Exploring the relationship between soil mesofauna, soil structure and N2O emissions

Soil Biology & Biochemistry 96 (2016) 55e64

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Exploring the relationship between soil mesofauna, soil structure and N2O emissions Rima J. Porre, Jan Willem van Groenigen, Gerlinde B. De Deyn, Ron G.M. de Goede, Ingrid M. Lubbers* Department of Soil Quality, Wageningen University, PO BOX 47, Droevendaalsesteeg 4, 6700 AA, Wageningen, The Netherlands

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 September 2015 Received in revised form 18 January 2016 Accepted 23 January 2016 Available online 8 February 2016

Agricultural soils are a large source of nitrous oxide (N2O) emissions. Soil mesofaunal species can accelerate, delay, increase or decrease N2O emissions. However, it is still unknown whether the soil fauna affect N2O emissions through trophic interactions or through their effect on soil structure. We explored the role of these two pathways in a 70 day microcosm experiment with a sandy loam subsoil with hay mixed in. Enchytraeids, fungivorous mites and predatory mites were added to the soil in a full factorial design to test for both single species effects as well as interactions between species. We measured N2O and CO2 fluxes and we analysed soil structural parameters using X-ray micro tomography. After 35 days of incubation, enchytraeid presence significantly increased the volumetric air content of the soil (0.049 e0.067 cm3 cm
3, P ¼ 0.010) as well as the abundance of pores with sizes similar to their body width. At the same time N2O emissions, NO3
and DOC concentrations were significantly higher when enchytraeids were present (3.6 to 8.4 mg N2O-N m
2, P < 0.001; 6.0 to 17.1 mg NO3
­N kg
1 soil, P < 0.001; 121.1 to 135.6 mg C kg
1 soil, and P ¼ 0.017, respectively). Neither fungivorous mites nor predatory mites nor their interactions had a significant effect on soil structure or N2O emissions. Enchytraeids accelerated peak N2O emissions (P ¼ 0.001), but did not increase cumulative N2O emissions on day 70. Structural equation modelling confirmed that enchytraeids enhanced nitrogen mineralisation directly and also indirectly by creating a higher volumetric air content, and thereby increased N2O emissions. We conclude that the soil structure pathway was important in driving N2O emissions, and that soil ecosystem engineers such as enchytraeids disproportionately affected N2O emissions as compared to other soil fauna. © 2016 Elsevier Ltd. All rights reserved.

Keywords: N2O emission Soil mesofauna Soil structure X-ray micro tomography Soil ecosystem engineers

1. Introduction Nitrous oxide (N2O) is the most significant anthropogenic ozone depleting substance in the atmosphere as well as the third most significant greenhouse gas: on a molecular basis it has a 298 times greater global warming potential than CO2 (Ravishankara et al., 2009; Paul et al., 2012; UNEP, 2013). The largest source of anthropogenic N2O emissions is agriculture. Combined, N2O emissions from agricultural soils and other agricultural practices constitute approximately two-thirds of global anthropogenic N2O emissions. This amounts to approximately 8% of the total greenhouse gas emissions in CO2 equivalents (Paul et al., 2012; UNEP, 2013). The three main biochemical processes in soils that can produce N2O are nitrification (N2O as a side product), denitrification (N2O as

* Corresponding author. Tel.: þ31 (0)317482350. E-mail address: [email protected] (I.M. Lubbers). http://dx.doi.org/10.1016/j.soilbio.2016.01.018 0038-0717/© 2016 Elsevier Ltd. All rights reserved.

an intermediate product that may escape to the atmosphere) and nitrifier denitrification (N2O as an intermediate product) (Wrage et al., 2001). The most important soil parameters driving these processes are carbon and nitrogen availability, (an)aerobicity, temperature and pH (Granli and Bøckman, 1994; Mosier et al., 1998; Kool et al., 2011). However, despite several decades of study, the dynamics of N2O in the soil profile are still poorly understood because of the complex interplay between physical, chemical and biological mechanisms affecting N2O production, reduction and emission (Blagodatsky and Smith, 2012). Since soil structure and water filled pore space (%WFPS) largely determine soil aeration and gas (N2O and O2) diffusion (albeit in different ways for the different biogeochemical pathways), they are very important factors in determining N2O production and emission (Pierret et al., 2002; Ball et al., 2008; Bessou et al., 2010; Kool et al., 2011; Laudone et al., 2011; Van Der Weerden et al., 2012). Nitrous oxide production by denitrification increases rapidly at a

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WFPS of 60% or more, whereas nitrification is the main source of N2O production at a WFPS of 35e60% (Bateman and Baggs, 2005; Laudone et al., 2011). Soil aggregation plays an additional role in the formation of anoxic microsites in which N2O production can take place (Mangalassery et al., 2013). Soil structural parameters such as total porosity, mean pore size and pore size distribution determine the rate of diffusivity and the amount of aeration in the soil, thereby affecting the fate of N2O (Deurer et al., 2009; Blagodatsky and Smith, 2012; Klefoth et al., 2014). The diffusivity of N2O gas through the soil profile determines the length of the path for N2O to reach the atmosphere. The longer this path, the higher the likelihood that it will be reduced to N2 (Chapuis-lardy et al., 2007; Klefoth et al., 2012, 2014; Paul et al., 2012). Soil fauna can affect these soil structural parameters and therefore play a role in N2O dynamics (Kuiper et al., 2013). Numerous studies have been conducted on the effect of macrofauna on soil structure as well as on N2O emissions, the large majority focussing on earthworms (Lee and Foster, 1991; Lubbers et al., 2013). Earthworms influence soil structure through their burrowing and casting activities (Didden, 1990; Capowiez et al., 2011). They have been shown to indirectly alter soil N2O formation through influencing substrate availability and by changing soil physical and chemical characteristics (Paul et al., 2012). A recent meta-analysis concluded that, on average, earthworm presence increased soil N2O emissions by 42% (Lubbers et al., 2013). However, there is growing evidence that other fauna (such as fungivorous mites, predatory mites, collembola, isopods and enchytraeids) can also influence N2O emissions (Kuiper et al., 2013; Thakur et al., 2014). Kuiper et al. (2013) showed that these species, in singlespecies treatments, can accelerate, delay, increase or decrease N2O emissions. Enchytraeid species have been shown to increase porosity and pore continuity in the soil, as well as to create soil aggregates of the same size as their fecal pellets (600- to 1000 mm) in the same manner as earthworms do (Didden, 1990; Van Vliet et al., 2004; Paul, 2007). Hence, it would seem likely that they, via their effect on soil structure, can also influence N2O emissions (Lubbers et al., 2011). Not only single faunal species but also interactions between fauna may alter N2O emissions, as shown by Thakur et al. (2014). In that study the authors focused on interactions within and between trophic levels and found that the interaction between enchytraeids, fungivorous mites and predatory mites resulted in an increase in N2O emissions of 580% compared to the control treatment. However, it is still unclear why this particular combination of soil mesofauna caused such a dramatic increase in N2O emissions. The increased NO3
concentration, combined with anoxic soil pores due to a relatively high WFPS of 70%, could have caused these higher N2O emissions (Williams et al., 1992). A potential explanation for the increased NO3
concentration is that the observed decline of fungivorous mites in the presence of predatory mites caused a change in the distribution of mineral nitrogen (Schmitz et al., 2010). Alternatively, the soil physical conditions could have been more favourable for N2O emissions as a result of soil physical disturbances caused by changes in the activity (spatial and/or temporal) of the enchytraeids and mites. These explanations were not directly tested and are highly speculative. Therefore the question remains whether the effect on N2O emissions is mainly through the effect of soil mesofauna on the microbial community; through their effect on soil structure; or both. We aimed to quantitatively analyse soil structural changes caused by soil mesofauna using X-ray micro tomography (XRT), and relate this to measured N2O emissions. We hypothesised that 1) Soil mesofauna have a measurable effect on soil structure; 2) Enchytraeids change the nature of porosity through burrowing, thereby creating a higher pore connectivity and increasing N2O emissions;

