Editor: Christoph Tebbe
Microbial communities and activities in alpine and subalpine soils
Article first published online: 1 DEC 2008
© 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Ecology
Volume 67, Issue 2, pages 208–218, February 2009
How to Cite
Margesin, R., Jud, M., Tscherko, D. and Schinner, F. (2009), Microbial communities and activities in alpine and subalpine soils. FEMS Microbiology Ecology, 67: 208–218. doi: 10.1111/j.1574-6941.2008.00620.x
- Issue published online: 22 DEC 2008
- Article first published online: 1 DEC 2008
- Received 22 June 2008; revised 5 August 2008; accepted 1 October 2008.First published online 1 December 2008.
- alpine soils;
- dehydrogenase activity;
Soil samples were collected along two slopes (south and north) at subalpine (1500–1900 m, under closed vegetation, up to the forest line) and alpine altitudes (2300–2530, under scattered vegetation, above the forest line) in the Grossglockner mountain area (Austrian central Alps). Soils were analyzed for a number of properties, including physical and chemical soil properties, microbial activity and microbial communities that were investigated using culture-dependent (viable heterotrophic bacteria) and culture-independent methods (phospholipid fatty acid analysis, FISH). Alpine soils were characterized by significantly (P<0.01) colder climate conditions, i.e. lower mean annual air and soil temperatures, more frost and ice days and higher precipitation, compared with subalpine soils. Microbial activity (soil dehydrogenase activity) decreased with altitude; however, dehydrogenase activity was better adapted to cold in alpine soils compared with subalpine soils, as shown by the lower apparent optimum temperature for activity (30 vs. 37 °C) and the significantly (P<0.01–0.001) higher relative activity in the low-temperature range. With increasing altitude, i.e. in alpine soils, a significant (P<0.05–0.01) increase in the relative amount of culturable psychrophilic heterotrophic bacteria, in the relative amount of the fungal population and in the relative amount of Gram-negative bacteria was found, which indicates shifts in microbial community composition with altitude.
Soil microorganisms play an essential role in soil organic matter turnover and biogeochemical cycling. Soil microbial activity and community composition are influenced by a number of biotic and abiotic factors, such as vegetation type, soil type and a range of environmental conditions, such as macro- and microclimate. The influence of climatic regimes on soil microorganisms has been rarely investigated, and mainly related to latitude (Sjogersten et al., 2003; Yergeau et al., 2007a, b), whereas little information is available on altitude. Temperature gradients in mountains can be similar to those related to latitude; the altitude-controlled vegetation belts on mountain slopes represent an analogue to the different latitudinally controlled climatic zones (Diaz et al., 2003). The annual average temperature decreases with increasing latitude; in mountain areas, temperature decreases with increasing altitude. While climate changes (e.g. temperature decrease) are spread over thousands of kilometers along latitude gradients, they occur on a comparatively small scale along altitude gradients: in the European Alps area in the northern direction, 1 km in altitude roughly equals 4000 km in latitude. Thus, the vegetation at an altitude of 2000 m above sea level (a.s.l.) corresponds to the tundra vegetation, and the vegetation at 3000 m altitude is similar to the one found in the High Arctic. This makes mountain regions useful for climate change studies (Diaz et al., 2003). Relations between climate (changing temperature conditions), vegetation and microbial activities, such as decomposition rates, along altitude gradients (on a comparatively small scale) can resemble those observed along latitude gradients (on a comparatively large scale).
Altitudinally defined climatic conditions, soil properties and vegetation may regulate community structure and metabolic rates in mountain soils (Whittaker, 1975; Schinner & Gstraunthaler, 1981). However, there is little information on the impact of altitude on soil microorganisms. Väre et al. (1997) and Männistöet al. (2007) investigated Arctic soils at altitudes ranging from 600 to 900–1020 m; Niklinska & Klimek (2007) focused on an altitude gradient (600–1200 m) in the Polish Carpathians. Subalpine and/or alpine soils were characterized by Uchida et al. (2000) in a Japanese forest (1500–2400 m), by Lipson (2007) in the Colorado Rocky Mountains and by Schinner & Gstraunthaler (1981) and Schinner (1982) in the European Alps, with the focus on litter degradation and filamentous fungi. A relation between altitude (increasing environmental harshness, i.e. lower annual temperature, lower soil nutrient contents) and a decrease in microbial population size (Ma et al., 2004; Giri et al., 2007), bacterial and fungal diversity (Schinner & Gstraunthaler, 1981; Lipson, 2007) as well as a decrease in microbial activities, such as respiration rate, microbial biomass and metabolic quotient (Schinner & Gstraunthaler, 1981; Väre et al., 1997; Niklinska & Klimek, 2007), has been reported.
Various methods are available to characterize soil microbial communities. Soil biological investigations, such as microbial counts and enzyme activities, provide information on the presence of viable microorganisms and on the impact of environmental conditions, such as temperature and nutrient conditions, on the metabolic activity of soil (Schinner et al., 1996; Kiss, 2001). Direct, molecular methods, such as profiling soil DNA, rRNA or phospholipid fatty acids (PLFA), provide information on the microbial community structure that is not based on culturing of microorganisms (Zelles, 1999; Hedrick et al., 2005). Examination of microbial populations using PLFA analysis is a well-characterized and powerful technique. Its suitability for characterizing soil microbial communities has been demonstrated (Zarda et al., 1997; Christensen et al., 1999; Männistöet al., 2007). FISH is based on the detection of rRNA. Because the rRNA content is associated with the metabolic state of microbial cells, FISH is a useful tool to describe the composition of the more active, ecologically relevant part of the microbial community (Amann et al., 1995, 2001; Wagner et al., 2003).
