Characterization, Identification and Metabolites of Fungi from the Soda Lakes in Kenya

Abstract

The soda lakes of Kenya developed as a consequence of geological and topological activities of the earth. These lakes are characterized by great variation in temperature, salt and pH extreme environment where diverse microorganisms inhabit. The physicochemical condtion in the lakes make them an extreme environment. The extreme conditions in the lakes may also provide microbial diversity as they adapt to this environment. However, Information on fungi from the Kenyan soda lakes environment is poorly documented or scanty. Little information is also available on the metabolic potential of the fungal diversity in the soda lakes. The main objective of this study was to isolate, and characterise fungi from the Kenyan Soda lakes and thereafter screen the isolates for enzymes and antimicrobial metabolites. The study sites were Lake, Elmentaita, Nakuru and Sonachi. Sediment samples were randomly collect from each lake, six sediment samples of 20g in polythene bags for each lake were pooled together to make a representative single sample of the lake sediments. The sediments were transported to JKUAT laboratory and stored in a freezer at minus 20 ºC. Serial diluted was used in isolation of fungi in triplicate on malt extract agar. The plates were incubated at 28 ºC for 48 hours. Purification of the isolates was done by subculturing on fresh malt extract medium. Cultural and morphological characterization was carried out to identify the isolates. Effect of growth, salt, pH, and temperature was done on malt extract agar agar and incubated at 28 ºC. Molecular characterization was done by amplifying the 18S gene using fungal primers. The amplicons were purified, quantified and checked for quality on an agarose gel and then sent for sequencing. Phylogenetic relationships were determined for the isolates in comparison with sequences retrieved from Nucleotide databse. Substrate utilization assays were carried out using different enzyme substrates to determine the ability of the fungus to degrade the substrate. Enzymatic activity was indicated by clearance zone around the fungal colony. The production of metabolites was carried out by grown isolates in liquid medium in conical flask. The medium was inoculated with a four millimetre agar fungal inoculums disc culture and incubated at 28 ºC in a shaker at 1000rpm for fourteen days. After the fourteen days of incubation the Crude filtrate was recovered through muslin filtration of the fermented fungal liquid culture. This was subjected to antimicrobial assay. Aportion of the crude filtrate was subjected to solvent extraction, three times.using ethylacetate and hexane (4:1-vol.) respectively. This constituted the solvent extract. This was dryried in a rotary vacuum evaporator. The dry extract was dissolved in 1ml ethylacetate and was used for antimicrobial assay.and GC-MS analysis for fungal metabolites A total of twenty six isolates were recovered, fourteen from Elmentaita, six from Nakuru and six from Sonachi. The recovered isolates were mainly anamorphic fungi in the phylum Ascomycota. Rhodotorula was the only genus recovered in the phylum Basidiomycota. Two species were recovered; the conspicuous one was Rhodotorula mucilaginosa, a yeast with valuable biotechnological feature and an emergent opportunistic pathogen that might cause disease in immunocompromised individuals. Effect of salt, pH, and temperature on the fungal isolates revealed different range for growth optimum for fungal isolates. Phylogenetic relationships aligned the isolates to the genera, Acrimonium, scopulariopsis, Verticillium, Rodotorula, Fusarium, Sarocladium, Paeciomyces, and Plectosphaerella among others. Two isolates were affiliated to the genus Rhodotorula in the phylum Basidiomycota. All the rest (24 isolates) were affiliated to the phylum Ascomycota. It has been reported that members of this phylum dominate in many extreme environments. Substrate utilization of the fungal isolates revealed different types of enzymes (cellulases, proteases, pectinases, lipases and esterase) as evidenced by the clearing zone. The percentage of isolates with enzymatic activity.were, Protease (58%), Esterase (39%), Lipase (31%) the list activity was amylase (4%). The solvent extract antimicrobial assay revealed that most fungal isolates exhibited antimicrobial activity against the test organisms, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aurogenosa, Candida albicans, Salmonella typhimurium and Streptococcus pneumoiae of which some of these microorganisms are potential human pathogens used in this study. GC-MS analysis revealed fungal metabolites in the groups (acids, ketones, quinones, alcohols, esters, aliphatic compounds). Among the revealed comounds like thujaplicin and thymoquinone have antifunal, and floropropione and maltol have antimicrobial effects, Isophorone are anticancer and antioxidants.The revealed high diversity of fungi has a potential of producing enzymes, antimicrobial agents and other metabolites of economic importance in food, agricultural or pharmaceutical industries.