Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition

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Soil Biology & Biochemistry xxx (2015) 1e9

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Q1,3

Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition

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ova  a, Zuzana Lhota kova  c, Toma 
s Cajthaml a Jan Frouz a, b, *, Alexandra Spaldo n a

Institute for Environmental Studies, Faculty of Science, Charles University in Prague, Czech Republic
 Institute of Soil Biology, Biology Centre AS CR, Cesk e Bud
ejovice, Czech Republic c  5, Prague, Czech Republic Department of Experimental Plant Biology, Faculty of Science, Charles University, Vini
cna b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 April 2015 Received in revised form 17 August 2015 Accepted 21 August 2015 Available online xxx

To understand why excrements of soil macrofauna often decompose more slowly than leaf litter, we fed Bibio marci larvae the litter of tree species differing in litter quality (Alnus glutinosa, Salix caprea, and Quercus robur) and then measured respiration induced by litter and excrements. We also measured respiration induced by the same litter artificially modified to mimic faunal effects; the litter was modified by grinding, grinding with alkalinization to pH ¼ 11, grinding with coating by kaolinite, and grinding with both alkalinization and coating. Decomposition of excrements tended to be slower for willow and was significantly slower for oak and alder than for the corresponding litter. With oak, decomposition was slower for all artificially modified litter than for non-modified litter. The reduction in the decomposition was similar for excrements and for alder and willow litter that was ground, coated, and alkalinized. In alder, a similar reduction was found in ground and alkalinized litter. 13C NMR indicated that gut passage increases aliphatic components and decreases polysaccharides. Pyrolysis indicated that gut passage increases the ratio of guaiacyl to hydroxymethyl derivatives in lignin. Our findings indicate that the decreased decomposition rate of excrements might result from the removal of easily available polysaccharides, the increase in aliphatic components, an increase in the resistant components of lignin, the accumulation of microbial cell walls, and the binding of nitrogen into complexes with aromatic components. Several of these mechanisms are supported or determined by litter alkalinization during gut passage. © 2015 Published by Elsevier Ltd.

Keywords: Alkalinization Bibio CN ratio Litter decomposition Mineralization Pyrolysis

1. Introduction With global warming, soil organic matter is receiving substantial attention as an important part of the global carbon (C) cycle. Soil contains three-times more C than the atmosphere and because of its dynamic nature, soil organic C could serve as either a significant sink or source for atmospheric carbon dioxide (Post et al., 1982). Most of the terrestrial net primary production enters the soil decomposer system as dead organic matter, namely leaf litter and dead roots (Wardle et al., 2004; García-Palacios et al., 2013). Leaf litter decomposition is affected by a complex interplay of climate, litter chemistry, soil properties, and activity of soil organisms (Schmidt et al., 2011).

* Corresponding author. Institute for Environmental Studies, Faculty of Science, Charles University in Prague, Czech Republic. E-mail address: [email protected] (J. Frouz).

Among soil organisms, research has more often focused on microorganisms than on fauna; the effect of fauna has been neglected in part because the assimilation efficiency of soil fauna is generally low (Anderson and Ineson, 1984; Lavelle et al., 1997; Kadamannaya and Sridhar, 2009), and consequently, most ingested organic matter is only transformed from litter into excrements. Fauna excrements, however, differ substantially from the original litter, and these differences greatly affect microbial activity and decomposition (Anderson and Ineson, 1984; Lavelle et al., 1997; Wolters, 2000). During faunal feeding or soon after excrements are produced, microbial activity is increased, and the decomposition rate is greater for the new excrements than for the original litter. As soil faunal excrements age, however, the decomposition rate decreases and becomes lower than that of the original litter (Van der Drift and Jansen, 1977; Hassall et al., 1986, 1987; Griffiths et al., 1989; Lavelle and Martin, 1992; Frouz et al., 1999; Frouz and
Simek, 2009; Kaneda et al., 2013; Frouz et al., 2014, 2015). Factors causing the increase of microbial activity in new faunal excrements

