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 d13C 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 d13C 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 d13C 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 d13C 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 d13C values of respired CO2.
Many studies have reported d13C values of respired CO2 that are lower than the d13C 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 eCO2-SOM, values, which are expected if a carbon isotope fractionation during respiration controls the increase in d13C values of SOM with depth. However, we suggest that negative eCO2-SOM values result from experimental artifacts. For instance, Wynn et al. (2006) observed negative eCO2-SOM values from incubation of minimally disturbed, 20cm long cores of loess-parented Alfisols. The negative eCO2-SOM values reported in that study were based on comparison of d13C values of CO2 respired during incubation of an entire 20 cm core with what are presumably weighted average d13C 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 d13C values and, presumably, the most labile organic matter might explain all or part of the negative eCO2-SOM values. Lerch et al. (2011) also observed negative eCO2-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 d13C values of soil microbial biomass were higher than d13C 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 d13C 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 eCO2-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 eCO2-SOM values reported by Wynn et al. (2006)).
Some studies have reported positive eCO2-SOM values. We argue that many of these positive eCO2-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 eCO2-SOM values. Mueller et al. (2014) disturbed their soil samples by physically mixing which could have resulted in positive eCO2-SOM values by exposing otherwise aggregate-protected labile organic compounds, which have higher d13C values than recalcitrant organic compounds (Benner et al., 1987). This is consistent with our observation that disturbance increases d13C values of respired CO2, even with a 3 week preincubation period (Table 2). Formánek and Ambus (2004) reported large positive eCO2-SOM values (+3.5 to +5‰). The d13C values of respired CO2 in that study were calculated using the keeling plot approach (d13Crespired CO2 taken as the y-intercept on a plot of d13C CO2 versus 1/CO2). The one keeling plot presented by Formánek and Ambus (2004, their figure 1) suggests that the d13C value of evolved CO2 decreased over the course of the experiment. However, the d13C values of respired CO2 were calculated using all the data form the entire course of the incubations, likely biasing the calculated d13C values toward processes occurring at the beginning of incubation experiments. Steady state d13C values probably more accurately reflect natural conditions and we therefore suggest the large positive d13C 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 d13C values of SOM in 3-5cm thick mor layers resulted in positive eCO2-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 d13C values of respired CO2 (Ekblad and Högberg, 2000). Furthermore, it should be noted that d13C values of CO2 emitted from the soil surface can depart substantially (~ 5‰) from d13C 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 eCO2-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. 12CO2 preferentially (compared to 13CO2) diffuses out of soils to the atmosphere leaving the residual pore space CO2 with higher d13C values than the respired CO2 (Cerling et al., 1991). Werth et al. (2006) almost certainly analyzed some soil CO2, with d13C values modified by diffusion prior to sealing the pots, resulting in measured d13C values that are greater than the actual d13C values of CO2 respired.
Several studies have reported variable eCO2-SOM values. For instance, incubation of A horizon samples collected from grassland soils resulted in eCO2-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 eCO2-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 eCO2-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 d13C values of headspace CO2 minimum estimates for the d13C values of respired CO2.
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