- Email: [email protected]
Measurements of microbial community activities in individual soil macroaggregates
Soil Biology & Biochemistry 48 (2012) 192e195
Contents lists available at SciVerse ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Measurements of microbial community activities in individual soil macroaggregates Vanessa L. Bailey a, *, Christina L. Bilskis a, Sarah J. Fansler a, Lee Ann McCue a, Jeffrey L. Smith b, Allan Konopka a a b
Pacific Northwest National Laboratory, Microbiology, 902 Battelle Boulevard, MSIN J4-18, Richland, WA 99354, USA USDA-ARS, 215 Johnson Hall, Washington State University, Pullman, WA 99164-6421, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2011 Received in revised form 4 January 2012 Accepted 8 January 2012 Available online 19 January 2012
The functional potential of single soil macroaggregates may provide insights into the localized distribution of microbial activities better than traditional assays conducted on bulk quantities of soil. Thus, we scaled down enzyme assays for b-glucosidase, N-acetyl-b-D-glucosaminidase, lipase, and leucine aminopeptidase to measure of the enzyme potential of individual macroaggregates (250e1000 mm diameter). Across all enzymes, the smallest macroaggregates had the greatest activity and the range of enzyme activities observed in all macroaggregates supports the hypothesis that functional potential in soil may be distributed in a patchy fashion. Paired analyses of ATP as a surrogate for active microbial biomass and b-glucosidase on the same macroaggregates suggest the presence of both extracellular b-glucosidase functioning in macroaggregates with no detectable ATP and also of relatively active microbial communities (high ATP) that have low b-glucosidase potentials. Studying function at a scale more consistent with microbial habitat presents greater opportunity to link microbial community structure to microbial community function. Published by Elsevier Ltd.
Keywords: Microscale Aggregates Enzyme activities ATP
Traditional soil microbiology studies the microbial community and processes in gram- and larger quantities of soil. However, studying soil aggregates as discrete entities may reveal more information about soil as a habitat for microbial communities (Young et al., 2008). Many studies have analyzed pools of aggregate size fractions (Busto and Perez-Mateos, 2000; Marx et al., 2005), and there has been some success reported in profiling C form and distribution in individual aggregates (Wan et al., 2007). We hypothesized that considering macroaggregates (defined here as 250e1000 mm diameter) individually would provide information about functional potential at a scale relevant to microbial communities, and potentially contribute to an understanding of the functional stability and spatial variability of a soil. Functional stability is considered here as the resistance and resilience of a measurable process to environmental perturbations (Griffiths et al., 2004). Thus, in order to assess the functional potential at the scale of soil macroaggregates, we developed an approach to conduct biomass and enzyme assays in individual (sub-millimeter) soil macroaggregates. Using soil from a minimally
* Corresponding author. Tel.: þ1 509 371 6965; fax: þ1 509 371 6955. E-mail address: [email protected] (V.L. Bailey). 0038-0717/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.soilbio.2012.01.004
disturbed location, we describe the variability in enzyme potential in individual macroaggregates for several important soil enzymes. We were also able to incorporate ATP measurement for biomass activity with the b-glucosidase assays to link biomass to cellulolytic potential. Soil was collected in a grassland field at the United States Department of Agriculture Conservation Field, near Pullman, Washington (Fine-silty, mixed, superactive, mesic Pachic Ultic Haploxeroll) where the soil contains approximately 750 mg biomass C g1 soil. Ten adjacent 5-cm diameter cores were collected and the 0e2 cm depths pooled and stored at 4 C until macroaggregate separation. The soil had 14% water (gravimetric) at the time of sampling. Macroaggregates were separated by dry-sieving to select individual macroaggregates in each of three size classes: 250e425 mm, 425e841 mm, and 841e1000 mm. Mass was determined for each macroaggregate in the two larger size classes on a Mettler-Toledo AX105 microbalance. The smallest size class could not be accurately weighed. Volumes were calculated for hypothetical spherical macroaggregates using the median diameter for each size class (Table 1). Enzyme activities and ATP were corrected for the bulk density of each medium and large macroaggregate and reported per unit volume (mm3) for the small macroaggregates.
V.L. Bailey et al. / Soil Biology & Biochemistry 48 (2012) 192e195 Table 1 Calculated size and biomass of study aggregates. Aggregate sizea
Volume,b mm3
Calculated mass,c mg
Measured mass, mg (std. dev.)
250e425 mm 425e841 mm 841e1000 mm
0.020 0.133 0.408
0.026 0.173 0.531
Below detection 0.17 (0.12) 0.60 (0.22)
a b c
Calculations use median of this range. Assumes spherical aggregates. Bulk density ¼ 1.3 g cm3.
