Interactive comment on “ Separation of soil respiration ; a site-specific comparison of partition methods

It is not an easy task to separate autotrophic and heterotrophic soil respiration. Five methods were compared and none was superior. Accuracy was defined by the difference with the ÉŮ13C-CO2 natural abundance. This is a risky approach because this method has a low precision. Fortunately an second low precision method (root regression) gave a similar estimate of the ratio between heterotrophic and total soil respiration. Strengths and weaknesses of each method are discussed and it is concluded that a combination of methods is needed. This is a thorough investigation on an important subject. The manuscript is well written and easy to read. The methods section is relatively long, but this is inherent to a methodical paper. Non accessible PhD and BSc theses, such as Farmer 2013 and Tong 2015, can be removed from the literature references.

were separated in four groups of different volumetric moisture content (i.e. 15, 25, 35 and 45). These moisture levels corresponded to the natural annual fluctuation in the field (i.e. from dry to moist season) (Cui and Lai, 2016). After moisturizing the samples, each individual soil core was placed into a hermetically sealed 2.9 dm 3 plastic container and left to stabilize in dark for two weeks at 25°C. After that, the experiment lasted four weeks and had four different incubation temperature levels (one per week; 14°C, 20°C, 26°C and 32°C) corresponding to the minimum, intermediate 110 and maximum soil temperature values in the field based on preliminary studies (Cui and Lai, 2016). At the beginning of each week, the soil cores were pre-incubated in their incubation box to their corresponding weekly temperature (i.e week #1, 14°C … week #4, 32°C) for 3 days and then opened and vented for one minute. From all the boxes gas samples were collected (20 ml) with an air-tight syringe (t= 0, 24, 72 hour) after box closure. The CO 2 concentrations were analyzed within 48 hours with a gas chromatograph (GC system 7890A, Agilent Technologies). The GC system 115 was equipped with a flame ionization detector and an electron capture detector to quantify CO 2 concentration. Between each measurement session, the boxes were opened to vent and the moisture of the soil cores was re-adjusted if needed.
Gaussian 3D regression fitted curve was derived as shown in equation 1. using SigmaPlot version 10.0 (Systat Software, San Jose, CA). (1) 120 where f(x,y) is the CO 2 efflux function; a, b and c are constant coefficients; x is the soil temperature (ºC); y is the soil moisture content (%); x 0 is the average temperature; y 0 is the average soil moisture.

Root exclusion bag methods
To partition the CO 2 efflux in-situ into Rs and Rh using mesh bags, two different approaches were followed: 1) the traditional dug soil with hand-sorted root removal and refilling method (HS) (Fenn et al., 2010;Hinko-Najera, 2015) 125 and 2) a variant of it with intact soil blocks (IB). The HS method consisted of digging a pit for each bag with a size matching the bag dimensions (20 × 20 cm, depth: 25 cm) where the soil is excavated in layers (to maintain soil horizons) and visible roots are removed before repacking the bag inside the pit with the removed soil. The IB variant of this technique consisted in extracting a cube as intact as possible from the soil (20 × 20 cm, depth: 25 cm). Then, tightly placing the soil block into the micromesh bag and inserting it back into its original pit. For both methods, the same type of micromesh bags (38µm nylon mesh), closed at the bottom but open at the top were used. This mesh size was used to impede roots from entering inside the bags, but allowed mycorrhiza to penetrate (Moyano et al., 2007). Collars measuring 10 cm diameter were installed on the soil in the center of each bag to a depth of 8 cm, for heterotrophic emissions sampling.
Seven plots were randomly distributed inside the study area. In each plot, an IB bag was paired with a HS bag with 135 space of 150 cm between them. The root exclusion bags were installed during the month of October 2016 and were let to stabilize for three months. At 1 m distance from each root exclusion bag, a collar was inserted into non-disturbed soil to measure Rs. To assess Rs and Rh without the influence of litterfall decomposition, the collars were cleared of leaves and flowers on a weekly basis.
From February 3 rd to April 19 th 2017 the collars were measured weekly with an IRGA (Environmental Gas Monitor,

