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Evaluating soil stabilisation by biological processes using step-wise aggregate fractionation
Soil & Tillage Research 102 (2009) 209–215
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Evaluating soil stabilisation by biological processes using step-wise aggregate fractionation M.R. Ashman a, P.D. Hallett b,*, P.C. Brookes a, J. Allen a a b
Soil Science Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Soil aggregate Hierarchy Carbon Microbial activity
Soil biological processes contribute stability against physical disruption. We present an approach of stepwise fragmentation to assess the role that microbes and organic matter have on soil aggregate stabilisation. Compared to slaking and ultrasound procedures, the approach has a low impact on the microbial biomass. It also does not impose a severe drying stress. Grassland soil was found to be more stable than arable soil. Further examination of the arable soil revealed that increased disruption by shaking caused unstable microaggregates 53–250 mm in size to fragment, leaving a higher proportion of stable microaggregates in this size range. Carbohydrates, C:N, and basal respiration were found to be higher in the stable microaggregates than the other size fractions. Our results indicate that a distinct size range of soil aggregates exists in which microbial stabilisation dominates. This contradicts other research and questions the usefulness of measuring the biological properties of aggregate size fractions without understanding the physical effects of the fractionation procedure. ß 2008 Elsevier B.V. All rights reserved.
1. Introduction Organic matter and biological activity levels have been linked to soil structural stabilisation in many studies (Abiven et al., 2007; Sainju, 2006; Puget et al., 1999; Oades and Waters, 1991; Six et al., 1998). It is now uncontested that microbial exudates have a dominant role in the aggregation of soil particles and the protection of carbon from further degradation (Six et al., 2006; Roberson et al., 1995). Through these processes, it has been postulated that microorganisms ‘engineer’ a superior habitat and soil structure leading to more productive and resilient soil systems (von Lutzow et al., 2006). The very important inter-relationship between soil structure and biology is now an intensively researched area in soil science. Most of this work is based around the concept of an aggregate hierarchy which Tisdall and Oades (1982) proposed to describe soil stabilisation by microorganisms over various aggregate size ranges. This concept suggests that larger aggregates are formed from assemblages of smaller aggregates. Studies based on the aggregate hierarchy concept generally examine the properties of differently sized soil aggregates obtained using a range of fractionation techniques (Ashman et al., 2003; Field et al., 2006;
* Corresponding author. Tel.: +44 1382 562731; fax: +44 1382 562426. E-mail address: [email protected] (P.D. Hallett). 0167-1987/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.still.2008.07.005
Plante and Mcgill, 2002; Puget et al., 1999; Jastrow et al., 1998; Six et al., 1998; Elliott, 1986). Soil aggregates able to withstand the disruptive effects of the fractionation technique are categorised as structurally stable. Aggregates are rarely individual structural entities in soil but volume elements released by the mechanical energy input from fractionation. The form and level of the energy input therefore has a significant influence on the properties and size distribution of the collected aggregates. The five major methods used to fractionate soil are (1) slaking of air-dried soil followed by wet-sieving (water stability test) (Jastrow et al., 1998; Six et al., 1998), (2) wet-sieving of capillary re-wetted or field-moist soil (Six et al., 1998; Wright and Upadhyaya, 1998), (3) dry sieving of soil (Seech and Beauchamp, 1988), (4) end-over-end shaking followed by wetsieving (Oades and Waters, 1991) and (5) ultrasonic dispersion (Tippkotter, 1994). Each approach imposes different forms of energy inputs on the soil and conflicting results between aggregate size and the abundance of stabilising agents have been found. Studies which fractionate soil using slaking tend to find carbon levels to be highest for macroaggregates (>250 mm) (Tisdall and Oades, 1982; Elliott, 1986; Gupta and Germida, 1988; Singh and Singh, 1995; Six et al., 1998; Puget et al., 1999). Seech and Beauchamp (1988) found that microaggregates (53–250 mm) have the greatest level of carbon if soil is fractionated using dry-sieving. Biological investigations of soil stabilisation are also affected by sample pre-treatment prior to fractionation. Both air-drying (Van
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Gestel et al., 1991; Birch, 1958) and ultrasonic dispersion (Stevenson, 1958) disturb the microbial community. Detailed investigations of the form and function of microbial communities associated with different aggregate sizes is an area requiring more research. Most research to date concerning biological stabilisation of soil, however, has fractionated soil using slaking (Puget et al., 1999) which is unsuitable for detailed microbial studies because the soil is dried. We propose that microbial disturbance can be reduced and a description of aggregation by biological processes improved by adopting end-over-end shaking of soil in water followed by wet sieving. The approach is adapted from Oades and Waters (1991) who showed that the aggregate hierarchy can be elucidated by different energy levels to fractionate soil. We improve the technique by controlling the energy input to achieve step-wise fragmentation of larger aggregates into smaller aggregates. This is achieved by a stepwise increase in imposed energy which is controlled by the shaking time and air:water volume in the shaking bottle. Our study first examines the effect shaking has on the microbial biomass in comparison to the other fractionation procedures. We then fractionate similar soils with different carbon levels using the shaking procedure. Measurements of the breakdown and biological properties of aggregates are made and discussed in the context of the soil aggregate hierarchy. 2. Methodology 2.1. Soils Large intact soil samples were collected from the top layer of one arable (0–100 mm depth) and one grassland (30–100 mm depth to exclude the turf layer) field experiment located close to each other at the Rothamsted Experimental Station. The Garden Plot (arable soil) has been under continuous wheat production with no pesiticide inputs for over 40 years. Highfield grassland soil has been under continuous grass since 1948. Properties of these soils that are relevant to this study are listed in Table 1. The preparation of the soil for further analysis depended on the test which is discussed below. 2.2. Microbial disturbance caused by fractionation In this experiment we investigate changes in microbial respiration caused by different forms of mechanical energy inputs used to breakdown soil into aggregate size fractions. The arable soil was selected for this study because it has the lower stability and is therefore probably more sensitive to mechanical disturbance. Approximately 10 kg of field moist soil was bulked and sieved to obtain a 1–2 mm aggregate size range. The matric potential of the Table 1 Soil characteristics Soil Series Classification USDA FAO Soil properties Particle size (g kg1) Sand (2000–60 mm) Silt (60–2 mm) Clay (<2 mm) Organic carbon (g kg1) pH
Batcombe Stagnogley Paleo-Argillic Brown Earth Aquic Paleudalf or Paleudlt Chromic Luvisol or Alisol Arable (Garden Plot)
Permanent grassland (Highfield)
190 580 230
110 660 230
13.5 4.9
51.2 4.8
aggregates was equilibrated to 5 kPa using a tension table. After water equilibration, 50 g masses of wet aggregates were transferred to 500 ml shaking bottles. A controlled energy input was applied to 10 replicate samples using one of the following treatments: (i) (ii) (iii) (iv) (v) (vi)
Aggregates (control), Aggregates + 250 ml water, Aggregates + 250 ml water + 20 s (end-over-end) shaking, Aggregates + 20 s (end-over-end) shaking, Aggregates + 250 ml water + ultrasonic dispersion, Aggregates (air-dried and slaked).
