Keywords

3.1 Introduction

Terrestrial hot springs, a type of extreme environments, existed on Earth for billions of years (Gold 1992). The physical and chemical characteristics of the hot springs make it a unique habitat which is quite different from the surrounding environments (Bisht et al. 2011). Hot springs are once perceived to be sterile environment (Chaudhuri et al. 2017), but the pioneer work of Thomas Brock’s in discovering Thermus aquaticus from thermal vents of Yellowstone National Park (Brock 1997) has completely changed our understanding of hot spring microbial diversity. Hot springs harbor unique microbial diversity that could be the source of commercially important products (Satyanarayana et al. 2005). Understanding how living communities survive and structured in hot spring conditions is important because hot springs are similar to the postulated early chemical environment on Earth (Li et al. 2015) and thus hot springs become a model ecosystems for research on the origin and evolution of life (Farmer 1998; Whitaker et al. 2003). Culture-dependent microbial analysis of hot springs reveals the presence of many new taxonomic and functional lineages (Xue et al. 2001; Huang et al. 2010). Although culture-dependent analysis is regarded as an effective method to understand the microbial diversity, these are not sufficient to explore the microbial diversity as it does not reveal a clear picture of the community diversity due to lack of cultivation of microorganisms in laboratory conditions (Kikani et al. 2017). Recent advances in culture-independent microbial diversity analysis have showed a remarkable progress in understanding community diversity. The application of culture-independent analysis has proved to be a promising tool to investigate detailed insight of hot spring microbial habitats in terms of diversity, adaptation, functions, and ecological significance (Badhai et al. 2015).

Geothermal Professional Committee of China Energy Research Society in 1986 has identified nearly 3398 hot springs distributed across China. The most concentrated regions of thermal springs are located in Yunnan and Tibet (Liao 2018). Extensive studies have been carried out in understanding microbial diversity of Yunnan and Tibet hot springs (Liu et al. 2016; Wang et al. 2014; Hou et al. 2013). In this chapter, we discuss several major hot springs of Yunnan and Tibet, their physicochemical properties, and their microbial diversity and bioactive molecules.

3.2 Terrestrial Hot Springs in Yunnan-Tibetan Geothermal Zone (YTGZ)

The YTGZ (Fig. 3.1) located between the Indian Plate and Eurasian Plate is well known for its volcanic activity and geothermal features (Wang et al. 2013). The northeastern edge of the YTGZ belongs to Himalayan Geothermal Belt (HGB) which resulted from the collision of the Indian Plate with the Eurasian Plate. HGB is more than 50 km wide and extends for 3000 km, distributed throughout India, Tibet, Yunnan, Myanmar, and Thailand, associated with at least 600 geothermal systems (Hochstein and Regenauer-Lieb 1998). The total amount of thermal springs in YTGB accounts for half of the total number of thermal springs in China with wide ranges of temperature and pH (Wang et al. 2013; Wu et al. 2015). Some of the important Yunnan and Tibet hot springs and their physicochemical parameters are listed in Table 3.1.

Fig. 3.1
figure 1

The red highlighted portion shows some of the important Yunnan and Tibet hot springs

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Table 3.1 Descriptions of some important hot springs of Yunnan and Tibet
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A total number of thermal springs in Tibet are 645, in which Nagqu Prefecture accounts for highest hot springs (187), followed by Qamdo Prefecture (126), Xigaze (117), Ngari Prefecture (88), Nyingchi Prefecture (49), Shangnan Prefecture (44), and Lhasa City (34). Tibet has 48 boiling springs (having temperature greater than 86 °C), 179 hot springs (having temperature less than 86 °C), 294 warm springs (having temperature less than 45 °C), and 127 tepid springs (having temperature less than 35 °C) (Liao 2018). Most of boiling springs found in Tibet discharge sodium chloride type of water which is boron rich. Some boiling springs discharge Cl–HCO3–Na-type water or HCO3–Cl–Na-type water due to blending of different degrees of cold water. Thermal springs in Tibet have high salinity (Liao 2018).

