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Microbial community structure in major habitats above 6000 m on Mount Everest LIU YongQin1, YAO TanDong1,2†, KANG ShiChang1,2, JIAO NianZhi3†, ZENG YongHui3, HUANG SiJun3 & LUO TingWei3 1

Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100085, China; State Key Laboratory of Cryospheric Science, Chinese Academy of Sciences, Lanzhou 730000, China; 3 State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China 2

Bacterial abundance in surface snow between 6600 and 8000 m a.s.l. on the northern slope of Mt. Everest was investigated by flow cytometry. Bacterial diversity in serac ice at 6000 m a.s.l., glacier meltwater at 6350 m, and surface snow at 6600 m a.s.l. was examined by constructing a 16S rRNA gene clone library. Bacterial abundance in snow was higher than that in the Antarctic but similar to other mountain regions in the world. Bacterial abundance in surface snow increased with altitude but showed no correlation with chemical parameters. Bacteria in the cryosphere on Mt. Everest were closely related to those isolated from soil, aquatic environments, plants, animals, humans and other frozen environments. Bacterial community structures in major habitats above 6000 m were variable. The Cytophaga-Flavobacterium-Bacteroides (CFB) group absolutely dominated in glacial meltwater, while β-Proteobacteria and the CFB group dominated in serac ice, and β-Proteobacteria and Actinobacteria dominated in surface snow. The remarkable differences among the habitats were most likely due to the bacterial post-deposition changes during acclimation processes. Mt. Everest, bacterial abundance, 16S rRNA, above 6000 m

Mt. Everest, the world’s highest mountain, plays an important role in global climate change[1,2]. Because it is so far away from human habitation, the area receives less impact from human activities, although the effects of global climate change have a large impact. Features of the snow and glaciers on Mt. Everest can be taken as a mirror of natural environmental change[3,4]. Physical and chemical characters of snow and glaciers in this region have been elaborated by glaciologists, but biological ― features have been little studied[1 7]. Proteolytic bacteria on the Gangotri glacier, Western Himalaya, and snow algae on the Yala glacier, on the south slope of Himalayan have been studied[8,9]. On the north slope of the Himalayas, only bacteria in the snow on the East Rongbuk glacier have been examined[10]. As one of the most sensitive indicators for the natural environmental changes, the microbial community of the high mountain regions www.scichina.com

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has drawn much attention. However, all the reported ― study sites were below 6000 m[11 14]. It was found that changes due to global warming at high-elevation sites were more pronounced as that at low elevations[15]. Regions above 6000 m a.s.l. on Mt. Everest are covered by snow and ice with little exposed rock. Snow accumulates to form ice and glaciers. At the edges of the glacier, are great seraces formed by partial melting of glacier. There is seasonally-dependent glacial meltwater, but no vegetation. The types of bacteria that are able to exist in this extreme environment, consisting of low temperaReceived March 29, 2007; accepted May 24, 2007 doi: 10.1007/s11434-007-0360-4 † Corresponding author (email: [email protected] or [email protected]) Supported by the Ministry of Science and Technology of China (Grant No. 2005CB422004), the National Natural Science Foundation of China (Grant Nos. 40121101 and 40401054), the Innovation Program (Grant No. KZCX3-SW-339), and the “Talent Project” of the Chinese Academy of Sciences, the Social Commonweal Research Project of Ministry of Science and Technology of China (2005DIA3J106)

Chinese Science Bulletin | September 2007 | vol. 52 | no. 17 | 2350-2357

1.1 Sample collection and measurement of environmental parameters

1.2 DNA extraction, PCR amplification, 16S rRNA gene clone library construction

All snow/ice samples were collected at different elevations on the north slope of Mt. Everest in April and May, 2005 (Figure 1). Surface snows (1―10 cm) were sampled along the climb route (6600―8000 m a.s.l.). About 2 L of surface snow at 6600 m a.s.l. was collected. One liter of glacial meltwater was collected at 6350 m a.s.l.. A block of ice was taken from the bottom of a serac (6000 m a.s.l.). Extreme caution was taken during the entire sampling process to avoid contamination. Poly-

