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Seasonal fluctuation in microbial biomass and activity along a natural nitrogen gradient in a drained peatland
Soil Biology & Biochemistry 36 (2004) 1047–1055 www.elsevier.com/locate/soilbio
Seasonal fluctuation in microbial biomass and activity along a natural nitrogen gradient in a drained peatland Hannamaria Potila*, Tytti Sarjala Parkano Research Station, The Finnish Forest Research Institute, Kaironiemente 54, FIN-39700 Parkano, Finland Received 23 June 2003; received in revised form 11 February 2004; accepted 23 February 2004
Abstract The effects of peat total N on the dissolved N and C concentrations and microbial biomass and activity and their range of seasonal fluctuation were studied in a drained peatland forest in Finland. Seasonal fluctuations in the concentrations of extractable dissolved organic (DON) and inorganic nitrogen (DIN) compounds and extractable dissolved organic carbon (DOC), microbial C and N, ergosterol, net and gross N mineralisation rates were investigated during two growing seasons along a natural peat N gradient in a drained peatland. Significant seasonal fluctuations in NHþ 4 and DOC concentrations, microbial C and N, but not in ergosterol or microbial C-to-N ratios in the peat, were observed during the 1999 and 2000 growing seasons. The peat total N concentration affected extractable DON and DOC, but not DIN concentrations in the peat. A negative correlation was found between total N concentration in peat and microbial N and C, and a positive correlation between total N and ergosterol, in peat with N concentrations of up to 2%. Gross mineralisation rates did not show any correlation, whereas net mineralisation rates showed a significant positive correlation with the total N concentration of the peat in both 1999 and 2000. q 2004 Elsevier Ltd. All rights reserved. Keywords: Dissolved N; Dissolved organic carbon; Ergosterol; Microbial biomass; Mineralisation; Peat
1. Introduction There are over 5 million ha of afforested drained mires in Finland. The potential tree growth in these peatland forests depends on the drainage efficiency and peat N content, as well as on the nutrient balance between N and especially K and P (Pa¨iva¨nen and Paavilanen, 1992; Kaunisto and Pietila¨inen, 2003). The majority of the N in the peat is in a bound form and not available for plants. A minor proportion of the N is in soluble form, most of which consists of dissolved organic N (DON) and low concen2 trations of inorganic N, NHþ 4 and NO3 (DIN). Slow mineralisation of soil organic N has earlier been assumed to be the main factor limiting plant growth in boreal forests. However, the uptake of amino acids in field conditions by ectomycorrhizal trees, ericaceous shrubs and herbaceous plants has been demonstrated (Na¨sholm et al., 1998). Thus, the availability of DON, and not only the mineralised inorganic pool, affects the overall N supply for trees. * Corresponding author. Tel.: þ 358-344-352; fax: þ358-344-35200. E-mail address: [email protected] (H. Potila). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.02.014
In the model presented by Lindahl et al. (2002), mineralisation and plant uptake of inorganic nutrients play a less important role. Peat consists of incompletely decomposed organic plant residues. Improved aeration in deeper peat layers after peatland drainage increases the populations of aerobic decomposers and thus enhances the decay of organic matter. Scheffer et al. (2001) studied decomposition and mineralisation rates in Sphagnum- and Carex-dominated peatlands, and concluded that nutrient availability and adaptation of the microbial community to nutritional and other environmental conditions may be the main regulators of C and nutrient transformations in these peatlands. Fungi are believed to be the main decomposers in acid mor soils of the boreal region (Frostega˚rd and Ba˚a˚th, 1996), and their contribution to nutrient transformations in peatland forest soils is dominant. Mycorrhizal plants and non-symbiotic soil micro-organisms may compete for both inorganic and organic soil N, since they potentially use the same N sources. Biotic immobilisation of N by free-living microbes requires a supply of available DOC produced by the decomposition
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of plant litter. According to Aber (1992), an increasing N concentration in plant litter reduces available DOC production and increases the utilization of existing pools of DOC. On peatlands, which have a high C content in the substrate, the factors controlling DON and DOC fluxes are poorly known. Our aim was to determine the seasonal fluctuation of DOC, dissolved N compounds and microbial biomass in peat. An additional aim was to investigate the influence of peat total N on dissolved N and C concentrations, microbial biomass and activity.