http://dx.doi.org/10.1016/j.soilbio.2015.08.024 0038-0717/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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(litter fragmentation, nutrient release due to the effects of the gut on the litter and to the killing of the ingested microflora) have often been studied (Hassall et al., 1986, 1987; Griffiths et al., 1989; Frouz et al., 1999), but the mechanisms causing a reduction in microbial activity in older excrement have been less studied. This phenomenon has received much more attention for earthworms (Lavelle and Martin, 1992; Zhang et al., 2003; Frouz et al., 2014) than for other litter-feeding macrofauna. This is despite the fact that soil macrofauna such as millipedes, isopods, and insect larvae can consume 20e100% of annual litter fall in many ecosystems and especially in temperate, broadleaf forests (Karpachevsky et al., 1968; Szabo, 1974; Tajovský, 1992; García-Palacios et al., 2013). The aim of this contribution is to explore mechanisms that may reduce the rate of microbial decomposition in older excrements of litter-feeding macroarthropods. To do so, we studied the decomposition (measured as loss of C in CO2) of litter and of excrements produced from this litter together with artificial litter treatments that mimic some of the modifications in litter caused by passage through the faunal gut. Bibio marci (Diptera: Bibionidae) was used as an example of a litter-feeding macroarthropod; in addition to dipteran larvae, such macroarthropods also include millipedes and terrestrial isopods. Bibionidae occur worldwide and are especially abundant in tropical and temperate regions, where larvae in litter and soil can achieve high biomasses (Frouz, 1999) and consume all of the annual litter fall (Karpachevsky et al., 1968; Szabo, 1974). The artificial treatments used in our study were litter fragmentation, litter alkalinization, coating by clay, and their combinations. Litter fragmentation is assumed to be the major reason for the initial increase in microbial respiration in excrements (Hassall et al., 1986, 1987; Griffiths et al., 1989; Frouz et al., 1999); therefore, we explored its long-term effect. Alkalinization was studied because many saprophagous arthropods including B. marci have a highly alkaline section of their gut (Johnson and Felton, 1996; €rlocher, 1999; Frouz et al., 2002). Brune, 1998; Graça and Ba Finally, clay coating has been suggested as one reason for the stabilization of soil organic matter processed by earthworms (Zhang et al., 2003; Frouz et al., 2014). 2. Materials and methods 2.1. Materials A litter bag experiment was conducted in microcosms (bottles) with litter of three tree species, excrements generated from that litter by a soil arthropod, and litter modified to mimic specific changes in litter during passage through the soil arthropod gut. Litter of alder (Alnus glutinosa), willow (Salix caprea), and oak (Quercus robur) was collected at post-mining sites near Sokolov (50140 N, 12 390 E). Litter of these species was chosen because of contrasting C:N ratios (Table 1). Fresh litter that had not contacted

soil was used. Part of the litter was air-dried; the rest was kept moist, was stored at 4  C, and was used to feed larvae of the soil arthropod B. marci, which were collected in an alder (A. glutinosa)
 Bude
jovice, Czech Republic (48 590 N, dominated forest near Cesk e 14 250 E), in October 2009. At the same time, samples of the fermentation layer were taken from each site where the litter was collected; the fermentation layer was kept at the original moisture and was stored at 4  C. To obtain excrements, larvae were maintained in a plastic container (10  20  5 cm) at 15  C with 95e100% relative humidity (RH) and in the dark. About 150 larvae were placed in each of the six containers (3 litter types  2 replicates) and fed with either alder, willow, or oak litter. To harvest excrements, the contents of each container were passed through a 1-mm sieve every other day. After excrements were separated, larvae were removed from the litter and returned to the container along with new litter. After they were collected by sieving, excrements were spread in a thin layer and air-dried. Excrements derived from the same kind of litter were pooled and stored in a dry, dark place. The first two collections were not used to ensure that the excrements were not contaminated by previously consumed litter. Besides litter and excrements, the following four artificial treatments were used in the experiment: ground litter (G); ground and alkalinized litter (GA); ground and coated litter (GC); and ground, coated, and alkalinized litter (GCA). These treatments were designed to mimic some of the following effects that fauna may have on litter: fragmentation; alkalinization due to gut passage; and coating by clay particles to mimic the situation when larvae ingest soil attached to litter surface. Ground litter was prepared by processing the dry litter in an electric blender and then passing the fragments through a 0.2-mm sieve. Ground litter was also used to prepare the other modified-litter treatments. For treatment G, the ground litter was not further treated. For treatment GA, the litter bags were placed in water alkalinized to pH 11 by addition of NaOH; after 8 h, the litter bags were washed in distilled water until the water had a neutral reaction, and then the litter bags and litter within were air-dried. For treatment GC, 0.1 g of kaolinite was mixed with the 0.5 g of ground litter before it was sealed in the litter bag. For treatment GCA, the ground litter was alkalinized before it was coated and sealed in litter bags. Excrements and all litter treatments including litter that was not modified were also placed in 2  2-cm litter bags (42-mm openings), with 0.5 g (dry weight) of litter or excrements per litter bag. Before the onset of the litter bag experiment, all litter bags prepared as above were rewetted by placing them for 24 h on sand saturated with a filtered soil suspension. The filtered soil suspension, which was made with water and fermentation layer soil (1:100, soil:water), provided autochthonous microorganisms that may have been lost during litter bag preparation. The litter bags were also sprayed with the suspension every 8 h during the 24 period.

