Communities of Archaea and Bacteria in a subsurface radioactive thermal spring in the Austrian ...

31 Pages·2006·4.69 MB·English

AEM Accepts, published online ahead of print on 3 November 2006
Appl. Environ. Microbiol. doi:10.1128/AEM.01570-06
Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Weidler et al. 1
1 Communities of Archaea and Bacteria in a subsurface radioactive thermal
2 spring in the Austrian Central Alps and evidence for ammonia oxidizing
3 Crenarchaeota
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5 Gerhard W. Weidler1), Marion Dornmayr-Pfaffenhuemer1), Friedrich W. Gerbl1), Wolfgang
6 Heinen2)* and Helga Stan-Lotter1)
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8 1)Division of Molecular Biology, Department of Microbiology, UniTversity of Salzburg,
9 Billrothstraße 11, A-5020 Salzburg, Austria
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10 2)Department of Microbiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, w
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11 The Netherlands a
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15 Running title: MCicrobial communities in a subsurface radioactive thermal spring ://
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17 *dAeceased June 30, 2006 s
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20 Corresponding author: Dr. Helga Stan-Lotter, Division of Molecular Biology, Department of A
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21 Microbiology, University of Salzburg, Billrothstraße 11, A-5020 Salzburg, Austria; Phone il
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22 [phone]; Fax: + 43 662 8044 7209; E-mail: [email protected]
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Weidler et al. 2
23 ABSTRACT
24
25 Scanning electron microscopy revealed great morphological diversity in biofilms from
26 several largely unexplored subterranean thermal Alpine springs, which contain radium
27 226 and radon 222. A culture independent molecular analysis of microbial communities D
28 on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, was
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29 performed. Four hundred fifteen clones were analyzed. One hundred thirty two
30 sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283
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31 with 4 archaeal OTUs. Rarefaction analysis indicated high diversity of bacterial
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32 sequences while archaeal sequences were less divePrse. The majority of the cloned o
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33 archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) n
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34 crenarchaeotic group; other representativEes belonged to the freshwater-wastewater-soil d
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35 (1.3b) group, except one clone, which was related to a group of uncultivated f
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36 Euryarchaeota. These findings support recent reports that Crenarchaeota exist not only

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37 in high-temperature environments. Most of the bacterial sequences were related to the tp
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38 Proteobacteria (αααα, ββββ, γγγγ and δδδδ), Bacteroidetes and Planctomycetes. One OTU was allied e
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39 with Nitrospina sp. (δδδδ-Proteobacteria) and three others grouped with Nitrospira. .a
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40 Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the .
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41 rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been /

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42 implicated recently in the nitrogen cycle, the spring environment was probed for the
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43 presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were ril
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44 obtained which were related to crenarchaeotic amoA genes from marine and soil ,
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45 habitats. The data suggested that nitrification processes are occurring in the
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46 subterranean environment and that ammonia may possibly be an energy source for the y

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47 resident communities. e
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48
49 INTRODUCTION
50
51 Bad Gastein, a village in the National Park "Hohe Tauern" in the Alps near Salzburg,
52 Austria (see Fig. 1) is known for its thermal mineral springs. A cluster of 17 major springs
53 (between 962 to 1029 m above sea level) with temperatures up to 47°C provide about 4 to 5
54 million liters of thermal mineral water per day (7, 10, 15). The water of the springs is
55 collected in such a way that nearly no surface water can mix with the warm spring water,
56 which is distributed to the spa hotels (10). Numerous medical determinations of the influence
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57 of radium 226 and radon (mainly Rn 222), which are present in the mineral water of the Bad
58 Gastein thermal springs, on the human organism have been made (10, 15). However, almost
59 no information about the microbial flora of these thermal springs exists (7, 18).
60 One of the major springs, located at the highest altitude (1029 m above sea level), is called
61 "Franz-Josef-Quelle" (designated as FJQ; Fig. 1); it consists of a total of 27 single water D
62 discharges, issuing directly from rock fissures (18). It is located at the end of a gallery that
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63 was driven horizontally about 100 – 150 m deep into the rock. It issues from an artificial
64 cavity from the chasms traversing the rock (7), delivering 295 m3 water per day (according to
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65 reports from 1997/98) with a Rn-222 quantity of 296 kBq/m3 water (7, 10, 18). Its average
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66 temperature is 45.6°C and the pH is about 8.0. The geoloPgical environment of the spring area, o
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67 consisting mainly of gneiss and crystalline slate, which are defined as primary rocks, prevents n
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68 rain water from trickling into the ground. AsE stated by Cudrigh (7), pure and unmixed thermal d
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69 mineral water possesses a mineralization degree of about 5000 µval/l and a temperature of f
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70 56°C. These properties, as well as the content of tritium - which should not occur in thermal