Yunnan Province is the largest province in terms of number of thermal springs in China with unbelievable physical and chemical features. More than 862 hot springs have been reported in Yunnan Province; among these springs, 20 are boiling springs, 314 are hot springs, 208 are warm springs, and 321 are tepid springs. All these high-temperature boiling springs have high fluorine and low boron content. In lower-temperature hot springs and warm springs, especially tepid springs, their water chemistry type is basically HCO3–Ca or HCO3–Mg type. TDS value of Yunnan springs is very low, except the Rehai geothermal field of Tengchong (Liao 2018).

3.3 Culture-Dependent Microbial Diversity Analysis

A large number of cultivation-dependent studies have been performed in Yunnan and Tibet hot springs (Hedlund et al. 2015; Xian et al. 2016; Khan et al. 2017a, b). Several new taxa (Table 3.2) and their bioactive potential have been reported from Yunnan and Tibet hot springs (Duan et al. 2014; Xian et al. 2016; Khan et al. 2017a, b; Chen et al. 2012).

Table 3.2 Novel bacterial and archaeal strains isolated from Yunnan and Tibet
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3.3.1 Physicochemical Factors Structuring Microbial Diversity

The seasonal dynamics of both the physicochemical conditions and the microbial communities inhabiting hot springs in Tengchong County, Yunnan Province, China, have been evaluated. The seasonal variation, especially the rainy season changed the physicochemical conditions and microbial communities. Monsoon samples showed increased concentrations of potassium, total organic carbon, ammonium, calcium, sodium, and total nitrogen with decreased ferrous iron relative to the dry season. High mesophilic community has been observed after monsoon which may be flushed into springs due to enhanced rain influx (Briggs et al. 2014).

Wang and co-workers have evaluated temporal changes of sediment and water microbial community in Tengchong hot springs, Yunnan Province, China. The authors suggest that the microbial communities were not transported into hot springs from the surroundings by increased surface runoff, but rather their occurrence or even dominance was due to large temporal variations of physicochemical conditions such as pH, temperature, and dissolved organic carbon. Water and sediment communities responded differently to temporal physicochemical changes. Water communities were found stable, while sediment communities were more responsive to temporal geochemical changes. Greater temporal variations were observed in individual taxa than at the whole community structure level (Wang et al. 2014).

3.3.2 Proteobacteria, Firmicutes, and Aquificae

Culture-dependent analysis showed that Proteobacteria, Firmicutes, and Aquificae were the prominent groups residing in the hot springs in India (Kumar et al. 2004; Sen and Maiti 2014; Pathak and Rathod 2014). Culture-dependent analysis of the samples from Yunnan and Tibetan hot springs also showed the presence of Proteobacteria, Firmicutes, and Aquificae groups.

Several novel genera such as Caldalkalibacillus (Caldalkalibacillus thermarum as type species) (Xue et al. 2015), Crenobacter (Crenobacter luteus as type species) (Dong et al. 2015), Crenalkalicoccus (Crenalkalicoccus roseus as type species) (Ming et al. 2016), Caldovatus (Caldovatus sediminis as type species) (Habib et al. 2017a), and Tibeticola (Tibeticola sediminis as type species) (Khan et al. 2017b) have been reported from Yunnan and Tibet hot springs. Genera, namely, Caldalkalibacillus, Crenobacter, Crenalkalicoccus, and Caldovatus have been reported from Tengchong (Yunnan province) hot springs, while Tibeticola has been reported from Tibet hot spring. The optimum temperature for growth of these novel genera varies; Caldalkalibacillus thermarum and Crenalkalicoccus roseus have been reported for optimum growth at 60 °C. Crenobacter luteus had optimum growth at 40–50 °C; Caldovatus sediminis and Tibeticola sediminis had optimum growth at 45 °C. Crenobacter luteus, Crenalkalicoccus roseus, Caldovatus sediminis, and Tibeticola sediminis were reported for their growth from slightly acidic to alkaline pH with optimum pH at 8.0–9.0, 8.0, 6.5–7.0, and 7.0, respectively (Xue et al. 2015; Dong et al. 2015; Ming et al. 2016; Habib et al. 2017a, b). Further, several novel aerobic species, namely, Laceyella sediminis (Chen et al. 2012), Brevibacillus sediminis (Xian et al. 2016), Altererythrobacter lauratis, and Altererythrobacter palmitatis (Yuan et al. 2017), and obligate anaerobes such as Thermoanaerobacter tengcongensis (Xue et al. 2001), and Thermoanaerobacterium calidifontis (Shang et al. 2013) have also been reported. In-depth analysis of novel species, Thermoanaerobacterium calidifontis, showed the ability to produce ethanol and ability to convert thiosulfate to elemental sulfur and reduce sulfite to hydrogen sulfide (Shang et al. 2013). Thermoanaerobacter tengcongensis reported to reduce thiosulfate and sulfur to hydrogen sulfide (Xue et al. 2001). Further, complete sequence of Thermoanaerobacter tengcongensis has been carried out which suggests Thermoanaerobacter tengcongensis has a genome size of 2,689,445 bp. The genome encodes 2588 predicted coding sequences. Among them, 1764 (68.2%) were similar to documented proteins, and the rest, 824 coding sequences (31.8%), were functionally unknown (Bao et al. 2002).