Ultrasonic dispersion was done for 20 s using a sonicator probe with a power output of 100 W and frequency of 22.5 kHz. After each treatment, the soil was transferred to a tension table set at 5 kPa and at 5 8C for 48 h. All water additions were made using distilled and deionized water. The soils were transferred to sealed conical flasks and incubated at 20 8C for three days before CO2 concentrations were measured by gas chromatography using 1.0% and 0.1% CO2 standard gas samples as references. 2.3. Sequential fractionation of soil aggregates by shaking Moist soil from the arable and grassland experiment was broken by hand to obtain 10–20 mm diameter aggregates. The grassland soil was too wet to break up without causing structural damage at the time of sampling so it was first dried to below field capacity at room temperature (20 8C). To minimise handling disturbance, reduce any potential slaking effects and standardise water properties across similar soils, the aggregates were capillary wetted on a tension table to obtain a water potential of 5 kPa. Upon immersion in water, the water potential adjusts to 0 kPa after a short time and the small amount of associated wetting will probably not induce slaking (Grant and Dexter, 1989). The water potential at the start of shaking is therefore 0 kPa for both soils. The shaking energy was imposed by rotating sample bottles filled with a specific amount of soil and water. The bottles were secured to a drum rotating at 30 rpm with an opposing bottle centre-line distance of 800 mm. Table 2 lists the different shaking times and air:water ratios used to impart energy to the soil. The grassland soil was the first tested. Upon extension of the procedure to the arable soils, the lower mechanical stability was accounted for by decreasing the shaking time to obtain a similar aggregate size distribution. To allow for accurate carbon measurements, the soil mass was doubled as well as the bottle volume. After shaking using the methods listed in Table 2, the bottles were removed (four replicates per treatment) and fractionated into aggregates by wet sieving (2000, 250, 125, and 53 mm mesh sizes) using the approach of Yoder (1936). Soil particles <53 mm were left to settle overnight before the supernatant was decanted and the soil sediment collected. Aggregates were washed from each sieve into a glass beaker. Any organic matter such as plant residues were removed by hand before the aggregates were oven-dried at 105 8C. After drying, aggregates were weighed and two subsamples were taken. One sub-sample was treated with hydrogen peroxide and sodium hexametaphosphate and then shaken endover-end overnight. Dispersed aggregate solutions were then poured through their corresponding sieve size. Any small stones, sands and silts were then dried at 105 8C overnight before their weighing. Final aggregate weights were then corrected for coarse mineral material. The other sub-sample was used to determine carbon and nitrogen levels.
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Table 2 Summary of the different treatments used to assess shaking in water as a method for fractionating soil aggregates Soil
Bottle volume (ml) Dry mass soil (kg) Water to bottle volume ratio Shaking time
Arable (Garden Plot)
Permanent grassland (Highfield)
500 200 0.5 10, 20, 30, 120, 600 s
250 100 0.1, 0.3, 0.5 0.5, 1, 2, 5 h
2.4. Differences in the biological characteristics of different sized aggregates A more detailed examination of the biological characteristics of the aggregates fractionated from the arable soil was conducted using a second set of samples. Soil was taken from the arable Garden Plot and fractionated into 10–20 mm aggregates. The soil was first saturated and then the water potential was equilibrated to 5 kPa cm using a tension table. Then, 200 g (wet weight) of aggregates were weighed into 500 ml shaking bottles and 250 ml of water was added. The bottles were shaken for 30 s before being separated into the aggregate size fractions using wet sieving. Aggregates were separated into the same aggregate size classes as the previous experiment: 2000, 250, 125, 53 and <53 mm using the same procedures listed previously. Care was taken to remove any plant material by washing and swirling the aggregates under running water. Each size fraction was then transferred to a tension table held at 5 kPa pressure for 48 h. Once the aggregate water potential equilibrated, the aggregates were placed in conical flasks for their separate treatments. Aggregates from each of the above size classes were incubated for 7 d at 20 8C. Concentrations of CO2 were measured using gas chromatography with 1% and 0.1% standard gas mixtures. Carbohydrates were measured using the method of Haynes and Francis (1993). Briefly, a sub-sample of soil was taken from each aggregate size class range from each site and bulked to from a composite sample. Three replicates from each size class fraction were then taken. The soil was dried at 20 8C and ground (<2 mm) before 2 g portions of soil were placed in centrifuge tubes. Carbohydrates were then sequentially extracted using hot water (80 8C), 1 M HCl, or 0.5 NaOH for 16 h using a water bath. At the end of each extraction samples were centrifuged for 15 min at 10,000 rpm. The supernatant was then decanted and frozen (18 8C) prior to analysis. Carbohydrates were determined using a 2% solution of Anthrone by the method of Brink et al. (1960). Total carbon and nitrogen were measured using a Leco combustion analyzer using standard soil reference material. Data were analysed using standard analysis of variance (ANOVA) with GenStat 8th edition (The GenStat Committee, 2005) and presented as means with an associated standard deviation. 3. Results and discussion 3.1. Microbial disturbance caused by fractionation The various methods used to disrupt the soil into aggregate size fractions had different effects on microbial respiration (Fig. 1). Airdrying, which is an essential step in determining aggregate stability in the most common approach of slaking, caused a large increase in respiration. More detailed investigations into the effects of air-drying on soil microbial activity have attributed the rise in respiration to organism mortality and the release of organic
Fig. 1. The effect of different forms of fractionation on microbial respiration measured as CO2 produced. The level of significance between the fractionation procedure and the control is indicated above each bar. Error bars represent the standard deviation.
compounds upon re-wetting; both of which provide an easily decomposable food source to surviving microbes (Van Gestel et al., 1991; Birch, 1958). Ultrasonic dispersion caused a similar increase in respiration, thus precluding its usefulness as a method for breaking down soil aggregates for subsequent biological study. The effect of ultrasonics on microbes is well documented and the approach is often used to lyse cells (Stevenson, 1958). Some organisms must have survived this aggressive stress, feeding off dead microbes and mechanically released organic compounds. The other methods used to disrupt soil into aggregates caused smaller changes in microbial respiration. Water added in isolation with no energy inputs only increased respiration slightly. In this treatment, saturation with water followed by draining, may have redistributed soluble carbon. Shaking in water followed by wet sieving did not cause a significant increase in respiration. This technique therefore offered a way in which a soil could be subjected to energy inputs and then fractionated into aggregate size classes using wet sieving with a low impact on the microbial biomass. 3.2. Sequential fractionation of soil aggregates by shaking Fig. 2 illustrates the effectiveness of shaking for the sequential fractionation of the grassland and arable soils tested. With increased shaking time, more fragmentation of soil aggregates resulted which is indicated by the declining mean weight diameter (MWD) values. The impact of shaking is enhanced by reducing the water to bottle volume ratio to 0.10, thus causing marked fragmentation at even the lowest shaking time of 0.5 h. The higher water to bottle volume ratios of 0.33 and 0.50 caused more gradual fragmentation which is favourable because it allows for more control. MWD values are useful for describing the degree of fragmentation but do not describe the distribution of different aggregate size classes. Table 3 shows the % soil retained on each sieve size at each shaking time. Apart from very large (>2000 mm) or very small aggregates (<53 mm), increasing the energy inputs did not significantly alter the distribution in the other aggregate size fractions. A similar fractionation pattern was obtained for the arable soil by reducing the shaking time significantly and using a water to bottle volume ratio of 0.50 (Tables 2 and 3; Fig. 2). Since shaking
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Fig. 2. Change in the MWD for the different fractionation procedures used. The symbols refer to the water:bottle volume ratios of 0.1 (*), 0.3 (*), and 0.5 (!) for the permanent grassland soil and 0.5 (~) for the arable soil. Error bars represent the standard deviation.