3) Fungivorous mites decrease the mineral nitrogen availability and therefore lower N2O emissions; and 4) When predatory mites are added to soil inhabited by enchytraeids and fungivorous mites, their resultant effect on soil structure will cause a large increase in N2O emissions. 2. Materials and methods 2.1. Experimental set-up In a 70 day microcosm experiment soil structural parameters as well as N2O and CO2 emissions as affected by soil mesofaunal species were measured. The experiment was set up as a full factorial design, consisting of seven mesofaunal treatments with hay as a feeding source and one control treatment (with hay and without mesofaunal species). The treatments included enchytraeids, fungivorous mites and predatory mites in single species treatments as well as all of their combinations (Table 1). Equal abundances of each mesofaunal group were added for independent analysis of single species- and species interaction effects. The soil mesofauna were collected from lab cultures. Prior to the start of the experiment, Enchytraeus albidus were fed with composting plant material. Fungivorous mite species Acarus siro and Rhizoglyphus robini were cultured on yeast. Predatory mite species Stratiolaelaps scimitus (Berlese, 1892) were bought commercially as Entomite-M (Koppert, Berkel en Rodenrijs, the Netherlands). Loamy sand subsoil (NH4 þ 0.8 mg N kg
1, NO3
0.6 mg N kg
1 soil, SON 14.7 mg N kg
1 soil, SOC 120.6 mg C kg
1 soil, DOC 115.4 mg C kg
1 soil, MBN 28.5 mg N kg
1 soil, pH-CaCl2 6.7) was collected from ‘Droevendaal Agricultural Farm’ near Wageningen University in the Netherlands (51590 N, 5 390 E), and was heated for 24 h at 70  C to eliminate meso- and macrofauna while maintaining a viable soil microbial population (Kaneda and Kaneko, 2011). Hay residue (32.8 g N kg
1 dry matter, 448.5 g C kg
1 dry matter, C:N ration ¼ 13.8) was cut into ~1.0 cm pieces, to make it better accessible for the soil mesofauna, and sterilised by autoclaving. To represent an N fertilization rate of 125 kg N ha
1, 1.34 g of dry hay was homogeneously mixed through the soil. Polypropylene microcosms (diameter ¼ 6.7 cm, height ¼ 14 cm, volume ¼ 500 cm3) were filled with a mixture of 229 g dry soil (sieved over a 2 mm mesh), 1.34 g hay (dry weight) and 53 ml distilled water. The soil was repacked to a bulk density of 1.2 g cm
3 with a soil depth of 5 cm, and was brought up to a gravimetric moisture content of 231 g kg
1 soil. Initially, this resulted in a water filled pore space (WFPS) of 53%. Water was gravimetrically adjusted every other day throughout the experimental period. After a week of preincubation to facilitate microbial colonisation, soil mesofauna were added according to the densities shown in Table 1. The ratio of adult to juvenile enchytraeids added was kept equal across all blocks. The microcosms were arranged in a randomised block design consisting of 10 replicates divided over five separate blocks. The microcosms were kept in a dark climate room (16  C, 60% humidity), covered with a black cloth to minimise water loss while allowing gas exchange. During the experimental period there were two destructive sampling moments where first the soil was scanned with XRT (including the control with hay and all treatments with enchytraeids present) and subsequently soil chemical properties were measured. Scanning and chemical analysis were done on day 35, at the first peak of N2O emissions, and on day 70 when peak emissions had subsided. At the start of the experimental period each treatment included 10 replicates (n ¼ 10). Five of these replicates were used for destructive soil sampling on day 35, the remaining 5 replicates were sampled on day 70. On both sampling moments the microcosms were first scanned with XRT (control with hay and

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57

Table 1 Treatments included in the microcosm study (n ¼ 10) with number of animals added per treatment. Faunal densities are according to De Ruiter et al. (1995). Code

a

CH E M P a EM a EP MP a,b EMP a

a b

Species

Control with hay Enchytraeus albidus Fungivorous mites (Rhizoglyphus robini and Acarus siro) Predatory mites (Stratiolaelaps scimitus) Enchytraeus albidus and Fungivorous mites Enchytraeus albidus and Stratiolaelaps scimitus Fungivorous mites and Stratiolaelaps scimitus Enchytraeus albidus, fungivorous mites and Stratiolaelaps scimitus

Faunal density # microcosm
1

# m
2

mg dwt g
1 dry soil

e 50 400 (300 robini, 100 siro) 3 450 (50 þ 400) 53 (50 þ 3) 403 (400 þ 3) 453 (50 þ 400 þ 3)

e

e 96.4 1.5 0.1 98 96.5 1.6 98

2596 20,769 156 23,365 2752 20,925 23,521

Scanned with X-ray micro tomography; n ¼ 5 at day 35 and n ¼ 5 at day 70. Replicated 5 times extra to ensure that the X-ray scanning had no negative influence on the survival of the introduced mesofauna; n ¼ 15.

enchytraeid treatments, n ¼ 5). Subsequently, the next day the soil (n ¼ 5) was destructively sampled for soil fauna count and chemical analysis. The treatment with all mesofaunal species present was replicated 5 times extra (EMP treatment as described in Table 1, total n ¼ 15) to ensure that the X-ray scanning had no negative influence on the survival of the introduced mesofauna. This was confirmed on day 35 when, after comparing the 5 scanned replicates with the other 5 replicates, no significant changes in soil mesofaunal numbers were recorded.

2.2. N2O and CO2 fluxes N2O and CO2 gas fluxes were measured with a static closed chamber technique, type 1302 multi gas monitor (Brüel & Kjaer, Naerum, Denmark) according to Velthof et al. (2002). Nitrous oxide and CO2 fluxes were measured for 5 consecutive days after soil fauna addition. Subsequently gas fluxes were measured two to three times a week for the remainder of the experimental period. Cumulative N2O and CO2 emissions were calculated assuming a linear change in emission rates between subsequent measurements, where the treatment with hay was used as the control treatment.