Many culture-independent analyses of microbial diversity in hot springs of Tengchong, Yunnan Province, were carried out suggesting Aquificales populations as the abundant group (Hou et al. 2013; Song et al. 2013a), but attempts to isolate Aquificales from Tengchong hot springs have never been made. Hedlund and his co-workers made an attempt to isolate diverse members of the Aquificales from geothermal springs in Tengchong, China. The authors have isolated five strains of Aquificales from diverse springs (temperature 45.2–83.3 °C and pH 2.6–9.1). Phylogenetic analysis showed that four of the strains belong to the genera Hydrogenobacter, Hydrogenobaculum, and Sulfurihydrogenibium, including some strains distinct enough to likely justify as new species of Hydrogenobacter and Hydrogenobaculum. They also suggested that one strain was distinct enough to represent as new genus in the Hydrogenothermaceae family. All strains were capable of aerobic respiration under microaerophilic conditions; however, they had variable capacity for chemolithotrophic oxidation of hydrogen and sulfur compounds and nitrate reduction (Hedlund et al. 2015).

Efforts to isolate acidophilic bacteria from Yunnan Province hot springs have been made. It was noticed that acidophilic mesophiles in these regions were more diverse, and several ferrous iron and sulfur-oxidizing genera such as Acidiphilum (Liu et al. 2007), Acidothiobacillus, Alicyclobacillus, Leptospirillum, and Sulfobacillus (Jiang et al. 2009) were present.

3.3.3 Deinococcus-Thermus

The genus Thermus has been regarded as models to investigate the mechanism of thermostability of thermophiles (Saiki et al. 1972). The diversity of Thermus has been evaluated in 15 hot springs of Rehai geothermal area, Tengchong, China. The isolation was carried out using Thermus and YIM14 medium. A total of 57 Thermus strains have been recovered. Strains from YIM14 medium were physiologically more diverse than strains from Thermus medium (Guo et al. 2003).

Several novel species, namely, Thermus rehai (Lin et al. 2002), Thermus caliditerrae (Ming et al. 2014), Thermus amyloliquefaciens (Yu et al. 2015), and Thermus caldifontis (Khan et al. 2017a), have been reported from Yunnan and Tibetan hot springs. All the above-described species reported for the growth till 70 °C (Lin et al. 2002; Ming et al. 2014; Yu et al. 2015; Khan et al. 2017a).

Thermus spp. isolated from Yunnan and Tibetan hot springs have been reported as a potential source for bioactive molecules. The novel species Thermus amyloliquefaciens was reported for its ability to liquify starch (Yu et al. 2015).

Gong and co-worker evaluated the diversity of thermostable alkaline phosphatase-producing bacteria in Tengchong (Yunnan province) hot springs. Sixty strains belonging to three genera have been isolated. Among them, one strain designated RHY12-2 had highest phosphatase activity. The 16S rRNA gene sequence of the strain RHY12-2 showed the strain was a member of the genus Thermus (highest similarity with Thermus scotoductus 96.3%) and probably new species. The enzyme had a single peptide with a molecular mass of about 52 kDa with highly specific activity and thermal resistance. The optimum enzyme activity is observed at pH 8.0–10.0 and temperature 70–80 °C (Gong et al. 2005). Thermus play a significant role in the cesium assembly. The bacterium Thermus sp. TibetanG7, isolated from hot springs in Tibet, China, has been examined for the ability to accumulate cesium from solutions. The accumulation of cesium by this microorganism was rapid with 40%–50% accumulation within the first 5 min (Wang et al. 2007).