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EGM-4, PP Systems, UK) attached to a soil respiration chamber (SRC-1, PP Systems, UK). Soil temperature and soil moisture were measured in the area located between the collar and the edge of the bag (to 10cm depth, HH2, Delta-T Devices, Cambridge-England). At the end of the study all the root exclusion bags were removed from the soil and inspected to ensure that no root had penetrated inside. The soil inside the measurement collars was then collected to assess bulk density (van Reeuwijk, 1992). Mathematical calculation and descriptive statistical analyses were done with

Lab incubations
For the lab incubations, undisturbed soil cores of volume 98 cm 3 (inner diameter 5 cm, height 5 cm) were collected using a stainless-steel core soil sampler from the upper part of the soil profile (0-5 cm). In the study area, four groups 160 of four soil cores were collected then pooled per group and brought to the lab. Subsequently all visible roots were removed but with special care to not destroy the small aggregates. The soil was then repacked to original bulk density in minimally disturbed soil microcosm cores of 45 cm 3 (inner diameter 3.5 cm, height 5 cm). The soil cores were separated in four groups of different volumetric moisture content (i.e. 15, 25, 35 and 45). These moisture levels corresponded to the natural annual fluctuation in the field (i.e. from dry to moist season) (Cui and Lai, 2016 (Cui and Lai, 2016). At the beginning of each week, the soil cores were pre-incubated in their incubation box to their corresponding weekly temperature (i.e week #1, 14°C … week #4, 32°C) for 3 days and then 170 opened and vented for one minute. From all the boxes gas samples were collected (20 ml) with an air-tight syringe (t= 0, 24, 72 hour) after box closure. The CO 2 concentrations were analyzed within 48 hours with a gas chromatograph (GC system 7890A, Agilent Technologies). The GC system was equipped with a flame ionization detector and an electron capture detector to quantify and CO 2 . Between each measurement session, the boxes opened to vent and the moisture of the soil cores was re-adjusted if needed.

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Gaussian 3D regression fitted curve was derived as shown in equation 1. using SigmaPlot version 10.0 (Systat Software, San Jose, CA).
where a, b and c are constant coefficients; x is the soil temperature (ºC); y is the soil moisture content (%); x 0 is the average temperature; y 0 is the average soil moisture. 180 2.45 δ 13 C natural abundance method Millard et al. (2010) have demonstrated that the natural abundance δ 13 C (‰) of Rs falls between the δ 13 C values of the Rh and Ra. The δ 13 C of Rs /Rh respiration was determined following Lin et al. (1999) and Millard et al. (2010). The isotopic partitioning experiment assessed values of the δ 13 C of the Rs, Ra and Rh. The sampling took place on March 15 th 2017. A closed chamber (10 cm diameter, 10 cm high) was positioned on each emissions measurement collar 185 (described in section 2.1). The chambers were flushed for 2 minutes with CO 2 -free air to remove all the atmospheric air trapped within the headspace. Chambers were left to incubate for 40 minutes to ensure the concentration of the chamber sample reached above 400 ppm of CO 2 from which a duplicate sample of the gas in the chamber headspace were extracted into evacuated vials to give the δ 13 C of the Rs. Subsequently, the soil under the chamber was dug and immediately brought to the lab (less than 30 minutes travel) where the soil and the roots were carefully separated. The

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roots were gently washed with water to remove adhered soil aggregates and slightly dried afterward with paper towels.
Samples of 5 g of root and 10 g of root-free soil per chamber were incubated in CO 2 free air in 250 ml airtight glass bottles to give the δ 13 C of the Ra and Rh respectively. The bottles were left to incubate for 90 minutes before duplicate extraction into evacuated vials. As recommended by Midwood et al., (2006), previous tobefore gas sample extraction, the butyl rubber septa used to seal the vials were heated at 105°C for 12 h. The C isotope ratio of the CO 2 in all samples was analyzed using a Gas-bench II connected to a DeltaPlus Advantage isotope ratio mass spectrometer (both Thermo Finnigan, Bremen, Germany) at the James Hutton Institute Scotland UK. The δ 13 C ratios, all expressed relative to Vienna-Pee-Dee Belemnite (VPDB), was calculated with respect to CO 2 reference gases injected with every sample and traceable to International Atomic Energy Agency reference material NBS 19 TS-Limestone. Measurement of the individual signatures of the natural abundance δ 13 C of the Rs, Rh and Ra allowed partitioning between the different 200 sources using the mass balance mixing model (Lin et al., 1999;Millard et al., 2010): where %Rh is the proportion of Rh from Rs, and δRs, δRh and δRa are the δ 13 C isotopic signatures.