Because little is known on the impact of altitude on soil microorganisms from the poorly investigated European Alps, it was the objective of this study to investigate soil microbial activities and communities at different altitudes by comparing subalpine soils (1500–1900 m) and alpine soils (2300–2530 m), sampled on two slopes (north and south) in the Grossglockner mountain area in the Austrian Central Alps. A number of methods, including soil physical and chemical characterization, enzyme activity measurements, culture-dependent (viable microbial numbers) and culture-independent techniques (PLFA, FISH), were used to determine the effect of altitude and to detect relations between the properties determined. The effect of temperature on a selected activity (dehydrogenase) was tested in order to find out whether altitude and the prevailing climate conditions had an effect on cold adaptation of enzymes.
Materials and methods
Sites and soils
Soil samples were collected in August 2006 along an altitude gradient over 1030 m (subalpine: 1500 m and 1900 m; alpine: 2300 m and 2530 ma.s.l.) in the Austrian Central Alps (Hohe Tauern/Grossglockner area) both on the south and the north slope. Details of the location, vegetation, climate and soil characteristics are provided in Tables 1 and 2. The parent material (C-horizon) was predominantly gneiss (silicate) for all soils. From each site, 10 soil samples (subsamples) were randomly collected from an area of 100 m × 100 m from the upper 5 cm, bulked to produce composite samples, transported in cooled boxes to the laboratory, sieved (<2 mm) and stored at 2 °C.
|Site||Altitude (m a.s.l.)||Slope||Vegetation type||Air temperature* (°C)||Soil temperature† (°C)||Precipitation* (mm)||Ice days (Tmin 0°C) per year||Frost days (Tmax 0°C) per year|
|Curvuletum||2300||South||Alpine sedge mat||−19.4/16.1/−2.1||6.8||1915||156||240|
|Hochtor Süd||2530||South||Cushion plant level||−25.2/14.7/−3.3||4.1||2100||205||290|
|Hochtor Nord||2530||North||Cushion plant level||−27.0/10.2/−3.8||2.8||2250||220||300|
|Fuschertörl||2300||North||Alpine sedge mat||−21.5/12.5/−2.5||4.9||2100||180||265|
|Altitude (m)||Slope||pH||CaCO3 (%)||Humus (%)||Total N (%)||C/N||Conductivity (μs cm−1)||Sand (%, (<2000–63 μm)||Silt (%, <63–2 μm)||Clay (%, <2 μm)|
Physical and chemical soil properties
Soil analysis included measurements of dry mass, pH (CaCl2), carbonate content [gas volumetric determination (after Scheibler) of CO2 released after the addition of 10% HCl], humus content (dry combustion of carbon), total nitrogen (Kjeldahl method: digestion with H2SO4), C/N, conductivity and particle size distribution (sedimentation) according to standard methods (Schinner et al., 1996; ÖNORM L1084, 1999; ÖNORM L1061-2, 2002; ÖNORM L1080, 2005; ÖNORM L1082, 2005).
Enumeration of culturable soil bacteria
Numbers of culturable soil bacteria were determined by the plate-count method for viable cells. Appropriate dilutions of soil suspensions were surface spread onto agar plates. R2A plates containing cycloheximide (400 μg mL−1) were used to determine the numbers of heterotrophic bacteria. CFU were counted after 7 days at 25 °C and after 21 days at 1 °C.
Total bacterial counts
Soil samples (5 g fresh mass) were shaken for 30 min at 180 r.p.m. with 45 mL of sodium pyrophosphate solution (0.1% w/v), further diluted (1 : 5) and filtered. Total counts of bacteria were determined in the filtrates by nonselective 4′,6′-diamino-2-phenylindole (DAPI; 1.5 μg mL−1) staining using DAPI/Vectashield® and epifluorescence microscopy as described below.
Soil filtrates that were used to determine the total bacterial counts were used for fixation. Fixation of soil bacteria was performed in 4% paraformaldehyde; ethanol fixation was used for Gram-positive target groups (Zarda et al., 1997; Daims et al., 2005). FISH was performed according to Daims et al. (2005), using oligonucleotide probes for different phylogenetic groups of the domain Bacteria (Loy et al., 2007): EUB338 (Eubacteria; Stahl & Amann, 1991), ALF1b (Alphaproteobacteria; Manz et al., 1992), BET42a (Betaproteobacteria; Manz et al., 1992), GAM42a (Gammaproteobacteria; Manz et al., 1992), CF319a (Cytophaga–Flavobacterium; Manz et al., 1996), HGC69a (Actinobacteria; Roller et al., 1994) and LGC354mix (Bacillus–Clostridium; Meier et al., 1999). The probe NONEUB (Wallner et al., 1993) was used as a negative control. Hybridizations were performed in parallel. After hybridization for 1.5 h at 46 °C, slides were washed, air-dried and mounted in DAPI/Vectashield®. This formulation contains DAPI as a nonselective counterstain for DNA. Group-specific cell counts were performed with these DAPI-stained samples simultaneously hybridized with the Cy3-labelled probe EUB338 and the respective group-specific Fam-6-labelled probe, to enable threefold staining of the target cells (Kobabe et al., 2004). The probe NONEUB was used as a negative control. Because soil particles interfered due to autofluorescence with counting by automated methods (Kobabe et al., 2004; Li et al., 2004), counting was performed manually using epifluorescence microscopy (Leica DM5000B) equipped with a digital camera by counting at least 20–30 images per hybridization approach. Bacterial cell numbers were calculated on an oven dry mass (105 °C) basis; quantification of group-specific cells was performed relative to the number of affiliated eubacterial cells.
