Research On Co-culture Of Marine-derived Microbes

In this thesis,marine microorganisms were selected as the research focus,and investigated for their ability to produce interesting secondary metabolites.After collection of marine-derived samples,isolation of pure microbial strains,and chemical and biological screening,eight bacteria and three fungi were identified as potentially interesting.Due to its diverse HPLC profile,the fungus LLT 3A-1-21 was selected for further research.Based on its 18 S r RNA gene sequence,the fungus was identified as Aspergillus neoellipticus.The fungus was grown in solid culture on rice.After extraction and purification by Diaion HP-20 resin column,silica gel column and semi-preparative HPLC,the structures of the compounds were elucidated by mass spectrometry and NMR spectroscopy.Twelve compounds were obtained,and for six of these the structures were elucidated,namely trypacidin(1),monomethylsulochrin(2),pseurotin A(3),fumiquinazolines C(4),verruculogen(5),and fumitremorgin C(6).All the compounds were subjected to antimicrobial activity assays,with trypacidin(1)showing weak antibacterial activity against S.aureus ATCC12600(MIC = 33 μg/m L),S.epidermidis ATCC14990(MIC = 17 μg/m L).Molecular networking analysis of the extracts of A.neoellipticus was conducted,using the MS/MS spectra to suggest structures for some other metabolites produced by the strain.In order to study the effect of microbial co-cultivation on the product,three fungi and eight bacteria obtained from the screening were used for co-cultivation.Through analysis using HPLC and comparison of the chromatograms of co-culture and pure cultures,the microbes Aspergillus neoellipticus LLT 3A-1-21 and Bacillus velezensis LLT 3A-4 were selected as a promising co-culture pair for further study.The large scale co-culture was fermented with PDB liquid medium,and supernatant and biomass were extracted using organic solvents.The extracts of the co-culture and pure cultures were subjected to antimicrobial activity testing,with the results showing that the co-culture extract had improved antimicrobial activities against Bacillus subtilis ATCC 6633,Aspregillus niger ATCC 34103,Staphylococcus aureus ATCC 12600,and Candida albicans ATCC 76615.After purification,8 compounds were isolated,including 6 compounds only produced under co-culture conditions.One of these was identified as the unusual terpene alkaloid pyripyropene E(7)was obtained.In additional,several compounds were obtained in co-culture with a distinctive UV spectrum.Preliminary NMR analysis suggested these might be members of the difficidins,a potent group of bacterial antibiotics,but structural elucidation is still ongoing.A molecular network was constructed among the co-culture and monocultures to analysis the relationship of their metabolites.The approach revealed metabolites from different sources and showed which products were only detected in co-culture.