Diversity and Biogeography of Fungi within the Hot Springs of Soda Lakes in Kenya

Abstract

The East African Rift valley soda lakes are sodium carbonate (Na2CO3)-dominated extreme environments of active volcanoes and differ from other soda lakes due to the fact that the surrounding hot springs supply water to the lake depressions. Study of microbial diversity of these soda lakes and hot springs are of great interest among microbiologists owing to their immense biotechnological applications. Hot springs harbor diverse groups of micro-organisms like fungi that have developed mechanisms to thrive at wide temperature ranges, according to their optimal growth requirements. Both culture-dependent and independent methods have been used to study the diversity of fungi from hot springs of soda lakes in Kenya. The main objective of this study was to use both culture dependent and culture independent methods to study the diversity and biogeography of fungal communities within hot springs of four selected soda lakes (Lake Bogoria, Elmenteita, Magadi and Little Magadi) in Kenya. Water, wet sediment and microbial mat samples were collected in triplicates from the hot springs of the four soda lakes. Culture independent method employed the use of 454 pyro sequencing to sequence amplicons of Internal Transcribed Spacer (ITS) gene region of the total community DNA in order to explore the fungal community composition in twenty four samples. Sequences were analyzed using QIIME pipeline Version 1.8.0, while hierarchical clustering, non-metric dimensional scaling (NMDS) and diversity indices were carried out using the R programming language version 3.1.3 and Vegan Community Ecology Package version 2.5.2. For culture-dependent approach, microbial mats and wet sediments were collected from three sampling points along the flow of the hot springs while water samples were collected from the mouth of the hot spring. Samples from the hot spring were isolated on four different media at pH 10 and at a temperature of 30 °C. From the culture independent approach, a total of 139,023 quality sequence reads were obtained from which, 2,179 operational taxonomic units (OTUs) were realized at 3% genetic distance. Three known phyla (Ascomycota [83.3%], Basidiomycota [15.8%], Glomeromycota [0.02%]) were identified. Richness, abundance and taxonomic analyses identified Agaricomycetes as the most abundant and diverse class within Basidiomycota. Sequences matching with Ascomycota had high affinities with seven known classes Dothideomycetes (91.7%) and Eurotiomycetes (70.8%) being the most abundant and diverse classes. The most abundant OTUs showed the highest sequence similarity to Cladosporium sp. (83.08%), Cladosporium cladosporioides (17.90%), Pleosporales sp. (86.07%), Aureobasidium pullulans (79.88 %) and Aspergillus oryzae (35.02%). From the culture dependent method, sixty one (61) fungal isolates were identified using morphological, physiological and molecular characters. Out of this, seventeen isolates were recovered from the hot spring on the shores of Lake Bogoria while twenty two isolates were from Lake Magadi and the same number from Lake Elmenteita. All the fungal isolates grew at pH ranging from 5 - 10, temperature range of 25 - 35 °C and sodium chloride range of 5 - 30 %. Substrate utilization of the sixty one fungal isolates revealed different types of enzymes (Amylases, proteases, pectinases, lipases, xylanases and esterases) as evidenced by the clearing zones. The percentage of isolates with enzymatic activity were; Esterase (85 %), amylases and xylanases (74 %), protease (66 %), lipase (59 %), while none showed production of cellulase enzyme. Analysis of partial sequences using Blastn showed that about 11.7 % and 29.1 % of the isolates from the hot spring on the shores of Lake Bogoria were affiliated with members belonging to the genera Penicillium and Aspergillus, respectively. A total of 12 % of the isolates belonged to the genera Alternaria and Fusarium while another 41 % of the isolates clustered closely with uncultured fungus. Fungal endophyte comprised 6 %. All the isolates from the hot spring at Lake Elmenteita were from the Phylum Ascomycota with 31.8 % of its members affiliated to the genus Aspergillus and 22.7 % were closer to the genus Alternaria while only 4.5 % were closely related to Debaryomyces hansenii, a yeast of the class Saccharomycetes. Contrary to the hot spring on the shores of Lake Bogoria, only 13.6 % of the isolates from Lake Elmenteita were affiliated with members belonging to the genus Penicillium. Blastn results from the hot spring on the shores of Lake Magadi showed 50 % of the isolates were closely clustered with members of the genus Aspergillus. An equal number of isolates representing 13.6 % were affiliated with members belonging to the genera Penicillium and Neurospora. Apart from isolate MM7, Parmastomyces transmutans, which belonged to the phylum Basidiomycota, the rest of the isolates from the hot spring at Lake Magadi were closely related to members of the Phylum Ascomycota. Although both culture dependent and independent methods did not reveal the true diversity of the fungal community, the isolates recovered were a representation of thermophilic, alkaliphilic and halophilic fungal taxa found in soda lake environments. These results suggest the ability of fungi to adapt to extremes of alkalinity, temperature and salinity. The isolates also have the potential to produce useful enzymes which could be exploited for future biotechnological applications.