Phospholipids were extracted from 2 g (fresh mass) of soil, fractionated and quantified using the procedures described (Frostegard et al., 1993; Bardgett et al., 1996). Separated fatty acid methyl-esters were identified using gas chromatography and a flame ionization detector. Fatty acid nomenclature was used as described (Frostegard et al., 1993). The fatty acids i15 : 0, a15 : 0, 15 : 0, i16 : 0, 16 : 1ω7c, 17 : 0, i17 : 0, cy17 : 0, 18 : 1ω7c and cy19 : 0 were chosen to represent bacterial biomass (bacterial PLFA), and 18 : 2ω6,9c (fungal PLFA) was chosen to indicate fungal biomass (Federle, 1986; Zelles, 1999). The ratio of bacterial PLFA to fungal PLFA was calculated to indicate shifts in the ratio between bacterial and fungal biomass. The Gram-positive specific fatty acids i15 : 0, a15 : 0, i16 : 0 and i17 : 0 and the Gram-negative specific fatty acids cy17 : 0 and cy19 : 0 (Haubert et al., 2006) were taken as a measure of the ratio between Gram-positive and Gram-negative bacteria. The fatty acid 20 : 5ω3c was used as an indicator for soil algae (Fleurence et al., 1994; Khotimchenko et al., 2002). PLFA concentrations (nmol g−1 soil) were calculated on an oven dry mass (105 °C) basis.
Ergosterol content was determined as a measure of fungal biomass as described by Schinner et al. (1996) with three replicates. Briefly, ergosterol was saponificated with KOH, extracted with n-hexane, dissolved in methanol after drying at 40 °C in a rotary evaporator and measured by HPLC at 282 nm (Zelles et al., 1987).
Effect of temperature on soil dehydrogenase activity
Soil dehydrogenase activity was carried out with three replicates as described in detail by Schinner et al. (1996) and Margesin & Schinner (2005), using iodonitrotetrazoliumchloride as a substrate and 1 M Tris-HCl, pH 7, as buffer. After 1 h of incubation at temperatures ranging from 10 to 40 °C, the reduced iodonitrotetrazoliumchloride formazan was extracted and measured spectrophotometrically.
Statistical data treatment
The properties of each of the composite samples were analyzed with three replicate determinations, and the mean values of replicate determinations were used for statistical calculations. Normal distribution was confirmed by the Kolmogorov–Smirnov test. Soil properties, activities and communities at subalpine (1500–1900 m) and alpine (2300–2530 m) altitudes as well as at the south and north slopes were compared by independent t-test analyses (P<0.05). Correlations between the determined properties were tested by Pearson's correlation.
Soils were classified as subalpine (1500–1900 m, under closed vegetation, up to the forest line) or alpine (2300–2530, under scattered vegetation, above the forest line).
Climate data (Table 1) demonstrate the conditions prevailing at each sampling site. Annual mean air and soil temperatures decrease with altitude on both slopes and are lower by c. 0.5 °C (air) or 1.5–2 °C (soil) on the north slope compared with the south slope. This temperature difference between the north and the south slope is, however, not statistically significantly different (P<0.05). Over the altitude gradient (1030 m), annual mean air temperatures range from 3.1 to −3.3 °C (south slope) and from 2.9 to −3.3 °C (north slope); the mean soil temperatures at a depth of 5-cm range from 12.1 to 4.1 °C (south) or from 10.1 to 2.8 °C (north). The investigated area is characterized by wide annual temperature variation. At 2530 m, temperatures vary from −25 °C (south) or −27 °C (nor th) to +15 °C (south) or +10 °C (north). Minimum and maximum air temperatures at the highest altitude investigated are lower by c. 10 °C compared with the lowest altitude investigated.
At the highest altitude (2530 m), soils are permanently covered with snow from October until May/June; at the south slope, this period may be interrupted by thawing periods and soils are subjected to frequent freezing and thawing. Notably, the sites at this altitude are characterized by 290 (south) or 300 (north) frost days, i.e. days with a minimum temperature of 0 °C. Two hundred and five (south) or 220 (north) days of the year are even ice days, i.e. days with a maximum temperature of 0 °C. At the lowest altitude investigated (1500 m), the annual number of frost and ice days is significantly lower.
