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Fungal and bacterial responses to phenolic compounds and amino acids in high altitude barren soils
Soil Biology & Biochemistry 34 (2002) 989±995
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Fungal and bacterial responses to phenolic compounds and amino acids in high altitude barren soils Ruth E. Ley, Steven K. Schmidt* Department of Environmental, Population and Organismic Biology, University of Colorado, Campus Box 334, Boulder, CO 80309-0334, USA Received 13 December 2000; received in revised form 11 January 2002; accepted 31 January 2002
Abstract The total, fungal and bacterial biomass of functional groups capable of growth on speci®c phenolic compounds and amino acids was determined for a high altitude (3750 m) unvegetated talus soil from the Colorado Rocky Mountains. Soils were incubated with 14C-labeled carbon substrates (salicylate, phenol, glutamate, or alanine) and either fungal inhibitors (cyclohexamide plus nystatin), bacterial inhibitors (ampicillin plus streptomycin), all four inhibitors or no inhibitors. The assays were carried out at 22 8C for soil collected in August 1998, and 10 8C for soils collected in July 1999 under snow, to determine if trends were consistent seasonally. Two important trends emerged. First, fungi and bacteria grew on different phenolic compounds. Growth on salicylate was entirely fungal, whereas growth on phenol was entirely bacterial. However growth on amino acids was by both bacteria and fungi. Second, the fungi appear to dominate labile C mineralization in spring, and bacteria dominate in summer. Glutamate-mineralizing fungi had a higher biomass than glutamate-mineralizing bacteria (600 vs. 200 ng C g 21) in spring, but lower biomass than bacteria in summer (2 vs. 26 mg C g 21). Salicylate-mineralizing fungi had higher biomass in spring than in summer (100 vs. 50 ng C g 21). These results suggest that fungi and bacteria are partitioning labile-C mineralization both by substrate (in the case of phenolic compounds) and seasonally. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Substrate-induced growth response; Substrate-induced respiration; Inhibition; Extreme environment; Talus soil; Fungi; Bacteria; Phenolics; Amino acids
1. Introduction Microbes have been found under glaciers (Sharp et al., 1999; Skidmore et al., 2000), in unvegetated soils of polar deserts (Horowitz et al., 1972) and in high altitude unvegetated soils (Ley et al., 2001). Heterotophic and autotrophic microbial activities have also been measured in some extreme soils (Williams et al., 1997; Wilson et al., 1997; Paerl and Priscu, 1998; Skidmore et al., 2000; Ley et al., 2001). The diversity of microbes from these extreme sites is becoming known (Priscu et al., 1998). Relating phylogenetic identity to metabolic function is a challenge for investigators of any environment under study. In extreme terrestrial soil environments, we do not know which kinds of organisms carry out speci®c biogeochemical functions, or even which domain (Eukarya, Bacteria, or Archaea) they might belong to. Demonstrating which metabolic functions are carried out by bacteria or fungi is a relatively simple ®rst approach to linking microbial metabolism to phylogeny. * Corresponding author. Tel.: 11-303-492-6248; fax: 11-303-492-8699. E-mail address: [email protected] (S.K. Schmidt).
Despite the presence of high mountains on most continents, very little is known about the biology of high altitude unvegetated soils. High altitude talus soils have low organic matter content, low water availability, and extremes of temperature in both winter and summer. Carbon inputs are very low because sources are dust deposition and CO2®xation by soil algae and cyanobacteria, and possibly lichens although these are scarce. Prolonged snow-cover throughout the year limits these inputs to a few months annually when soil are exposed to the atmosphere and light (Ley and Schmidt, unpublished). Because their composition is loose sand and gravel that is cryogenically disturbed, these soils lack the cohesive structure of vegetated soils that provide multiple niches for a diversity of microorganisms. Whether the physical and chemical conditions result in microbial communities substantially different from those in vegetated soil is not known, since the composition and metabolic capacities of the microbial communities in these unvegetated soils are largely undescribed. In an initial study (Ley et al., 2001) of the microbial communities of high elevation talus soils, we quanti®ed the biomass of speci®c microbial functional groups using the substrate-induced growth response (SIGR) assay
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(02)00032-9
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(Schmidt, 1992; Colores and Schmidt, 1999). We found that salicylate-mineralizing microbes made up a larger proportion of the total biomass of unvegetated (no plants with roots) soils than of nearby vegetated soils and that total biomass did not correlate with soil organic matter content in unvegetated talus soils (Ley et al., 2001). The present study was undertaken to characterize the microbial community in one of the harshest unvegetated sites studied by Ley et al. (2001). To do so, we determined the relative contributions of fungi and bacteria to the mineralization of various carbon sources, i.e. two amino acids (alanine and glutamate) and two phenolic compounds (salicylate and phenol). In addition, we did a subset of these assays on a seasonal basis to ascertain whether bacteria and fungi were predominately active at different times of the year in unvegetated talus soils. 2. Materials and methods 2.1. Site We conducted this research on the slopes of Green Lakes Valley, part of the City of Boulder, CO, watershed in the Front Range of the Colorado Rocky Mountains (40803 0 N, 105835 0 W). Green Lakes Valley drains the eastern slope of the continental divide and the southern aspect of the Niwot Ridge Long Term Ecological Research/UNESCO Biosphere Reserve. Green Lakes Valley is a typical high elevation valley except that it is closed to the public and therefore minimally affected by human activity. Our study site is located on a south-facing talus slope at 3750 m. About 80% of the 930-mm mean annual precipitation occurs as snow (Caine, 1996). Three distinct periods describe the microclimate of these soils: (1) frozen under snow, from October to May, with mean soil temperatures of 21.3 8C, and mean soil water content of 4%; (2) thawed under snow during the spring melt of the snow-pack that lasts from May to early August, with mean soil temperatures of 0 8C and saturated soil water content of 10%; and (3) exposed during summer, from August to the ®rst permanent snow in October, with soil temperatures ranging diurnally from 5± 30 8C, and soil water content varying from 1 to 10% depending on rain (R. Ley, PhD Thesis, University of Colorado, Boulder, 2001). The soils are granite-derived and very coarse (roughly 90% sand and 10% silt) with very low soil organic matter content (0.6±2%) (Ley et al., 2001), and a water holding capacity of 10%. The carbon sources for these soils are primarily dust inputs from deposition and melting snow, with some CO2 ®xation by soil algae, cyanobacteria and the scarce lichens during the short summer months (Ley and Schmidt, unpublished). 2.2. Soil collection We collected soils in August 1998 during the exposed period and in July 1999 during the spring thaw period