Snow and glacial meltwater samples were thawed overnight at 4℃, and meltwater was filtered through a 0.22-μm filter (Millipore). The ice sample surface was cut two times successively, using two clean, sterilized knives, and then washed with cold 95% ethanol. The remaining inner sample was allowed to thaw at 4℃. Approximately 1 L of meltwater was filtered through a 0.22-μm filter (Millipore). The membrane was incubated with 1 mL GTE buffer (25 mmol/L Tris, 10 mmol/L

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1 Materials and methods

propylene Nalgene bottles used as sampling containers were washed and heat-sterilized in laboratory before their use in the field. All samples were kept frozen during transportation. Total bacterial abundance was analyzed by flow cytometry (Beckman Coulter, Epics Altra II) in the State Key Laboratory for Marine Environmental Science, Xiamen University. Major ions were analyzed via suppressed ion chromatography (Dionex 320 series instrument) in the laboratory of the Institute of Tibetan Plateau Research, Chinese Academy of Sciences.

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tures, extreme oligotrophy and strong UV radiation, is an interesting and important topic to both glaciologists and biologists. In the present study, we used flow cytometry to enumerate the total number of cells, and constructed a clone library of PCR-amplified bacterial 16S rRNA sequences to investigate the bacterial community structure in snow, glacial meltwater, and serac ice. We focused on the microbial features of different habitats above 6000 m.

Figure 1

Location map of serac, glacial meltwater and snow sampling sites above 6000 m on the north slope of Mt. Everest.

LIU YongQin et al. Chinese Science Bulletin | September 2007 | vol. 52 | no. 17 | 2350-2357

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EDTA, 50 mmol/L Sucrose, 20 mg/mL, pH 8.0) at 37℃ for 2 h. Then, proteinase K, NaCl and sodium dodecyl sulfate were added at final concentrations of 0.2 mg/mL, 0.7 mol/L and 1%, respectively. The mixture was incubated at 65℃ for 1.5 h. After extraction with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1), DNA in the aqueous phase was precipitated with an equal volume of isopropanol overnight at −20℃. To prevent contamination in each step, a negative parallel control was established by filtering 1 L of autoclaved deionized water with the same method as described above. After centrifugation and washing with 75% ethanol, DNA was dissolved in TE buffer. DNA preparations from both the sample and negative control were used as templates to amplify bacterial 16S rDNA genes. A primer pair, consisting of 27F (forward, 5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1392R (reverse, 5′-ACG GGC GGT GTG TRC-3′) was used. The PCR program was as follows: after an initial incubation at 94℃ for 5 min, 30 cycles were run with 94℃ for 1 min, 56℃ for 1 min and 72℃ for 1.5 min, followed by a final extension at 72℃ for 8 min. 1.3 16S rRNA gene clone libraries and statistical analysis The PCR products were purified using an Agarose gel DNA purification kit (TaKaRa Co., Dalian, China), ligated into a pGEM-T vector (TaKaRa Co., Dalian, China), and then transformed into E. coli DH5α. The presence of inserts was checked by ampicillin-resistance selection and colony PCR. 70―100 clones were selected randomly for re-amplification and restriction digestion by enzymes Hha I and Afa I. The digested fragments were visualized on a 3% agarose gel, and different clones were distinguished according to the RFLP patterns. One clone of each RFLP type was sequenced. Genetic diversity obtained by RFLP analysis was subTable 1

1.4 Homogenous analysis of 16S rRNA sequences All sequences obtained were checked for chimeric artifacts using the CHIMERA_CHECK program[17]. The nearest neighbors were retrieved from the NCBI (http://www.ncbi.nih.gov/ BLAST) through a BLAST search. All sequences were assigned to genus level grouping with 80% confidence by the “Classifier” program of RDP[17]. The nucleotide sequences of partial 16S rRNA genes have been deposited in the GenBank database with the accession numbers: EF190114 to EF190144, DQ DQ675465, DQ675469, DQ675500, DQ675487, DQ675470, DQ675471.