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71 water - indicated an intermixture of the spring FJQ with colder ground or subsurface water of tp
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72 about 10 %, which was corroborated by measurements of d18O and deuterium (7). a
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73 Furthermore, the subterranean water cycle was measured by the 14C method, which suggested .
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74 a retention time of 3600 to 3800 years (7, 15). Currently little is known of such subsurface
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75 environments and in particular, about possible energy and carbon sources in such niches, but g
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76 the interest in the microbiology of subterranean environments is clearly rising (14, 19, 20, 33, n

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77 46). r
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78 Caves, especially, harbor an interesting composition and variety of mineral utilizing ,

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79 microorganisms, which may contribute to the formation of unusual netlike microstructures as 1
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80 described by Boston et al. (4, 33). Very similar arrangements were detected by scanning y

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81 electron microscopy in the springs of Bad Gastein (Heinen et al., unpublished). Therefore, the u
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82 spring FJQ was chosen for examination of its microbial composition. The inadequacy of
83 culture-based methods for estimation of species numbers in natural environments has been
84 well documented (1, 35), so culture independent techniques were applied. These included 16S
85 rRNA gene sequence analyses and restriction fragment length polymorphism (20). In
86 addition, the search for crenarchaeal ammonia oxidation (48) related genes was applied to
87 examine this subsurface habitat, because early results in this study revealed the presence of a
88 large number of crenarchaeal clones and, as mentioned, the energy supply for microbial cave
89 communities is, with some exceptions, entirely unknown.
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91 MATERIALS AND METHODS
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93 Sampling and site description. The samples were taken from water discharges number 21,
94 23 and 24 which flow into a small basin (Fig. 1). The water temperature at the point of
95 sampling was 42.3°C with a pH value of 8.1; the air temperature was 34°C with a humidity D
96 near 100 %. Water samples and bacterial mats (biofilms), designated FJQF and FJQB,
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97 respectively, were taken under aseptic conditions, using sterile equipment. Water samples
98 were collected in aluminum-foil wrapped one-liter bottles. Spatulas were used for removal of
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99 biofilm samples, which were placed into 50 ml reaction tubes. Glass slides (75 mm x 25 mm
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100 x 1 mm) were mounted into a metal rack, which held 10 sPlides and were placed into the spring o
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101 FJQ for up to 11 days. Following collection, the slides were placed into 50 ml reaction tubes. n
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102 Samples from glass slides were designatedE as FJQOT. Water samples were stored at 4°C. d
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103 Biofilm samples and glass slides were stored at – 70°C until further use. f
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104 Some specific data for the spring FJQ are as follows (7): the mineralization degree of the

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105 spring water is 4825 µval/l; the O content is up to 0.3 mg/kg water; cations (in mval %/kg tp
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106 water) are Na+, 76 %; Ca2+, 21 %; anions are SO 2-, 57 %; HCO -, 21 %; Cl-, 16 %; and F-, 6 a
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107 %. Mn2+ is present as trace element in a concentration of up to 0.03 mg/kg water. NH3, NH4+, .a
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108 NO - and NO - are present in traces (< 0.01 to 0.1 mg/kg water). m
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109 g
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110 DNA preparation from filtered water and from biofilm on glass slides. Two liters of n

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111 mineral water were passed through a 0.22 µm pore-size autoclaved membrane filter r
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112 (Durapore filters, Millipore, Bedford, MA, USA), using a filtration unit (SM 16201/19/20; ,

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113 Sartorius, Vienna, Austria) with low-pressure vacuum. Filters were kept at – 70°C until 1
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114 further use. Glass slides that were incubated in the spring for 11 days were broken into small y

