The five hot springs investigated in the present study were highly diverse in their environmental attributes, encompassing a temperature range from 49.0 to 100 °C and salinity from 3 to 32 as well as wide ranges of water and sediment geochemistry. Maiwooi and Akwar have low salinity levels and are located at the main eastern escarpment in Gahtelai along the Asmara-Massawa highway with temperatures ranging from 50 °C to 70 °C. These low energy hot springs discharge near-neutral bicarbonate waters . Gelti and Garbanabra are located on the seashore of the Red Sea near the Gulf of Zula. The salinity (3.2 and 3.1%, respectively) is much higher than Maiwooi and Akwar (0.4 and 0.7%, respectively) due to mixing with the sea water. Elegedi is located about 30 km away from the seashore of the Gulf of Zula and is associated with a high temperature geothermal system underlying the Alid volcanic centre in the Northern Danakil depression of Eritrea . The bubbling water discharged from this hot spring is typical of the fumarolic steam condensate with high temperatures.
The majority of the sequences (95.1%) were observed to belong to the kingdom bacteria. These results suggest that bacteria are the most dominant taxa in all five hot springs. The phylotypes Proteobacteria, Firmicutes, Deinococcus-Thermus, Planctomycetes, Bacteroidetes, Chloroflexi and Aquificea have also been reported to inhabit hot springs in India [15, 16, 17], Yunnan and Tibet in China , Siloam in South Africa , and Malaysian hot spring . The abundance of Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Cyanobacteria and Chloroflexi was also reported in the hypersaline hot springs of Lake Magadi and Little Magadi in Kenya .
The phylum Proteobacteria was the most abundant phylum observed in all the five hot springs. Proteobacteria has also been reported from many studies based on the 16S rRNA analysis of hot springs with moderately high and very high temperatures (44–110 °C) at various geographical locations [15, 17, 20, 21, 24, 36, 37, 38]. The relative abundance of α-Proteobacteria and γ-Proteobacteria from the water samples was higher in the saline hot springs compared to β-Proteobacteria. Previous investigation in the Baltic Sea revealed transitions in bacterial communities along the 2000 km salinity gradient . The relative abundance of α-Proteobacteria and γ-Proteobacteria increased with salinity, whereas Actinobacteria and β-Proteobacteria displayed the opposite trend. The phylum Firmicutes was the second most abundant phylum observed. It was abundant in the microbial mat samples compared to wet sediment and water samples. This could imply that the Firmicutes sequences retrieved in the present study probably represent bacteria embedded in the microbial mat sample. Recently, high relative abundance of Firmicutes (23%) were observed from microbial mat samples at 45 °C .
Sequence affiliated with Euryarchaeota, Crenoachaeota and Thaumarchaeota were detected from the archaeal kingdom. The most abundant archaeal group Euryarchaeota, particularly the genus Methermicoccus, a thermophilic and methylotrophic methanogen , was predominantly recovered from the wet sediment sample in Akwar. Within the order Thermoproteales, the terrestrial genera Thermofilumwas recovered only from the wet sediment sample of Akwar. Thermofilum was observed in YNP springs with mildly acidic pH (5.5–6.1) and moderate to high temperature, which is consistent with the physiology of this genus . Acidianus, was the only genus observed from the order Sulfolobales. Acidianus, unlike the obligate aerobic Sulfolobus, is facultative anaerobic and can utilize sulfur either as an electron donor (in aerobic metabolism) or electron acceptor (in anaerobic metabolism). Garbanabra and Gelti, situated at the sea shore, where high levels of salinity was registered, harbored the genus Halobacterium (“salt” or “ocean bacterium”). This was confirmed by Mantel test, where the relative abundance of Halobacterium increased with increased salinity levels (r = 0.75, p = 0.02). The abundance of Halobacterium in these two hot springs was observed from the wet sediment samples.
At genus level, the microbial community composition clustered according to the three locations. In Location 1, euryarchaeal genus Methanomicrobium as well as bacterial genera Proteiniphilium from the phylum Bacteroidetes and Proteiniclasticum from Firmicutes were dominant. Location 2 were mainly associated with unclassified Thaumarchaeota, Actinobacteria and Cyanobacteria indicating possible occurrence of novel microorganisms in these springs. The locations of these two saline springs to the Gulf of Zula, suggest that a marine influence may be a possible reason for the occurrence of novel microorganisms in these springs. Similar observation was noted in hot springs in Philippines . They observed large sequence reads in the two saline hot springs located close to Albay Gulf which were mainly associated with unclassified Archaea (3–19%), Crenarchaeota (63–82%), and Bacteria (25–58%), indicating possible occurrence of novel microorganisms in these springs.
