北臺灣酸性熱泉中微生物群集的總體基因體分析

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୯ҥᆵ᡼εᏢғڮࣽᏢଣ୏ނᏢࣴز܌

ᅺγፕЎ

Institute of Zoology, College of Life Science

National Taiwan University Master Thesis

чᆵ᡼ለ܄዗ࢨύ༾ғނဂ໣ޑᕴᡏ୷Ӣᡏϩ݋

!

Metagenomic Analysis of Microbial Communities from an Acidic Hot Spring in Northern Taiwan

֖݅ࠪ

Kuei-Han Lin

ࡰᏤ௲௤ǺΪֻᔾ റγ Advisor: Hon-Tsen Yu, Ph.D.

ύ๮҇୯ 102 ԃ 1 Д Jan, 2013

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ᇞ ᇞᖴ!

ܭԜࣁයΟԃъޑਔ໔ǴҁጇፕЎளаֹԋѸ໪ᘜфܭ೚ӭΓǶவӦౚࣽᏢၠຫ ډྕࢨ༾ғނᕴᡏ୷ӢᡏǴྍܭਜൔ૸ፕਔ݅ҥहԴৣ܌໒௴ޑߐǴᅟࡕΞӢጔ ሞ཮ӦуΕ୏ނ܌ΪֻᔾԴৣޑჴᡍ࠻ǶΪԴৣࢂ᏾ঁၸำύനख़ाޑࡰЇޣа ϷЍ࡭ޣǴдаيբ߾ǵόᒪᎩΚӦᏤ҅ᆶ௲ᇧǴаϷӧځдӚБय़ޑڐշᆶᡏ ፊǴᏢғుϪགۺǶགᖴӦ፦ࣽᏢسླྀᔾ൏Դৣჴᡍ࠻ഋΏᅼǵྕЈ܃ᆶഋՆ಼

ӧྕࢨ௖୎΢ޑڐշǶྕࢨНޑεໆ௦໣ࢂҁԛჴᡍ׷਑ޑٰྍǴགᖴᗬ଻Κǵ ቅܳӹϷՖठ動ǴڐշཚၮԭϦϲޑࢨНǴаϷගᒬך໒ًόाᅵ๱Ƕჴᡍᏹբ

΢Ǵᆶ೭ࢤВηаٰჴᡍ࠻ғࢲޑελ٣Ǵ੝ձाჹ໳᐀ᖚᏢۊठ΢ལཀǴགᖴ Ꮲۊ௲Ꮴך܌Ԗख़ाޑჴ଺ૈΚǴಒЈှเᅪൽǴѳВΨᕴࢂᓨᓨᜢЈךǶགᖴ ֈ᐀؊ᏢۊǴਔதᆶךϩ٦Ӵޑ࿶ᡍᆶـှǴගٮཥޑགྷݤک௴ҢǴӕਔΨࢂό ёલޑᆒઓუՔаϷᏢಞڂጄǶ୯ৎፁғࣴزଣޑᄃҁඦԴৣǵ஭৥ᒆӃғǴҁ ჴᡍ࠻ޑЦػரᏢߏǴдॺޑڐշࢂԜࣴزளаֹԋޑќ΋ᜢᗖǶғނၗૻܭך

ࢂӄฅढ़ғޑሦୱǴᄃԴৣᆶ஭৥ᒆӃғό໻ऐЈЇᏤךࡘԵϩ݋ၗ਑ޑБݤǴ ගٮ೬ฯᡏӚБय़ޑЍජǴΨ຤ΑࣗӭЈઓᆶך૸ፕǶঃऩؒԖдॺǴࣴزԋ݀

ჴᜤၲډႣයޑ፦ໆǶќѦӆԛགᖴΪֻᔾԴৣǴӧ᏾ᡏࣴزࢎᄬޑ೛གྷаϷቸ

܄΢ፓࡋԖБǴ٬ளӭኧୢᚒᕴࢂૈᕇளှ،Ƕགᖴ܌Ԗα၂ہ঩ჹܭፕЎኗቪ ޑࡰ҅ᆶࡌ᝼ǴЀځགᖴ໳᐀ᖚᏢۊᆶᄃҁඦԴৣǶགᖴӦ፦܌݅ҥहԴৣᆶੇ

ࣴ܌Ц੦࣓ԴৣǴᕴࢂ኷ཀӧךሡाᔅշޑਔং໼᠋ךޑୢᚒаϷϩ٦дॺޑЈ ளǶགᖴᕉπ܌ูЈݒԴৣჴᡍ࠻ڐշྕࢨНύԖᐒᅹᐚࡋޑෳໆǶགᖴѦᝤғ

ௗࡑ୍ܺကπޗǵ୯ሞ٣୍ೀک܌Ԗම࿶ӝբၸޑܻ϶ᆶߏ፸ॺǴᆶғࣽଣޑ୯ ሞҬࢬံշǴдॺࣁך໒Α΋৻Ԗ๱όӕ॥ඳޑืǶགᖴӳᎃۚቅܳӹکԴӕᏢ Ֆठ動Ƕགᖴᗬ଻ΚǴᕴࢂࣁךံкததό୼Ҕޑ׆ఈǶགᖴৎᓫکৎХޑഉՔǶ!

!

നࡕǴགᖴךޑৎΓǺҁጇፕЎ᝘๏գॺǶ!

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ᄔ ᄔा!

