Screening of Mangrove Endophytic Fungi for Bioactive Compounds by ONN MAY LING A thesis presented in fulfillment of the requirements for the degree of Masters of Science (by Research) at Swinburne University of Technology 2013 Abstract Endophytic fungi are an underexplored group of microorganisms as only a few plants have been studied with regards to this community. They live inside the tissues of other organisms, such as mangrove plants that provide protection to them and in return endophytic fungi support their hosts by fighting off pathogens through the production of antimicrobial compounds. These bioactive compounds are secondary metabolites which are often produced as waste- or by-products. Besides, endophytic fungi also help the host plant in adapting to (extreme) environments, for example by removing harmful heavy metals. In Malaysia, mangrove forests continue to be threatened by heavy metal pollution, resulting from industrial waste water pollution and urbanization.The presence of heavy metals can lead to severe damage as they are bioaccumulative and toxic. In the present study, endophytic fungi isolated frommangrove plants were characterized and assessed for their antimicrobial, cytotoxicity activity and heavy metal biosorption potential. Twelve endophytic fungi were isolated and identified (using molecular methods) to belong to 7 families: Penicillium, Curvularia, Diaporthe, Aspergillus, Guignardia, Neosartorya and Eupenicillium. Antimicrobial activities of these 12 fungal endophytes were tested against gram positive bacteria (Bacillus subtilis and Staphylococcus aureus among others), gram negative bacteria (Escherichia coli among others), yeast (Saccharomyces cerevisiae) and fungi (Candida albicans and Aspergillus niger). Two strains; Isolate 7 and Isolate 13 (related to Guignardia sp. and Neosartoya sp., respectively) showed strong antimicrobial (and antifungal) activity which was indicated by the formation of clear zone of inhibition, whereas the rest showed no activity. Compounds were isolated from the extracts of both isolates and screened using HPLC. Whereas for cytotoxicity assay, two strains; Isolate 3 and Isolate 9 (related to Diaporthe sp. and Eupenicillium sp., respectively) displayed toxicity against the matured brine shrimps at concentrations of 500 ppm after 24 hours incubation. For heavy metal biosorption, Isolate 2, which is closely related to Curvularia sp., is the most efficient in removing Cu, up to 25mg Cu/g biomass (using dead biomass). On the other hand, Isolate 8 and Isolate 13 (both related to Neosartorya sp.) are the most efficient in removing zinc (also using dead biomass), with a removal of up to 24 mg Zn/g biomass.The findings clearly indicate the potential of mangrove endophytic fungi to be used for drug development and also in wastewater bioremediation. ACKNOWLEDGEMENT I would like to express the deepest appreciation to my supervisor, Dr. Moritz Mueller, who has the attitude and substance of a genius: he continually and convincingly conveyed a spirit of adventure in regard to research, and an excitement in regard to teaching his students. Without his guidance and persistent help, this research as well as dissertation would not have been possible. I would also like to thank my co-supervisors, Dr. Lim Po Teen and Dr. Aazani Mujahid, whose work demonstrated that science and technology should always transcend academia. In addition, a thank you to my senior lab mate, Noreha Mahidi, who gave the permission to use her required equipment and the necessary materials to complete the benchwork. Besides, her stimulating suggestions and encouragement definitely has helped me to coordinate my project. I would also like to thank my fellow colleagues of the lab, Felicity Kuek, Chua Jia Ni, Nurul and Fika for the guidance and help throughout my benchwork period. Deepest appreciation for the time spent helping me out with some equipments as well as other tasks. Lastly, I would like to thank Professor Peter Proksch from the Institut für Pharmazeutische Biologie und Biotechnologie, University of Düsseldorf,Germany for the HPLC analysis conducted on my research samples. DECLARATION I, Miss Onn May Ling, Masters of Science (By Research), Faculty of Engineering, Computing and Science, hereby declare that my project work titled “Screening of Mangrove Endophytic Fungi For Bioactive Compounds” is original and contains no material which has been accepted for the award to the candidate of any other degree or diploma, except where due reference is made in the text of the examinable outcome; to the best of candidate’s knowledge contains no material previously published or written by another person except where due reference is made in the text of the examinable outcome; and where the work is based on joint research or publications, discloses the relative contributions of the respective workers or authors. All the given information is true to best of my knowledge. …………………………… (ONN MAY LING) DATE: 1.7.2013 I Table of Contents List of Figures ...................................................................................................................... VI List of Tables......................................................................................................................VIII 1. Introduction................................................................................................................ 1 1.1 Infectious diseases, drug resistance, and bioactive compounds................................... 1 1.2 Sources of bioactive compounds.................................................................................. 4 1.3 Fungi ............................................................................................................................ 6 1.3.1 Fungi as sources of bioactive compounds............................................................. 7 1.4Endophytic fungi........................................................................................................... 7 1.4.1 Endophytic fungi as sources of bioactive compounds .......................................... 9 1.5 Mangroves.................................................................................................................. 17 1.5.1 Mangrove endophytic fungi ................................................................................ 19 1.5.2Threats to mangroves ........................................................................................... 21 1.5.3 Heavy metal pollution......................................................................................... 21 1.5.4 Heavy metal uptake and removal........................................................................ 23 1.5.5Biosorption by Marine Fungi............................................................................... 24 1.6Aim of the project and scope of study ........................................................................ 25 2. Materials and methods ............................................................................................. 26 2.1 Sampling .................................................................................................................... 26 2.1.1 Field site sampling .............................................................................................. 26 2.2 Isolation of mangrove endophytic fungi .................................................................... 27 2.2.1 Plant samples....................................................................................................... 27 2.2.2 Soil samples ........................................................................................................ 28 2.3 Fungal Cultivation...................................................................................................... 31 2.3.1 Fungal Culture for Short Term Storage .............................................................. 31 2.3.2 Fungal Culture for Long Term Storage............................................................... 31 2.3.3 Fungal Culture for Extraction of Bioactive Compounds .................................... 31 2.4 Endophytic fungi identification.................................................................................. 33 2.5 Extraction of bioactive compounds............................................................................ 36 2.5.1 Solvent-solvent extraction................................................................................... 36 II 2.6 Biological Assays....................................................................................................... 40 2.6.1 Primary Screening of antimicrobial activity ....................................................... 40 2.6.2 Secondary screening of antimicrobial activity.................................................... 41 2.6.3 General Cytotoxicity assay ................................................................................. 41 2.7 Heavy Metal Analysis................................................................................................ 45 2.7.1 Determination of heavy metal-resistant fungi..................................................... 45 2.7.2 Heavy metal biosorption by dead fungal cells .................................................... 45 3. Results and Discussion............................................................................................. 48 3.1 Fungi identification .................................................................................................... 48 3.2 Biological assays.......................................................................................................