Editor: Clive Edwards
Microbial community structure in moraine lakes and glacial meltwaters, Mount Everest
Version of Record online: 18 OCT 2006
FEMS Microbiology Letters
Volume 265, Issue 1, pages 98–105, December 2006
How to Cite
Liu, Y., Yao, T., Jiao, N., Kang, S., Zeng, Y. and Huang, S. (2006), Microbial community structure in moraine lakes and glacial meltwaters, Mount Everest. FEMS Microbiology Letters, 265: 98–105. doi: 10.1111/j.1574-6968.2006.00477.x
- Issue online: 18 OCT 2006
- Version of Record online: 18 OCT 2006
- Received 9 July 2006; revised 11 September 2006; accepted 11 September 2006.First published online 18 October 2006
- Mount Everest;
- moraine lake;
- glacial meltwater;
- bacterial diversity and abundance
- Top of page
- Materials and methods
The bacterial diversity and abundance in two moraine lakes and two glacial meltwaters (5140, 5152, 5800 and 6350 m above sea level, respectively) in the remote Mount Everest region were examined through 16S rRNA gene clone library and flow cytometry approaches. In total, 247 clones were screened by RFLP and 60 16S rRNA gene sequences were obtained, belonging to the following groups: Proteobacteria (8% alpha subdivision, 21% beta subdivision, and 1% gamma subdivision), Cytophaga–Flavobacteria–Bacteroides (CFB) (54%), Actinobacteria (4%), Planctomycetes (2%), Verrucomicrobia (2%), Fibrobacteres (1%) and Eukaryotic chroloplast (3%), respectively. The high dominance of CFB distinguished the Mount Everest waters from other mountain lakes. The highest bacterial abundance and diversity occurred in the open moraine lake at 5152 m, and the lowest in the glacial meltwater at 6350 m. Low temperature at high altitude is considered to be critical for component dominancy. At the same altitude, nutrient availability plays a role in regulating population structure. Our results also show that the bacteria in Mount Everest may be derived from different sources.
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- Materials and methods
Remote high mountain lakes, being far from habitation and located in extreme environments, receive less impact from human activities but magnify the effects of global climate changes, and can thus be taken as a mirror of natural environmental changes (Pechlaner, 1971; Patrick et al., 1998). Since the 1990s, microbial community structure in such special waters has drawn much attention from scientists and considerable work has been carried out in the Pyrenees, Alpine regions and Yunnan Plateau, China (Alfreider et al., 1996; Jiang & Xu, 1996; Felip et al., 1999; Felip et al., 2002). A typical case was the EU MOLAR Project on ‘Measuring and modeling the dynamic response of remote mountain lake ecosystems to environmental change’, in which a systematic investigation was conducted (Callieri et al., 1999; Pugnetti & Bettinetti, 1999; Marchetto et al., 2004). It was found that changes due to global warming at high-elevation sites were more pronounced than that at low elevations (Giorgi et al., 1997; Liu & Chen, 2000). However, all the reported study sites were below 3000 m, and, therefore, investigations at higher locations are desirable.
Mount Everest plays an important role in global climate change (Qin et al., 2000; Ren et al., 2004). There is about 800 km2 of glacial area with a total volume of about 82 km3 in the Everest region (Shi, 2000). Glacial melting supplies water for inhabitants and livestock along the Rongbuk River. A string of moraine lakes is situated at the terminus of the Rongbuk Glacier. Owing to harsh environmental conditions, sampling is extremely difficult, and therefore, the bacterial community structure in these aquatic systems (moraine lake and glacial meltwater) remained unknown. To fill these blanks, we explored the bacterial abundance, diversity and community structure in two moraine lakes at 5140 m and 5152 m, and two glacial meltwater sites at 5800 m and 6350 m above sea level (a.s.l.) in the Rongbuk Glacier area (Fig. 1). This should provide a better understanding of the linkage between microbial features and environmental conditions in such special waters above 5000 m a.s.l.