time is proportional to the energy applied to the soil that causes fragmentation, the results illustrate that the arable soil is much less stable than the grassland. As with the grassland soil, the aggregate size range distribution for the arable soil is bimodal with the largest (>2000 mm) and smallest (<53 mm) aggregates comprising the majority of the sieved soil. In Fig. 3, the data has been renormalized to illustrate the mass change associated with shaking time for the arable soil. Aggregates 125 mm were unable to withstand the energy applied by shaking and thus fragment to form smaller aggregates. This trend is more apparent after the stone mass is removed. There are probably two different effects that contribute to the transition between a loss and gain in the mass of different aggregate sizes with increased shaking time. The first effect is the relationship between aggregate size, the kinetic energy applied by shaking, and the energy required for
Fig. 3. Proportional change in aggregate mass with increased shaking time for the arable soil. The data is renormalized by dividing the mass fraction after shaking by the mass fraction after the shortest shaking time of 10 s. Shaking times are represented as (*) 10 s, (*) 30 s, (!) 120 s, and (5) 600 s.
fragmentation. Research on larger size soil aggregates has shown that the energy required for fracture increases exponentially with decreasing aggregate size (Hallett et al., 1998). It would therefore be expected that larger aggregate sizes would be more likely to
Table 3 Aggregate size distribution expressed as percent total mass caused by the different fractionation treatments listed in Table 2 and for the detailed biological investigation of the aggregate properties Water:bottle volume
Permanent grassland (Highfield) 0.1 0.3 0.5 0.1 0.3 0.5 0.1 0.3 0.5 0.1 0.3 0.5 Arable (Garden Plot) 0.5 0.5 0.5 0.5 0.5 Biological investigation 0.5 a
Mean standard deviation.
Shaking time
0.5 h 0.5 h 0.5 h 1.0 h 1.0 h 1.0 h 2.0 h 2.0 h 2.0 h 5.0 h 5.0 h 5.0 h
10 s 20 s 30 s 120 s 600 s
30 s
Sieve size (mm) 2000
250
125
53
<53
35.19 3.24a 65.86 4.16 72.73 7.38 17.58 2.67 44.93 2.72 61.54 5.90 9.52 1.25 27.97 7.07 38.54 2.57 2.89 0.31 13.74 4.49 19.76 2.00
16.57 5.24 16.95 5.85 15.41 1.39 11.75 2.83 14.77 0.26 14.91 3.22 15.31 1.49 16.87 2.72 14.76 2.78 13.60 1.95 14.34 3.64 18.27 3.48
9.19 1.08 6.49 3.13 7.02 0.92 7.26 3.55 8.73 0.37 7.33 0.74 9.75 0.73 8.67 1.12 7.99 0.52 9.53 0.66 8.84 0.99 8.17 0.83
18.89 0.87 14.52 0.96 13.12 2.33 15.75 9.33 17.81 0.95 13.45 3.97 16.71 8.54 18.87 0.93 17.63 0.89 20.70 2.38 21.14 3.28 18.55 0.90
20.57 2.38 1.44 2.72 0.15 0.31 52.30 10.98 13.73 2.97 2.86 1.43 48.76 9.19 27.52 9.48 21.10 1.30 53.77 3.39 41.96 4.28 32.57 7.54
56.50 1.23 46.85 0.49 43.93 3.49 32.54 2.21 10.08 2.72
14.44 0.83 11.96 2.01 12.93 0.54 7.66 0.95 1.14 1.37
3.47 0.49 3.20 0.69 2.40 0.51 2.58 0.72 1.70 0.38
2.70 1.42 2.66 1.15 3.55 0.74 6.49 1.31 2.36 0.57
22.90 0.72 35.34 0.32 37.21 3.36 50.74 2.12 84.72 2.71
52.31 10.14
7.12 0.37
2.06 0.09
3.22 0.16
35.29 7.84
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Fig. 4. Change in the concentration of carbon in different aggregate sizes caused by increased shaking time. The symbols represent the sieve sizes 2000 mm (*), 250 mm (&), 125 mm (*), and 53 mm (&). Error bars represent the standard deviation.
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Fig. 6. Concentrations of hot water (black bars), alkali (white bars) and acid (striped bars) extractable carbohydrates in the different size aggregates of the arable soil fractionated by shaking for 30 s in a bottle filled 50% with water. Note that the values for acid extractable carbohydrates are indicated on the second y-axis. Error bars represent the standard deviation.
3.3. Differences in the biological characteristics of different sized aggregates
fragment, leading to an increase in the mass of smaller size aggregates. Coupled with the fragmentation energy vs. aggregate size property is the second effect caused by the stabilisation of soil by decomposed carbon. Oades and Waters (1991) showed using electron microscopy that aggregation by decomposing plant fragments occurs for aggregates <250 mm. Aggregates of this form are called ‘stable microaggregates’ because they have a far higher resistance to fragmentation. As a result, the rise in the proportion of aggregate size fractions <250 mm with increased shaking time that was observed in our experiment may be accentuated. To provide further support for this argument, concentrations of carbon in aggregates of different size were measured for the different shaking times (Fig. 4). The concentrations increase in 53– 250 mm aggregates with increasing shaking time which supports Oades and Waters (1991) conclusion that decomposed carbon stabilises soil. This trend continues until the soil is fractionated into its primary particles by the addition of glass balls. Fragmentation appears to be step-wise in both Figs. 3 and 4 with large aggregates breaking to become small aggregates. Although this suggests a hierarchical aggregate structure, it appears that carbon stabilisation is enhanced in the 53–250 mm aggregate size range.
The results from the second set of arable samples provides further information on the biological characteristics of different sized aggregates in the soil tested. We found no significant difference in either total carbon (P = 0.185) or total nitrogen (P = 0.515) in the any aggregate size fraction (Fig. 5). However, the C:N ratio was significantly different between aggregate size fractions (P < 0.001) with the 53–250 mm size range having the highest values (Fig. 5). This suggests that although the concentration of carbon in each size was similar, the quality of SOM associated with different sized aggregates was different. Although there was no link between C concentration and aggregate size, hot water and acid extractable carbohydrate differed significantly with aggregate size (P < 0.005). Aggregates in the 125–250 mm size class range had significantly higher concentrations of hot water and acid extractable carbohydrate (Fig. 6). Differences in alkali extractable carbohydrate were not as significant (P = 0.122). Carbohydrates are composed partially of microbial extracellular polysaccharides (EPS) and have been linked to aggregate stability (Roberson et al., 1995; Ball et al., 1996). Our results provide further evidence of a ‘stable microaggregate’ fraction produced by microbial activity in soil. The levels of microbial activity, measured as basal respiration patterns (Fig. 7), were
Fig. 5. Concentrations of nitrogen (clear bars) and carbon (diagonally striped bars) and the carbon:nitrogen (filled bars) in different size aggregates of the arable soil fractionated by shaking for 30 s in a bottle filled 50% with water. Error bars represent the standard deviation.
Fig. 7. Basal respiration levels in different size aggregates of the arable soil fractionated by shaking for 30 s in a bottle filled 50% with water. Error bars represent the standard deviation.