ཱུᆄᕉნ༾ғނғᄊسޑ௖ࢗǴኧΜԃٰࢂࣁӦౚᐕўǵғނᄽϯǵϺᡏғނᏢ ሦୱว৖ޑ୷ᘵࣴزϐ΋Ƕྕࢨࢂᆵ᡼தـޑཱུᆄᕉნғᄊس಍ǴԐයӧч೽ε Ђξ୔ჹྕࢨ༾ғނғᄊسޑፓࢗୃख़٬Ҕᆫӝ䁙ೱᙹϸᔈ(polymerase chain

reaction, PCR) ୀෳচਡғނ 16 S rRNA ୷ӢǴӆуаᙯ෗ᆶۓׇǴჹܭ༾ғނ௼

ဂ่ᄬᆶфૈޑ௖زుࡋϷቶࡋၨࣁόىǶ߈ϖԃٰǴԛШжۓׇޑמೌ೴ᅌԋ ዕᆶදϷǴ٬ׇӈၗ਑ޑᕇள׳Ԗਏ౗ǴΨૈளޕ׳ௗ߈੿ჴޑԾฅࣚኬᇮǶӧ ೭ጇࣴزύǴᒧۓܭεЂξ୔ѤᕘڳӦ୔ྕࢨ຾Չ௦ኬǴճҔᕴᡏ୷ӢᡏᏢ่ӝ

Illumina^TMۓׇѳᆵϩ݋εЂОξဂޑѤᕘڳለ܄዗ࢨύޑ༾ғނဂ໣Ǵ׆ఈΑ

ှځЬाޑဂ໣่ᄬᆶфૈǶ

࿶ၸፓࢗǴѤᕘڳ୔ୱޑྕࢨǴࢨྕѳ֡ࣁ 73°CǴpH ѳ֡ࣁ 2.8Ǵ՗ीྕࢨНύ

ੌෞচਡғނஏࡋऊࣁ 7.3×10⁵/mLǴեܭ΋૓ഌӦ఩НதྕНᡏᆶᡶ෫εऊΜ७Ƕ

ੌෞ༾ғނᕴᡏ୷Ӣᒿࡕࡌᄬࣁ΋х֖ΐίᎩਲ਼ኬҁޑ fosmid libraryǴԾ library ύڗ΋ίᎩਲ਼ኬҁᆶќ΋ץޔௗԾѤᕘڳࢨН๧ڗޑᕴᡏ୷Ӣӕਔ຾ՉԛШж ۓׇǶ࿶җғނၗૻϩ݋ۓ೚่݀Ǵว౜ಒ๵ࣁԜྕࢨғᄊسޑம༈ᅿǴ՗ीᆶ ђಒ๵ޑКٯऊࣁ 7:3 ډ 8:2 ϐ໔Ƕಒ๵ЬाаϯᏢԾᔼ܄ಒ๵ phylum Aquificae ԋ঩ࣁЬǴځԛࣁ Proteobacteria ک Firmicutesǹђಒ๵߾а phylum Crenarchaeota

՞ЬाՏ࿼ǶӧҁԛፓࢗύǴ೽ϩϩ݋ᡉҢ Aquificae ϐΠޑ Hydrogenobaculum ឦ՞Ԗ࣬྽ଯޑ࣬ჹᙦࡋǶӧКჹኳԄᅿ୷ӢᡏׇӈࡕǴว౜คݤֹ᏾ख़ࡌҁ୔

Hydrogenobaculumᜪ՟ނᅿޑ୷ӢᡏǴёૈསҢ๱ԜΒޣ໔ӸԖࢌᅿำࡋ΢ޑ

ׇӈৡ౦܄Ƕᙖҗ Protein COG ޑϩթኳԄϩ݋Ǵ٠ᆶځд࣬՟ਢٯКၨǴ௢ෳ

Ԝ዗ࢨ༾ғނဂ໣ޑ᏾ᡏ୷Ӣᡏಔԋୃख़ܭжᖴ࣬ᜢфૈǶ

ᜢᗖӷǺྕࢨǴ༾ғނǴӭኬ܄Ǵᕴᡏ୷ӢᡏǴԛШжۓׇǴғނၗૻ

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Abstract

Researches of ecosystems in extreme environments have been critical to the

accumulation of fundamental knowledge in fields of earth history, evolution biology and astrobiology. Novel enzymes, molecules possessed by microorganisms in extreme environments also provide sources for potent molecular biological and biotechnical applications. Extreme environments such as hot springs are commonly seen in Taiwan.

Previous studies relevant to hot spring microbial ecosystems in the Tatun Volcanic Group area (TVG, northern Taiwan) relied mainly on SSU rRNA gene sequence

analyses, which were unilateral and could be possibly biased. To obtain a more realistic view of prokaryotic diversity by acquiring sufficient sequence information,

next-generation sequencing (NGS) was applied to this study with the aid of

bioinformatics. Through the investigations on metagenomes from microbial community in a hot spring in SHP area, this study was aimed to obtain an overview on prokaryotic diversity, community structure and function in the hydrothermal spring system.

According to the results, the average pH of the hot spring in SHP area was 2.8, and average temperature was 73°C. Cell density of planktonic prokaryotes was estimated to be 7.3×10⁵/mL, which was about 10 times lower than that of terrestrial fresh water and salt lakes. Metagenomic DNAs extracted from SHP hot spring were used for

construction of a fosmid library containing 9481 clones. DNAs extracted from hot spring water and DNAs of 1485 clones from fosmid library were sequenced using Illumina^TM platform. Analyses of community composition revealed dominance of bacteria over archaea by 7:3 to 8:2. Members of bacterial phylum Aquificae were

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abundant, followed by Proteobacteria and Firmicutes; members of phylum Crenarchaeota were most abundant among archaea. Sequences from predominant members of genus Hydrogenobaculum, affiliated with Aquificae, were mapped to the reference genome and certain level of sequence difference was found in the comparison.

By analysis of the protein COG distribution pattern and comparing results from the other research, the hot spring prokaryotic community in SHP displayed a higher proportion of metabolism-related functions encoded in their genomes.