(described earlier). At each date, we collected three randomly located soil cores, each with a volume of about 100 cm 3, to a depth of 10 cm. Soil was kept on ice and immediately returned to the laboratory on the day of collection. All soil was stored at 3 8C and processed within 24 h of sampling. We bulked and homogenized the replicate samples for each date, and sieved the soils to 8 mm. Soil water content was determined gravimetrically. 2.3. Substrate-induced growth response (SIGR) assays Soil samples of 20 gdw were added to sterilized biometer ¯asks, and two replicate ¯asks were randomly assigned to each treatment. Treatments consisted of a carbon source addition in combination with either (1) fungal inhibitors alone, (2) bacterial inhibitors alone, (3) fungal and bacterial inhibitors together, (4) no inhibitors. The carbon substrates and their concentrations consisted of: glutamate (100 mg C g 21), alanine (1000 mg C g 21), salicylate (50 mg C g 21) and phenol (50 mg C g 21). The inhibitor types and concentrations were: the fungal inhibitors cyclohexamide (2000 mg g 21) and nystatin (400 mg g 21), and the bacterial inhibitors streptomycin (3000 mg g 21) and ampicillin (840 mg g 21). The assays using the August 1998 soils included all of the treatments and were conducted at 22 8C; the assays using the July 1999 soils included glutamate and salicylate as carbon sources, and were conducted at 10 8C. The carbon substrates were dissolved in double-distilled (DD)-H2O and ®ltered (0.2 mm pore size) prior to addition. The inhibitors were suspended in sterile DD-H2O and added as 400 ml ¯ask 21 suspensions by rapid vortexing followed by immediate pipetting. The inhibitors were added in two equal doses, the ®rst half 6 h before the addition of the carbon substrate, and the second half of the dose concurrently with the carbon substrate (Schmidt and Gier, 1990). All ¯asks received the same ®nal volume (1 ml) of sterile water bringing the ®nal water content to about 15%. The amended soils were thinly spread (5 mm) over the bottom of the ¯ask to permit aeration. Soils that were not amended with both inhibitors received commensurate volumes of water at the same time as those receiving inhibitors. We used uniformly labeled 14C-substrates (Sigma Biochemical Co.) as tracers of the carbon substrates such that the ®nal radioactivity range was 0.7±1.4 mCi per ¯ask. The CO2 evolved during the assays was trapped in 1 ml of 0.5 M NaOH in the sidearm of the biometer ¯ask. Periodically (2±3 h for the 22 8C assays, and 12 h for the 10 8C assays) the NaOH was removed, mixed with 2.5 ml of Scintiversee scintillation ¯uid and the radioactivity measured with a scintillation counter. Fresh NaOH was immediately added back to the sidearm after each sampling. We continued sampling until mineralization rates had returned to initial values or lower following the exponential growth phase of the microbial biomass. The duration of the assay ranged from 75 h (glutamate, 22 8C) to 500 h (salicylate, 10 8C).
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Fig. 1. Rates of glutamate mineralization in summer soils. `Fungi' refers to the response with bacterial inhibitors added (D-dotted line); `Bacteria' refers to the response with fungal inhibitors added (B-dashed line); `All Inhib' refers to the response with all four inhibitors added (fungal and bacterial) (X-solid line) and `Control' refers to the response of the whole community with no inhibitors added ( £ -broken line). Values are means of two replicates, error bars are standard errors.
2.4. Analyses To derive initial biomass estimates for each functional group, respiration data were analyzed with Kaleidographe software using equations derived by Colores et al. (1996). To convert the units of mg C±CO2 g 21 to mg C-biomass g 21, growth yields (Yc) were used in the equation Xa X1 Yc =1 2 Yc ; where Xa is the actual biomass in mg Cbiomass g 21, and X1 is the biomass in units of mg C± CO2 g 21 (Colores et al., 1996; Lipson et al., 1999). To derive these growth yields, the cumulative curves of CO2 formation were analyzed using nonlinear regression and an integrated logistic equation (Hess et al., 1990; M. Fisk, PhD Thesis, University of Colorado, Boulder, 1995): P S0 2 S0 Yc 1 1 2 Yc { S0 1 X0 S0 e2rt = X0 1 S0 e2rt } where P is product (CO2) formation, S0 is the initial concentration of substrate, X0 is the initial population size in the same units as substrate, r is the maximum rate of growth achieved during the incubation, Yc is the asymptotic fraction of S0 incorporated into biomass and t is time. The means of the two replicate biomass estimates for each group (fungi, bacteria, whole community) within a treatment were compared by analysis of variance with SAS software (Judd and McClelland, 1989). 2.5. Active cell counts Active counts of active ®lamentous fungi, bacteria and yeasts were counted in soil after the salicylate SIGR treatments (500 h) by staining with ¯uorescein diacetate (SoÈderstroÈm, 1977; Ingham and Klein, 1984; Stamatiadis
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et al., 1990). Fungal and bacterial biomass were estimated from hyphal lengths, and bacterial and yeast from numbers g 21 soil. Sub-samples (10 g) of soil from the SIGR incubations were used to make 1000-fold dilutions in buffer (1 M MgSO4) by vigorous agitation in a blender followed by serial 10-fold dilutions. Fluorescein diacetate (40 mg ml 21) was added to 1 ml aliquots of the dilutions and 2 ml buffer (9 g l 21NaCl, pH 7.6) and after 4 min passed through non¯uorescent ®lters (Millipore MF Black ®lters, 0.2 mm) held in a Pyrex microanalysis ®lter holder. After rinsing with H2O, the ®lters were mounted on glass slides and immediately studied by epi¯uorescent microscopy (Zeiss Axioskop). For this 125 ®elds/slide were examined for total hyphal length at 400 £ total magni®cation, and 100 ®elds/slide were examined for bacteria and yeasts at 1000 £ total magni®cation. The majority of bacteria observed were cocci of ca 1 mm dia, and hyphal dia were ca 1 mm. Coryneform cells 10 mm in length were counted as yeasts. Biovolumes were calculated assuming ®lamentous fungi were cylinders, bacteria were spheres and yeasts had the shape of two cones end-to-end. For biomass estimates, a biovolumeto-biomass conversion factor of 130 mg C mm 23 was used, which assumes a wet density of 1.1 g cm 23, a dry matter content of 25% and a C content of 47% (Jenkinson and Ladd, 1981). The variance in the cell counts was estimated empirically at 1/210%.