The boiling hot spring in Location 3 displayed a diverse thermophilic population. Two genera of Aquificae, Hydrogenobacter and Thermocrinis were detected. Hydrogenobacter was the most dominant genus. Likewise, In Tengchong springs, Hydrogenobacter was the dominant genus from Aquificae in high temperature (73.8–93.6 °C) and circumneutral to alkaline pH (6.7–9.4) springs . Thermocrinis were recovered at much lower abundance than Hydrogenobacter from Elegedi. The genus Thermocrinis was commonly found in other hot springs worldwide with near-neutral pH, high temperature (75–92 °C), and low sulfide concentrations (usually, 1 mg/L) [42, 43, 44]. The high sulphate concentration (949 mg/L) at Elegedi might have been the reason for the low numbers of Thermocrinis. Meiothermus, the dominant genera from Deinococcus-Thermus, and Thermus were also detected in relatively high abundances from the boiling hot spring. Meiothermus was the dominant genus (19.5%) in the mat samples of Elegedi. Genera Thermus and Meiothermus have been detected in hot springs with moderate-high temperatures of 50–99 °C and slightly acidic to alkaline pH, such as in Iceland , Kamchatka in Russia , Long Valley Caldera , the Great Basin of the United States , and Thailand . However, Thermus and Meiothermus are usually not dominant in terrestrial hot springs except for the hot springs of Tibetan plateau  and a few springs in Iceland . The predominance of Meiothermus over Thermus in the boiling hot spring is surprising, because most species of Thermus have been shown to grow anaerobically in the presence of nitrate with an optimal growth temperature of 65–75 °C, whereas Meiothermus spp. have a lower optimal growth temperature 50–65 °C and exhibit O2 respiration . The genus Chlorogloeopsis were also recovered predominantly from the mat and wet sediment samples of the boiling hot spring. The upper temperature limit for Chlorogloeopsis was previously determined to be between 60 and 65 °C , while the water temperature of the boiling hot spring during sampling was 100 °C. Further investigation may be necessary to explain their occurrence.
The archaeal community composition in Location 3 contained Ignisphaera, Acidianus and Pyrobaculum. The abundance of Desulfurococcales was much higher than Thermoproteales and Sulfolobales. The predominance of Desulfurococcales in the boiling hot spring with high temperature (100 °C) and near-neutral pH (7.19) is consistent with the known hyperthermophily of these organisms . Similar to Desulfurococcales, Thermoproteales, mainly Pyrobaculum relatives were the most abundant in Elegedi with high temperature and near-neutral pH. The high temperature of Elegedi hot spring is consistent with the hyperthermophilic nature of the genus Pyrobaculum (74–102 °C) with optimum 100 °C .
Surprisingly, sequences affiliated with Thermomonas, member of the γ-proteobacteria, as well as Nitrosomonas, from β-proteobacteria, were recovered from the water sample of Elegedi. Strains affiliated to the genus Thermomonas has been described previously as ferrous iron-oxidizing and nitrate-reducing bacteria . Thermomonas has been described to grow between 37 and 50 °C  and moderately thermophilic Nitrosomonas at 50 °C  hence their detection in the boiling hot springs is surprising. Earlier, a culture of thermophilic nitrifying bacteria belonging to the genus Nitrosomonas, isolated from geothermal springs of Kamchatka (50–86 °C, pH 6.3–7.5) and Tajig (50–85 °C, pH 7.3–8.5), was shown to oxidize ammonium nitrogen at 40–70 °C . Some previous studies have also reported other mesophilic bacterial species in high-temperature hot spring in the Philippines , where sequences affiliated to Sulfobacillus predominated in high-temperature spring BAL-0 (90.8 °C) and its occurrence was confirmed by the full-length bacterial 16S rRNA gene. The genus Acinetobacter, reported to have an optimum growth temperature between 33 and 35 °C , was recovered predominantly (92.1%) from extremely high temperature Tattapani hot spring (98 °C) in Central India . Recently, Alishewanella, a genus from γ-proteobacteria, was also detected predominantly (4.1%) from a high temperature (95 °C) Soldhar hot spring in India using high throughput sequencing . This might suggest the possible higher temperature tolerance of some bacteria known to be mesophiles. In the natural environment, biofilms enable bacterial consortium tolerate extreme temperatures and other environmental factors. Further investigations on the presence of high temperature tolerant Nitrosomonas, detected in boiling hot spring with high ammonium concentrations, are necessary to rule out dead or inactive cells carried in from the fringes of the boiling hot spring.
The NMDS analysis of sample types showed an overlap in microbial communities between water and wet sediment samples. While, microbial mat samples formed a separate cluster. Even though, there was notable overlapping between the wet sediment and water samples, the microbial community secured from water samples were commonly found elsewhere in the environment, whereas an appreciable part of the microbial community from the wet sediment samples remained unidentified and therefore might be environmentally unique. Of the 16S rRNA filtered reads recovered from the wet sediment samples, 4.5% could not be classified into any phylum under the Bacteria domain while only 1.2% from the water samples could not be classified. A similar observation was made in a study conducted in China which compared planktonic and sedimentary bacteria diversity of the Lake Sayram during summer . In contrast to the planktonic bacteria, they detected an appreciable portion of the sedimentary bacteria that could not be classified into any known taxonomic unit.