Interactive comment on “ Bacterial diversity in Himalayan glacial ice and its relationship to dust ” by S .

Question 1: The main focus of the paper is on bacterial diversity, DGGE by itself can definitely provide subjective information on changes in bacterial diversity. But if this information is to be used effectively by microbiologists, it would be essential also to clarify what are the different types of microorganisms present at the various depths. At least those microorganisms which are dominating and predominant at each depth could be identified by PCR and sequencing of the DGGE bands.


Introduction
The relationship between concentrations of microorganisms and dust in glacial ice has been studied for polar regions and the Tibetan Plateau (TP).For instance, Mitskevich et al. (2001) analyzed 10 samples from the Antarctic glacial ice over Lake Vostok.They observed that the maximum diversity of bacterial cells was correlated with organic detritus, and was presented in samples with maximum density of microorganisms.By using confocal microscopy, Priscu et al. (1998) identified cyanobacterial cells in the sediment of the ice cover of Lake Bonney, Antarctica.Zhang et al. (2003) suggested that the concentrations of total and culturable microorganisms from 23 samples of the Tibetan Malan ice core were correlated with dust content.By using these samples and δ 18 O as a proxy for temperature, Yao et al. (2006) further suggested that more microorganisms were associated with cold periods, and fewer with warm periods.It is suggested that microorganisms in glacial ice depend on the transportation of micro-particles during glacier growth (Zhang et al., 2003).Sediment particles can serve as nutrient-enriched (inorganic and organic) microzones for the establishment of a physiologically and ecologically complex microbial consortium that is capable of contemporaneous photosynthesis, N 2 fixation, and decomposition (Priscu et al., 1998).However, in a shallow ice core from the Himalayan Yala Glacier in the Langtang region of Nepal (Yoshimura et al., 2006), algal biomass in each annual layer from 1984 to 1994 AD was negatively, but not significantly correlated with micro-particle concentrations (r=−0.345,p>0.2).
Results of samples from the upper 12.5 m of a 22.4 m long ice core drilled from Muztagh Ata in the north-western TP also showed insignificant correlation (r=0.104, n=43) between concentrations of microparticle (1-30 µm in diameter) and bacteria (Xiang et al., 2006).These imply that, although microparticles may serve as the source and nutrition for bacteria, it is only one of the factors that determines bacterial concentrations in glacial ice.Therefore more efforts are necessary to verify the feasibility of reconstructing past climatic and environmental changes by means of glacial microorganism.
In addition, most of the above work suffered from limited samples, and some of the Tibetan ice cores are not well dated.Here we investigate the bacterial concentrations of 50 samples collected along a 108.83 m ice core recovered from the ER Glacier on the northeast slope of Mt.Qomolangma (Everest).Among them, 13 samples were analyzed for bacterial community diversity.We wish to extend (with more reliable statistics) our current understanding of bacterial concentrations and community diversity in the Himalayan glacial ice and to study their relationship with dust content.This work extends our previous results of the seasonal variations of abundance and species diversity of culturable bacteria recovered from Himalayan glacial ice (Zhang et al., 2007a).Introduction

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2 Materials and Methods

The ice core
A 108.83 m ice core, diameter 9.4 cm, was drilled to the bedrock on the col of the ER Glacier (Fig. 1) by using an electromechanical drill in a dry hole in September-October 2002.The ER Glacier covers an area of 48.45 km 2 with a length of 14 km.
Its equilibrium line of 6250 m above sea level is believed to be the highest among all glaciers on Earth.Repeat surveys with a Sokkia GSS1, a Global Positioning System (GPS), in 1998 and 2002 did not identify horizontal movement at the drilling site.Visible stratigraphy shows no hiatus features, and the ice layers are horizontal.The average annual net accumulation is around 400 mm water equivalent as determined by snow pit and an 80.36 m ice core study (Hou et al., 2002).Bore-hole temperature ranged from a minimum of −9.6 • C at 20 m depth to −8.9 • C at the bedrock.The relatively high annual accumulation and low temperature throughout the glacial ice ensure the preservation of a high resolution environmental and biological record.After drilling, the ice core was transported in a frozen state to a cold room at about −20 Laboratoire des Sciences du Climat et de l'Environnement, France (Hou et al., 2004), trace metals at Korea Polar Research Institute, lead isotopes at Department of Applied Physics, Curtin University of Technology, Australia, and bacteria (presented in this paper) at the State Key Laboratory of Cryospheric Science, Chinese Academy of Sciences, China.A total of 50 samples were collected for bacterial cell concentrations and community diversity, roughly proportionally but discontinuously along this core.
The vertical length of these samples was in the range 0.25-0.48m (Fig. 2) The ice core was annually dated to 1534 AD to a depth of 98.0 m using seasonal variations of δD and soluble ions, and the timescale was verified by identifying large volcanic horizons from the first high-resolution measurements of Bismuth on an Asian ice core (Kaspari et al., 2007).The bottom 2 m of the ice core was dated to 1498-2055 years BP using methane and isotopic composition of atmospheric O 2 (Hou et al., 2004).Below 98.0 m annual layer counting is not possible due to layer thinning, thus prior to 1534 AD the ice core was dated using a flow model.The details of dating were described by Kaspari et al. (2007;2008).