2.3. X-ray micro tomography (XRT) On day 35 and day 70 the control treatment with only hay and the treatments in which enchytraeids were present were scanned (n ¼ 5), as indicated in Table 1, with XRT. The microcosms were scanned over a time span of 2 days, block by block and the different treatments within each block in a random order. The microcosms were kept as long as possible in the controlled climate room (16  C, 60% humidity). The scans were performed using the v[tome]x m (Phoenix X-ray/General Electric). The XRT system is equipped with two X-ray sources. For optimal contrast and spatial resolution the microfocus transmission X-ray tube (240 kV/320 W), equipped with a tungsten target, was used. The microcosms were placed in the tomography chamber on a rotating stage between the X-ray source and the detector (40  40 cm GE DXR detector array, 200 mm pixel size, 2024  2024 pixels). The voltage was set on 200 kV with a filament current of 0.15 mA, resulting in a power of the Tungstentarget of 30 W. The microcosms were placed at a distance of 203.92 mm from the target corresponding to a voxel size of 50 mm, allowing to detect macropores (>80 mm) (Brady and Weil, 2008). Xray projection images were taken of the sample at 1500 different equidistant rotation angles (around the vertical axis) between 0 and 360 . Acquisition time for each image was 250 ms, resulting in a total acquisition time of 27 min for each microcosm.

Reconstruction of the tomographic data was performed using the Phoenix datosjx CT software (filtered back-projection algorithm) yielding a 3D 16 bit grayscale image. In order to calculate soil structural data such as volumetric air content, average pore sizes and the number of pores the 3D images were processed and analysed using the Avizo 3D software (Avizo 8.1, FEI, USA). First, the 3D image was filtered with a median filter and subsequently the soil profile was identified using thresholding (the threshold was set manually) to set the limits of the microcosm and the soil material (‘Image A’). By using the built-in image processing ‘closing’ routine, the total soil volume (including the inside pores) could be determined (‘Image B’). By using the built-in routine ‘fill holes’ and subsequent subtraction of Image A, the inner pores that are not in contact with the environment could be detected (‘Image C’). The volumetric air content was calculated as the total number of voxels of Image C
B divided by that of Image C. 2.4. Soil chemical analysis At both destructive sampling moments, the day after scanning with X-ray micro tomography, the soil was sampled for chemical analysis (n ¼ 5). Each microcosm was divided into two vertical halves, one of which was used for faunal extractions and the other half was used for soil analysis. After mixing the latter one, a subsample was dried at 40  C for 48 h and analysed for dissolved organic carbon (DOC) and pH in a 0.01 M CaCl2 extraction (Houba et al., 2000). Another subsample was used to determine microbial biomass nitrogen (MBN), following the chloroform fumigation and extraction technique (kEC ¼ 0.54) (Brookes et al., 1985). Subsequently, total dissolved N (Nts), ammonia (NH4 þ ), nitrate and nitrite (NO3
þ NO2
) concentrations were measured colorimetrically in a K2SO4 extract. To calculate the dissolved organic nitrogen (DON) content, NH4 þ and (NO3
þ NO2
) were subtracted from Nts. 2.5. Fauna abundances Faunal species were extracted from the other vertical half of the microcosms using different extraction techniques for the mites and the enchytraeids, following Thakur et al. (2014). Enchytraeids were extracted with a Baermann funnel, a wet extraction with temperature increasing from 20  C to 45  C within 3 h (Petersen and Luxton, 1982). Both mite species were extracted with a Berlese funnel (Tullgren funnel), with a gradual temperature increase from 20  C to 45  C in 5 days (Petersen and Luxton, 1982). 2.6. Data processing and statistical analysis Analysis of variance was carried out with the general ANOVA (analysis of variance) module in SPSS (IBM SPSS Statistics 22.0).

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The dependent variables used in a two-way ANOVA with blocking were gaseous emissions and soil parameters. The three independent factors were the presence or absence of enchytraeids, the presence or absence of fungivorous mites, and the presence or absence of predatory mites. For further analysis of the effects of soil mesofauna, treatment effects on gas emission data and soil parameters were also tested by a one-way blocked analysis of variance (ANOVA) with post hoc Tukey's honestly significant difference test (HSD) to check for differences between treatments. In addition, a two-way repeated measures ANOVA was conducted to test for an interaction between two independent factors: time (26 levels: 26 measurement days) and treatment (2 levels: presence and absence of enchytraeids) on gaseous emissions of N2O. As our data violated the assumption of sphericity (Mauchly's Test), we used the P-value of the Greenhouse-Geisser correction. To test for differences between treatments with and without enchytraeids in the XRT results, the independent T-test (in SPSS) was used. For all analyses, P-values of <0.05 were considered statistically significant. The Lavaan R package was used for structural equation modelling (SEM). SEM was used to identify the dominant pathways in the system and to improve its mechanistic understanding. The initial conceptual model included parameters and relationships between parameters based on our knowledge of soil processes in relation to N2O production and emission. Prior to the SEM analyses the units of the predictor and dependent parameters were adjusted to obtain comparable parameter variances. The quality of the SEM model was assessed by using the X2 goodness of fit statistic (P-values >0.05 indicate statistically significant model fit), the root mean square error of approximation value (RMSEA; values < 0.08 indicate reasonable error of approximation), and the Akaike information criteria (AIC) (Kline, 2011). The modification index (mi) was used to investigate whether omitted pathways could improve our initial conceptual model (Rosseel, 2012). Pathways with a P-value <0.05 were considered significant.

3. Results 3.1. Soil fauna Enchytraeids reproduced during the experiment, their numbers increasing from 50 microcosm-1 to an average of 375 microcosm-1 by day 35, and an average of 805 microcosm-1 by day 70 (Table 2). Enchytraeid numbers were not significantly affected by the presence of fungivorous mites or predatory mites. In contrast, fungivorous mite abundance declined over time with the largest reduction in the presence of enchytraeids (60 to 80% reduction,

Table 3 Cumulative N2O and CO2 fluxes on day 35 and day 70. Code CH E EM EMP EP M MP P

N2O (mg N2O-N m
2)

CO2 (g CO2-C m
2)

Day 35

Day 70

Day 35

Day 70

176.2 109.9 189.8 153.9 181.5 140.9 133.6 122.9

62.3 68.1 70.4 69.3 65.6 66.1 66.2 66.6

87.2 89.8 88.5 87.5 87.4 87.0 85.2 83.9

1.93 7.90 9.95 8.23 7.66 3.62 4.80 3.92

(±1.13) (±0.80) (±2.26) (±1.53) (±1.64) (±0.74) (±0.43) (±1.16)

a ab b b ab a a a

ANOVA full factorial E <0.001*** M 0.178ns P 0.747ns E*P 0.183ns E*M 0.987ns P*M 0.547ns E*M*P 0.859ns

(±24.4) (±18.7) (±61.2) (±36.1) (±61.7) (±27.6) (±9.8) (±8.0)

0.556ns 0.791ns 0.812ns 0.360ns 0.463ns 0.556ns 0.148ns

(±4.1) (±2.1) (±1.0) (±2.6) (±3.4) (±1.8) (±2.0) (±1.7)

0.093ns 0.191ns 0.901ns 0.272ns 0.726ns 0.697ns 0.441ns

(±1.5) (±3.9) (±3.8) (±0.7) (±2.0) (±1.8) (±1.4) (±1.9)

0.152ns 0.974ns 0.213ns 0.807ns 0.744ns 0.653ns 0.984ns

Standard errors are shown between brackets (n ¼ 5). Letters represent significant differences between the different treatments with post-hoc Tukey HSD test after significant one-way ANOVA (P < 0.05). Levels of significance: * <0.05; ** <0.01; *** <0.001; ns ¼ not significant. Codes refer to treatments listed in Table 1.

