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Properties of dissolved organic matter derived from silver birch and Norway spruce stands: Degradability combined with chemical characteristics
Soil Biology & Biochemistry 43 (2011) 421e430
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Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Properties of dissolved organic matter derived from silver birch and Norway spruce stands: Degradability combined with chemical characteristics Oili Kiikkilä*, Veikko Kitunen, Aino Smolander Finnish Forest Research Institute, Vantaa Research Center, P.O. Box 18, FIN 01301 Vantaa, Finland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 July 2010 Received in revised form 4 November 2010 Accepted 9 November 2010 Available online 23 November 2010
Dissolved organic matter (DOM) derived from the humus layer under silver birch (Betula pendula Roth), Norway spruce (Picea abies (L.) Karst.), and mixed stands, and from senescent birch leaves and from spruce needles of the four oldest year-growth were characterized microbiologically and chemically. Samples were collected in the autumn and the solutions were obtained by centrifugation-drainage technique. The degradability of DOM, the availability of DOM to bacteria and fungi, concentrations of phenolic compounds and carbohydrates, and the distribution of carbon and nitrogen into fractions according to the chemical nature and the molecular size were studied. DOM derived from leaves and needles was clearly more labile than DOM derived from the humus layer indicating the importance of studying the DOM originating from fresh litter when assessing the turnover of DOM. DOM derived from spruce needles appeared to differ chemically greatly from all other samples. It had very high concentrations of carbohydrates, probably due to the sampling time, and phenolic compounds. The chemical composition of DOM derived from humus layer did not reflect the composition of DOM derived from needles and leaves. DOM derived from birch leaves degraded more than DOM derived from spruce needles and DOM derived from humus layer collected at the birch sites degraded more than DOM derived from humus layer collected at the spruce sites. The degradability of different compound groups of DOC and DON was studied in a short-term incubation (20 d) of DOM solutions by characterizing the solutions initially and after the incubation. Almost all compound groups appeared to degrade but weak hydrophobic acids, bases, hydrophilic neutrals, the smallest molecular size compounds, carbohydrates, and phenolic compounds degraded the most. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: DOC DON Degradation Chemical composition Molecular size distribution Tree species
1. Introduction The quality and quantity of DOM should play a key role in the dynamics of C and N in forest soils because dissolved organic matter (DOM) may be the most important energy and nutrient source for microorganisms (Marchner and Kalbitz, 2003). The quality of DOM in forest soils is affected by tree species. In general conifer litter contains more recalcitrant, hydrophobic, aromatic compounds, whereas deciduous litter contains more labile, hydrophilic, low molecular weight compounds (Hongve et al., 2000; Kaiser et al., 2001, 2002; Kalbitz et al., 2003a). In most studies, it seems that despite significant differences in the composition of the source materials, the DOM chemistry is relatively similar in decomposed material (Smolander and Kitunen, 2002; Yano et al., 2005; Traversa et al., 2008). One of the main
* Corresponding author. Tel.: þ358 45 638 5254, þ358 10 211 2450. E-mail address: oili.kiikkila@metla.fi (O. Kiikkilä). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.11.011
problems in studying DOM is that, in practice, a great part of DOM is turned over so quickly (van Hees et al., 2005; Boddy et al., 2007) that it is never measured. The fast turnover of labile compounds can explain why DOC chemistry in soil solution has not been reflective of DOC chemistry in the litter layer (Kiikkilä et al., 2006; Wickland et al., 2007) and CO2 production has been higher than the pool of carbon (Bengtson and Bengtsson, 2007). More recalcitrant compounds accumulate in the soil solution where labile compounds have been consumed (Qualls et al., 2002; Hagedorn et al., 2004; Wickland et al., 2007; Sanderman et al., 2008). DOM in soil is generated by different pathways: it may originate from fresh litter, older humified organic material, root exudates and microorganisms. The proportional contribution of these DOC sources to soils is, however, unclear. It has been suggested that labile carbon pools, typically including root and leaf litter (Park et al., 2002; Uselman et al., 2009; Hansson et al., 2010) and root exudates (Yano et al., 2000, 2005; Giesler et al., 2007; Kramer et al., 2010), are considerable sources of DOM in soil, and it has been emphasized that they are also important sources for microbial
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activities in soil (van Hees et al., 2005). In other studies, fresh litter, throughfall or root exudates have made minor contributions to DOC in soil whereas the greatest part of DOM have been suggested to originate from the older organic matter in organic soil horizons (Park and Matzner, 2003; Hagedorn et al., 2004; Kalbitz et al., 2007; Fröberg et al., 2007, 2009; Müller et al., 2009). However, increased DOM production from organic soil horizons has been suggested as being a consequence of increased microbial activities that were stimulated by labile compounds delivering from fresh litter (Kalbitz et al., 2007). Thus studying the quality of DOM derived from fresh litter, instead of older organic matter, the litter layer, or humus layer, may increase the understanding of dynamics of C and N in forest soils. The aim of the study was to characterize DOM derived from the humus layer under silver birch and Norway spruce and respective leaves and needles. In order to measure the DOM originating from senescent litter as fresh as possible, without loosing labile compounds, we collected leaves and needles directly from the trees and compared to humus layer samples. We characterized DOM according to the molecular size distribution and the chemical nature i.e. adsorption properties. In order to assess the turnover of DOM in forest soil we measured the degradability of DOM and the availability of DOM to bacteria and fungi. The aim was also to assess the degradability of different compound groups of DOC and DON. For that purpose we characterized DOM before and after a shortterm incubation. 2. Materials and methods 2.1. Study site and sampling The study site was a replicated silver birch-Norway spruce (Betula pendula RothePicea abies (L.) Karst.) experiment in Eno, central-eastern Finland described earlier by Smolander et al. (2005). Briefly, the soil was a podsol and humus type mor. The site type was Vaccinium myrtillus type (Cajander, 1949). The study site had originally been a birch stand, which had been clear-cut in 1964. On spruce plots, 3e4-year-old Norway spruce seedlings were planted in autumn 1964, and 2-year-old silver birch seedlings were planted on birch plots in spring 1965. The birch plots were thinned in autumn 1985. The size of the plots was 40 m 40 m. Of the whole field experiment, which covered a total area of 6.7 ha, only part of the plots were used in this study; three adjacent plots of both tree species and two mixed species plots were selected. Birch plots were single-species stands and, of the total stem number, the spruce plots, still regarded as spruce plots, contained 1.6% and 7% birch. The mixed plots were mainly spruce, containing 22% and 37% birch of total stem number. Stand characteristics of birch and spruce plots were determined in 2002 and were presented in detail by Smolander et al. (2005). On birch plots, the understorey vegetation (under 3 m tall) consisted mainly of grey alder (Alnus incana) and rowan (Sorbus aucuparia), whereas on spruce plots the understorey was very sparse. Ground vegetation in the stands clearly differed: herbs and grasses were abundant in birch plots while the spruce plots were covered almost solely by moss and a layer of needle litter. The ground vegetation of mixed plots resembled that of spruce plots. Samples were collected in September 2007 during the senescence of birch. Composite samples (20e25 cores, core diameter 58 mm) were taken systematically from the humus layer (F þ H) of all plots, 8 altogether. After green plant material was removed, the samples were sieved through a 4.0 mm mesh and stored in plastic bags at 4 C. Leaves and needles were collected from birch and spruce plots, 5 trees per sample plot. Leaf litter was collected from birch trees by
shaking. A great part of the leaves were yellow, but some were still green. Leaves were air dried at room temperature in a laboratory for two days. Leaves were cut into ca. 10 mm2 pieces and stored in 4 C. At the same time, the lowest branches of spruce were taken into a laboratory. The branches were air dried for two days, needles were separated and crushed lightly using a mortar and stored in 4 C. The needles consisted of the four oldest annual growth and were mostly green. 2.2. Collection of DOM To obtain the DOM solution, the centrifugation-drainage technique described by Giesler and Lundstöm (1993) was used. Samples were centrifuged at 5000 g for 20 min. Humus layer samples were adjusted to 100% water holding capacity (WHC) by ultrapure water and allowed to stand overnight at 4 C before centrifugation. To 10 g dry weight of leaves and needles 100 ml of ultrapure water was added and allowed to stand overnight at 4 C before centrifugation. Finally the solutions were filtered through a 0.45 mm polyethersulfone membrane and stored in a freezer until further analysis. After thawing the solutions were kept at 4 C for 3e10 days in order to allow them to stabilize chemically. 2.3. Determination of the properties of DOM The molecular size (MS) distribution of DOC and DON was determined by tangential ultrafiltration apparatus (MinimateÔ TFF, Pall Corporation). After thawing the solutions were filtered through 0.45 mm. Membranes with nominal weight cut-offs at 100, 10 and 1 kD were used. The solution (ca. 25 mg l1 C), 210 ml, was filtered through 100 kD membrane and concentrated to 40 ml. This retentate, i.e. the solution that was left in the container, was collected as a sample >100 kD. Next, the filtered solution (<100 kD), 170 ml, was filtered through 10 kD and concentrated to 90 ml (sample 10e100 kD). The filtered solution (<10 kD), 80 ml, was then filtered through 1 kD membrane and concentrated to 40 ml (sample 1e10 kD). The sample <1 kD was the final filtrate through 1 kD membrane. The diafiltration procedure was not performed because in pre-testing it was found to increase remarkably the loss of DOC from samples. Instead, it was observed that the concentration procedure was fairly steady with the used volumes. Thus the sample of 1e10 kD was assumed to include smaller (<1 kD) compounds in the same concentration as the sample of <1 kD and the concentration of DOC or DON in <1 kD sample was subtracted from 1e10 kD sample. The same calculation, i.e. the concentration of the smaller sample was subtracted from the larger, was made with 10e100 kD and >100 kD samples. The DOC and DON of solutions were fractionated into compound groups according to their chemical nature i.e. adsorption properties using the procedure described by Aiken and Leenheer (1993) and Smolander and Kitunen (2002). The compound groups were hydrophilic acids (phiA), bases (phiB) and neutrals (phiN), weak hydrophobic acids (weak phoA), hydrophobic acids (phoA), bases (phoB) and neutrals (phoN). XAD-8 (Amberlite XAD-8, Merck), MSC-1 cation-exchange (Sigma Chemical Co.) and AG-MP1 anionexchange (BioRad Laboratories) resin columns were used. The concentrations of carbohydrates and phenolic compounds in DOM were measured spectrophotometrically. The concentration of carbohydrates was measured using anthrone reagent (Brink et al., 1960) and the concentration of total phenolic compounds was measured with the FolineCiocalteu method, as described in Suominen et al. (2003). The pH was measured from the DOM solution where the DOC concentration was 25 mg l1. To assess the availability of DOM to bacteria (Tdr) the 3 H-thymidine incorporation technique (Bååth et al., 2001) modified