Four enzyme assays, b-glucosidase (EC 3.2.1.21), N-acetyl-b-Dglucosaminidase (EC 3.2.1.30), lipase (EC 3.1.1.3), and leucine aminopeptidase (EC 3.4.11.1) were modified from bulk-scale fluorescent protocols and applied to90 discrete macroaggregates. Assay reaction times were varied to optimize the detection of enzyme potential in these small volumes of soil. We conducted the assays for b-glucosidase and total ATP (a surrogate for active microbial biomass (Alef and Nannipieri, 1995b)) sequentially on the same macroaggregates. b-glucosidase (modified from Saiya-Cork et al., 2002) was scaled down to 125 mL volume: 100 ml of acetate buffer (50 mM, pH 5.0) was added to an macroaggregate in a single 500-mL Eppendorf tube and vortexed. Twenty-five microliters of 4-methylumbelliferyl b-Dglucopyranoside (200 mM) was added and the reaction was incubated at 22 C in the dark for 2 h for the 250e425 mm macroaggregates, and 1 h for the 425e841 mm and 841e1000 mm macroaggregates. The reactions were centrifuged (10 000 g) for 2 min, the supernatant was transferred into a 96-well plate and 5 mL NaOH (1.0 M) was added to stop the reaction. Fluorescence was measured immediately, using a Wallac Victor2 1420 Multilabel Counter (PerkineElmer, Waltham, Massachusetts, USA). The pellets from this procedure were kept on ice for the ATP assay. ATP was assayed with the Bac Titer kit (Promega Inc, Madison, WI). Soil pellets were resuspended in 100 mL EDTA (20 mM, pH 7.5) and sonicated for 15 min (Branson Ultrasonics Corp, Danbury, CT). The soil was centrifuged (10 000 g) for 2 min and 100 mL of the supernatant transferred into a well of a 96-well plate. Five microliters of MgCl2 (0.4 M) and 100 mL ATP assay reagent were added in a dark room. Plates were shaken gently for 5 min and luminescence was read on the Wallac Victor 2 1420 Multilabel Counter. N-acetyl-b-D-glucosaminidase and lipase assays were similarly scaled down and fluorescence analyzed. N-acetyl-b-D-glucosaminidase was assayed for 1 h with the model substrate 4methylumbelliferyl-N-acetyl-b-D-glucosaminide. Lipase was assayed for 10 min using the substrate 4-methylumbelliferone heptanoate (Alef and Nannipieri, 1995a). The leucine aminopeptidase assay was based on 7-amino-4-methyl coumarin liberation from L-leucine-7amino-4-methylcoumarin (Saiya-Cork et al., 2002) over 1 h. Preliminary studies indicated that fluorescence-based assays were significantly more sensitive than paranitrophenol-based colorimetric assays for analysis of the smallest macroaggregates. Several modifications were evaluated. First, macroaggregates were crushed prior to the assay; similar activities were seen in crushed and intact sets of macroaggregates. Thus, macroaggregates were left intact. Second, incubation times were adjusted for each assay. For b-glucosidase, two hour incubations were long enough that all of the smallest macroaggregates tested were within the linear range of the standard curve. For medium and large macroaggregates (425e1000 mm), 1-hour incubations were sufficient. Tests were run to 4- and 6-hours to confirm that product formation was a linear function of incubation time. In a similar fashion, 1 h was determined adequate for N-acetyl-b-D-glucosaminidase and leucine aminopeptidase. Ten minutes was used for lipase.