P < 0.001, Table 2). Predatory mites did not have an effect on either fungivorous mite or enchytraeid abundances. We did not find any fungivorous mites, predatory mites or enchytraeids in treatments where they were not introduced at the start of the experiment. 3.2. N2O and CO2 emissions Enchytraeids significantly increased cumulative N2O emissions from 3.6 (Control with hay treatment, CH) to 8.4 mg N2O-N m
2 (average of all treatments including enchytraeids, E) after 35 days (P < 0.001, Table 3). Enchytraeids accelerated peak N2O emissions by approximately 10 days compared to the control treatment (peak emissions on day 42 and 52, respectively, Fig. 1). This temporal effect of enchytraeids on peak N2O emissions was further corroborated by a repeated measures analysis (P ¼ 0.001, Appendix V). Thus, enchytraeids accelerated N2O emissions but they did not increase cumulative N2O emissions after 70 days. Around the time of peak N2O emissions, CO2 emissions were higher from the enchytraeid treatments compared to the treatments without enchytraeids (68.3 compared to 65.3 g CO2-C m
2, respectively). However, this difference was not significant (P ¼ 0.093). The presence of fungivorous mites, predatory mites or their interactions had no significant effect on the cumulative N2O and CO2 emissions.

Table 2 Soil mesofauna abundance on two destructive sampling moments (day 35 and 70). Code

CH E EM EMP EP M MP P ANOVA

Fungivorous mites (# microcosm
1)

Enchytraeids (# microcosm
1)

Predatory mites (# microcosm
1)

Day 35

Day 70

Day 35

Day 70

Day 35

Day 70

141 (±21) ab 77 (±6) a

142 (±48) ab 47 (±16) a

430 345 342 380

843 (±156) 750 (±103) 609 (±63) 1016 (±153)

0 (±0) 0 (±0)

3 (±2) 0 (±0)

297 (±46) b 207 (±60) ab

435 (±58) c 297 (±26) bc

<0.05

<0.05

ns

0 (±0) 0.4 (±0.4) ns

7 (±4) 0 (±0) ns

ns

(±54) (±47) (±73) (±67)

Codes refer to the treatments listed in Table 1. Standard errors are shown between brackets (n ¼ 5). Letters represent significant differences between the different treatments with post-hoc Tukey HSD test after significant one-way ANOVA (P < 0.05).

R.J. Porre et al. / Soil Biology & Biochemistry 96 (2016) 55e64

59

Fig. 1. Average (n ¼ 5) N2O flux from the soil in mg N2O-N h
1 m
2 as affected by the soil fauna treatments. Codes refer to the treatments listed in Table 1.

found on day 70 when both fungivorous mites and enchytraeids independently decreased MBN concentrations (P < 0.01). The pH decreased over time, from 6.7 at the start to 6.2 on day 70, and remained unaffected by soil mesofauna. In their single species treatment, predatory mites did not have a significant effect on any of the soil chemical properties.

3.3. Soil chemical properties The average NH4 þ concentrations in all treatments first increased from 0.8 mg N kg
1 soil to 31.3 mg N kg
1 soil by day 35 and decreased to 0.4 mg N kg
1 soil NH4 þ on day 70 (averaged over all treatments; Table 4). The presence of fungivorous mites slightly increased the NH4 þ concentration on day 35 (P ¼ 0.045). Enchytraeids and predatory mites did not affect the NH4 þ concentrations. Nitrate concentrations increased over time, from 0.6 mg N kg
1 soil at the start of the experiment to 57.2 mg N kg
1 soil on day 70 (averaged over all treatments; Table 4). Only the presence of enchytraeids significantly increased NO3
concentrations, from 6.0 to 17.1 mg N kg
1 soil on day 35 (P < 0.001). This effect disappeared by day 70, when all treatments had an equally elevated level of NO3
. Similarly enchytraeids significantly increased DOC concentrations (121.1 to 134.7 mg C kg
1 soil, P < 0.05) on day 35, but not on day 70. Significant effects of soil mesofauna on MBN were only

3.4. Soil structure Averaged over all microcosms, bulk density increased over time from 1.2 g cm
3 at the start of the experiment to 1.5 g cm
3 on days 35 and 70. Assuming a particle density of 2.60 g cm
3 for the soil mineral fraction, this corresponds to a decrease in porosity from 53% at the start of the experiment to 43% on both days 35 and 70, and an increase in WFPS from 53% to 79%. However, none of these parameters were significantly different between treatments. X-ray tomography showed a significant increase in the volumetric air

Table 4 Soil chemical properties on day 0, 35 and 70 in the experimental period. NH4 þ (mg N kg
1 soil)

NO3 (mg N kg
1 soil)

DOC (mg C kg
1 soil)

MBN (mg N kg
1 soil)

pH (CaCl2)

Day 0

0.8 (±0.2)

0.6 (±01)

115.4 (±3.6)

28.5 (±4.3)

6.7 (±0.02)

Code

Day 35

Day 70

Day 35

Day 70

Day 35

Day 70

Day 35

Day 70

Day 35

Day 70

CH E EM EMP EP M MP P

24.3 31.4 38.4 34.2 29.7 32.9 32.1 27.7

0.4 0.4 0.4 0.4 0.4 0.3 0.3 0.3

5.5 (±2.4) a 16.1 (±2.3) b 16.1 (±4.9) b 19.9 (±2.8) b 16.4 (±1.6) b 6.0 (±1.6) a 6.7 (±0.9) a 5.6 (±1.5) a

47.0 55.8 54.6 68.3 66.7 59.9 49.4 56.1

121.2 135.4 132.2 128.2 142.8 120.0 117.2 126.0

85.8 98.8 89.8 85.2 87.2 87.6 91.0 86.2

19.4 25.5 14.4 22.5 25.8 26.4 17.0 25.7

15.2 (±0.9) ab 16.8 (±4.9) ab 4.4 (±1.7) a 4.1 (±4.0) a 5.5 (±1.8) a 7.9 (±2.7) ab 13.0 (±1.9) ab 21.0 (±3.6) b

6.7 6.7 6.7 6.7 6.7 6.7 6.8 6.7

6.3 6.2 6.2 6.1 6.1 6.2 6.3 6.2

<0.001*** 0.426ns 0.447ns 0.614ns 0.784ns 0.512ns 0.644ns

0.109ns 0.743ns 0.255ns 0.200ns 0.774ns 0.404ns 0.271ns

0.005** 0.002** 0.924ns 0.014* 0.855ns 0.238ns 0.192ns

0.703ns 0.956ns 0.329ns 0.415ns 0.279ns 0.479ns 0.743ns

(±6.9) (±1.6) (±7.8) (±2.7) (±3.3) (±3.3) (±3.3) (±3.2)