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to DOM studies by Kiikkilä et al. (2005) was applied. A bacterial inoculum was prepared as follows: 3 g fresh weight of humus (a mixture of birch and spruce humus layer) was mixed with 100 ml of water and shaken (200 rpm) for 1 h, followed by low-speed centrifugation (750 g for 10 min) and filtration through quartz wool. The bacterial suspension (5 ml) was then mixed with DOM solution, which had ca. 30 mg l1 DOC, (10 ml) or water (control). Thymidine incorporation rate, i.e. the bacterial growth rate, was measured after the solution was pre-incubated at 20 C for 24 h. An aliquot (3.5 ml, 0.1 MBq) of methyl-3H-thymidine (740 GBq mmol1, Moravek Biochemicals) was added and the samples were incubated for 2 h. Washing to remove excess tracer, and measurement of radioactivity were described in detail by Bååth et al. (2001). Availability of DOM to fungi (Ac-erg) was assessed using the 14 C-acetate-in-ergosterol technique (Bååth, 2001) modified by Kiikkilä et al. (2006). A piece of humus (a mixture of birch and spruce humus layer, 0.03 g fresh weight) was added as fungal inoculum to 30 ml of DOM solution (ca. 10 mg l1 DOC), or water (control) in Erlenmeyer flasks. Streptomycin and ampicillin were added in order to diminish the bacterial growth in solutions. The pH was adjusted to 4.5 in order to give fungi favourable growing conditions (Rousk et al., 2010). The bottles were pre-incubated in darkness at 20 C. After 24 h the solutions were filtered through Whatman GF/D glass fiber filter. The soil and the filter were transferred to a test tube with 1.5 ml of the DOM solution. In order to measure the fungal growth rate, 0.13 mmol (0.3 MBq) of 14Cacetate solution (1-14C-acetic acid, sodium salt, 2.2 GBq mmol1, Moravek Biochemicals) and 0.35 mmol of 1 mM non-radioactive acetate were added. After incubating the mixture for 20 h at 20 C, formalin was added, the test tubes were centrifuged, and the supernatant discarded. The ergosterol was then extracted as in Bååth (2001) and measured with HPLC (Hitachi, Merck) and 14Cergosterol with the HPLC radioactivity monitor (Berthold, LB 506 C-1). The relative availability of a DOM solution to bacteria or fungi was calculated by dividing the incorporation rate of the DOM solution by the incorporation rate of the control solution. The mineralization rate of DOC was measured in a long-term incubation (110 days). For microbial inoculum the mixture of birch and spruce humus layer (10 g fresh weight) was shaken (200 rpm, 1 h) in 100 ml water. 100 ml of this soil suspension was added to 30 ml of DOM solution (25 2.3 mg l1 DOC, mean SD, n ¼ 14), which was filtered through 0.2 mm in 120 ml glass bottles. To assess also the potential mineralization rate of DOC, nutrients were added to half of the bottles, to give a final concentration of 0.1 mM NH4NO3 and 0.1 mM K2HPO4. The solutions were shaken (120 rpm) in the dark at 20 C and the CO2 evolution was measured by gas chromatography every other day and finally every two weeks. After CO2 measurements, the bottles were aerated. CO2eC loss was calculated, and the proportion of labile DOC and the half-life of labile DOC and were estimated (see data analysis). 2.4. Assessing the degradability of compound groups of DOM In addition to the long-term incubation, we made a separate short-term incubation in order to study the degradation of different compound groups of DOM. The solutions were filtered through 0.2 mm and diluted to a concentration of 25.6 2.3 mg l1 DOC (mean SD, n ¼ 14). The initial solutions were frozen until further analysis. The solutions were placed in sterile 3 l Erlenmeyer flasks, 650 ml in each flask, in two replicates. To provide extra surface area for microbial growth, a glass fiber filter (GF/D) was added to each flask. An inoculum was prepared from the humus layers as described above and added 1 ml l1. Control solutions, without an inoculum, were prepared of four randomly chosen solutions in order to detect the possible precipitation of DOM. The flasks were
423
kept in the dark at 20 C and gently shaken by hand daily. After 20 days the two replicates were combined, the solutions were filtered (0.2 mm) and frozen until further analysis. The concentration of DOC and DON was determined as described in Smolander and Kitunen (2002) initially and after the incubation. The proportional loss of DOC (DOC loss%) and DON (DON loss%) during 20-days incubation was calculated as difference between initial and incubated solutions subtracted by the mean loss of DOC (8.7%) and DON (10.0%) in control samples. The concentration of bioavailable DON was calculated as the actual loss of DON in incubation solutions equalized to C concentration. The relative availability of DOM to bacteria and fungi, concentration of carbohydrates and phenolic compounds and the distribution of DOC and DON according to chemical nature and molecular size were determined from initial and incubated solutions as described above. 2.5. Data analysis To normalise the distribution of the variables necessary logarithmic transformations were made. The statistical significance for all tests was set at p < 0.05. One-way ANOVA was used to determine the differences in the properties of DOM between birch and spruce. Mixed plots were not included in ANOVA, because n ¼ 2. Pearson and partial correlations were calculated. The mineralization rate of DOC was analyzed by fitting a double exponential model (Kalbitz et al., 2003a) to the data of the longterm incubation estimating an easily degradable (labile) and a more stable pool of DOC. Mineralized DOC ¼ (100-s)(1-eklt) þ s(1-ekst) where t ¼ time (days); following estimated parameters (100-s) ¼ the part of DOC that is rapidly mineralizable ¼ labile DOC (%); s ¼ the part of DOC that is slowly mineralizable ¼ stable DOC (%); kl ¼ mineralization rate constant of labile DOC (day1); ks ¼ mineralization rate constant of stable DOC (day1). The curves were fitted using the sequential quadratic programming algorithm. We calculated the half-life of the labile DOC ¼ ln2/kl (days). The degradation of different compounds and compound groups i.e. changes in the short-term incubation was analyzed by three separate principal component analyses (PCA) using correlation matrix: 1) The concentrations of DOC, DON, carbohydrates and phenolic compounds and the relative availability of DOM to bacteria and fungi, 2) DOC and DON divided into fractions according to the chemical nature, 3) molecular size distribution of DOC and DON. In the datasets of chemical fractions and molecular size classes the total initial concentration of carbon and nitrogen have been calculated to equal 100 mg l1 and the incubated values to decrease from that concentration. The variables most responsible for the ordination of samples are described as vectors (loadings). Separate variables were subjected to a paired samples t-test in order to detect significant differences between initial and incubated samples. 3. Results 3.1. Properties of DOM The concentrations of compounds were calculated to equal the concentration of 100 mg l1 DOC in the solution. Chemical and microbiological properties of DOM derived from humus layer (humus DOM) did not differ much between birch, spruce and mixed plots whereas many large differences were detected in DOM derived from birch leaves and spruce needles (plant DOM). The pH
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of spruce DOM was lower than that of birch DOM in both humus and plant material (Table 1). The concentration of inorganic N (mostly NH4) was very low in plant DOM whereas in humus DOM it was higher for birch than for spruce. The concentration of DON was higher and thus the DOC-to-DON ratio was lower for birch than for spruce in both humus and plant DOM (Table 1). The concentrations of carbohydrates and phenolic compounds did not differ significantly in humus DOM but were much higher for spruce needle than for birch leaf DOM (Fig. 1a,c). The chemical nature (Fig. 2) and the molecular size (MS) distribution (Fig. 3) of compounds did not differ much between birch, spruce and mixed plots in humus DOM. In plant DOM, spruce had clearly higher concentrations of C in weak phoA and phiN fractions and in small MS compounds (<1 kD) than birch. Instead, spruce had lower concentrations of C in phoA and large MS compounds (>100 kD) than birch. Most of plant DON was in phoA, phiB and phiN although spruce needle had no N in phoA. DON derived from birch leaves was biased towards large MS and that of spruce needles towards small MS compounds (Fig. 3). The degradability of DOC and DON i.e. the proportional losses of DOC and DON in the short-term incubation (20 days) were clearly higher for plant than for humus DOM (Table 2). DOC loss% was significantly higher for birch than for spruce in both humus and plant DOM (Table 2) whereas the DON loss% did not differ significantly between birch and spruce. The concentration of bioavailable DON in the incubation solution was significantly higher for birch than for spruce. The mineralization rate of DOC derived from the long-term incubation did not differ significantly between the tree species (Fig. 4). The potential mineralization rate of DOC (NPK added solutions) was higher for spruce than for birch in both humus and plant DOM. For plant DOM the variables calculated from the degradation curve (labile DOC and the half-life of labile DOC) (Fig. 4) indicated the same; there were no significant differences without NPK addition whereas in NPK addition solutions spruce had a higher proportion of labile DOC than birch, and the half-life of labile DOC was shorter for spruce than for birch.