3. Results 3.1. Amino acids In exposed summer soils, bacteria were the dominant glutamate mineralizers. Bacteria grew faster on glutamate than did fungi (Fig. 1). Indeed, the bacteria and the whole community exhibited almost identical growth responses, whereas fungal growth was delayed and the peak rates achieved were lower. The biomass of bacteria that grew on glutamate was comparable to the biomass of the whole community, whereas the fungal biomass was considerably less (Table 1). Bacterial growth on alanine also followed the growth of the whole community, and the fungi were also delayed on alanine relative to these two groups (Fig. 2). However, the estimated biomass of alanine-mineralizing bacteria and fungi were not signi®cantly different (Table 1). In soils collected in spring under snow, bacteria and fungi appear to have comparable roles in glutamate mineralization. Bacteria and fungi grew at similar rates on glutamate (Fig. 3). Interestingly, the growth responses of these two groups lagged behind the response of the whole community by about 100 h. The biomass of the glutamate mineralizing fungi and bacteria were low compared to the whole community (Table 1). Either the inhibitors had non-target effects in this case, or they interfered with a synergism between bacteria and fungi that is operative in the absence of inhibitors.
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Table 1 SIGR-biomass estimates (mg C g 21) of Bacteria alone (fungal inhibitors added), Fungi alone (bacterial inhibitors added), the whole community (no inhibitors added) and the community with all inhibitors added, of organisms that grow on amino acids and phenolic compounds in summer and spring. The biomass estimates are derived from the mineralization rate curves presented in Figs. 1±6. Values are means of n 2 with standard errors in parentheses. Nd: no data Substrate addition (season)
All inhibitors
Bacteria
Fungi
Whole community
Glutamate (summer) Fig. 1 Alanine (summer) Fig. 2 Glutamate (spring) Fig. 3 Salicylate (spring) Fig. 4 Salicylate (summer) Fig. 5 Phenol (summer) Fig. 6
0 0 0 0 0 0
26.58 (nd) 0.13 (0.06) 0.20 (0.01) 0 0.01 (0) 0.20 (0.01)
2.17 (0.15) 0.13 (0.03) 0.57 (0.02) 0.14 (0) 0.05 (0.02) 0
23.42 (nd) 0.07 (0.01) 4.70 (1.17) 0.15 (0.02) 0.08 (0.01) 0.27 (0.02)
3.2. Phenolic compounds In spring soils collected under snow only fungi grew in response to salicylate addition. This was evident because the addition of both inhibitors or just fungal inhibitors totally inhibited growth on salicylate (Fig. 4). The biomass of the fungi that grew on salicylate and the whole community were not signi®cantly different (Table 1). In the summer soils, the mineralization patterns were the same as for spring soils (Figs. 4 and 5) and the biomass estimates for fungi and the whole community were also similar (Table 1). In the case of phenol mineralization, the addition of both inhibitors again suppressed all substrate-induced growth, however so did the bacterial inhibitor mixture alone (Fig. 6). The bacterial biomass and the biomass of the whole community that grew on phenol were equivalent (Table 1). Therefore, in contrast to salicylate, bacteria are the organisms responding to the phenol additions in these soils.
counts. Active fungal biomass was an order of magnitude higher in the treatments without fungal inhibitors relative to the treatments with fungal inhibitors. The yeast biomass was two orders of magnitude higher in the antibacterial treatment, and one order of magnitude higher in the treatment without inhibitors. Filamentous fungi and yeasts were not present in the treatment with both inhibitor types. Bacterial counts were of the same order of magnitude in all treatments, but highest when fungal inhibitors were present. This suggests that salicylate did not stimulate the growth of bacteria as much as the death or competitive exclusion of fungi. 4. Discussion 4.1. Niche partitioning between fungi and bacteria
We con®rmed the speci®city of the inhibitors by directly counting active fungi and bacteria in the spring in the salicylate SIGR assays after the growth period (500 h) (Table 2). Both ®lamentous fungi and yeasts appeared to be released from competition by the addition of bacterial inhibitors, since this treatment resulted in the greatest biomass
The most surprising result of this study was that fungi and bacteria are specialized to grow on separate types of phenolic compounds in unvegetated soil. The summer biomass of the salicylate functional group and the phenol functional group were comparable. However the growth on salicylate was fungal and the growth on phenol was bacterial. This could be evidence of different roles for bacteria and fungi in soil organic matter mineralization in unvegetated soil.
Fig. 2. Rates of alanine mineralization in summer soils. Symbols are the same as in Fig. 1.
Fig. 3. Rates of glutamate mineralization in spring soils. Symbols are the same as in Fig. 1.
3.3. Active cell counts
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Fig. 4. Rates of salicylate mineralization in spring soils. Symbols are the same as in Fig. 1.
There is no obvious reason for the exclusive growth of bacteria on phenol and fungi on salicylate in this soil. Phenolic compounds in soil are derived from plants and are precursors to humic acids (Hedges, 1988). In unvegetated soils at high altitude, plant material is deposited by wind (Litaor, 1987) and may contribute to humi®cation. The organic matter in these soils, though sparse compared to most soils, could be a signi®cant source of carbon for the soil microbes given the lack of plants. Indeed we have found that salicylate mineralizers were proportionally more abundant in unvegetated soils than in vegetated soils (Ley et al., 2001). Some bacteria can metabolize both phenol and salicylate, such as Pseudomonas putida, which converts both to catechol prior to ring cleavage (Bayly and Barbour, 1984). P. putida is a common soil bacterium that has been found in these soils by rDNA analysis (Ley and Schmidt, unpublished). This and other metabolically similar bacteria may indeed be present in this soil, but fungi out-compete them for salicylate in the growth assay. There are several possible interpretations of these results: (1) only fungi can grow on
Fig. 5. Rates of salicylate mineralization in summer soils. Symbols are the same as in Fig. 1.