2.2. Dissolved N and DOC
2. Material and methods
Extractable dissolved N and C were determined by extracting 10 g of fresh sample with 100 ml of 0.5 M K2SO4 (2 h shaking), and filtering the suspension through filter paper and further through a 0.45 mm membrane filter (Williams et al., 1995). The extract was frozen (2 20 8C) 2 until analysis. Total dissolved N (TDN), NHþ 4 and NO3 in the extracts were analysed by flow injection analysis (FIA Star 5020, Tecator). DON in the extracts was obtained by 2 deducting the NHþ 4 and NO3 -N concentration from TDN. DOC in the extract was analysed on a TOC-5000 analyser (Schimadzu). The moisture content (overnight at 105 8C) of the peat samples was also determined.
2.1. Sampling
2.3. Ergosterol, Cmic and Nmic
Fluctuations in the concentrations of soluble nitrogen 2 compounds (NH þ 4 , NO 3 , DON), DOC, ergosterol, microbial N (Nmic) and C (Cmic) were followed during the 1999 and 2000 growing seasons in a field experiment (62890 N, 228520 E) in a Scots pine stand in Western Finland. The field experiment (109 A and B) was established by the Finnish Forest Research Institute in 1974 (Kaunisto, 1982; Kaunisto et al., 1986) on a mire drained in 1969. Five non-fertilised and 10 PK-fertilised plots (42 kg P ha21, 50 kg K ha21) were chosen to represent a natural gradient in the total N concentration of the peat (1.2 – 2.7% N). An influx of spring water from surrounding mineral soils before drainage of the mire has created a natural N gradient in this area. There were only minor differences between the plots in the C content of the peat, which ranged from 45.4 to 50.5%. Therefore, N was the determinant of the peat C-to-N ratio, which varied from 17 to 42. The plots were afforested with Scots pine in 1973, but the non-fertilized plots are now almost treeless. The thickness of the peat layer varied between 0.34 and 2 m. The average pH of the peat was 4.3 ^ 0.2 in the nonfertilised plots and 4.1 ^ 0.2 in the PK-fertilised plots. The effect of PK fertilisation 30 years ago has now disappeared from the peatland, because there were no significant differences in the P and K concentrations between the non-fertilised and PK-fertilised plots. Samples were collected from the 0 – 10 cm peat layer on eight occasions between 18 May and 8 October, 1999, and between 23 May and 3 October, 2000. On the first sampling date in summer 1999 only the non-fertilised plots were sampled because the other plots were still frozen. Five cores were taken from the acrotelm with an auger (6 £ 6 cm), and bulked to give one composite sample per plot. The mean daily temperature and precipitation in the area during the two growing seasons are presented in Fig. 1. In 1999 there was a dry period during 5 – 18 July. The effective temperature sum (threshold þ 5 8C) in the area in 1999 (1139 d.d.) and in 2000 (1136 d.d.) was close to the longterm average (1110 d.d.).