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115 pieces and DNA extraction was performed as described by Radax et al. (38). u
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117 DNA preparation from natural biofilm samples. During DNA extraction presumptive
118 organic or inorganic compounds, e. g. humic acids, phenolic compounds and heavy metals,
119 which can inhibit or decrease the sensitivity of PCR (8, 45), appeared as a brownish-reddish
120 color, or as yellow color after DNA extraction. To eliminate these interfering compounds, the
121 DNA extraction protocol described by Glenn (Glenn, T., 2001. MUD-DNA extraction
122 protocol. www.uga.edu/srel/DNA_Lab/protocols.htm) was used with the following
123 modifications: about 2 g of brownish-white biofilm were centrifuged at 6,000 x g for 5 min at
124 4 °C. After centrifugation the watery supernatant was removed and 900 µl of TE buffer (100
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125 mM Tris-Cl, pH 7.6, 10 mM EDTA) and 50 µl of lysozyme (10 mg/ml) were added. Samples
126 were mixed for 2 min and incubated for 1 h at 37°C. Following boiling for 10 min, 100 µl of
127 proteinase K (10 mg/ml) were added and samples were incubated for 1 h at 56°C. To each
128 sample, guanidinium thiocyanate (Sigma, Saint Louis, MI, USA) and diatomaceous earth
129 (Sigma), prepared as described by Glenn (2001), were added. Subsequent sample preparation D
130 was done as recommended in the protocol by Glenn (2001). DNA was further cleaned with
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131 the Gene Clean II kit (Bio-101, Carlsbad, CA, USA) according to the instructions of the
132 manufacturer. The yellow color was nearly completely eliminated using this protocol.
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133
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134 PCR and clone library construction. Several prPimer pairs were tested for PCR o
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135 amplification of partial 16S rRNA gene fragments of the extracted community DNA from n
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136 filtered water, glass slide and biofilm samplEes by using a standard PCR approach. The primer d
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137 pairs A21F (5’ TTC CGG TTG ATC C(CT)G CCG GA) and A958R (5’ (C/T)CC GGC GTT f
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138 GA(A/C) TCC AAT T) for Archaea (11) and P797F (5’ C(A/G)A A(C/T)(A/C) GGA TTA

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139 GAT ACC C) and E1492R (5’ TAC GG(C/T) TAC CTT GTT ACG ACT T) for Bacteria tp
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140 (42), respectively, were chosen, since they yielded the highest amounts of PCR product. The a
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141 reactions were performed in a total volume of 50 µl with 1.25 U of Taq DNA-polymerase .
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142 (Fermentas, St. Leon-Rot, Germany). The PCR products were excised following separation
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143 by gel electrophoresis and recovered from the gel slices using the QIAquick® Gel Extraction g
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144 Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s instructions. n

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145 Subsequently, two separate clone libraries were created, one for Bacteria and one for r
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146 Archaea, and for each sample type (water, biofilm and glass slides). Purified PCR products ,

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147 were ligated into the pGEM®-T vector system (Promega, Madison, WI, USA) and 1
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148 transformed into competent E. coli JM 109 cells as recommended by the manufacturer. y

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149 Positive clones were cultivated overnight in selective media and vectors were purified using a u
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150 "TENS-Mini Prep" protocol (50) with slight modifications as follows: To resuspended E. coli
151 pellets 300 µl of TENS-buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 0.1 N NaOH, 0.5 %
152 SDS) were added and incubated for 15 min at room temperature. Following cell lysis, 150 µl
153 of 3 M sodium acetate pH 5.5 were mixed with the cell lysate by inverting the reaction tubes
154 several times. Samples were incubated on ice for 15 min, centrifuged for 10 min at 17,530 x g
155 at 4°C, and again for 15 min after precipitation of DNA. Samples were washed with 75 %
156 ethanol, air dried, and plasmid DNA was dissolved in 50 µl ddH O.
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158 RFLP analysis and grouping of clones. Purified vectors were digested for 16 h at 37 °C
159 with DdeI (Promega) for RFLP analysis, in order to separate the clones into groups according
160 to their restriction patterns. Because DdeI cuts at four restriction sites in the pGEM®-T vector,
161 it was decided not to amplify the inserted DNA by PCR, but to digest the cloned insert
162 together with the vector. The digests were analyzed on 3 % agarose gels. Inserted fragments, D
163 which were not cut by DdeI, were amplified with Archaea or Bacteria specific primers by a
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164 standard PCR protocol. PCR products were digested for 16 h at 37°C with HpaII (Fermentas)
165 and separated on 3 % agarose gels. Each RFLP group was preliminarily defined as an
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166 operational taxonomic unit (OTU). As representative sequences of each OTU were available,
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167 sequences were subjected to similarity matrix analysis Pusing Similarity Matrix version 1.1 o
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168 (27) of the RDP II (28, 29), to support OTU definition and RFLP grouping, respectively. The n
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169 clones were clustered into operational taxoEnomic units at a level of sequence similarity of d
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170 >99% (30, 43) in order to quantify diversity. f
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171