Soil general characterization
Four soil profiles were dug in the study area, characterizing the different landforms present at the site. Morphological 205 description was done according to Jahn (2006) and the soil was classified with the World Reference Base (IUSS-Working-Group-WRB, 2014). Soil pH was determined with a glass-calomel electrode pH meter (McLean, 1982).
Rainfall and air temperature were recorded hourly with a HOBO Weather station (rain gauge, S-RGB-M002; air temperature/RH, sensor S-THB-M008, Onset Computer Corp., USA). Water holding capacity was assessed by saturating the soils, allowing them to freely drain for 24 h and determining gravimetric water content after oven-drying 210 at 105 ºC following Arcand et al. (2016). Root biomass was measured by collecting soil cores (inner diameter 5 cm, height 5 cm) and determined using the approach of Tufekcioglu et al. (1999). The soil was dried, finely ground, and subsequently analyzed for total C and N content using a CNS Analyzer System (Perkin Elmer 2400 Series II CHNS/O Analyzer, USA).

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Isotopic partitioning methods are recognized as a more accurate approach to segregation of Rh/Rs than non-isotopic techniques (Paterson et al., 2009;Kuzyakov, 2006). Therefore, the soil δ 13 C natural abundance method was used as reference point for segregation accuracy. Partition methods that had Rh%: <10, 10-20 and >20 lower or larger than the δ 13 C-CO 2 natural abundance were categorized as high, intermediate and low accuracy, respectively. The level of precision of the segregation methods was determined with the statistical variance associated with the Rh/Rs averages.

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High, intermediate and low precision were attributed to Rh% standard errors of <10, 10-20 and >20, respectively. The level of complexity was evaluated with the number of steps required to complete each method. For example, the handsorted root exclusion bags technique was judged as a four steps method (pit excavation, root removal, bag/pit refiling, and CO 2 efflux measurements). Methods with five steps or less were deemed simple and six steps or more deemed as complex. The time inversion needed to set up the experiment was assessed by counting the number of working hours

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(eight hours equal one day) required prior to the start of the measurements. The time inversion needed to produce seasonal trends was the number of months of measurements (in the field or in the lab) required to characterize the Rh at the different temperature and moisture levels of the year.

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According to their morphology and diagnostic properties, the soil was classified as Alic Umbrisol (Nechic) and Haplic Alisol (Nechic) (IUSS-Working-Group-WRB, 2014). The difference between the two soil groups was the thickness of humus-containing horizon (between 20 and 30 cm for the Umbrisol; while, 10 to 20 cm for the Alisol). The A horizon had high organic C content (3.2 ±0.2%) and high acidity (pH H2O 4.2) ( Table 1). The sub-superficialsurface soil was represented by clayey yellow-colored profiles with an argic horizon. Soil texture was heavier in the argic horizon than 235 in the topsoil and parent material. The structure in all the soil profiles was predominantly granular in the upper horizons, whereas the argic horizon was characterized by subangular blocky structure (Table 1). The argic horizon was deemed to be of high-activity clays and low cation base status based toon previous results in the area (Tong, 2015), along with soil acidity, type parent material and level of mineralization of the bedrock in the soil pits.

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The 22 quadrats used for the root regression assessment yielded average Rs of 0.46 ±0.04 g CO 2 m 2 h -1 . The regression of the CO 2 efflux against root density produced a statistically significant slope correlation of 0.08 ±0.04 g CO 2 m 2 h -1 per mg root cm -3 (p=0.03), and set the intercept at 0.25 ±0.10 g CO 2 m 2 h -1 (p=0.02) which represented the basal efflux in absence of root i.e. the Rh (Fig. 2 and Table 3). The Rh measured when the root regression technique was performed (October 2016) was 6.0 ±2.4 Mg C ha -1 y -1 , equivalent to 54% of the Rs 245 (Table 6).