193
When ATP and b-glucosidase were analyzed sequentially on the same 90 individual macroaggregates, biomass activity (ATP) had a broader spread around the mean than did enzyme activity (Table 2). b-glucosidase (Fig. 1) and lipase had similar degrees of variation across the 90 macroaggregates assayed for each enzyme, while N-acetyl-b-D-glucosaminidase was more narrowly distributed. Leucine aminopeptidase had the least variation, however activity levels were very low. A broad distribution of biomass and enzyme activities were measured among individual macroaggregates, that in each case ranged from below the detection limit to >4 times the mean value for ATP and lipase. Beta-glucosidase enzyme activity was not wellcorrelated with biomass in the macroaggregates with R2 < 0.36 for the whole set (Fig. 2); in medium and large macroaggregate classes, up to 96% of the macroaggregates had beta-glucosidase levels >1.5 or <0.67 than expected from biomass. This suggests that microbial community composition has a significant impact upon functional potential at the macroaggregate scale. The activities of N-acetyl-b-D-glucosaminidase, lipase, and leucine aminopeptidase were similarly widely spread across their respective means (Table 2). When activities were presented by macroaggregate volume, the small macroaggregates had the greatest activities and also the lowest %CV. The large variability in activities among the populations of individual macroaggregates demonstrates the heterogeneity of these functions in soils. In 0.05 g pooled samples of each macroaggregate size class (Table 3), the small macroaggregates have the greatest activity when normalized to soil mass, with the large macroaggregates having the lowest activity. Given the aggregate hierarchy hypothesis (Six et al., 2000), in which large macroaggregates are composed of smaller aggregates, one might anticipate that larger macroaggregates would have greater absolute activities, but that these differences would not be apparent when normalized to volume or mass. As this is not the case, it suggests that the surface area of the aggregates is a factor in its potential enzyme activity; perhaps the most dynamic and active microbes are located on macroaggregate surfaces. Most studies of soil microbiology make analyses at a bulk scale, and this carries an implicit assumption that microbes and their activity are evenly distributed. However, microbial colonization and Table 2 Descriptive statistics for total ATP and activities of enzymes (b-glucosidase, N-acetylb-D-glucosaminidase, lipase, and leucine aminopeptidase) in individual aggregates, normalized by mean aggregate volume, calculated for each size class. Units for bglucosidase and N-acetyl-b-D-glucosaminidase (MUB) are mmol methylumbelliferone mm3 h1, units for lipase are nmol methylumbelliferone mm3 10 min1, and units for leucine aminopeptidase are mmol aminomethylcoumarin mm3 h1. Small aggregates are 250e425 mm, medium aggregates are 425e841 mm, and large aggregates are 841e1000 mm in diameter. Aggregate Min. Max. Mean Median Std. %CV size dev. ATP (pmol)
Small Medium Large
0 0 0.1
30.2 16.7 5.9
7.0 5.3 2.0
4.8 4.4 1.7
6.3 3.7 1.1
89.6 69.4 54.2
b-glucosidase
Small Medium Large
9.3 1.4 0.6
35.8 13.4 11.2 4.4 3.5 1.7
12.6 3.6 1.6
4.2 2.2 0.7
30.9 50.5 42.7
N-acetyl-b-D-glucosaminidase Small Medium Large
8.8 1.3 0.4
16.8 3.7 1.4
9.6 1.7 0.6
9.4 1.7 0.5
0.9 0.3 0.2
9.7 18.1 28.5
Lipase
Small Medium Large
8.3 1.3 0
30.3 15.0 12.2 4.1 14.5 2.5
13.9 3.6 2.4
4.6 2.3 1.8
30.9 56.6 70.9
Leucine aminopeptidase
Small Medium Large
3.4 0.5 0.2
4.0 0.6 0.2
0.2 0.04 0.01
5.3 6.3 6.2
4.7 0.6 0.2
3.9 0.6 0.2
194
V.L. Bailey et al. / Soil Biology & Biochemistry 48 (2012) 192e195
Small 250 425µm
40
100%
20
Medium 425 841µm
15
80% 60%
20
More
10.1
9.0
7.9
0%
6.8
0 5.7
20% 4.6
5 3.5
40%
2.5
10
100%
C Large 841 1000µm
15
80% 60%
10 40% 5
20%
β glucosidase activity (μg methylumbelliferone mm 3 h 1 )
More
3.2
2.8
2.5
2.2
1.9
1.6
1.2
0.9
0% 0.6
0
Fig. 1. Histogram of b-glucosidase activities in 90 macroaggregates assayed individually. (A) Small macroaggregates are 250e425 mm, (B) medium macroaggregates are 425e841 mm, and (C) large macroaggregates are 841e1000 mm in diameter. Note different absolute scaling of both x- and y-axes between graphs.