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Fig. 6. Rates of phenol mineralization in summer soils. Symbols are the same as in Fig. 1.
salicylate in this soil, (2) the fungi are secreting antibiotics that suppress the bacteria, (3) the growth of the bacteria is too slow relative to that of fungi, and they are out-competed. The direct counts of bacteria and fungi after the 500 h incubation with salicylate (Table 2) show that the bacteria did not respond to the salicylate alone, but grew in the presence of fungal inhibitors. We observed what appeared to be lysed yeast cells in the fungal inhibitor treatments alone, and conjectured that the bacteria were growing on the cellular contents of these dead cells. The direct counts of the fungi showed a growth response to salicylate alone, but a greater yield when bacterial inhibitors were present. This could be due to the nutrients released by dying bacteria during the course of the treatment, either because of the direct effect of the inhibitors, or because of natural cell turnover in the absence of new bacterial growth. 4.2. Seasonal patterns in fungal and bacterial abundances Our results indicate that fungi may dominate when snow is present, whereas bacteria may dominate when the soils are snow-free. The assays with both glutamate and salicylate support this pattern. Among the glutamate mineralizers, the fungal biomass is greater than bacterial biomass in spring, but the reverse is true in summer. This suggests that fungi would be less likely to compete effectively with bacteria for labile C in the summer. Furthermore the salicylate mineralizers, which are fungi, have greater biomass in the spring than in the summer. Microbial activity is known to occur under snow in cold soils (Sommer®eld et al., 1993; Brooks et al., 1996, 1997; Fahnestock et al., 1998; Fahnestock et al., 1999; Schmidt et al., 1999) and there is evidence that winter communities are different from summer communities in high altitude soils (Lipson et al., 1999; Maire et al., 1999). Fungi have been shown to have higher biomass in winter in some oligotrophic soils (Nilsson and Rulcker, 1992), and to respond to phenolic compounds only in winter in forest soil (Souto et al., 2000). These
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Table 2 Biomass estimates from direct counts of yeast, ®lamentous fungi and bacteria in soils after the SIGR treatments with salicylate with or without inhibitors
No inhibitors Bacterial inhibitors Fungal inhibitors All inhibitors
Filamentous fungi (mg C g 21)
Yeasts (mg C g 21)
Bacteria (mg C g 21)
1.86 6.37 0.15 0
0.80 1.60 0.05 0
2.00 4.00 5.00 8.00
studies together with our results suggest that very different suites of organisms may carry out the turnover of carbon in soil at different times of the year in a number of soil systems. 4.3. The use of inhibitors in SIGR and substrate-induced respiration assays The patterns of substrate mineralization with different inhibitors were particularly easy to interpret in this study because of the striking effectiveness of inhibitors in unvegetated soil. In vegetated soils the mixture of the bacterial and fungal inhibitors depresses, but does not suppress, substrate-induced growth (Anderson and Domsch, 1975; Wardle and Parkinson, 1990; Lin and Brookes, 1999a,b). The lack of suppression can be attributed to the sorption of the inhibitors to soil particles, which reduces the effective concentration of the inhibitor, and to the possible resistance of soil microbes to inhibitors. The coarse texture of the unvegetated soils (Ley et al., 2001) ensures that inhibitors will not be bound by organic matter. The microbial biomass does not appear to be resistant to the inhibitors we used, as our cell counts after the salicylate SIGR con®rmed the interpretations of the mineralization patterns. The use of the SIGR assay with inhibitors has advantages over the substrate-induced respiration (SIR) assay with inhibitors. SIR rates produced in the presence of inhibitors have been used to determine fungal-to-bacterial ratios (Anderson and Domsch, 1975, 1978; Beare et al., 1990; Lin and Brookes, 1999a,b; West, 1986). In these studies, the ratio of bacteria-to-fungi is the ratio of their initial respiration rates, which are derived from the fractions of antibiotic susceptible respiration that is inhibited by one antibiotic alone. One drawback to this interpretation is that inhibitors will not affect the fraction of biomass that can mineralize the substrate and is already active in soil. The SIGR method does not have this problem because it addresses only the growth-inducible fraction of the biomass. Furthermore, the SIGR assay yields individual biomass estimates for bacterial and fungal portions of the biomass assayed without requiring conversion factors. The SIGR assay, like other forms of enrichments, probably favors fast-growing, r-selected microbes (Eilers et al., 2000). Yet the initial mineralization of the speci®c compounds may be carried out by a diversity of slowgrowing fungi and bacteria (StentroÈm et al., 1998). In order to differentiate between the speci®c microbes that grow on added carbon substrates and those that may
mineralize these compounds and grow too slowly to be detected, a molecular approach based on extraction and analysis of labeled rRNA is necessary. 4.4. Conclusion Using the SIGR assay in combination with speci®c inhibitors, we determined the total, fungal and bacterial biomass of speci®c functional groups in soil. Our results suggest niche partitioning is occurring among the different constituents of the microbial community, both by substrate and seasonally. We found that fungi grow on salicylate, whereas bacteria grow on phenol in this soil. The fungi had higher biomass overall in spring soils. This implies that fungi might be more competitive with bacteria in colder months than warm months in unvegetated high elevation soils. Acknowledgements This study was funded by a National Science Foundation LExEn grant to S.K. Schmidt, and a National Science Foundation Biosphere-Atmosphere Research Training Fellowship to R.E. Ley. We thank Gamelyn Dykstra for help collecting samples and Kirsty Johnston for laboratory assistance. References Anderson, J.P.E., Domsch, K.H., 1975. A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology and Biochemistry 10, 215±221. Anderson, J.P.E., Domsch, K.H., 1978. Measurement of bacterial and fungal contributions to respiration of selected agricultural and forest soils. Canadian Journal of Microbiology 21, 314±322. Bayly, R.C., Barbour, M.G., 1984. The degradation of aromatic compounds by the meta and gentisate pathways, biochemistry and regulation. In: Gibson, D.T. (Ed.). Microbial Degradation of Organic Compounds. Marcel Dekker, New York, pp. 253±294. Beare, M.H., Neely, C.L., Coleman, D.C., Hargrove, W.L., 1990. A substrate-induced respiration (SIR) method for measurement of fungal and bacterial biomass on plant residues. Soil Biology and Biochemistry 22, 585±594. Brooks, P.D., Williams, M.W., Schmidt, S.K., 1996. Microbial activity under alpine snowpacks. Biogeochemistry 32, 93±113. Brooks, P.D., Williams, M.W., Schmidt, S.K., 1997. Winter production of CO2 and N2O from Alpine tundra: environmental controls and relationship to inter-system C and N ¯uxes. Oecologia 110, 403±413. Caine, N., 1996. Stream¯ow patterns in the alpine environment of North Boulder Creek, Colorado Front Range. Zeitschrift Geomorphologie 104, 27±42.