Ice core decontamination
In order to obtain reliable microbiological data, it is essential to eliminate the potential contamination caused during drilling, transportation, storage and sampling.This procedure was established according to Willerslev et al. (1999)  face.The remaining inner core was then placed into a sterile glass beaker for melting completely at 4 • C in the dark.At the same time, the scraped surface ice was collected and used as controls.The same procedure was used to process an artificial ice core of ultrapure water as an analytical blank.

Direct cell counts
Treatment of the samples by SYBR Green-II (Molecular Probes, Inc.) was modified from Yamagishi et al. (2003).Fifty milliliters of each sample were fixed in 2% (vol/vol) glutaraldehyde buffered with phosphate-buffered saline (PBS) and then filtered onto 25 mm, 0.2 µm black track-etch membrane filters (Whatman).Dimethyl sulfoxide (DMSO) was diluted with PBS to 5% (vol/vol) as stock solution for SYBR Green II.Both SYBR Green II and the diluted DMSO were passed through 0.2 µm filters to remove extraneous particles and cells prior to analysis.Filtered samples were incubated with DMSO-buffered 1 g/L SYBR Green II for at least 15 min at 20 • C in the dark.Afterwards the filters were mounted onto a glass slide treated with mounting medium (1:1 mixture of PBS diluted glycerin and 0.1% p-phenylenediamine).Cells on the filters were counted using an Olympus BH-2 microscope with a 405 nm argon laser at the final magnification of 1000×.The total number of cocci and rods in 60 fields of view was determined, and the number of cells per mL was calculated by computing the cumulative average cell per field (field of view area at 1000× is 16 741 µm 2 ).

DNA extraction
Thirteen samples selected from the above 50 were employed for DNA extraction.The remaining 200-1100 mL melted ice core samples were filtrated through 0.22 µm hydrophilic polyethersulfone membranes (Pall) with a vacuum pump (Ntengwe, 2005).Microorganisms on the membranes were eluted by agitation for 2 min by hand, then by sonication for 2 min with a sonicator (model 14; Branson Ultrasonics Corp) (Uga et al., 2003) and then suspended in 2.0 mL PBS.Half of the suspension was used for extrac-Introduction

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Full  (1996).The pellets of crude nucleic acid were finally dried and resuspended in 30 µL of sterile deionized water and quantified by UV spectrophotometer (UV 7501, Techcomp).

PCR Amplification
Bacterial specific primers, 357F-GC (5 -CGCCCGCCGCGCGCGGCGGGCG GGGCGGGGGCACGGGGGG-CCTACGGGAGGCAGCAG-3 ) and 518R (5 -ATTACCGCGGCTGCTGG-3 ) were used to amplify the V3 hypervariable region (Crump et al., 2003) of the 16S rDNA gene by PCR with the following conditions: 50 µg genomic DNA solution, 0.5 µM each primer, 1×PCR buffer (Promega), 1.5 mM MgCl 2 (Promega), 0.2 mM each deoxyribonucleotide triphosphate, 0.5 U Taq DNA Polymerase (Promega), and sterile deionized water to a final volume of 25 µL.Negative controls, with 1 µL of sterile deionized water as template, were included in all sets of PCR to provide a contamination check.The amplification program consisted of 94 • C for 5 min; 30 cycles of 94 • C for 30 s, 56 • C for 30 s, and 72 • C for 60 s; and a final extension step of 72 • C for 8 min.All the PCR products were analyzed on 30 g/L agarose gels in 1×TAE buffer, purified with QIA quick PCR mini kit (QIAGEN, Germany) and quantified by UV spectrophotometer (UV 7501, Techcomp).