Alpine soils are characterized by significantly (P<0.01–0.001) colder climate conditions, i.e. lower mean annual air and soil temperatures, more frost and ice days and higher precipitation, compared with subalpine soils (Table 3). The climatic differences between soils at the two slopes are, however, not statistically (P<0.05) significant.
|Subalpine soils (n=4)||Alpine soils (n=4)||Significance level (P)|
|Mean annual soil temperature (°C)||10.2||4.7||0.003**|
|Mean annual air temperature (°C)||2.2||–2.9||<0.000***|
|Annual frost days||193||274||0.009**|
|Annual ice days||100||190||0.004**|
|Annual precipitation (mm)||1571||2091||0.003**|
|Total N (%)||0.448||0.175||0.037*|
|Viable bacteria counts at 1°C [log (CFU g−1 dry soil)]||6.20||6.59||0.041*|
|Ratio CFU (25°C/1°C)||11.1||2.1||<0.001**|
|Total bacterial cells (DAPI) [log (cells g−1 dry soil])||7.96||7.77||0.035*|
|FISH: EUB338-detected cells [log (cells g−1 dry soil)]||7.60||7.42||0.017*|
|FISH: Gram-positive bacteria† (% of affiliated Eubacteria)||38.2||31.6||0.040*|
|FISH: Gram-negative bacteria‡ (% of affiliated Eubacteria)||61.8||68.5||0.040*|
|FISH: ratio Gram-negative/Gram-positive bacteria||1.6||2.2||0.044*|
|PLFA: ratio bacteria/fungi||17.7||10.9||0.022*|
|PLFA: algae (nmol g−1 dry soil)||2.32||0.87||0.020*|
|Activity at 30°C (μg INTF g−1 h−1)||184||97||0.041*|
|Activity at 37°C (μg INTF g−1 h−1)||239||90||0.006**|
|Relative activity at 10°C (%)||9.6||35.9||0.002**|
|Relative activity at 20°C (%)||25.9||53.1||<0.000***|
|Relative activity at 30°C (%)||76.8||100||<0.000***|
|Relative activity at 37°C (%)||100||92.9||0.001**|
Physical and chemical soil properties
The soils investigated were acidic sandy soils (64–76% sand) with low contents of clay (1–5%) and pH values below pH 6. Two of the soils had a pH of 4.4 and 4.3. The contents of carbonate, humus and total nitrogen varied considerably; two soils had comparatively high carbonate contents, and two other soils had high humus contents (Table 2). The C/N ratio was in the range of 10.2–15.7 for seven of the eight soils, except for the soil from 2530 m north, which had a low C/N ratio of 7.4 and was characterized by low contents of humus (0.7%) and total N (0.06%). Subalpine soils had significantly (P<0.05) higher contents of total nitrogen and clay than alpine soils (Table 3), which contributes to lower activities and microbial biomass in alpine soils.
Bacterial biomass and communities
Viable numbers of culturable soil bacteria, grown at 25 °C, were (6.1–8.2) × 106 CFU g−1 soil dry mass in alpine soils and about twice as high [(1.2–2.3) × 107 CFU g−1 soil dry mass] in subalpine soils (Fig. 1). The opposite was observed for psychrophiles, grown at 1 °C, which were significantly (P<0.05) more numerous in alpine soils [(2.1–8.2) × 106 CFU g−1 soil] than in soils from lower altitudes [(0.95–2.1) × 106 CFU g−1 soil]. Consequently, the ratio between bacteria growing at 25 and 1 °C was 11.1±2.8 in subalpine soils and only 2.1±1.0 and thus significantly lower (P<0.01) in soils from higher altitudes (Table 3). No significant difference was noted between viable numbers at 25 °C in alpine and subalpine soils, which can be explained by the ability of several cold-adapted microorganisms to grow at 25 °C. Thus, the relative fraction of psychrophiles among culturable heterotrophic bacteria increased with altitude, while the opposite was observed for bacteria cultured at 25 °C. This indicates the ecological importance of psychrophiles at high altitudes, i.e. in cold climates. In addition, psychrophiles were more numerous (although not significant) in alpine soils from the north slope [(4.3±2.9) × 106 CFU g−1 soil] compared with the south slope [(1.9±0.8) × 106 CFU g−1 soil], which can be explained by adaptation to colder temperatures prevailing on the north slope and also confirms data obtained on cold adaptation of soil dehydrogenase activity.
Total bacterial counts
Total bacterial counts, as determined by nonselective DAPI staining, ranged from 4.7 × 107 to 1.2 × 108 cells g−1 dry soil. These numbers were comparable to cells found in arctic soils (Schmidt, 1999; Kobabe et al., 2004) and soils along altitude gradients of three Chinese locations (Ma et al., 2004). Total bacterial cell numbers were significantly (P<0.05) higher in subalpine compared with alpine soils (Tables 3 and 4); they decreased with altitude to a stronger extent in soils from the north slope compared with soils from the south slope (data not shown). However, no significant difference was detected between total bacterial cells when the two slopes were compared.
|Soils||Total cell counts (× 106 g−1 dry soil)||EUB338 (× 106 g−1 dry soil)||Relative amount (%) of affiliated Eubacteria, hybridized with specific probes|
|ALF1b||BET42a||GAM42a||CF319a||LGCmix||HGC69a||Gram- positive*||Gram- negative†||Ratio G−/G+|
|Subalpine (n=4)||93.6 ± 19.4||40.1 ± 2.9||19.1 ± 1.6||12.8 ± 1.7||15.9 ± 4.4||14.1 ± 0.9||15.8 ± 3.5||22.4 ± 2.7||38.2 ± 3.5||61.8 ± 3.5||1.6 ± 0.2|
|Alpine (n=4)||60.3 ± 16.8||27.0 ± 6.7||23.0 ± 1.8||17.4 ± 5.2||14.4 ± 5.2||13.7 ± 5.0||12.6 ± 5.8||18.9 ± 4.1||31.6 ± 3.7||68.5 ± 3.7||2.2 ± 0.4|
|South slope (n=4)||87.5 ± 23.8||35.7 ± 6.6||20.6 ± 3.0||13.6 ± 2.7||17.1 ± 3.9||13.8 ± 4.1||13.7 ± 3.1||21.1 ± 4.7||34.8 ± 4.5||65.2 ± 4.5||1.9 ± 0.4|
|North slope (n=4)||66.3 ± 23.0||31.4 ± 10.6||21.4 ± 2.5||16.5 ± 5.6||13.1 ± 4.7||14.0 ± 3.0||14.7 ± 6.5||20.2 ± 3.1||34.9 ± 5.9||65.1 ± 5.9||1.9 ± 0.5|
Bacterial phylogenetic diversity (FISH)
Similar to total bacterial counts, the fraction of cells detectable with the FISH-probe EUB338 (specific for members of the domain Bacteria), which represented 36–56% of DAPI-stained cells, was also significantly (P<0.01) higher in subalpine than in alpine soils (Table 3).