15
y = 0.32x + 11.17 R² = 0.24
10
β-glucosidase activities (μg methylumbelliferone mm-3 h-1)
More
32.9
0%
29.9
0
27.0
20% 24.0
10 21.1
20
18.1
40%
15.2
20
1.4
Frequency
30 25
B
A
35
80% 60%
12.2
30
25
Frequency
40
100%
A
9.3
Frequency
50
5 0 0 12
10
20
30
40
B
10 8 6 4
y = 0.36x + 2.49 R² = 0.35
2 0 0 4
5
10
15
20
C
3 2 y = 0.24x + 1.17 R² = 0.14
1 0 0 40
2
4
6
8
D
35 30 25 20
activity may be patchy, as reported at scales that range from micrometers (Dandurand et al., 1997), to millimeters (Pallud et al., 2004), to meters (Pen-Mouratov et al., 2006), as well as in association with particular locations within aggregates (Ranjard et al., 2000). Physical and chemical considerations such as macroaggregate size (surface area), mineral composition, and carbon content (Denef et al., 2004) would imply that not all aggregates may host equally abundant or active microbial communities (Simpson et al., 2004). In addition, the distribution of specific enzymatic activities may be skewed with respect to the distribution of biomass or active microbes. For example, the few macroaggregates in which the ATP content was very low but had relatively high b-glucosidase activities can be attributed either to the intrinsic community being dominated by particularly b-glucosidase responsive microorganisms, or to the presence of extracellular b-glucosidase. This is not resolvable within the scope of work conducted, though reports of extracellular enzymes, including b-glucosidase are abundant (Allison and Vitousek, 2005; Murashima et al., 2002; Semedo et al., 2000; Singh and Kumar, 1998) and can be contrasted with the wide range of glycosyl hydrolase capabilities among soil microorganisms (Eivazi and Tabatabai, 1988). Enzymes associated with particular soil size fractions have kinetic parameters that vary with the size fraction (Marx et al., 2005). This suggests that there is a functional redundancy in that capability within the soil microbial community wherein one form
15 y = 0.69x + 3.20 R² = 0.32
10 5 0 0
10
20
30
40
ATP (pmol) Fig. 2. Correlation of b-glucosidase activities and ATP in 90 macroaggregates assayed individually and consecutively on the same individual macroaggregates. (A) Small macroaggregates are 250e425 mm, (B) medium macroaggregates are 425e841 mm, (C) large macroaggregates are 841e1000 mm in diameter, and (D) all macroaggregates reported together. For all relationships, P < 0.001.
Table 3 Enzyme activities measured in whole soil and 0.05 g pooled aggregates from each size class. Units for b-glucosidase and N-acetyl-b-D-glucosaminidase are mmol methylumbelliferone g1 soil h1, units for lipase are nmol methylumbelliferone g1 soil 10 min1, and units for leucine aminopeptidase are mmol aminomethylcoumarin g1 soil h1. Small aggregates are 250e425 mm, medium aggregates are 425e841 mm, and large aggregates are 841e1000 mm in diameter.
b-glucosidase N-acetyl-b-D-glucosaminidase Lipase Leucine aminopeptidase
Whole soil
Small aggregates
Medium aggregates
Large aggregates
15.4 2.9 19.9 0.06
18.9 3.7 19.0 0.06
17.8 3.0 17.7 0.06
14.0 2.0 13.3 0.06
V.L. Bailey et al. / Soil Biology & Biochemistry 48 (2012) 192e195
of the enzyme is preferentially expressed in the different habitat types. Such fine-scale questions in functional soil ecology cannot be definitively resolved using bulk samples. For this reason, we suggest that soil microbial ecologists consider whether bulk analyses or habitat-scaled analyses best answer their unique research questions.
Acknowledgements This research was funded by the Pacific Northwest National Laboratory’s Lab Directed Research and Development program, and is a contribution of the PNNL Microbial Communities Initiative. PNNL is operated for the DOE by Battelle under contract DE-AC0576RL01830.
References Alef, K., Nannipieri, P., 1995a. Enzyme activities. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, San Diego, pp. 311e373. Alef, K., Nannipieri, P., 1995b. Microbial biomass. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, San Diego, pp. 375e417. Allison, S.D., Vitousek, P.M., 2005. Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biology & Biochemistry 37, 937e944. Busto, M.D., Perez-Mateos, M., 2000. Characterization of b-D-glucosidase extracted from soil fractions. European Journal of Soil Science 51, 193e200. Dandurand, L.M., Schotzko, D.J., Knudsen, G.R., 1997. Spatial patterns of rhizoplane populations of Pseudomonas fluorescens. Applied and Environmental Microbiology 63, 3211e3217. Denef, K., Six, J., Merckx, R., Paustian, K., 2004. Carbon sequestration in microaggregates of no-tillage soils with different clay mineralogy. Soil Science Society of America Journal 68, 1935e1944. Eivazi, F., Tabatabai, M.A., 1988. Glucosidases and galactosidases in soils. Soil Biology & Biochemistry, 601e606.