ANOVA full factorial E 0.164ns M 0.045* P 0.776ns E*P 0.467ns E*M 0.895ns P*M 0.573ns E*M*P 0.895ns

(±0.09) (±0.06) (±0.06) (±0.07) (±0.07) (±0.02) (±0.04) (±0.01)

0.467ns 0.897ns 0.676ns 0.591ns 0.384ns 0.654ns 0.394ns

(±5.7) (±11.7) (±10.8) (±6.5) (±3.0) (±2.3) (±4.8) (±4.2)

(±9.1) (±4.0) (±6.7) (±6.1) (±6.7) (±7.7) (±6.6) (±12.0)

0.017* 0.205ns 0.803ns 0.948ns 0.719ns 0.383ns 0.861ns

(±1.7) (±1.2) (±3.8) (±3.0) (±3.8) (±3.2) (±5.7) (±2.1)

0.279ns 0.644ns 0.199ns 0.042ns 0.072ns 0.298ns 0.675ns

(±4.5) (±3.5) (±1.3) (±3.8) (±5.1) (±3.3) (±4.7) (±3.6)

0.976ns 0.137ns 0.621ns 0.281ns 0.239ns 0.466ns 0.034*

(±0.01) (±0.03) (±0.05) (±0.02) (±0.01) (±0.02) (±0.04) (±0.01)

(±0.06) (±0.12) (±0.12) (±0.05) (±0.03) (±0.04) (±0.05) (±0.06)

0.345ns 0.870ns 0.385ns 0.235ns 0.870ns 0.666ns 0.509ns

Standard errors are shown between brackets (n ¼ 5). Letters represent significant differences between the different treatments with post-hoc Tukey HSD test after significant one-way ANOVA (P < 0.05). Levels of significance: * <0.05; ** <0.01; *** <0.001; ns ¼ not significant, MBN ¼ Microbial biomass nitrogen, DOC ¼ Dissolved organic carbon. Codes refer to treatments listed in Table 1.

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content (0.049e0.067 cm3 cm
3, P ¼ 0.010) (Table 5). Also, there was a clear increase in the percentage of air-filled macropores in the 0.5e0.75 mm size class due to the presence of enchytraeids (Table 5: day 35 P ¼ 0.012, day 70 P < 0.001). The control treatment had relatively more air-filled macropores in the >1.25 mm size class compared to the treatments with enchytraeids present (Fig. 2). Using XRT it was possible to visualise the effects of enchytraeids on soil structure (burrows; Fig. 3). In the enchytraeid treatments a visually larger fraction of the soil volume was occupied by air-filled macropores. Also, more pores were observed to be connected to the soil surface, compared to the enchytraeid-free controls. Addition of fungivorous and predatory mites to treatments with enchytraeids had no additional effect on soil structure.

volumetric air content all contribute to the level of aeration in the soil and thereby N2O emissions (Blagodatsky and Smith, 2012; Klefoth et al., 2014). In addition, we visually observed a more continuous pore network in the soil microcosms with enchytraeids present (Fig. 3), which would further affect N2O losses to the atmosphere. In fact, enchytraeids affect soil structure in a similar manner as earthworms do, albeit on a finer scale, confirming that enchytraeids are soil ecosystem engineers (Capowiez et al., 2011; Wall et al., 2012). In our experiment enchytraeids created a large number of burrows with a diameter of 0.50e0.75 mm (Table 5, Figs. 2 and 3), which correlates closely to their body width (Chan and Heenan, 1995; Schmelz and Collado, 2010). As with the earthworm drilosphere (i.e. earthworm gut, burrows, and casts), the enchytraeid drilosphere may also stimulate interactions with soil denitrifiers and offer microsites with precisely the conditions that are optimal for N2O production (anearobic conditions in combination with available NO3
or NO2
and election-rich C) (Ihssen et al., 2003; Drake and Horn, 2007).

3.5. Structural equation modelling (SEM) Only treatments that were scanned with XRT (the control with hay and all the treatments with enchytraeids) were included in the SEM. Statistical analysis showed that our initially conceptualised model was a good fit to our data (chi-square P-values ¼ 0.314, RMSEA ¼ 0.077, Fig. 4). Modification index values were low, indicating that our model could not be further improved by adding omitted relationships. Model statistics show that only the DOC and the NO3
concentrations directly influenced N2O emissions (P < 0.001). The volumetric air content did not have a direct effect on N2O emissions, instead, the SEM model shows that the volumetric air content increased the NO3
concentrations. The volumetric air content also had a significant effect on CO2 emissions (P < 0.010). Enchytraeids did not directly increase N2O emissions, rather they increased the volumetric air content, the pore size, the DOC (P ¼ 0.054), and NO3
concentrations directly and also indirectly via the increased volumetric air content. Addition of fungivorous or predatory mites to treatments with enchytraeids had no effect on N2O emissions.

4.2. Enchytraeids increase substrate (NO3
and DOC) availability Enchytraeids not only altered soil structure but also increased NO3
and DOC concentrations on day 35 (Table 4), in accordance with Van Vliet et al. (2004). Structural equation modelling indicated a significant pathway where enchytraeids indirectly increased NO3
concentrations via their effect on the volumetric air content (Fig. 4). These results are a confirmation of earlier suggestions that enchytraeids increase the NO3
concentration via their effect on soil structure caused by burrowing activities (Van Vliet et al., 2004). The observed increased DOC concentrations and CO2 emissions are also in line with increased decomposition and mineralisation (Cole et al., 2000; Roithmeier and Pieper, 2009). This is most likely caused by increased aeration of the soil (indicated by the increase in volumetric air content), stimulating the saprotrophic microbial community (Williams et al., 1992).

4. Discussion 4.3. Enchytraeids increase cumulative N2O emissions on day 35 4.1. Enchytraeids have an effect on soil structure Enchytraeids increased cumulative N2O emissions on day 35 (Table 3, Fig. 1), when WFPS values increased to levels where denitrification is the dominant pathway for N2O production (Kool et al., 2011). Structural equation modelling indicated that the most important controls over N2O emissions in that phase of the experiment were DOC and NO3
concentrations (Fig. 4) which are indeed very important parameters influencing N2O production through denitrification (Granli and Bøckman, 1994; Wrage et al., 2001; Huang et al., 2004). However, nitrate in our system could only originate from decomposition and mineralisation, which

Using XRT it was possible to detect changes in soil structure caused by soil mesofauna, confirming our first hypothesis. Although porosity calculations based on changes in bulk density did not yield significant differences, using XRT we showed that enchytraeids significantly increased the volumetric air content and altered the pore size distribution on day 35 (Table 5, Figs. 2 and 4). This corroborates previous findings on the effects of enchytraeids on soil structure by Didden (1990), Paul (2007) and Van Vliet et al. (2004). These changes in soil structure; pore size distribution and

Table 5 Soil structural properties measured by X-ray micro tomography (XRT) on day 35 and day 70. Code

Volumetric air content cm3 cm
3 0.0e0.25 mm % pores

CH E EM EMP EP ANOVA T-test CH vs. E

0.049 (±0.005) 0.071 (±0.006) 0.062 (±0.004) 0.079 (±0.007) 0.063 (±0.007) <0.05 0.010*