Table 4 shows the distribution of major bacterial phylogenetic groups among affiliated Eubacteria. The relative amount of Gram-positive bacteria, Firmicutes (low GC content) and Actinobacteria (high GC content) decreased with increasing altitude and was significantly (P<0.05) higher in subalpine than in alpine soils, while the opposite was observed for the relative amount of Gram-negative bacteria (Alpha-, Beta- and Gammaproteobacteria, and members of Cytophaga–Flavobacterium). This fraction as well as consequently the ratio between Gram-negative and Gram-positive bacteria was significantly (P<0.05) higher in alpine than in subalpine soils (Table 3). Among FISH-detected Gram-negative bacteria, the relative amount of Proteobacteria was the highest in the soil from the highest altitude, while the relative fraction of members of Cytophaga–Flavobacterium was the lowest in this soil (data not shown because they are statistically not evaluable). Significant differences between the soils from the two slopes could not be detected.
PLFA analyses demonstrated a general decrease of PLFA representing bacteria, fungi and algae with altitude (Table 5). Among bacterial PLFA, those specific for Gram-positive bacteria decreased with increasing altitude to a higher extent than PLFA indicative for Gram-negative bacteria; consequently, the ratio between PLFA specific for Gram-positive and Gram-negative bacteria tended to be lower in alpine compared with subalpine soils (Table 5), although not to a significant extent. This indicates a shift of the bacterial population toward the increase of the gram-negative population with altitude, which confirmed the results obtained by FISH.
|Soils||Subalpine (n=4)||Alpine (n=4)||South slope (n=4)||North slope (n=4)|
|PLFA (nmol g−1 dry soil)|
|Total bacteria||258.3 ± 114.7||106.9 ± 71.6||171.0 ± 71.6||194.2 ± 167.6|
|Total G+||98.1 ± 32.3||40.6 ± 35.4||70.2 ± 35.3||68.5 ± 56.9|
|Total G−||19.5 ± 10.1||7.8 ± 6.0||12.9 ± 6.8||14.4 ± 13.5|
|G+/G−||5.5 ± 1.2||4.7 ± 1.0||5.5 ± 0.8||4.7 ± 1.4|
|Soil fungi||14.7 ± 6.6||9.3 ± 5.6||11.4 ± 2.9||12.6 ± 9.2|
|Total bacterial/fungal PLFA||17.7 ± 4.1||10.9 ± 1.7||15.1 ± 5.8||13.5 ± 3.8|
|Soil algae||2.3 ± 0.8||0.9 ± 0.4||1.6 ± 0.8||1.6 ± 1.3|
|Ergosterol (μg g−1 dry soil)||13.8 ± 7.5||8.4 ± 5.9||9.1 ± 4.6||13.1 ± 8.8|
PLFA specific for fungi decreased with altitude. Also, the relation between PLFA specific for total bacteria and fungi decreased with altitude (Table 5) and was significantly lower (P<0.05) in subalpine (17.7±4.1) than in alpine (10.9±1.7) soils (Table 3), which demonstrates the relative increase in fungi with altitude.
Soil fungal biomass was determined by measuring the ergosterol content. The determination of viable counts (CFU) for fungi is associated with a number of problems because CFU may result from spores and thus are not an indication of the number of originally present individual fungal cells. Ergosterol is the predominant sterol of most fungi, and the ergosterol content of soils is directly related to fungal biomass (Zelles et al., 1987; Schinner et al., 1996). Fungal biomass decreased with altitude, as already observed with fungal PLFA, and tended to be lower in soils from the south slope compared with the north slope (Table 5); statistically significant (P<0.05) differences, however, could not be detected.
Microbial activity: effect of temperature on soil dehydrogenase activity
The measurement of potential biological activities in soils from different altitudes, and thus different climate conditions, is critical because the results are influenced by the incubation temperature at which reactions are carried out. Thus, the use of one single incubation temperature is not appropriate to reflect activity in soils from different altitudes. To investigate whether enzymes in soils from higher altitudes (colder climate) are better adapted to the cold environment than those from lower altitudes, the effect of temperature on a selected enzyme, soil dehydrogenase activity, was tested.
Dehydrogenases are important components of the enzyme system of all microorganisms; dehydrogenase activity reflects a broad range of microbial oxidative activities, and is a measure of the intensity of microbial metabolism in soil (Schinner et al., 1996). A significantly negative effect of altitude on this activity could be recognized, with significantly (P<0.05–0.01) higher values in subalpine soils than in alpine soils when activity was measured at 30 or 37 °C (Fig. 2, Table 3). There was also a trend towards lower (although not significant) activity in soils from the north slope compared with soils from the south slope, which can be attributed to the colder (although not significantly) climate conditions prevailing at the north slope.