Denaturing gradient gel electrophoresis (DGGE)
Two hundred and fifty ng of purified amplicons were used in DGGE analysis in 1× TAE buffer with 80 g/L polyacrylamide gels containing a linear denaturant gradient from 25% to 60% of urea (Merck) and formamide (Saarchem) (100% denaturant contains 7 M urea and 40% formamide (vol/vol)) in a D-Code system (Bio-Rad, Hercules, CA, USA) according to McCaig et al. (2001).Gels were run at a constant temperature of 60 • C, with 100 V for 14 h.After completion of electrophoresis, gels were fixed overnight (10% ethanol (vol/vol), 0.5% glacial acetic acid (vol/vol), 89.followed by incubation in fresh developing solution (15 g/L NaOH, 0.5% formaldehyde (vol/vol)) until bands appeared.The gels were then fixed for 10 min in 7.5 g/L Na 2 CO 3 , and preserved in ethanol-glycerol preservative (25% ethanol (vol/vol), 10% glycerol (vol/vol), 65% H 2 O (vol/vol)) for at least 15 min (McCaig et al., 2001).Images of each gel were documented with ImageScanner (Amersham Pharmacia Biotech).To ensure the reproducibility and reliability of the DGGE banding patterns, three independent PCR amplification and subsequent DGGE analysis were performed in independent trials.DGGE banding patterns were exactly the same for each replicate (Fig. 3).

Precautions and controls
DNA extraction was carried out in a fully equipped and physically isolated clean laboratory dedicated solely to ancient DNA work with separate ventilation systems, nightly exposure of surfaces to UV irradiation, and weekly cleaning of surface with bleach.The PCR setup was in a flow clean bench, exposed to UV light during the night.Both extraction and pre-PCR work were carried out with their own sets of lab clothes, lab tools, and unopened reagents (Lydolph et al., 2005).Tools for pre-PCR work were washed in 5% sodium hypochlorite (Willerslev et al., 1999) and exposed to UV light overnight.Full-body suits, facemasks, and sterile surgical gloves were used.Mock extraction and PCR controls were made to check for possible contamination by extraneous DNA (Willerslev et al., 1999).

Statistical analysis of DGGE profiles
Bacterial community diversity was calculated from the DGGE band profiles.The Shannon-Weaver index of general diversity (H ) was analyzed with the software of Labworks 4.0 (UVP Bioimaging system) based on the number and relative intensity of the bands in a gel lane and was calculated according to the equation (Shannon and Weaver, 1963) where P i is the relative intensity of the bands in a gel lane and is calculated as where n i is the height of a peak or the relative intensity of the bands in a gel lane as the result of Labworks 4.0.N is the sum of all peak heights in the densitometric curve.

Decontamination criteria
To make sure that each inner ice core sample was free of contamination, we analyzed separately each veneer layer and inner core of half of the 50 samples to monitor changes of bacterial concentrations from the surface to the center.Figure 4a shows an example for the change in bacterial concentrations from surface to inner section of the ice core for the depth interval 59.46-59.76m, indicating apparent contamination for the most outer veneer layer.Bacterial concentrations reduced to a stable low level from the third veneer layer to the center.Thus the low value of the inner core represents an authentic bacterial concentration result.
A control core constructed with Milli-Q-purified water was processed by methods identical with those of the samples.Bacterial concentrations were 14, 11, 9 and 6 cells/mL from surface to inner, respectively (Fig. 4b), and they are not significantly different from each other (using Chi-Square test: p=0.334>0.05).Moreover, the bacterial concentration in each layer of the control core was, at least, one order of magnitude lower than those measured in the ice core samples.Thus the potential contribution to the contamination during the process is the existence of a small DNA segment after autoclaving of the redistilled water, and therefore negligible.
All the mock extraction and PCR controls made to test for false-positive results were negative, demonstrating the authenticity of our DGGE data.Introduction