Day 35 a ab ab b ab

Day 70

Day 35

0.040 (±0.003) 0.050 (±0.007) 0.042 (±0.006) 0.047 (±0.008) 0.055 (±0.012) ns 0.291ns

5.7 (±0.3) 2.7 (±0.5) 3.6 (±0.6) 2.7 (±0.3) 3.2 (±0.3) <0.05 <0.001***

Day 70 a b b b b

6.9 (±0.8) 3.5 (±0.8) 3.6 (±0.6) 3.4 (±0.9) 3.7 (±0.6) <0.05 <0.001***

a b b b b

0.25e0.5 mm % pores

0.5e0.75 mm % pores

Day 35

Day 70

Day 35

Day 70

32.9 (±1.7) 31.5 (±7.4) 41.6 (±1.9) 34.5 (±1.6) 39.6 (±2.3) ns 0.368ns

32.3 (±2.4) 34.0 (±2.7) 28.0 (±4.1) 32.0 (±3.0) 37.5 (±3.0) ns 0.873ns

25.0 (±0.4) 29.1 (±5.6) 35.9 (±1.2) 33.7 (±0.6) 34.3 (±1.6) ns 0.012*

24.4 (±0.8) 32.5 (±1.3) 28.0 (±1.9) 31.2 (±0.7) 31.8 (±1.5) <0.05 <0.001***

a b ab b b

0.75e1.0 mm % pores

1.0e1.25 mm % pores

Day 35

Day 70

Day 35

17.3 (±0.5) 15.7 (±2.1) 14.7 (±1.0) 18.4 (±0.5) 15.7 (±1.3) ns 0.204ns

17.3 (±1.0) 18.5 (±1.0) 20.3 (±1.3) 19.5 (±1.3) 16.9 (±1.1) ns 0.272ns

7.0 (±0.4) 5.8 (±1.9) 2.8 (±0.2) 4.9 (±0.5) 3.8 (±0.5) <0.05 0.022*

Day 70 a ab b ab ab

6.9 (±0.6) 5.4 (±0.9) 7.7 (±1.4) 6.2 (±0.9) 4.7 (±1.0) ns 0.427ns

Standard errors are shown between brackets (n ¼ 5). Letters represent significant differences between the different treatments with post-hoc Tukey HSD test after significant one-way ANOVA (P < 0.05). The T-test represents the difference between the control with hay (CH) and the treatments with enchytraeids (E, EM, EMP, EP). Levels of significance: * <0.05; ** <0.01; *** <0.001; ns ¼ not significant. Codes refer to treatments listed in Table 1.

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61

Fig. 2. Relative percentages of pores of different sizes (determined by X-ray micro tomography) as affected by the soil fauna on day 35. Error bars represent SE (n ¼ 5). Codes refer to the treatments listed in Table 1.

progresses faster under more aerobic conditions. It is therefore likely that the slight but significant increase in the volumetric air content in the presence of enchytraeids further stimulated decomposition and mineralisation and thereby NO3
availability for subsequent denitrification. Based on this, we confirm that through burrowing enchytraeids can increase N2O emissions by changing the volumetric air content as well as pore connectivity. 4.4. Enchytraeids accelerate N2O emissions On day 70 the cumulative N2O emissions were no longer increased in the enchytraeid treatments (Table 3). Also, increased NO3
and DOC concentrations by enchytraeids activity ceased to exist (Table 4). The volumetric air content decreased over time in all treatments (Table 5). A possible explanation for the receding N2O emissions in presence of enchytraeids is that the volumetric air content was now too low to allow for gas diffusion of N2O to the soil surface. Alternatively, the reduced volumetric air content at this stage decreased O2 availability and could have been restrictive to decomposition and mineralisation processes, which could explain the decreased DOC and NO3
concentrations. However, concentrations of DOC and NO3
in the soil by day 70 were still relatively high, so this was not likely to be the limiting factor for N2O production. Possibly the decreased decomposition and mineralisation

processes would have resulted in more oxic conditions in the soil and thus limited denitrification rates and N2O production. The reduced porosity of the soil is likely to have resulted from the decomposition of hay and because of watering the soil. We added hay to a mineral soil and the enchytraeids most likely increased the decomposition rate of the hay through their feeding activities and by thoroughly mixing the soil and litter; during destructive sampling it was visible that hay was further decomposed in treatments with enchytraeids present. Kuiper et al. (2013) already showed that enchytraeids accelerated N2O production, simultaneously leading to an increased rate of food depletion. These increased decomposition rates most likely caused the higher substrate (NO3
and DOC) availability with enchytraeids present on day 35. Enchytraeids therefore seemed to accelerate rather than increase N2O emissions in the long run. 4.5. Fungivorous mites Fungivorous mites did not significantly decrease NO3
concentrations or lower N2O emissions, therefore our third hypothesis was rejected (Table 4). Since fungivorous mites graze on soil microbes it is reasonable that this would decrease mineralisation rates because of a decrease in microbes. However, when grazing pressure on fungi is in balance with the speed of fungal regrowth, no impact

Fig. 3. Representative X-ray micro tomography images (CH on the left and E on the right). The linear colour scale shows a minimum size of air-filled macropore space of 0.1 mm and a maximum of 2.0 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Structural equation model (SEM) results for the effects of enchytraeids and mites on N2O and CO2 emission on day 35. The model is a good fit to the data: chi-square ¼ 0.251, P ¼ 0.304; RMSEA ¼ 0.097; AIC ¼ 920.953. Dashed arrows represent non-significant relationships (ns ¼ not significant), continuous arrows represent significant relationships (*P < 0.05; **P < 0.01; ***P < 0.001). Numbers next to the arrows represent the (positive or negative) path coefficients.

or even a positive effect on nitrogen mineralisation is possible (Osler and Sommerkorn, 2007; Crowther et al., 2012). In treatments where both enchytraeids and mites were present a possible explanation is that the grazing of fungivorous mites on the microbial population was negligible compared to the grazing of the enchytraeids. Enchytraeid abundances had increased whereas fungivorous mite abundances decreased significantly (Tables 1 and 2). This might be because the wet environmental conditions as well as the relatively low temperatures (16  C) were more conducive for enchytraeids than for mites. The extra decline in fungivorous mite abundances in the presence of enchytraeids could be caused by the burrowing activities of enchytraeids; these burrows could potentially be destructive for the mite habitat in the same manner as earthworm burrows affect mite communities (Maraun and Scheu, 2000). An alternative explanation is that enchytraeids, apart from being able to feed on particulate organic matter (POM), can feed on bacteria and fungi and might therefore compete with fungivorous mites for their food source (Osler and Sommerkorn, 2007).