The apparent optimum temperature for activity was 30 and 37 °C for the alpine and subalpine soils, respectively (Fig. 2). The relative enzyme activity in the temperature range 10–30 °C was considerably, and, therefore, significantly (P<0.01–0.001) higher (by a factor of 3–5 and 2 at 10 and 20 °C, respectively) in the alpine soils (Fig. 2, Table 3).
Various methods were used in this study to describe the microbial communities in the investigated soils. Significant positive correlations (P<0.05–0.01) were found between total bacterial cell numbers (DAPI counts), bacterial numbers obtained from FISH using the probe EUB338 and PLFA specific for bacteria (r=0.73–0.88; P<0.05–0.01; n=8). For viable numbers of heterotrophic bacteria, no significant correlation was detected with PLFA, but with numbers of EUB338-hybridized cells (P<0.05). The two methods used to determine fungal biomass correlated highly significantly (r=0.96; P<0.001, n=8).
The soil contents of humus, total nitrogen and clay were significantly (P<0.01–0.05) positively correlated with microbial (bacterial and fungal) communities as determined by total bacterial counts, the bacterial FISH-probe EUB338, PLFA specific for bacteria and fungi and ergosterol content; significant negative correlations were found with soil dry mass (data not shown). Contrary to Männistöet al. (2007), no correlation between fungal biomass (determined via ergosterol or via PLFA) and soil pH was detected in our study.
The soil properties mentioned above (contents of humus, total nitrogen and clay) also correlated significantly with soil dehydrogenase activity, determined at various temperatures. There were also significant correlations between soil microbial activity (dehydrogenase) and microbial communities. Relative soil dehydrogenase activity determined at low temperatures (10 and 20 °C) correlated significantly positively (r=0.73–0.76; P<0.05, n=8) with viable numbers of psychrophiles, indicating the contribution of psychrophiles to low-temperature activity, while no significant correlation was found between dehydrogenase activity and fungal biomass. Thus, the activity measured in this study may mainly be attributed to soil bacteria rather than fungi.
The comparison of data obtained in subalpine (1500–1900 m) and alpine soils (2300–2530 m) revealed a number of significant differences that were related to altitude, whereas significant differences between the two slopes could not be recognized at all. Alpine soils were characterized by highly significant (P<0.01) colder climate conditions, which included lower air and soil temperatures, more frost and ice days and higher precipitation. Higher altitudes are associated with increasing environmental harshness, i.e. cold climate, unfavorable nutrient conditions and modified vegetation (decreased plant biomass and soil cover), which in turn influence microbial activities and communities (Ma et al., 2004), as shown in this study by significant correlations or differences (Table 3). Subalpine soils had significantly higher contents of total nitrogen and clay than alpine soils, which also explains lower activities and microbial biomass in these soils. Nitrogen is generally a limiting factor for soil biological processes (microbial growth and activity). High clay contents in soils are favorable for the immobilization of nutrient ions and enzymes.
In our study, microbial, bacterial and fungal communities were significantly influenced by altitude. Culture-dependent and culture-independent techniques all demonstrated a decrease of microbial (bacterial and fungal) biomass with altitude. Total bacterial cell numbers and FISH-detected Eubacteria were significantly higher in subalpine than in alpine soils; microbial biomass also decreased (although not to a significant extent) with altitude as demonstrated with PLFA specific for total bacteria and fungi and with viable bacterial numbers. Other studies also generally observed a decrease in microbial (bacterial and fungal) biomass and activity with increasing altitude and colder climate conditions (Väre et al., 1997; Uchida et al., 2000; Lipson, 2007). Variations in vegetation and soil properties also influence microbial community composition; Männistöet al. (2007) recognized soil pH as the most significant factor for influencing soil bacterial biodiversity in acidic Arctic soils.
On a relative basis, our data indicate several shifts in the microbial community composition with altitude:
- 1There was a significant shift within the bacterial population, with a significant increase in the proportion of FISH-detected Gram-negative bacteria, as well as of the ratio between Gram-negative and Gram-positive bacteria, in alpine soils compared with subalpine soils, while the relative amount of Gram-positive bacteria decreased with altitude. This shift was also confirmed by PLFA specific for Gram-negative and Gram-positive bacteria (although not significant). Because FISH is a useful tool to detect the physiologically active fractions of bacterial cells (Amann et al., 1995; Zarda et al., 1997; Wagner et al., 2003), our data indicate that Gram-negative bacteria are not only relatively more numerous at high altitudes but they are also active. Unfortunately, only few data are available from the literature for comparison. Lipson (2007) observed that subalpine and alpine bacterial communities were markedly distinct from each other and reported a lower fraction of the Cytophaga—Flavobacterium–Bacteroides group in alpine soils compared with subalpine soils (Lipson & Schmidt, 2004). This result seems to contradict the hypothesis of the r-K scheme, which assumes that evolution favors either adaptation to high rates of reproduction (r strategists, Gram-negative bacteria grow under substrate-rich conditions) or optimal utilization of environmental resources (K strategists, Gram-positive bacteria tend to be more successful in resource-limited areas) (Atlas & Bartha, 1998). According to our study, Gram-negative bacteria seem to be more competitive under the prevailing conditions at high altitudes (low temperatures, low pH, low nutrient contents, and changed composition of vegetation); this may be attributed to their ability to be more tolerant to freeze–thaw cycles and to grow better at lower pH values than Gram-positive bacteria (Aislabie et al., 2006).