The peat ergosterol concentration was determined by a method modified from Nylund and Wallander (1992) consisting of ethanol extraction, saponification with KOH and further extraction in pentane, followed by HPLC with a UV – VIS detector (Merck-Hitachi) (wavelength 280 nm), LiCrospher 100 RP-18 column and methanol as eluent. Cmic and Nmic were determined from fresh peat samples with the fumigation-extraction (FE) method by fumigating 10 g of fresh sample for 18 h in chloroform vapour (Brookes et al., 1985), followed by extraction and analysis in the same way as dissolved N and DOC. Nmic was calculated from the difference between fumigated and fresh concentrations of TDN using the value 0.54 for kN (Brookes et al., 1985). Cmic was obtained by deducting the DOC concentration of the fresh peat sample from the DOC concentration of the fumigated peat sample using the recovery factor of 0.45 derived for peat soils by Sparling et al. (1990). 2.4. Mineralisation The gross N mineralisation in the field was measured on nine plots (five sampling points per plot), using the 15N isotopic dilution technique in soil cores (Davidson et al., 1991). The total N concentration in the peat in these plots varied from 1.2 to 1.9%. Two samples were taken at each sampling point, and 10 ml of a dilute (15NH4)2SO4 solution, containing 20 mg (NH4)2SO4 98% atom% 15N (SigmaAldrich) l-1 distilled water, was injected into each sample. The other subsample was taken to the laboratory and kept at þ 5 8C until the next day. The other subsample was incubated for 3 d between 19 and 22 July 1999, and 10 and 13 July 2000 in the field in a plastic cylinder (105 cm3 volume) sealed at the top and bottom with PVC lids and inserted in the peat layer. Four holes in the upper part of the tubes were made for aeration. Ammonium was extracted by shaking 20 g peat with 100 ml of 0.5 M K2SO4 for 2 h and filtering the suspension through filter paper and further through a 0.45 mm membrane filter. For the 15N analyses, 10 ml of extract
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Fig. 1. The mean daily precipitation (mm) (bars) and temperature (8C) (lines) during the 1999 and 2000 growing seasons at a weather station of the Finnish Meteorological Institute 6 km from the experimental area in Alkkia.
and ca. 500 mg MgO powder were reacted for 3 d at þ 30 8C in airtight plastic jars together with an open vial containing 10 mM H2SO4. The sulphuric acid was dried at þ 45 8C. The 15N/14N ratio was determined by mass spectrometry at MTT Agrifood Research Finland, and the gross N mineralisation rates were calculated according to the equations presented in Kirkham and Bartholomew (1954) and Davidson et al. (1991). Net N and C mineralisation were measured in the field in the same plots as gross N mineralisation (total N 1.2– 1.9%). Five samples per plot (total of nine plots) were placed in plastic cylinders similar to those used to measure gross mineralisation and then returned intact for exposure in situ between 14 June and 24 August 1999, and between 6 June and 3 October 2000. The other five samples per plot were taken to the laboratory in the beginning of the field exposure to determine the initial inorganic N and TDN concentration. During the 2000 growing season samples from four plots (total N 1.2, 1.4, 1.8, and 1.9%) were taken on 4 July and 8 August to follow possible seasonal fluctuations in net N mineralisation. Field-exposed cores were taken to the laboratory and the samples were extracted and analysed in the same way as the samples for determination of dissolved N. Net ammonification and nitrification for each period 2 were calculated by subtracting the initial NHþ 4 and NO3 þ 2 concentrations from the final NH4 and NO3 concentrations. Net N mineralisation was calculated as the sum of net ammonification and net nitrification. Negative values for this difference are referred to as net immobilisation (Williams, 1992). The net C mineralisation was calculated as the difference between the post-field exposure and prefield exposure TDC concentrations.
2.5. Statistical analyses Data on seasonal fluctuation and effects of the N gradient and fertilization on dissolved N and C concentrations, ergosterol and Cmic and Nmic were tested with analysis for repeated measures of General Linear Model and linear regression analysis. In the case that the assumption of sphericity was violated (ergosterol), the Greenhouse-Geisser test was used. The N gradient was set as a covariate and fertilisation as a between-subject factor when testing the differences between years, sampling dates and interaction year x date. The average of five subsamples from each plot was used to analyse the mineralisation data. A linear regression model was used to test the influence of the N gradient on net or gross mineralisation, and one-way ANOVA to test the differences between years and fertilisations. All the analyses were made using SPSS 10.0.