The genus Meiothermus which was reclassified from the genus Thermus (Nobre et al. 1996) is the most common genus isolated from hot spring (Chan et al. 2015). Tengchong hot springs (Yunnan, China) served as potential source for harboring novel species of the genus Meiothermus. Several novel species such as Meiothermus rosaceus (Chen et al. 2002), Meiothermus roseus (Ming et al. 2015), and Meiothermus luteus (Habib et al. 2017b) have been reported.

3.3.4 Actinobacteria

Actinobacteria have been widely concerned due to their ability of producing various kinds of antibiotics and bioactive molecules (Liu et al. 2016). Actinobacteria residing in hot springs exhibits unique metabolic activity. (Xu et al. 1998). Recently, our group has evaluated actinobacterial diversity in ten hot springs distributed over three geothermal fields, namely, Hehua, Rehai, and Ruidian. A total of 58 thermophilic actinobacterial strains have been isolated, and the 16S rRNA gene sequence result showed that these strains shared high similarities with actinobacterial genera, namely, Actinomadura, Micromonospora, Microbispora, Micrococcus, Nocardiopsis, Nonomuraea, Promicromonospora, Pseudonocardia, Streptomyces, and Verrucosispora. Some of the strains had low sequence similarity which suggested that these hot springs harbor many novel strains. The isolated strains showed good antimicrobial activity. Fifty-three strains exhibited antimicrobial activities against Acinetobacter baumannii. Eighteen and three strains showed inhibitory activities against Micrococcus luteus and Staphylococcus aureus, respectively. Further, 22 strains were positive for PCR amplification of at least 1 of the 3 biosynthetic gene clusters (PKS-I, PKS-II, and NRPS) (Liu et al. 2016).

Many novel strains have been reported from Yunnan province. During our investigation on thermophilic actinobacterial diversity from hot springs, strain YIM 78087 has been isolated from a sediment sample collected from the Hehua hot spring in Yunnan Province, southwest China. The strain can grow up to 50 °C temperature and tolerate NaCl up to 9% (w/v). The strain was reported for its growth in acidic as well as basic conditions (pH 4.0–10.0). The 16S rRNA gene sequence result showed that strain YIM 78087 belonged to the genus Streptomyces and was closely related to Streptomyces fimbriatus DSM 40942 (97.18%), Streptomyces marinus DSM 41968 (97.05%), and Streptomyces qinglanensis DSM 42035 (97.1%). When the taxonomic position of strain YIM 78087 was evaluated, it represented as a novel species of genus Streptomyces, for which the name Streptomyces calidiresistens has been proposed (Duan et al. 2014). Similarly, strain Y-14046 isolated from a hot spring in Eryuan, Yunnan, China, has been described as new species (Streptomyces thermogriseus) of the genus Streptomyces (Xu et al. 1998).

3.3.5 Thermophilic Archaea

Compared with bacteria, studies on cultivation of archaea in Yunnan and Tibetan hot springs were rather limited. Xiang and co-workers isolated and characterized novel species Sulfolobus tengchongensis RT8-4 from acidic hot spring located in Tengchong, Yunnan Province, China (Xiang et al. 2003). Sulfolobus tengchongensis RT8-4 had long and curved peritrichous flagella with aerobic growth either a lithotrophic or heterotrophic mode. It grew fastest at 85–90 °C and was capable of slow growth at 95 °C. Growth has been observed at various pH values ranging from 1.7 to 6.5 with optimum growth at pH 3.5 (Xiang et al. 2003).

A facultatively aerobic novel species, Acidianus tengchongensis, has been isolated from a Tengchong acidothermal spring. The optimal pH and temperature for growth reported were 2.5 and 70 °C, respectively. Acidianus tengchongensis cells were non-motile, and under anaerobic conditions Acidianus tengchongensis reduces elemental sulfur with molecular hydrogen, producing hydrogen sulfide. Under aerobic conditions, it oxidizes elemental sulfur and produces sulfuric acid. No growth reported when cultivated in iron medium, indicating that ferrous iron not be used as an energy source (He et al. 2004).