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highly correlated to carbohydrate levels (Fig. 6) and mirrored the C:N ratio (Fig. 5). Aggregates within the 53–250 mm size class range had significantly higher levels of respiration (P < 0.005); aggregates in the 125 mm fraction had the highest respiration rates of all (Fig. 7). 3.4. General discussion Investigations in which the properties of different aggregate sizes are examined are generally based on the concept of a soil aggregate hierarchy (Singh and Singh, 1995; Gupta and Germida, 1988; Puget et al., 1999; Elliott, 1986). There is an assumption that aggregates are inherent structural units of soil that are composed of smaller assemblages of smaller aggregates. In reality, a soil aggregate is rarely a unique structural entity in soil under field conditions, but a stronger volume element that is released from larger and weaker elements by mechanical disruption. The form of mechanical energy input is therefore a dominant factor in the observed aggregate fraction characteristics. Beare and Bruce (1993), for instance, found large differences in fractionation patterns between pretreatment conditions and wet-sieving procedures. Our results show, using a more controlled fractionation procedure, that higher energy inputs enhance soil fragmentation. This is not surprising and is observed for most materials and larger sized (>5 mm) soil aggregates (Hallett et al., 1998). The procedure of collecting wet-sieved soil aggregates of different sizes to describe the aggregate hierarchy concept is therefore not worthwhile unless the effect of the energy input on fragmentation is understood. We found that shaking caused a preferential fragmentation of aggregates >2000 mm into particles 53 mm in size. However, very strong microaggregates were present which caused a shift in the distribution of carbon between different aggregate sizes with increased shaking time (Fig. 4). Although carbon levels were highest in macroaggregates before shaking, they were similar for all aggregate sizes after moderate shaking. Wet-sieving of slaked soil provides only one level of energy input and it is generally found that the concentrations of carbon are highest in the largest aggregate size fractions (Singh and Singh, 1995; Gupta and Germida, 1988; Puget et al., 1999; Elliott, 1986). However, this probably has more to do with the disruptive effects of slaking than the inherent structure of the soil. Carbon has been linked to hydrophobicity which reduces the rate of water infiltration and probably slaking caused by rapid wetting (Hallett et al., 2001; von Lutzow et al., 2006). The disruptive input from slaking is also dependent on the length of the water transmission pathways in which air-pressure build-up occurs. Therefore, only the weakest elements will break down to form smaller aggregates (<250 mm) and there is a chance that the stable microaggregates observed in our study and by Oades and Waters (1991) will remain trapped in larger aggregates. These properties of soil raise questions about the validity of relating the biological stabilisation of soil to discrete aggregate sizes. Field et al. (2006) also questioned the validity of compartmentalizing aggregates into discrete size fractions rather than considering gradual breakdown mechanisms. They controlled mechanical energy input with ultrasonic dispersion, which is not suited for biological studies. 4. Conclusion Sequential fractionation of soil by applying different amounts of energy through shaking in water has elucidated various important aspects about the microbial stabilisation of soil aggregates. Soil is not air-dried, in contrast with the approach based on slaking, so the microbial biomass is not severely disrupted. Moreover, drying
affects the extent of aggregation and many soils in temperate are never completely air-dry under natural conditions. In the sequential fractionation procedure, one form of mechanical stress was applied in the form of shaking. Soil is disrupted if the mechanical stress imposed by shaking is sufficient to disrupt interparticle bonds. By increasing the energy input to soil, unstable larger aggregates fragment to form smaller aggregates. Arable soil required far lower energy inputs than grassland soil to cause fragmentation suggesting lower stability. In the 53–250 mm size range of ‘stable microaggregates’ we found increased levels of biological activity, carbohydrate, and carbon:nitrogen. This contradicts research in which slaking was used to fractionate soil that finds macroaggregates to be hotspots of biological activity. A direct comparison between the two procedures was reported by Ashman et al. (2003). Acknowledgements This research was funded by the Biotechnology and Biological Sciences Research Council (Grant No. 206/SGO 4490). IACR Rothamsted receives Grant-in-Aid support from the BBSRC. SCRI receives Grant-in-Aid support from the Scottish Government, Rural and Environment Research Analysis Directorate. References Abiven, S., Menasseri, S., Angers, D.A., Leterme, P., 2007. Dynamics of aggregate stability and biological binding agents during decomposition of organic materials. Eur. J. Soil Sci. 58 (1), 239–247. Ashman, M.R., Hallett, P.D., Brookes, P.C., 2003. Are the links between soil aggregate size class, soil organic matter and respiration rate artefacts of the fractionation procedure? Soil Biol. Biochem. 35 (3), 435–444. Ball, B.C., Cheshire, M.V., Robertson, E.G., Hunter, E.A., 1996. Carbohydrate composition in relation to structural stability, compatibility and plasticity of two soils in a long-term experiment. Soil Till. Res. 39, 143–160. Beare, H.M., Bruce, R.R., 1993. A comparison of methods for measuring water stable aggregates: implications for determining environmental effects on soil structure. Geoderma 56, 87–104. Birch, H.F., 1958. The effect of soil drying on humus decomposition and nitrogen availability. Plant Soil. X, 9–31. Brink, R.H., Dubach, P., Lynch, D.L., 1960. Measurement of carbohydrates in soil hydrolysates with anthrone. Soil Sci. 89, 157–166. Elliott, E.T., 1986. Aggregate structure and carbon, nitrogen and phosphorus in native and cultivated soils. Soil Sci. Soc. Am. J. 50, 627–633. Field, D.J., Minasny, B., Gaggin, M., 2006. Modelling aggregate liberation and dispersion of three soil types exposed to ultrasonic agitation. Aust. J. Soil Res. 44, 497–502. Grant, C.D., Dexter, A.R., 1989. Generation of microcracks in molded soils by rapid wetting. Aust. J. Soil Res. 27, 169–182. Gupta, V.V.S.R., Germida, J.J., 1988. Distribution of microbial biomass and its activity in different soil aggregate size classes as affected by cultivation. Soil Biol. Biochem. 20, 777–786. Hallett, P.D., Bird, N.R.A., Dexter, A.R., Seville, J.P.K., 1998. Application of fractals to the scaling of aggregate structure and strength. Eur. J. Soil Sci. 49, 203–211. Hallett, P.D., Baumgartl, T., Young, I.M., 2001. Subcritical water repellency of aggregates from a range of soil management practices. Soil Sci. Soc. Am. J. 65, 184–190. Haynes, R.J., Francis, G.S., 1993. Changes in biomass C, soil carbohydrate composition and aggregate stability induced by growth of selected crop and forage species under field conditions. J. Soil Sci. 44, 665–675. Jastrow, J.D., Miller, R.M., Lussenhop, J., 1998. Contributions of interacting biological mechanisms to soil aggregate stabilization in restored prairie. Soil Biol. Biochem. 30, 905–916. Oades, J.M., Waters, A.G., 1991. Aggregate hierarchy in soils. Aust. J. Soil Res. 29, 815–828. Plante, A.F., Mcgill, W.B., 2002. Soil aggregate dynamics and the retention of organic matter in laboratory-incubated soil with differing simulated tillage frequencies. Soil Till. Res. 66, 79–92. Puget, P., Angers, D.A., Chenu, C., 1999. Nature of carbohydrates associated with water-stable aggregates of two cultivated soils. Soil Biol. Biochem. 31, 55–63. Roberson, E., Sarig, S., Shennan, C., Firestone, M.K., 1995. Nutritional management of microbial polysaccharide production and aggregation in an agricultural soil. Soil Sci. Soc. Am. J. 59, 1587–1594. Sainju, U.M., 2006. Carbon and nitrogen pools in soil aggregates separated by dry and wet sieving methods. Soil Sci. 171, 937–949. Seech, A.G., Beauchamp, E.G., 1988. Denitrification in soil aggregates of different sizes. Soil Sci. Soc. Am. J. 52, 1616–1621.
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Tisdall, J.M., Oades, J.M., 1982. Organic matter and water-stable aggregates in soils. J. Soil Sci. 33, 141–163. Van Gestel, M., Ladd, J.N., Amato, M., 1991. Carbon and nitrogen mineralization from two soils of contrasting texture and microaggegate stability: influence of sequential fumigation, drying and storage. Soil Biol. Biochem. 23, 313–322. von Lutzow, M., Kogel-Knabner, I., Ekschmitt, K., Matzner, E., Guggenberger, G., Marschner, B., Flessa, H., 2006. Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions—a review. Eur. J. Soil Sci. 57, 426–445. Wright, S.F., Upadhyaya, A., 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant Soil. 198, 97–107. Yoder, R.E., 1936. A direct method of aggregate analysis of soils and a study of the physical nature of erosion losses. J. Am. Soc. Agron. 28, 337–351.
