Prior to pulse labelling each soil core, the RSMS at each soil depth was connected through a 3-way stopcock to a long PVC tube and a glass tube (internal diameter 2.2 mm), creating a 'U' bend, which was used as a manometer. A syringe ( 1 ml) was also attached to the 3-way stopcock for creating suction and taking soil solution samples through RSMS by manipulation of the 3-way stopcock. Immediately before sampling soil solution, the change in the manometer water level with reference to the sampling depths was recorded at 8, 1 2, 1 6, 20, 24, 30, 36 and 44 h after pulse labelling to monitor the diurnal change in the root activity (in terms of the soil water potential) at the root-soil water interface. The manometer readings recorded for all the depths were then adjusted for the rise of water level in the tube due to capillarity. Recording of manometer readings was stopped 44 h after pulse labelling because of the appearance of bubbles in the 3-way stopcock, which interfered with the measurements.
5.2. 6 Sampling and harvesting
5.2. 6. 1 Soil solution sampling
Samples of the soil solution (0.5 ml) were taken from both the lysimeters through the RSMS placed at 2, 4, 8 and 12 cm soil depths by creating a vacuum produced by a 1 ml syringe attached to the 3-way stopcock on the L-L connector of the RSMS (see Plate 7.4). These samples were collected periodically at 1 , 2, 4, 8, 1 2, 1 6, 20, 24, 30, 36, 44 and 48 h after exposing pastures to 14C02 atmosphere. The 0.5 ml samples of soil solution were added to polypropylene mini vials containing 1 ml of 0. 1 7 M NaOH as a 14C02-trap solution. These vials were sealed immediately and held for scintillation counting.
5.2. 6.2 Herbage, root and soil sampling and their preparation for analysis
Herbage samples (8 to 10 shoots/leaves per sampling) were taken randomly from the pasture swards growing on the lysimeter at 59, 79, 103.5, 1 29.5, 1 52.5 and 1 72.5 h after exposure to 14C02 pulse, dried at 70°C, finely chopped and stored for analyses of total C
and 14e. This enabled the specific activity (DPS mg-1 C) of herbage/shoots to be monitored during the one-week period of growth from the time of 1 4C02 exposure.
A week after pulse labelling, the lysimeters were removed from the sand bath (connected to water supply) and the RSMS's were gently pulled out of the lysimeters. The soil core was removed intact from the PVC cylinder. The shoots were excised at the soil surface and collected in paper bags, dried at 70°C in an oven, weighed and stored for total
C and 1 4C analysis. Also the clover/grass sterns below the soil surface, along with some adhering soil, were removed from the soil and dried at 70°C in an oven for total C and 14C
analysis. The soil cores were sectioned at 2, 4, 8, 12, 1 6 cm depth using a sharp knife and circular sections of soi1+roots were divided into eight equal sections. Two opposite sections (4 sections in all) were dried to a constant weight at 1 000e for soil moisture content determination and then ground and stored for analysis of total C and 14C in soi1+roots. The soil+root samples were ground to <0.2 mm firstly with a pestle and mortar and then roots and soil were finely chopped using a domestic electric mini food processor in a fume cupboard. Any dead, brownlblack material was discarded and a sub-sample (approximately 50 g) of the above processed soil samples were ring ground using a RockLabs ring grinder (for 10 seconds). A subsequent evaluation of the ring grinding procedure has been conducted in Section 6.2.5. 1 (Figure 6.1b).
The roots were carefully separated from the remaining two halves of soil section by wet sieving (> 0.5 mm
).
The roots were dried at 700e in an oven, weighed, finely chopped and stored for total C and 14C determination. The root mass in each soil slice is calculated from the dry weight of the roots in the sub-sampled wet soil and the total net weight of wet soil in each slice.All operations using 1 4C labelled solutions, gas, herbage, roots and soil materials were
5.2. 7 A nalysis
5.2. 7. 1 Basic chemical analysis of soil
Soil pH in distilled water ( 1 :2.5 w/w) was detennined usmg a glass electrode according to Blakemore et al. ( 1987). Olsen P was detennined by extracting soil samples for 30 minutes with 0.5 M NaHC03 at pH 8.5 (Olsen et al., 1 954) and measuring the phosphate concentration colorimetrically using the Murphy and Riley ( 1962) method as described by Blakemore et al. ( 1987). Total S in the soil detennined using the NaHC03 fusion method of Steinberg et al. ( 1962) (see Section 3.2.4). Total N and P in the soil were detennined by Kjeldahl digestion (McKenzie and Wallace, 1 954). The Kjeldahl digests were analysed for ammonium and phosphate using a Technicon II autoanalyser.
5.2. 7.2 Scintillation cocktail recipe
The scintillation cocktail used was prepared by mIxmg 8 g of PPO (2,5- diphenyloxazole) as a primary scintillant, 0.2 g of PO POP [ 1 ,4-Di- {2-(4-methyl-5-phenyl oxazolyl)} -benzene] as a secondary scintillant, 2000 ml of toluene (solvent) and 1 250 ml of surfactant triton-X 100 (Middleboe et al., 1 976). The mixture was stirred overnight by a magnetic stirrer and kept in a dark glass bottle. The recipe for making the cocktail solution was slightly modified from the original recipe i.e. an additional 250 ml of the surfactant was added (surfactant volume used in the original recipe is 1000 ml) to ensure complete covering of salt and water molecules and provide a clear suspension of the scintillation cocktail and different soil extracts obtained in this study.
5. 2. 7. 3
14
C in soil solutionScintillation cocktail (5 ml) was added to the soil solution (0.5 ml from RSMS), which was mixed with 1 ml of 0. 17 M NaOH in the mini vials. Vials were shaken vigorously to produce a clear solution and placed in dark for 30-60 minutes to minimise the chemiluminescence before 14C counting. The 14C counts were measured using a Wallac 1414 WinSpectral™ liquid scintillation counter (LSC) with standard parameters for 14C counting, and automatic quench correction.
14C02 from root/microbial respiration and/or diffusion of 14C-C02 into the soil through gaseous exchange process or b) the release of soluble !4C-carbohydrate, amino acids etc. as root exudes, the suspension of the alkaline soil solution and cocktail solution was acidified to pH :::;4.5 with addition of 0.14 ml of I M HCl. The vials were shaken well after the addition of the acid and placed open in the fume hood for three days so that any 14C02 formed upon acidification of the suspension could escape to the atmosphere. After three days, all the vials were placed in an ultrasonic bath (power rating - 200 watts) for about 20 minutes to remove any 14C02 entrapped in gas bubbles. The volume was readjusted to compensate the loss of toluene from the cocktail solution. The vials were capped and recounted for !4C counts using LSC as mentioned above.