2 Results 2.1 Bacterial abundance and major ion concentrations in surface snow on the north slope of Mt. Everest Bacterial abundance and major ion concentrations in surface snow are listed in Table 1. Bacterial abundance tended to increase with altitude, but showed no obvious correlation with ion concentrations. 2.2 Bacterial community structures in the major habitats Three bacterial 16S rRNA gene clone libraries of serac ice (ESE), glacier meltwater (L), and surface snow at 6600 m (ESS) were constructed. A total of 240 clones were grouped by RFLP analysis, with 71 clones in ESE, 77 clones in L and 92 clones in ESS. Thirty-seven unique patterns were obtained. Chimera check results showed that all of the sequences obtained were normal. Coverage values of clone libraries were all over 80%, meaning that the clone numbers screened in each library were statistically sufficient for diversity analysis.

Bacterial abundance and major ion concentrations in surface snow along elevations on the north slope of Mt. Everest −1 −1 −1 − 3− − Cell abundance (104 cells/mL) Ca2+(μL·L−1) Mg2+(μL·L−1) NO3 (μL·L ) PO4 (μL·L ) NH4 (μL·L )

Altitude (m) 6600 6700 6800 6900 7000 7500 8000

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jected to statistical analysis with Coverage using the equation Coverage=1−(N/Individuals)[16]. Here, N represents the number of clones that occurred only once, and Individuals is the total clone numbers examined.

2.97 3.06 4.03 4.74 6.98 2.11 9.44

3978 206 2671 111 84 6182 −

160 12 111 4.42 4.18 189 −

638 63 431 40 80 66 −

220 2 0.94 3 6 34 −


12 6 29 16 17 34 −

SO2− (μL·L−1) 4 599 35 259 23 27 291 −

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New Zealand (AY315162) and soil (AY960773). Deinococcus is a remarkable bacterium and possesses the ability to tolerate very high doses of ionizing radiation[18]. Deinococcus-like clones accounted for 10%― 20% of the total snow bacteria in Antarctic. 2.3 Homogenous analysis of 16S rRNA gene sequences Nearest sequences and isolates of all the 16S rRNA gene sequences were retrieved from the GenBank database through gaped-Blast analysis. Sequences in the cryosphere at high elevation regions on Mt. Everest were similar to environmental 16S rRNA gene sequences in the database with identity values of 93%― 100%, and to those from pure cultures with identity values of 89% ― 100%. Among 37 sequences, 23 sequences’ identity values were more than 98%―100%, accounting for 62% of total sequences. Twenty-one sequences’ identity values with isolates were more than 98%―100%, accounting for 58%. Source environment information for nearest neighbors was retrieved from the GenBank database for 37 clones with identity values of more than 97% (Table 2). Eight sequences had nearest neighbors isolated from the cryosphere such as Antarctica, the Arctic, permafrost and glaciers; 12 sequences from aquatic environments; 14 sequences from soil and sediment; and 14 sequences from plants, animals or humans. Eleven sequences’ identity values with their nearest neighbor were lower than 97%.

3 Discussion 3.1 Relationship between snow bacterial abundance and environment Bacterial abundance and major ion concentrations in surface snow from 6600 to 8000 m on the north slope of Mt. Everest are listed in Table 1. Bacterial abundance in surface snow increased with altitude, but was not correlated with chemical parameters reflecting nutrient levels. Snow with higher nutrient levels (higher concentrations of PO43−, NO3−, NH4+) did not contain more bacteria. This indicated that nutrition was not the determining factor of bacterial abundance. The atmosphere has been reported to serve as carrier of organisms, and bacteria could be contained in ice nuclei which are suspended in the atmosphere[18,19]. Bacteria in the Malan and Puruogangri