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172 Sequencing and phylogenetic analysis. At least two sequences representing each OTU tp
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173 group - if OTUs had more than one representative - were amplified with the primers SP6 (5’ a
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174 TAT TTA GGT GAC ACT ATA G) and T7 (5’ TAA TAC GAC TCA CTA TAG GG). .a
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175 Sequences were determined by automated dideoxynucleotide methods with the ABI Prism
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176 Big-Dye Terminator Cycle Sequencing Kit on an ABI Prism 310 Genetic Analyzer (Perkin- g
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177 Elmer Applied Biosystems, Vienna, Austria), as recommended by the manufacturer. n

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178 Sequence comparisons to identify database sequences similar to those from clones were done r
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179 using the FASTA3 (37) web interface from the European Molecular Biology Laboratory ,

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180 (EMBL). The potential presence of chimeric sequences was examined with the 1
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181 CHECK_CHIMERA program (27) available through the RDP II, release 8.1, with the y

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182 chimera check program of the Bellerophon server (21), and in addition, the phylogenetic u
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183 affiliations of their 5’ and 3’ ends were compared manually. No chimeras were recognized
184 within archaeal sequences, but 3 potential chimeras were seen within bacterial sequences.
185 Selected sequences were aligned with ClustalX (47). Subsequently the alignment was
186 subjected to phylogenetic analysis using distance-based (22, 40) maximum parsimony and
187 maximum likelihood methods, with programs from the PHYLIP package, version 3.5.1c
188 (Felsenstein, J., University of Washington, Seattle, USA. PHYLIP (phylogenetic interference
189 package), distributed by the author) and the ClustalX software. Trees were visualized with the
190 Treeview PPC program version 1.6.0 (36). In addition, the significance levels of interior
191 branch points were determined by bootstrap analysis using 1000 data resamplings.
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192 Phylogenetic trees shown in this work were constructed by neighbor-joining algorithm using
193 ClustalX version 1.81 (40, 47).
194
195 Amplification of amoA (ammonia monooxygenase subunit A) related sequences. PCR
196 was performed with a standard touchdown approach using the primer pair amo111F and D
197 amo643R (48), which are specific for crenarchaeal ammonia monooxygenase subunit A
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198 related genes. The primers were a gift from Christa Schleper and coworkers, University of
199 Bergen, Norway. PCR products from filtered water, glass slides and biofilm material were
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200 designated as F, OT and BF, respectively, and were recovered from the gel slices using the
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201 QIAquick® Gel Extraction Kit (QIAGEN) in accordance Pwith the manufacturer’s instructions. o
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202 PCR products of each sample type were cloned into the pGEM®-T vector and five randomly n
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203 chosen clones of each sample were sequenEced. Cloning and sequencing were performed as d
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204 described above. f
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205

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206 Statistical analysis. The screening process was tested by statistical analyses to evaluate tp
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207 whether total diversity was covered by screening 415 clones. Two types of analyses were a
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208 used: Coverage values were calculated by use of the equation C = 1 – (n/N) x 100, where n is .
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209 the number of unique OTUs and N is the total number of clones examined (see reference (39),
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210 for a discussion of terms). In addition, a rarefaction analysis was performed to determine the g
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211 number of unique OTUs as proportion of the estimated total diversity. Calculations were n

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212 performed using the freeware program Analytic Rarefaction version 1.3 (Holland, S. M., r
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213 2003, www.uga.edu/strata/software/software.html). ,

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214 1
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215 Scanning electron microscopy (SEM). Material for scanning electron microscopy was y

