International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia) Selective Isolation of Actinomycetes from Mangrove Forest of Pahang, Malaysia Nurfathiah Abdul Malek, Ahmed Jalal Khan Chowdhury, Zarina Zainuddin, and Zaima Azira Zainal Abidin uneconomical rediscovery of known compounds. Thus, a new resource is essential to circumvent this issue. Thereby, researchers are now looking forward on the underexplored habitats such as marine ecosystem, dunes, volcanic zones, deserts and mangrove forests as new reservoirs of putative actinomycetes. Mangrove forests are highly productive ecosystems which comprise of unique woody plant communities and located in tropical and subtropical coastal area [1],[8]. According to Ara [9], mangroves form unique saline environments under the influence of tidal flow, hence the muddy alluvial soil due to the intermittent flooding. Mangrove ecosystems are nutritionally versatile as they are highly rich in organic matter, nitrogen and sulfur content which can be used by living microorganisms. Thus, it is believed that mangrove ecosystems have the potential of becoming new reservoir for highly diverse actinomycetes as demonstrated by the isolation of Micromonospora rifamycinica [10] and Verrucosispora wenchangensis [11]. Located in the world tropical belt, Malaysia is blessed with abundant mangrove forest, particularly in the west coast of Peninsular Malaysia [12]. Pahang mangrove forest which is situated on the east coast of Penisular Malaysia, is a poorly studied ecosystem. Assessment of actinomycetes diversity in Pahang mangrove forest is not attempted so far, hence this location is foreseen to be able to provide rich source of actinomycetes. The primary aim of this study was to determine the diversity of culturable actinomycetes from mangrove forest of Pahang. The efficiency of various selective isolation procedures employed were also discussed. Abstract—Actinomycetes which abundantly found in nature are renowned for their unique capabilities to produce biologically active metabolites. Poorly studied mangrove ecosystem offers a promising natural resource of new actinomycetes due to their high species diversity. This study aims to assess the diversity of actinomycetes from mangrove forest of Pahang, Malaysia. Various selective isolation media and pre-treatments were employed for maximum isolation of mangrove actinomycetes. A total of 4850 actinomycete isolates had been successfully recovered. It was observed that inorganic-salt starch agar (ISP4) displayed the highest percentage of actinomycetes recovery (31.7 %). Starch-yeast extract (SYE) and starch casein agar (SCA) were found to be more efficient in isolating wide variety of actinomycetes based on their morphological appearance. It was also observed that wet heat pre-treatment was more effective in isolating mangrove actinomycetes as 81.4 % of total isolates were yielded using this technique. On the other hand, dry heat treatment was better in the enumeration of spore-forming actinomycetes despite lower number of isolates recovered. Of both pre-treatments, station 1 which is located within the deep mangrove forest area recorded the highest recovery (23.7 %), with Streptomyces and Micromonospora-like isolates being the most predominant observed from all sampling sites. The presence of relatively large number of isolates suggests that Pahang mangrove forest is a potentially rich source of actinomycetes. Keywords—Mangrove actinomycetes, Pahang, pre-treatments, selective isolation. I. INTRODUCTION Filamentous bacteria within the order of Actinomycetales or else known as actinomycetes are aerobic and Gram-positive bacteria that belong to the class Actinobacteria [1], [2]. These slow growing prokaryotes are widely distributed in nature. They can be found virtually in every natural substrate; the water, air, soil, foodstuffs, manure and composts [3]. Actinomyetes have been continuously reported as the main prolific producers of microbial bioactive secondary metabolites for potential agricultural, pharmaceutical and industrial applications [4]-[6]. More than 70% of the commercially available active compounds originated from actinomycetes [7]. Extensive isolation and screening of actinomycetes from soil however, has lowered the rate of discovery novel biological active compounds owing to the II. MATERIAL AND METHODS A. Study Area and Location of Sampling