Materials and methods
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- Materials and methods
Sample collection and measurement of environmental parameters
Surface water samples were collected at four sites (L1–L4) on the northern slope of Mount Everest in April and May, 2005 (Fig. 1). L1 and L2 are moraine lakes, located at the terminus of the Rongbuk Glacier, at altitudes of 5140 m and 5152 m, respectively. L3 (5800 m a.s.l.) and L4 (6350 m a.s.l.) are glacial meltwaters in the ablation area of the East Rongbuk Glacier. Lake L1 is clear, small and closed, about 4 m in depth with a stone bottom and a surrounding moraine. Lake L2's area is greater than L1, with a depth of 6.7 m and a soil and silt surrounding. Waters in lake L2 are turbid and outflow to the Rongbuk River. Extreme caution was exercised at all times during the entire sampling process to avoid contamination. Prewashed and sterilized polypropylene Nalgene bottles were used as sample containers. All instruments and solutions were sterilized twice. All samples were kept frozen during the transport process.
Total bacterial abundance was analyzed by flow cytometry (Beckman Coulter, Epics Altra II). SYBR Green (Molecular Probes) was used as the nucleic acid stain. Parallel samples of every station were measured. Water samples (250 mL) from L1, L2 and L4 were filtered through a GF/F filter (Whatman), and chlorophyll a (Chl a) was measured using a Fluoro spectrophotometer (Shimadzu Corp., Japan). Chl a was not measured at L3 due to insufficient sample volume. Total organic carbon was analyzed by TOC-Vcph (Shimadzu Corp., Japan). Each sample was measured three times with a relative standard deviation lower than 10%. Particle number concentration was measured by a Backman Coulter Counter Multisizer with a precision of 3.9% (N=20).
DNA extraction, PCR amplification, 16S rRNA gene clone library construction and statistical analysis of clone libraries
Approximately 200–400 mL of each frozen sample was melted at 4°C, and filtered through a 0.22 μm filter (Millipore). Community DNA was extracted according to the procedures described by Gordon and Giovannoni (1996). To prevent contamination in any step of the procedure, a negative control was established by filtering 400 mL of autoclaved deionized water using the same method as described above. DNA preparations from samples and the negative control were used as templates to amplify the bacterial 16S rRNA genes. The primers used were 27F (forward, 5′-AGAGTTTGATCMTGGCTCAG-3′) and 1392R (reverse, 5′-ACGGGCGGTGTGTRC-3′). The PCR program was as follows: after an initial incubation at 94°C for 5 min, 30 cycles were run at 94°C for 1 min, 56°C for 1 min and 72°C for 1.5 min, followed by a final extension at 72°C for 8 min.
The PCR products were purified using an Agarose gel DNA purification kit (TaKaRa Co., Japan), ligated into a pMD18-T vector (TaKaRa Co., Japan) and then transformed into competent Escherichia coli DH5α. The presence of inserts was checked by ampicillin resistance selection and colony PCR.
For each clone library, 75–100 clones were selected randomly for reamplification and restriction digest by enzymes HhaI and AfaI (TaKaRa Co., Japan). The digested fragments were visualized on a 3% agarose gel, and different clones were discriminated according to the RFLP patterns. One clone of each RFLP type was sequenced.
Genetic diversity obtained by RFLP analysis was subjected to statistical analysis including the following indexes: (1) Taxa, the total numbers of RFLP patterns in each library; (2) Individuals, the total clone numbers examined; (3) Coverage, which was derived from the equation Coverage=1−(N/Individuals), N being the number of clones that occurred only once (Kemp & Aller, 2004); and (4) Diversity indexes (Dominance, Evenness, Shannon, Simpson), calculated using the statistical program PAST (http://folk.uio.no/ohammer/past).
Sequencing and phylogenetic analysis
All sequences obtained were checked for chimeric artifacts using the CHIMERA_CHECK program (Maidak et al., 2001). The nearest neighbors were retrieved from the NCBI (http://www.ncbi.nih.gov/BLAST) through the blast search and from the Ribosomal Database Project 9.0 (RDP, http://rdp.cme.msu.edu) through the Sequence Match tool. All sequences were assigned to the genus level grouping with 80% confidence by the ‘Classifier’ program of RDP (Cole et al., 2005).