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Fungal and bacterial responses to phenolic compounds and amino acids in high altitude barren soils Ruth E. Ley, Steven K. Schmidt* Department of Environmental, Population and Organismic Biology, University of Colorado, Campus Box 334, Boulder, CO 80309-0334, USA Received 13 December 2000; received in revised form 11 January 2002; accepted 31 January 2002
Abstract The total, fungal and bacterial biomass of functional groups capable of growth on speci®c phenolic compounds and amino acids was determined for a high altitude (3750 m) unvegetated talus soil from the Colorado Rocky Mountains. Soils were incubated with 14C-labeled carbon substrates (salicylate, phenol, glutamate, or alanine) and either fungal inhibitors (cyclohexamide plus nystatin), bacterial inhibitors (ampicillin plus streptomycin), all four inhibitors or no inhibitors. The assays were carried out at 22 8C for soil collected in August 1998, and 10 8C for soils collected in July 1999 under snow, to determine if trends were consistent seasonally. Two important trends emerged. First, fungi and bacteria grew on different phenolic compounds. Growth on salicylate was entirely fungal, whereas growth on phenol was entirely bacterial. However growth on amino acids was by both bacteria and fungi. Second, the fungi appear to dominate labile C mineralization in spring, and bacteria dominate in summer. Glutamate-mineralizing fungi had a higher biomass than glutamate-mineralizing bacteria (600 vs. 200 ng C g 21) in spring, but lower biomass than bacteria in summer (2 vs. 26 mg C g 21). Salicylate-mineralizing fungi had higher biomass in spring than in summer (100 vs. 50 ng C g 21). These results suggest that fungi and bacteria are partitioning labile-C mineralization both by substrate (in the case of phenolic compounds) and seasonally. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Substrate-induced growth response; Substrate-induced respiration; Inhibition; Extreme environment; Talus soil; Fungi; Bacteria; Phenolics; Amino acids
1. Introduction Microbes have been found under glaciers (Sharp et al., 1999; Skidmore et al., 2000), in unvegetated soils of polar deserts (Horowitz et al., 1972) and in high altitude unvegetated soils (Ley et al., 2001). Heterotophic and autotrophic microbial activities have also been measured in some extreme soils (Williams et al., 1997; Wilson et al., 1997; Paerl and Priscu, 1998; Skidmore et al., 2000; Ley et al., 2001). The diversity of microbes from these extreme sites is becoming known (Priscu et al., 1998). Relating phylogenetic identity to metabolic function is a challenge for investigators of any environment under study. In extreme terrestrial soil environments, we do not know which kinds of organisms carry out speci®c biogeochemical functions, or even which domain (Eukarya, Bacteria, or Archaea) they might belong to. Demonstrating which metabolic functions are carried out by bacteria or fungi is a relatively simple ®rst approach to linking microbial metabolism to phylogeny. * Corresponding author. Tel.: 11-303-492-6248; fax: 11-303-492-8699. E-mail address: [email protected] (S.K. Schmidt).
Despite the presence of high mountains on most continents, very little is known about the biology of high altitude unvegetated soils. High altitude talus soils have low organic matter content, low water availability, and extremes of temperature in both winter and summer. Carbon inputs are very low because sources are dust deposition and CO2®xation by soil algae and cyanobacteria, and possibly lichens although these are scarce. Prolonged snow-cover throughout the year limits these inputs to a few months annually when soil are exposed to the atmosphere and light (Ley and Schmidt, unpublished). Because their composition is loose sand and gravel that is cryogenically disturbed, these soils lack the cohesive structure of vegetated soils that provide multiple niches for a diversity of microorganisms. Whether the physical and chemical conditions result in microbial communities substantially different from those in vegetated soil is not known, since the composition and metabolic capacities of the microbial communities in these unvegetated soils are largely undescribed. In an initial study (Ley et al., 2001) of the microbial communities of high elevation talus soils, we quanti®ed the biomass of speci®c microbial functional groups using the substrate-induced growth response (SIGR) assay
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(02)00032-9
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(Schmidt, 1992; Colores and Schmidt, 1999). We found that salicylate-mineralizing microbes made up a larger proportion of the total biomass of unvegetated (no plants with roots) soils than of nearby vegetated soils and that total biomass did not correlate with soil organic matter content in unvegetated talus soils (Ley et al., 2001). The present study was undertaken to characterize the microbial community in one of the harshest unvegetated sites studied by Ley et al. (2001). To do so, we determined the relative contributions of fungi and bacteria to the mineralization of various carbon sources, i.e. two amino acids (alanine and glutamate) and two phenolic compounds (salicylate and phenol). In addition, we did a subset of these assays on a seasonal basis to ascertain whether bacteria and fungi were predominately active at different times of the year in unvegetated talus soils. 2. Materials and methods 2.1. Site We conducted this research on the slopes of Green Lakes Valley, part of the City of Boulder, CO, watershed in the Front Range of the Colorado Rocky Mountains (40803 0 N, 105835 0 W). Green Lakes Valley drains the eastern slope of the continental divide and the southern aspect of the Niwot Ridge Long Term Ecological Research/UNESCO Biosphere Reserve. Green Lakes Valley is a typical high elevation valley except that it is closed to the public and therefore minimally affected by human activity. Our study site is located on a south-facing talus slope at 3750 m. About 80% of the 930-mm mean annual precipitation occurs as snow (Caine, 1996). Three distinct periods describe the microclimate of these soils: (1) frozen under snow, from October to May, with mean soil temperatures of 21.3 8C, and mean soil water content of 4%; (2) thawed under snow during the spring melt of the snow-pack that lasts from May to early August, with mean soil temperatures of 0 8C and saturated soil water content of 10%; and (3) exposed during summer, from August to the ®rst permanent snow in October, with soil temperatures ranging diurnally from 5± 30 8C, and soil water content varying from 1 to 10% depending on rain (R. Ley, PhD Thesis, University of Colorado, Boulder, 2001). The soils are granite-derived and very coarse (roughly 90% sand and 10% silt) with very low soil organic matter content (0.6±2%) (Ley et al., 2001), and a water holding capacity of 10%. The carbon sources for these soils are primarily dust inputs from deposition and melting snow, with some CO2 ®xation by soil algae, cyanobacteria and the scarce lichens during the short summer months (Ley and Schmidt, unpublished). 2.2. Soil collection We collected soils in August 1998 during the exposed period and in July 1999 during the spring thaw period