Sites Pahang mangrove forest which covers an area of 343 ha is located in Kuantan, the capital state of Pahang, Malaysia. It is situated within Kuantan estuary which mainly influenced by Kuantan river that flows out to South China Sea. This study area experience semidiurnal tides that lies in wet tropical area [12]. According to Jalal et al. [13], the brackish water ecosystem surrounding the Pahang mangrove forest serves as a natural resource of diverse species of microorganism communities. Seven locations (S1, S2, S3, S4, S5, S6 and S7) were identified as sampling sites as shown in Fig. 1. The locations of the sampling sites were noted using Global Positioning System (GPS). Nurfathiah Abdul Malek, Ahmed Jalal Khan Chowdhury, Zarina Zainuddin, Zaima Azira Zainal Abidin, are with Kulliyyah of Science, International Islamic University Malaysia, Malaysia. (Email Id: [email protected], [email protected], [email protected], [email protected]) http://dx.doi.org/10.17758/IAAST.A1214007 9 International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia) Fig. 1 Location of the Sampling Sites III. RESULTS AND DISCUSSION B. Collection of Samples At each location, three sediment core samples were collected at a depth of 0 – 30 cm. The sediment samples were then immediately transported back to the laboratory using sterile polythene bag. A total of 4850 actinomycete isolates had been successfully recovered from all seven sites of Pahang mangrove forests. These dull, tough, chalky, leathery in appearance and earthly smell colonies were enumerated using 2 pretreatment procedures and 8 selective isolation media supplemented with nystatin (50 µg ml-1) to combat the growth of fungus. An average of 19 mycelium-forming colonies per plate was observed when only considering those plates that yielded actinomycetes. These relatively large numbers of isolates suggested that Pahang mangrove forest is a prominent resource of actinomycetes. The percentage of actinomycetes recovered from each sites was presented in Fig. 2. It was clearly observed that highest number of isolates were obtained from S1 (23.7%), which is located within the deep untapped Kuantan Mangrove Reserve Forest having rich alluvial soil, while the lowest number are recovered from both S6 and S7 (6.9%). Similar observation was reported by Mitra et al. [14], whereby isolation from the intertidal region furthest from the coastal line yielded maximum number of isolates. According to Kathiresan and Bingham [15], such forests were influenced with the movement of higher amount of freshwater with fluvial nutrients and consequently making it an eminently productive ecosystem. Low recovery of actinomycete colonies were observed from S6 and S7; the sites that are near the fishing village occupied with fishing activity. Contaminants from domestic wastewater and urban runoff from nearby fishing village might be deposited to these two mangrove sites [12], subsequently lowering the number of bacterial counts recovered. C. Pre-treatments of the Sediment Samples A total of 21 sediment samples were air dried at room temperature for 1 week to constant weight. The sediments were then ground and sieved to exclude large mineral and organic matter particles before pre-treatment. For wet heat pre-treatment; soil suspension containing 1 gm of dried soil diluted in 9mL of sterilized sea water was heated in a 60 oC water bath for 20 minutes. Meanwhile, for dry heat pretreatment, 1gm of dried sediment was heated at 120 oC for 60 minutes in a hot air oven. D. Selective Isolation of Actinomycetes from the Sediment Samples The pre-treated sediment samples were diluted 1:10 v/v with sterile saline, serially diluted down to 10-6 and plated onto a set of selective isolation plates in triplicate. The selective media that were used in this study include starchyeast extract agar (SYE), yeast extract-malt extract agar (ISP2), oatmeal agar (ISP3), inorganic salt-starch agar (ISP4), starch-casein agar (SCA), glucose asparagine agar (GAA), actinomycete isolation agar (AIA) and marine agar (MA). All media were supplemented with nystatin (50 µg ml-1) as antifungal agent. The inoculated plates were incubated at 28 – 30 oC for 2 – 3 weeks. Typical actinomycete colonies on the isolation plates were picked out based on morphological bases, streaked and purified on the original media. http://dx.doi.org/10.17758/IAAST.A1214007 