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Griffiths, B.S., Kuan, H.L., Ritz, K., Glover, L.A., McCaig, A.E., Fenwick, C., 2004. The relationship between microbial community structure and functional stability, tested experimentally in an upland pasture soil. Microbial Ecology 47, 104e113. Marx, M.C., Kandeler, E., Wood, M., Wermbter, N., Jarvis, S.C., 2005. Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle-size fractions. Soil Biology & Biochemistry 37, 35e48. Murashima, K., Nishimura, T., Nakamura, Y., Koga, J., Moriya, T., Sumida, N., Yaguchi, T., Kono, T., 2002. Purification and characterization of new endo-1,4-bD-glucanases from Rhizopus oryzae. Enzyme and Microbial Technology 30, 319e326. Pallud, C., Dechesne, A., Gaudet, J.P., Debouzie, D., Grundmann, G.L., 2004. Modification of spatial distribution of 2,4-dichloro-phenoxyacetic acid degrader microhabitats during growth in soil columns. Applied and Environmental Microbiology 70, 2709e2716. Pen-Mouratov, S., Rakhimbaev, M., Steinberger, Y., 2006. Spatio-temporal effect on soil respiration in fine-scale patches in a desert ecosystem. Pedosphere 16, 1e9. Ranjard, L., Poly, F., Combrisson, J., Richaume, A., Gourbiere, F., Thioulouse, J., Nazaret, S., 2000. Heterogeneous cell density and genetic structure of bacterial pools associated with various soil microenvironments as determined by enumeration and DNA fingerprinting approach (RISA). Microbial Ecology 39, 263e272. Saiya-Cork, K.R., Sinsabaugh, R.L., Zak, D.R., 2002. The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest soil. Soil Biology & Biochemistry 34, 1309e1315. Semedo, L.T.A.S., Gomes, R.C., Bon, E.P.S., Soares, R.M.A., Linhares, L.F., Coelho, R.R.R., 2000. Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil. Applied Biochemistry and Biotechnology 84-6, 267e276. Simpson, R.T., Frey, S.D., Six, J., Thiet, R.K., 2004. Preferential accumulation of microbial carbon in aggregate structures of no-tillage soils. Soil Science Society of America Journal 68, 1249e1255. Singh, V.K., Kumar, A., 1998. Production and purification of an extracellular cellulase from Bacillus brevis VS-1. Biochemistry and Molecular Biology International 45, 443e452. Six, J., Elliott, E.T., Paustian, K., 2000. Soil macroaggregate turnover and microaggregate formation: a mechanism for C sequestration under no-tillage agriculture. Soil Biology & Biochemistry 32, 2099e2103. Wan, J., Tyliszczak, T., Tokunaga, T.K., 2007. Organic carbon distribution, speciation, and elemental correlations within soil micro aggregates: applications of STXM and NEXAFS spectroscopy. Geochimica et Cosmochimica Acta 71, 5439e5449. Young, I.M., Crawford, J.W., Nunan, N., Otten, W., Spiers, A., 2008. Microbial distribution in soils: physics and scaling. Advances in Agronomy 100, 81e121.
Contents lists available at SciVerse ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Measurements of microbial community activities in individual soil macroaggregates Vanessa L. Bailey a, *, Christina L. Bilskis a, Sarah J. Fansler a, Lee Ann McCue a, Jeffrey L. Smith b, Allan Konopka a a b
Pacific Northwest National Laboratory, Microbiology, 902 Battelle Boulevard, MSIN J4-18, Richland, WA 99354, USA USDA-ARS, 215 Johnson Hall, Washington State University, Pullman, WA 99164-6421, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2011 Received in revised form 4 January 2012 Accepted 8 January 2012 Available online 19 January 2012
The functional potential of single soil macroaggregates may provide insights into the localized distribution of microbial activities better than traditional assays conducted on bulk quantities of soil. Thus, we scaled down enzyme assays for b-glucosidase, N-acetyl-b-D-glucosaminidase, lipase, and leucine aminopeptidase to measure of the enzyme potential of individual macroaggregates (250e1000 mm diameter). Across all enzymes, the smallest macroaggregates had the greatest activity and the range of enzyme activities observed in all macroaggregates supports the hypothesis that functional potential in soil may be distributed in a patchy fashion. Paired analyses of ATP as a surrogate for active microbial biomass and b-glucosidase on the same macroaggregates suggest the presence of both extracellular b-glucosidase functioning in macroaggregates with no detectable ATP and also of relatively active microbial communities (high ATP) that have low b-glucosidase potentials. Studying function at a scale more consistent with microbial habitat presents greater opportunity to link microbial community structure to microbial community function. Published by Elsevier Ltd.