3. Results 3.1. Dissolved N and DOC The TDN concentration varied between 0.067 – 0.194 mg g21 DW in the plots. TDN was mainly composed 2 of NH þ 4 and DON (Fig. 2). The extractable NO 3 concentration was usually very low, and often no NO2 3 was detected. The NHþ 4 concentration showed considerable seasonal fluctuation. A significant difference was found between the years in the extractable DOC concentrations (Table 1). A decrease in DOC was observed in July
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Fig. 2. Seasonal fluctuation in 1999 and 2000 in total dissolved N, NHþ 4 , dissolved organic C and N, microbial C and N, ergosterol and microbial C-to-N ratios in the experimental plots ðn ¼ 15Þ:
and August 1999 (Fig. 2), but this was only a temporary change and coincided with a dry period. Concentrations of DON and DOC in the K2SO4 extract decreased significantly along the N gradient, which ranged
from 1.2 to 2.7% N (Fig. 3, Table 1). A negative correlation was found between the N gradient and DON or DOC concentrations (1999: r ¼ 20:667; P , 0:01; r ¼ 20:544; P , 0:05; 2000: r ¼ 20:734; P , 0:01; r ¼ 20:688;
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Table 1 The F-values and significances of differences ðPÞ in the average total dissolved N, NHþ 4 , dissolved organic N and C, microbial C, N and ergosterol concentrations and microbial C-to-N ratio of General Linear Model for repeated measurements between year, sampling dates and interaction year £ date, N gradient and fertilisation ðn ¼ 15Þ Source of variation
Year Dates Year x dates N gradient Fertilisation
df
1 6 6 1 1
F-value TDN
NHþ 4
DON
DOC
Cmic
Nmic
Ergosterol
C-to-N ratio
0.0 2.1 3.3 5.3* 3.5
0.7 1.6 3.8 1.3 5.9*
0.5 1.1 2.1 16.9*** 2.0
29.1*** 2.1 1.9 14.7* 16.2*
4.8* 4.1** 9.0*** 11.6** 1.7
8.9* 4.5** 3.2** 14.8** 2.4
1.1 0.6 0.5 7.0* 0.0
0.1 1.3 0.7 3.3 0.9
P , 0:01). The total peat N concentration had a significant effect on the extractable NHþ 4 concentration at only one sampling: in the beginning of the 1999 growing season when NHþ 4 correlated positively with total N (r ¼ 0:939;
P , 0:001). The total peat N concentration had no significant effect on the extractable DOC-to-DON ratio in this study. Higher NHþ 4 concentrations were found in the non-fertilised plots.
Fig. 3. Dissolved organic N and C, microbial C and N, ergosterol and microbial C-to-N ratio in peat along an N gradient in 1999 and 2000 ðn ¼ 15Þ:
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3.2. Ergosterol, Cmic and Nmic Significant seasonal fluctuation of Cmic and Nmic were observed (Fig. 2, Table 1). There was a significant difference between the two growing seasons, and also interaction between the years and dates. However, no significant variation in the Cmic to Nmic ratio was found (Fig. 2, Table 1). The N gradient significantly affected the ergosterol, Cmic and Nmic, but not the Cmic-to-Nmic ratio (Fig. 3, Table 1). The microbial biomass decreased and ergosterol increased along the N gradient up to a N concentration of over 2%. The correlation between peat total N and the ergosterol or Cmic and Nmic changed to the opposite direction, although
not significantly, when the total N concentration in the peat was over 2% (Fig. 3). Fertilisation did not affect the Cmic, Nmic or ergosterol (Table 1).
3.3. Mineralisation The net N mineralisation rate increased significantly along the N gradient from 1.2 to 1.9% N. This was true for both growing seasons (Fig. 4, Table 2). In 2000 a significant negative correlation was observed between the net C mineralisation rate and peat total N concentration (Fig. 4, Table 2), whereas in 1999 the C mineralisation rates were
Fig. 4. Net C and N mineralisation along N gradient in 1999 and 2000 ðn ¼ 5Þ:
H. Potila, T. Sarjala / Soil Biology & Biochemistry 36 (2004) 1047–1055 Table 2 Coefficients of correlation and significances ðPÞ of linear regression between peat total N concentrations and net N and C mineralisation and gross N mineralisation and immobilisation ðn ¼ 9Þ Year
Net N miner.
Net C miner.
Gross N miner.
Gross N immob.