Table 1 CN ration of litter, treated litter, and excrements prepared from the same litter (alder, oak, or willow) before and after 54 weeks of decomposition. G ¼ ground litter, GC ¼ ground and coated litter, GA ¼ ground and alkalinized litter, GCA ¼ ground, coated, and alkalinized litter. Values are means (with SD below), statistically homogenous groups of CN values are marked by the same letter (one-way ANOVA, LSD, p > 0.05). Litter Before

Excrements After

Alder (Alnus glutinosa) 14.6a (0.6) 14.7a (1.0) Willow (Salix caprea) 29.4b (2.6) 36.2c (2.7) Oak (Quercus robur) 58.0d (3.2) 59.0d (5.5)

GA

G

GC

GA

GCA

Before

After

Before

After

After

After

After

15.4a (0.1)

17.4a (0.3)

14.6a (0.1)

15.7a (0.0)

15.5e (0.2)

16.8 (1.6)

16.0a (0.3)

14.5a (0.1)

23.9b (1.4)

28.5b (1.1)

28.8b (0.5)

28.9b (1.7)

29.2b (1.3)

29.7b (0.2)

17.3a (0.1)

23.0b (0.9)

58.8b (5.9)

59.7d (2.4)

70.4e (0.1)

60.9d (0.8)

63.4de (2.5)

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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2.2. Experimental design and analysis Each litter bag was placed separately in one 250-ml glass bottle with 40 g of fine sand on the bottom. The sand was free of organic matter and was saturated with distilled water to field capacity. The bottles were sealed and kept in the dark at 15  C. Every 2 weeks, the bottles were weighed, and water was supplied as needed to maintain the moisture content of the sand. Each treatment was represented by four replicate bottles. Respiration was measured for 54 weeks. Each week, a measurement was initiated by placing a small beaker containing 3 ml of 0.5 M NaOH in each sealed bottle. The CO2 produced in the bottle was trapped in the NaOH. The beaker with NaOH was removed and replaced with a new beaker and fresh NaOH every 7 days. The bottles were kept sealed except when the NaOH was exchanged and when water was added. The quantity of CO2 trapped in the NaOH was determined by 0.05 M HCl titration after the addition of 2 ml of BaCl2. The experiment was terminated after 54 weeks, giving 54 weekly measurements of CO2 production. Total C and nitrogen (N) contents in litter, ground and alkalinized litter, and excrements were measured before the experiment and in all the materials at the end of the experiment using a CN analyzer (The Elemental Analyzer 1108, Carlo Erba Instruments). At the start and end of the experiment, total soluble phenolics were extracted from these same materials with 80% methanol; the extracted soluble phenolics were spectrophotometrically quantified using FolineCiocalteu reagent (Singleton et al., 1998). The litter and excrements before and after the experiment were subjected to thermochemolysiseGCeMS and 13C CP/MAS NMR. Samples analyzed before the experiment included litter, excrements, and ground and alkalinized litter because there was no reason to believe that grinding or coating would change the litter chemistry. Four replicates were used for TMAH-Py-GC MS but these were pooled for 13C CP/MAS NMR in order to obtain sufficient material for the determination. NMR spectra were measured with a Bruker Avance 500 WB/US NMR spectrometer (Karlsruhe, Germany, 2003) in a 4-mm ZrO2 rotor. The magic angle spinning (MAS) speed was 9 kHz in all cases, with a notation frequency of B1 (1H) and B1 (13C) fields for crosspolarization u1/2p ¼ 62.5 kHz. Repetition delay and number of scans was 4 s and 1024, respectively. TPPM (two-pulse phasemodulated) decoupling was applied during evolution and both detection periods. The phase modulation angle was 15 , and the flip-pulse length was 4.8e4.9 ms. The applied notation frequency of the B1 (1H) field was u1/2p ¼ 89.3 kHz. The 13C scale was calibrated using glycine as the external standard (176.03 ppm; low-field carbonyl signal). The resulting 13C NMR spectra were used to quantify three basic fractions (aromatic, aliphatic, and polysaccharide) according to Wilson (1987) and Keeler and Maciel (2000), and based on the area of the appropriate peak relative to the total area. TMAH-Py-GC MS analyses were performed as previously described (Sampedro et al., 2009). Briefly, 1-mg samples of ground litter were treated with an excess of tetramethylammonium hydroxide (25% aqueous solution). The samples were then placed on Wolfram wire spirals and dried in a desiccator overnight at room temperature. Pyrolysis was performed with a PYR-01 pyrolyzer (Labio, Czech Republic). Pyrolysis was performed directly in the injector of a GC/MS system (Varian 3400/Finnigan ITS 40 ion trap detector). The GC instrument was equipped with a split injector (split ratio 1/40). An HP-5 column was used for separation (30 m, inner diameter 0.25 mm, 0.25 mm film thickness), and the carrier gas was helium (1 ml min1). The temperature program started at 45  C, and the oven was heated to 240  C at a rate of 5  C min1. The detector delay time was 2 min. The injector and transfer line