(described earlier). At each date, we collected three randomly located soil cores, each with a volume of about 100 cm 3, to a depth of 10 cm. Soil was kept on ice and immediately returned to the laboratory on the day of collection. All soil was stored at 3 8C and processed within 24 h of sampling. We bulked and homogenized the replicate samples for each date, and sieved the soils to 8 mm. Soil water content was determined gravimetrically. 2.3. Substrate-induced growth response (SIGR) assays Soil samples of 20 gdw were added to sterilized biometer ¯asks, and two replicate ¯asks were randomly assigned to each treatment. Treatments consisted of a carbon source addition in combination with either (1) fungal inhibitors alone, (2) bacterial inhibitors alone, (3) fungal and bacterial inhibitors together, (4) no inhibitors. The carbon substrates and their concentrations consisted of: glutamate (100 mg C g 21), alanine (1000 mg C g 21), salicylate (50 mg C g 21) and phenol (50 mg C g 21). The inhibitor types and concentrations were: the fungal inhibitors cyclohexamide (2000 mg g 21) and nystatin (400 mg g 21), and the bacterial inhibitors streptomycin (3000 mg g 21) and ampicillin (840 mg g 21). The assays using the August 1998 soils included all of the treatments and were conducted at 22 8C; the assays using the July 1999 soils included glutamate and salicylate as carbon sources, and were conducted at 10 8C. The carbon substrates were dissolved in double-distilled (DD)-H2O and ®ltered (0.2 mm pore size) prior to addition. The inhibitors were suspended in sterile DD-H2O and added as 400 ml ¯ask 21 suspensions by rapid vortexing followed by immediate pipetting. The inhibitors were added in two equal doses, the ®rst half 6 h before the addition of the carbon substrate, and the second half of the dose concurrently with the carbon substrate (Schmidt and Gier, 1990). All ¯asks received the same ®nal volume (1 ml) of sterile water bringing the ®nal water content to about 15%. The amended soils were thinly spread (5 mm) over the bottom of the ¯ask to permit aeration. Soils that were not amended with both inhibitors received commensurate volumes of water at the same time as those receiving inhibitors. We used uniformly labeled 14C-substrates (Sigma Biochemical Co.) as tracers of the carbon substrates such that the ®nal radioactivity range was 0.7±1.4 mCi per ¯ask. The CO2 evolved during the assays was trapped in 1 ml of 0.5 M NaOH in the sidearm of the biometer ¯ask. Periodically (2±3 h for the 22 8C assays, and 12 h for the 10 8C assays) the NaOH was removed, mixed with 2.5 ml of Scintiversee scintillation ¯uid and the radioactivity measured with a scintillation counter. Fresh NaOH was immediately added back to the sidearm after each sampling. We continued sampling until mineralization rates had returned to initial values or lower following the exponential growth phase of the microbial biomass. The duration of the assay ranged from 75 h (glutamate, 22 8C) to 500 h (salicylate, 10 8C).
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Fig. 1. Rates of glutamate mineralization in summer soils. `Fungi' refers to the response with bacterial inhibitors added (D-dotted line); `Bacteria' refers to the response with fungal inhibitors added (B-dashed line); `All Inhib' refers to the response with all four inhibitors added (fungal and bacterial) (X-solid line) and `Control' refers to the response of the whole community with no inhibitors added ( £ -broken line). Values are means of two replicates, error bars are standard errors.
2.4. Analyses To derive initial biomass estimates for each functional group, respiration data were analyzed with Kaleidographe software using equations derived by Colores et al. (1996). To convert the units of mg C±CO2 g 21 to mg C-biomass g 21, growth yields (Yc) were used in the equation Xa X1 Yc =1 2 Yc ; where Xa is the actual biomass in mg Cbiomass g 21, and X1 is the biomass in units of mg C± CO2 g 21 (Colores et al., 1996; Lipson et al., 1999). To derive these growth yields, the cumulative curves of CO2 formation were analyzed using nonlinear regression and an integrated logistic equation (Hess et al., 1990; M. Fisk, PhD Thesis, University of Colorado, Boulder, 1995): P S0 2 S0 Yc 1 1 2 Yc { S0 1 X0 S0 e2rt = X0 1 S0 e2rt } where P is product (CO2) formation, S0 is the initial concentration of substrate, X0 is the initial population size in the same units as substrate, r is the maximum rate of growth achieved during the incubation, Yc is the asymptotic fraction of S0 incorporated into biomass and t is time. The means of the two replicate biomass estimates for each group (fungi, bacteria, whole community) within a treatment were compared by analysis of variance with SAS software (Judd and McClelland, 1989). 2.5. Active cell counts Active counts of active ®lamentous fungi, bacteria and yeasts were counted in soil after the salicylate SIGR treatments (500 h) by staining with ¯uorescein diacetate (SoÈderstroÈm, 1977; Ingham and Klein, 1984; Stamatiadis
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et al., 1990). Fungal and bacterial biomass were estimated from hyphal lengths, and bacterial and yeast from numbers g 21 soil. Sub-samples (10 g) of soil from the SIGR incubations were used to make 1000-fold dilutions in buffer (1 M MgSO4) by vigorous agitation in a blender followed by serial 10-fold dilutions. Fluorescein diacetate (40 mg ml 21) was added to 1 ml aliquots of the dilutions and 2 ml buffer (9 g l 21NaCl, pH 7.6) and after 4 min passed through non¯uorescent ®lters (Millipore MF Black ®lters, 0.2 mm) held in a Pyrex microanalysis ®lter holder. After rinsing with H2O, the ®lters were mounted on glass slides and immediately studied by epi¯uorescent microscopy (Zeiss Axioskop). For this 125 ®elds/slide were examined for total hyphal length at 400 £ total magni®cation, and 100 ®elds/slide were examined for bacteria and yeasts at 1000 £ total magni®cation. The majority of bacteria observed were cocci of ca 1 mm dia, and hyphal dia were ca 1 mm. Coryneform cells 10 mm in length were counted as yeasts. Biovolumes were calculated assuming ®lamentous fungi were cylinders, bacteria were spheres and yeasts had the shape of two cones end-to-end. For biomass estimates, a biovolumeto-biomass conversion factor of 130 mg C mm 23 was used, which assumes a wet density of 1.1 g cm 23, a dry matter content of 25% and a C content of 47% (Jenkinson and Ladd, 1981). The variance in the cell counts was estimated empirically at 1/210%.