- 2Among viable culturable heterotrophic bacteria, psychrophiles (grown at 1 °C) were significantly more numerous in alpine than in subalpine soils, while the opposite was observed for mesophiles (grown at 25 °C). Psychrophiles are known to dominate in environments that are subjected to permanently low temperatures (for reviews, see Margesin et al., 2008).
- 3The fraction of the fungal biomass (ratio of bacterial/fungal PLFA) was significantly higher in alpine than in subalpine soils, which demonstrates the relative increase in fungal biomass with altitude. In cold environments, fungi are characterized by lower optimum and maximum temperatures for growth and activity compared with bacteria (Margesin et al., 2003) and are thus well adapted to cold climate conditions. Hart (2006) observed a relative increase in the ratio of bacterial/fungal biomass after experimental soil warming and the opposite after experimental cooling, which confirms our results. Altitude has an effect on the biodiversity of fungi; Schinner & Gstraunthaler (1981) observed decreasing diversity of filamentous fungi with increasing altitude and a change in the dominating species at different altitudes.
Microbial activity (soil dehydrogenase) decreased with altitude; however, relative activities at low temperatures were significantly higher in alpine soils than in subalpine soils, which means that enzymes from soils from higher altitudes are better adapted to the prevailing cold climate conditions. These results confirm earlier studies reporting decreased microbial activity (enzyme activities, respiration rates) with increasing altitude (Schinner, 1982; Väre et al., 1997; Niklinska & Klimek, 2007). This can be attributed partly to the influence of altitude on the physicochemical properties; for example, lower contents of clay, humus and nitrogen due to unfavorable conditions for soil formation with increasing altitude. Slower nutrient cycling at high altitudes due to cold temperatures could possibly affect organic matter structure and quality. Another important determining factor is the decreasing number of microbial cells with altitude, as shown in this study for both cultured and uncultured microorganisms. Low temperature is not a limiting factor for microbial activity in cold climates; microbial activity in soil has been reported at subzero temperatures down to −20 °C (Lipson & Schmidt, 2004; Panikov & Sizova, 2007) and substantial carbon mineralization has been described in cold soils during winter months (Clein & Schimel, 1995).
In conclusion, the results reported in this study show that environmental (climate) conditions determine the composition and activity of soil microorganisms. Several studies have reported that a change in temperature affects soil microbial communities and nutrient cycling (Uchida et al., 2000; Hart, 2006). With increasing altitude, and thus colder climate conditions, microorganisms are better adapted to the cold with regard to community composition and activity. Psychrophilic bacteria and fungi able to grow and to be active at low temperatures are of ecological significance at high altitudes.
R.M. thanks M. Wagner and his group for valuable introduction to the FISH technique. We also acknowledge K. Weber and S. Rudolph for technical assistance, and E. Kandeler for providing the laboratory facilities for PLFA analysis.
- 2006) Culturable aerobic heterotrophic bacteria from high altitude, high latitude soil of La Gorce Mountains (86°30′S, 147°W), Antarctica. Antarct Sci 18: 313–321. , & (
- 1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59: 143–169. , & (
- 2001) The identification of microorganisms by fluorescence in situ hybridisation. Curr Opin Biotechnol 12: 231–236. , & (
- 1998) Microbial Ecology: Fundamentals and Applications, 4th edn. Benjamin/Cummings Publication, Menlo Park, CA. & (
- 1996) Changes in soil fungal: bacterial biomass ratios following reductions in the intensity of management of an upland grassland. Biol Fert Soils 22: 261–264. , & (
- 1999) Counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rRNA oligonucleotide probe. Appl Environ Microbiol 65: 1753–1761. , & (
- 1995) Microbial activity of tundra and taiga soils at sub-zero temperatures. Soil Biol Biochem 27: 1231–1234. & (
- 2005) Fluorescence in situ hybridization for the detection of prokaryotes. Advanced Methods in Molecular Microbial Ecology (OsbornM & SmithC, eds), pp. 213–239. Routledge, UK. , & (
- 2003) Climate variability and change in high elevation regions: past, present and future. Climatic Change 59: 1–4. , & (
- 1986) Microbial distribution in soil: new techniques. Perspectives in Microbial Ecology (MegusarF & GantarM, eds), pp. 493–498. Slovene Society for Microbiology, Ljubljana. (
- 1994) Fatty acids from 11 marine macroalgae of the French Brittany coast. J Appl Phycol 6: 527–530. , , & (
- 1993) Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis. Soil Biol Biochem 25: 723–730. , & (
- 2007) Variation in methanotrophic bacterial population along an altitude gradient at two slopes in tropical dry deciduous forest. Soil Biol Biochem 39: 2424–2426. , , , , & (
- 2006) Potential impacts of climate change on nitrogen transformations and greenhouse gas fluxes in forests: a soil transfer study. Glob Change Biol 12: 1032–1046. (
