Short Communication: Quantifying and Correcting for Pre-Assay CO2 Loss in Short-Term Carbon Mineralization Assays

Short Communication: Quantifying and Correcting for Pre-Assay CO2 Loss in Short-Term Carbon Mineralization Assays Matthew A. Belanger, Carmella Vizza, G. Philip Robertson, and Sarah S. Roley W.K. Kellogg Biological Station, Michigan State University, Hickory Corners, MI 49060, USA School of the Environment, Washington State University, Richland, WA 99354, USA 5 Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA


Introduction
The pulse of CO2 following the re-wetting of dried soils Franzluebbers et al., 2000) has been widely 20 used to indicate soil C availability because of its association with soil microbial biomass C and the active fraction of SOC.
This method is derived from the "Birch Effect," whereby re-wetted dry soils release a pulse of CO2 resulting from increased microbial activity (Birch, 1958). Drought stress drives microbial communities to dormancy or death (Borken and Matzner, 2009), and following the reintroduction of moisture, microbes burst or release solutes to avoid bursting (Schimel et al., 2007), which stimulates C mineralization (Kim et al., 2012). 25 Although the short-term pulse of CO2 following the re-wetting of dry soils is a widely used method for assessing soil C availability (e.g., Culman et al., 2013;Ladoni et al., 2016;Morrow et al., 2016;Sprunger and Robertson, 2018), we are unaware of efforts to quantify the potential bias introduced by assaying soils of different moisture contents at the time of sampling.
Soils that differ in moisture will dry down at different rates, potentially losing different amounts of available C prior to the 30 start of the assay. If sufficiently large, differential pre-assay losses could complicate comparisons of C availability across field treatments or landscape catenas. https://doi.org/10.5194/soil-2020-55 Preprint. Discussion started: 29 September 2020 c Author(s) 2020. CC BY 4.0 License.
Here we investigate the influence of different initial soil moisture levels on pre-assay CO2 release during drying for an Alfisol soil in the upper Midwest, USA. We test the hypothesis that moister soil will have higher pre-assay CO2 loss because a longer dry-down period results in more time for such losses to occur. 35 2 Materials and methods
Average annual rainfall at KBS is 1005 mm, average annual snowfall is 1300 mm, and mean annual temperature is 10.1°C (Robertson and Hamilton, 2015). The site was in various corn-soybean-wheat rotations for the past 40 years and before that, corn-soybean-small grain rotations for at least 60 years. 45

Experimental design
To examine the influence of initial soil moisture on the pre-assay loss of CO2 during dry-down, we pre-wet recently collected soil to three different initial water-filled pore space (WFPS) levels: 30, 50, and 70%. Then we measured gravimetric soil moisture (GSM) and CO2 loss while soil was air-drying, after which we re-wet them and measured the 24-hr CO2 pulse by standard methods Franzluebbers et al., 2000). 50

Laboratory analyses
After collection, soil was sieved through a 4-mm mesh and mixed. We measured gravimetric soil moisture (GSM) and calculated the target volumetric water content (VWC, g H2O cm -3 soil) for each treatment following Eq. 1 (Elliott et al., 1999): where soil bulk density (SBD) was 1.5 g soil cm -3 , a previously assessed value from KBS soils . Then we 55 divided VWC by SBD to obtain a target GSM and thereby determined the amount of water to add to the field-moist soil (11% WFPS; GSM = 0.032 g H2O g -1 dry soil). We then weighed 40 g of soil into each of 75 specimen cups. Each cup was randomly assigned to an initial WFPS treatment (30, 50, or 70%), for a total of 25 replicates per treatment. We added sufficient deionized water to each cup to achieve the target initial WFPS and stirred to evenly distribute water. After soil was wet and stirred in the specimen cups, the contents of each cup were transferred to a labeled paper bag. The soil was spread evenly across the bottom 60 of the bag, and the top portion of the bag was cut off to increase air flow. Afterwards, the soil was immediately weighed and set on a laboratory bench to air-dry.
Immediately after wetting, as well as 1, 3, and 8 days later, we assessed GSM and CO2 loss rates for five replicates per initial WFPS treatment. GSM, which was determined after drying the soil at 105°C for 24 hrs, stabilized at 1.5% in the air-dried soil 65 ( Fig. 1a), but did not reach zero even when soil was completely air-dry. Because soil in all initial WFPS treatments were airdry by day 3, with CO2 loss rates close to zero, we terminated GSM and CO2 measurements after day 8.
CO2 loss rates at each sampling interval were measured by placing 10 g of soil into a 235 mL mason jar equipped with a gassampling septum. Then we sampled 5 mL of headspace from each jar at 4 intervals (0, 0.5, 1, and 2 hr), injected it into an 70 evacuated 3 mL exetainer (Labco Limited, Lampeter, Wales, United Kingdom), and replaced the jar headspace with laboratory air. CO2 samples were analyzed within 24 hrs using a LI-820 CO2 Gas Analyzer (LI-COR Biosciences, Lincoln, NE, USA).