3. Results 3.1. Amino acids In exposed summer soils, bacteria were the dominant glutamate mineralizers. Bacteria grew faster on glutamate than did fungi (Fig. 1). Indeed, the bacteria and the whole community exhibited almost identical growth responses, whereas fungal growth was delayed and the peak rates achieved were lower. The biomass of bacteria that grew on glutamate was comparable to the biomass of the whole community, whereas the fungal biomass was considerably less (Table 1). Bacterial growth on alanine also followed the growth of the whole community, and the fungi were also delayed on alanine relative to these two groups (Fig. 2). However, the estimated biomass of alanine-mineralizing bacteria and fungi were not signi®cantly different (Table 1). In soils collected in spring under snow, bacteria and fungi appear to have comparable roles in glutamate mineralization. Bacteria and fungi grew at similar rates on glutamate (Fig. 3). Interestingly, the growth responses of these two groups lagged behind the response of the whole community by about 100 h. The biomass of the glutamate mineralizing fungi and bacteria were low compared to the whole community (Table 1). Either the inhibitors had non-target effects in this case, or they interfered with a synergism between bacteria and fungi that is operative in the absence of inhibitors.
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Table 1 SIGR-biomass estimates (mg C g 21) of Bacteria alone (fungal inhibitors added), Fungi alone (bacterial inhibitors added), the whole community (no inhibitors added) and the community with all inhibitors added, of organisms that grow on amino acids and phenolic compounds in summer and spring. The biomass estimates are derived from the mineralization rate curves presented in Figs. 1±6. Values are means of n 2 with standard errors in parentheses. Nd: no data Substrate addition (season)
All inhibitors
Bacteria
Fungi
Whole community
Glutamate (summer) Fig. 1 Alanine (summer) Fig. 2 Glutamate (spring) Fig. 3 Salicylate (spring) Fig. 4 Salicylate (summer) Fig. 5 Phenol (summer) Fig. 6
0 0 0 0 0 0
26.58 (nd) 0.13 (0.06) 0.20 (0.01) 0 0.01 (0) 0.20 (0.01)
2.17 (0.15) 0.13 (0.03) 0.57 (0.02) 0.14 (0) 0.05 (0.02) 0
23.42 (nd) 0.07 (0.01) 4.70 (1.17) 0.15 (0.02) 0.08 (0.01) 0.27 (0.02)
3.2. Phenolic compounds In spring soils collected under snow only fungi grew in response to salicylate addition. This was evident because the addition of both inhibitors or just fungal inhibitors totally inhibited growth on salicylate (Fig. 4). The biomass of the fungi that grew on salicylate and the whole community were not signi®cantly different (Table 1). In the summer soils, the mineralization patterns were the same as for spring soils (Figs. 4 and 5) and the biomass estimates for fungi and the whole community were also similar (Table 1). In the case of phenol mineralization, the addition of both inhibitors again suppressed all substrate-induced growth, however so did the bacterial inhibitor mixture alone (Fig. 6). The bacterial biomass and the biomass of the whole community that grew on phenol were equivalent (Table 1). Therefore, in contrast to salicylate, bacteria are the organisms responding to the phenol additions in these soils.
counts. Active fungal biomass was an order of magnitude higher in the treatments without fungal inhibitors relative to the treatments with fungal inhibitors. The yeast biomass was two orders of magnitude higher in the antibacterial treatment, and one order of magnitude higher in the treatment without inhibitors. Filamentous fungi and yeasts were not present in the treatment with both inhibitor types. Bacterial counts were of the same order of magnitude in all treatments, but highest when fungal inhibitors were present. This suggests that salicylate did not stimulate the growth of bacteria as much as the death or competitive exclusion of fungi. 4. Discussion 4.1. Niche partitioning between fungi and bacteria
We con®rmed the speci®city of the inhibitors by directly counting active fungi and bacteria in the spring in the salicylate SIGR assays after the growth period (500 h) (Table 2). Both ®lamentous fungi and yeasts appeared to be released from competition by the addition of bacterial inhibitors, since this treatment resulted in the greatest biomass
The most surprising result of this study was that fungi and bacteria are specialized to grow on separate types of phenolic compounds in unvegetated soil. The summer biomass of the salicylate functional group and the phenol functional group were comparable. However the growth on salicylate was fungal and the growth on phenol was bacterial. This could be evidence of different roles for bacteria and fungi in soil organic matter mineralization in unvegetated soil.
Fig. 2. Rates of alanine mineralization in summer soils. Symbols are the same as in Fig. 1.
Fig. 3. Rates of glutamate mineralization in spring soils. Symbols are the same as in Fig. 1.
3.3. Active cell counts
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Fig. 4. Rates of salicylate mineralization in spring soils. Symbols are the same as in Fig. 1.
There is no obvious reason for the exclusive growth of bacteria on phenol and fungi on salicylate in this soil. Phenolic compounds in soil are derived from plants and are precursors to humic acids (Hedges, 1988). In unvegetated soils at high altitude, plant material is deposited by wind (Litaor, 1987) and may contribute to humi®cation. The organic matter in these soils, though sparse compared to most soils, could be a signi®cant source of carbon for the soil microbes given the lack of plants. Indeed we have found that salicylate mineralizers were proportionally more abundant in unvegetated soils than in vegetated soils (Ley et al., 2001). Some bacteria can metabolize both phenol and salicylate, such as Pseudomonas putida, which converts both to catechol prior to ring cleavage (Bayly and Barbour, 1984). P. putida is a common soil bacterium that has been found in these soils by rDNA analysis (Ley and Schmidt, unpublished). This and other metabolically similar bacteria may indeed be present in this soil, but fungi out-compete them for salicylate in the growth assay. There are several possible interpretations of these results: (1) only fungi can grow on
Fig. 5. Rates of salicylate mineralization in summer soils. Symbols are the same as in Fig. 1.