1999 2000
0.846** 0.828**
NS 20.750*
NS NS
0.721* NS
low and showed no significant correlation with peat N. Fertilisation did not affect N or C mineralisation. The fluctuation in net N mineralisation on four plots with different peat N concentrations in 2000 was measured four times from the beginning of June to October (Fig. 5). In October the cumulative net N mineralisation increased with increasing N concentrations. The dynamics of the mineralisation during the growing season did not show any notable differences between the plots (Fig. 5). The gross N mineralisation rate was greater than immobilisation in both years (Fig. 6). The gross N mineralisation rate in July 2000 was significantly ðP , 0:05Þ higher than that in July 1999 (Fig. 6). The gross N mineralisation did not vary along the N gradient, whereas the gross N immobilisation increased significantly ðP , 0:05Þ along the N gradient in 1999 (Fig. 6, Table 2).
4. Discussion 4.1. Dissolved N The concentrations of dissolved N (mainly NHþ 4 and DON) fluctuated significantly within and between the growing seasons. Seasonal fluctuations in the concentrations of dissolved N in peatland have been reported by Williams (1992) and Williams and Silcock (2000). Their results are only partly in agreement with those of the present study. Owing to the varying weather conditions in different years,
Fig. 5. Cumulative net N mineralisation during the growing season 2000 in peats with four different N concentrations (1.17, 1.42, 1.83, 1.86%) ðn ¼ 5Þ:
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it is not possible to find any consistent patterns in the fluctuation of dissolved N during the growing season. Our study suggests that more active mineralisation of DON by the microbes at higher peat N concentrations, or more efficient uptake of DON by the increasing fungal biomass in the peat along the N gradient, may be the reasons for the negative correlation between extractable DON and total N concentrations in the peat. We observed a positive correlation between the net N mineralisation and N concentration of the peat between 1.2 and 1.9% N. Extractable NHþ 4 did not show any positive correlation with the N gradient except in early June 1999. However, a positive correlation between the net mineralisation and N gradient in the same samples reported here indicates that although more NHþ 4 has been formed at higher peat N concentrations, this has evidently been efficiently utilized by the plants. 4.2. DOC Williams and Silcock (2000) reported significant changes in the concentration of extractable DOC with time, but DOC showed less seasonal variation than DON. They noticed that extractable DOC reached higher values during March and August. In our study the extractable DOC concentrations increased slightly towards autumn, but the seasonal variation was greater than that in DON. Williams and Edwards (1993) postulated that both hydrological factors and the chemical and mineralogical properties of the soil affect DOC concentrations. This is supported by our study, because there was a large temporary decrease in DOC concentrations during July 1999 at the same time as a dry period. Disturbances to fungal mycelia as a result of drying/ wetting events are known to cause mycelial senescence (Pulleman and Tietema, 1999), and this may lead to a mineral N flush. An increase in inorganic N was also observed in our study after the dry period in 1999. According to Gundersen et al. (1998), DOC fluxes are correlated to the amount of litterfall, not to the N status of the sites. In this study the decreasing DOC concentrations along the N gradient may be due to increased consumption of C by the microbes, because N limitation of the microbial community may turn into C limitation as N availability increases (Aber, 1992; Gundersen et al., 1998). This would explain the decline in Nmic concentrations along the N gradient up to a peat N concentration of 2%. Also a decrease in the production of available DOC, when the N concentration in plant litter increases (Aber, 1992), may explain the lower DOC concentrations at higher peat N concentrations. Microbial activity is a key factor affecting N mineralisation and the availability of dissolved N compounds in peat. Lindahl et al. (2002) emphasised in their model that microorganisms, and fungi in particular, play an important role in the transformations of C and nutrients in the soil, as well as the cycling between the atmospheric and plant biomass pool.