Statistical analyses and correction factor
CO2 pulses were calculated as the positive slope of the linear regression of CO2 concentrations through time after accounting for headspace dilution, and then converted to a standardized rate using the ideal gas law. In 17 of 75 cases, we omitted one of the four data points within a jar, which were clear visual outliers. In two cases, we rejected jars with leaks. CO2 loss rates during the dry-down period were analyzed with a two-way analysis of covariance (ANCOVA), where initial WFPS treatment 80 and days elapsed since wetting were factors and GSM at the time of sampling was a covariate. Additionally, a one-way analysis of variance (ANOVA) was used to determine whether initial WFPS treatment had an effect on the 24-hr CO2 pulses upon rewetting the air-dried soil.
We also calculated a correction factor to account for pre-assay CO2 loss prior to the 24-hr CO2 pulse assay. To calculate the 85 total amount of CO2 loss during dry-down for each initial WFPS treatment, we calculated a best-fit exponential decay curve (Y = α βX + θ), where Y = daily CO2-C loss and X = length of dry-down period, until soil was air-dry (i.e., immediately after wetting through day 3). Total C loss was equivalent to the area under the curve.
Because we used sacrificial sampling, we could not calculate standard deviation or standard error in the usual way. Instead, 90 we used a bootstrapping approach in which we computed predicted values for CO2 losses (Ŷi) and residuals (ei = Yi -Ŷi). All zeroes for CO2 losses were set to 1 for the sake of fitting the regression because an exponential decay curve can approach but never attain 0 and because 1 was lower than any value we observed. Then we created a bootstrap sampling of residuals specific to each dry-down interval (0, 1, or 3 days), sampled randomly from each interval with replacement, and added randomly sampled residuals to predicted values (Yi * = Ŷi + ei * ) for each dry-down interval (after Hesterberg, 2015). Residuals were bootstrapped 10,000 times to derive multiple estimates of coefficients for the exponential decay curve (α, β, and θ). We also integrated under the curve 10,000 times to get an error estimate (i.e., coefficient of variation) associated with the total amount of pre-assay CO2 loss during dry-down.
Then we divided the total CO2 loss by three days to obtain the daily rate used to calculate a correction factor following Eq. 2: 100 CF = (daily CO2 loss during dry-down / 24-hr CO2 pulse after re-wetting) + 1 ( 2) The correction factor for each treatment was then multiplied by each replicate's 24-hr CO2 pulse following re-wetting. Finally, we verified that the correction factors worked by conducting a one-way ANOVA to determine whether initial WFPS treatment still had an effect on the corrected pulses. For all analyses, we confirmed that assumptions of normality and homogeneity of variance were not violated. 105

Results
Soil in the 50 and 70% WFPS treatments took longer to dry than did soil in the 30% WFPS treatment (Fig 1a). A day after wetting, soil from the 30% WFPS treatment was completely air-dry, but soil had lost only 79% and 68% of its initial moisture in the 50 and 70% WFPS treatments, respectively. All soil was air-dry by three days after wetting. Pre-assay CO2 losses mirrored soil moisture loss, reaching zero for all WFPS treatments by day 3 (Fig 1b). Both GSM at the time of sampling and day had effects on pre-assay CO2 loss rates (P < 0.0001), but initial WFPS treatment did not (P = 0.28) probably because GSM captures more variation in soil moisture as the soil dries than WFPS treatment. However, there was an interaction between treatment and day (P = 0.0005). Soil of even the lowest initial WFPS treatment lost C as CO2 over three days of drying (26 µg 115 https://doi.org/10.5194/soil-2020-55 Preprint. Discussion started: 29 September 2020 c Author(s) 2020. CC BY 4.0 License.