lLab incubation
The 22 quadrats used for the root regression assessment yielded average Rs of 0.46 ±0.04 g CO 2 m 2 h -1 . The regression of the CO 2 efflux against root density produced a statistically significant slope correlation of 0.08 ±0.04 g CO 2 m 2 h -1 per mg root cm -3 (p=0.03), and set the intercept at 0.25 ±0.10 g CO 2 m 2 h -1 (p=0.02) which represented the basal efflux 250 in absence of root i.e. the Rh (Fig. 2 and Table 3). The Rs measured when the root regression technique was performed (October 2016) was 11.1 ±1 Mg C ha -1 y -1 ( Mg CO 2 -C ham 21 yh -1 , respectively (Fig. 3). The exponential relationship between CO 2 efflux, soil temperature and 255 moisture is presented in Table 4.

Root exclusion bag methods
During the root exclusion bags measurements period (Feb-Apr 2017), the average air temperature was 16ºC and the total rainfall 107 mm. and theDuring that period the Rs averaged 6.1 Mg C ha -1 y -1 (Fig. 1). One of the requirements for the suitability of root exclusion bag methods to estimate Rh is that soil bulk density, soil temperature and moisture are 260 statistically equal inside and outside of the bags. In this experiment, no significant differences were detected regarding the bulk density and soil temperature (p=0.87 and p=0.15, respectively) but the volumetric soil moisture in the HS bags was on average 17% lower than outside the root exclusion bags (p=0.04) ( Table 2). As would be expected, all Rh IB and Rh HS efflux rates were lower than the Rs efflux at each measurement date. Throughout the experiment, the Rh IB was repetitively lower than the Rh HS except on March 31 st (Fig. 1b).

Root regression and lab incubation
The 22 quadrats used for the root regression assessment yielded average Rs of 0.46 ±0.04 g CO 2 m 2 h -1 . The regression of the CO 2 efflux against root density produced a statistically significant slope correlation of 0.08 ±0.04 g CO 2 m 2 h -1 per mg root cm -3 (p=0.03), and set the intercept at 0.25 ±0.10 g CO 2 m 2 h -1 (p=0.02) which represented the basal efflux in absence of root i.e. the Rh (Fig. 2 and Table 3). The Rs measured when the root regression technique was 270 performed (October 2016) was 11.1 ±1 Mg C ha -1 y -1 (Table 6), equivalent to 54% of the Rs.

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The δ 13 C-CO 2 natural abundance determination satisfactorily segregated the three respiration components ( Table 5).
The fact that the δ 13 C-CO 2 of the Rh HS, Rh IB and Rh lab were in a very close range indicated that in the field the efflux measured in the root exclusion bags were not contaminated with root respiration. Based on the δ 13 C-CO 2 of the Rs, the Rh lab and the Ra lab the percentage of heterotrophic respiration was 61 ±39% (Table 6). The notably large standard error of the percentage of heterotrophic respiration was due to the large variance in the δ 13 C-CO 2 of the three 280 respiration components.

Root and carbon dioxide efflux regression technique
As demonstrated by Gupta and Singh (1981) the intercept of the regression line between the independent variable (i.e. root biomass) and the dependent variable (i.e. Rs) corresponds to soil respiration in absence of root (i.e. Rh) (Fig. 2). In this study the regression had ten points (45%) outside the confidence interval but the intercept and slope were still statistically significant . This uncertainty in the regression fit was likely caused in large part by the older roots which are bulkier but respire less than fine and young roots (Behera et al., 1990). However, this method had the closest average Rh/Rs to the δ 13 C natural abundance technique. Consequently the root regression technique was assessed as high accuracy and low precision (Table 7). Previous studies also highlighted large variation of CO 2 efflux and root biomass which causes relatively low coefficient of determinations (Behera et al., 1990;Farmer, 2013). In accordance to Kuzyakov (2005), this method was comparatively simple (Table 7).