Table 1 Means (SD, n ¼ 3) of pH, concentration of inorganic N, and DON, and DOC-to-DON ratio in DOM solutions derived from birch leaves, spruce needles and humus layer of birch, spruce and mixed plots.a Humus
Plant
Birch Spruce Mixedb
pH 5.7 (0.2)a 5.0 (0.1)b 5.1 (0.1)
6.8 (0.1)a 4.5 (0.1)b
Birch Spruce Mixedb
Inorganic N, mg l1 3.6 (1.4)a 0.7 (0.6)b 1.3 (0.5)
0.2 (0.1) 0.2 (0.3)
Birch Spruce Mixedb
DON, mg l1 4.3 (0.1)a 3.1 (0.3)b 3.1 (0.1)
2.5 (0.2)a 0.6 (0.2)b
Birch Spruce Mixedb
DOC-to-DON ratio 23 (1)a 31 (3)b 32 (1)
41 (3)a 185 (55)b
The different letters (ANOVA) indicate significant differences between birch and spruce. a Results are calculated in solution where the concentration of DOC would initially be 100 mg l1. b n ¼ 2, not included in ANOVA.
The relative availability of humus DOM to bacteria (Tdr) and fungi (Ac-erg) did not differ much between the tree species. In plant DOM Tdr was higher for birch than for spruce whereas Ac-erg did not differ much between birch and spruce (Fig. 5). Mixed plots were not included in ANOVA (n ¼ 2). Concentrations of inorganic N and DON, the DOC-to-DON ratio and pH in humus DOM from mixed plots seemed to be nearer spruce than birch (Table 1). DOC loss%, DON loss%, bioavailable DON, carbohydrates, phenolic compounds, fractions according to chemical nature, mineralization rate of DOC, Tdr or Ac-erg in humus DOM from mixed plots seemed not to differ from either spruce or birch (Table 2, Figs. 1, 2 and 4e6c). The molecular size distribution of compounds for mixed humus DOM seemed to differ slightly from both spruce and birch (Figs. 3 and 6e). 3.2. Degradability of compound groups of DOM The degradation of compound groups of DOM i.e. changes during the short-term incubation were analyzed with three separate PCA analyses. The variance explained by PC 1 and PC 2 was 66, 63 and 77% for the first, second and third PCA (Fig. 6), respectively. Figures are rotated so that the initial and incubated samples of spruce needle DOM (mean) separate along x-axis. Also all other initial and incubated samples separate along the x-axis (Fig. 6a,c,e) and in addition they (except spruce needle DOM) separate slightly also along the y-axis. Thus both x and y-axis indicate changes during the incubation. The change is larger in plant than humus DOM since in all Fig. (6a,c,d) the distances between initial and incubated samples of plant DOM are longer than those of humus DOM. The variables that have the loading value 0.5 have been drawn as vectors in the Fig. 6b,d,f. The variables that have high values on the x-axis decrease in all samples. The variables that that have high values only on the y-axis decrease in all but spruce needle DOM samples. High negative values on the y-axis do not refer to the degradability of compounds but the variables in question have clearly higher values either in spruce or litter samples compared to birch and humus samples, respectively. Both total DOC and DON degraded in the incubation (6a,b) (proportional losses are presented in Section 3.1.). Carbohydrates and phenolic compounds degraded (Fig. 6a,b), and Tdr and Ac-erg decreased in the incubation. These were supported by the paired t-tests on separate variables. Tdr (Fig. 5) and the concentration of carbohydrates (Fig. 1) decreased significantly in all treatments. The concentration of phenolic compounds decreased significantly in plant DOM (Fig. 1). Ac-erg decreased significantly only for birch plant DOM (Fig. 5). PCA (Fig. 6d) of the chemical nature of compounds indicates that C and N in PhiN, weak phoA, phiB and phoB fractions degraded. The vectors of C and N in phoA have high loading on the y-axis and negative loading on the x-axis indicating that phoA degraded in all except spruce needle DOM, where it increased (Fig. 6d). PCA of MS distribution (Fig. 6f) indicates that all size classes degraded. Only C and N > 100 kD, of which vectors have loading near zero on the xaxis, did not degrade in spruce needle DOM. The actual values for the decrease in incubation support the results of PCAs, but are not presented because many of the differences were not significant in the paired t-test. 3.3. Correlation between compound groups Correlations between the concentrations of C in weak phoA and phiN, phenolic compounds, and carbohydrates were calculated. Because phenolic compounds and carbohydrates correlated strongly positively, the partial correlation was performed. When
O. Kiikkilä et al. / Soil Biology & Biochemistry 43 (2011) 421e430
a
b
c
d
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Fig. 1. Concentrations of a) carbohydrates and c) phenolic compounds in DOM derived from birch, spruce and mixed humus and from needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The values are calculated to equal the solution 100 mg l1 DOC initially. The different letters (ANOVA) indicate significant differences between birch and spruce. The respective values after the 20-days incubation and significant differences between initial and incubated values are indicated by * (paired samples t-test) in the figures b and d.
phenolic compounds were kept as a controlling variable, carbohydrates and phiN correlated positively (r ¼ 0.78, n ¼ 25). Instead, phenolic compounds and weak phoA did not correlate when carbohydrates were kept as a controlling variable. 4. Discussion DOM originating from fresh litter seems to belong to a great extent to the labile part of the DOM (Kalbitz et al., 2003a; Hagedorn et al., 2004; Kiikkilä et al., 2006; Müller et al., 2009). In our study, DOM derived from needles and leaves was clearly more degradable than DOM derived from humus layer. Plant DOM had at least twofold mineralization rate of DOC in 110 days incubation and also the loss of DOC in 20-days incubation was much higher compared to humus DOM. The degradability of the most labile DOM, which was assessed with the availability of DOM to bacteria (Tdr) and fungi (Ac-erg), was over 30% higher for plant DOM than humus DOM. The most labile DOM can stimulate microbial activities and further DOM production in organic horizon as suggested by Kalbitz et al. (2007). Microbial activities of soil have often had a positive
relation to the concentration of DOC and DON (Marchner and Kalbitz, 2003; Neff and Hooper, 2002; Moore et al., 2008; Smolander and Kitunen, submitted for publication). More important than the concentration may be the degradability and thus the turnover of DOM. Earlier at the same sites higher microbial activities in humus layer have been detected under birch than under spruce (Smolander et al., 2005). Thus it seems that microbial activities and the degradability of DOM have a positive relationship at this site. However, more data is needed in order to assess the relationship between the microbial activities of soil and the degradability of DOM in general. As a conclusion, in assessing the properties and turnover of DOM in soil it seems essential also to characterize the DOM derived from fresh litter although it seems to make a minor contribution to the total steady state concentration of DOM in the humus layer. Measuring the steady state concentrations of DOM alone does not detect the actual turnover of labile compounds. Also the chemical measurements indicated that labile compounds were in general more abundant in the litter layer DOM (Kiikkilä et al., 2006) and in plant DOM (Fig. 6) than in humus DOM. The most degradable compound groups of DOM, which have also
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a
b
Fig. 2. Distribution of the chemical fractions of a) DOC and b) DON in birch, spruce and mixed humus and in needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The values are calculated to equal the solution 100 mg l1 DOC (note the different scales in humus and plant DON). Weak hydrophobic acids (W PhoA), hydrophobic bases (PhoB), acids (PhoA) and neutrals (PhoN), and hydrophilic bases (PhiB), acids (PhiA) and neutrals (PhiN). The different letters (ANOVA) indicate significant differences between birch and spruce.