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- Materials and methods
Bacterial abundance, chlorophyll concentration, total organic carbon and particle number concentration
Remarkable differences in ecological variables were found among the four sampling sites (Table 1). Except for L1, the total bacterial abundance varied from nearly 105 cells mL−1 in L2 to 103 cells mL−1 in L4, showing a decreasing trend with altitude. Particles of 1–15 μm showed a pattern similar to that of bacterial abundance. The chlorophyll concentration in L2 was also the highest, which was fourfold and eightfold those in L1 and L4, respectively. The highest total organic carbon also occurred in L2, followed by L1 and L4. L1 was distinguished from the other sites by the much lower values of all four parameters relative to L2, which is located at essentially the same altitude. The four ecological variables showed the same tendency among these stations, indicating the close relationship between biological community and physicochemical characteristics in the lakes and glacial meltwaters, Mount Everest.
|Station||Alt (m/a.s.l)||Location||Chlorophyll a (μg L−1)||Bacterial abundance (104 cells mL−1)||TOC (μg L−1)||Particles (105) (1.13–14.93 μm)|
|L1||5140||N 28°07′, E 86°51′||0.04||1.81||1.79||1.25|
|L2||5152||N 28°07′, E 86°51′||0.17||8.41||2.15||4.01|
|L3||5800||N 28°05′, E 86°54′||–||3.85||–||1.88|
|L4||6350||N 27°59′, E 86°55′||0.02||0.29||0.39||0.29|
16 rRNA gene clone libraries and statistical analysis
Four 16S rRNA gene clone libraries for moraine lakes and glacial meltwaters in Mount Everest were constructed. In total, 247 clones were subjected to RFLP screening. Negative control did not yield the amplification products, confirming the credibility of the results. The coverage values of all four clone libraries were greater than 70%, meaning that the clone numbers screened in each library were statistically enough for diversity analysis. After RFLP screening, all unique patterns were subjected to sequencing analysis. In total, 60 sequences were obtained and identified as normal 16S rRNA gene sequences using the CHIMERA_CHECK program. Table 2 lists the taxa, individuals and diversity indexes of sites L1–L4. The number of unique RFLP patterns that occurred in the library was 11 (17% in total patterns, 10% in total clones) in L1, 26 (40% in total patterns, 16% in total clones) in L2, 13 (20% in total patterns, 21% in total clones) in L3 and five (8% in total patterns, 30% in total clones) in L4, also consistent with the highest Evenness value for L2 and the lowest for L4. The Shannon and Simpson indexes were both at their highest value at L2 and lowest at L4, suggesting the highest overall diversity at L2 and the lowest at L4, L1 and L2 shared seven common RFLP patterns, accounting for 60% and 43% of total clones, respectively, which means that the two moraine lakes share a large number of common groups.
Sequences of moraine lakes and glacial meltwaters in Mount Everest were subjected to Classifier analysis through the RDP database to determine their phylogenetic affiliation. The classifier result of each sequence was verified by construction of a phylogenetic tree (data not shown), and the results indicated that the genus determined with a confidence value of more than 80% was phylogenetically convincible. All the sequences obtained in this work were affiliated with nine phylogenetic groups, i.e. alpha, beta, gammaproteobacteria, Actinobacteria, Cytophaga–Flavobacterium–Bacteroides (CFB), Fibrobacteres, Planctomycetes, Verrucomicrobia and E. chroloplast. Table 3 lists all the sequences and the nearest neighbors. The dominant groups were the phylum CFB and subclass Betaproteobacteria, accounting for 54% and 21% of the total clones. Clones that fell into the groups Alphaproteobacteria, Actinobacteria, E. chroloplast, Gammaproteobacteria, Planctomycetes, Verrucomicrobia and Fibrobacteres accounted for 8%, 4%, 3%, 2%, 2%, 2% and 1% of total clones, respectively.