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(i) Bacterial community of serac ice. The sequences were affiliated with 7 phylogenetic groups: α-, β-, γ-Proteobacteria, Actinobacteria, Firmicutes, CFB and Cyanobacteria. The dominant group was β-Proteobacteria, accounting for 44% of the total clones. All clones were from an unclassified genus in the Oxalobacteraceae family. Among 4 sequences of β-Proteobacteria, 3 sequences’ nearest neighbors were isolated from glacier or tundra, and 1 was identified from soil. Sequence ESE-2 accounted for 40% of total clones, and its nearest neighbor was isolated from the Canada glacier, Antarctica (AF479326, identity value 98%). Clones that fell into the groups, CFB, γ-Proteobacteria and α-Proteobacteria, accounted for 21%, 14% and 13% of total clones, respectively. The nearest neighbors of most clones were isolated from soil. Sequence ESE-68 (genus Devosia, α-Proteobacteria) was similar to sequences isolated from an Antarctic lake (UBA440974, identity value 97%), Arctic sea ice (AF468359, identity value 97%), and the Franz Josef Glacier, New Zealand (AY315165, identity value 98%). (ii) Bacterial community of glacial meltwater. This clone library was almost completely dominated by the genus Flavobacterium of the CFB group (94.8% of clones). Only two genera, Acidovorax and Polaromonas, belonging to β-Proteobacteria were found, represented by 4 clones. Among the 4 sequences affiliated with the genus Flavobacterium, three sequences’ nearest neighbors were isolated from an Antarctic lake and the other is derived from a river. Clones of sequence L-4-59 accounted for 85%, and the nearest neighbors (AJ551150, identity value 97%) were from lake sediment in Ardley Island, Antarctica. The nearest neighbors of 2 sequences belonging to β-Proteobacteria were from Arctic sea ice (AF468326) and bottle water (AF442523), with identity values of 99%. This library featured the lowest diversity among all samples and showed dominance by psychrophilic bacteria. (iii) Bacterial community of snow at 6600 m. Nearly half (46%) of clones from the EFS clone library belonged to β-Proteobacteria. Others belonged to Actinobacteria (34.78%), α-Proteobacteria (13.04%), CFB (2.17%), Deinococci (2.17%) or were unclassified (2.17%). The nearest neighbors were isolated from glacier, soil and aquatic environments. Sequence ESS-2 was classified to genus Deinococci in RDP, and was nearest to Deinococci bacteria isolated from the Franz Josef Glaciers,

Table 2 Affiliation of bacterial 16S rRNA gene sequences in the cryosphere above 6000 m on the north slope of Mt. Everest and the isolated environment of their nearest neighborsa) RDP classified Isolated environment of the nearest neighbor in NCBI genus confidence value aquatic environments soil/sediment cryosphere other ESE-7 EF190117 Agrobacterium 98% α-Proteobacteria ESE-27 EF190122 Agrobacterium 93% α-Proteobacteria ESE-59 EF190128 unclassified 66% + +T α-Proteobacteria ESE-68 EF190129 Devosia 100% + +G + α-Proteobacteria ESE-29 EF190124 unclassified 64% β-Proteobacteria ESE-2 EF190114 unclassified 64% + +G + β-Proteobacteria ESE-14 EF190119 unclassified 52% + + +G + β-Proteobacteria ESE-19 EF190120 unclassified 52% + + +G,P β-Proteobacteria ESE-33 EF190126 Acinetobacter 100% + + γ-Proteobacteria ESE-8 EF190118 Acinetobacter 100% + γ-Proteobacteria ESE-54 EF190127 Acinetobacter 100% + γ-Proteobacteria ESE-72 EF190130 Acinetobacter 100% + γ-Proteobacteria ESE-21 EF190121 Actinobacteria unclassified 48% + ESE-78 EF190132 Actinobacteria Brevibacterium 100% + + ESE-28 EF190123 Firmicutes Anoxybacillus 100% + + Anoxybacillus 100% + + ESE-75 EF190131 Firmicutes ESE-3 EF190115 CFB unclassified 38% + ESE-4 EF190116 CFB Pedobacter 100% ESE-32 EF190125 Cyanobacteria unclassified 17% L-4-25 DQ675500 Acidovorax 100% + + + β-Proteobacteria L-4-30 DQ675487 Polaromonas 100% + + +G,A,An β-Proteobacteria L-2-12 DQ675465 CFB Flavobacterium 100% L-4-24 DQ675469 CFB Flavobacterium 100% L-4-59 DQ675470 CFB Flavobacterium 100% L-4-62 DQ675471 CFB Flavobacterium 100% ESS-45 EF190143 unclassified 75% + + α-Proteobacteria ESS-31 EF190141 Methylobacterium 100% α-Proteobacteria ESS-9 EF190134 unclassified 52% β-Proteobacteria ESS-13 EF190135 Polaromonas 100% + + β-Proteobacteria ESS-14 EF190136 Polaromonas 96% β-Proteobacteria ESS-22 EF190139 Polaromonas 100% +A β-Proteobacteria ESS-21 EF190138 unclassified 56% + + β-Proteobacteria ESS-27 EF190140 Actinobacteria Kocuria 100% + + + ESS-7 EF190133 Actinobacteria unclassified 58% ESS-19 EF190137 CFB Flavobacterium 100% +G ESS-32 EF190142 Deinococci Deinococcus 100% ESS-81 EF190144 unclassified 57% + + a) +T, Tibetan Plateau; +An, Antarctic; +A, Arctic; +G, Glacier; +P, permafrost. Clone

