M aterials and methods

2.3.1 Study site a n d so il sam pling

The experim ental site w as located at the T eagasc Research Centre, Oak Park, Carlow, Ireland (52°51' N 6°54' W , 50 m a.s.l.). The soil at this site has a loam y

sand texture, with a pH o f 6.9. Mean annual precipitation and annual temperature are 830 mm and 9.3 °C, respectively. At the experimental site, we established three sampling plots (15x30 m) in a Miscanthus plantation and three plots on adjacent arable land. The Miscanthus field (30x120 m) was established in 1994. Although a detailed historical record o f past land use is not available, the entire experimental site had been under cultivation for at least 15 years before the Miscanthus plantation was established, and has been cropped exclusively with C3

crops. Aboveground standing biomass o f the Miscanthus was harvested annually in March - April.

In order to collect undisturbed soil cores, two different soil sampling procedures were used. In June 2007, four cores ( 0 10 cm) were taken down to 60 cm in each plot and were divided in four soil layers (0-15 cm, 15-30 cm, 30-45 cm and 45-60 cm). These samples were used for bulk soil analyses. In addition, four cores ( 0 5.6 cm) per plot were collected and divided into 0-15 and 15-30 cm soil layers. These samples were used in the soil fractionation procedure. All soil samples were combined by depth increment. Soil samples were air-dried and soil density and gravel content were measured by conventional methodologies. The four cores were sieved to pass through an 8 mm sieve by gently breaking apart the soil. Aboveground biomass production in the Miscanthus plots was determined by harvesting 2x2 m quadrats in each plot. The mean peak dry matter yield (November 2007) was 16 t ha'' and the mean harvest dry matter yield (April 2008) was 13 t h a''. Plant, litter and root material were collected from the Miscanthus plots three times during the growing season o f 2007 for isotopic analyses. All plant samples were combined per plot and were air-dried.

Bulk soil M acroaggregates M (>250 (jm) M icroaggregates (53-250 |jm) Itl C o a rse POM Intra-microaggregate POM (>1.85 g cm IPOIVI_m M icroaggregates (53-250 ^im) mlVI Intra-microaggregate POM (>1.85 g cm 3) IPOM_mM

F ig u r e 1: Fractionation sch em e adopted in this study, m o d ified after S ix et al. (1 9 9 8 , 2 0 0 2 ). M = m acroaggregates (> 2 5 0 |im siz e aggregates); m = m icroaggregates (5 3 -2 5 0 |im siz e aggregates); SC = silt& clay53 |j.m siz e cla ss fraction; coarse POM = > 2 5 0 jim siz e particulate organic matter; m M = m icroaggregates w ithin m acroaggregates; SC _M = silt& clay w ithin m acroaggregates; iPO M _m M = intra-aggregate particulate organic matter w ithin mM ; iPO M _m = intra-aggregate particulate organic matter w ithin m.

2.3.2 Soil fractionation

All sam ples from the 0-15 cm and 15-30 cm soil layers were fractionated by size and density (Fig. 1). Briefly, tw o sieves (250 and 53 [im mesh size) were used to separate m acroaggregates (>250 |im; M), m icroaggregates (53-250 |im ; m) and the silt & clay fraction (<53 p-m; SC). A subsam ple was subm erged for 5 m in in room -tem perature-deionized water, on top o f a 250 |j,m sieve. A ggregate separation was achieved by m anually m oving the sieve up and dow n 3 cm w ith 50 repetitions during a period o f 2 m in. A fter the 2 m in cycle, the M fraction was gently backw ashed o ff the sieve into an alum inum pan. W ater plus soil that w ent through the sieve w as poured onto a 53 |xm sieve and the sieving procedure w as repeated. M aterial <53 |im w as left to settle for 24 hours in plastic bottles, the supernatant w as poured o ff and the SC fraction were w ashed into another alum inum pan. All the fractions w ere oven dried at 50 °C.

In the second step, the M fraction w as separated into coarse particulate organic m atter (>250 jj.m; coarse PO M ), m icroaggregates (53-250 [im; m M ) and silt and clay (<53 |im ; SC_M ) by using the m ethodology described in Six et al. (2000). Subsam ples o f fraction M w ere im m ersed in deionized w ater on top o f a 250 |j,m m esh screen and gently shaken w ith 50 glass beads ( 0 5.4 m m ) for 10 min. The coarse POM w as retained on the 250 (xm screen, w hile a continuous and steady w ater flow ensured that the m M were flushed onto a 53 |im sieve. O nce the M fraction was broken up entirely, the m aterial on the 53 |im sieve w as wet- sieved. The fraction retained on the 53 (j.m sieve (i.e. w ater-stable m M ) and the fraction that passed through the 53 |j,m sieve (i.e. SC_M ) w ere collected and dried at 50 °C.

Density fractionation was carried out by follow ing the m ethod described in Six et al. (1998). Subsam ples (5 g) o f the oven-dried m and mM fractions w ere suspended in 35 mL o f a 1.85 g cm'^ o f sodium polytungstate (SPT) solution and slow ly shaken by hand. M aterial rem aining on the cap and sides o f the centrifuge tube w as w ashed into suspension w ith 10 mL o f the SPT solution. A fter 20 min o f vacuum (138 kPa), the sam ple w as centrifuged (1250 g) at 20 °C for 60 m in. The floating m aterial was aspirated onto a 20 mm nylon filter, thereby rem oving all undecom posed litter and root fragm ents from the soil sam ples. The heavy fraction w as rinsed tw ice w ith 50 mL o f deionized w ater and dispersed by shaking it w ith 30 mL o f 0.5% sodium hexam etaphosphate for 18 h. The dispersed heavy fraction w as passed through a 53 |i.m sieve and the m aterial rem aining on the sieve, i.e. the intra-aggregate particulate organic m atter (iPO M ), w as dried (50 °C) and w eighed.