest potential of being further studied for antibiotic productions against various microbial pathogens and the chemical structure analyses. Preliminary results, it was found that the most suitable cultural time for enriching the Aspergillus sp.1 was days in Sabouraud dextrose broth medium and Staphylococcus aureus ATCC was the most susceptible human pathogen. Sabouraud s dextrose broth medium that good growth and sporulation of Aspergillus spp. with Sabouraud s dextrose broth might be due to presence of peptone as a source of nitrogen. To optimize the growth for 868

11 Aspergillus sp.1, we found that the glucose and yeast extract were the most suitable carbon and nitrogen, respectively, to obtain highest biomass production and highest antimicrobial activities. 5. Acknowledgement The authors thank Dr. Monthon Lertworapreecha Department of Biology, Faculty of Science, Thaksin University, Phatthalung for his kind assistance to provide some research facilities and suggestions. 6. References Arunpanichlert, J., Rukachaisirikul, V., Tadpetch, K., Phongpaichit, S., Hutadilok-Towatana, N., Supaphon, O., Sakayaroj. J., (2010). A dimeric chromanone and a phthalide : Metabolites from the seagrassderivedfungus Bipolaris sp. PSU-ES64, Journal of Phytochemistry Letters. 4, p Food and Drug Administration., (2012). Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, Second Edition. Mathan, S., Subramanian, V., Nagamony, S., Ganapathy, k., (2013) Isolation of Endophytic Fungi Marine Algae and its Bioactivity, Journal of Res. Pharm. 4, p Mathan, S., Subramanian, V., Nagamony, S. (2013) Optimizition and antimicrobial metabolite production from Endophytic Fungi Aspergillus terreus KC , Journal of Experimental Biology. 3(4), p Merlin, J., N., Christhudas, I.,V.,S.,N., Kumar, P., P., Agastian, P., (2013). Optimizition of growth and bioactive metabolite production Fusarium solani, Journal of Pharmaceutical and Clinical Research. 6, p Sajitha, N., Vasuki, S., Suja, M., Kokilam, G., Gopinath, M., (2013). Screening of L-glutaminase from seaweed endophytic fungi, Journal of Pharmaceutical and Applied Science. 3, p Suryanarayanan, T., S., Venkatachalam, A., Ravishankar, J., P., Mukesh, D., Geetha, V., (2010) Internal mycobiota of marine macroalgae from the Tamilnadu coast: distribution, diversity and biotechnological potential, Journal of Botanica Marina. 53, p Tanuttawongcharoen, R. (2012). Flavoring agent and antioxidant agent from hydrolyzed Ulva intestinalis protein, Master of science Biochemical Technology field of study school of bioresources and tecnology, King Mongkut's University of Technology Thonburi. Thirunavukkarasu, N., Suryanarayanan, T., S., Murali, T., S., Ravishankar, J., P., Gummadi, S., N., (2011). L- asparaginase from marine derived fungal endophytes of seaweeds, Mycosphere. 2, p Zhu, T., J., Du, L., Hao, P., F., Lin, Z., J., Gu, Q., Q., (2009). Cirtinal A, a novel tricyclic derivative of citrinin from an algicolous fungus Penicillum sp. i-1-1, Journal of Chinese Chemical Letters. 20, p