3

temperature was set to 240  C. Mass spectra were recorded at 1 scan s1 under an electron impact at 70 eV, mass range 50e450 amu. Pyrolysis products were identified both by comparing mass spectra with data in the NIST02 library and by interpreting the fragmentation pattern. Values presented are the means of triplicate runs, and the percentages of pyrolysis products were calculated from the relative areas of the peaks after recalculation according to the exact weight of samples. Reproducibility of the sample introduction exceeded 95%. The individual chromatograms were integrated, and the peaks representing lignin-related structures (guaiacyl, syringyl, and hydroxymethyl) were compared. 2.3. Data analysis One-way ANOVA was used to compare various treatments (untreated and treated litter and excrements) produced from the same litter species. To evaluate the effect of individual litter parameters on decomposition, these parameters measured before decomposition experiment were correlated with C loss in CO2 during the decomposition experiment, which was used as a proxy for decomposition in this study. This was done either for raw data of the parameters or for relative changes of the parameters in comparison with untreated litter by dividing values of individual parameters in all treatments by those of untreated litter. All computations were made using Statistica 10.0. 3. Results Respiration in all treatments gradually decreased with time (Fig. 1). Even though the C:N ratio of the litters differed substantially (Table 1), the total C lost by respiration over the 54-week experiment did not significantly differ among the three kinds of litter (one-way ANOVA, F ¼ 1.924, p ¼ 0.2016, df ¼ 9) (Fig. 2). With oak and alder, C loss was significantly lower from excrements than from litter. With willow, this decrease was only marginally significant (t-test, p ¼ 0.0913) (Fig. 2). The C loss from excrements relative to the C loss from litter was 29, 77, and 89% for oak, alder, and willow, respectively (Fig. 2). For oak, C loss was significantly lower for all artificial litter treatments than for untreated litter (Fig. 2); C loss was less for these artificial litter treatments, however, than for the excrements. For alder, C loss was significantly lower for both alkalinized treatments (ground and alkalinized; and ground, coated, and alkalinized) than for untreated litter but did not differ from C loss for excrements. For willow, C loss was significantly lower for both alkalinized treatments than for untreated litter or for excrements (Fig. 2).

Fig. 1. Carbon lost as CO2 from litter (L) and excrements (E) during the 54-week litter bag experiment. The litter was from oak (Q) or alter (A), and the excrements were obtained by feeding Bibio marci larvae with Q or A litter as indicated.

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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Fig. 2. Total carbon lost as CO2 from litter, treated litter, and excrements after 54 weeks of decomposition. The litter was from alder, willow, and oak, and the excrements were obtained by feeding Bibio marci larvae with alder, willow, or oak litter as indicated. Values are means þ SD. Columns marked with the same letter within the same litter species are not statistically different (one-way ANOVA, LSD, p > 0.05).

The C:N ratio of excrements produced from litter with a high C:N ratio (oak and willow) significantly decreased but remained about the same in excrements produced from litter with a low C:N ratio (alder). As a consequence, excrements had a similarly low C:N ratio regardless of the litter source. None of the artificial litter treatments caused a decrease in the C:N ratio for any litter type (Table 1). 13C NMR did not indicate any difference in the content of aliphatic, polysaccharide, or lignin components between alder litter and excrements produced from alder litter. In oak and willow, aliphatic components were increased and polysaccharides were decreased in excrements relative to untreated litter. In willow, the chemical properties of alkalinized litter were similar to those of excrements (Table 2). Pyrolysis was used to determine whether the untreated litter and excrements derived from that litter differed in the major components of lignin (derivatives of guaiacyl, syringyl, and hydroxymethyl). For alder, the content of guaiacyl derivatives did not differ significantly between litter and excrements, whereas the contents of syringyl and hydroxymethyl derivatives were lower in excrements than in untreated litter (Table 3). For oak, the content of guaiacyl derivatives was significantly higher, the content of syringyl derivatives was not significantly changed, and the content of hydroxymethyl derivatives was significantly lower in excrements relative to untreated litter (Table 3). As a consequence, the guaiacyl:syringyl ratio was significantly higher in excrements than in

litter for alder, and the guaiacyl:hydroxymethyl ratio was significantly higher in excrements than in litter for oak (Fig. 3). These ratios did not differ among treatments for willow (Fig. 3). The content of phenolic compounds was significantly greater in undecomposed litter than in all other treatment including litter subjected to 54 weeks of decomposition (Table 4). Alder had significantly more phenolic compounds than oak, which had significantly more phenolics than willow. Phenolic content was significantly lower in excrements and artificially alkalinized litter than in untreated litter (Table 4). To assess the importance of chemical changes on the rate of decomposition, C loss (Fig. 2) was correlated with the chemical properties of litter, excrements, or litter treatments (Tables 1, 2, and 4, and Fig. 3) at the beginning of experiment. This was done either by comparing the mean values of these parameters for each of the 18 treatments or by comparing relative values (so each litter had value 1) (Table 5). All ground treatments as well as excrements were assumed to be fragmented. Based on absolute values, only the guaiacyl to hydroxymethyl ratio (G:H) and the fragmentation of litter (either by grinding or by soil fauna) were correlated with C loss, and the correlations were negative. Based on relative values, C loss was positively correlated with the C:N ratio, polysaccharide content, and phenol content, and was negatively correlated with the G:H ratio and the grinding of litter.