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Soil & Tillage Research journal homepage: www.elsevier.com/locate/still
Evaluating soil stabilisation by biological processes using step-wise aggregate fractionation M.R. Ashman a, P.D. Hallett b,*, P.C. Brookes a, J. Allen a a b
Soil Science Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Soil aggregate Hierarchy Carbon Microbial activity
Soil biological processes contribute stability against physical disruption. We present an approach of stepwise fragmentation to assess the role that microbes and organic matter have on soil aggregate stabilisation. Compared to slaking and ultrasound procedures, the approach has a low impact on the microbial biomass. It also does not impose a severe drying stress. Grassland soil was found to be more stable than arable soil. Further examination of the arable soil revealed that increased disruption by shaking caused unstable microaggregates 53–250 mm in size to fragment, leaving a higher proportion of stable microaggregates in this size range. Carbohydrates, C:N, and basal respiration were found to be higher in the stable microaggregates than the other size fractions. Our results indicate that a distinct size range of soil aggregates exists in which microbial stabilisation dominates. This contradicts other research and questions the usefulness of measuring the biological properties of aggregate size fractions without understanding the physical effects of the fractionation procedure. ß 2008 Elsevier B.V. All rights reserved.
1. Introduction Organic matter and biological activity levels have been linked to soil structural stabilisation in many studies (Abiven et al., 2007; Sainju, 2006; Puget et al., 1999; Oades and Waters, 1991; Six et al., 1998). It is now uncontested that microbial exudates have a dominant role in the aggregation of soil particles and the protection of carbon from further degradation (Six et al., 2006; Roberson et al., 1995). Through these processes, it has been postulated that microorganisms ‘engineer’ a superior habitat and soil structure leading to more productive and resilient soil systems (von Lutzow et al., 2006). The very important inter-relationship between soil structure and biology is now an intensively researched area in soil science. Most of this work is based around the concept of an aggregate hierarchy which Tisdall and Oades (1982) proposed to describe soil stabilisation by microorganisms over various aggregate size ranges. This concept suggests that larger aggregates are formed from assemblages of smaller aggregates. Studies based on the aggregate hierarchy concept generally examine the properties of differently sized soil aggregates obtained using a range of fractionation techniques (Ashman et al., 2003; Field et al., 2006;
* Corresponding author. Tel.: +44 1382 562731; fax: +44 1382 562426. E-mail address: [email protected] (P.D. Hallett). 0167-1987/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.still.2008.07.005
Plante and Mcgill, 2002; Puget et al., 1999; Jastrow et al., 1998; Six et al., 1998; Elliott, 1986). Soil aggregates able to withstand the disruptive effects of the fractionation technique are categorised as structurally stable. Aggregates are rarely individual structural entities in soil but volume elements released by the mechanical energy input from fractionation. The form and level of the energy input therefore has a significant influence on the properties and size distribution of the collected aggregates. The five major methods used to fractionate soil are (1) slaking of air-dried soil followed by wet-sieving (water stability test) (Jastrow et al., 1998; Six et al., 1998), (2) wet-sieving of capillary re-wetted or field-moist soil (Six et al., 1998; Wright and Upadhyaya, 1998), (3) dry sieving of soil (Seech and Beauchamp, 1988), (4) end-over-end shaking followed by wetsieving (Oades and Waters, 1991) and (5) ultrasonic dispersion (Tippkotter, 1994). Each approach imposes different forms of energy inputs on the soil and conflicting results between aggregate size and the abundance of stabilising agents have been found. Studies which fractionate soil using slaking tend to find carbon levels to be highest for macroaggregates (>250 mm) (Tisdall and Oades, 1982; Elliott, 1986; Gupta and Germida, 1988; Singh and Singh, 1995; Six et al., 1998; Puget et al., 1999). Seech and Beauchamp (1988) found that microaggregates (53–250 mm) have the greatest level of carbon if soil is fractionated using dry-sieving. Biological investigations of soil stabilisation are also affected by sample pre-treatment prior to fractionation. Both air-drying (Van
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Gestel et al., 1991; Birch, 1958) and ultrasonic dispersion (Stevenson, 1958) disturb the microbial community. Detailed investigations of the form and function of microbial communities associated with different aggregate sizes is an area requiring more research. Most research to date concerning biological stabilisation of soil, however, has fractionated soil using slaking (Puget et al., 1999) which is unsuitable for detailed microbial studies because the soil is dried. We propose that microbial disturbance can be reduced and a description of aggregation by biological processes improved by adopting end-over-end shaking of soil in water followed by wet sieving. The approach is adapted from Oades and Waters (1991) who showed that the aggregate hierarchy can be elucidated by different energy levels to fractionate soil. We improve the technique by controlling the energy input to achieve step-wise fragmentation of larger aggregates into smaller aggregates. This is achieved by a stepwise increase in imposed energy which is controlled by the shaking time and air:water volume in the shaking bottle. Our study first examines the effect shaking has on the microbial biomass in comparison to the other fractionation procedures. We then fractionate similar soils with different carbon levels using the shaking procedure. Measurements of the breakdown and biological properties of aggregates are made and discussed in the context of the soil aggregate hierarchy. 2. Methodology 2.1. Soils Large intact soil samples were collected from the top layer of one arable (0–100 mm depth) and one grassland (30–100 mm depth to exclude the turf layer) field experiment located close to each other at the Rothamsted Experimental Station. The Garden Plot (arable soil) has been under continuous wheat production with no pesiticide inputs for over 40 years. Highfield grassland soil has been under continuous grass since 1948. Properties of these soils that are relevant to this study are listed in Table 1. The preparation of the soil for further analysis depended on the test which is discussed below. 2.2. Microbial disturbance caused by fractionation In this experiment we investigate changes in microbial respiration caused by different forms of mechanical energy inputs used to breakdown soil into aggregate size fractions. The arable soil was selected for this study because it has the lower stability and is therefore probably more sensitive to mechanical disturbance. Approximately 10 kg of field moist soil was bulked and sieved to obtain a 1–2 mm aggregate size range. The matric potential of the Table 1 Soil characteristics Soil Series Classification USDA FAO Soil properties Particle size (g kg1) Sand (2000–60 mm) Silt (60–2 mm) Clay (<2 mm) Organic carbon (g kg1) pH
Batcombe Stagnogley Paleo-Argillic Brown Earth Aquic Paleudalf or Paleudlt Chromic Luvisol or Alisol Arable (Garden Plot)
Permanent grassland (Highfield)
190 580 230
110 660 230
13.5 4.9
51.2 4.8
aggregates was equilibrated to 5 kPa using a tension table. After water equilibration, 50 g masses of wet aggregates were transferred to 500 ml shaking bottles. A controlled energy input was applied to 10 replicate samples using one of the following treatments: (i) (ii) (iii) (iv) (v) (vi)
Aggregates (control), Aggregates + 250 ml water, Aggregates + 250 ml water + 20 s (end-over-end) shaking, Aggregates + 20 s (end-over-end) shaking, Aggregates + 250 ml water + ultrasonic dispersion, Aggregates (air-dried and slaked).