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Fig. 6. Gross mineralisation rates and immobilisation along N gradient in July 1999 and 2000 ðn ¼ 5Þ:
4.3. Microbial biomass and activity Microbial biomass values showed both seasonal fluctuations and differences between the years. In the 1999 the microbial biomass decreased from June to a minimum in July, and increased again to the highest value in October. The dry period in July 1999 probably had an adverse effect on the microbial population in the peat, causing a lower gross N mineralisation rate in July 1999 than in 2000. Lund and Goksøyr (1980) reported that disruption of the microbial community, for example as a result of a drying and rewetting event, induced a 20-fold increase in the numbers of culturable bacteria. Furthermore, a 3-fold increase in net N mineralisation in corresponding conditions has been reported by Pulleman and Tietema (1999). Wardle (1998) noted that the temporal variability of Cmic in forest ecosystems was most closely related to the soil N concentration. In our study the Cmic and Nmic decreased along the N gradient up to the value of 2% N, which was also reflected in the extractable DOC and DON concentrations. In our study decreasing decomposition, measured as C mineralisation, along the N gradient followed
the reduction in microbial biomass. The rate of C mineralisation was much less or even turned to immobilisation in 1999 compared to 2000. Possible changes in litter quality along the N gradient may explain the decreasing decomposition because, among other factors, the N or C-toN ratios of litter (Koenig and Cochran, 1994; Scheffer et al., 2001) have been shown to control decomposition. Mycorrhizal fungi, that have access to recent photosynthates from the plant, will not be limited by the availability of organic C in the soil. The concurrent decrease in microbial biomass and increase in fungal biomass up to a peat N concentration of 2% may indicate a competitive advantage for fungi in the microbial community. When the peat total N concentration was more than 2% there was a shift in the opposite direction in the correlations between the N gradient and ergosterol concentration, as well as between the N and Cmic and Nmic. This indicates a change in the balance in the microbial community that was less favourable for the fungi. Also Arnebrant (1994) recognised that the growth of the extramatrical mycelium of an unidentified ectomycorrhizal fungus was totally inhibited once it was exposed to a peat total N concentration of
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2 mg g21, and growth of the extramatrical mycelium decreased at a peat total N concentration above 1 mg g21. Increase in fungal biomass in Scots pine roots along an increasing N gradient up to 2% peat N concentration in drained peatland was reported by Sarjala and Kaunisto (2000). Wallenda et al. (1996) have hypothesized that ectomycorrhizal fungi may become C limited at higher N availabilities, because of the lower amounts of fungusspecific sugars.
5. Conclusions Our study revealed that production and availability of dissolved N forms and microbial biomass in peat in a drained peatland varies considerably during the growing season, but without a consistent pattern. Dissolved inorganic N fluctuated much more than the relatively stable fraction of DON. The total N content of peat affected the dissolved N, especially DON, DOC and microbial biomass and activity, but NHþ 4 in the 0 –10 cm peat layer did not show any correlation. With a higher peat N concentration (. 2%) the fungal biomass declined with a simultaneous increase in microbial biomass C and N. According to our results, the relative importance of bacteria and fungi in the C and N fluxes in drained peatland may vary depending on the peat N concentration.
Acknowledgements We are grateful to Ms Eeva Pihlajaviita, Mr Kari Honka and Ms Helena Railo for technical assistance, and to John Derome and Professor Rauni Stro¨mmer for critical reading of the manuscript. This work was supported by Ministry of Agriculture and Forestry in Finland.
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Seasonal fluctuation in microbial biomass and activity along a natural nitrogen gradient in a drained peatland Hannamaria Potila*, Tytti Sarjala Parkano Research Station, The Finnish Forest Research Institute, Kaironiemente 54, FIN-39700 Parkano, Finland Received 23 June 2003; received in revised form 11 February 2004; accepted 23 February 2004