Table 2 Chemical composition of litter, treated litter, and excrements prepared from the same litter (alder, oak, or willow) before and after 54 weeks of decomposition. Values indicate the proportion of peak areas relative to the entire spectra based on 13C CP/MAS NMR. G ¼ ground litter, GC ¼ ground and coated litter, GA ¼ ground and alkalinized litter, GCA ¼ ground, coated, and alkalinized litter. Property

Treatment Litter

Alder (Alnus glutinosa) Aliphatics Polysaccharides Lignin Oak (Quercus robur) Aliphatics Polysaccharides Lignin Willow (Salix caprea) Aliphatics Polysaccharides Lignin

GC

GA

GCA

Before

After

Before

Excrements After

G Before

After

After

After

After

29.3 39.3 16.0

28.4 36.1 19.5

30.0 39.2 16.6

30.2 36.8 19.0

27.2 40.1 18.4

28.7 36.4 20.0

29.0 37.5 18.1

29.1 38.1 19.0

30.6 35.7 18.6

21.4 58.3 12.1

19.8 60.6 10.4

28.6 44.6 12.5

23.9 45.9 20.2

23.3 52.2 14.8

14.4 65.5 12.4

15.4 63.6 14.0

18.4 60.2 13.8

14.9 63.5 12.3

26.9 45.8 15.5

29.0 48.3 12.3

34.5 35.8 15.5

33.9 37.6 15.8

39.2 36.0 9.3

33.9 40.7 14.3

29.5 41.1 17.9

26.4 42.1 18.7

32.8 48.6 10.5

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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Table 3 Relative quantities of guaiacyl, syringyl, and hydroxymethyl in litter, treated litter, and excrements prepared from the same litter. Values are means (with SD below) and indicate the peak areas divided by mass of the sample based on TMAH-Py-GC MS. The litter was from alder, oak, and willow, and the excrements were obtained by feeding Bibio marci larvae with alder, oak, or willow as indicated. For treated litter: G ¼ litter that was ground; GC ¼ litter that was ground and coated; GA ¼ litter that was ground and alkalinized; and GCA ¼ litter that was ground, coated, and alkalinized. Data were collected before and after 54 weeks of decomposition for litter, excrements, and ground litter but only after 54 weeks of decomposition for the other litter treatments. For each litter type, means in a row followed by the same letter are not statistically different (one-way ANOVA, LSD, p > 0.05). Lignin component Treatment and time of determination Litter

Litter

Excrem.

Excrem.

G

G

GC

GA

GCA

Before

After

Before

After

Before

After

After

After

After

Alder (Alnus glutinosa) Guaiacyl 4976a Syringyl 3388c Hydroxymethyl 4344bc Willow (Salix caprea) Guaiacyl 6608d Syringyl 2377abc Hydroxymethyl 4508 Oak (Quercus robur) Guaiacyl 8450b Syringyl 3068b Hydroxymethyl 6296de

(558) (347) (830)

7163c (834) 2266b (220) 4929bcd (756)

4603a (531) 1652a (96) 2742a (256)

6942bc (420) 2103ab (60) 4672bcd (513)

6539bc (488) 2236ab (85) 4389bc (349)

6932bc (1491) 4718a (31) 2218ab (391) 1540ab (46) 5794d (991) 3936ab (208)

6664bc (261) 2140ab (93) 5379cd (263)

(1480) (395) (609)

6496cd (344) 5155abcd (492) 2157ab (475) 1410a (412) 3782 (409) 3248 (673)

4829abc (980) 5806bcd (216) 4835abcd (985) 4498ab (848) 5210abcd (490) 1443a (291) 3293c (593) 1834ab (739) 1826ab (595) 2806bc (629) 2881 (965) 4926c (1537) 5078 (2029) 2630 (380) 3078 (462)

(1328) (474) (828)

7659b (384) 3031b (77) 5504dc (234)

8461b (265) 3054b (30) 4059abc (500)

10533c (1678) 3823b (463) 4181abc (760)

8312b (516) 3140b (174) 7358d (786)

3864a (306) 1877a (412) 3563a (694)

3465a (34) 1797a (287) 4097ab (226)

5702ab (142) 1705ab (87) 4142ab (380) 3486a (1259) 1517a (140) 2346 (579)

7316b (937) 3232a (582) 4833c (1094) 1907a (240) 5118bcd (935) 4101ab (821)

Fig. 3. Ratios of guaiacyl to syringyl (G:S) and guaiacyl to hydroxymethyl (G:H) in litter (L), treated litter, and excrements (E). The litter was from alder, willow, and oak, and the excrements were obtained by feeding Bibio marci larvae with alder, willow, or oak litter as indicated. For treated litter: G ¼ litter that was ground; GC ¼ litter that was ground and coated; GA ¼ litter that was ground and alkalinized; and GCA ¼ litter that was ground, coated, and alkalinized. Data were collected before (be) and after (af) 54 weeks of decomposition for litter, excrements, and the GA treatment but only after 54 weeks of decomposition for G, GC, and GCA treatments. Values are means ± SD. For each litter species, bars with the same letter are not statistically different, and there were no significant differences with willow (one-way ANOVA, LSD, p > 0.05).