4.6. Interactions between enchytraeids, fungivorous mites and predatory mites Interactions between enchytraeids, fungivorous mites and predatory mites (EMP) resulted in a reduction of fungivorous mite abundance, however no conclusive effects on either soil structural parameters or on soil substrate availability and N2O emissions were measured. We did not find predatory mites in most treatments after soil sampling (Table 2). Also, we did not find the large increase in N2O emissions caused by EMP that Thakur et al. (2014) reported. The EMP treatment had a higher N2O flux than the other treatments around day 40 (Fig. 1), but this did not result in increased cumulative N2O emissions for the combination of EMP. In general, high N2O emission variability within mesofaunal treatments was observed. This large variation in N2O emission can be explained by

the fact that we mixed hay through the soil, causing a large heterogeneity in the soil with respect to the food source for the mesofauna and microbes (Van Vliet et al., 2004). The only difference in experimental setup with Thakur et al. (2014) was that we mixed the hay through the entire soil profile rather than only in the top layer. This may have altered the behaviour of the soil mesofauna. The NO3
concentrations (DOC concentrations were not mentioned) in the study of Thakur et al. (2014) were lower than what we found for corresponding treatments; possibly this relative increase in NO3
concentration in the EMP treatment of Thakur et al. (2014) had a large effect on N2O emissions since NO3
was the limiting factor. 4.7. Research recommendations We used XRT to characterize the three-dimensional structure of the soil and to quantify soil structural parameters such as volumetric air content and the pore size distribution. We showed that enchytraeids can influence these parameters and thereby affect N2O emissions. However, additional research is still needed to determine the connectivity of the (enchytraeid-induced) pores to the soil surface and its link to diffusivity, since this link has been challenged in the literature (e.g. Mangalassery et al., 2013). In addition to the change in pore size distribution in the presence of enchytraeids, visual observations suggested that a larger fraction of the soil volume is occupied by air-filled macropores (Fig. 3). Because these pores are formed by burrowing enchytraeids it is likely (and visual observations suggest) that this also results in a larger pore connectivity. However, this should be further studied in the future, and we therefore suggest modelling of the pore connectivity and tortuosity in order to further quantify diffusivity in the soil profile. Our experimental setup was focused on mechanistically unravelling pathways through which soil mesofauna can influence N2O emissions. For this reason our experiment was set up under strictly controlled laboratory conditions. To create conditions

R.J. Porre et al. / Soil Biology & Biochemistry 96 (2016) 55e64

that resemble field conditions more closely, further research could focus on more realistic soil meso- and macro-faunal community compositions. This could also include a higher functional diversity of species and combining soil mesofauna with soil macrofauna such as earthworms. 5. Conclusion Enchytraeids had a significant effect on the soil structure. Similar to earthworms, they are soil ecosystem engineers that can alter the soil structure and increase the volumetric air content, as well as the number of pores similar to its body width (size class 0.5e0.75 mm). These changes in the volumetric air content in combination with soil respiration most likely provided anoxic microsites which could therefore promote N2O emissions. However, over the entire experimental period of 70 days enchytraeids accelerated but did not increase cumulative N2O emissions. Fungivorous mites and predatory mites had a negligible effect on soil structure and N2O emissions. Structural equation modelling confirmed that enchytraeids, by creating a higher volumetric air content, increased decomposition rates, as indicated by higher CO2 emissions and mineral nitrogen availability. This strongly suggests that an increased volumetric air content in combination with high mineral nitrogen and DOC concentrations accelerated N2O emissions. We conclude that from the soil fauna taxa we tested, only enchytraeids, as soil ecosystem engineers, have an important effect on the soil structure pathway, resulting in enhanced nitrogen mineralisation and accelerated N2O emissions. Acknowledgements This study was supported by a grant from the Netherlands Organization for Scientific Research/Earth and Life Sciences (NWOALW, grant number 823.01.016). The X-ray micro tomography scans (XRT) were obtained using CAT-AgroFood equipment and we thank Remco Hamoen for his assistance with scanning, reconstructing and analyzing. We are grateful to Isa Lesna for providing Acarus siro; to Isabel Smallegange for providing Rhizoglyphus robini; and to
s Sala
nki for providing Enchytraeus albidus. In addition, we Tama
s would like to thank Jaap Nelemans, Willeke van Tintelen, Tama
nki, Gerlinde Vink for their assistance with laboratory work, Sala and Lijbert Brussaard, Hannah Vos and Susan Klinkert for comments on earlier versions of this manuscript. Furthermore, we thank Gerrit Gort for his advice on statistics. Finally, we are grateful to two anonymous reviewers for their detailed and constructive comments on a previous version of this manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2016.01.018. References Ball, B.C., Crichton, I., Horgan, G.W., 2008. Dynamics of upward and downward N2O and CO2 fluxes in ploughed or no-tilled soils in relation to water-filled pore space, compaction and crop presence. Soil and Tillage Research 101, 20e30. Bateman, E.J., Baggs, E.M., 2005. Contributions of nitrification and denitrification to N2O emissions from soils at different water-filled pore space. Biology and Fertility of Soils 41, 379e388.
onard, J., Roussel, M., Gre
han, E., Gabrielle, B., 2010. Modelling Bessou, C., Mary, B., Le soil compaction impacts on nitrous oxide emissions in arable fields. European Journal of Soil Science 61, 348e363. Blagodatsky, S., Smith, P., 2012. Soil physics meets soil biology: towards better mechanistic prediction of greenhouse gas emissions from soil. Soil Biology and Biochemistry 47, 78e92.