Abstract The effects of peat total N on the dissolved N and C concentrations and microbial biomass and activity and their range of seasonal fluctuation were studied in a drained peatland forest in Finland. Seasonal fluctuations in the concentrations of extractable dissolved organic (DON) and inorganic nitrogen (DIN) compounds and extractable dissolved organic carbon (DOC), microbial C and N, ergosterol, net and gross N mineralisation rates were investigated during two growing seasons along a natural peat N gradient in a drained peatland. Significant seasonal fluctuations in NHþ 4 and DOC concentrations, microbial C and N, but not in ergosterol or microbial C-to-N ratios in the peat, were observed during the 1999 and 2000 growing seasons. The peat total N concentration affected extractable DON and DOC, but not DIN concentrations in the peat. A negative correlation was found between total N concentration in peat and microbial N and C, and a positive correlation between total N and ergosterol, in peat with N concentrations of up to 2%. Gross mineralisation rates did not show any correlation, whereas net mineralisation rates showed a significant positive correlation with the total N concentration of the peat in both 1999 and 2000. q 2004 Elsevier Ltd. All rights reserved. Keywords: Dissolved N; Dissolved organic carbon; Ergosterol; Microbial biomass; Mineralisation; Peat
1. Introduction There are over 5 million ha of afforested drained mires in Finland. The potential tree growth in these peatland forests depends on the drainage efficiency and peat N content, as well as on the nutrient balance between N and especially K and P (Pa¨iva¨nen and Paavilanen, 1992; Kaunisto and Pietila¨inen, 2003). The majority of the N in the peat is in a bound form and not available for plants. A minor proportion of the N is in soluble form, most of which consists of dissolved organic N (DON) and low concen2 trations of inorganic N, NHþ 4 and NO3 (DIN). Slow mineralisation of soil organic N has earlier been assumed to be the main factor limiting plant growth in boreal forests. However, the uptake of amino acids in field conditions by ectomycorrhizal trees, ericaceous shrubs and herbaceous plants has been demonstrated (Na¨sholm et al., 1998). Thus, the availability of DON, and not only the mineralised inorganic pool, affects the overall N supply for trees. * Corresponding author. Tel.: þ 358-344-352; fax: þ358-344-35200. E-mail address: [email protected] (H. Potila). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.02.014
In the model presented by Lindahl et al. (2002), mineralisation and plant uptake of inorganic nutrients play a less important role. Peat consists of incompletely decomposed organic plant residues. Improved aeration in deeper peat layers after peatland drainage increases the populations of aerobic decomposers and thus enhances the decay of organic matter. Scheffer et al. (2001) studied decomposition and mineralisation rates in Sphagnum- and Carex-dominated peatlands, and concluded that nutrient availability and adaptation of the microbial community to nutritional and other environmental conditions may be the main regulators of C and nutrient transformations in these peatlands. Fungi are believed to be the main decomposers in acid mor soils of the boreal region (Frostega˚rd and Ba˚a˚th, 1996), and their contribution to nutrient transformations in peatland forest soils is dominant. Mycorrhizal plants and non-symbiotic soil micro-organisms may compete for both inorganic and organic soil N, since they potentially use the same N sources. Biotic immobilisation of N by free-living microbes requires a supply of available DOC produced by the decomposition
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of plant litter. According to Aber (1992), an increasing N concentration in plant litter reduces available DOC production and increases the utilization of existing pools of DOC. On peatlands, which have a high C content in the substrate, the factors controlling DON and DOC fluxes are poorly known. Our aim was to determine the seasonal fluctuation of DOC, dissolved N compounds and microbial biomass in peat. An additional aim was to investigate the influence of peat total N on dissolved N and C concentrations, microbial biomass and activity.
2.2. Dissolved N and DOC
2. Material and methods
Extractable dissolved N and C were determined by extracting 10 g of fresh sample with 100 ml of 0.5 M K2SO4 (2 h shaking), and filtering the suspension through filter paper and further through a 0.45 mm membrane filter (Williams et al., 1995). The extract was frozen (2 20 8C) 2 until analysis. Total dissolved N (TDN), NHþ 4 and NO3 in the extracts were analysed by flow injection analysis (FIA Star 5020, Tecator). DON in the extracts was obtained by 2 deducting the NHþ 4 and NO3 -N concentration from TDN. DOC in the extract was analysed on a TOC-5000 analyser (Schimadzu). The moisture content (overnight at 105 8C) of the peat samples was also determined.