The diversity of Sulfolobus in acidic hot springs of Tengchong of Yunnan, China, has been evaluated by Jian and co-workers. Eleven thermoacidophilic strains from six acidic hot springs have been isolated. The 16S rRNA gene sequence result showed that these strains belong to the genus Sulfolobus but are distinct enough to designate as new species (Jian et al. 2010).

Many studies have been carried out to use acidothermophilic archaeal species isolated Yunnan hot springs for bioleaching. Zou and co-workers have isolated thermoacidophilic archaea (Sulfolobus acidocaldarius) from hot sulfur spring in the Yunnan Province and conducted bioleaching activity in both laboratory batch bioreactors and leaching columns on low-grade chalcopyrite ore. They reported that bioreactor experiments showed 97% rate of copper bioleaching (in 12 days). In the case of column leaching, tests of a two-phase leaching have been conducted. In the first phase, Thiobacillus ferrooxidans has been used, followed by a 140-day thermoacidophilic archaeal leaching in the second phase. The average leaching rate of copper achieved by thermoacidophilic archaea has found to be 195 mg/(L·d), while for the control experiments (for the Thiobacillus ferrooxidans), it was 78 mg/(L·d), indicating thermoacidophilic archaea possesses a more powerful oxidizing ability than Thiobacillus ferrooxidans (Zou et al. 2006). Two novel acidothermophilic archaeal species Metallosphaera tengchongensis (Peng et al. 2015) and Metallosphaera cuprina (Liu et al. 2011a) reported for oxidizing metal sulfide ores, showing their potential in bioleaching. These two species were isolated from muddy water samples collected at the edge of the hot springs of Tengchong, Yunnan Province, China. Metallosphaera tengchongensis and Metallosphaera cuprina were aerobic and facultatively chemolithoautotrophic with growth at temperature ranging from 55 to 75 °C (Peng et al. 2015; Liu et al. 2011a). For better understanding of bioleaching potential, complete genome sequence of Metallosphaera cuprina has been carried out. The genome in total carried 2029 open reading frames (ORFs). Genome annotation and metabolic reconstruction supported the idea that Metallosphaera cuprina lived a facultative life. Metallosphaera cuprina strain fixed CO2 via the 3-hydroxypropionate/4-hydroxybutyrate cycle, and this strain assimilated carbohydrates via the nonphosphorylated Entner-Doudoroff pathway. It had a complete tricarboxylic acid (TCA) cycle and an incomplete phosphate pentose pathway. Oxidation of RISCs by the heterodisulfide reductase complex, sulfide/quinone oxidoreductase, thiosulfate/quinone oxidoreductase, tetrathionate hydrolase, and sulfite/acceptor oxidoreductase in Metallosphaera cuprina has been proposed (Liu et al. 2011b).

3.3.6 Thermophilic Virus

Viruses are the most abundant biological entities in every ecosystem, even in hot springs (Lopez-Lopez et al. 2013). They are probably the only predators in these communities and may be involved in the control of host mortality (Lopez-Lopez et al. 2013). In this section, we provided insights into the latest study being carried out to understand viruses in Yunnan and Tibetan hot springs.