earlier been reported to be labile (Qualls and Haines, 1992; Kalbitz et al., 2003b; Qualls, 2005; Wickland et al., 2007), were carbohydrates, phenolic compounds, phiN, weak phoA, nitrogen containing compounds (bases) and smaller MS compounds. These compounds decreased in the short-term incubation and their concentrations were positively related to the DOC loss and the availability of DOM to bacteria and fungi (Fig. 6). DOM, either a degradation product or leached from litter, is degraded or stabilized by sorption to the organic and mineral soil layers. More stable compounds seem to adsorb preferentially,
leaving the more easily degradable compounds in solution (Kalbitz et al., 2005; Hunt et al., 2008; Schneider et al., 2010). Kalbitz and Kaiser (2008) estimated that 19%e50% of soil total C derives from DOM. Thus especially refractory DOM significantly contributes to the accumulation of C in soil. DOM derived from birch stands seemed to be more degradable than DOM derived from spruce stands whereas mixed plots resembled more spruce than birch. Birch DOM, originating either from plant material or humus, had a higher loss of DOC and DON during the 20-days incubation than the respective spruce DOM. The
a
b
Fig. 3. Distribution of the molecular size classes of a) DOC and b) DON in birch, spruce and mixed humus and from needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The values are calculated to the solution 100 mg l1 DOC (note the different scales in humus and plant DON). The different letters (ANOVA) indicate significant differences between birch and spruce.
O. Kiikkilä et al. / Soil Biology & Biochemistry 43 (2011) 421e430 Table 2 Means (SD, n ¼ 3) of loss of DOC and DON during a 20-day incubation of DOM derived from birch leaves, spruce needles and humus layer of birch, spruce and mixed plots. Humus
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a
b
c
d
Plant
DOC loss% Birch Spruce Mixeda
14.6 (1.3)a 9.2 (2.8)b 11.5 (7.8)
Birch Spruce Mixeda
DON loss% 21.1 (6.0) 8.5 (6.4) 11.4 (13.9)
Birch Spruce Mixeda
Bioavailable DON, mg l1b 0.9 (0.3)a 0.3 (0.2)b 0.4 (0.4)
48.3 (3.5)a 40.4 (5.7)b
43.3 (8.6) 32.7 (17.9)
1.1 (0.2)a 0.2 (0.2)b
The different letters (ANOVA) indicate significant differences between birch and spruce. a Not included in ANOVA (n ¼ 2). b Calculated DON loss in solution where the concentration of DOC would initially be 100 mg l1.
degradability of the most labile DOM, measured as availability of DOM to bacteria, was also higher for birch than for spruce. However, when the mineralization rate of DOC was followed by CO2 evolution in the long-term incubation, only very small differences between tree species were detected. We cannot exclude the possible effect of different pH; it was higher for birch than for spruce especially in plant DOM. Therefore it is possible that birch DOM gave too low CO2 values due to the higher dissolved CO2. Phenolic compounds left in spruce needle DOM after the shortterm incubation had perhaps degraded in a longer run and increased the mineralization rate of spruce needle DOC in the longterm incubation, although this is neither supported by the CO2 evolution curve at the moment of 20 days nor the estimated proportion of the labile compounds (Fig. 4). DOM derived from the green spruce needles also had a high concentration of carbohydrates that were labile, and would have been less abundant in senescent needles. We suggest that the degradability of senescent spruce needle DOM would be lower than senescent birch leaf DOM. Nutrient addition increased the mineralization rate of spruce DOC clearly over birch. This is not surprising because the N content was very low in spruce DOM and probably a limiting factor for
Fig. 5. The relative availability of DOM to a) bacteria (Tdr) and c) fungi (Ac-erg) in birch, spruce and mixed humus and in needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The different letters (ANOVA) indicate significant differences between birch and spruce. The respective values after the 20-days incubation are presented in figures b and d. * indicates significant differences between initial and incubated values (paired samples t-test).
microorganisms. Thus it seems that the degradation of DOM derived from spruce needles and humus is clearly N limited. Our aim was to measure the DOM as fresh as possible to be able to detect also the most labile part of DOM, and therefore we took needle and leaf samples directly from the trees. All the samples were taken in the autumn during the birch senescent whereas the main senescent time for spruce is in the spring. Spruce needles were of the four oldest year-growth. Leaves were cut in pieces and needles were lightly crushed, which released much DOM especially
Fig. 4. Cumulative CO2eC loss (% of calculated total) during incubation of DOM solutions derived from birch, spruce and mixed humus and from needles and leaves (litter) of birch and spruce without and with NPK addition (mean and SD). Estimated proportion of labile DOC (%) and half-life of the labile DOC (days). The different letters (ANOVA) indicate significant differences between birch and spruce.
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a
b
c
d
e
f
Fig. 6. PCA analysis of chemical and microbiological characteristics of DOM derived from leaves, needles and humus layer in birch, spruce and mixed forest stands. a), c), e) Ordination of the sample plots (mean and SD, n ¼ 3); b), d), f) Vectors describing the importance of the variables in constructing the ordination (loadings > 0.5 are presented). a), b) The concentrations of DOC, DON, inorganic N, carbohydrates and phenolic compounds, DOC-to-DON ratio, pH, and the relative availability of DOM to bacteria (Tdr) and fungi (Ac-erg) in the incubation solution are used. In the datasets of different chemical fractions of C and N (c,d, see abbreviations in Fig. 2) and molecular size distribution (e,f) the total initial concentration of DOC or DON have been calculated to equal 100 mg l1 and the incubated values decreased from that concentration. Initial samples are denoted with circles, incubated samples with squares, humus samples are denoted with empty and plant samples with grey symbols. The letters indicate birch (b), spruce (s) and mixed (m) samples.
from needles. These aspects give uncertainty to the comparison between birch and spruce plant material. Spruce needle DOM appeared to differ much from all other samples in almost all chemical measurements. Spruce needle DOM had higher concentrations of weak phoA and phiN, phenolic compounds, carbohydrates, and small MS (<1 kD) compounds, compared to birch leaf DOM. As discussed above, differences could be partly due to the crushing of needles and sampling.
Carbohydrates are transported from needles to stem in senescence and thus the concentration of carbohydrates would have probably been lower in brown, senescent needles. This is confirmed with the unpublished data of Kiikkilä et al. where freshly fallen, senescent needles were studied. DOM derived from spruce needles had also a low proportion of C in PhoA that seemed to contain no N. PhoA, which was here and has been also earlier (Smolander et al., 2005) the most abundant fraction in humus DOM, is suggested to
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represent intermediates in organic matter degradation (Guggenberger et al., 1994). DOM derived from green needles contained very little PhoA probably because needles had not started to degrade, and during the incubation phoA increased as a degradation product. In contrast, phoA decreased during the incubation in humus and birch leaf DOM meaning that these solutions degraded further. Total carbohydrates determined with anthrone reagent correlated moderately with hydrophilic neutral (phiN) fraction. This is in accordance with Qualls and Haines (1991). The anthrone method as a simple technique could be used to assess concentrations of total carbohydrates in DOM solutions. Another fraction, weak hydrophobic acids (phoA) are often called phenols (Qualls and Haines, 1991). Total phenolic compounds measured spectrophotometrically did not correlate with weak phoA indicating that weak phoA is a mixture of hydrophobic compounds. In conclusion, most degradable compound groups were carbohydrates, phenolic compounds, hydrophilic neutrals, weak hydrophobic acids, N-containing and small molecular size compounds. DOM derived from leaves and needles was more degradable and contained a greater amount of labile compounds than DOM derived from the humus layer. We suggest that the labile compounds of fresh litter affect the properties of the underlying humus layer. In assessing the turnover of DOM in soil it seems essential to characterize also the DOM derived from fresh litter in order to detect the most labile DOM.
Acknowledgements We are grateful to R. Lievonen for collecting the samples, A. Rautiainen for laboratory work, to A. Siika and S. Elomaa for the figures and M. Waller for checking the English. The research was supported by the Academy of Finland.