|Category||Clone||Nearest neighbor||RDP classifier|
|Sequence||Identity (%)||Isolated environment||Genus||Value (%)|
|Alphaproteobacteria||L-1-17||AJ227793||98||Freshwater slough adjacent to Lake Washington, USA||Brevundimonas||100|
|L-3-30||AJ303009||98||Cold fluidized-bed process treating chlorophenol-contaminated groundwater in Finland||Novosphingobium||99|
|L-1-27||AJ785998||95||Sediments of Taihu Lake, China||Phenylobacterium||81|
|L-1-42||AY584572||96||Crater Lake, Oregon, USA (ultraoligotrophic lake, at the crest of the Cascade Mountains)|
|L-3-51||AY662025||98||Groundwater contaminated with high levels of nitric acid-bearing uranium waste|
|L-3-80||AY792291||95||Shallow humic lake in Wisconsin, USA|
|L-2-8||AJ290043||96||Gossenkoellesee Lake, Austria (a small oligotrophic high-mountain lake)|
|Betaproteobacteria||L-4-25||AF442523||99||Bromate-reducing microorganisms from drinking water||Acidovorax||100|
|L-3-58||AJ582191||99||Uranium mining wastes||Hydrogenophaga||100|
|L-3-40||AF523046||99||Natural mineral water after bottling||Polaromonas||97|
|L-4-30||AF468326||99||Melt pond on Arctic sea ice floe||Polaromonas||81|
|L-1-68||AY584576||98||Crater Lake, Oregon, USA||Polaromonas||100|
|L-3-20||AY315174||98||Subglacial sediments and ice from two Southern Hemisphere glaciers||Polaromonas||100|
|L-3-52||AY584576||97||Crater Lake, Oregon, USA||Polaromonas||100|
|L-3-21||AJ421913||98||Elbe River,Germany (polluted river)||Rhodoferax||100|
|L-3-64||AJ422162||99||Elbe River, Germany (polluted river)||Rhodoferax||100|
|L-3-65||AY315172||98||Subglacier sediments and ice from two Southern Hemisphere glaciers||Rhodoferax||100|
|L-2-62||AY863102||94||Wastewater treatment pools|
|L-2-69||DQ066954||96||Sediment of Lake Washington, USA|
|L-1-85||DQ088796||99||High-pressure, alkaline, saline groundwater at 2.8 km below land surface in a South African gold mine|
|L-2-1||AY212692||96||Water 10 m downstream of equine manure pile|
|L-2-21||AY145571||94||Freshwater section of the Weser estuary, Germany|
|L-2-32||AJ224990||99||Gossenkoellesee Lake, Austria (a small oligotrophic high-mountain lake)|
|Gammaproteobacteria||L-3-81||AY212714||97||Water downstream of equine manure pile|
|Actinobacteria||L-2-79||AJ575508||99||Lake North Mamry, Poland|
|CFB||L-2-6||AY752122||96||Massif Central, Sep Reservoir, France||Chitinophaga||100|
|L-1-16||AF433173||97||China No. 1 glacier||Flavobacterium||83|
|L-4-24||AF493656||97||Epilitho from River Taff, UK||Flavobacterium||100|
|L-1-48||AB180738||97||Granules used in a wastewater treatment plant||Flavobacterium||100|
|L-3-50||AJ290033||97||Gossenkoellesee Lake, Austria (a small oligotrophic high-mountain lake)||Flectobacillus||100|
|L-2-4||AF289149||97||Parker River, USA (a small woodland river, mesotrophic)||Flectobacillus||100|
|L-3-25||AF414577||97||Uranium Mine Sediment||Pedobacter||100|
|L-2-14||AY509263||99||Swedish lake (Eutrophic lakes)|
|L-4-59||AJ551150||97||Lake sediments of Ardley Island, Antarctica||Flavobacterium||100|
|L-3-23||AJ601392||96||Microbial mats in Antarctic lakes||Flavobacterium||100|
|L-2-12||AJ585428||96||Microbial mats in Antarctic lakes||Flavobacterium||100|
|Planctomycetes||L-2-15||AF391976||92||Extreme thermal soil, Yellowstone Park||Gemmata||83|
|Verrucomicrobia||L-2-5||AF316729||98||Crater Lake, Oregon, USA|
|Eukaryotic chroloplast||L-2-51||AF316703||93||Crater Lake, Oregon, USA|
Figure 2 shows the bacterial community structures in L1–L4. Bacteria in moraine lake L2 belonged to eight groups comprising 11 genera, while those in L1 belonged to seven groups comprising 10 genera. Bacterial diversities in glacial meltwaters were much lower than those in moraine lakes: those in L3 belonged to four groups comprising nine genera (alpha, beta, Gammaproteobacteria, CFB), and those in L4 only belonged to two groups comprising three genera (Betaproteobacteria, CFB), with the lowest diversity.