Thermus bacteriophage named TSP4 has been isolated from Tengchong hot springs, China. TSP4 belonged to the Siphoviridae family and had a hexagonal head of 73 nm in diameter, an extremely long and flexible tail of 785 nm in length and 10 nm in width (Lin et al. 2010). The first reported Meiothermus phage, MMP17 (Meiothermus Myoviridae phage 17), has been isolated from hot spring in Eryuan County, Yunnan. MMP17 was a typical myovirus with an icosahedral head (42 nm in diameter) and a tail of 120 nm in length and 17 nm in width. Its DNA was about 33.5–39.5 kb in size. MMP17 was very stable at 55–60 °C and pH 6–7. An average of 15 phages were released from each infected cell (Lin et al. 2011). A virus, denoted STSV1 infecting the hyperthermophilic archaeon Sulfolobus tengchongensis, has been isolated from acidic hot springs located in Tengchong, China. The virus STSV1 was spindle (230 by 107 nm) with a tail of variable length (68 nm on average) at one end. STSV1 shape was similar to the members of the family Fuselloviridae but much larger than known fuselloviruses. After infecting its host, STSV1 multiplied rapidly to high titers (>1010 PFU/ml). Replication of the virus retards host growth, but does not lyse host cells. STSV1 do not integrate into the host chromosome and existed in a carrier state. The STSV1 DNA modifies in an unusual fashion, presumably by virally encoded modification systems. STSV1 harbors a double-stranded DNA genome of 75,294 bp, which shares no significant sequence similarity to those of fuselloviruses. The viral genome contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs that encode viral structural proteins, including a putative coat protein of high abundance, have been noticed. The products of the other nine ORFs have been mentioned to be involved in polysaccharide biosynthesis, nucleotide metabolism, and DNA modification (Xiang et al. 2005).

3.4 Culture-Independent Microbial Diversity Analysis

Culture-dependent method revealed immense limitation for addressing microbial diversities. Majority of microbes in various environments, including hot springs, are still not isolated using traditional cultivation methods (Streit and Schmitz 2004). Hence, culture-dependent microbial analysis does not give clear idea about the microbial diversity residing in a particular environment. In the past few years, the application of culture-independent analysis has proved to be a promising tool to investigate the population diversity, gene content, function, and ecological significance of microbial communities living in diverse hot spring environments (Hou et al. 2013; Badhai et al. 2015). Several culture-independent analyses have been carried out in Yunnan and Tibet hot springs (Song et al. 2009, 2010, 2013a, b; Hou et al. 2013).

A comprehensive cultivation-independent census of microbial communities in 37 samples collected from Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, has been evaluated using 16S rRNA gene pyrosequencing to understand microbial diversity. The temperature and pH of the samples sites ranged from 55.1 to 93.6  °C and 2.5 to 9.4, respectively. Richness found low in all samples, with 21–123 species-level operational taxonomic units (OTUs). The bacterial phylum Aquificae and archaeal phylum Crenarchaeota dominated in Rehai samples, yet the dominant taxa within these phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5–2.6), high temperature (85.1–89.1  °C), and high sulfur contents favored Sulfolobales, whereas lower temperature (55.1–64.5  °C) with low pH (2.6–4.8) favored the Aquificae (genus Hydrogenobaculum). Rehai springs with neutral-alkaline pH (7.2–9.4) and high temperature (80 °C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae (genus Hydrogenobacter). Ruidian water samples harbored a single Aquificae (genus Hydrogenobacter), whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with high abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii,” and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy (Hou et al. 2013).

Diversity of Crenarchaeota has been investigated in eight terrestrial hot springs (pH 2.8–7.7; temperature 44–96 °C) located in Tengchong, China, using 16S rRNA gene phylogenetic analysis. A total of 826 crenarchaeotal clones were sequenced, and a total of 47 OTUs were identified. About 93% of the OTUs were identical to those retrieved from hot springs and other thermal environments. The result suggests that temperature predominates over pH in affecting crenarchaeotal diversity in Tengchong hot springs. Crenarchaeotal diversity in moderate-temperature (59–77 °C) hot springs was the highest, indicating that the moderately hot-temperature springs may provide optimal conditions for speciation of Crenarchaeota (Song et al. 2010).

Investigation on the community diversity and composition in Yunnan and Tibetan hot springs using a barcoded 16S rRNA gene-pyrosequencing approach has been carried out. 16 hot spring samples from five thermal fields, namely, Tengchong, Longling, and Eryuan in Yunnan Province and Gulu and Qucai in Tibet, have been collected. Hot spring samples had a range of temperature (47–96 °C) and pH (3.2–8.6) conditions. Proteobacteria, Aquificae, Firmicutes, Deinococcus-Thermus, and Bacteroidetes comprised the large portion of the bacterial communities in acidic hot springs (in Yunnan). Nonacidic hot springs (both Yunnan and Tibet) harbor more and variable bacterial phyla than acidic springs; the major phyla of Tibetan hot springs were similar to the Yunnan nonacidic samples but showed different relative abundances. For example, Bacteroidetes in Tibetan nonacidic hot springs shows higher abundance than Yunnan. Desulfurococcales and unclassified Crenarchaeota were the dominated groups in archaeal populations from most of the nonacidic hot springs, whereas the archaeal community structure in acidic hot springs was simpler and dominated by Sulfolobales and Thermoplasmata. The phylogenetic analyses showed that Aquificae and Crenarchaeota were predominant in the investigated springs and possessed many phylogenetic lineages that have never been detected in other hot springs in the world (Song et al. 2013a).