Lab incubation method
Interpreting soil respiration processes in response to seasonal changes is generally challenging because soil temperature and moisture regularly covary (Carbone et al., 2011;Davidson et al., 1998). The lab incubation technique was the only method capable of dividing the effect of soil temperature and moisture on Rh and to produce a significant Gaussian regression fit (Table 3). However, the microcosm incubation produced Rh values notably lower than the other 300 techniques (Table 5). This might mainly be due to three different causes. First, the fact that the soil column in the incubation microcosms were 5 cm high while the A horizon in the field (i.e. where the Rh assessments from the other techniques were made) was 10 cm thick (Table 2). Second, to prevent potential shifts in the microbial community during the incubations (i.e. adapting to lower resource availability), previous to the beginning of the experiment the microcosms were left stabilize for two weeks. Accordingly, the fresh and label organic residues that would in the other 305 segregation methods contribute to the soil respiration had already decomposed before the beginning of the incubations.
Third, the low Rh of the lab incubation method could also be attributed in part to the fact that this technique did not contain any rhizomicrobial respiration and its priming effect. That is, this method produced Rh from basal microbial respiration which is considered to be from stabilized SOM with slow turnover rates (Kuzyakov, 2006Neff et al., 2002. In view of that, with additional field and lab methods development it would be possible to further segregate Rh 310 assessments into percentage of rhizomicrobial respiration, decomposition of plant residues and basal decomposition of SOM. Overall, the lab incubation technique was slightly more complex than the non-isotopic field Rh assessment methods but allowed a prompt determination of Rh whilst simulating year round field environment (Table 6). Further studies should test the effect of microcosm height on Rh in relation to field measurements and determine microbial C use efficiency by isothermal microcalorimetry during the incubations.

Root exclusion bags methods
The HS and IB methods had %Rh of 79 ±3 and 49 ±7 %, respectively. These variances around the means (i.e. ±3 and ±7, respectively) were the lowest of all the field segregation methods tested (Table 5). Comparing the %Rh of the HS and IB with the δ 13 C natural abundance technique, they resulted 18% above and 12% below, respectively. Thus the root exclusion bags methods were judged of intermediate accuracy and high precision. Also, the HS and IB methods were 320 fast and simple to setup ( Table 6).
The micromesh size used in the root exclusion bags was 38µm which was reported to impede root penetration but to allow arbuscular mycorrhizal to spread inside the bags (Moyano et al., 2007;Rühr and Buchmann, 2010). In turn, Fenn et al. (2010) stated that in the mycorrhizal structures the arbuscules exist within roots, and therefore, the CO 2 efflux from these bags could contain some portions of the roots respiration. Contrary to this, the IB and HS air samples 325 analyzed for δ 13 C had an isotopic signature close and not statistically different from the gas samples collected in the lab airtight glass bottle of fresh soil without roots. This indicates that the root exclusion bags (both IB and HS) did not comprise traces of root respiration that had a significantly larger δ 13 C-CO 2 signature (Table 4). After the three months of soil stabilization period, both bag methods for partitioning total soil respiration and root-free soil respiration components successfully produced Rs>Rh in every sampling dates indicating that efflux rates within the bags had which causes relatively low coefficient of determinations (Behera et al., 1990;Farmer, 2013). In accordance to Kuzyakov (2005), this method was comparatively simple (Table 7).

Lab incubation method
Interpreting soil respiration processes in response to seasonal changes is generally challenging because soil temperature and moisture regularly covary (Carbone et al., 2011;Davidson et al., 1998). The lab incubation technique was the only 405 method capable of dividing the effect of soil temperature and moisture on Rh and to produce a significant Gaussian regression fit (Table 4). However, the microcosm incubation produced Rh values notably lower than the other techniques (Table 6). This might be due to the fact that the soil column in the incubation microcosms were 5 cm high while the A horizon in the field (i.e. where the Rh assessments from the other techniques were made) was 10 cm thick (Table 2). Further studies should test the effect of microcosm height on Rh in relation to field measurements. The low

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Rh of the lab incubation method could also be attributed in part to the fact that this technique did not contain any rhizomicrobial respiration and its priming effect. That is, this method produced Rh from basal microbial respiration which is considered to be from stabilized SOM with slow turnover rates (Kuzyakov, 2006Neff et al., 2002. In view of that, with additional field and lab methods development it would be possible to further segregate Rh assessments into percentage of rhizomicrobial respiration, decomposition of plant residues and basal decomposition of SOM. Overall,

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the lab incubation technique was slightly more complex than the non-isotopic field Rh assessment methods but allowed a prompt determination of Rh whilst simulating year round field environment (Table 7).