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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Table 4 Content of phenolic compounds (mg g1) in litter, treated litter, and excrements prepared from the same litter (alder, oak, or willow) before and after 54 weeks of decomposition. G ¼ ground litter, GC ¼ ground and coated litter, GA ¼ ground and alkalinized litter, GCA ¼ ground, coated, and alkalinized litter. Values are means (with SD below), statistically homogenous groups of CN values are marked by the same letter (one-way ANOVA, LSD, p > 0.05). Litter Before

Excrements After

Alder (Alnus glutinosa) 177.2a (2.5) 14.3ef (0.2) Willow (Salix caprea) 42.9c (2.0) 4.1klm (0.6) Oak (Quercus robur) 90.8b (6.2) 28.9d (0.3)

GA

G

GC

GA

GCA

After

After

After

Before

After

Before

After

14.6g (1.7)

10.8ghi (0.4)

11.8ghi (0.8)

13.5ghi (1.0)

7.0ijk (0.1)

10.4ghj (0.9)

10.4ghj (0.9)

12.1g (5.1)

0.4m (0.1)

3.9klm (0.9)

4.5jklm (1.6)

3.1efghklm (0.8)

3.9klm (1.7)

8.7gh (5.0)

8.5gh (0.6)

2.2lm (0.7)

8.7gh (2.0)

30.0d (0.5)

20.6ef (0.7)

19.1gh (0.8)

11.6ghijk (0.2)

Table 5 Correlation between total C loss from the system and the initial chemical properties of the litter based on absolute values and relative values. Relative values for C loss were calculated by dividing C loss in the given treatment by the C loss in untreated litter of the same species. Relative values for chemical properties were calculated by dividing the property's value for any given treatment by its value in untreated litter of the same species. Fragmentation refers to all litter treatments prepared by grinding and excrements fragmented by larvae. Significant correlation coefficients (p < 0.05) are in bold. Property

C:N Aliphatics N initial Polysaccharides Lignin G:S G:H Phenols Fragmentation

Correlation coefficients between C loss and chemical property based on absolute and relative values Absolute value

Relative value

0.334 0.250 0.164 0.258 0.112 0.024 ¡0.585 0.239 ¡0.526

0.518 0.425 0.068 0.482 0.040 0.037 ¡0.564 0.458 ¡0.526

4. Discussion In agreement with previous reports (Frouz et al., 1999; Frouz


ov and Simek, 2009; Kaneda et al., 2013; Spaldo n a and Frouz, 2014; Frouz et al., 2015), the results of this study show that excrements of a species of soil macrofauna (B. marci) tend to decompose more slowly than original litter from which the excrements were produced. This is consistent with the observation that excrements of litter-feeding macrofauna may accumulate in soil and form distinct layers in some soils (Ponge, 2003; Frouz et al., 2007). Excrements may also serve as food for earthworms and other invertebrates (Frouz et al., 1999, 2007), which makes the biology more complex. As shown by a recent meta-analysis of experiments with various enclosures (Frouz et al., 2015), this reduced decomposition rate for excrements could have a significant impact on C cycling in many ecosystems. Because of the complexity of the issue, however, more research is needed to evaluate the impact of this phenomenon on global C cycling. In the current research, we focused on mechanisms that may cause this slowing of excrement decomposition. The extent of reduction in microbial decomposition caused by conversion of litter to excrements differed among the litter species (Fig. 2). Moreover, all of the artificial litter modifications used in our study decreased the overall C loss for at least one litter type; however, the effect of individual treatments again differed among litter species. This suggests that several mechanisms contribute to the decrease in microbial respiration associated with excrements and that these contributions may vary with conditions. Based on

ova  and Frouz, 2014), removal of highly previous studies (Spaldo n available polysaccharides and the dominance of aliphatic and

lignin as well as changes in lignin structure may contribute to the process. The correlations between C loss and litter chemistry indicated that changes in lignin composition rather than overall lignin quantity affect C loss. More specifically, C loss was negatively correlated with the increase in the guaiacyl to hydroxymethyl ratio (G:H). Changes in lignin composition during faunal gut passage

ova  and Frouz (2014). Hydroxwere also observed by Spaldo n ymethyl is generally assumed to be a relatively decomposable part of lignin; therefore, it is possible that its lower content can make the material less decomposable. The mechanisms underlying this change in the G:H ratio during gut passage are unclear, but they may include the selective removal of more readily decomposed fractions of lignin as well as the condensation of tannins. Despite these correlations between decomposition rate and major components of lignin, we did not find any correlation between decomposition and the total amount of lignin, which differs from many other studies that found litter decomposition to be highly correlated with lignin content (Melillo et al., 1982; Rahman et al., 2013). Perhaps the range of lignin concentrations was not large enough in our data set, such that the quality of lignin appeared more important than the absolute amount. In contrast to Hopkins et al. (1998), we did not observe that lignin content was higher in B. marci excrements than in the original litter. Reduction in polysaccharide content can reduce decomposition in two ways. Polysaccharides are more easily decomposed than lignin, and reduction in their content can thus reduce the amount of easily decomposed substrate and thus the decomposition rate. This effect is likely to be more pronounced in early stages of decomposition. In later stages of decomposition, microorganisms may decompose some recalcitrant substances to obtain N. This decomposition of recalcitrant materials can be stimulated by addition of easily decomposed substrates, such as simple sugars or polysaccharides, a phenomenon termed the priming effect (Kuzyakov et al., 2000). In intact leaves, polysaccharides are stored in cells and are gradually made available as individual cells break open. This may provide easily available C and result in a local, internal priming effect. This internal priming effect resulting from the heterogeneity in litter decomposition at the cellular level may be absent in excrements because polysaccharides have been removed as a result of litter fragmentation. When litter is fragmented and passes through the gut of a soil invertebrate, a substantial portion of the carbohydrates is used by the invertebrate and or by microorganisms that grow rapidly in the posterior parts of invertebrate guts or in fresh excrements (Frouz et al., 2003). For litter with a high C:N ratio, the C:N ratio is lower in excrements than in the ingested litter. The mechanism for this C:N decrease is unclear. Gunnarsson and Tunlid (1986), who observed a similar decrease in C:N ratio, attributed it to the accumulation of products of microbial metabolism, namely cell walls of dead microorganisms. As stated by Wolters (2000), this may be associated