Key words: hot spring, microorganism, diversity, metagenomics, next-generation sequencing, bioinformatics

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List of Figures and Tables

Figure 1. Workflow of this study. ... 109

Figure 2. Tangential flow system for spring water concentration. ... 110

Figure 3. Contig length distribution of fosmid and DSS sample ... 110

Figure 4. Relative abundance distribution of DSS sample. ... 111

Figure 5. Mapping status of reference Hydrogenobaculum sp. Y04AAS1. ... 113

Figure 6. Relationship between sequence coverage and GC content. ... 114

Figure 7. COG distribution of gene functions. ... 115

Table 1. Hot spring classification based on temperature ... 116

Table 2. Hot spring classification based on physical characters ... 116

Table 3. Hot spring classification based on chemical properties ... 117

Table 4. Average geochemical and physical parameters in SHP (Ѥᕘڳ) ... 118

Table 5. Sequencing and assembly of fosmid clones (Fos) ... 119

Table 6. Direct shotgun sequencing and assembly of sample (DSS) ... 120

Table 7. Percentage of DSS reads used in each analysis on community structure ... 120

Table 8. Assessments on relative abundance of DSS sample through 4 approaches .... 121

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1. Introduction

1.1 Extreme Environments and Extremophiles 1.1.1 Definitions

From an ecological view, the whole Earth system encompasses numerous niches with unique environmental settings and each of them has its own biota that consists of functional related consortia of organism. For not only survival but also prosperous growth and reproduction, moderate environments of comparatively less evolutionary stress are preferred by vast majority of life.

The criteria for moderate conditions are somehow anthropocentric, referred to those adequate physical and chemical property that are close to the range favored by human beings: pH value around neutral, temperatures between 20 to 40°C, air pressure about 1 atm, with sufficient water activity (better higher than 0.7) and nutrients accessibility, suitable concentration of salts (between normal fresh water and sea water), without excessive exposure to radiation (compared with the average amount that is received on the surface of the Earth), and lower levels of heavy metal or toxic compounds (such as organic solvents). In contrast, those harsh environments which are featured by unusual physicochemical conditions, such as high acidity and alkalinity, extreme temperatures, high pressure or vacuum state, low water activity, low amount of nutrition, high salinity, intensive radiation, and places with high concentration of heavy metal or toxic substance, are defined as extreme environments (Rothschild and Mancinelli, 2001; Wilson and Brimble, 2009).

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Survival and reproduction are the most important goals to achieve for all kinds of life. In order to adapt to the dynamic material surroundings, organisms are usually capable of tolerating a certain degree of physical and chemical fluctuations in environments. However, a state of tolerating is different from optimal growth; thus, organisms which can survive in extreme environments are sometimes just extremotolerant instead of extremophilicɡthe latter describes the characteristics that an organism has its optimal growth under extreme conditions, and the organism per se is called an extremophile

(Macelroy, 1974; Kristjansson and Hreggvidsson, 1995; Wilson and Brimble, 2009). Accordingly, extremophiles thriving in environments with multiple harsh properties are grouped as “polyextremophiles”.

Increasing interest and efforts in researches relevant to extreme environments and organisms started around the 1950’s. Biotopes such as deep sea,

hypersaline environments, hot springs, deserts, ice, permafrost and atmosphere are the most focused regions for studying and all of them have various

combinations of geochemical backgrounds. For instance, general features of deep sea environment are low temperature around 1 to 2°C, anoxic and lack of photosynthesis. However, at some specific locations, when hydrothermal vents or ancient evaporite beddings exist, environmental conditions would then be a mixture of its original geochemical context plus individual variables:

hydrothermal vents heat up surrounding sea water and provide an additional chemical source, and dissolution of evaporite beddings would greatly increase

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the local salinity. The same is true for other locales: environmental conditions are results of interplays of regional backgrounds and local variables.

In the conventional classification of extremophiles, the main categories are divided based on physical and chemical conditions optimal for growth. Those commonly mentioned are thermophiles (high-temperature-loving),

psychrophiles (low-temperature-loving), acidophiles (low-pH-loving), alkaliphiles (high-pH-loving), halophiles (high-salinity-loving), xerophiles (low-water-activity-loving) and barophiles (pressure-loving, also called piezophiles). In some cases, those being able to resist particular stringent environmental factors are included into extremophiles, such as radioresistant and endolithic (living in cracks or pores of rocks or minerals) organisms, although this is somehow incongruent with the definition of extremophiles.

To view extremophiles as a whole, interest of research are of three categories:

(1) evolution and earth history, (2) astrobiology and extraterrestrial trace of life and (3) biotechnological applications (Wilson and Brimble, 2009). From the formation of the planet Earth to the existence of life, the

proto-environments are regarded to be analogous to some current extreme biotopes, which are generally anaerobic with extreme high temperature, low organic matters, and sparse nutrients. Through the expansion of knowledge on extreme biogeochemical systems, mechanisms of evolution, and biodiversity, the paleoclimatic change along the geological time could be better deduced. In

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a broader scale, the current search for extraterrestrial life within the solar system is supported by knowledge from studying environment analogues on the Earth. In practical aspects, extremozymes (Hough and Danson, 1999) produced by extremophiles with distinct functions have tremendous potential for the biotechnology industry, bioremediation, chemistry and pharmacyɡ the well-known Taq polymerase isolated from Thermus aquaticus (Brock and Freeze, 1969) used in polymerase chain reaction has served as a remarkable example.

1.1.2 The Underexplored Microbial Extreme Ecosystems

Microbial ecosystems, compared with other ecosystems of macro-organisms, are still poorly understood. Microscopic size made them inconspicuous for human eyes, therefore receiving less than sufficient attention. Furthermore, their almost-universal existence, difficulties in cultivation and technical constraints (although great progression has been made molecularly and bioinformatically, abundance and diversity of microorganism are not

completely assessable within a short period of time) have made the discovery and in-depth understanding of microorganisms in a slower process than their significance deserved. The same situation occurs in the exploration of microbial extreme ecosystems.

Extreme biotopes, located only in limited geographic regions with unusual physical and chemical characteristics, usually harbor a significantly lower

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amount of cells compared to normal niches and thus make it more laborious to collect sufficient amount of samples (Ferrer et al., 2009) for adequate analyses.