GenBan Accession No.

Classified

glaciers on the Tibetan Plateau were shown to be transported by the atmosphere[20,21]. Bacteria in snow on Mt. Everest were possibly imported by atmospheric transportation from other areas. During precipitation, bacterial cells are deposited within snow. Bacterial abundance was closely related with dust concentration in Malan ice cores, and clearly correlated with Ca2+ concentration in Puruogangri ice cores[20,21]. However, bacterial abundance showed no correlation with Ca2+ concentration on Mt. Everest. This result is possibly due to changes in Ca2+ concentration caused by local rock (i.e. exposed rock around 7500 m). Influenced by temperature, nutri2354

tion and radiation, snow algal biomass decreased with altitude on the south slope of Himalaya[8], but bacterial abundance in snow increased with altitude on the north slope. Bacterial abundance in the snow is controlled by many factors, such as the number of bacteria in a snowfall and bacterial growth conditions after deposition in the glacier. Bacteria in situ growth conditions in high altitude regions on Mt Everest are still unclear even now. We only analyzed possible factors that could influence bacterial abundance in the course of transportation and precipitation. Bacteria deposited first as snowfall in the higher altitudes may cause the greater


3.2 Comparison of snow bacterial abundance to Antarctica and other high mount regions Remarkable differences in bacterial abundance between snow on Mt. Everest and Antarctica was observed, with those on Mt. Everest 10―100 fold those found in Antarctica. Bacterial population densities were reported from 200 to 5000 cells/mL in South Pole snow[22], and 3000 cells in Rose ice shelf[23]. This difference was possibly due to the fact that Mt. Everest is closer to major sources of airborne microorganisms, such as exposed soils and tropical ecosystems. This difference was also observed in polar and non-polar glacial ice[24]. Snow bacterial abundance on Mt. Everest was similar to that on other high mountains. Bacterial abundance in surface snow on Mount Sonnblick (3106 m, Austria)[19] was from 9.5×103 to 1.3×104 cells/mL, and bacterial abundance in snow pit on Tateyama Mountains, Japan (2450 m, a.s.l.) was from 6.0×103 to 2.3×105 cells/mL, averagely 0.84×104 cells/mL[14]. In spite of similar bacterial abundance, the bacterial community structures on Mt. Everest were remarkably different from those on the Tateyama Mountains due to differences in the geographical environment. Various bacterial communities existed in snow deposited in March, June and September on the Tateyama Mountains. Bacillus/Clostridium and Actinobacteria were dominant groups, accounting for 36.1% and 34.8%, and α, β, γ-Proteobacteria accounted for 10.6%, 3% and 15.1%, respectively, in snow deposited at March. β-Proteobacteria dominated in June snow, accounting for 67.7%, while CFB and γ-Proteobacteria accounted for 12.5% and 7.3% in June snow. β-Proteobacteria also dominated in Augest snow, accounting for 52.9%, but CFB bacteria increased to 35.6%. Snow samples on Mt. Everest in this study were collected in April and had different bacterial community structures than those from all 3 months on the Tateyama Mountains. Different atmospheres presumably transferred different bacteria to these mountains, and rapid growth of some genera during the melting season in the Tateyama Mountains may change