Ultrasonic dispersion was done for 20 s using a sonicator probe with a power output of 100 W and frequency of 22.5 kHz. After each treatment, the soil was transferred to a tension table set at 5 kPa and at 5 8C for 48 h. All water additions were made using distilled and deionized water. The soils were transferred to sealed conical flasks and incubated at 20 8C for three days before CO2 concentrations were measured by gas chromatography using 1.0% and 0.1% CO2 standard gas samples as references. 2.3. Sequential fractionation of soil aggregates by shaking Moist soil from the arable and grassland experiment was broken by hand to obtain 10–20 mm diameter aggregates. The grassland soil was too wet to break up without causing structural damage at the time of sampling so it was first dried to below field capacity at room temperature (20 8C). To minimise handling disturbance, reduce any potential slaking effects and standardise water properties across similar soils, the aggregates were capillary wetted on a tension table to obtain a water potential of 5 kPa. Upon immersion in water, the water potential adjusts to 0 kPa after a short time and the small amount of associated wetting will probably not induce slaking (Grant and Dexter, 1989). The water potential at the start of shaking is therefore 0 kPa for both soils. The shaking energy was imposed by rotating sample bottles filled with a specific amount of soil and water. The bottles were secured to a drum rotating at 30 rpm with an opposing bottle centre-line distance of 800 mm. Table 2 lists the different shaking times and air:water ratios used to impart energy to the soil. The grassland soil was the first tested. Upon extension of the procedure to the arable soils, the lower mechanical stability was accounted for by decreasing the shaking time to obtain a similar aggregate size distribution. To allow for accurate carbon measurements, the soil mass was doubled as well as the bottle volume. After shaking using the methods listed in Table 2, the bottles were removed (four replicates per treatment) and fractionated into aggregates by wet sieving (2000, 250, 125, and 53 mm mesh sizes) using the approach of Yoder (1936). Soil particles <53 mm were left to settle overnight before the supernatant was decanted and the soil sediment collected. Aggregates were washed from each sieve into a glass beaker. Any organic matter such as plant residues were removed by hand before the aggregates were oven-dried at 105 8C. After drying, aggregates were weighed and two subsamples were taken. One sub-sample was treated with hydrogen peroxide and sodium hexametaphosphate and then shaken endover-end overnight. Dispersed aggregate solutions were then poured through their corresponding sieve size. Any small stones, sands and silts were then dried at 105 8C overnight before their weighing. Final aggregate weights were then corrected for coarse mineral material. The other sub-sample was used to determine carbon and nitrogen levels.
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Table 2 Summary of the different treatments used to assess shaking in water as a method for fractionating soil aggregates Soil
Bottle volume (ml) Dry mass soil (kg) Water to bottle volume ratio Shaking time
Arable (Garden Plot)
Permanent grassland (Highfield)
500 200 0.5 10, 20, 30, 120, 600 s
250 100 0.1, 0.3, 0.5 0.5, 1, 2, 5 h
2.4. Differences in the biological characteristics of different sized aggregates A more detailed examination of the biological characteristics of the aggregates fractionated from the arable soil was conducted using a second set of samples. Soil was taken from the arable Garden Plot and fractionated into 10–20 mm aggregates. The soil was first saturated and then the water potential was equilibrated to 5 kPa cm using a tension table. Then, 200 g (wet weight) of aggregates were weighed into 500 ml shaking bottles and 250 ml of water was added. The bottles were shaken for 30 s before being separated into the aggregate size fractions using wet sieving. Aggregates were separated into the same aggregate size classes as the previous experiment: 2000, 250, 125, 53 and <53 mm using the same procedures listed previously. Care was taken to remove any plant material by washing and swirling the aggregates under running water. Each size fraction was then transferred to a tension table held at 5 kPa pressure for 48 h. Once the aggregate water potential equilibrated, the aggregates were placed in conical flasks for their separate treatments. Aggregates from each of the above size classes were incubated for 7 d at 20 8C. Concentrations of CO2 were measured using gas chromatography with 1% and 0.1% standard gas mixtures. Carbohydrates were measured using the method of Haynes and Francis (1993). Briefly, a sub-sample of soil was taken from each aggregate size class range from each site and bulked to from a composite sample. Three replicates from each size class fraction were then taken. The soil was dried at 20 8C and ground (<2 mm) before 2 g portions of soil were placed in centrifuge tubes. Carbohydrates were then sequentially extracted using hot water (80 8C), 1 M HCl, or 0.5 NaOH for 16 h using a water bath. At the end of each extraction samples were centrifuged for 15 min at 10,000 rpm. The supernatant was then decanted and frozen (18 8C) prior to analysis. Carbohydrates were determined using a 2% solution of Anthrone by the method of Brink et al. (1960). Total carbon and nitrogen were measured using a Leco combustion analyzer using standard soil reference material. Data were analysed using standard analysis of variance (ANOVA) with GenStat 8th edition (The GenStat Committee, 2005) and presented as means with an associated standard deviation. 3. Results and discussion 3.1. Microbial disturbance caused by fractionation The various methods used to disrupt the soil into aggregate size fractions had different effects on microbial respiration (Fig. 1). Airdrying, which is an essential step in determining aggregate stability in the most common approach of slaking, caused a large increase in respiration. More detailed investigations into the effects of air-drying on soil microbial activity have attributed the rise in respiration to organism mortality and the release of organic
Fig. 1. The effect of different forms of fractionation on microbial respiration measured as CO2 produced. The level of significance between the fractionation procedure and the control is indicated above each bar. Error bars represent the standard deviation.
compounds upon re-wetting; both of which provide an easily decomposable food source to surviving microbes (Van Gestel et al., 1991; Birch, 1958). Ultrasonic dispersion caused a similar increase in respiration, thus precluding its usefulness as a method for breaking down soil aggregates for subsequent biological study. The effect of ultrasonics on microbes is well documented and the approach is often used to lyse cells (Stevenson, 1958). Some organisms must have survived this aggressive stress, feeding off dead microbes and mechanically released organic compounds. The other methods used to disrupt soil into aggregates caused smaller changes in microbial respiration. Water added in isolation with no energy inputs only increased respiration slightly. In this treatment, saturation with water followed by draining, may have redistributed soluble carbon. Shaking in water followed by wet sieving did not cause a significant increase in respiration. This technique therefore offered a way in which a soil could be subjected to energy inputs and then fractionated into aggregate size classes using wet sieving with a low impact on the microbial biomass. 3.2. Sequential fractionation of soil aggregates by shaking Fig. 2 illustrates the effectiveness of shaking for the sequential fractionation of the grassland and arable soils tested. With increased shaking time, more fragmentation of soil aggregates resulted which is indicated by the declining mean weight diameter (MWD) values. The impact of shaking is enhanced by reducing the water to bottle volume ratio to 0.10, thus causing marked fragmentation at even the lowest shaking time of 0.5 h. The higher water to bottle volume ratios of 0.33 and 0.50 caused more gradual fragmentation which is favourable because it allows for more control. MWD values are useful for describing the degree of fragmentation but do not describe the distribution of different aggregate size classes. Table 3 shows the % soil retained on each sieve size at each shaking time. Apart from very large (>2000 mm) or very small aggregates (<53 mm), increasing the energy inputs did not significantly alter the distribution in the other aggregate size fractions. A similar fractionation pattern was obtained for the arable soil by reducing the shaking time significantly and using a water to bottle volume ratio of 0.50 (Tables 2 and 3; Fig. 2). Since shaking
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Fig. 2. Change in the MWD for the different fractionation procedures used. The symbols refer to the water:bottle volume ratios of 0.1 (*), 0.3 (*), and 0.5 (!) for the permanent grassland soil and 0.5 (~) for the arable soil. Error bars represent the standard deviation.