In the study of extreme ecosystems, prokaryotic organisms have long been the major target of interest because of their higher abundance and diversity over other multicellular or eukaryotic life in extreme environments. Prokaryotic microorganisms, which consist of members from bacteria and archaea

domains, are thought to be the most wide-spread form of life on Earth and the number of prokaryotes was about 10³⁰ cells in total (Turnbaugh and Gordon, 2008). As inhabitants and decomposers in ecosystems, their remarkable physiological functions are closely connected to the biogeochemical cycles of the Earth.

As progress of microbiology started from traditional culture-based methods into a culture-independent era and by the aid of extensive global explorations, rapid analysis and large amount of sequence data also revealed the

underestimation of prokaryotic diversity in extreme environments (Hugenholtz et al., 1998; Reysenbach et al., 2000; Takai et al., 2001b; Huber et al., 2002b;

Satyanarayana et al., 2005; Sogin et al., 2006; Yim et al., 2006; Huber et al., 2007; Wilson et al., 2008; Kato et al., 2011). Cultivation had identified 10 or 12 divisions in Bacteria domain and 2 or 3 divisions in Archaea domain (Woese, 1987). However, more than 40 divisions in bacteria (Pace, 1997) and more than 12 divisions in archaea (DeLong and Pace, 2001) are now

discovered through culture-independent approaches and the total number of

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genospecies approximates 10⁶ to 10⁸ (Sleator et al., 2008) with 1316 complete prokaryotic genomes available in online databases (updated in October 2012).

According to statistics, in recent prokaryotic sequencing projects only c.a. 4%

of them belong to extremophiles (Ferrer et al., 2007), which strongly indicates that there is still room and needs in the field of extreme ecosystem

explorations.

1.2 General Features of Hot Spring Environments

1.2.1 Geological, Physical and Chemical Conditions

A spring is a concentrated discharge of groundwater that appears at the surface as a current of flowing water (Todd, 1980). Conventionally, hot springs are terrestrial. When geothermal water issues under the sea, although it is also a kind of hot spring under water, a better term is hydrothermal or geothermal vent. Therefore, in this research we followed the general principle and declined the usage of “hot spring” in continental regime. To date, there is no single definition for hot springs. Usually the term is applied when the spring water meets one of the three descriptions (Todd, 1980; LaMoreaux and Tanner, 2001): (1) spring water with temperatures higher than that of its local

underground water; (2) spring water that is 5°C higher than the average temperature of its local annual air average temperature or (3) springs with warm water above body temperature.

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Hot springs are classified according to different purposes: temperatures (Table 1), geological backgrounds (such as volcanic, sedimentary or metamorphic), physical features (such as fluid states, activity patterns and geomorphic

appearance)(Table 2) and chemical properties (pH and dissolved ions in spring water)(Table 3). Geothermally and volcanically active zones are locations suitable for hot spring development. Precipitations of meteoric water and underground water are usually the sources of hot spring water, but

hydrological properties of each hot spring varies because of the influences by various geochemical environments.

1.2.2 Lives and Biological Activities

Hot springs in some regions are part of local cultures (hydrotherapy, bathing etiquettes, etc.) and recreations, and its geothermal nature is also close related to the mining of resources in peripheral areas (elemental sulfur, silica,

bentonite, pyrite and gold). Generally speaking, the variety and intensiveness of biological activity depend almost always on environmental conditions a hot spring possesses. It is possible for large animals, plants and invertebrates such as insects to thrive nearby the hot spring. However, taking high temperature or acidic spring water as an example, there are only a few kinds of life that might survive—most of them are microorganisms. For photosynthetic

microorganisms, the maximum temperature for survival is 73 to 75°C (Brock, 1967; Cox et al., 2011) and better with low sulfide concentration (Cox et al., 2011). While the environment becomes harsher, certain branches of

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prokaryotes could be found under certain conditions (Satyanarayana et al., 2005).

1.3 Microbial Activities in Acidic Hot Springs in Tatun Volcanic Area 1.3.1 Regional Background

Sitting on the lively collision zone between the Eurasian Plate and the Philippine Sea Plate, Taiwan is an island featured by frequent geothermal activities. The mountain belt has been well-accepted to be a result from collision between the Luzon arc and the Asian continent (Chai, 1972). Our study focused on prokaryotic microorganisms in hot springs of Tatun Volcanic Group (TVG), the largest volcanic group in Taiwan (Wang and Chen, 1990) (400 km², including more than 20 volcanoes), which is dormant in modern times. TVG has an average elevation of 800 to 1000 m and consists of two principal volcanic ridges, E–W and SW-NE; each is about 15 km long.

Previous dating data (Wang and Chen, 1990; Song et al., 2000) suggested the volcanic activity started from 2.8 to 2.5 Ma with relatively mild eruptions due to the regional compressive stress field. The massive explosive event occurred between 0.8 to 0.2 (some suggested after 0.1) Ma, while collision between plates in northern Taiwan had been weakened or even stopped and thus created a divergent stress environment which facilitated the extrusion and release of magma through surface cracks generated by normal faults. The latest evidence

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specifically indicated the last eruption record was around 6000 years ago (Belousov et al., 2010).

Although generally being considered dormant or even extinct, TVG still displays several traces that strongly suggest the existence of a magma chamber and its active geothermal activity: frequent shallow micro-earthquakes,

harmonic codas, seismic tremors (Lin et al., 2005; Konstantinou et al., 2007), high heat flows, measurements of volcanic gases (Yang et al., 1999) and abnormally high helium ratios (Song et al., 2000). Many distinguishing volcanic characters such as solfataras, fumaroles, sulfur crystals (yellowish needle or dendritic shaped) and irritating sulfuric gases are common scenes in this area.