Keywords: Microscale Aggregates Enzyme activities ATP
Traditional soil microbiology studies the microbial community and processes in gram- and larger quantities of soil. However, studying soil aggregates as discrete entities may reveal more information about soil as a habitat for microbial communities (Young et al., 2008). Many studies have analyzed pools of aggregate size fractions (Busto and Perez-Mateos, 2000; Marx et al., 2005), and there has been some success reported in profiling C form and distribution in individual aggregates (Wan et al., 2007). We hypothesized that considering macroaggregates (defined here as 250e1000 mm diameter) individually would provide information about functional potential at a scale relevant to microbial communities, and potentially contribute to an understanding of the functional stability and spatial variability of a soil. Functional stability is considered here as the resistance and resilience of a measurable process to environmental perturbations (Griffiths et al., 2004). Thus, in order to assess the functional potential at the scale of soil macroaggregates, we developed an approach to conduct biomass and enzyme assays in individual (sub-millimeter) soil macroaggregates. Using soil from a minimally
* Corresponding author. Tel.: þ1 509 371 6965; fax: þ1 509 371 6955. E-mail address: [email protected] (V.L. Bailey). 0038-0717/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.soilbio.2012.01.004
disturbed location, we describe the variability in enzyme potential in individual macroaggregates for several important soil enzymes. We were also able to incorporate ATP measurement for biomass activity with the b-glucosidase assays to link biomass to cellulolytic potential. Soil was collected in a grassland field at the United States Department of Agriculture Conservation Field, near Pullman, Washington (Fine-silty, mixed, superactive, mesic Pachic Ultic Haploxeroll) where the soil contains approximately 750 mg biomass C g1 soil. Ten adjacent 5-cm diameter cores were collected and the 0e2 cm depths pooled and stored at 4 C until macroaggregate separation. The soil had 14% water (gravimetric) at the time of sampling. Macroaggregates were separated by dry-sieving to select individual macroaggregates in each of three size classes: 250e425 mm, 425e841 mm, and 841e1000 mm. Mass was determined for each macroaggregate in the two larger size classes on a Mettler-Toledo AX105 microbalance. The smallest size class could not be accurately weighed. Volumes were calculated for hypothetical spherical macroaggregates using the median diameter for each size class (Table 1). Enzyme activities and ATP were corrected for the bulk density of each medium and large macroaggregate and reported per unit volume (mm3) for the small macroaggregates.
V.L. Bailey et al. / Soil Biology & Biochemistry 48 (2012) 192e195 Table 1 Calculated size and biomass of study aggregates. Aggregate sizea
Volume,b mm3
Calculated mass,c mg
Measured mass, mg (std. dev.)
250e425 mm 425e841 mm 841e1000 mm
0.020 0.133 0.408
0.026 0.173 0.531
Below detection 0.17 (0.12) 0.60 (0.22)
a b c
Calculations use median of this range. Assumes spherical aggregates. Bulk density ¼ 1.3 g cm3.
Four enzyme assays, b-glucosidase (EC 3.2.1.21), N-acetyl-b-Dglucosaminidase (EC 3.2.1.30), lipase (EC 3.1.1.3), and leucine aminopeptidase (EC 3.4.11.1) were modified from bulk-scale fluorescent protocols and applied to90 discrete macroaggregates. Assay reaction times were varied to optimize the detection of enzyme potential in these small volumes of soil. We conducted the assays for b-glucosidase and total ATP (a surrogate for active microbial biomass (Alef and Nannipieri, 1995b)) sequentially on the same macroaggregates. b-glucosidase (modified from Saiya-Cork et al., 2002) was scaled down to 125 mL volume: 100 ml of acetate buffer (50 mM, pH 5.0) was added to an macroaggregate in a single 500-mL Eppendorf tube and vortexed. Twenty-five microliters of 4-methylumbelliferyl b-Dglucopyranoside (200 mM) was added and the reaction was incubated at 22 C in the dark for 2 h for the 250e425 mm macroaggregates, and 1 h for the 425e841 mm and 841e1000 mm macroaggregates. The reactions were centrifuged (10 000 g) for 2 min, the supernatant was transferred into a 96-well plate and 5 mL NaOH (1.0 M) was added to stop the reaction. Fluorescence was measured immediately, using a Wallac Victor2 1420 Multilabel Counter (PerkineElmer, Waltham, Massachusetts, USA). The pellets from this procedure were kept on ice for the ATP assay. ATP was assayed with the Bac Titer kit (Promega Inc, Madison, WI). Soil pellets were resuspended in 100 mL EDTA (20 mM, pH 7.5) and sonicated for 15 min (Branson Ultrasonics Corp, Danbury, CT). The soil was centrifuged (10 000 g) for 2 min and 100 mL of the supernatant transferred into a well of a 96-well plate. Five microliters of MgCl2 (0.4 M) and 100 mL ATP assay reagent were added in a dark room. Plates were shaken gently for 5 min and luminescence was read on the Wallac Victor 2 1420 Multilabel Counter. N-acetyl-b-D-glucosaminidase and lipase assays were similarly scaled down and fluorescence analyzed. N-acetyl-b-D-glucosaminidase was assayed for 1 h with the model substrate 4methylumbelliferyl-N-acetyl-b-D-glucosaminide. Lipase was assayed for 10 min using the substrate 4-methylumbelliferone heptanoate (Alef and Nannipieri, 1995a). The leucine aminopeptidase assay was based on 7-amino-4-methyl coumarin liberation from L-leucine-7amino-4-methylcoumarin (Saiya-Cork et al., 2002) over 1 h. Preliminary studies indicated that fluorescence-based assays were significantly more sensitive than paranitrophenol-based colorimetric assays for analysis of the smallest macroaggregates. Several modifications were evaluated. First, macroaggregates were crushed prior to the assay; similar activities were seen in crushed and intact sets of macroaggregates. Thus, macroaggregates were left intact. Second, incubation times were adjusted for each assay. For b-glucosidase, two hour incubations were long enough that all of the smallest macroaggregates tested were within the linear range of the standard curve. For medium and large macroaggregates (425e1000 mm), 1-hour incubations were sufficient. Tests were run to 4- and 6-hours to confirm that product formation was a linear function of incubation time. In a similar fashion, 1 h was determined adequate for N-acetyl-b-D-glucosaminidase and leucine aminopeptidase. Ten minutes was used for lipase.