Fig 3. Daily CO2 production rates for each initial water-filled pore space (WFPS) treatment. Lined bars represent the average daily rate of pre-assay CO2 loss during a 3-day dry-down period and solid bars represent the 24-hr CO2 pulses after re-wetting the airdried soil. Together both bars represent the 24-hr CO2 pulse corrected for pre-assay losses of CO2 during dry-down. Error bars
130 represent standard deviation as described in the "Statistical analyses" section.

Discussion
Initial soil moisture levels played a significant role in our ability to accurately characterize soil C availability (Fig 2) via the conventional 24-hr CO2 pulse assay Franzluebbers et al., 2000). Wetter soil lost more C during drydown, presumably because soil microbes remained active for a longer period of time. These losses decreased the short-term 135 CO2 pulses and therefore the final estimates of soil C availability.
Without knowledge of these losses, one might erroneously conclude that soil from the 30% WFPS treatment had about 35% higher soil C availability than the others (Fig 2), but this trend is instead due to higher pre-assay CO2 losses during the drydown period for wetter soil (Fig 3). It is striking that even short drying intervals (i.e., 1 versus 3 days) can affect soil C 140 availability as deduced from the 24-hr CO2 pulse after re-wetting air-dried soil. However, we were able to account for the preassay CO2 losses for our soil with a correction factor that made C availability approximately equivalent across all initial WFPS treatments.
These trends suggest that efforts to characterize C availability via short-term CO2 pulses following the re-wetting of dry soil 145 should exercise caution if comparisons involve soils with a range of initial soil moistures. This includes soils compared across seasons; across drought, precipitation, or irrigation gradients; across landscape catenas; across crop, grazing, or forest https://doi.org/10.5194/soil-2020-55 Preprint. Discussion started: 29 September 2020 c Author(s) 2020. CC BY 4.0 License. management practices; and as well in cross-site comparisons and meta-analyses that include soils collected at different initial soil moistures.

150
A correction factor that accounts for pre-assay CO2 losses may help to normalize such comparisons. In our soil, pre-assay CO2 losses led to a C mineralization bias as high as 32%, for which we could confidently correct by applying a correction factor based on measured rates of pre-assay CO2 loss (Eq. 2). Other soils with moisture contents sufficient to oxidize available C during dry-down will require different correction factors. A soil-specific correction factor can be calculated by measuring CO2 loss during dry-down on a subset of samples, as we described above (Eq. 2). 155 An alternate solution is to minimize the dry-down period such that little available C is lost prior to the assay. Strategies to minimize pre-assay CO2 loss might include exposing soils to temperatures high enough to speed evaporation, but low enough to avoid sterilization (Jager, 1968) or otherwise artificially disrupt the microbial community (Evans and Wallenstein, 2012).
This could be performed in a closed vented chamber such as a soil incubator. Alternatively, faster and more even drying might 160 be achieved with a steady flow of air (i.e., a fan or vented system) over exposed soil samples. Even with faster drying, however, a correction factor may be needed.
Overall, our results demonstrate that using the 24-hr CO2 pulse following the re-wetting of a dried soil to evaluate soil C availability can be misleading for soils with different moisture contents at time of sampling. For such soils a correction factor 165 based on pre-assay CO2 losses can be applied with confidence.

Data availability
Data will be made publicly available upon publication at Dryad; a pre-publication version is available at https://datadryad.org/stash/share/o4a-ESVy8wxpnk-kbrZicM9eMuMrX2F5oJpg039DWGQ.

Author contributions 170
CV and GPR designed this study, MAB and CV performed the laboratory assays, MAB analyzed the CO2 samples, CV conducted the statistical analyses, MAB and CV wrote the paper with contributions from SSR and GPR.