Homogenous analysis of 16S rRNA gene sequences
Nearest neighbors of all the 16S rRNA gene sequences in L1–L4 were retrieved from the GenBank database through gaped-blast analysis (Table 3). The corresponding source information of neighbors was also obtained from the database. Among 60 sequences, 35 were nearest to the clones collected from rivers, lakes and water, with 11 sequences' identity values of 93–96%, and 24 sequences' identity values of more than 97%. Seven sequences were nearest to the clones from the sediments of lakes, rivers and springs with identity values of 93–97%. Four sequences had a high identity value with the clones that were isolated from glaciers and sea ice with identity values of 97–99%. Thirteen sequences were nearest to clones in environments contaminated by organic substances or mining wastes with identity values of 94–99%. These results show that the bacteria in Mount Everest may be from versatile sources.
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- Materials and methods
Lower bacterial abundance in moraine lakes and glacial meltwaters in Mount Everest
Bacterial abundance in the elevated moraine lakes and glacial meltwaters in Mount Everest was lower than those in mountain lakes at altitudes less than 5000 m a.s.l. in other regions. The highest abundance occurred in L2 (8.41 × 104 cells mL−1), which was still low compared with other mountain lakes such as the Gossenlollesee Lake at 2417 m in the Alps (3 × 105 cells mL−1) (Pernthaler et al., 1997) and the case of 12 selected mountain lakes in the MOLAR project (6 × 104–9.79 × 106 cells mL−1) (Straškrabováet al., 1999). The decreasing trend in bacterial abundance with increasing altitude found in the present study suggests that altitude is an important factor controlling bacterial abundance. The abundance values also correlated positively with particle concentration and total organic carbon at each sampling site.
Quite different dominant component in the moraine lakes and glacial meltwaters of Mount Everest
Although bacterial community composition in the moraine lakes and glacial meltwaters of Mount Everest were similar to those in other freshwater habitats, the dominant component was quite different. Zwart et al. (2002) analyzed bacterial 16S rRNA gene sequences from 11 lakes and four rivers worldwide and found that the major bacterial divisions represented in freshwater were the Alpha, Betaproteobacteria, CFB group, Actinobacteria and Verrucomicrobia (Zwart et al., 2002). Most of the bacteria in the aquatic system of Mount Everest belonged to these groups, but the dominant bacteria in moraine lakes and glacial meltwaters were different from those in other mountain or polar regions. The Betaproteobacteria were dominant in small oligotrophic lakes in the Adirondack Mountain, USA, Alfreider Mountain, Germany, and the Arctic (Hiorns et al., 1997; Zwart et al., 2002). In the case of Mount Everest, the dominant group was the CFB group, accounting for 57% of the total clones. Betaproteobacteria were the second largest group, accounting for 21% of total clones. Although Actinobacteria are distributed widely in lakes and rivers, being the dominant group in the oligotrophic Crater Lake, USA, this group only existed in moraine lake L2, where it accounted for 13% of total clones.
Low temperature at high altitude is critical for dominant component
Four sites in our studies shared common genera, although they displayed remarkable differences in habitat condition. The altitude difference was more than 1200 m between the lowest and highest sites. Flavobacterium, in the CFB group, existed in all four sites. Bacteria belonging to this genus were isolated from other cryospheres, i.e. snow in the East Rongbuk Glacier, Mount Everest; Malan and Guliya ice core from the Tibetan Plateau; Muztag Ata Glacier; and Tianshan No.1 Glacier from western China (Christner et al., 2000; Zhang et al., 2003; Zhu et al., 2003; Xiang et al., 2004; Xiang et al., 2005; Liu et al., 2006). Flavobacterium species have a distinct predilection to low salinity, cool to cold environments, and are commonly isolated from polar lakes, and from streams, rivers, lakes and muddy soils in other cold environments (Dworkin). Most Flavobacterium species are psychrotolerant, and grow well at 4°C. Research on bacteria in Arctic sea ice showed an increase of CFB abundance at lower temperatures (Junge et al., 2004). Owing to the psychrotolerant character of Flavobacterium, bacteria belonging to this genus occurred in cold lake and glacial meltwater in Mount Everest. Flavobacterium was dominant, accounting for 94% of total clones, in glacial meltwater at 6350 m (a.s.l.), where the temperature was the lowest. Polaromonas (Betaproteobacteria) occurred in L1, L3 and L4 in a low proportion (1–4% of total clone). Bacteria of the genus Polaromonas are psychrophilic, and have been isolated from snow in the East Rongbuk Glacier, Mount Everest and in Antarctic sea ice (Irgens et al., 1996; Liu et al., 2006).