193
When ATP and b-glucosidase were analyzed sequentially on the same 90 individual macroaggregates, biomass activity (ATP) had a broader spread around the mean than did enzyme activity (Table 2). b-glucosidase (Fig. 1) and lipase had similar degrees of variation across the 90 macroaggregates assayed for each enzyme, while N-acetyl-b-D-glucosaminidase was more narrowly distributed. Leucine aminopeptidase had the least variation, however activity levels were very low. A broad distribution of biomass and enzyme activities were measured among individual macroaggregates, that in each case ranged from below the detection limit to >4 times the mean value for ATP and lipase. Beta-glucosidase enzyme activity was not wellcorrelated with biomass in the macroaggregates with R2 < 0.36 for the whole set (Fig. 2); in medium and large macroaggregate classes, up to 96% of the macroaggregates had beta-glucosidase levels >1.5 or <0.67 than expected from biomass. This suggests that microbial community composition has a significant impact upon functional potential at the macroaggregate scale. The activities of N-acetyl-b-D-glucosaminidase, lipase, and leucine aminopeptidase were similarly widely spread across their respective means (Table 2). When activities were presented by macroaggregate volume, the small macroaggregates had the greatest activities and also the lowest %CV. The large variability in activities among the populations of individual macroaggregates demonstrates the heterogeneity of these functions in soils. In 0.05 g pooled samples of each macroaggregate size class (Table 3), the small macroaggregates have the greatest activity when normalized to soil mass, with the large macroaggregates having the lowest activity. Given the aggregate hierarchy hypothesis (Six et al., 2000), in which large macroaggregates are composed of smaller aggregates, one might anticipate that larger macroaggregates would have greater absolute activities, but that these differences would not be apparent when normalized to volume or mass. As this is not the case, it suggests that the surface area of the aggregates is a factor in its potential enzyme activity; perhaps the most dynamic and active microbes are located on macroaggregate surfaces. Most studies of soil microbiology make analyses at a bulk scale, and this carries an implicit assumption that microbes and their activity are evenly distributed. However, microbial colonization and Table 2 Descriptive statistics for total ATP and activities of enzymes (b-glucosidase, N-acetylb-D-glucosaminidase, lipase, and leucine aminopeptidase) in individual aggregates, normalized by mean aggregate volume, calculated for each size class. Units for bglucosidase and N-acetyl-b-D-glucosaminidase (MUB) are mmol methylumbelliferone mm3 h1, units for lipase are nmol methylumbelliferone mm3 10 min1, and units for leucine aminopeptidase are mmol aminomethylcoumarin mm3 h1. Small aggregates are 250e425 mm, medium aggregates are 425e841 mm, and large aggregates are 841e1000 mm in diameter. Aggregate Min. Max. Mean Median Std. %CV size dev. ATP (pmol)
Small Medium Large
0 0 0.1
30.2 16.7 5.9
7.0 5.3 2.0
4.8 4.4 1.7
6.3 3.7 1.1
89.6 69.4 54.2
b-glucosidase
Small Medium Large
9.3 1.4 0.6
35.8 13.4 11.2 4.4 3.5 1.7
12.6 3.6 1.6
4.2 2.2 0.7
30.9 50.5 42.7
N-acetyl-b-D-glucosaminidase Small Medium Large
8.8 1.3 0.4
16.8 3.7 1.4
9.6 1.7 0.6
9.4 1.7 0.5
0.9 0.3 0.2
9.7 18.1 28.5
Lipase
Small Medium Large
8.3 1.3 0
30.3 15.0 12.2 4.1 14.5 2.5
13.9 3.6 2.4
4.6 2.3 1.8
30.9 56.6 70.9
Leucine aminopeptidase
Small Medium Large
3.4 0.5 0.2
4.0 0.6 0.2
0.2 0.04 0.01
5.3 6.3 6.2
4.7 0.6 0.2
3.9 0.6 0.2
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V.L. Bailey et al. / Soil Biology & Biochemistry 48 (2012) 192e195
Small 250 425µm
40
100%
20
Medium 425 841µm
15
80% 60%
20
More
10.1
9.0
7.9
0%
6.8
0 5.7
20% 4.6
5 3.5
40%
2.5
10
100%
C Large 841 1000µm
15
80% 60%
10 40% 5
20%
β glucosidase activity (μg methylumbelliferone mm 3 h 1 )
More
3.2
2.8
2.5
2.2
1.9
1.6
1.2
0.9
0% 0.6
0
Fig. 1. Histogram of b-glucosidase activities in 90 macroaggregates assayed individually. (A) Small macroaggregates are 250e425 mm, (B) medium macroaggregates are 425e841 mm, and (C) large macroaggregates are 841e1000 mm in diameter. Note different absolute scaling of both x- and y-axes between graphs.
