Appendix
Recommended procedure for incubation of soil samples
The variance among soils is substantially large that it is unlikely any single study
will isolate all important phenomena regarding the incubation of soils. Rather, patterns
will likely emerge from comparison among multiple studies. Comparison among studies
is facilitated by use of common methods, but there seems to be no such standards in the
literature. Therefore, we make the following recommendations for soil incubation studies
and encourage others to follow this protocol and improve upon it.
1) Minimal disturbance: Incubation of minimally disturbed soil samples most closely
approximates field conditions. If the goal of the study is to investigate naturally occurring
processes, soil samples should be minimally disturbed before incubation.
2) Preincubation: Given the known influence of disturbance on elevated respiration rates
and the common practice of preincubation for respiration rate studies (Lerch et al., 2011),
we recommend a preincubation period of at least 1 week.
3) Duration of incubation: For many soils at least 2 weeks of incubation are required
before steady state 13C values of headspace CO2 are achieved. Ideally, the required
incubation time is determined for each soil and experimental conditions.
4) Small water/gas volume ratios: Incubation vials should have small water/gas volume
ratios in order to minimize the effect of fractionation associated with dissolution of CO2
in the water (Figure 4). A first order correction for this effect can be made using
temperature dependent carbon isotope fractionation factors between CO2 and DIC species
(e.g., Deines et al., 1974; Mook et al., 1974) if the amount of water in the sample is
known, the head space volume is known, and the pH of the soil solution is known or
approximated. This is especially important for high pH soils (e.g., high CEC soils).
5) Flushing with CO2 free air: Flushing with CO2-free air is easy and overcomes the need
to calculate the 13C value of respired CO2 with a mixing model (otherwise a source of
error). However, flushing with CO2 free air likely induces desorption and/or degassing of
CO2 and therefore still requires time for the biological flux to overcome this initial CO2
pulse. A comparison of results from the same soils incubated with and without flushing
with CO2-free air would be useful for method development.
6) Calibration of carbon isotope ratio measurements: Multiple point calibration and
application of resulting stretching factors for 13C values of both SOM and CO2 improve
the accuracy of comparisons between the two. Multiple point calibration is perhaps more
important for SOM analysis. Another option is to directly compare the microbially
respired CO2 with the CO2 generated from total oxidation of SOM (i.e. one in each
bellows of a mass spectrometer).
7) Avoid soils with carbonates: The dissolution of carbonates in incubation vials will
influence measured apparent respiration rates and 13C values of respired CO2.
Quantitatively removing carbonates without influencing organic matter is difficult.
Therefore, unless the objective is to investigate the influence of carbonates, soil samples
with carbonates should be avoided.
Explanation of potential artifacts in previous soil incubation experiments
For instance, Andrews et al. (2000) collected headspace gas samples 1 hour after
flushing with CO2 free air, which may have influenced their results given our
observations of CO2 desorption following flushing incubation vials with CO2 free air
(Figure 1). Stevenson et al. (2005) incubated calcium carbonate-bearing soils for a short
period of time (1 week) and it is unclear whether incubated soils were acidified to remove
carbonates and if so, whether acidification affected the 13C values of respired CO2.
Many studies have reported 13C values of respired CO2 that are lower than the
13C values of substrate. In most of these studies the substrate was either pure
biochemical compounds or fresh plant matter (Blair et al., 1985; Mary et al., 1992;
Schweizer et al., 1999; Fernandez and Cadisch, 2003; Fernandez et al., 2003) which is
not immediately relevant to the more complex relationship between respired CO2 and
SOM. Several bulk soil studies have documented negative CO2-SOM, values, which are
expected if a carbon isotope fractionation during respiration controls the increase in 13C
values of SOM with depth. However, we suggest that negative CO2-SOM values result
from experimental artifacts. For instance, Wynn et al. (2006) observed negative CO2-SOM
values from incubation of minimally disturbed, 20cm long cores of loess-parented
Alfisols. The negative CO2-SOM values reported in that study were based on comparison
of 13C values of CO2 respired during incubation of an entire 20 cm core with what are
presumably weighted average 13C values of SOM for each core. Disproportionately (in
comparison to organic carbon concentrations) high respiration rates in the tops of the soil
cores, which contained the SOM with the lowest 13C values and, presumably, the most
labile organic matter might explain all or part of the negative CO2-SOM values. Lerch et al.