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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with a higher mortality of fungi than bacteria in the gut and the lower decomposability of fungal than bacterial residues (Saito, 1966). Another mechanism that may decrease the C:N ratio in excrements may involve interactions between proteins and phenolic compounds. After a plant cell dies, a substantial part of its proteins is bound in insoluble complexes with tannins and other phenolic compounds (Johnson and Felton, 1996). The stability of these complexes decreases in an alkaline environment (Johnson and Felton, 1996). That would help explain why many herbivorous and saprophagous invertebrates have highly alkaline guts (Johnson and Felton, 1996; Brune, 1998; Graça and B€ arlocher, 1999; Frouz et al., 2002), i.e., alkaline conditions help invertebrates utilize N bound in phenoleprotein complexes (Johnson and Felton, 1996; Ji and Brune, 2001, 2005). Because pH decreases in the posterior part of the gut or in excrements, however, insoluble phenoleprotein complexes reestablish and can again bind N. Frouz et al. (2011) showed that a significantly larger amount of added radio-labeled protein was bound in a humic acid fraction in B. marci excrements than in litter that was not eaten by the larvae. Rice (1982) found that during decomposition, a substantial part of proteins becomes bound to phenolics in humic-like substances. This idea is also supported by the observation that the content of phenolic compounds in litter decreased as a result of gut passage. Similarly, Coulis et al. (2009) observed that tannins dramatically decrease as a result of gut passage. These decreases in phenols or tannins during gut passage are probably not explained by leaching and are probably explained by the binding of phenols or tannins into larger molecular complexes. Hence, binding of proteins by phenolics may be another reason why N can be stored in excrements. We expect that phenoleprotein complexes bind more amino acids in excrements than in original litter because additional proteins may be available in the gut. In addition to protein in the litter, microbial protein released by the dying ingested microorganisms and residuals of invertebrate enzymes are available for binding. Moreover, the presence of protease in the digestive tract of the soil invertebrates is likely to result in the decomposition of proteins and an increase in the concentration of free amino acids. In litter, phenols and proteins are bound in individual cells and cell compartments. The crushing of cells during invertebrate feeding plus the action of enzymes, alkaline pH, and material mixing during gut passage may cause most available phenols and proteins to be released into the gut content. Consequently, a larger amount of proteins and phenols present in the gut material may participate in binding of phenol and amino acids and formation of humic-like complexes in the posterior part of the gut. Finally, the peroxidases that are widely distributed in the digestive tracts of invertebrates (Hartenstein, 1982) may favor humification (Schnitzer, 1976). Interestingly, laboratory conditions similar to those in the B. marci gut are used in the synthesis of model humic compounds similar to natural humic acids (Kappler et al., 2000). This indicates that soil fauna can make it difficult to detect simple correlations between phenolic content and decomposition. Phenolics are generally expected to slow microbial decomposition €ttenschwiler and Vitousek, 2000). In our study, however, a (Ha relative decrease in polyphenol content reduced the decomposition rate. As explained above, we suspect that the polyphenol decrease resulted from polyphenol condensation and N binding rather than from an absolute loss of phenolics from the system. The coexistence of two mechanisms that may presumably decrease microbial decomposition, a high concentration of polyphenols as well as polyphenol condensation (which decreases the polyphenol concentration), may explain why polyphenol concentration per se was not correlated with decomposition. A low C:N ratio may slow decomposition by two possible mechanisms. First, a low C:N ratio may indicate a high availability