10 International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia) isolation recovery (31.7%), followed by AIA (24.1%) as seen in Fig. 3. A total of 1538 colonies were picked from ISP4 agar based on actinomycete-like morphology, of which around 735 isolates formed powdery colonies with well-developed aerial hyphae fragmented into spore-chains. As described by Bredholt et al. [20], these isolates are tentatively called as Streptomyces-like colonies. ISP4 contains starch, calcium carbonate and ammonium sulphate that support good growth of actinomycetes, particularly the Streptomyces-like group [23]. Enhancement of actinomycetes growth by addition of calcium carbonate to the culture medium has also been suggested previously by Arifuzzaman et al. [24] and Kitouni et al. [25]. Fig. 2 Recovery of Actinomycetes at Different Sampling Sites of Pahang Mangrove Forest Previous studies had reported that pre-heating regimes generally stimulate the isolation of actinomycetes by eliminating the dominance of unwanted microorganisms which could inhibit the colonization of slow growing actinomycetes [16]-[19]. Treatment of the samples might also promote the germination of rare genera as previously reported by Bredholt et al. [20]. Hence, pre-heating treatment had become a useful standard practise for routine isolation. In this study, two pre-heat procedures; wet heat and dry heat pretreatment were employed for maximum isolation of actinomycetes. It was stipulated that wet heat pre-treatment at 60 oC for 20 minutes was more effective in enumerating actinomycetes from Pahang mangrove forest. More than twothird (84.11%) of the total isolates have been successfully yielded using this moist heating technique. This was in line with the findings of Naikpatil and Rathod [21] whereby recovery of actinomycetes increased up to 50% of the total microorganisms upon mild heating treatment of the sediment suspension. On the other hand, dry heat treatment was better in the isolation of spore-forming actinomycetes and greater in suppressing the growth of bacterial colonies, despite of low actinomycetes recovery. Elevated temperature may promote the germination of highly resistant endospores; subsequently support their survival through this heating procedure at 120 oC for 60 minutes. Nutrient availability is one of the main factors that determine the growth of actinomycetes. Most actinomycetes can use wide variety of compounds such as glucose, starch, proteins and amino acids as their energy source; unlike other bacterial groups that only favour simple carbon and nitrogen compound [22]. Various types of selective isolation media were employed in order to increase the number of nonSteprtomyces actinomycetes relative to the non-actinomycetes microbial flora [20]. In this sudy, the efficiency of culture media was evaluated using eight different selective isolation media to assess the optimal nutrient conditions for isolation of actinomycetes of Pahang mangrove forest. ISP4 was the most effective medium in the isolation of Pahang mangrove actinomycetes as it showed the best http://dx.doi.org/10.17758/IAAST.A1214007 Fig. 3 Percentage of Actinomycetes Recovery Using Various Selective Isolation Media However, the use of SCA and SYE agar result in better recovery of wide variety of actinomycetes, based on their morphological characteristics. This was also supported by the finding of Naikpatil and Rathod [21], whereby SCA improved the enumeration of actinomycetes from mangrove sediment of Karwar, India. According to Kim [26], SCA possesses high carbon-to-nitrogen ratio and contains resistant complex carbon such as starch and casein which was very suitable for actinomycete isolation as these organisms are able to utilize such high molecular weight polymers. Despite containing simple carbon and nitrogen source; the presence of seawater in SYE medium might promote the growth of actinomycetes by mimicking the high salinity mangrove environment. Actinomycetes isolated from SYE agar should be further tested for their sea water requirement that could be a unique and crucial component for their growth condition. GAA was the least effective medium for the isolation of Pahang mangrove actinomycetes with 3.0% of recovery (Fig. 3). This clearly showed that L-asparagine, the main component of GAA might not be a good nitrogen source of mangrove actinomycetes. Of all isolation procedures, 140 representative isolates have been selected based on their distinct morphology appearances, from seven sediment samples of Pahang mangrove forest. These isolates are further categorized into seven series according to the colour of mature aerial mycelium; namely white, grey, red, yellow, green, black and orange series. 