At the same altitude, nutrients play a role in regulating bacterial community structure
L1 and L2 were both moraine lakes at the terminus of glaciers with similar altitude and temperature. L1 and L2 shared seven common genera, and bacteria belonging to these common genera accounted for about 60% and 40% of the total, respectively. The nearest clone neighbors of sequences from L1 and L2 were mostly isolated from lakes and rivers. Although homogeneous features existed in the two moraine lakes, the bacterial abundance and diversity were different. Abundance in L2 was almost four times that in L1, and diversity indexes in L2 were higher than those in L1. The nutrient condition is most likely responsible for these differences in bacterial community structure between L1 and L2. The area of L2 is five times larger than that of L1. L1 is a small closed lake, and the water source is the melt water from ice under the moraine. L2 waters are from glacial meltwater, precipitation and a spring, which have been influenced by local rocks and soil on the northern slope of Mount Everest. Thus, the drainage of L2 is much larger than that of L1, which causes greater nutrient input into L2. Eight sequences in L2 had nearest neighbors from eutrophic lakes, more than those in L1 (1 sequence). Similarly, both bacterial abundance and diversity in L2 with its high Chl a concentration were higher than in L1 (Table 1), supporting the previous conclusion that bacterial diversity was positively related to lake productivity (Yannarell & Triplett, 2005).
Natural differences affecting the bacterial community structure and diversity
Bacterial community structure in the glacial meltwaters L3 and L4 was distinct from that in moraine lakes L1 and L2. Planctomycetes and Verrucomicrobia, which occurred in moraine lakes (L1 and L2) and other freshwaters (Dworkin; Zwart et al., 2002), did not occur in L3 and L4. L3 and L4 are situated in the high-elevation serac region without vegetation cover. Nutrients for the bacteria came from atmospheric wet and dry deposition, and had much lower values than at L1 and L2. Bacteria in L3 and L4 were exposed to harsh abiotic conditions, such as lower temperature, stronger radiation, lower oxygen concentration and nutrients. Only those bacteria with the ability to tolerate extreme living conditions could survive, which resulted in lower bacterial diversity in L3 and L4. Bacterial diversity was the lowest at the highest altitude site L4, with only three genera. Bacteria in L4 exhibited the greater resistance to cold with four sequences nearest to clones from sea ice or sediment in the Antarctic and Arctic. Bacteria in L4 were closely related to those in ambient snow and all genera also existed in snow in the East Rongbuk Glacier, Mount Everest (Liu et al., 2006).
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This is the first report on bacterial community structure in the high elevated waters of Mount Everest. We found that the bacterial community structures in moraine lakes and glacial meltwaters were similar to high mountain lakes in other regions. The study area is characterized by a distinctly lower abundance of total bacteria and by dominance of the CFB group. Within the region, remarkable differences in bacterial abundance and community structure existed between moraine lakes (5140 m and 5152 m) and glacial meltwaters (5800 m and 6350 m). A low temperature at high altitude is critical for the dominance of a component. At the same altitude, nutrients play a role in regulating bacterial community structure.
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This work was supported by the Ministry of Science and Technology of the People's Republic of China (Grant No. 2005CB422004), the National Natural Science Foundation of China (Grant Nos. 40121101, 40401054), the Innovation Program (Grant No. KZCX3-SW-339) and the ‘Talent Project’ of the Chinese Academy of Sciences, the Social Commonwealth Research Project of Ministry of Science and Technology of the People's Republic of China (2005DIA3J106).
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