3.3 Variable bacterial community structure in major habitats Snow, serac ice and glacial meltwater represented major bacterial habitats, and revealed bacteria carried by the atmosphere and their post-deposition change in this extreme environment. Thus, the snow sample at 6600 m directly reflects the bacterial community of snowfall (atmosphere), and bacterial features represent the type of bacteria that are transported and deposited in this region. Serac ice forms by compression and recrystallization of snow that has accumulated on the glacier surface, and so the bacteria in serac ice represent the community that can endure harsh environments and survive for a long time (maybe hundreds years). Glacial meltwater at 6350 m only occurred in summer and, therefore, the bacteria in meltwater represents the community that is well adapted to the aquatic niche resulting from melted ice/snow. The bacterial community composition and the dominant component of each of these samples were quite different (Figure 2). The CFB group dominated in glacial meltwater, while β-Proteobacteria was the dominant group in serac ice and snow. The bacteria in serac ice were different from those in snow, and showed a decrease of Actinobacteria and an increase of the CFB and γ-Proteobacteria groups. Meanwhile, serac ice bacteria were more psychrophilic than snow bacteria, with more clones similar to those isolated from the cryosphere. These results imply that bacteria may undergo environmental selection or acclimation after deposition in the snow. Psychrophilic or psychrotrophic bacteria, such as those affiliated with β-Proteobacteria and the CFB group in serac ice and snow, may endure low temperatures and survive or even grow after they are captured in serac ice. Other bacteria, i.e. Actinobacteria, may be out-competed due to non-adaptation to this harsh environment. The bacteria in meltwater were remarkably different from those found in ice and snow, in that they exhibited low diversity and were almost completely dominated by a psychrophilic genus, Flavobacterium. The notable distinction in the type of bacterial community was most likely caused by the change in niche from snow/ice to water and, consequently, differences in selection pressure on the bacterial community. Our research revealed the distinctive differences among bacterial communities in major habitats in high altitude regions on Mt. Everest, however the factors that lead to


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the bacterial community.

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bacterial abundance observed at higher altitudes. In addition, more snowfall at higher altitudes could possibly account for the greater bacterial abundance. However, the relationship between bacterial abundance and the environment needs to be more carefully researched in the future.

these differences are still unknown and future work on the possible causes is required.

and Antarctic ice cores, high mountain glaciers and other frozen environments, were also found in the ― cryosphere on Mt. Everest[24 28].

4 Conclusion

Figure 2 Comparisons of microbial community structures in major habitats above 6000 m on the north slope of Mt. Everest.

Although bacterial community structures were different among snow, ice and glacial meltwater, homogenous analysis indicated that bacteria in the cryosphere on Mt. Everest were closely related to those obtained from other environments (Table 2). This result suggested that most of bacteria on Mt. Everest were most likely carried by the atmosphere, and derived from different sources. Bacteria in major habitats on Mt. Everest also showed psychrophilic characteristics. Most of clones were similar to those isolated from cold environments. Psychrophilic and psychrotolerant bacteria, such as Flavobacterium (in the CFB group), found in the Arctic 1

Bacterial abundance in surface snow ranged from 2.11×104 to 9.44×104 cells/mL between 6600 and 8000 m a.s.l. on Mt. Everest, 10―100 fold that found in Antarctic snow. Surface snow bacterial abundance increased with altitude but showed no correlation with chemical parameters, suggesting that nutrient level was unlikely to be the determining factor of bacterial abundance. Bacterial abundance in snow on Mt. Everest was similar to other mountain regions in the world, however the community structure was different. The results show that bacteria varied depending on the geographic environment. Most of bacteria in the cryosphere on Mt. Everest were closely related to those obtained from other environments, showing the bacteria source from various environments. But most bacteria were near to those isolated from cold environment. Bacterial community structures were different among snow, ice and glacial meltwater. The CFB group dominated in glacier meltwater, while β-Proteobacteria dominated in serac ice and snow. The remarkable differences in bacterial community structure among the habitats were most likely due to post-deposition changes in bacterial abundance during the acclimation processes. The authors thank Prof. John Hodgkiss for his assistance with English.

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