11 International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia) IV. ACKNOWLEDGMENTS This research was funded by Fundamental Research Grant Scheme (FRGS13-039-0280). The authors would like to acknowledge Ministry of Higher Education for the scholarship given. Fig. 4 Some Actinomycetes Colonies Successfully Enumerated from Pahang Mangrove Forest Members of white and grey series tend to have unfragmented substrate mycelium and abundant aerial mycelium with long spore chains, suggesting that they are members of Streptomyces-like group as seen in Fig. 4(a) – Fig. 4(f). They represented the highest number of isolates being 67 out of 140 representative isolates (47.7%). The orange series or else known as Micromonospora-like group (tiny, solid colonies with unusual aerial hyphae) (Fig. 4(g), Fig. 4(h)) were also found in all sampling sites representing 25.0% of the total isolates. The relative abundance of these two groups compared to the other series, indicated that Streptomyces-like and Micromonospora-like actinomycetes were dominant in Pahang mangrove forest. Red (2.1%), yellow (2.1%) and green (0.7%) series were the least abundant actinomycete isolates where they were only found in some sampling sites. As proposed by Hong et al. [1], mangrove forest might become an important source for discovering novel actinomycetes owing to their adaptation towards salinity and tidal gradients. Presence of huge numbers of actinomycete with various distinct morphological appearances (Fig. 4) suggests that Pahang mangrove forest is a promising resource of actinomycetes. This study was not intended to construct a detailed inventory of actinomycetes kinds found in the mangrove ecosystem, but it serves as a foreground to study the biocapability of mangrove actinomycetes. Nevertheless, this study provide the most comprehensive survey of culturable actinomycetes present in Pahang mangrove forest, resulting from the use of broad selective isolation procedures. http://dx.doi.org/10.17758/IAAST.A1214007 V. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] 12 K. Hong, A. Gao, Q. Y. Xie, H. Gao, L. Zhuang, H. P., Lin, H. P. Yu, H. P. Yu, X. S. Yao, M. Goodfellow and J. S. Ruan, “Actinomycetes for marine drug discovery isolated from mangrove soil and plants in China,” Marine Drugs, vol 7, pp 24-44, 2009. http://dx.doi.org/10.3390/md7010024 G. Y. Bais, T. P. Nimbekar, B. R. Wanjari and S. P. Timande, “Isolation of antibacterial compound from marine soil actinomycetes,” International Journal of Biomedical and Advanced Research, vol 3, pp. 193-196, 2012. A. Bizuye, F. Moges and B. Andualem, “Isolation and screening of antibiotic producing actinomycetes from soils in Gondar town, North West Ethiopia,” Asian Pacific Journal of Tropical Disease, vol 3, pp. 371-375, 2013. http://dx.doi.org/10.1016/S2222-1808(13)60087-0 V. Gesheva and R. Gesheva, “Physiological and antagonistic potential of actinomycetes from loquat rhizosphere,” Microbiological Research, vol 155, pp. 133-135, 2000. http://dx.doi.org/10.1016/S0944-5013(00)80049-X V. R. R. K. Dasari, M. K. K. Muthyala, M. Y. Nikku, and S. R Donthireddy, “Novel pyridinium compound from marine actinomycete, Amycolatopsis alba var. nov. DVRD4 showing antimicrobial and cytotoxic activities in vitro,” Microbiological Research, vol 167, pp. 346-351, 2012. http://dx.doi.org/10.1016/j.micres.2011.12.003 C. Balachandran, V. Duraipandiyan, and S. Ignacimuthu, “Purification and characterization of protease enzyme from actinomycetes and its cytotoxic effect on cancer cell line (A549),” Asian Pacific Journal of Tropical Biomedicine, vol 1, pp. S392-S400, 2012. http://dx.doi.org/10.1016/S2221-1691(12)60195-6 A. L. Demain, and S. Sanchez, “Microbial drug discovery: 80 years of progress,” The Journal of Antibiotics, vol 62, pp. 5-16, 2009. http://dx.doi.org/10.1038/ja.2008.16 S. Hunadanamra, A. Akaracharanya, and S. Tanasupawat, “Characterization and antimicrobial activity of Streptomyces strains from Thai mangrove soils,” International Journal of Bioassays, vol 2, pp. 775-779, 