Hydrothermal systems in TVG area, primarily with water of meteoric origin, are heated by the geothermal environment and react with peripheral rocks and gases. Therefore, the water property is mostly determined by the regional geological background. TVG is an andesitic volcanic unit with volcanic gases comprising mainly of H₂O, CO₂, SO₂, HCl (Lee et al., 2005) and its hot springs are of principally three types defined by properties of host rocks, pH values, and the major elements: (1) SO42-

acidic water (Type I); (2) HCO3-

a nearly neutral water (Type II); and (3) Cl^- -rich acidic water (Type III) (Liu et al., 2011). The three types of hot springs are scattered in TVG. Type I is usually with pH and temperature around 1.5 to 3.2 and 42 to 93°C, TDS (total

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dissolved solids) 245 to 12900 mg/L; type II is generally with near neutral pH, 40 to 62°C, and TDS concentration about 957 to 1149 mg/L; type III has its pH, temperature and TDS in between 1.2 to 1.9, 71 to 91°C, 17400 to 19800 mg/L, respectively.

Although there are some neutral carbonate hot springs in the TVG area, sulfuric acidic hot springs are predominant in this area. The sampling site of this research belongs to Type I, featured by sulfur crystalized on the rim of wide-spread fumaroles. This type of hot springs is geochemically

characterized by a high concentration of SO₄^2-, but with low HCO₃^-and Cl^- anions and low Na⁺, K⁺, Mg²⁺ and Ca²⁺ cations.

1.3.2 Previous Surveys on Microbial Diversity in TVG Area

Researches related to thermophilic microorganisms in Taiwan have been started since 1960s. Most of them were focused on physiological and biochemical aspects through cultivation or selective functions of specific genes; only a few had put the interest on a broad spectrum of surveys on microbial diversity and community function in TVG hydrothermal systems (ഋ ᔌࡏ, 2002; Ֆ੦ཹ, 2004; Ng et al., 2005; ׵ख़က, 2006; ᎄ൛Ў and ݅ ҥह, 2007; ׵ߞጎ, 2009; ׵ख़က and ஭܃༠, 2009; Cheng et al., 2013;

Lu et al., unpublished data).

In environments such as deep sea hypersaline basins (Ferrer et al., 2012),

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hydrothermal vents (Takai et al., 2001b; Nakagawa et al., 2005b), mud volcanoes (Cheng et al., 2012) and continental geothermal springs (Macur et al., 2004; Cheng et al., 2013), it is well-documented that microbial community structures are dynamic in accordance with physical and chemical profiles of the environment. In a terrestrial hydrothermal spring or hot pond, sediment and water layer are viewed as two distinguished habitats for microbes due to many differences of environmental variables such as photic/aphotic,

aerobic/anaerobic, etc. Therefore, it would be more realistic to separate the sediment and the water layer when the microbial diversity in a hot pond is to be investigated.

Earlier works on microbial diversity of acidic thermal springs in TVG area were done by Li’s group (׵ख़က, 2006; ׵ख़က and ஭܃༠, 2009) using 1:1 shallow layer sediments (10 cm at depth) mixed with spring water as material. They pointed out that each sample site has a unique microbial community composition with specific patterns of archaea and bacteria constituents.

Applying the 16 S rDNA sequence amplification to water-sediment samples from three sites DRG (Ӧ዗ك), LHG (౷ᕘك) and GZP (۪ηڳ), ׵ (2006) constructed libraries and set up an indicative reference for comparing the relative amount of microbes. The author observed that in low pH

environments bacteria were more abundant than archaea, whereas an

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increasing amount of archaea was found in mildly low pH environments.

However, there were no strict criteria for defining the environment conditions and for quantifying the amount of microorganisms. Conclusively, two orders of Crenarcheota, Thermoprotei and Sulfolobales were discovered in four selected springs in the report: Caldisphaera sp., Metallosphaera sp. and Sulfolobus sp.. About 78% were uncultured archaeon clones. In bacteria

domain, microbes such as Aquabacterium sp., Delftia sp., Desulfurella sp., Methylophilus sp., Pseudomonas sp., Stenotrophomonas sp. and Thiobacillus

sp. were detected, which belong to classes Beta-proteobacteria

(Burkholderiales, Methylophilales, Hydrogenophilales), Delta-proteobacteria (Desulfurellales) and Gamma-proteobacteria (Pseudomonadales,

Xanthomonadales). Approximately 45% of bacteria clones were uncultured.

In the research conducted by Cheng et al. (2013), samples from the two locations HS (ᕘξ) and LHG (౷ᕘك) in TVG acidic hot springs

demonstrated that diversity and segregated niches (water body and sediment layer) of hot spring microbes were likely correlated with the oxygen level in the system. In the water column, microbial community structures were homogeneous in different depth (since the hot ponds were not deep) but remarkably different from that in the sediment layer: archaea displayed different community structures between water and sediment layers, while those for bacteria did not differ as much. According to the PCR amplification on 16 S rDNA sequences, this research also indicated that at least during this

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sampling period some sample sites contained no bacteria. Based on the conclusion derived from the clone library screening and sequencing, they concluded that community structures in the two sample sites were relatively simple, only 12 phylotypes discovered. Crenarchaeal orders Sulfolobales (Sulfolobus sp. and Acidianus sp.), Thermoproteales (Vulcanisaeta sp.),

Caldisphaerales (Caldisphaera sp.) and euryarchaeal order Thermoplasmatales (Thermoplasma sp.) were identified to be the closest cultivated representatives, and bacterial order Aquificales (Hydrogenobaculum sp.) was predominant.

About 40% and 12% in the archaeal and bacterial clone libraries, respectively, were unclassified phylotypes.

