A water body connotes the portion of the earth’s surface covered with water (such as a lake, river or ocean). Two kinds of salt water are classified on the earth’s surface, and they include epicontinental (inland surface) salt lakes and marine waters (the ocean) [1] . However, this research is slanted towards the latter type, specifically, ephemeral or permanent water bodies with salinities greater than 3 g/L. Lakes are water bodies surrounded by land but are not as a result of distributary from an ocean [2] ; they are relatively still water bodies when compared to rivers which are known for flowing waters [3] . They can also contain either salt water or fresh water, and are basically larger than ponds. Water bodies are subject to seasonal variations which impact microbial distributions found in them per time [4] - [6] .

Hypersaline lakes, typified by low oxygen concentrations, could either be thalassohaline or athalassohaline (from the Greek word, thalassa, meaning sea) [7] - [12] . Thalassohaline lakes are creations of evaporation of seawater and as such contain sodium chloride as the major salt, with a salinity that surpasses that of seawater by a factor of 5 - 10 and a neutral or slightly alkaline pH [13] . Examples include the Great Salt Lake of Utah, playas, salt mine drainage waters, brine springs from underground salt deposits, natural coastal splash zones and tide pools, and solar salterns [14] . Athalassohaline lakes on the other hand stem from non-seawater sources and are made up of high concentrations of ions such as magnesium and calcium and sundry other ions viz potassium, or sodium in trace amounts and are often the sources of potash, magnesium metal, soda, and even borax if the waters have high boron content. Good examples are the alkaline soda lakes of Egypt (Wadi El-Natrun), soda lakes of Antarctica, Dead Sea, and Big Soda Lake and Mono Lake in California [14] - [18] . De-salination of salt water bodies using either membrane or thermal technologies have been proposed to be a promising approach to unending water supply in the world [19] , and goes to portray the potential usefulness of salt lakes amidst their perceived toxicity.

Uburu and Okposi salt lakes are situated in Ohaozara Local Governmeant Area of Ebonyi State, Nigeria and remain under-studied till today. Much of the research done on these lakes by [20] - [23] , has only focused broadly on its geology, geochemistry and geophysics with special attention on its physicochemical parameters, hydrochemistry, water types and facie evolution, gross alpha and beta concentrations, ground water exploration using aeromagnetic and electromagnetic geophysical methods, respectively. Unlike other salt lakes across the world, Uburu and Okposi salt lakes have not been classified as either thalassohaline or athalassohaline. More recent biological studies have only concentrated on the initiation of oxidative stress in the antimicrobial susceptibility of Ebonyi salt mining sites, reproductive parameters and reduction of sperm number, motility and morphology of adult male Sprague-Dawley rats and radiological health risks due to gamma dose rates around these lakes [24] - [28] . However, some scientists have studied the halophilic archaea, bacteria, cyanobacteria and fungi of other hypersaline ecosystems in various countries of the world such as India (Goa solar salterns, Gujarat coast, Kerala and Sambhar salt lakes); Israel/Jordan (Dead sea); USA (Great salt lake of Utah). None has studied those of Uburu and Okposi salt lakes of Nigeria.

In 1978, Donn Kushner put forward what remains today as the most acceptable definition and classification of microorganisms based on their salt requirement and tolerance. He divided halophiles into: extreme halophiles (optimum growth at 2.5 - 5.2 M NaCl), borderline extreme halophiles (having their optimum at 1.5 - 4.0 M NaCl), moderate halophiles (preferring media with 0.5 - 2.5 M NaCl), slight halophiles (having optimum growth at 0.2 - 0.85 M NaCl) and halotolerant microorganisms that do not require salt for growth but tolerate salt often in high concentrations; and extremely halotolerant if the salt requirement exceeds 2.5 M NaCl [29] . Again, the mercantile importance of halophiles has been noted and benefitted from since antiquity. For instance, Roman soldiers were given “salt money” (salarium argentum) as a part of their payment; and from this practice, the English word “salary” (sal meaning salt, ary pertaining to or connected with money) was coined. According to evidences from small subunit rRNA sequence-based phylogenetic tree of life, halophiles can be found in each of the three domains of life: Archaea, Bacteria and Eukarya [30] . However, bacterial diversity is greatly decreased with increased salinity, while fungal diversity appears to be more complex, which led to this study being mainly fungal-based. Water chemistry of salt lakes impact on the diversity of microorganisms found in them, with seasons also contributing to the microbial niches per fluctuation in water volumes in both wet and dry seasons. For fungi, they basically have a modified plasma membrane fluidity that enables them alleviate salinity stress and also perform symbiosis with plant rhizospheres or as endophytes [30] . Some of these fungi possess the ability to solubilize insoluble phosphates from calcium, aluminium and iron which plays vital roles in nutrient sampling and remediation of heavy metals. Thus, it becomes important to study the fungal diversity in this extreme environment for reasons centred on possible biotechnology usefulness and climate change modulation at large.