Culture-independent approach that combines CARD-FISH, qPCR, and 16S rRNA gene clone library has been carried out to investigate the abundance, community structure, and diversity of microbes along a steep thermal gradient in the Tengchong geothermal field named Shuirebaozhaqu. The authors observed a remarkable change in bacterial and archaeal abundance with temperature changes. Under low-temperature conditions (52.3–74.6 °C), the microbial community that dominated was bacteria. The community was dominated by five phyla, namely, Proteobacteria, Firmicutes, Nitrospirae, Thermotogae, and Cyanobacteria. The greatest diversity was observed in the phylum Proteobacteria, with 11 genera belonging to the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Archaea dominant at 74.6 °C and 90.8 °C, the number of cells was lowest, but the archaea accounted more than 90% of the total number of cells. Additionally, the microbial communities at high temperatures (74.6–90.8 °C) were substantially simpler than those at the low-temperature sites. Only a few bacterial genera, namely, Caldisericum, Thermotoga, and Thermoanaerobacter, and archaeal genera Vulcanisaeta and Hyperthermus dominated at high temperature. Some bacteria were observed at both low temperature and high temperature but with different abundance. Genera such as Hippea, Syntrophus, and Geobacter were more adapted to hyperthermal environments, whereas genera such as Methylobacterium, Novosphingobium, Achromobacter, Desulfomonile, Rubrivivax, Haemophilus, Sorangium, and Thauera were only detected at low temperatures (Li et al. 2015).

Microbial community composition and diversity in hot springs of the Tibetan Plateau across a wide range of temperatures have been evaluated. Thirteen hot spring samples from Nima, Gulu, Naqu, Guozu, and Qucai in Naqu County have been collected and evaluated its microbial diversity using the 16S rRNA gene-pyrosequencing approach. The temperature of these springs ranged from 22.1 to 75 °C. The results suggested that bacteria (42 bacterial phyla) in Tibetan hot springs were more abundant and far more diverse than archaea (5 archaeal phyla). The dominant bacterial phyla systematically varied with temperature. Moderate temperatures (75–66 °C) favored Aquificae, whereas low temperatures (60–22.1 °C) favored Deinococcus-Thermus, Cyanobacteria, and Chloroflexi. The relative abundance of Aquificae was correlated positively with temperature, but the abundances of Deinococcus-Thermus, Cyanobacteria, and Chloroflexi were negatively correlated with temperature. Cyanobacteria and Chloroflexi were abundant in Tibetan hot springs, and their abundances were positively correlated at low temperatures (55–43 °C) but negatively correlated at moderate temperatures (75–55 °C). Most archaeal sequences were related to Crenarchaeota with only a few related to Euryarchaeota and Thaumarchaeota (Wang et al. 2013).

The first culture-independent report specifically to actinobacterial diversity in three hot springs located in Tengchong (Frog Mouth hot spring) in China, Kamchatka (Robb Flag hot spring) in Russia, and Nevada boiling spring in the USA has been carried out using denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP), and actinobacterial 16S rRNA gene phylogenetic analysis. The authors noticed very diverse actinobacterial populations, and most of the retrieved actinobacterial 16S rRNA gene sequences were affiliated with uncultured Actinobacteria. The actinobacterial clone sequences retrieved were affiliated to Actinomycetales, Rubrobacterales, uncultured Candidatus Microthrix, and unclassified Actinobacteria. The actinobacterial diversity was noticed at high temperature. Unexpected high actinobacterial diversity was observed in Tengchong hot spring where temperature was 81 °C suggesting these Actinobacteria might have an extraordinary capability to adapt to hot spring environments. In this study, authors for the first time were able to retrieve sequences affiliated to Frankineae and uncultured Candidatus Microthrix in hot spring with temperature as high as 81 °C (Song et al. 2009).