time is proportional to the energy applied to the soil that causes fragmentation, the results illustrate that the arable soil is much less stable than the grassland. As with the grassland soil, the aggregate size range distribution for the arable soil is bimodal with the largest (>2000 mm) and smallest (<53 mm) aggregates comprising the majority of the sieved soil. In Fig. 3, the data has been renormalized to illustrate the mass change associated with shaking time for the arable soil. Aggregates 125 mm were unable to withstand the energy applied by shaking and thus fragment to form smaller aggregates. This trend is more apparent after the stone mass is removed. There are probably two different effects that contribute to the transition between a loss and gain in the mass of different aggregate sizes with increased shaking time. The first effect is the relationship between aggregate size, the kinetic energy applied by shaking, and the energy required for
Fig. 3. Proportional change in aggregate mass with increased shaking time for the arable soil. The data is renormalized by dividing the mass fraction after shaking by the mass fraction after the shortest shaking time of 10 s. Shaking times are represented as (*) 10 s, (*) 30 s, (!) 120 s, and (5) 600 s.
fragmentation. Research on larger size soil aggregates has shown that the energy required for fracture increases exponentially with decreasing aggregate size (Hallett et al., 1998). It would therefore be expected that larger aggregate sizes would be more likely to
Table 3 Aggregate size distribution expressed as percent total mass caused by the different fractionation treatments listed in Table 2 and for the detailed biological investigation of the aggregate properties Water:bottle volume
Permanent grassland (Highfield) 0.1 0.3 0.5 0.1 0.3 0.5 0.1 0.3 0.5 0.1 0.3 0.5 Arable (Garden Plot) 0.5 0.5 0.5 0.5 0.5 Biological investigation 0.5 a
Mean standard deviation.
Shaking time
0.5 h 0.5 h 0.5 h 1.0 h 1.0 h 1.0 h 2.0 h 2.0 h 2.0 h 5.0 h 5.0 h 5.0 h
10 s 20 s 30 s 120 s 600 s
30 s
Sieve size (mm) 2000
250
125
53
<53
35.19 3.24a 65.86 4.16 72.73 7.38 17.58 2.67 44.93 2.72 61.54 5.90 9.52 1.25 27.97 7.07 38.54 2.57 2.89 0.31 13.74 4.49 19.76 2.00
16.57 5.24 16.95 5.85 15.41 1.39 11.75 2.83 14.77 0.26 14.91 3.22 15.31 1.49 16.87 2.72 14.76 2.78 13.60 1.95 14.34 3.64 18.27 3.48
9.19 1.08 6.49 3.13 7.02 0.92 7.26 3.55 8.73 0.37 7.33 0.74 9.75 0.73 8.67 1.12 7.99 0.52 9.53 0.66 8.84 0.99 8.17 0.83
18.89 0.87 14.52 0.96 13.12 2.33 15.75 9.33 17.81 0.95 13.45 3.97 16.71 8.54 18.87 0.93 17.63 0.89 20.70 2.38 21.14 3.28 18.55 0.90
20.57 2.38 1.44 2.72 0.15 0.31 52.30 10.98 13.73 2.97 2.86 1.43 48.76 9.19 27.52 9.48 21.10 1.30 53.77 3.39 41.96 4.28 32.57 7.54
56.50 1.23 46.85 0.49 43.93 3.49 32.54 2.21 10.08 2.72
14.44 0.83 11.96 2.01 12.93 0.54 7.66 0.95 1.14 1.37
3.47 0.49 3.20 0.69 2.40 0.51 2.58 0.72 1.70 0.38
2.70 1.42 2.66 1.15 3.55 0.74 6.49 1.31 2.36 0.57
22.90 0.72 35.34 0.32 37.21 3.36 50.74 2.12 84.72 2.71
52.31 10.14
7.12 0.37
2.06 0.09
3.22 0.16
35.29 7.84
M.R. Ashman et al. / Soil & Tillage Research 102 (2009) 209–215
Fig. 4. Change in the concentration of carbon in different aggregate sizes caused by increased shaking time. The symbols represent the sieve sizes 2000 mm (*), 250 mm (&), 125 mm (*), and 53 mm (&). Error bars represent the standard deviation.
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Fig. 6. Concentrations of hot water (black bars), alkali (white bars) and acid (striped bars) extractable carbohydrates in the different size aggregates of the arable soil fractionated by shaking for 30 s in a bottle filled 50% with water. Note that the values for acid extractable carbohydrates are indicated on the second y-axis. Error bars represent the standard deviation.
3.3. Differences in the biological characteristics of different sized aggregates
fragment, leading to an increase in the mass of smaller size aggregates. Coupled with the fragmentation energy vs. aggregate size property is the second effect caused by the stabilisation of soil by decomposed carbon. Oades and Waters (1991) showed using electron microscopy that aggregation by decomposing plant fragments occurs for aggregates <250 mm. Aggregates of this form are called ‘stable microaggregates’ because they have a far higher resistance to fragmentation. As a result, the rise in the proportion of aggregate size fractions <250 mm with increased shaking time that was observed in our experiment may be accentuated. To provide further support for this argument, concentrations of carbon in aggregates of different size were measured for the different shaking times (Fig. 4). The concentrations increase in 53– 250 mm aggregates with increasing shaking time which supports Oades and Waters (1991) conclusion that decomposed carbon stabilises soil. This trend continues until the soil is fractionated into its primary particles by the addition of glass balls. Fragmentation appears to be step-wise in both Figs. 3 and 4 with large aggregates breaking to become small aggregates. Although this suggests a hierarchical aggregate structure, it appears that carbon stabilisation is enhanced in the 53–250 mm aggregate size range.
The results from the second set of arable samples provides further information on the biological characteristics of different sized aggregates in the soil tested. We found no significant difference in either total carbon (P = 0.185) or total nitrogen (P = 0.515) in the any aggregate size fraction (Fig. 5). However, the C:N ratio was significantly different between aggregate size fractions (P < 0.001) with the 53–250 mm size range having the highest values (Fig. 5). This suggests that although the concentration of carbon in each size was similar, the quality of SOM associated with different sized aggregates was different. Although there was no link between C concentration and aggregate size, hot water and acid extractable carbohydrate differed significantly with aggregate size (P < 0.005). Aggregates in the 125–250 mm size class range had significantly higher concentrations of hot water and acid extractable carbohydrate (Fig. 6). Differences in alkali extractable carbohydrate were not as significant (P = 0.122). Carbohydrates are composed partially of microbial extracellular polysaccharides (EPS) and have been linked to aggregate stability (Roberson et al., 1995; Ball et al., 1996). Our results provide further evidence of a ‘stable microaggregate’ fraction produced by microbial activity in soil. The levels of microbial activity, measured as basal respiration patterns (Fig. 7), were
Fig. 5. Concentrations of nitrogen (clear bars) and carbon (diagonally striped bars) and the carbon:nitrogen (filled bars) in different size aggregates of the arable soil fractionated by shaking for 30 s in a bottle filled 50% with water. Error bars represent the standard deviation.
Fig. 7. Basal respiration levels in different size aggregates of the arable soil fractionated by shaking for 30 s in a bottle filled 50% with water. Error bars represent the standard deviation.