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Brady, N.C., Weil, R.R., 2008. The Nature and Properties of Soils, 14th ed. Pearson Education, Inc., New Jersey, p. 158. Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology and Biochemistry 17, 837e842. Capowiez, Y., Sammartino, S., Michel, E., 2011. Using X-ray tomography to quantify earthworm bioturbation non-destructively in repacked soil cores. Geoderma 162, 124e131. Chan, K.Y., Heenan, D.P., 1995. Occurrence of enchytraeid worms and some properties of their casts in an Australian soil under cropping. Australian Journal of Soil Research 33, 651e657. Chapuis-lardy, L., Wrage, N., Metay, A., Chotte, J.L., Bernoux, M., 2007. Soils, a sink for N2O? A review. Global Change Biology 13, 1e17. Cole, L., Bardgett, R.D., Ineson, P., 2000. Enchytraeid worms (Oligochaeta) enhance mineralization of carbon in organic upland soils. European Journal of Soil Science 51, 185e192. Crowther, T.W., Boddy, L., Hefin Jones, T., 2012. Functional and ecological consequences of saprotrophic fungusegrazer interactions. The ISME Journal 6, 1992e2001. De Ruiter, P.C., Neutel, M., Moore, J.C., 1995. Energetics, patterns of interaction strengths, and stability in real ecosystems. Science (New York, N.Y 269, 1257e1260. Deurer, M., Grinev, D., Young, I., Clothier, B.E., Müller, K., 2009. The impact of soil carbon management on soil macropore structure: a comparison of two apple orchard systems in New Zealand. European Journal of Soil Science 60 (6), 945e955. Didden, W.A.M., 1990. Involvement of Enchytraeidae (Oligochaeta) in soil structure evolution in agricultural fields. Biology and Fertility of Soils 9, 152e158. Drake, H.L., Horn, M.A., 2007. As the worm turns: the earthworm gut as a transient habitat for soil microbial biomes. Annual Review of Microbiology 61, 169e189. Granli, T.O.C.B., Bøckman, O.C., 1994. Nitrous oxide from agriculture. Norwegian Journal of Agricultural Sciences 7e128. Supplement 12. Houba, V.J.G., Temminghoff, E.J.M., Gaikhorst, G.A., Van Vark, W., 2000. Soil analysis procedures using 0.01 M calcium chloride as extraction reagent. Communications in Soil Science and Plant Analysis 31, 1299e1396. Huang, Y., Zou, J., Zheng, X., Wang, Y., Xu, X., 2004. Nitrous oxide emissions as influenced by amendment of plant residues with different C: N ratios. Soil Biology and Biochemistry 36, 973e981. € ssner, A., Drake, H.L., 2003. N2O-producing Ihssen, J., Horn, M.A., Matthies, C., Go microorganisms in the gut of the earthworm Aporrectodea caliginosa are indicative of ingested soil bacteria. Applied and Environmental Microbiology 69 (3), 1655e1661. Kaneda, S., Kaneko, N., 2011. Influence of collembola on nitrogen mineralization varies with soil moisture content. Soil Science and Plant Nutrition 57, 40e49. Klefoth, R., Oenema, O., van Groenigen, J.W., 2012. A novel method for quantifying nitrous oxide reduction in soil. Vadose Zone Journal 11. Klefoth, R.R., Clough, T.J., Oenema, O., Van Groenigen, J.W., 2014. Soil bulk density and moisture content influence relative gas diffusivity and the reduction of nitrogen-15 nitrous oxide. Vadose Zone Journal 13. Kline, R.B., 2011. Principles and Practice of Structural Equation Modeling, third ed. The Guilford Press, New York, p. 427. Kool, D.M., Dolfing, J., Wrage, N., Van Groenigen, J.W., 2011. Nitrifier denitrification as a distinct and significant source of nitrous oxide from soil. Soil Biology and Biochemistry 43, 174e178. Kuiper, I., De Deyn, G.B., Thakur, M.P., Van Groenigen, J.W., 2013. Soil invertebrate fauna affect N2O emissions from soil. Global Change Biology 19, 2814e2825. Laudone, G.M., Matthews, G.P., Bird, N.R.A., Whalley, W.R., Cardenas, L.M., Gregory, A.S., 2011. A model to predict the effects of soil structure on denitrification and N2O emission. Journal of Hydrology 409, 283e290. Lee, K.E., Foster, R.C., 1991. Soil fauna and soil structure. Australian Journal of Soil Research 29, 745e775. Lubbers, I.M., Brussaard, L., Otten, W., Van Groenigen, J.W., 2011. Earthworminduced N mineralization in fertilized grassland increases both N2O emission and crop-N uptake. European Journal of Soil Science 62, 152e161. Lubbers, I.M., Van Groenigen, K.J., Fonte, S.J., Six, J., Brussaard, L., Van Groenigen, J.W., 2013. Greenhouse-gas emissions from soils increased by earthworms. Nature Climate Change 3, 187e194. €gersten, S., Sparkes, D.L., Sturrock, C.J., Mooney, S.J., 2013. The Mangalassery, S., Sjo effect of soil aggregate size on pore structure and its consequence on emission of greenhouse gases. Soil and Tillage Research 132, 39e46. Maraun, M., Scheu, S., 2000. The structure of oribatid mite communities (Acari, Oribatida): patterns, mechanisms and implications for future research. Ecography 23, 374e383. Mosier, A., Kroeze, C., Nevison, C., Oenema, O., Seitzinger, S., Van Cleemput, O., 1998. Closing the global N2O budget: nitrous oxide emissions through the agricultural nitrogen cycle: OECD/IPCC/IEA phase II development of IPCC guidelines for national greenhouse gas inventory methodology. Nutrient Cycling in Agroecosystems 52, 225e248. Osler, G.H.R., Sommerkorn, M., 2007. Toward a complete soil C and N cycle: incorporating the soil fauna. Ecology 88, 1611e1621. Paul, E.A., 2007. Soil Microbiology, Ecology, and Biochemistry, third ed. 532 pp. Paul, B.K., Lubbers, I.M., Van Groenigen, J.W., 2012. Residue incorporation depth is a controlling factor of earthworm-induced nitrous oxide emissions. Global Change Biology 18, 1141e1151.

64

R.J. Porre et al. / Soil Biology & Biochemistry 96 (2016) 55e64

Petersen, H., Luxton, M., 1982. A comparative analysis of soil fauna populations and their role in decomposition processes. Oikos 39, 287e388. Pierret, A., Capowiez, Y., Belzunces, L., Moran, C.J., 2002. 3D reconstruction and quantification of macropores using X-ray computed tomography and image analysis. Geoderma 106, 247e271. Ravishankara, A.R., Daniel, J.S., Portmann, R.W., 2009. Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326, 123e125. Roithmeier, O., Pieper, S., 2009. Influence of Enchytraeidae (Enchytraeus albidus) and compaction on nutrient mobilization in an urban soil. Pedobiologia 53, 29e40. Rosseel, Y., 2012. Lavaan: an R package for structural equation modeling. Journal of Statistical Software 48. Schmelz, R.M., Collado, R., 2010. A guide to European terrestrial and freshwater species of Enchytraeidae (Oligochaeta). Senckenberg Museum für Naturkunde €rlitz 82 (1), 1e176. Go Schmitz, O.J., Hawlena, D., Trussell, G.C., 2010. Predator control of ecosystem nutrient dynamics. Ecology Letters 13, 1199e1209. Thakur, M.P., van Groenigen, J.W., Kuiper, I., De Deyn, G.B., 2014. Interactions between microbial-feeding and predatory soil fauna trigger N2O emissions. Soil Biology and Biochemistry 70, 256e262.

UNEP, 2013. Drawing Down N2O to Protect Climate and the Ozone Layer. A UNEP Synthesis Report. United Nations Environment Programme (UNEP), Nairobi, Kenya. Van Vliet, P.C.J., Beare, M.H., Coleman, D.C., Hendrix, P.F., 2004. Effects of enchytraeids (Annelida: Oligochaeta) on soil carbon and nitrogen dynamics in laboratory incubations. Applied Soil Ecology 25, 147e160. Velthof, G.L., Kuikman, P.J., Oenema, O., 2002. Nitrous oxide emissions from soils amended with crop residues. Nutrient Cycling in Agroecosystems 62, 249e261. Wall, D.H., Bardgett, R.D., Behan-Pelletier, V., Herrick, J.E., Jones, T.H., Ritz, K., Six, J., Strong, D.R., Van der Putten, W.H., 2012. Soil Ecology and Ecosystem Services. Oxford University Press, Oxford, 424 p. Van Der Weerden, T.J., Kelliher, F.M., De Klein, C.A.M., 2012. Influence of pore size distribution and soil water content on nitrous oxide emissions. Soil Research 50, 125e135. Williams, E.J., Hutchinson, G.L., Fehsenfeld, F.C., 1992. NOx and N2O emissions from soil. Global Biogeochemical Cycles 6, 351e388. Wrage, N., Velthof, G.L., Van Beusichem, M.L., Oenema, O., 2001. Role of nitrifier denitrification in the production of nitrous oxide. Soil Biology and Biochemistry 33, 1723e1732.