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Properties of dissolved organic matter derived from silver birch and Norway spruce stands: Degradability combined with chemical characteristics Oili Kiikkilä*, Veikko Kitunen, Aino Smolander Finnish Forest Research Institute, Vantaa Research Center, P.O. Box 18, FIN 01301 Vantaa, Finland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 July 2010 Received in revised form 4 November 2010 Accepted 9 November 2010 Available online 23 November 2010
Dissolved organic matter (DOM) derived from the humus layer under silver birch (Betula pendula Roth), Norway spruce (Picea abies (L.) Karst.), and mixed stands, and from senescent birch leaves and from spruce needles of the four oldest year-growth were characterized microbiologically and chemically. Samples were collected in the autumn and the solutions were obtained by centrifugation-drainage technique. The degradability of DOM, the availability of DOM to bacteria and fungi, concentrations of phenolic compounds and carbohydrates, and the distribution of carbon and nitrogen into fractions according to the chemical nature and the molecular size were studied. DOM derived from leaves and needles was clearly more labile than DOM derived from the humus layer indicating the importance of studying the DOM originating from fresh litter when assessing the turnover of DOM. DOM derived from spruce needles appeared to differ chemically greatly from all other samples. It had very high concentrations of carbohydrates, probably due to the sampling time, and phenolic compounds. The chemical composition of DOM derived from humus layer did not reflect the composition of DOM derived from needles and leaves. DOM derived from birch leaves degraded more than DOM derived from spruce needles and DOM derived from humus layer collected at the birch sites degraded more than DOM derived from humus layer collected at the spruce sites. The degradability of different compound groups of DOC and DON was studied in a short-term incubation (20 d) of DOM solutions by characterizing the solutions initially and after the incubation. Almost all compound groups appeared to degrade but weak hydrophobic acids, bases, hydrophilic neutrals, the smallest molecular size compounds, carbohydrates, and phenolic compounds degraded the most. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: DOC DON Degradation Chemical composition Molecular size distribution Tree species
1. Introduction The quality and quantity of DOM should play a key role in the dynamics of C and N in forest soils because dissolved organic matter (DOM) may be the most important energy and nutrient source for microorganisms (Marchner and Kalbitz, 2003). The quality of DOM in forest soils is affected by tree species. In general conifer litter contains more recalcitrant, hydrophobic, aromatic compounds, whereas deciduous litter contains more labile, hydrophilic, low molecular weight compounds (Hongve et al., 2000; Kaiser et al., 2001, 2002; Kalbitz et al., 2003a). In most studies, it seems that despite significant differences in the composition of the source materials, the DOM chemistry is relatively similar in decomposed material (Smolander and Kitunen, 2002; Yano et al., 2005; Traversa et al., 2008). One of the main
* Corresponding author. Tel.: þ358 45 638 5254, þ358 10 211 2450. E-mail address: oili.kiikkila@metla.fi (O. Kiikkilä). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.11.011
problems in studying DOM is that, in practice, a great part of DOM is turned over so quickly (van Hees et al., 2005; Boddy et al., 2007) that it is never measured. The fast turnover of labile compounds can explain why DOC chemistry in soil solution has not been reflective of DOC chemistry in the litter layer (Kiikkilä et al., 2006; Wickland et al., 2007) and CO2 production has been higher than the pool of carbon (Bengtson and Bengtsson, 2007). More recalcitrant compounds accumulate in the soil solution where labile compounds have been consumed (Qualls et al., 2002; Hagedorn et al., 2004; Wickland et al., 2007; Sanderman et al., 2008). DOM in soil is generated by different pathways: it may originate from fresh litter, older humified organic material, root exudates and microorganisms. The proportional contribution of these DOC sources to soils is, however, unclear. It has been suggested that labile carbon pools, typically including root and leaf litter (Park et al., 2002; Uselman et al., 2009; Hansson et al., 2010) and root exudates (Yano et al., 2000, 2005; Giesler et al., 2007; Kramer et al., 2010), are considerable sources of DOM in soil, and it has been emphasized that they are also important sources for microbial
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activities in soil (van Hees et al., 2005). In other studies, fresh litter, throughfall or root exudates have made minor contributions to DOC in soil whereas the greatest part of DOM have been suggested to originate from the older organic matter in organic soil horizons (Park and Matzner, 2003; Hagedorn et al., 2004; Kalbitz et al., 2007; Fröberg et al., 2007, 2009; Müller et al., 2009). However, increased DOM production from organic soil horizons has been suggested as being a consequence of increased microbial activities that were stimulated by labile compounds delivering from fresh litter (Kalbitz et al., 2007). Thus studying the quality of DOM derived from fresh litter, instead of older organic matter, the litter layer, or humus layer, may increase the understanding of dynamics of C and N in forest soils. The aim of the study was to characterize DOM derived from the humus layer under silver birch and Norway spruce and respective leaves and needles. In order to measure the DOM originating from senescent litter as fresh as possible, without loosing labile compounds, we collected leaves and needles directly from the trees and compared to humus layer samples. We characterized DOM according to the molecular size distribution and the chemical nature i.e. adsorption properties. In order to assess the turnover of DOM in forest soil we measured the degradability of DOM and the availability of DOM to bacteria and fungi. The aim was also to assess the degradability of different compound groups of DOC and DON. For that purpose we characterized DOM before and after a shortterm incubation. 2. Materials and methods 2.1. Study site and sampling The study site was a replicated silver birch-Norway spruce (Betula pendula RothePicea abies (L.) Karst.) experiment in Eno, central-eastern Finland described earlier by Smolander et al. (2005). Briefly, the soil was a podsol and humus type mor. The site type was Vaccinium myrtillus type (Cajander, 1949). The study site had originally been a birch stand, which had been clear-cut in 1964. On spruce plots, 3e4-year-old Norway spruce seedlings were planted in autumn 1964, and 2-year-old silver birch seedlings were planted on birch plots in spring 1965. The birch plots were thinned in autumn 1985. The size of the plots was 40 m 40 m. Of the whole field experiment, which covered a total area of 6.7 ha, only part of the plots were used in this study; three adjacent plots of both tree species and two mixed species plots were selected. Birch plots were single-species stands and, of the total stem number, the spruce plots, still regarded as spruce plots, contained 1.6% and 7% birch. The mixed plots were mainly spruce, containing 22% and 37% birch of total stem number. Stand characteristics of birch and spruce plots were determined in 2002 and were presented in detail by Smolander et al. (2005). On birch plots, the understorey vegetation (under 3 m tall) consisted mainly of grey alder (Alnus incana) and rowan (Sorbus aucuparia), whereas on spruce plots the understorey was very sparse. Ground vegetation in the stands clearly differed: herbs and grasses were abundant in birch plots while the spruce plots were covered almost solely by moss and a layer of needle litter. The ground vegetation of mixed plots resembled that of spruce plots. Samples were collected in September 2007 during the senescence of birch. Composite samples (20e25 cores, core diameter 58 mm) were taken systematically from the humus layer (F þ H) of all plots, 8 altogether. After green plant material was removed, the samples were sieved through a 4.0 mm mesh and stored in plastic bags at 4 C. Leaves and needles were collected from birch and spruce plots, 5 trees per sample plot. Leaf litter was collected from birch trees by
shaking. A great part of the leaves were yellow, but some were still green. Leaves were air dried at room temperature in a laboratory for two days. Leaves were cut into ca. 10 mm2 pieces and stored in 4 C. At the same time, the lowest branches of spruce were taken into a laboratory. The branches were air dried for two days, needles were separated and crushed lightly using a mortar and stored in 4 C. The needles consisted of the four oldest annual growth and were mostly green. 2.2. Collection of DOM To obtain the DOM solution, the centrifugation-drainage technique described by Giesler and Lundstöm (1993) was used. Samples were centrifuged at 5000 g for 20 min. Humus layer samples were adjusted to 100% water holding capacity (WHC) by ultrapure water and allowed to stand overnight at 4 C before centrifugation. To 10 g dry weight of leaves and needles 100 ml of ultrapure water was added and allowed to stand overnight at 4 C before centrifugation. Finally the solutions were filtered through a 0.45 mm polyethersulfone membrane and stored in a freezer until further analysis. After thawing the solutions were kept at 4 C for 3e10 days in order to allow them to stabilize chemically. 2.3. Determination of the properties of DOM The molecular size (MS) distribution of DOC and DON was determined by tangential ultrafiltration apparatus (MinimateÔ TFF, Pall Corporation). After thawing the solutions were filtered through 0.45 mm. Membranes with nominal weight cut-offs at 100, 10 and 1 kD were used. The solution (ca. 25 mg l1 C), 210 ml, was filtered through 100 kD membrane and concentrated to 40 ml. This retentate, i.e. the solution that was left in the container, was collected as a sample >100 kD. Next, the filtered solution (<100 kD), 170 ml, was filtered through 10 kD and concentrated to 90 ml (sample 10e100 kD). The filtered solution (<10 kD), 80 ml, was then filtered through 1 kD membrane and concentrated to 40 ml (sample 1e10 kD). The sample <1 kD was the final filtrate through 1 kD membrane. The diafiltration procedure was not performed because in pre-testing it was found to increase remarkably the loss of DOC from samples. Instead, it was observed that the concentration procedure was fairly steady with the used volumes. Thus the sample of 1e10 kD was assumed to include smaller (<1 kD) compounds in the same concentration as the sample of <1 kD and the concentration of DOC or DON in <1 kD sample was subtracted from 1e10 kD sample. The same calculation, i.e. the concentration of the smaller sample was subtracted from the larger, was made with 10e100 kD and >100 kD samples. The DOC and DON of solutions were fractionated into compound groups according to their chemical nature i.e. adsorption properties using the procedure described by Aiken and Leenheer (1993) and Smolander and Kitunen (2002). The compound groups were hydrophilic acids (phiA), bases (phiB) and neutrals (phiN), weak hydrophobic acids (weak phoA), hydrophobic acids (phoA), bases (phoB) and neutrals (phoN). XAD-8 (Amberlite XAD-8, Merck), MSC-1 cation-exchange (Sigma Chemical Co.) and AG-MP1 anionexchange (BioRad Laboratories) resin columns were used. The concentrations of carbohydrates and phenolic compounds in DOM were measured spectrophotometrically. The concentration of carbohydrates was measured using anthrone reagent (Brink et al., 1960) and the concentration of total phenolic compounds was measured with the FolineCiocalteu method, as described in Suominen et al. (2003). The pH was measured from the DOM solution where the DOC concentration was 25 mg l1. To assess the availability of DOM to bacteria (Tdr) the 3 H-thymidine incorporation technique (Bååth et al., 2001) modified