(2011) also observed negative CO2-SOM after 15 days from the incubation of sieved,
physically mixed and then preincubated (3 weeks) Luvisol plow layer samples. In
addition, Lerch et al. (2011) determined that 13C values of soil microbial biomass were
higher than 13C values of SOM. Given the evidence for microbial biomass as a precursor
for SOM (Dijkstra et al., 2006; Simpson et al., 2007; Miltner et al., 2009), the results of
Lerch et al. (2011) satisfy, at least qualitatively, the mass balance required for carbon
isotope fractionation during microbial decomposition to explain the increase in the 13C
values of SOM with depth. However, Lerch et al. (2011) incubated mixtures of soil
collected from a large depth interval (0-30 cm). Therefore their negative CO2-SOM values
may have resulted from preferential respiration of rapidly cycling SOM from the
shallowest parts of the profile (i.e. the same experimental artifact that may have
controlled the negative CO2-SOM values reported by Wynn et al. (2006)).
Some studies have reported positive CO2-SOM values. We argue that many of these
positive CO2-SOM values also resulted from experimental artifacts. Incubation of size
fractions from the Ah horizon of a Norway Spruce forest Luvisol (Mueller et al., 2014)
and samples collected from the O, A and Bw horizons of a beech forest Inceptisol
(Formánek and Ambus, 2004) resulted in positive CO2-SOM values. Mueller et al. (2014)
disturbed their soil samples by physically mixing which could have resulted in positive
CO2-SOM values by exposing otherwise aggregate-protected labile organic compounds,
which have higher 13C values than recalcitrant organic compounds (Benner et al., 1987).
This is consistent with our observation that disturbance increases 13C values of respired
CO2, even with a 3 week preincubation period (Table 2). Formánek and Ambus (2004)
reported large positive CO2-SOM values (+3.5 to +5‰). The 13C values of respired CO2 in
that study were calculated using the keeling plot approach (13Crespired CO2 taken as the y-
intercept on a plot of 13C CO2 versus 1/CO2). The one keeling plot presented by
Formánek and Ambus (2004, their figure 1) suggests that the 13C value of evolved CO2
decreased over the course of the experiment. However, the 13C values of respired CO2
were calculated using all the data form the entire course of the incubations, likely biasing
the calculated 13C values toward processes occurring at the beginning of incubation
experiments. Steady state 13C values probably more accurately reflect natural conditions
and we therefore suggest the large positive 13C values of respired CO2 reported by
Formánek and Ambus (2004) may be too high. Ekblad et al. (2002) and Ekblad and
Högberg (2000) measured CO2 emitted from the soil surface in the field. Comparison
with 13C values of SOM in 3-5cm thick mor layers resulted in positive CO2-SOM values
for their control experiments. However, CO2 emitted from the soil surface integrates
respiration from the entire soil profile, including root respiration by water stressed plants,
which might explain the elevated 13C values of respired CO2 (Ekblad and Högberg,
2000). Furthermore, it should be noted that 13C values of CO2 emitted from the soil
surface can depart substantially (~ 5‰) from 13C values of respired CO2 if the soil CO2
profile is not at steady state (e.g., Moyes et al., 2010). Werth et al. (2006) also reported
large, positive CO2-SOM values from incubation of a loess-parented loamy haplic Luvisol;
however, these values are likely biased by diffusive loss of pore space CO2. In that study,
experimental pots containing moist soil were open to the atmosphere and then sealed one
day prior to purging in order to collect pore space CO2 for carbon isotope measurements.
12
CO2 preferentially (compared to 13CO2) diffuses out of soils to the atmosphere leaving
the residual pore space CO2 with higher 13C values than the respired CO2 (Cerling et al.,
1991). Werth et al. (2006) almost certainly analyzed some soil CO2, with 13C values
modified by diffusion prior to sealing the pots, resulting in measured 13C values that are
greater than the actual 13C values of CO2 respired.
Several studies have reported variable CO2-SOM values. For instance, incubation of
A horizon samples collected from grassland soils resulted in CO2-SOM with an average
value near zero but substantial variability from -4.2 to +1.5 ‰ for individual samples
(Santrucková et al., 2000). In that study, soils were disturbed by sieving and it is not clear
whether there was a preincubation period. The incubation period was relatively short (10
days) and respired CO2 was collected in NaOH, which probably absorbed all of the CO2
initially sorbed onto the soil particles, which would have magnified the effect we
observed in the present study when flushing with CO2 free air. Some soil incubation
studies found that the sign of CO2-SOM varied with incubation time; respired CO2 was
initially lighter than SOM (Andrews et al., 2000; Crow et al., 2006; Mueller et al., 2014)
or vice versa (Lerch et al., 2011). Crow et al. (2006) incubated density fractions and it is
unclear how their CO2-SOM values relate to those for undisturbed soil. It should also be
noted that the soil samples occupied roughly half the volume of the incubation vials in
that study. Depending on pH, the relatively large volume of water used by Crow et al
(2006) could have contained a substantial percentage of total respired CO2, which would
make measured 13C values of headspace CO2 minimum estimates for the 13C values of
respired CO2.
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