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highly correlated to carbohydrate levels (Fig. 6) and mirrored the C:N ratio (Fig. 5). Aggregates within the 53–250 mm size class range had significantly higher levels of respiration (P < 0.005); aggregates in the 125 mm fraction had the highest respiration rates of all (Fig. 7). 3.4. General discussion Investigations in which the properties of different aggregate sizes are examined are generally based on the concept of a soil aggregate hierarchy (Singh and Singh, 1995; Gupta and Germida, 1988; Puget et al., 1999; Elliott, 1986). There is an assumption that aggregates are inherent structural units of soil that are composed of smaller assemblages of smaller aggregates. In reality, a soil aggregate is rarely a unique structural entity in soil under field conditions, but a stronger volume element that is released from larger and weaker elements by mechanical disruption. The form of mechanical energy input is therefore a dominant factor in the observed aggregate fraction characteristics. Beare and Bruce (1993), for instance, found large differences in fractionation patterns between pretreatment conditions and wet-sieving procedures. Our results show, using a more controlled fractionation procedure, that higher energy inputs enhance soil fragmentation. This is not surprising and is observed for most materials and larger sized (>5 mm) soil aggregates (Hallett et al., 1998). The procedure of collecting wet-sieved soil aggregates of different sizes to describe the aggregate hierarchy concept is therefore not worthwhile unless the effect of the energy input on fragmentation is understood. We found that shaking caused a preferential fragmentation of aggregates >2000 mm into particles 53 mm in size. However, very strong microaggregates were present which caused a shift in the distribution of carbon between different aggregate sizes with increased shaking time (Fig. 4). Although carbon levels were highest in macroaggregates before shaking, they were similar for all aggregate sizes after moderate shaking. Wet-sieving of slaked soil provides only one level of energy input and it is generally found that the concentrations of carbon are highest in the largest aggregate size fractions (Singh and Singh, 1995; Gupta and Germida, 1988; Puget et al., 1999; Elliott, 1986). However, this probably has more to do with the disruptive effects of slaking than the inherent structure of the soil. Carbon has been linked to hydrophobicity which reduces the rate of water infiltration and probably slaking caused by rapid wetting (Hallett et al., 2001; von Lutzow et al., 2006). The disruptive input from slaking is also dependent on the length of the water transmission pathways in which air-pressure build-up occurs. Therefore, only the weakest elements will break down to form smaller aggregates (<250 mm) and there is a chance that the stable microaggregates observed in our study and by Oades and Waters (1991) will remain trapped in larger aggregates. These properties of soil raise questions about the validity of relating the biological stabilisation of soil to discrete aggregate sizes. Field et al. (2006) also questioned the validity of compartmentalizing aggregates into discrete size fractions rather than considering gradual breakdown mechanisms. They controlled mechanical energy input with ultrasonic dispersion, which is not suited for biological studies. 4. Conclusion Sequential fractionation of soil by applying different amounts of energy through shaking in water has elucidated various important aspects about the microbial stabilisation of soil aggregates. Soil is not air-dried, in contrast with the approach based on slaking, so the microbial biomass is not severely disrupted. Moreover, drying
affects the extent of aggregation and many soils in temperate are never completely air-dry under natural conditions. In the sequential fractionation procedure, one form of mechanical stress was applied in the form of shaking. Soil is disrupted if the mechanical stress imposed by shaking is sufficient to disrupt interparticle bonds. By increasing the energy input to soil, unstable larger aggregates fragment to form smaller aggregates. Arable soil required far lower energy inputs than grassland soil to cause fragmentation suggesting lower stability. In the 53–250 mm size range of ‘stable microaggregates’ we found increased levels of biological activity, carbohydrate, and carbon:nitrogen. This contradicts research in which slaking was used to fractionate soil that finds macroaggregates to be hotspots of biological activity. A direct comparison between the two procedures was reported by Ashman et al. (2003). Acknowledgements This research was funded by the Biotechnology and Biological Sciences Research Council (Grant No. 206/SGO 4490). IACR Rothamsted receives Grant-in-Aid support from the BBSRC. SCRI receives Grant-in-Aid support from the Scottish Government, Rural and Environment Research Analysis Directorate. References Abiven, S., Menasseri, S., Angers, D.A., Leterme, P., 2007. Dynamics of aggregate stability and biological binding agents during decomposition of organic materials. Eur. J. Soil Sci. 58 (1), 239–247. Ashman, M.R., Hallett, P.D., Brookes, P.C., 2003. Are the links between soil aggregate size class, soil organic matter and respiration rate artefacts of the fractionation procedure? Soil Biol. Biochem. 35 (3), 435–444. Ball, B.C., Cheshire, M.V., Robertson, E.G., Hunter, E.A., 1996. Carbohydrate composition in relation to structural stability, compatibility and plasticity of two soils in a long-term experiment. Soil Till. Res. 39, 143–160. Beare, H.M., Bruce, R.R., 1993. A comparison of methods for measuring water stable aggregates: implications for determining environmental effects on soil structure. Geoderma 56, 87–104. Birch, H.F., 1958. The effect of soil drying on humus decomposition and nitrogen availability. Plant Soil. X, 9–31. Brink, R.H., Dubach, P., Lynch, D.L., 1960. Measurement of carbohydrates in soil hydrolysates with anthrone. Soil Sci. 89, 157–166. Elliott, E.T., 1986. Aggregate structure and carbon, nitrogen and phosphorus in native and cultivated soils. Soil Sci. Soc. Am. J. 50, 627–633. Field, D.J., Minasny, B., Gaggin, M., 2006. Modelling aggregate liberation and dispersion of three soil types exposed to ultrasonic agitation. Aust. J. Soil Res. 44, 497–502. Grant, C.D., Dexter, A.R., 1989. Generation of microcracks in molded soils by rapid wetting. Aust. J. Soil Res. 27, 169–182. Gupta, V.V.S.R., Germida, J.J., 1988. Distribution of microbial biomass and its activity in different soil aggregate size classes as affected by cultivation. Soil Biol. Biochem. 20, 777–786. Hallett, P.D., Bird, N.R.A., Dexter, A.R., Seville, J.P.K., 1998. Application of fractals to the scaling of aggregate structure and strength. Eur. J. Soil Sci. 49, 203–211. Hallett, P.D., Baumgartl, T., Young, I.M., 2001. Subcritical water repellency of aggregates from a range of soil management practices. Soil Sci. Soc. Am. J. 65, 184–190. Haynes, R.J., Francis, G.S., 1993. Changes in biomass C, soil carbohydrate composition and aggregate stability induced by growth of selected crop and forage species under field conditions. J. Soil Sci. 44, 665–675. Jastrow, J.D., Miller, R.M., Lussenhop, J., 1998. Contributions of interacting biological mechanisms to soil aggregate stabilization in restored prairie. Soil Biol. Biochem. 30, 905–916. Oades, J.M., Waters, A.G., 1991. Aggregate hierarchy in soils. Aust. J. Soil Res. 29, 815–828. Plante, A.F., Mcgill, W.B., 2002. Soil aggregate dynamics and the retention of organic matter in laboratory-incubated soil with differing simulated tillage frequencies. Soil Till. Res. 66, 79–92. Puget, P., Angers, D.A., Chenu, C., 1999. Nature of carbohydrates associated with water-stable aggregates of two cultivated soils. Soil Biol. Biochem. 31, 55–63. Roberson, E., Sarig, S., Shennan, C., Firestone, M.K., 1995. Nutritional management of microbial polysaccharide production and aggregation in an agricultural soil. Soil Sci. Soc. Am. J. 59, 1587–1594. Sainju, U.M., 2006. Carbon and nitrogen pools in soil aggregates separated by dry and wet sieving methods. Soil Sci. 171, 937–949. Seech, A.G., Beauchamp, E.G., 1988. Denitrification in soil aggregates of different sizes. Soil Sci. Soc. Am. J. 52, 1616–1621.
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