In another study, Lu et al. (unpublished data) analyzed thermal spring planktonic microbial community structures at five locations (DRGӦ዗ك, LHG౷ᕘك, LFG ᓪስك, HS ᕘξ, FY ൤ྍ and HG ᕘෝ) along the west-east ridge of TVG area. At the east-most parts of the sampling region no bacterial 16 S rRNA gene sequences were amplified by PCR. Although diversity and community structures varied from one another, Caldisphaerales, Desulfurococcales, Sulfolobales and Thermoproteales were common archaeal orders seen in those sites, and Aquificales, Alphaproteobacteria,

Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria and Clostridia were phylogenetic groups harbored the sampled bacterial members.

Uncultured archaeal were 33% of total archaeal phylotypes, and 21% was that of bacterial ones.

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The research completed by׵ख़က and ஭܃༠ (2009) collected

water-sediment samples from five sites (LHG౷ᕘك, ZSL ύξኴ, MC ଭኲ, DYKεݨ֞ and GZP ۪ηڳ) for microbial diversity analysis. Significant differences of bacterial and archaeal community compositions among

sampling sites were again confirmed, which revealed that dominant microbes differed among locations. Most of the archaea and bacteria were uncultivable species. Of archaea, 4 orders were discovered under phylum Crenarchaeota:

Caldisphaerales (Caldisphaera sp., lagunensis sp.), Desulfurococcales (Acidilobus sp.), Sulfolobales (Acidianus sp., Metallosphaera sp., Sulfolobus sp.) and Thermoproteales; only order Thermoplasmatales was of

Euryarcharchaeota. In bacteria, 9 phyla were found in this research:

Acidobacteria (Acidobacterium sp.), Actinobacteria, Aquificae

(Hydrogenobaculum sp.), Chlorobi (Phylum), Chloroflexi (Anaerolinea thermophila), Firmicutes (Alicyclobacillus sp., Thermoanaerobacter sp.),

Proteobacteria (Sphingomonas sp., Ralstonia sp., Thiomonas sp., Thiobacillus sp., Acidithiobacillus sp., Rheinheimera sp., Desulfurella sp. Desulfurella kamchatkensis and Desulfovibrio sp.), Spirochaetes and Thermotogae (Thermotoga sp.).

1.3.3 Previous Research from Hot Springs Worldwide

Thermophilic communities are assumed to be geographically highly isolated from one another due to thermal barriers between habitats, but certain genera appear to be quite common to many continents. So far the most studied

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microbial ecosystem of terrestrial hot spring environments are in the Yellowstone National Park (YNP) of the United States (Barns et al., 1994;

Pace, 1997; Meyer-Dombard et al., 2005; Hall et al., 2008) , Kamchatka of Russia (Bonch-Osmolovskaya et al., 1999; Reigstad et al., 2010), Japan (Yamamoto et al., 1998; Nakagawa and Fukui, 2002), Tibet (Yim et al., 2006;

Lau et al., 2009; Huang et al., 2011), Iceland (Marteinsson et al., 2001;

Reigstad et al., 2010), New Zealand (Hetzer et al., 2007; Childs et al., 2008), Bulgaria (Tomova et al., 2010; Ivanova et al., 2011), Indonesia (Baker et al., 2001), Thailand (Kanokratana et al., 2004) and South Africa (Tekere et al., 2011). Due to the different regional geological history, each hot spring has its unique physiochemical features which are relevant to the components, structures and functions of its microbiomes.

Although detailed tectonic histories vary, hydrothermal systems hotspots share one important characteristic: the existence of geothermal energy sources.

Yellowstone National Park (YNP), featured by Yellowstone Caldera, is the largest volcanic system in North America. Several volcanic eruptions driven by North American Plate drifting over a stationary mantle hotspot have created diversified geothermal environments with active hydrothermal in YNP. Iceland lies on the mid-Atlantic ridge (also: mid-ocean ridge) rift zone, the boundary between the North American and Eurasian tectonic plates. Upwelling of high-temperature mantle material generates the geothermal energy and results in active volcanic events. Most of the geothermal springs are with pH around 8

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to 9, mild-acidic to neutral are of the minority. Na⁺ and Cl^- ions are usually abundant (Arnórsson et al., 1983). The extremely isolated Tibetan geothermal region has recently become a focus of microbial diversity exploration. Among the youngest mountain ranges on the planet, the Himalaya was formed as a result of a continental collision or orogeny along the convergent boundary between the Indo-Australian Plate and the Eurasian Plate since Upper Cretaceous (70 Ma).

Almost all eukaryotic microorganisms cannot thrive in environments with temperature higher than 68°C. In terrestrial hydrothermal systems, regardless of pH, Crenarchaeota of archaea domain are more prevalent than

Euryarchaeota and other archaeal phylum (Thaumarchaeota, Korarchaeota and Nanoarchaeota) in hydrothermal systems. In bacteria domain, Cyanobacteria are widely distributed and commonly seen to be associated with Chloroflexi (green non-sulfur bacteria) in environments with temperatures under 73 to 75°C (Ferris and Ward, 1997) where photosynthesis are still possible. At temperatures above 75°C to boiling degree, chemolithoautotrophic communities appear as gray or pink filaments/streamers in waters, often tolerating high dissolved sulfide levels (Reysenbach et al., 1994). Other bacteria such as Proteobacteria (α, β, γ, δ), Aquificae, Firmicutes,

Acidobacteria, Actinobacteria, Thermodesulfobacteria, Deinococcus-Thermus, Bacteroidetes, Nitrospirae, Spirochaetes and Verrucomicrobia were also discovered in hydrothermal systems (although not all of these groups of

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microbes occurred in every hot spring).

For acidic hot springs, well-studied research area were more restricted to YNP (Burton and Norris, 2000; Jackson et al., 2001; Inskeep et al., 2010), and other studies are in Andes (Bohorquez et al., 2012), Japan (Kato et al., 2011), New Zealand (Ellis et al., 2005) and Tibet. Bacteria phyla such as Proteobacteria, Firmicutes, Planctomycetes, Spirochaetes and Aquificae were found; archaeal phylum Crenarcheota is still in general more prevalent than Euryarchaeota and others. In some cases, especially when the environments were more acidic (pH value lower than 5) and had higher temperatures (above 65°C), archaea

populations are sometimes become dominant over those of bacteria (Inskeep et al., 2010). Within Crenarchaea, Sulfolobales were found to be dominant in many surveys (Ellis et al., 2005; Inskeep et al., 2010). In addition to

widely-distributed Proteobacteria, deep-branched Aquificae is another phylum which was revealed to be dominant among bacteria.

