The fresh weights of all cores used for the incubation experiment were recorded on removing rubber lids. The lower opening of incubation cores was covered with plastic
mesh and filter paper to prevent loss of material, before mounting on a plastic draining plate. Intact cores were placed upright in 1L Mason jars for experimental incubation (Fig. 4.1).
For each field sampling point (three per plot), each of the three cores collected was randomly assigned to one of three experimental moisture treatments: 1) control (average field % WHC); 2) mild drought; 3) severe drought. Cores were separated into three experimental blocks (A, B and C) according to their respective plot sampling point (i.e.
one true replicate per block), and arranged by moisture treatment in alternating CCF and SLG pairs (Fig. 4.2).
Figure 4.1 Side (A) and top (B) view of 8 x 4 cm intact soil core installed in mason jar for experimental incubation.
A B
Cores were installed in a controlled temperature room maintained at 24 ˚C for the duration of the study. This was the average soil temperature across all CCF and SLG plots measured in situ during the previous soil survey (Chapter 2). Jars were placed on one shelf at the same height to maximise uniformity in temperature across all cores (Fig.
4.3), with the nested block design providing additional control of small variations in local microclimate conditions within the room. When not under drought conditions, jars were covered with a moisture-resistant flexible film (Parafilm; Bemis, USA) punctured with air holes, to reduce soil evaporation rates while allowing gas exchange to avoid anaerobic conditions.
Figure 4.2 Schematic of experimental incubation setup. Cores were arranged in blocks of paired closed-canopy forest (F) and selective logging gap (G) cores by plot (1-6) and moisture treatment (control, mild drought and severe drought).
For soil % WHC adjustment, synthetic rain was prepared using autoclaved deionised water with additions of sodium chloride (NaCl: 0.29 g l-1), calcium chloride dihydrate (CaCl2.2H20: 0.09 g l-1), calcium sulphate dihydrate (CaSO4.2H2O: 0.07 g l
-1), magnesium sulphate heptahydrate (MgSO4.7H2O: 0.13 g l-1) and sulphuric acid (98
% H2SO4: 0.23 g l-1) based on available chemical composition data of rain collected at Danum Valley Field Station, Danum Valley Conservation Area, Sabah, Borneo (4.95°, 117.79°), in the same region as the present study plots (data were obtained from the World Data Centre for Precipitation Chemistry; http://wdcpc.org; Vet et al., 2014).
Synthetic rain was used rather than pure deionised water to minimise leaching of nutrients from soils during the incubation, and more closely represent field conditions.
60 % maximum soil WHC was chosen to represent average field moisture conditions, using the approximate % WHC of all cores measured for WHC (CCF and SLG). The weights of soil cores at 60 % WHC was determined by estimating the mass of dry soil Figure 4.3 Soil cores mounted in mason jars installed at equal height in a controlled temperature (CT) room.
average soil moisture content for each plot, then multiplying by average maximum WHC for each plot.
All cores were adjusted to and maintained at 60 (± 3) % soil WHC (monitored every three days minimum) for an equilibration period of one week prior to the measurement of baseline CO2 efflux and experimental drought treatments. This was to allow microbial communities to revive after refrigeration, and stabilisation of soil CO2
efflux. The start of the equilibration period (and of all subsequent experiment time points) was staggered by one day between blocks, to allow time for % WHC monitoring and adjustment, and gas sampling throughout the duration of the incubation experiment.
After equilibration, only control treatment cores were maintained at 60 % WHC. The paraffin film was removed from jars of mild and severe drought treatment cores, and % WHC was monitored daily. When drought treatment cores reached approximately half the control % WHC when averaged across all CCF and SLG cores (i.e. 30 % WHC;
after seven days of drying), mild drought treatment cores were readjusted back to 60 % WHC and re-covered with paraffin film. Severe drought treatment cores were left to dry for approximately double the drying period of mild treatment cores (a total of 15 days) before readjusting back to 60 % WHC and re-covering with paraffin film. After rewetting, both mild and severe drought treatment cores were maintained at 60 % WHC for a recovery period of 11 days.
CO2 efflux rates were measured at four experimental time points: 1) before drought (BD), i.e. after stabilisation period for baseline CO2 efflux; 2) after drought (AD), for mild and extreme drought treatments and respective controls; 3) initial recovery (IR), three days after rewetting of mild and extreme drought treatment cores;
4) final recovery (FR), eleven days after rewetting of mild and extreme drought
treatment cores. CO2 sampling was conducted using a Picarro gas analyser (Picarro Instruments, USA). Jars were sealed using a custom lid fitted with inlet and outlet pipes in a closed system with the Picarro analyser, using silicon grease to ensure an airtight seal. Increase in CO2 concentration was allowed to stabilise for approximately one minute, based on real-time visualisation using the analyser display. CO2 concentrations were then recorded in ppm at one second intervals for six minutes. CO2 concentration data for each measurement were trimmed to the last 350 points to retain a period of linear increase over time, and rate of change in ppm was calculated using linear regression. CO2 efflux rates were then calculated in µg CO2-C cm-2 hour-1 using the following formula:
𝐶𝑂2 𝑒𝑓𝑓𝑙𝑢𝑥 𝑟𝑎𝑡𝑒 =3,600 ∗ 𝑚 ∗ 𝑉 ∗ 𝐶𝑀∗ 𝑃 𝐴 ∗ 𝑅 ∗ 𝑇
where m is the rate of change on CO2 concentration (ppm s-1), V is the volume of chamber used during measurement corrected for soil core volume (m3), CM is the molecular mass of C (g mol-1), P is absolute gas pressure, A is surface area of core (m2), R is the universal gas constant and T is temperature (Kelvin). As intact cores were used
to represent the inherent properties of the entire soil matrix, CO2 efflux rates were expressed on a per area rather than per g soil basis. This retains integrated soil physicochemical and structural properties influencing soil water and gas dynamics along the soil profile (see for example Briones et al., 2014).