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Bacterial concentrations
Figure 5 shows the change of bacterial concentrations along the 108.83 m ice core, with a fluctuation of 0.02×10 3 -6.4×10 3 cells/mL (mean=0.71×10 3 cells/mL, SD=0.95×10 3 cells/mL, n=50).Because Ca 2+ can be regarded as a good proxy for atmospheric dust transport in Himalayan snow and ice (Kang et al., 2002;Kaspari et al., 2007), we compared the bacterial concentrations with Ca 2+ concentrations corresponding to the same ice core sections as shown in Fig. 5 The Ca 2+ concentrations fluctuate in the range 52.18-745.69ppb (mean=155.77ppb, SD=118.93 ppb, n=50).Figure 5 showed obvious difference of distribution trend between the bacterial and Ca 2+ concentrations.This is further confirmed by their weakly negative correlation (r=−0.135,P =0.351) (Fig. 6).
Although Yao et al. (2006) suggested that more microorganisms were associated generally with dust layers in the Malan ice core, they did not perform a correlation between the bacterial concentrations and insoluble particles.Our further analysis from their reported data (Yao et al., 2006) of 6 dirty layers for mineral measurement showed a weakly negative correlation between these two parameters (r=−0.022,p=0.967,

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Printer-friendly Version Interactive Discussion n=6).Moreover, a weakly negative correlation (r=−0.031, p=0.896, n=20) was also found between the bacterial and Ca 2+ concentrations of 20 samples collected from an 83.45 m Puruogangri ice core from the reported data by Zhang (2006).For all the ice core drilling sites (Fig. 1) in the Himalaya (ER and Yala glaciers), the central TP (Malan and Puruogangri glaciers), or the northern TP (Muztagh Ata Glacier), the correlation between bacterial concentrations/biomass and micro-particle concentrations is insignificant.Therefore, there might be other factors, together with micro-particles serving as a nutrition source for bacteria, that determines bacterial concentrations in glacial ice.For instance, algal biomass in annual layers of a shallow ice core from the Yala Glacier was found to be correlated significantly with two indices of snow-cover thickness, which determines the intensity of light available for algal growth, as well as the amount and period of availability of snow meltwater (Yoshimura et al., 2006).These results demonstrate that not only the relatively perplexing source of microorganisms, but also the complex growth mechanisms of bacterial community can be promoted by different nutrilites or environmental conditions in glaciers (Segawa et al., 2005).Given the ambiguous relationship between bacterial concentrations and ice core parameters, more efforts are needed for the reconstruction of past climatic and environmental changes by means of microbiological concentrations.

Bacterial community diversity with DGGE and H index analysis
Among the 50 samples for DNA work, the extraction was successful only for the 13 samples that were not evenly spaced along the core (Fig. 2).The H index, reflecting the structural diversity of bacterial community, fluctuates in the range of 0-0.75 (mean=0.41,SD=0.19, n=13), and the Ca 2+ average is 132.65 ppb (SD=78.87ppb, n=13) in response to these 13 samples (Fig. 7).Considering Ca 2+ as a proxy for the atmospheric dust transport to our study site, there are two high dust periods from 32.11 to (Fig. 7).A significantly positive correlation (r=0.486, p=0.092, n=13) is also observed between the two parameters (Fig. 8), which is consistent with that of the Puruogangri ice core (r=0.71,P <0.01, n=13) (Zhang et al., 2006).
In the Himalaya and southern TP, the periods of high dust concentrations generally coincide with warm climate, while that of low dust concentrations with cold climate (Wang et al., 2006).Dust storm outbreaks over the TP are associated with the movement of westerlies (Xu et al., 2007).During the cold periods of Little Ice Age (LIA), the westerlies were enhanced resulting in higher than normal precipitation and large seasonal snow cover on central Asia, thus the dust concentrations decreased.However, during the relatively warm periods of LIA, the intensity of westerlies weakened resulting in low precipitation in the winter and spring seasons and small seasonal snow cover (Xu et al., 2007  1 ).As a consequence, the dust concentrations increased, resulting in desiccation reduction and protection of microorganisms necessary for their survival (Priscu et al., 1998).The rise of air temperature not only increases the outbreak of the dust storm which may possibly influence the physiological state of microbes, but also provides a favorable condition for bacterial production, e.g.favorable temperature and sufficient light.Our previous research also demonstrated that bacterial diversity is higher in the temperate glacier of Hailuogou than in the ER glacier (Zhang et al., 2007b).The common distribution of cryoconite holes in temperate glaciers of the midlatitudes (McIntyre, 1984;Margesin et al., 2002) and sediments in small patches on the glacier surface (Fountain et al., 2004) indicated that both temperature and sediment are crucial for cold weather critters.Our result that bacterial diversity is higher during warm periods further illuminates the complicated environments for glacial microorganisms.Full and Christner et al. (2005) with minor modification.The ice core was sampled in a class-100 unidirectional flow clean bench, which was located inside a −20• C class-1000 dedicated cold room.Personnel involved wore clean room garbs and polyethylene gloves.At first, the potentially contaminated surface layer was removed (about 2 mm thick) by shaving with a sterile stainless steel scalpel.Afterwards, the operator changed to a new pair of gloves and the second veneer layer was shaved off by using a new scalpel.This procedure was repeated three times and about 6 mm thick veneer layer was removed.Then still in the cold room (temperature was switched to 4• C), the remaining inner core was rinsed with Introduction DNA according to Zhou et al.