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216 taken randomly from the spring and its vicinity. A sample holder with two round 1.5-cm- u
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217 diameter coverslips was placed for several days at a defined location, e.g. 2, 25, 45 and 55 cm
218 from the rock fissures, allowing formation of a biofilm and deposition of small rock particles.
219 The part of the coverslip that was covered by the holder was not colonized and thus served as
220 a control. The coverslips as well as pieces of rock surface were immediately air dried and
221 transported in a closed and dry container to the laboratory. The samples were mounted on
222 aluminum SEM holders and coated with 1.5 nm Au/Pd (40:60) using a 208 high resolution
223 (HR) Cressington sputter coater (Watford, England) in order to enhance the conductance of
224 the material and to prevent charging artifacts during scanning. Electron microscopy was
225 carried out with a JEOL JSM-6330F field emission instrument (Tokyo, Japan) operated at 3.0
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226 kV. This instrument is hosted by the Department of General Instrumentation of the Radboud
227 University Nijmegen.
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229 Accession numbers. All small-subunit rRNA gene sequences as well as the amoA
230 sequences were deposited in the EMBL/EBI nucleotide sequence database under the D
231 following accession numbers: AM039528 - AM039550 for partial 16S rRNA gene sequences,
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232 AM233905 and AM260487 – AM260489 for the crenarchaeal amoA sequences.
233
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234 RESULTS
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236 Sampling and 16S rRNA gene analysis using restriction fragment length n
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237 polymorphism (RFLP). Water samples andE bacterial mats (biofilms) were designated FJQF d
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238 and FJQB, respectively. The microbes, which cover the surface of the submerged boulders in f
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239 the spring, seemed to exist in two different forms (18), both of which were collected as

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240 samples designated FJQB: an older colonization stage of these mats adhered strongly to the tp
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241 rock surfaces, whereas the second form appeared as a whitish slimy substance, which a
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242 probably represented the glycocalyx of diverse microorganisms, and seemed to be less firmly .
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243 attached to the rocks (18). Archaeal and bacterial 16S rRNA gene fragments were amplified
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244 with primers A21F/A958R and P797F/E1492R, respectively. A total number of 415 clones g
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245 was analyzed; 283 clones were of archaeal, 132 clones of bacterial origin. RFLP analysis n

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246 resulted in 7 archaeal groups, designated FJQGA, and 16 bacterial groups, designated r
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247 FJQGB, or FJQGBL, if inserts were not cut by DdeI. This was the case for 5 of the 16 ,

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248 bacterial RFLP groups, and therefore the grouping of those five was based on the digestion of 1
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249 the amplified insert sequence with HpaII. When representative sequences of each group y

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250 became available, sequences were compared using the Similarity Matrix version 1.1 of the u
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251 RDP II. This method reduced the archaeal groups to 4 and bacterial groups to 14, since the
252 similarity of some sequences was higher than 99 %. These final groups were defined as one
253 OTU each. Representative sequences of these merged groups were treated as single
254 phylotypes included in one OTU (see Table 1) and were also submitted to the EMBL/EBI
255 nucleotide sequence database. The procedure was applied to the archaeal phylotypes
256 FJQFA13, FJQBAA3 and FJQBAA20 (OTU FJQGA2), because these sequences were more
257 than 99 % similar to each other (data not shown), and to phylotypes FJQBAA5 and FJQFA2
258 (OTU FJQGA1). This procedure was also applied to bacterial phylotypes FJQOTB21,
259 FJQFB23 and FJQBIB7 for the same reasons (FJQGBL2; see Table 1).
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261 Archaeal community composition and phylogenetic analysis. The composition of
262 archaeal OTUs clearly showed the abundance of crenarchaeal clones in all three sample types:
263 filtered water, biofilm (microbial mats) and glass slides, which confirmed the common
264 suggestion that mesophilic Crenarchaeota represent a stable and specific component in D
265 terrestrial habitats (2, 12, 34, 41). FJQGA1 and FJQGA2 consisted of clones from all three
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266 sample types, while the dominating clones of FJQGA1 originated from filtered water and
267 FJQGA2 stemmed from the biofilm (Table 1). The database sequence that was most similar to
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268 FJQGA1 was clone SAGMA-2 of waters from a South African gold mine (46), but the most
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269 similar database sequence of FJQGA2 was an unculturPed crenarchaeon, clone Gitt-GR-39 o
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270 from uranium mill tailings. This result is of interest to our examinations, since the presence of n
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271 radioactivity (radium, radon and uranium) Eis characteristic for the thermal springs of Bad d
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272 Gastein (18). Table 2 shows that crenarchaeal clones of FJQGA1 and FJQGA2 were the most f
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273 frequently recovered archaeal groups in this habitat, representing 99.65 % of all archaeal

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274 clones. A single clone, recovered from a glass slide, may have been of euryarchaeal origin, tp
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275 but was only 82.15 % similar to its nearest neighbor sequence, which was from a deep-sea a
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276 hydrothermal vent site (Table 1). FJQGA3, which belonged to the freshwater-wastewater-soil .
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277 (1.3b) group, appeared to be a subdominant crenarchaeal group in this habitat (Table 2, Fig.
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278 2). Four clones, recovered from glass slides, may represent this group in the FJQ subsurface g
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279 thermal spring, but were only 94.75 % similar to its nearest neighbor sequence, which was n