During the global metagenomic survey in geothermal springs, our group found a new bacterial candidate phylum, Candidatus Kryptonia with two genera Candidatus Chrysopegis kryptomonas and Candidatus Kryptobacter tengchongensis from Yunnan hot springs. This lineage had remained hidden as a taxonomic blind spot because of mismatches in the primers commonly used for ribosomal gene surveys. The discovery of a new candidate phylum from the Yunnan hot springs emphasizes that extraordinary microbial novelty is still waiting for the discovery (Eloe-Fadrosh et al. 2016).

3.5 Function Genes and Ecology

Microbial diversity in the hot springs plays a major role in controlling the cycling of organic and inorganic compounds, thereby directly affecting characteristics of environments (Huang et al. 2010; Wu et al. 2015). Various studies have been carried out to understand microbial diversity controlling the cycling of organic and inorganic compounds in Yunnan and Tibetan hot springs (Wu et al. 2015; Song et al. 2013b).

3.5.1 Ammonia-Oxidizing Microorganisms

Ammonium is considered as the major source of inorganic nitrogen in most geothermal springs (Zhang et al. 2008). The abundance of ammonia-oxidizing microorganisms (AOM) and effect of environmental variables in 13 hot springs located in Yunnan Province, China, have been studied. Ammonia-oxidizing archaeal (AOA) abundance ranged 0.02–1.32%, whereas no ammonia-oxidizing bacteria were detected. AOA abundance was significantly correlated with concentrations of NH3, NO2−, NO3−, pH, and temperature, but not related to salinity and concentrations of Fe2+ and salinity (Huang et al. 2010). Studies in Tengchong hot springs showed that the amoA gene of aerobic ammonia-oxidizing archaea can be transcribed at temperatures higher than 74 °C and up to 94 °C, suggesting that archaeal nitrification can potentially occur at near boiling temperatures (Jiang et al. 2010).

3.5.2 Archaeal accA Gene Genes

Archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway (Berg et al. 2007; Song et al. 2013b). The abundance and diversity of archaeal accA gene in Yunnan hot springs have been studied using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66–96 °C) and pH (4.3–9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate (Song et al. 2013b).

3.5.3 Arsenite-Oxidizing Microorganisms

Arsenic is widely distributed in nature and can exist in four oxidation states, As(III), As(0), As(III), and As(V). Arsenic oxyanions could be used for energy generation of prokaryotes, either by oxidizing arsenite or by respiring arsenate (Oremland and Stolz 2003). There are certain microbes like arsenite-oxidizing microorganisms containing arsenite oxidase that catalyzes the transformation of arsenite [As(III)] to arsenate [As(V)] (Lett et al. 2012). aioA gene is a molecular biomarker for studying the distribution and activity of arsenite-oxidizing bacteria in various environments.

The abundance and diversity of arsenite-oxidizing bacteria in the geothermal features of Tengchong County of Yunnan Province, Dachaidan County of Qinghai Province, and Tibet have been investigated. The results showed that the aioA gene abundance increased as temperature decreased, whereas its diversity at the OTU level (97% cutoff) increased with increase in temperature. This suggests that temperature played an important role in affecting aioA gene distribution and thus arsenic speciation. The aioA gene population (at OTU level) differed among the studied regions, indicating geographic isolation may be an important factor controlling aioA gene distribution in hot springs (Wu et al. 2015).

3.6 Conclusion and Future Perspectives

The investigations have demonstrated that huge diverse and novel thermophilic bacterial and archaeal communities with bioactive potentials thrive in Yunnan and Tibet hot springs. Most of the microbial communities are still unclassified or unknown, which awaits further exploration. Environmental factors play an important role in structuring microbial communities, and hence these factors should be considered in future analysis. Only few reports have been described in studying thermophilic virus in Yunnan and Tibet hot springs. Hence, attempts to identify the distribution pattern and host-virus interaction in these hot springs have to be conducted.