Figure 1 .
Figure 1.Location map of ice core drilling site.

Fig. 5 .Figure 6 .
Fig. 5. Profiles of bacterial and Ca 2+ concentrations along the ER ice core.Dashed lines through the data sets indicate the third-order regression.

Fig. 6 .Figure 7 .
Fig. 6.The linear correlation between the bacterial and Ca2+ concentrations of the ER ice core samples.

Fig. 7 .Figure 8 .
Fig. 7. Vertical distribution of Shannon-Weaver index and Ca 2+ concentrations along the ER ice core.Dotted lines through the data sets indicate their mean values.

Fig. 8 .
Fig. 8.The linear correlation between Ca 2+ concentrations and bacterial community diversity of the ER ice core samples.
Ivask et al. (2001), USA, and oxygen isotopes (δ 18 O) at the State Key Laboratory of Cryospheric Science, Chinese Academy of Sciences, China.Ca 2+ analysis was performed via suppressed ion chromatography (Dionex 4000 series instruments) with a CS12 column, 125 mL loop, which was described in detail byIvask et al. (2001).The other half core was sampled discontinuously for measurement of gases at Laboratoire de Glaciologie et G éophysique de l'Environnement, France, gas stable isotopes at Figures • C and was stored there until analysis.2.2 Ice core measurements and datingAt first, this core was split axially into two equivalent portions for different measurements.One portion was melted continuously into 3123 samples and analyzed for major soluble ions, hydrogen isotopes (δD), and Bismuth at the Climate Change Institute, The quantitative distribution of bacterial population preserved in a Himalayan glacier displays a dissimilar variation trend to Ca 2+ concentrations, and a weakly negative correlation was observed between them.However, the Shannon-Weaver index showed a certain similarity with Ca 2+ concentrations.Therefore, dust concentrations might be more important in determining bacterial community diversity than bacterial concentrations in the Himalayan glaciers.Because only 13 samples were employed for deducing such a relationship between Ca 2+ concentrations and bacterial community diversity, further detailed investigation with more samples are needed to better understand the distribution mechanism of bacterial abundance and community diversity in the glacial ice.Hou, S., Qin, X.,Wang, Y., and Du, W.:Relation between bacteria in glacial snow with climate and environment in the Tibetan Plateau, Res.Environ.Sci., 20(5), 39-44, 2007b, (in Chinese).Zhang, X.: Diversity of microorganisms entrapped in Puruogangri ice core and its relation to environment, Ph.D. thesis, Cold and Arid Reg.Environ.Eng.Res.Inst., Chin.Acad. of Sci., Lanzhou, China, 2006.Zhang, X., Yao, T., An, L., Tian, L., and Xu, S.: A study on the vertical profile of bacterial DNA structure in the Puruogangri (Tibetan Plateau) ice core using denaturing gradient gel electrophoresis, Ann.Glaciol., 43, 160-166, 2006.Zhang, X., Ma, X., Yao, T., and Zhang, G.: Diversity of 16S rDNA and environmental factor influencing bacteria in Malan ice core, Chinese Sci.Bull., 48(11), 1146Bull., 48(11),  -1151Bull., 48(11),  , 2003.Zhou, J., Bruns, M. A., and Tiedje, J. M.: DNA recovery from soils of diverse composition, Appl.