The aim of this study is to investigate the seasonal microbiological changes of halophiles isolated from Uburu and Okposi salt lakes, with a view of specifically investigating and ascertaining the fungal dynamics/variations in both lakes over a one-year period; also to characterize and identify the halophilic molds associated with the lakes, carry out halotolerance evaluation of the isolates and classifying these halophiles accordingly that are believed to have possible cutting edge biotechnological applications.

1) Study Locations

The images of Uburu and Okposi Salt lakes are presented in Plate 1 and Plate 2 as shown below.

2) Seasonal Microbiological Analyses/Dynamics of Uburu and Okposi Salt Lakes

Fourteen fungal isolates labeled UBA, UBD, UBE, OKA, OKC, UA, UB, UC, OA, OD, UE, OKB, OB, and OKPb were obtained from the lakes and characterized. All isolates were identified colonially and microscopically. Thus, seven best salt tolerant isolates were selected were identified to species level using ITS rDNA sequencing tool as shown in Table 1 and Table 2 . The best salt tolerant fungus was selected for further studies. However, it can be concluded from Table 1 and Table 2 that fungi belonging to the genera of Aspergillus and Penicillium occurred in both lakes throughout the study period.

A little more than 20 years ago, scientists believed that hypersaline environments were predominantly inhabited by archaea, bacteria and a eukaryote—the alga named Dunaliella salina. But currently, several researchers such as [15] [38] - [42] isolated and reported a plethora of predominant eukaryotic fungal species like Cladosporium, different species of anamorphic Aspergillus and Penicillium, the teleomorphic Emericella and Eurotium, certain species of non-melanized yeasts represented by black, yeast-like hyphomycetes (mainly Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, and Aureobasidium pullulans). Likewise, phylogenetically closely related species of Cladosporium belonging to the order Dothideales, and Wallemia sp from different hypersaline waters of the world. Findings from this work corroborated these initial published findings by isolating diverse halophilic fungi from Uburu and Okposi salt lakes ( Table 1 and Table 2 ). This present study carried out on Uburu and Okposi salt lake spanned throught out the 4 quarters and two seasons of a year, giving rise to a total of 14 different fungal isolates with varying halotolerance profiles viz: Aspergillus sydowi (28 mm at 30%), Aspergillus flavipes (13 mm at 40%), Alternaria sp. (22 mm at 25%), Aspergillus ochraceus (9 mm at 40%), Microsphaeropsis arundinis (12 mm at 35%), Mucor sp. (13 mm at 30%), Aureobasidium sp. (12 mm at 30%), Penicillium citrinum (10 mm at 40%), Penicillium janthinellum (26 mm at 30%), Trichoderma sp. (9 mm at 30%), Aspergillus sp. (18 mm at 25%), Penicillium sp. (20 mm at 20%), Aspergillus nomius (15 mm at 35%), and Cladosporium sp. (7 mm at 15%) ( Figures 1-14 ). The seasonal study was also divided into rainy season (April-September) and dry season (October-March). Penicillium and Aspergillus species were predominant throughout the study period in both lakes. However, the top 7 extreme halophilic fungi—Microsphaeropsis arundinis, Aspergillus flavipes, Aspergillus ochraceus, Aspergillus nomius, Penicillium citrinum, Aspergillus sydowi and Penicillium janthinellum were characterized and identified phenotypically and genotypically; while the rest were characterized only phenotypically as seen in Table 1 and Table 2 . Twelve isolates from the fourteen total isolates were classified as extremely halophilic, whereas one was a borderline extreme halophile, and the remaining isolate was a moderate halophile ( Table 3 ). Plemenitaš and Gunde-Cimerman [43] stated that fungi isolated from hypersaline habitats of 1.7 M (~10%) salt concentration and can grow in vitro at or above 3 M (~18%) salt concentration should be considered as a halophilic fungus, and this served as the basis of the classification reported on Table 3 . All the organisms isolated in this study tolerated this salinity range, and some of them exceeded the salinity range as well, and no slight halophile was isolated in this work. Fungal salt tolerance classification findings from this work partly correspond with the work of Mansour [44] who examined sandstone samples from the Medamoud, Egypt, and halophilic fungi (Aspergillus nidulans, Aureobasidium pullulans and Cladosporium sphaerospermum) from a local culture bank. Mansour estimated salinity tolerance by growing these fungi at 0% to 25% of NaCl concentration supplemented on potato dextrose agar and broth and opined that 5% of NaCl was the best suited growth supplement for halophilic fungi; and halophiles showed better tolerance to salt as compared to the solid media. They also reported that, 25% of NaCl concentration was found to inhibit any fungal growth. In this research however, solid medium was used and salt concentrations above 15% inhibited Cladosporium sp., but Aureobasidium and other species of Aspergillus grew well above borderline extreme salt ranges of 9% - 23%, as opposed to the findings of Mansour [44] , as seen in Figures 1-14 . The fact that no slight halophilic fungus was isolated in this work is also agrees with the reports of [9] [37] [45] , who opined that microorganisms belonging to the slight halophiles are mostly marine bacteria such as Vibrio. The most halophilic fungi that was isolated in this study was Aspergillus flavipes which had a colony diameter of 13 mm at 40% salt concentration; hence it was used for further studies namely enzymatic and biodegradation studies which is not covered in the scope of this present publication. Enzyme complexes expressed by A. flavipes made it a useful biological agent in cancer research as an anti-tumor agent on the basis of its cytochalasin production [46] . This further shows the biotech and medical applications of this very important halophilic fungus. Other halophilic fungi have been reported to express enzyme complexes useful in textile manufacturing, detergent and surfactant production, bioremediation of industrial waste water contaminated with metal ions, agricultural waste water bioremediation, and also soy sauce production [47] . In contrast, halophilic archaea and bacterium are not left out in their biotechnology usefulness. Halobacterium salinarium and Haloferax volcanii have been useful in biosensor technology for environmental toxicity monitoring studies [48] . Bacillus megaterium and Halobacillus sp. have been useful as metal ion bioremediators through nanoparticle formation technology [48] . Citing these few examples, it could be said that salt lakes do not just contain lethality and toxicity only, but also contain well-adapted useful extremophiles that serve the good interest of man and his environment.