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Fig. 6. Rates of phenol mineralization in summer soils. Symbols are the same as in Fig. 1.
salicylate in this soil, (2) the fungi are secreting antibiotics that suppress the bacteria, (3) the growth of the bacteria is too slow relative to that of fungi, and they are out-competed. The direct counts of bacteria and fungi after the 500 h incubation with salicylate (Table 2) show that the bacteria did not respond to the salicylate alone, but grew in the presence of fungal inhibitors. We observed what appeared to be lysed yeast cells in the fungal inhibitor treatments alone, and conjectured that the bacteria were growing on the cellular contents of these dead cells. The direct counts of the fungi showed a growth response to salicylate alone, but a greater yield when bacterial inhibitors were present. This could be due to the nutrients released by dying bacteria during the course of the treatment, either because of the direct effect of the inhibitors, or because of natural cell turnover in the absence of new bacterial growth. 4.2. Seasonal patterns in fungal and bacterial abundances Our results indicate that fungi may dominate when snow is present, whereas bacteria may dominate when the soils are snow-free. The assays with both glutamate and salicylate support this pattern. Among the glutamate mineralizers, the fungal biomass is greater than bacterial biomass in spring, but the reverse is true in summer. This suggests that fungi would be less likely to compete effectively with bacteria for labile C in the summer. Furthermore the salicylate mineralizers, which are fungi, have greater biomass in the spring than in the summer. Microbial activity is known to occur under snow in cold soils (Sommer®eld et al., 1993; Brooks et al., 1996, 1997; Fahnestock et al., 1998; Fahnestock et al., 1999; Schmidt et al., 1999) and there is evidence that winter communities are different from summer communities in high altitude soils (Lipson et al., 1999; Maire et al., 1999). Fungi have been shown to have higher biomass in winter in some oligotrophic soils (Nilsson and Rulcker, 1992), and to respond to phenolic compounds only in winter in forest soil (Souto et al., 2000). These
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Table 2 Biomass estimates from direct counts of yeast, ®lamentous fungi and bacteria in soils after the SIGR treatments with salicylate with or without inhibitors
No inhibitors Bacterial inhibitors Fungal inhibitors All inhibitors
Filamentous fungi (mg C g 21)
Yeasts (mg C g 21)
Bacteria (mg C g 21)
1.86 6.37 0.15 0
0.80 1.60 0.05 0
2.00 4.00 5.00 8.00
studies together with our results suggest that very different suites of organisms may carry out the turnover of carbon in soil at different times of the year in a number of soil systems. 4.3. The use of inhibitors in SIGR and substrate-induced respiration assays The patterns of substrate mineralization with different inhibitors were particularly easy to interpret in this study because of the striking effectiveness of inhibitors in unvegetated soil. In vegetated soils the mixture of the bacterial and fungal inhibitors depresses, but does not suppress, substrate-induced growth (Anderson and Domsch, 1975; Wardle and Parkinson, 1990; Lin and Brookes, 1999a,b). The lack of suppression can be attributed to the sorption of the inhibitors to soil particles, which reduces the effective concentration of the inhibitor, and to the possible resistance of soil microbes to inhibitors. The coarse texture of the unvegetated soils (Ley et al., 2001) ensures that inhibitors will not be bound by organic matter. The microbial biomass does not appear to be resistant to the inhibitors we used, as our cell counts after the salicylate SIGR con®rmed the interpretations of the mineralization patterns. The use of the SIGR assay with inhibitors has advantages over the substrate-induced respiration (SIR) assay with inhibitors. SIR rates produced in the presence of inhibitors have been used to determine fungal-to-bacterial ratios (Anderson and Domsch, 1975, 1978; Beare et al., 1990; Lin and Brookes, 1999a,b; West, 1986). In these studies, the ratio of bacteria-to-fungi is the ratio of their initial respiration rates, which are derived from the fractions of antibiotic susceptible respiration that is inhibited by one antibiotic alone. One drawback to this interpretation is that inhibitors will not affect the fraction of biomass that can mineralize the substrate and is already active in soil. The SIGR method does not have this problem because it addresses only the growth-inducible fraction of the biomass. Furthermore, the SIGR assay yields individual biomass estimates for bacterial and fungal portions of the biomass assayed without requiring conversion factors. The SIGR assay, like other forms of enrichments, probably favors fast-growing, r-selected microbes (Eilers et al., 2000). Yet the initial mineralization of the speci®c compounds may be carried out by a diversity of slowgrowing fungi and bacteria (StentroÈm et al., 1998). In order to differentiate between the speci®c microbes that grow on added carbon substrates and those that may
mineralize these compounds and grow too slowly to be detected, a molecular approach based on extraction and analysis of labeled rRNA is necessary. 4.4. Conclusion Using the SIGR assay in combination with speci®c inhibitors, we determined the total, fungal and bacterial biomass of speci®c functional groups in soil. Our results suggest niche partitioning is occurring among the different constituents of the microbial community, both by substrate and seasonally. We found that fungi grow on salicylate, whereas bacteria grow on phenol in this soil. The fungi had higher biomass overall in spring soils. This implies that fungi might be more competitive with bacteria in colder months than warm months in unvegetated high elevation soils. Acknowledgements This study was funded by a National Science Foundation LExEn grant to S.K. Schmidt, and a National Science Foundation Biosphere-Atmosphere Research Training Fellowship to R.E. Ley. We thank Gamelyn Dykstra for help collecting samples and Kirsty Johnston for laboratory assistance. References Anderson, J.P.E., Domsch, K.H., 1975. A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology and Biochemistry 10, 215±221. Anderson, J.P.E., Domsch, K.H., 1978. Measurement of bacterial and fungal contributions to respiration of selected agricultural and forest soils. Canadian Journal of Microbiology 21, 314±322. Bayly, R.C., Barbour, M.G., 1984. The degradation of aromatic compounds by the meta and gentisate pathways, biochemistry and regulation. In: Gibson, D.T. (Ed.). Microbial Degradation of Organic Compounds. Marcel Dekker, New York, pp. 253±294. Beare, M.H., Neely, C.L., Coleman, D.C., Hargrove, W.L., 1990. A substrate-induced respiration (SIR) method for measurement of fungal and bacterial biomass on plant residues. Soil Biology and Biochemistry 22, 585±594. Brooks, P.D., Williams, M.W., Schmidt, S.K., 1996. Microbial activity under alpine snowpacks. Biogeochemistry 32, 93±113. Brooks, P.D., Williams, M.W., Schmidt, S.K., 1997. Winter production of CO2 and N2O from Alpine tundra: environmental controls and relationship to inter-system C and N ¯uxes. Oecologia 110, 403±413. Caine, N., 1996. Stream¯ow patterns in the alpine environment of North Boulder Creek, Colorado Front Range. Zeitschrift Geomorphologie 104, 27±42.
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