15
y = 0.32x + 11.17 R² = 0.24
10
β-glucosidase activities (μg methylumbelliferone mm-3 h-1)
More
32.9
0%
29.9
0
27.0
20% 24.0
10 21.1
20
18.1
40%
15.2
20
1.4
Frequency
30 25
B
A
35
80% 60%
12.2
30
25
Frequency
40
100%
A
9.3
Frequency
50
5 0 0 12
10
20
30
40
B
10 8 6 4
y = 0.36x + 2.49 R² = 0.35
2 0 0 4
5
10
15
20
C
3 2 y = 0.24x + 1.17 R² = 0.14
1 0 0 40
2
4
6
8
D
35 30 25 20
activity may be patchy, as reported at scales that range from micrometers (Dandurand et al., 1997), to millimeters (Pallud et al., 2004), to meters (Pen-Mouratov et al., 2006), as well as in association with particular locations within aggregates (Ranjard et al., 2000). Physical and chemical considerations such as macroaggregate size (surface area), mineral composition, and carbon content (Denef et al., 2004) would imply that not all aggregates may host equally abundant or active microbial communities (Simpson et al., 2004). In addition, the distribution of specific enzymatic activities may be skewed with respect to the distribution of biomass or active microbes. For example, the few macroaggregates in which the ATP content was very low but had relatively high b-glucosidase activities can be attributed either to the intrinsic community being dominated by particularly b-glucosidase responsive microorganisms, or to the presence of extracellular b-glucosidase. This is not resolvable within the scope of work conducted, though reports of extracellular enzymes, including b-glucosidase are abundant (Allison and Vitousek, 2005; Murashima et al., 2002; Semedo et al., 2000; Singh and Kumar, 1998) and can be contrasted with the wide range of glycosyl hydrolase capabilities among soil microorganisms (Eivazi and Tabatabai, 1988). Enzymes associated with particular soil size fractions have kinetic parameters that vary with the size fraction (Marx et al., 2005). This suggests that there is a functional redundancy in that capability within the soil microbial community wherein one form
15 y = 0.69x + 3.20 R² = 0.32
10 5 0 0
10
20
30
40
ATP (pmol) Fig. 2. Correlation of b-glucosidase activities and ATP in 90 macroaggregates assayed individually and consecutively on the same individual macroaggregates. (A) Small macroaggregates are 250e425 mm, (B) medium macroaggregates are 425e841 mm, (C) large macroaggregates are 841e1000 mm in diameter, and (D) all macroaggregates reported together. For all relationships, P < 0.001.
Table 3 Enzyme activities measured in whole soil and 0.05 g pooled aggregates from each size class. Units for b-glucosidase and N-acetyl-b-D-glucosaminidase are mmol methylumbelliferone g1 soil h1, units for lipase are nmol methylumbelliferone g1 soil 10 min1, and units for leucine aminopeptidase are mmol aminomethylcoumarin g1 soil h1. Small aggregates are 250e425 mm, medium aggregates are 425e841 mm, and large aggregates are 841e1000 mm in diameter.
b-glucosidase N-acetyl-b-D-glucosaminidase Lipase Leucine aminopeptidase
Whole soil
Small aggregates
Medium aggregates
Large aggregates
15.4 2.9 19.9 0.06
18.9 3.7 19.0 0.06
17.8 3.0 17.7 0.06
14.0 2.0 13.3 0.06
V.L. Bailey et al. / Soil Biology & Biochemistry 48 (2012) 192e195
of the enzyme is preferentially expressed in the different habitat types. Such fine-scale questions in functional soil ecology cannot be definitively resolved using bulk samples. For this reason, we suggest that soil microbial ecologists consider whether bulk analyses or habitat-scaled analyses best answer their unique research questions.
Acknowledgements This research was funded by the Pacific Northwest National Laboratory’s Lab Directed Research and Development program, and is a contribution of the PNNL Microbial Communities Initiative. PNNL is operated for the DOE by Battelle under contract DE-AC0576RL01830.
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