7

of N, which may decrease the need for microbial mining of N (Moorhead and Sinsabaugh, 2006; Craine et al., 2007) and may consequently slow the later stages of decomposition. Second, most of the N that remains in feces may become unavailable, as observed by (Gunnarsson and Tunlid, 1986), and this may slow decomposition. Reduced availability of N may result from binding of protein to phenol (Johnson and Felton, 1996; Ji and Brune, 2001, 2005) as explained above. It is even possible that both of these mechanisms may apply but may be important at different times. In early stages of decomposition, when easily available resources are preferentially used, binding of proteins in protein-phenol complexes reduces N availability and hence microbial decomposition. In later stages of decomposition, when the microbial community increases its N mining, less material needs to be “mined” (decomposed) because of the low C:N ratio. In our study, the increase in the aliphatic fraction in excrements agrees with the data of Hopkins et al. (1998). Although aliphatics were only marginally correlated with decomposition in our data set, aliphatic components are assumed to be generally less decomposable than other components in litter (Lützow et al., 2006), and their accumulation in excrements may thus slow decomposition. An increase in aliphatic components may be caused by the low decomposability of plant waxy substances in the gut, which may consequently accumulate in the excrements. Another mechanism that may contribute to the increased proportion of aliphatics in excrements is the accumulation of microbial cell walls (Gunnarsson and Tunlid, 1986). Regarding the artificial litter treatments, grinding may increase microbial access to plant cell contents, including polysaccharides, and thus increase microbial biomass, which may in turn increase aliphatic components as mentioned above. Grinding can also accelerate leaching of tannins and other phenolics. Researchers previously observed that microbial respiration is lower in ground than in unground oak litter (Kaneda et al., 2013). This has been explained by the leaching of phenolics from ground litter that may directly slow microbial activity. Phenolics may also condense with the available protein and slow decomposition in the long term. This may explain why grinding of litter greatly reduced the overall rate of oak litter decomposition (Fig. 2). Interestingly, grinding with coating caused the largest decrease in the rate of decomposition of oak litter. In this case, coating may have slowed the leaching of released phenolics, which could then condense with available protein and thereby directly reduce microbial activity. Coating by clay is a major consequence of litter passage through earthworms, which ingest large amounts of mineral soil and may isolate fine organic particles within soil particles; such isolation slows the diffusion into and from the organic particles. This may reduce leaching of wastes of microbial metabolism from the fragment or limit the input of some nutrients from the surroundings, which may finally slow the rate of decomposition (Six et al., 1998; Zhang et al., 2003). Litter-feeding macrofauna may ingest some mineral soil with litter but much less than earthworms. Whereas the fragments of ground litter in this study were placed loosely in litter bags, macrofauna excrements are often compacted in distinct pellets and may be surrounded by a residue of peritrophic membrane (Tajovský et al., 1991; Frouz et al., 2002). These pellets are quite persistent and accumulate in soil; in some forest soils, they may even form a distinct layer (Ponge, 2003; Frouz et al., 2007). Compaction in pellets may have a similar effect as coating on diffusion and clearly warrants future investigation. The alkalinization of litter caused the greatest reduction in C loss from litter, i.e., alkalinization stabilized the organic matter. We propose a conceptual model summarizing how temporary alkalinization of litter followed by a shift back to neutral or slightly

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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acidic pH in excrements may contribute to organic matter stabilization (Fig. 4). We expect that alkaline conditions in the gut contribute to the solubilization of complexes between proteins and tannins and or other phenolics and to the release of N from these €rlocher, 1999; Ji complexes (Johnson and Felton, 1996; Graça and Ba and Brune, 2001, 2005). Alkaline conditions in the B. marci gut help kill ingested bacteria (Frouz et al., 2003; Oravecz et al., 2004), which may be partly digested by the larva. pH decreases in the posterior parts of the gut and in excrements, such that bacterial biomass increases again (Frouz et al., 2003; Oravecz et al., 2004). This may i) increase the amount of aliphatics, ii) promote the use of remaining available organic matter in excrements, and iii) possibly bind N in difficult to decompose residues as proposed by Gunnarsson and Tunlid (1986). Because the stability of phenoleprotein complexes increases with decreasing pH (Johnson and Felton, 1996), a decrease in pH may cause condensation of phenols into complexes with proteins released from litter and also with proteins from killed but unassimilated microflora. This may contribute to the decrease

in C:N ratio and at the same time transfer N into hardly decomposable substances. Alkaline conditions together with digestive enzymes that have alkaline pH optima break down polysaccharides, which can then be digested by the larva (Frouz et al.,
2002; Sustr and Frouz, 2002). This decrease in polysaccharide content in excrements was observed here but also in another study (Hopkins et al., 1998). During gut passage, the proportion of hydroxymethyl to other, more resistant parts of the lignin decreases. The effect of alkaline treatment on lignin composition is unclear and should be studied. In conclusion, our results suggest that short-term alkalinization, litter fragmentation, and the action of digestive enzymes explain the lower rate of decomposition for excrements than for the original litter. More specifically, they cause depletion of polysaccharides, condensation of polyphenols, and binding of N. These changes result in a decrease in the C:N ratio and an increase in aliphatic components, which correspond with the reduced rate of decomposition for excrements (Fig. 4).

Fig. 4. Conceptual model of the major mechanisms that may cause stabilization of organic matter after passage through the gut of larvae of the soil macroarthropod Bibio marci. The drawing of gut morphology and the values of gut pH are according to Frouz et al. (2003).

Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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Acknowledgment This study was supported by a grant from the Czech Science Foundation GAP504/12/1288. We thank Dr. B. Jaffee (JaffeeRevises, Davis CA, USA) for reading the manuscript and for language cor is thanked for technical assistance. rections. Jitka Huba
cova

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Please cite this article in press as: Frouz, J., et al., Major mechanisms contributing to the macrofauna-mediated slow down of litter decomposition, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.08.024

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