Microbial composition in communities is thought to be correlated with environmental factors; however so far there are no universal quantitative standards to clarify the definite relationship between them. According to Mathur et al. (2007), phylogenetic diversity of bacterial communities seems to be more related to temperature.

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1.4 Study of Microbial Community

1.4.1 Metagenomics and DNA Sequencing

The largest proportion of individual organisms on earth is represented by prokaryotes, which were estimated to comprise 10⁶ to 10⁸ genospecies (Sleator et al., 2008). At present, only a few thousand species of microorganisms have been formally described, while more than 99% are still under discovery and most of them cannot be readily cultured (Amann et al., 1995; Rappe and Giovannoni, 2003; DeLong, 2005). Basic microbiology stemmed in the

accumulation of knowledge from species which had been isolated and cultured.

Life history, morphological, physiological and metabolic characteristics were described in detail.

Based on sequence similarity of small subunit ribosomal RNA gene, Woese and Fox constructed the first tree of life that portrayed the concept of three domains of life in 1977. A decade later, the utilization of polymerase chain reaction (PCR) started (Mullis and Faloona, 1987). These key revolutionary methods combined with gene cloning technique (Weisburg et al., 1991;

Reysenbach et al., 1992a), molecular identification and phylogenetic analysis of prokaryotes thus became another core procedure in microbiology studies that offered a culture-independent option for systematic assessments of microbes.

With the aid of development in modern molecular biotechnology and

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methodology, metagenomics (Handelsman et al., 1998), a study of a whole microbial community through genetic material recovered directly from field samples, further expanded the realm of microbiology to the ecological scale. It allowed scientists to look into the microbial diversity and interactions with a broad view bypassing cultivations of single isolates, and has gradually become a regular strategy for probing into the structure and function of

microscopic-biospheres. From genomics to metagenomics, an enlarged frame of research brought increasing amount of specimens and genetic information as well. DNA sequencing is one of those critical innovations that help the effective transformation from hardly visible microorganism specimens to readable genetic codes.

DNA-sequencing techniques, first appeared in 1968 and became prevalent after 1977 (Maxam and Gilbert, 1977; Sanger et al., 1977), has played a key role concurrently with the trend of culture-independent microbiology research.

In its early stage, the focus of metagenomics (Schmidt et al., 1991) was the analyses of single representative genes (e.g. the small subunit ribosomal RNA gene) in a community of interest. Routine procedures included target gene amplification, cloning, library construction, and sequencing. To explore the ecological diversity (Biddle et al., 2008) or metabolic profile (Tringe et al., 2005), sequences generated were often compared with references from well-studied cultivable relative species, in order to depict possible

physiological or metabolic traits as well as evolutionary distances. In addition

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to genetic information, metagenomic libraries constructed in the process are themselves valuable reservoirs of novel enzymes and molecules ready to be discovered by means of function- or sequence-based screening (Handelsman, 2004; Simon and Daniel, 2011).

Automated Sanger DNA-sequencing was the chief approach used in

metagenomic research in the early stage. Due to technical constraints, efforts were mainly put on certain gene markers or genes encoding molecules with essential functions. However, discoveries driven by only a few marker genes provided restrictive or sometimes even biased information. Taxonomically, information tends to suffer from loss or distortion during PCR and cloning steps; functionally, microbial ecology analysis represented by only a few genes is far from convincing. Therefore, completeness of every genome in the

surveyed community was then pursued in attempt to create a more objective and systematic vision in metagenomic studies. One of the strategies to achieve this is environmental shotgun sequencing (ESS), which sequences massive small pieces from a fragmented genomes, and assemble the sequences by the overlapping regions through the assist of algorithms and computational power (Venter et al., 2004; Edwards et al., 2006). Evidence had supported that,

compared to traditional ribosomal RNA–based investigation, direct sequencing of metagenomic DNA (ESS) provides the most accurate approach for

assessing taxonomic composition (von Mering et al., 2007). By now ESS has been one of the most conventional ways for environmental microbiology

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studies and the general concept of shotgun sequencing has also been the core strategy involved in genome sequencing projects.

1.4.2 Next Generation Sequencing

Regardless of what purposes to achieve, the genetic sequence has so far been a standard information format to access the world of microbiology and most of the fields relevant to biology. Conventional Sanger DNA-sequencing

technology (Sanger et al., 1977), the so-called first-generation sequencing, is capable of recovering up to 1 kb of sequence data from an individual sample at a time. The most advanced Sanger sequencers can elevate the efficiency to processing 96 samples at a time with up to 1 kb sequence for each sample.

Since one reaction allows only one specimen to be analyzed with Sanger sequencing, it is quite time-consuming, labor-intensive and expensive for metagenomic studies with huge amount of clones to be sequenced.

In 2005, the “next-generation” sequencing (NGS) technology (also: second generation sequencing, massive parallel sequencing or high-throughput sequencing) was first commercially available with the 454 sequencing system (Margulies et al., 2005). Being developed in 1996 (Ronaghi et al., 1996;

Kawashima et al., 1998) and are now with several derived core technologies (Mardis, 2008; Shokralla et al., 2012), NGS possesses some revolutionary features that are distinguishable from capillary-based Sanger sequencing. First is the throughput of data, which is at least an order of magnitude higher than

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Sanger method, measured by base pairs per day of run time. Second, the cost of NGS by base pair is at least an order of magnitude lower. Third, the reads from NGS are generally shorter than those generated by capillary sequencing.

