2.2. Experimental design and soil incubation
The soil from the OF treatment (15 year-old) represents active SOC. The
soils from the BF treatment for 15 years and BF treatment for 23 years
plus an extra 815 days of laboratory incubation (BF+, the additional
incubation is to further consume active organic carbon) represent
relatively resistant SOC (Zhang et
al., 2017). Therefore, we refer to the OF SOC as active C, the BF and
the BF+ SOC as relatively resistant C in this study. Although all three
soils may still contain various components with different turnover times
and protection mechanisms, here we operationally used the three soils to
represent active and relatively resistant SOC
(Conant
et al., 2008; Townsend et al., 1997), and compared the relative
vulnerability of these SOC pools to the direct and legacy effects of
drying-wetting cycles for two reasons.
First, we measured the proportion
of active C (dissolved organic carbon/total carbon, DOC/TC) and found
that the proportion of active C in the OF soil is the highest, and that
in the BF+ soil is the lowest (Table 1). Second, we measured the
chemical composition of SOC of these three soils and found that the
molecular index of SOC stability (or resistance) in the OF soil is the
lowest, and that in the BF+ soil is the highest (Fig. 2). In addition,
Barré et al. (2010) constrained a three-pool-model using observed soil
organic C data in long-term BF soils and found that most of the organic
C are stable C (turnover time of several centuries or more). Basic
properties of these three soils can be found in Table 1 and
Zhang
et al.
(2017).
The incubation experiment consisted of a nine 10-d DWC period and a 28-d
extended period, with a 10-d stabilization period between them (Fig. 1).
In the DWC period (0-90 d), soils were subjected to three water regimes,
including one constant-moisture control (mean moisture content: 60%
WHC), one mild DWC treatment (80-40% WHC), and one strong DWC treatment
(100-20% WHC), each with three replicates. After the 90-d DWC period,
all treatments were adjusted to constant moisture of 60% WHC for ten
days to a consistent state (90-100 d). All soils were kept at 60% WHC
during the subsequent 28-d extended period (100-128 d).
For each soil, a subsample of 60 g (dry weight basis) was placed in a
1-L incubation jar at 60% WHC with deionized water, and the soils were
pre-incubated at 22°C for 10 days in the dark. To achieve soil drying,
45 g and 20 g silica gel were placed in the incubation jars for 100-20%
WHC and 80-40% WHC treatments, respectively (Fig. S1). These two
weights were chosen because our pre-experimental data showed that these
two weights of silica gel could accelerate the loss of soil moisture to
the expected levels (i.e. 20% and 40% WHC) in 10 days. Inside each
jar, 10 ml 0.5 M NaOH solution was used to trap CO2 from
SOC decomposition. At the end of each drying period, the NaOH solution
and silica gel were replaced, and the soil samples were rapidly rewetted
by adding deionized water with a syringe. The trapped
CO2-C in the NaOH solutions were immediately measured in
the form of total inorganic C using a Lotix combustion TOC analyzer
(Teledyne Tekmar, USA), and the silica gel was dried in an oven at 105°C
overnight to regenerate it. After replacing the silica gel and NaOH
solution, the jars were sealed immediately with gas-tight lids and kept
at 22°C in the dark. A previous study proved that silica gel does not
absorb CO2(Harrison-Kirk
et al., 2013). Three blanks without soil were incubated at the same
condition to quantify the amount of C in the air and initial NaOH
solution. Prior to measuring the total inorganic C, the NaOH solutions
in the DWC treatments were adjusted back to 10 ml with deionized water.
After the 90-d DWC period, all soils were adjusted to a constant
moisture content of 60% WHC and maintained for 10 days (stabilization
period, 90-100 d). Note that we did not measure respiration for the
stabilization period. On day 100, we did a destructive soil sampling.
Specifically, 8 g subsamples were used to determine dissolved organic
carbon (DOC) and microbial biomass carbon (MBC), 10 g subsamples were
stored at -80°C until the measurement of microbial biomass, and 10 g
subsamples were incubated for another 28 d (extended period, 100-128 d)
in which the CO2-C in the NaOH solutions were measured
on day 103, 107, 114, 121 and 128.
In total, we incubated 30 jars ((3 × 3 + 1) × 3) consisting of three
soils (OF, BF and BF+ soils), two DWC treatments (100-20% and 80-40%
WHC) and one constant-moisture control treatment (60% WHC), one blank,
and three replicates for each treatment.