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to DOM studies by Kiikkilä et al. (2005) was applied. A bacterial inoculum was prepared as follows: 3 g fresh weight of humus (a mixture of birch and spruce humus layer) was mixed with 100 ml of water and shaken (200 rpm) for 1 h, followed by low-speed centrifugation (750 g for 10 min) and filtration through quartz wool. The bacterial suspension (5 ml) was then mixed with DOM solution, which had ca. 30 mg l1 DOC, (10 ml) or water (control). Thymidine incorporation rate, i.e. the bacterial growth rate, was measured after the solution was pre-incubated at 20 C for 24 h. An aliquot (3.5 ml, 0.1 MBq) of methyl-3H-thymidine (740 GBq mmol1, Moravek Biochemicals) was added and the samples were incubated for 2 h. Washing to remove excess tracer, and measurement of radioactivity were described in detail by Bååth et al. (2001). Availability of DOM to fungi (Ac-erg) was assessed using the 14 C-acetate-in-ergosterol technique (Bååth, 2001) modified by Kiikkilä et al. (2006). A piece of humus (a mixture of birch and spruce humus layer, 0.03 g fresh weight) was added as fungal inoculum to 30 ml of DOM solution (ca. 10 mg l1 DOC), or water (control) in Erlenmeyer flasks. Streptomycin and ampicillin were added in order to diminish the bacterial growth in solutions. The pH was adjusted to 4.5 in order to give fungi favourable growing conditions (Rousk et al., 2010). The bottles were pre-incubated in darkness at 20 C. After 24 h the solutions were filtered through Whatman GF/D glass fiber filter. The soil and the filter were transferred to a test tube with 1.5 ml of the DOM solution. In order to measure the fungal growth rate, 0.13 mmol (0.3 MBq) of 14Cacetate solution (1-14C-acetic acid, sodium salt, 2.2 GBq mmol1, Moravek Biochemicals) and 0.35 mmol of 1 mM non-radioactive acetate were added. After incubating the mixture for 20 h at 20 C, formalin was added, the test tubes were centrifuged, and the supernatant discarded. The ergosterol was then extracted as in Bååth (2001) and measured with HPLC (Hitachi, Merck) and 14Cergosterol with the HPLC radioactivity monitor (Berthold, LB 506 C-1). The relative availability of a DOM solution to bacteria or fungi was calculated by dividing the incorporation rate of the DOM solution by the incorporation rate of the control solution. The mineralization rate of DOC was measured in a long-term incubation (110 days). For microbial inoculum the mixture of birch and spruce humus layer (10 g fresh weight) was shaken (200 rpm, 1 h) in 100 ml water. 100 ml of this soil suspension was added to 30 ml of DOM solution (25 2.3 mg l1 DOC, mean SD, n ¼ 14), which was filtered through 0.2 mm in 120 ml glass bottles. To assess also the potential mineralization rate of DOC, nutrients were added to half of the bottles, to give a final concentration of 0.1 mM NH4NO3 and 0.1 mM K2HPO4. The solutions were shaken (120 rpm) in the dark at 20 C and the CO2 evolution was measured by gas chromatography every other day and finally every two weeks. After CO2 measurements, the bottles were aerated. CO2eC loss was calculated, and the proportion of labile DOC and the half-life of labile DOC and were estimated (see data analysis). 2.4. Assessing the degradability of compound groups of DOM In addition to the long-term incubation, we made a separate short-term incubation in order to study the degradation of different compound groups of DOM. The solutions were filtered through 0.2 mm and diluted to a concentration of 25.6 2.3 mg l1 DOC (mean SD, n ¼ 14). The initial solutions were frozen until further analysis. The solutions were placed in sterile 3 l Erlenmeyer flasks, 650 ml in each flask, in two replicates. To provide extra surface area for microbial growth, a glass fiber filter (GF/D) was added to each flask. An inoculum was prepared from the humus layers as described above and added 1 ml l1. Control solutions, without an inoculum, were prepared of four randomly chosen solutions in order to detect the possible precipitation of DOM. The flasks were
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kept in the dark at 20 C and gently shaken by hand daily. After 20 days the two replicates were combined, the solutions were filtered (0.2 mm) and frozen until further analysis. The concentration of DOC and DON was determined as described in Smolander and Kitunen (2002) initially and after the incubation. The proportional loss of DOC (DOC loss%) and DON (DON loss%) during 20-days incubation was calculated as difference between initial and incubated solutions subtracted by the mean loss of DOC (8.7%) and DON (10.0%) in control samples. The concentration of bioavailable DON was calculated as the actual loss of DON in incubation solutions equalized to C concentration. The relative availability of DOM to bacteria and fungi, concentration of carbohydrates and phenolic compounds and the distribution of DOC and DON according to chemical nature and molecular size were determined from initial and incubated solutions as described above. 2.5. Data analysis To normalise the distribution of the variables necessary logarithmic transformations were made. The statistical significance for all tests was set at p < 0.05. One-way ANOVA was used to determine the differences in the properties of DOM between birch and spruce. Mixed plots were not included in ANOVA, because n ¼ 2. Pearson and partial correlations were calculated. The mineralization rate of DOC was analyzed by fitting a double exponential model (Kalbitz et al., 2003a) to the data of the longterm incubation estimating an easily degradable (labile) and a more stable pool of DOC. Mineralized DOC ¼ (100-s)(1-eklt) þ s(1-ekst) where t ¼ time (days); following estimated parameters (100-s) ¼ the part of DOC that is rapidly mineralizable ¼ labile DOC (%); s ¼ the part of DOC that is slowly mineralizable ¼ stable DOC (%); kl ¼ mineralization rate constant of labile DOC (day1); ks ¼ mineralization rate constant of stable DOC (day1). The curves were fitted using the sequential quadratic programming algorithm. We calculated the half-life of the labile DOC ¼ ln2/kl (days). The degradation of different compounds and compound groups i.e. changes in the short-term incubation was analyzed by three separate principal component analyses (PCA) using correlation matrix: 1) The concentrations of DOC, DON, carbohydrates and phenolic compounds and the relative availability of DOM to bacteria and fungi, 2) DOC and DON divided into fractions according to the chemical nature, 3) molecular size distribution of DOC and DON. In the datasets of chemical fractions and molecular size classes the total initial concentration of carbon and nitrogen have been calculated to equal 100 mg l1 and the incubated values to decrease from that concentration. The variables most responsible for the ordination of samples are described as vectors (loadings). Separate variables were subjected to a paired samples t-test in order to detect significant differences between initial and incubated samples. 3. Results 3.1. Properties of DOM The concentrations of compounds were calculated to equal the concentration of 100 mg l1 DOC in the solution. Chemical and microbiological properties of DOM derived from humus layer (humus DOM) did not differ much between birch, spruce and mixed plots whereas many large differences were detected in DOM derived from birch leaves and spruce needles (plant DOM). The pH
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of spruce DOM was lower than that of birch DOM in both humus and plant material (Table 1). The concentration of inorganic N (mostly NH4) was very low in plant DOM whereas in humus DOM it was higher for birch than for spruce. The concentration of DON was higher and thus the DOC-to-DON ratio was lower for birch than for spruce in both humus and plant DOM (Table 1). The concentrations of carbohydrates and phenolic compounds did not differ significantly in humus DOM but were much higher for spruce needle than for birch leaf DOM (Fig. 1a,c). The chemical nature (Fig. 2) and the molecular size (MS) distribution (Fig. 3) of compounds did not differ much between birch, spruce and mixed plots in humus DOM. In plant DOM, spruce had clearly higher concentrations of C in weak phoA and phiN fractions and in small MS compounds (<1 kD) than birch. Instead, spruce had lower concentrations of C in phoA and large MS compounds (>100 kD) than birch. Most of plant DON was in phoA, phiB and phiN although spruce needle had no N in phoA. DON derived from birch leaves was biased towards large MS and that of spruce needles towards small MS compounds (Fig. 3). The degradability of DOC and DON i.e. the proportional losses of DOC and DON in the short-term incubation (20 days) were clearly higher for plant than for humus DOM (Table 2). DOC loss% was significantly higher for birch than for spruce in both humus and plant DOM (Table 2) whereas the DON loss% did not differ significantly between birch and spruce. The concentration of bioavailable DON in the incubation solution was significantly higher for birch than for spruce. The mineralization rate of DOC derived from the long-term incubation did not differ significantly between the tree species (Fig. 4). The potential mineralization rate of DOC (NPK added solutions) was higher for spruce than for birch in both humus and plant DOM. For plant DOM the variables calculated from the degradation curve (labile DOC and the half-life of labile DOC) (Fig. 4) indicated the same; there were no significant differences without NPK addition whereas in NPK addition solutions spruce had a higher proportion of labile DOC than birch, and the half-life of labile DOC was shorter for spruce than for birch.