- 2006) Trophic shift of stable isotopes and fatty acids in Collembola on bacterial diets. Soil Biol Biochem 38: 2004–2007. , , , & (
- 2005) Interpretation of fatty acid profiles of soil microorganisms. Manual of Soil Analysis, Soil Biology, Vol. 5 (MargesinR & SchinnerF, eds), pp. 252–259. Springer Verlag, Berlin. , & (
- 2002) Fatty acids of marine algae from the pacific coast of North California. Bot Mar 45: 17–22. , & (
- 2001) Advances in soil enzymology (Parts I–III). Stud Univ Babes-Bolyai Biol 46: 1–48. (
- 2004) Characterisation of microbial community composition of a Siberian tundra soil by fluorescence in situ hybridisation. FEMS Microbiol Ecol 50: 13–23. , & (
- 2004) Fluorescence microscopy for visualization of soil microorganisms – a review. Biol Fert Soils 39: 301–311. , & (
- 2007) Relationships between temperature responses and bacterial community structure along seasonal and altitudinal gradients. FEMS Microbiol Ecol 59: 418–427. (
- 2004) Seasonal changes in an alpine soil bacterial community in the Colorado Rocky Mountains. Appl Environ Microbiol 70: 2867–2879. & (
- 2007) probeBase – an online resource for rRNA-targeted oligonucleotide probes: new features 2007. Nucleic Acids Res 35: D800–D804. , , & (
- 2004) Microbial community structure along an altitude gradient in three different localities. Folia Microbiol 49: 105–111. , , & (
- 2007) Bacterial communities in Arctic fjelds of Finnish Lapland are stable but highly pH-dependent. FEMS Microbiol Ecol 59: 452–465. , & (
- 1992) Phylogenetic oligonucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst Appl Microbiol 15: 593–600. , , , & (
- 1996) Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum Cytophaga–Flavobacter–Bacteroides in the natural environment. Microbiology 142: 1097–1106. , , , & (
- MargesinR & SchinnerF (eds) (2005) Manual for Soil Analysis, Soil Biology, Vol. 5. Springer-Verlag, Berlin Heidelberg.
- 2003) Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts. Extremophiles 7: 451–458. , , , & (
- MargesinR, SchinnerF, MarxJC & GerdayC (eds) (2008) Psychrophiles: From Biodiversity to Biotechnology. Springer Verlag, Berlin Heidelberg.
- 1999) Specific oligonucleotide probes for in situ detection of a major group of Gram-positive bacteria with low DNA G+C content. Syst Appl Microbiol 22: 186–196. , , & (
- 2007) Effect of temperature on the respiration rate of forest soil organic layer along an elevation gradient in the Polish Carpathians. Biol Fert Soils 43: 511–518. & (
- ÖNORM L1084 (1999) Determination of Carbonate. Austrian Standards Institute, Vienna, Austria.
- ÖNORM L1061-2 (2002) Determination of Grain Size Distribution of Soils. Austrian Standards Institute, Vienna, Austria.
- ÖNORM L1080 (2005) Chemical Analysis of Soils: Determination of Humus by Dry Combustion of Carbon. Austrian Standards Institute, Vienna, Austria.
- ÖNORM L1082 (2005) Chemical Analysis of Soils: Determination of Total Nitrogen. Austrian Standards Institute, Vienna, Austria.
- 2007) Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to −35 °C. FEMS Microbiol Ecol 59: 500–512. & (
- 1994) In situ probing of Gram-positive bacteria with high G+C content using 23S rRNA-targeted oligonucleotides. Microbiology 140: 2849–1858. , , , & (
- 1982) Soil microbial activities and litter decomposition related to altitude. Plant Soil 65: 87–94. (
- 1981) Adaptation of microbial communities to the environmental conditions in alpine soils. Oecologia 50: 113–116. & (
- SchinnerF, ÖhlingerR, KandelerE & MargesinR, (eds) (1996) Methods in Soil Biology. Springer-Verlag, Berlin.
- 1999) Microbial properties and habitats of permafrost soils an Taymir peninsula, central Siberia. Reports Polar Res 340: 1–164. (
- 2003) Soil organic matter biochemistry and potential susceptibility to climatic change across the forest-tundra ecotone in the Fennoscandian mountains. Glob Change Biol 9: 759–772. , , , & (
- 1991) Development and application of nucleic acid probes. Nucleic Acid Techniques in Bacterial Systematics (StackebrandtE & GoodfellowM, eds), pp. 205–248. Wiley, New York. & (
- 2000) Altitudinal differences in organic matter mass loss and fungal biomass in a subalpine coniferous forest, Mt. Fuji, Japan. Arct Antarct Alp Res 32: 262–269. , , , & (
- 1997) Shifts in mycorrhiza and microbial activity along an oroarctic altitudinal gradient in Northern Fennoscandia. Arct Alp Res 29: 93–104. , & (
- 2003) Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr Opin Microbiol 6: 302–309. , & (
- 1993) Optimizing fluorescent in situ hybridisation with rRNA targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14: 136–143. , & (
- 1975) Communities and Ecosystems, 2nd edn. Macmillan, New York. (
- 2007a) Size and structure of bacterial, fungal and nematode communities along an Antarctic environmental gradient. FEMS Microbiol Ecol 59: 436–451. , , , , & (
- 2007b) Patterns of bacterial diversity across a range of Antarctic terrestrial habitats. Environ Microbiol 9: 2670–2682. , , & (
- 1997) Analysis of bacterial community structure in bulk soil by in situ hybridisation. Arch Microbiol 168: 185–192. , , , , , & (
- 1999) Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biol Fert Soils 29: 111–129. (
- 1987) Methoden zur relativen Quantifizierung der pilzlichen Biomasse im Boden. J Plant Nutr Soil Sci 150: 249–252. , & (