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280 from an oceanic crust (Table 1). r
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281 ,

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282 Bacterial community composition and phylogenetic analysis. Bacterial 16S rRNA gene 1
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283 composition and abundance of OTUs showed a higher diversity in community composition y

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284 compared to the archaeal representatives of this subsurface environment. Several OTUs were u
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285 represented by clones of only one sample type, e. g. clones obtained from filtered water
286 appeared in FJQGB1, FJQGB2 and FJQGB3, but FJQGB2 was represented by only one
287 clone. FJQGB7, FJQGB8, FJQGB9 and FJQGBL4 were represented exclusively by clones
288 from bacterial mats, but only by one or two clones each. The clones obtained from glass slides
289 represented OTUs FJQGB6 and FJQGBL3, where the latter again consisted of only one clone
290 (Table 2). The most prominent OTUs of bacterial sequences were FJQGBL2 (25 % of
291 bacterial clones) and FJQGB5 (28 % of bacterial clones) followed by FJQGBL1, FJQGB4
292 and FJQGB1 with about 10 % of all bacterial clones each (Table 2). Other groups were
293 represented by 1 to 4 clones (Table 2). The two OTUs accounting for the largest percentages
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294 of clones were most similar to sequences recovered from a microbial mat (FJQGBL2) and
295 from a trichloroethene contaminated site (FJQGB5). The less dominating groups (about 10 %
296 of the abundance of bacterial clones) were composed of OTU FJQGB1, which was only 89 %
297 similar to an uncultured strain isolated from subtropical freshwater marsh (Table 1). OTU
298 FJQGB4 had only 87 % similarity to its next relative, a clone isolated from continental shelf D
299 sediments (Antarctica). FJQGBL1 (99 % sequence similarity), like OTU FJQGB7 (94 %
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300 similarity), was affiliated to a cluster of thermophilic methanotrophic Bacteria and to cultured
301 Methylocaldum - Methylococcus strains (Table 1, Fig. 4). The less frequently recovered OTUs
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302 (1 to 4 clones) possessed sequences which were similar to those of isolates from diverse
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303 habitats like soils, e. g. uranium contaminated soil,P uranium mine sediments waters, o
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304 ANAMMOX (anaerobic ammonium oxidation) sludges and artificial microcosms (FJQGBL3, n
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305 FJQGBL4, FJQGBL5, FJQGB2, FJQGB3, EFJQGB6 and FJQGB8), as well as one extreme d
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306 habitat like extremely thermal soil (FJQGB9). f
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307

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308 Statistical analysis. Eighteen different OTUs were represented by two clone libraries, for tp
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309 Bacteria and Archaea each, consisting of 415 clones. Coverage, calculated as in reference a
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310 (39), reached a value of 95.66 %, indicating that nearly the total diversity was covered by the .
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311 two clone libraries. However, since coverage is based on the number of unique OTUs relative
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312 to total richness, evenness is not taken into account, and thus, this value should be seen as a g
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313 rough estimate of diversity. The coverage value for Archaea (n = 4, N = 283) was 98.58 % n

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314 and for Bacteria (n = 14, N = 132) was 89.39 %. As a second approach, a rarefaction analysis r
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315 was done to determine if screening of 415 clones was sufficient for an estimation of diversity ,

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316 in the clone libraries of the spring FJQ. The expected number of the unique OTUs was plotted 1
9

b
317 against the number of clones (Fig. 6). The calculated rarefaction curve of total OTUs showed y

g
318 a slight tendency to saturation; the curve for archaeal OTUs showed saturation but the one for u
e
s
t
319 bacterial OTUs did not reach saturation (Fig. 6), indicating that diversity was not completely
320 covered by screening of 415 clones.
321
322 Analyses of amoA (ammonia monooxygenase subunit A) related genes. Five clones
323 recovered from each sample type were randomly selected and sequenced. Two sequences
324 affiliated with amoA genes from the soil/sediment group (Fig. 5), whereas 13 clones were
325 more similar to amoA genes of marine water column or sediments. The sequences of amoA
326 related genes from Crenarchaeota showed similarities of 80.7 % (clone F2), 78 % (clone B2)
327 and 77.2 % (clone OT2), respectively, to candidatus Nitrosopumilus maritimus (26), using