Table 1 Means (SD, n ¼ 3) of pH, concentration of inorganic N, and DON, and DOC-to-DON ratio in DOM solutions derived from birch leaves, spruce needles and humus layer of birch, spruce and mixed plots.a Humus
Plant
Birch Spruce Mixedb
pH 5.7 (0.2)a 5.0 (0.1)b 5.1 (0.1)
6.8 (0.1)a 4.5 (0.1)b
Birch Spruce Mixedb
Inorganic N, mg l1 3.6 (1.4)a 0.7 (0.6)b 1.3 (0.5)
0.2 (0.1) 0.2 (0.3)
Birch Spruce Mixedb
DON, mg l1 4.3 (0.1)a 3.1 (0.3)b 3.1 (0.1)
2.5 (0.2)a 0.6 (0.2)b
Birch Spruce Mixedb
DOC-to-DON ratio 23 (1)a 31 (3)b 32 (1)
41 (3)a 185 (55)b
The different letters (ANOVA) indicate significant differences between birch and spruce. a Results are calculated in solution where the concentration of DOC would initially be 100 mg l1. b n ¼ 2, not included in ANOVA.
The relative availability of humus DOM to bacteria (Tdr) and fungi (Ac-erg) did not differ much between the tree species. In plant DOM Tdr was higher for birch than for spruce whereas Ac-erg did not differ much between birch and spruce (Fig. 5). Mixed plots were not included in ANOVA (n ¼ 2). Concentrations of inorganic N and DON, the DOC-to-DON ratio and pH in humus DOM from mixed plots seemed to be nearer spruce than birch (Table 1). DOC loss%, DON loss%, bioavailable DON, carbohydrates, phenolic compounds, fractions according to chemical nature, mineralization rate of DOC, Tdr or Ac-erg in humus DOM from mixed plots seemed not to differ from either spruce or birch (Table 2, Figs. 1, 2 and 4e6c). The molecular size distribution of compounds for mixed humus DOM seemed to differ slightly from both spruce and birch (Figs. 3 and 6e). 3.2. Degradability of compound groups of DOM The degradation of compound groups of DOM i.e. changes during the short-term incubation were analyzed with three separate PCA analyses. The variance explained by PC 1 and PC 2 was 66, 63 and 77% for the first, second and third PCA (Fig. 6), respectively. Figures are rotated so that the initial and incubated samples of spruce needle DOM (mean) separate along x-axis. Also all other initial and incubated samples separate along the x-axis (Fig. 6a,c,e) and in addition they (except spruce needle DOM) separate slightly also along the y-axis. Thus both x and y-axis indicate changes during the incubation. The change is larger in plant than humus DOM since in all Fig. (6a,c,d) the distances between initial and incubated samples of plant DOM are longer than those of humus DOM. The variables that have the loading value 0.5 have been drawn as vectors in the Fig. 6b,d,f. The variables that have high values on the x-axis decrease in all samples. The variables that that have high values only on the y-axis decrease in all but spruce needle DOM samples. High negative values on the y-axis do not refer to the degradability of compounds but the variables in question have clearly higher values either in spruce or litter samples compared to birch and humus samples, respectively. Both total DOC and DON degraded in the incubation (6a,b) (proportional losses are presented in Section 3.1.). Carbohydrates and phenolic compounds degraded (Fig. 6a,b), and Tdr and Ac-erg decreased in the incubation. These were supported by the paired t-tests on separate variables. Tdr (Fig. 5) and the concentration of carbohydrates (Fig. 1) decreased significantly in all treatments. The concentration of phenolic compounds decreased significantly in plant DOM (Fig. 1). Ac-erg decreased significantly only for birch plant DOM (Fig. 5). PCA (Fig. 6d) of the chemical nature of compounds indicates that C and N in PhiN, weak phoA, phiB and phoB fractions degraded. The vectors of C and N in phoA have high loading on the y-axis and negative loading on the x-axis indicating that phoA degraded in all except spruce needle DOM, where it increased (Fig. 6d). PCA of MS distribution (Fig. 6f) indicates that all size classes degraded. Only C and N > 100 kD, of which vectors have loading near zero on the xaxis, did not degrade in spruce needle DOM. The actual values for the decrease in incubation support the results of PCAs, but are not presented because many of the differences were not significant in the paired t-test. 3.3. Correlation between compound groups Correlations between the concentrations of C in weak phoA and phiN, phenolic compounds, and carbohydrates were calculated. Because phenolic compounds and carbohydrates correlated strongly positively, the partial correlation was performed. When
O. Kiikkilä et al. / Soil Biology & Biochemistry 43 (2011) 421e430
a
b
c
d
425
Fig. 1. Concentrations of a) carbohydrates and c) phenolic compounds in DOM derived from birch, spruce and mixed humus and from needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The values are calculated to equal the solution 100 mg l1 DOC initially. The different letters (ANOVA) indicate significant differences between birch and spruce. The respective values after the 20-days incubation and significant differences between initial and incubated values are indicated by * (paired samples t-test) in the figures b and d.
phenolic compounds were kept as a controlling variable, carbohydrates and phiN correlated positively (r ¼ 0.78, n ¼ 25). Instead, phenolic compounds and weak phoA did not correlate when carbohydrates were kept as a controlling variable. 4. Discussion DOM originating from fresh litter seems to belong to a great extent to the labile part of the DOM (Kalbitz et al., 2003a; Hagedorn et al., 2004; Kiikkilä et al., 2006; Müller et al., 2009). In our study, DOM derived from needles and leaves was clearly more degradable than DOM derived from humus layer. Plant DOM had at least twofold mineralization rate of DOC in 110 days incubation and also the loss of DOC in 20-days incubation was much higher compared to humus DOM. The degradability of the most labile DOM, which was assessed with the availability of DOM to bacteria (Tdr) and fungi (Ac-erg), was over 30% higher for plant DOM than humus DOM. The most labile DOM can stimulate microbial activities and further DOM production in organic horizon as suggested by Kalbitz et al. (2007). Microbial activities of soil have often had a positive
relation to the concentration of DOC and DON (Marchner and Kalbitz, 2003; Neff and Hooper, 2002; Moore et al., 2008; Smolander and Kitunen, submitted for publication). More important than the concentration may be the degradability and thus the turnover of DOM. Earlier at the same sites higher microbial activities in humus layer have been detected under birch than under spruce (Smolander et al., 2005). Thus it seems that microbial activities and the degradability of DOM have a positive relationship at this site. However, more data is needed in order to assess the relationship between the microbial activities of soil and the degradability of DOM in general. As a conclusion, in assessing the properties and turnover of DOM in soil it seems essential also to characterize the DOM derived from fresh litter although it seems to make a minor contribution to the total steady state concentration of DOM in the humus layer. Measuring the steady state concentrations of DOM alone does not detect the actual turnover of labile compounds. Also the chemical measurements indicated that labile compounds were in general more abundant in the litter layer DOM (Kiikkilä et al., 2006) and in plant DOM (Fig. 6) than in humus DOM. The most degradable compound groups of DOM, which have also
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a
b
Fig. 2. Distribution of the chemical fractions of a) DOC and b) DON in birch, spruce and mixed humus and in needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The values are calculated to equal the solution 100 mg l1 DOC (note the different scales in humus and plant DON). Weak hydrophobic acids (W PhoA), hydrophobic bases (PhoB), acids (PhoA) and neutrals (PhoN), and hydrophilic bases (PhiB), acids (PhiA) and neutrals (PhiN). The different letters (ANOVA) indicate significant differences between birch and spruce.