2013. International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia) [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] I. Ara, M. A. Bakir, W. N. Hozzein, and T. Kudo, “Population, morphological and chemotaxonomical characterization of diverse rare actinomycetes in the mangrove and medicinal plant rizhosphere,” African Journal of Microbiological Research, vol 7, pp. 1480-1488, 2013. H. Huang, L. Jiasen,Y. Hu, Z. Fang, K. Zhang,and S. Bao, “Micromonopora rifamycinica sp. Nov., a novel actinomycete from mangrove sediment,” International Journal of Systematic and Evolutionary Microbiology, vol 58, pp. 17-20, 2008. http://dx.doi.org/10.1099/ijs.0.64484-0 Q. Y. Xie, H. P. Lin, L. Li, R. Brown, M. Goodfellow, Z. Deng, and K. Hong, “Verrucosispora wenhangensis sp. nov., isolated from mangrove soil,” Antonie van Leeuwenhoek, vol 102, pp. 1-7, 2012. http://dx.doi.org/10.1007/s10482-012-9707-1 Y. Kamaruzzaman, M. Y. Nurul Nadia, S. M. Noor Azhar, M. C. Ong, S. Shahbuddin, K. C. Jalal, and B. Joseph, “Heavy metal concentration in the surface sediment of Tanjung Lumpur mangrove forest, Kuantan, Malaysia. Sains Malaysiana, vol 40, pp. 89-92, 2011. K. C. A. Jalal, Y. Kamaruzzaman, A. Fairuz, B. Akbar, S. Shahbuddin, and Y. Faridah, “Bacterial communities in Kuantan Estuary of Pahang Malaysia.” Journal of Applied Sciences, vol 10, pp. 652-657, 2010. http://dx.doi.org/10.3923/jas.2010.652.657 A. Mitra, S. C. Santra, and J. Mukherjee. “Distribution of actinomycetes, their antagonistic behavious and the physico-chemical characteristics of the world’s largest tidal mangrove forest,” Applied Microbiology Biotechnology, vol 80, pp. 685-695, 2008. http://dx.doi.org/10.1007/s00253-008-1626-8 K. Kathiresan, and B. L. Bingham, “Biology of mangrove and mangrove ecosystem,” Advances in Marine Biology, vol 40, pp. 81-250, 2001. http://dx.doi.org/10.1016/S0065-2881(01)40003-4 M. Hayakawa, T. Sadakata, T. Kajiura, and H. Nonomura, “New methods for the highly selective isolation of Micromonospora and Microbispora from soil,” Journal of Fermentation and Bioengineering, vol 72, pp. 320-326, 1991. http://dx.doi.org/10.1016/0922-338X(91)90050-Q C. N. Seong, J. H. Choi,and K. S. Baik, “An improved selective isolation of rare actinomycetes from forest soil,” The Journal of Microbiology, vol 39, pp. 17-23, 2001. R. Baskaran, R. Vijayakumar, and P. M. Mohan, “Enrichment method for the isolation of bioactive actinomycetes from mangrove sediments of Andaman Islands, India,” Malaysian Journal of Microbiology, vol 7, pp. 26-32, 2011. N. Niyomvong, W. Pathom-aree, A. Thamchaipenet, and K. Duangmal, “Actinomycetes from tropical limestone caves,” Chiang Mai Journal of Science, vol 39, pp. 373-388, 2012. H. Bredholt, E. Fjarvik, G. Johsen, and S. B. Zotchev, “Actinomycetes from sediments in the Trondheim Fjord, Norway: diversity and biological activity,” Marine Drugs, vol 6, pp. 12-24, 2008. http://dx.doi.org/10.3390/md6010012 S. V. Naikpatil, and J. L. Rathod, “Selective isolation and antimicrobial activity of rare actinomycetes from mangrove sediment of Karwar,” Journal of Ecobiotechnology, vol 3, pp. 48-53, 2008. V. G. Gil, S. Pastor, and G. J. March, “Quantitative isolation of biocontrol agents Trichoderma spp., Gliocladium spp. and actinomycetes from soil with culture media,” Microbiological Research, vol 164, pp. 196-205, 2009. http://dx.doi.org/10.1016/j.micres.2006.11.022 F. L. Rabah, A. Elshafei, M. Saker, B. Cheikh, and H. Hocine, “Screening, isolation and characterization of a novel antimicrobial producing actinomycete, strain RAF10,” Biotechnology, vol 6, pp. 489496, 2007. http://dx.doi.org/10.3923/biotech.2007.489.496 M. Arifuzzaman, M. R. Khatun, and H. Rahman, “Isolation and screening of actinomycetes from Sundarbans soil for antibacterial activity,” African Journal of Biotechnology, vol 9, pp. 4615-4619, 2010. M. Kitouni, A. Boudemagh, F. Boughachiche, H. Hamdiken, L. Oulmi, S. Reghioua, H. Zerizer, A. Couble, D. Mouniee, A. Boulahrouf, and P. Boiron, “Isolation of actinomycetes producing bioactive substances from water, soil and tree bark samples of the north-east of Algeria,” Journal de Mycologie Medicale, vol 15, pp. 45-51, 2005. http://dx.doi.org/10.1016/j.mycmed.2004.12.005 http://dx.doi.org/10.17758/IAAST.A1214007 [26] S. K. Kim, Marine microbiology: Bioactive compounds and biotechnological applications. Weinheim: Wiley-VCH.], pp. 281-285. 13
© Copyright 2024