Last, an environmental sample can be recovered and analyzed directly through NGS and bypass the cloning procedure as well as the PCR amplification.

Suppose that one human genome is to be sequenced by the two approaches.

By utilizing ABI 3700 (Mitchelson, 2001) of Sanger capillary sequencing technology (could read up to 1 million base per day), eight years of non-stop processing is needed to complete the task. In contrast, only 2 hours are required if an Illumina HiSeq 2000 (Ajay et al., 2011) of NGS technology is applied (which is capable of producing 50 Gbp per day).

Bypassing the cloning step in NGS sequencing makes it possible to obtain result in an objective way: DNA fragments are usually not cloned into host with equal possibility, some may lost during processing, some may subject to selective bias such as GC content (Temperton et al., 2009). However, for NGS samples which have no library archives, genes are discovered only as a series of sequences, their actual physiological functions or relative gene locations are so far impossible to be re-accessed from picking up clones that harbor the desired inserts. NGS has advantages over capillary-based method mainly in its affordable price for high-throughput data production, but reduction in average read length has always been a drawback even advances in bioinformatics are gradually narrowing the gap between immense data and proper solutions for

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analysis and interpretation.

Since 2005, researches incorporating NGS technique have increased dramatically. Unlike first generation sequencing, NGS constitutes various strategies that rely on complex interplay of enzymology, chemistry, high-resolution optics, hardware and software engineering. By now,

454-prosequencing GS FLX + system (Roche) and HiSeq system (Illumina) are the main players in high throughput sequencing despite the fact that there are some other competitors and new comers.

1.4.3. From Sequencing, Genetic Codes to Information

From capillary-based sequencing to the era of NGS massive parallel sequencing, output data has dramatically expanded in size but meanwhile shortened in the read length. Careful considerations must be taken when evaluating if a sample is suitable to be analyzed through the shotgun

sequencing approach, since the output sequences from all members within the community will be fragmented and mixed. To choose a community with less structural complexity (such as one dominant species and low diversity) can effectively reduce the difficulty during assembly, especially when shotgun sequencing is to be carried out by an NGS sequencer. Supporting evidences came from several metagenomic studies on extreme or oligotrophic

environments (although were sequenced through capillary-based method) such as acidophilic biofilm (Tyson et al., 2004), acid mine drainage (Edwards et al.,

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2000) and the Sargasso Sea (Venter et al., 2004), which were successfully profiling the community structures or even recovered whole genomes of some dominant species (Baker and Banfield, 2003).

Sufficient quantity and high quality of extracted DNA is critical to sequencing.

In general, for pyrosequencing or cloning, some micrograms of genomic DNA are required. Although library construction is not necessary for other NGS systems such as Illumina, at least 20 μg would be suggested. For low-biomass samples derived from environments with harsh conditions, usually it would be the major challenge to obtain enough metagenomic material for starting up (Ferrer et al., 2009).

Once the sequencing stage has been finished, reads are base-called from signals generated by sequencer, screened to remove vectors (if vectors were used) and barcodes and then undergo a quality check to trim off bases or reads with poor-quality. The next step is to assemble reads according to overlapping regions into longer fragments termed contigs. Assembly is a process which consumes enormous computing power and also a step which might introduce artifacts to the final output (Kunin et al., 2008). The finished length of contig can vary widely from around 100 bp to more than 100 kb, depending on microbial community composition and sample quality.

After contigs are generated, the diversity, gene functions and metabolic

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pathways of the sample are three basic directions for further analyses.

Databases such as SILVA (Pruesse et al., 2007), Greengenes (DeSantis et al., 2006), or Ribosomal Database Project II (RDP II) (Cole et al., 2003) offer useful resources for rRNA gene-based classification of microorganisms so that sequences containing taxonomic information or sequences derived directly from 16 S rRNA gene amplicon can be analyzed readily.

For gene function annotation and metabolic pathway investigation, the Gene Ontology (GO) database (Ashburner et al., 2000), Clusters of Orthologous Groups (COG) database (Tatusov et al., 2001), Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa et al., 2008), Pfam (Finn et al., 2010), NCBI (Sayers et al., 2009), SEED (Overbeek et al., 2005) are

commonly used databases for metagenomic data sets generated by NGS. To compare data on hand with other known species, reference genomes deposited in database like IMG (Joint Genome Institute) can be downloaded to a local server and mapped with reads or contigs.

After the sequence information was annotated, taxonomic relative abundance, main metabolic functions and unique pathways are usually integrated with environmental factors and cross-compared with other studies. Public domain depositions of all available data are also essential for environmental

metagenomic research; the accumulation of results from surveys facilitates later cross-comparison and also contributes to the discovery of the whole

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picture of microbial biosphere on Earth.

1.5 Motivations and Aims of This Study

Hot springs, one of extreme environments that had been proven to be a reservoir of valuable knowledge to the past (earth history), present (microbial ecology) and future (bioprospecting and astrobiology), are worth being further explored and understood. In the past few decades, studies of this field in Taiwan have accumulated a certain amount of knowledge that provided a rough image of local hot spring ecosystems. However, more thorough and systematic researches on prokaryotic diversity are needed. In this study, the high-throughput sequencing technique was applied to obtain sufficient sequence data that facilitated a more comprehensive analysis on prokaryotic community structure in an acidic hot spring ecosystem.

1.6 Framework of Research

Samples of hot spring water were collected, concentrated and from which metagenomic DNAs were extracted for fosmid library construction and direct shotgun sequencing. Results of sequencing were analyzed for assessments of prokaryotic community composition, genome mapping and community gene function distribution. Prokaryotic community structure analysis and gene functions were the main topics of this study (blue and green boxes of Figure 1).

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Prior to the sample collection, a general survey on the bacteria, archaea and nanoarchaea distribution of six candidate hydrothermal springs in TVG area was done (white box of Figure 1 and Appendix 1).

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