earlier been reported to be labile (Qualls and Haines, 1992; Kalbitz et al., 2003b; Qualls, 2005; Wickland et al., 2007), were carbohydrates, phenolic compounds, phiN, weak phoA, nitrogen containing compounds (bases) and smaller MS compounds. These compounds decreased in the short-term incubation and their concentrations were positively related to the DOC loss and the availability of DOM to bacteria and fungi (Fig. 6). DOM, either a degradation product or leached from litter, is degraded or stabilized by sorption to the organic and mineral soil layers. More stable compounds seem to adsorb preferentially,
leaving the more easily degradable compounds in solution (Kalbitz et al., 2005; Hunt et al., 2008; Schneider et al., 2010). Kalbitz and Kaiser (2008) estimated that 19%e50% of soil total C derives from DOM. Thus especially refractory DOM significantly contributes to the accumulation of C in soil. DOM derived from birch stands seemed to be more degradable than DOM derived from spruce stands whereas mixed plots resembled more spruce than birch. Birch DOM, originating either from plant material or humus, had a higher loss of DOC and DON during the 20-days incubation than the respective spruce DOM. The
a
b
Fig. 3. Distribution of the molecular size classes of a) DOC and b) DON in birch, spruce and mixed humus and from needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The values are calculated to the solution 100 mg l1 DOC (note the different scales in humus and plant DON). The different letters (ANOVA) indicate significant differences between birch and spruce.
O. Kiikkilä et al. / Soil Biology & Biochemistry 43 (2011) 421e430 Table 2 Means (SD, n ¼ 3) of loss of DOC and DON during a 20-day incubation of DOM derived from birch leaves, spruce needles and humus layer of birch, spruce and mixed plots. Humus
427
a
b
c
d
Plant
DOC loss% Birch Spruce Mixeda
14.6 (1.3)a 9.2 (2.8)b 11.5 (7.8)
Birch Spruce Mixeda
DON loss% 21.1 (6.0) 8.5 (6.4) 11.4 (13.9)
Birch Spruce Mixeda
Bioavailable DON, mg l1b 0.9 (0.3)a 0.3 (0.2)b 0.4 (0.4)
48.3 (3.5)a 40.4 (5.7)b
43.3 (8.6) 32.7 (17.9)
1.1 (0.2)a 0.2 (0.2)b
The different letters (ANOVA) indicate significant differences between birch and spruce. a Not included in ANOVA (n ¼ 2). b Calculated DON loss in solution where the concentration of DOC would initially be 100 mg l1.
degradability of the most labile DOM, measured as availability of DOM to bacteria, was also higher for birch than for spruce. However, when the mineralization rate of DOC was followed by CO2 evolution in the long-term incubation, only very small differences between tree species were detected. We cannot exclude the possible effect of different pH; it was higher for birch than for spruce especially in plant DOM. Therefore it is possible that birch DOM gave too low CO2 values due to the higher dissolved CO2. Phenolic compounds left in spruce needle DOM after the shortterm incubation had perhaps degraded in a longer run and increased the mineralization rate of spruce needle DOC in the longterm incubation, although this is neither supported by the CO2 evolution curve at the moment of 20 days nor the estimated proportion of the labile compounds (Fig. 4). DOM derived from the green spruce needles also had a high concentration of carbohydrates that were labile, and would have been less abundant in senescent needles. We suggest that the degradability of senescent spruce needle DOM would be lower than senescent birch leaf DOM. Nutrient addition increased the mineralization rate of spruce DOC clearly over birch. This is not surprising because the N content was very low in spruce DOM and probably a limiting factor for
Fig. 5. The relative availability of DOM to a) bacteria (Tdr) and c) fungi (Ac-erg) in birch, spruce and mixed humus and in needles and leaves (Plant) of birch and spruce (mean and SD, n ¼ 3). The different letters (ANOVA) indicate significant differences between birch and spruce. The respective values after the 20-days incubation are presented in figures b and d. * indicates significant differences between initial and incubated values (paired samples t-test).
microorganisms. Thus it seems that the degradation of DOM derived from spruce needles and humus is clearly N limited. Our aim was to measure the DOM as fresh as possible to be able to detect also the most labile part of DOM, and therefore we took needle and leaf samples directly from the trees. All the samples were taken in the autumn during the birch senescent whereas the main senescent time for spruce is in the spring. Spruce needles were of the four oldest year-growth. Leaves were cut in pieces and needles were lightly crushed, which released much DOM especially
Fig. 4. Cumulative CO2eC loss (% of calculated total) during incubation of DOM solutions derived from birch, spruce and mixed humus and from needles and leaves (litter) of birch and spruce without and with NPK addition (mean and SD). Estimated proportion of labile DOC (%) and half-life of the labile DOC (days). The different letters (ANOVA) indicate significant differences between birch and spruce.
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a
b
c
d
e
f
Fig. 6. PCA analysis of chemical and microbiological characteristics of DOM derived from leaves, needles and humus layer in birch, spruce and mixed forest stands. a), c), e) Ordination of the sample plots (mean and SD, n ¼ 3); b), d), f) Vectors describing the importance of the variables in constructing the ordination (loadings > 0.5 are presented). a), b) The concentrations of DOC, DON, inorganic N, carbohydrates and phenolic compounds, DOC-to-DON ratio, pH, and the relative availability of DOM to bacteria (Tdr) and fungi (Ac-erg) in the incubation solution are used. In the datasets of different chemical fractions of C and N (c,d, see abbreviations in Fig. 2) and molecular size distribution (e,f) the total initial concentration of DOC or DON have been calculated to equal 100 mg l1 and the incubated values decreased from that concentration. Initial samples are denoted with circles, incubated samples with squares, humus samples are denoted with empty and plant samples with grey symbols. The letters indicate birch (b), spruce (s) and mixed (m) samples.
from needles. These aspects give uncertainty to the comparison between birch and spruce plant material. Spruce needle DOM appeared to differ much from all other samples in almost all chemical measurements. Spruce needle DOM had higher concentrations of weak phoA and phiN, phenolic compounds, carbohydrates, and small MS (<1 kD) compounds, compared to birch leaf DOM. As discussed above, differences could be partly due to the crushing of needles and sampling.
Carbohydrates are transported from needles to stem in senescence and thus the concentration of carbohydrates would have probably been lower in brown, senescent needles. This is confirmed with the unpublished data of Kiikkilä et al. where freshly fallen, senescent needles were studied. DOM derived from spruce needles had also a low proportion of C in PhoA that seemed to contain no N. PhoA, which was here and has been also earlier (Smolander et al., 2005) the most abundant fraction in humus DOM, is suggested to
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represent intermediates in organic matter degradation (Guggenberger et al., 1994). DOM derived from green needles contained very little PhoA probably because needles had not started to degrade, and during the incubation phoA increased as a degradation product. In contrast, phoA decreased during the incubation in humus and birch leaf DOM meaning that these solutions degraded further. Total carbohydrates determined with anthrone reagent correlated moderately with hydrophilic neutral (phiN) fraction. This is in accordance with Qualls and Haines (1991). The anthrone method as a simple technique could be used to assess concentrations of total carbohydrates in DOM solutions. Another fraction, weak hydrophobic acids (phoA) are often called phenols (Qualls and Haines, 1991). Total phenolic compounds measured spectrophotometrically did not correlate with weak phoA indicating that weak phoA is a mixture of hydrophobic compounds. In conclusion, most degradable compound groups were carbohydrates, phenolic compounds, hydrophilic neutrals, weak hydrophobic acids, N-containing and small molecular size compounds. DOM derived from leaves and needles was more degradable and contained a greater amount of labile compounds than DOM derived from the humus layer. We suggest that the labile compounds of fresh litter affect the properties of the underlying humus layer. In assessing the turnover of DOM in soil it seems essential to characterize also the DOM derived from fresh litter in order to detect the most labile DOM.
Acknowledgements We are grateful to R. Lievonen for collecting the samples, A. Rautiainen for laboratory work, to A. Siika and S. Elomaa for the figures and M. Waller for checking the English. The research was supported by the Academy of Finland.
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