Salt lakes are abundant, widely distributed geographically, and play a vital role in inland aquatic ecosystems worldwide. Their aesthetic, cultural, economic, recreational, scientific, conservation, and ecological values make them significant natural resources. Their microbiota composition uniquely distinguishes them from other aquatic ecosystems. Salt lakes develop as the termini of inland drainage basins where hydrological inputs and outputs are balanced. These conditions occur in arid and semi-arid regions (approximately one-third of the total world land area). Many human activities threaten or have already impacted salt lakes, especially surface inflow diversions, salinization and other catchment activities, mining, pollution, biological introduction, and anthropogenically induced climatic and atmospheric changes. In spite of these facts, Uburu and Okposi salt lakes have been less studied hitherto. In fact, there is paucity of research and data on the microbiological properties of both lakes, which has culminated in the disappearance of both lakes in the annals of salt lakes of the world, as have been noticed in numerous literatures and texts on saline lakes. This study presented seasonal fungal analysis of water samples obtained from Uburu and Okposi salt lakes, with subsequent identification using ITS rDNA sequencing (Macrogen, South Korea). The results revealed representatives of moderately to extremely halophilic fungi within both lakes. No slight halophile was isolated, out of the 14 fungi isolated. These fungi have abilities to adapt to a multi-extreme environment owing to the near-neutral to moderate alkaline and high salinity conditions of both lakes, as evidenced in the halotolerance curves of the recovered isolates. Culture dependent studies in future will help us unravel the survival mechanisms used by these polyextremophilic fungi.

Our profound gratitude goes to Microbial Identification Service, Macrogen Inc., 10 F, 254 Beotkkot-ro, Geumcheon-gu, Seoul, Republic of South Korea, for their efforts in characterizing some of the isolates to the species level using the ITS rDNA sequencing analysis and a BLAST search using the GenBank sequence database.

Authors specially thank Petroleum Technology Development Fund (PTDF) for providing funding for this study as a Ph.D. scholarship.