Comparison of methods and recommendations
The analysis of the five different Rh/Rs partitioning methods examined in this study shows that none of them was fully satisfactory. That is, each technique had strengths and weaknesses (Table 76). Using δ 13 C-CO 2 is acknowledged as the 420 preeminent way to segregate Rh/Rs (Cheng, 1996;Kuzyakov, 2006); and accordingly accuracy was defined by the difference with this method. We recognize this was a precarious approach because the δ 13 C-CO 2 method had a large variation. Fortunately, the root regression, which is also recognized in the literature as a reliable method (Kuzyakov, 2006), gave a similar %Rh estimate. However, we found several other shortcomings with the δ 13 C-CO 2 method. First, the conjunction of field and lab procedures makes it difficult to complete this method in one day as needed. Second, the 425 air flushing with CO 2 free gas in the field (to prevent ambient δ 13 CO 2 contamination) makes that technique more complex than the other methods to assess Rh%. Third, the ability to perform this technique in remote areas is limited because the δ 13 C-CO 2 needs to be quickly assessed with a calibrated and accurate spectrometer (Midwood et al., 2006).
Fourth, in our study we foundthe large variationnce in δ 13 C-CO 2 of the respiration components (i.e. Ra, Rh and Rs) that impeded the assessment of Rh% per individual collar. Accordingly, further studies should analyze the spatial relationships of δ 13 C-CO 2 with soil properties and root characteristics. As standalone, the δ 13 C-CO 2 technique was unable to produce assessment of soil CO 2 efflux; thus needed to be performed in conjunction with field Rs measurements. In this regards, the δ 13 C-CO 2 complemented well with root exclusion bags methods because it allowed to determine if the buried bags had teared and been invaded by roots and to standardized Rh% determination.
The root regression method had the advantage to be simple, to produce an average Rh% close to the δ 13 C-CO 2 natural 435 abundance and the disadvantage to require a high number of replicates due to low coefficient of determination between CO 2 efflux and root biomass. Another disadvantage of the root regression technique is that in order to produce seasonal trends, the labor intensive procedures (i.e. pit digging, CO 2 measurements and root counting) need to be reinitiated several times during the years. This shortcoming can be particularly impractical in small plot experiments.
Complementary studies should assess thresholds of root size to be included in the regression fit in order to optimize the 440 correlation fit and use the δ 13 C-CO 2 natural abundance method to determine the effect of root size on the isotopic signature.
The root exclusion bags methods (i.e. HS and IB) had the advantage to be easy to monitor throughout the year. That is, because the % of Rh is unlikely to be constant in time it is important to assess it periodically. The bags methods can be considered as a miniaturization of the traditional soil trenching method. However, contrasting with large trenches (e.g. 445 Comeau et al., 2016;Fisher and Gosz, 1986) the root exclusion bags had the advantage to be simpler to establish and to allow mycorrhiza development inside the mesh bags (Moyano et al., 2007). Conversely, due to the relatively small bag sizes, root webs on the outside edge could potentially contaminate Rh assessment. In this study, the δ 13 C-CO 2 determination made with the collars located in the center of the bags showed no isotopic signature of root respiration.
Similarly with the trenching method, the root exclusion bag methods had the disadvantages to require several months 450 of soil stabilization before starting CO 2 efflux measurements. Compared with the δ 13 C-CO 2 natural abundance method, the HS and IB overestimated and underestimated %Rh by 18 and 12%, respectively. The divergences were likely caused by soil disturbances, alteration in root demise dynamic and lack of root exudates. Correspondingly, Carbone et al. (2016) found 11% differences in Rh% assessment between an isotopic partition method and the trenching technique.
high amount of sand, gravel or rock because the intact blocks would collapse.
The lab incubation with minimally disturbed microcosms was the only method that had absolutely no influence of root or mycorrhiza. Thus the results from this method exclusively represented the CO 2 efflux originating from the mineralization of the slow turnover SOC pool (i.e. basal soil respiration) (Pell et al., 2006). Assessment of basal soil respiration in relationship with the total Rh is of great importance in evaluating the dynamic of the stabilized SOC. In

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Values are means and standard error, n = 14 for Rs and Ra and n = 7 for HS, IB and Rh lab.

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Values followed by a different Greek letter (α, β) are significantly different from each other at α=0.05.         .