2.1. Sampling
2.3. Ergosterol, Cmic and Nmic
Fluctuations in the concentrations of soluble nitrogen 2 compounds (NH þ 4 , NO 3 , DON), DOC, ergosterol, microbial N (Nmic) and C (Cmic) were followed during the 1999 and 2000 growing seasons in a field experiment (62890 N, 228520 E) in a Scots pine stand in Western Finland. The field experiment (109 A and B) was established by the Finnish Forest Research Institute in 1974 (Kaunisto, 1982; Kaunisto et al., 1986) on a mire drained in 1969. Five non-fertilised and 10 PK-fertilised plots (42 kg P ha21, 50 kg K ha21) were chosen to represent a natural gradient in the total N concentration of the peat (1.2 – 2.7% N). An influx of spring water from surrounding mineral soils before drainage of the mire has created a natural N gradient in this area. There were only minor differences between the plots in the C content of the peat, which ranged from 45.4 to 50.5%. Therefore, N was the determinant of the peat C-to-N ratio, which varied from 17 to 42. The plots were afforested with Scots pine in 1973, but the non-fertilized plots are now almost treeless. The thickness of the peat layer varied between 0.34 and 2 m. The average pH of the peat was 4.3 ^ 0.2 in the nonfertilised plots and 4.1 ^ 0.2 in the PK-fertilised plots. The effect of PK fertilisation 30 years ago has now disappeared from the peatland, because there were no significant differences in the P and K concentrations between the non-fertilised and PK-fertilised plots. Samples were collected from the 0 – 10 cm peat layer on eight occasions between 18 May and 8 October, 1999, and between 23 May and 3 October, 2000. On the first sampling date in summer 1999 only the non-fertilised plots were sampled because the other plots were still frozen. Five cores were taken from the acrotelm with an auger (6 £ 6 cm), and bulked to give one composite sample per plot. The mean daily temperature and precipitation in the area during the two growing seasons are presented in Fig. 1. In 1999 there was a dry period during 5 – 18 July. The effective temperature sum (threshold þ 5 8C) in the area in 1999 (1139 d.d.) and in 2000 (1136 d.d.) was close to the longterm average (1110 d.d.).
The peat ergosterol concentration was determined by a method modified from Nylund and Wallander (1992) consisting of ethanol extraction, saponification with KOH and further extraction in pentane, followed by HPLC with a UV – VIS detector (Merck-Hitachi) (wavelength 280 nm), LiCrospher 100 RP-18 column and methanol as eluent. Cmic and Nmic were determined from fresh peat samples with the fumigation-extraction (FE) method by fumigating 10 g of fresh sample for 18 h in chloroform vapour (Brookes et al., 1985), followed by extraction and analysis in the same way as dissolved N and DOC. Nmic was calculated from the difference between fumigated and fresh concentrations of TDN using the value 0.54 for kN (Brookes et al., 1985). Cmic was obtained by deducting the DOC concentration of the fresh peat sample from the DOC concentration of the fumigated peat sample using the recovery factor of 0.45 derived for peat soils by Sparling et al. (1990). 2.4. Mineralisation The gross N mineralisation in the field was measured on nine plots (five sampling points per plot), using the 15N isotopic dilution technique in soil cores (Davidson et al., 1991). The total N concentration in the peat in these plots varied from 1.2 to 1.9%. Two samples were taken at each sampling point, and 10 ml of a dilute (15NH4)2SO4 solution, containing 20 mg (NH4)2SO4 98% atom% 15N (SigmaAldrich) l-1 distilled water, was injected into each sample. The other subsample was taken to the laboratory and kept at þ 5 8C until the next day. The other subsample was incubated for 3 d between 19 and 22 July 1999, and 10 and 13 July 2000 in the field in a plastic cylinder (105 cm3 volume) sealed at the top and bottom with PVC lids and inserted in the peat layer. Four holes in the upper part of the tubes were made for aeration. Ammonium was extracted by shaking 20 g peat with 100 ml of 0.5 M K2SO4 for 2 h and filtering the suspension through filter paper and further through a 0.45 mm membrane filter. For the 15N analyses, 10 ml of extract
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Fig. 1. The mean daily precipitation (mm) (bars) and temperature (8C) (lines) during the 1999 and 2000 growing seasons at a weather station of the Finnish Meteorological Institute 6 km from the experimental area in Alkkia.
and ca. 500 mg MgO powder were reacted for 3 d at þ 30 8C in airtight plastic jars together with an open vial containing 10 mM H2SO4. The sulphuric acid was dried at þ 45 8C. The 15N/14N ratio was determined by mass spectrometry at MTT Agrifood Research Finland, and the gross N mineralisation rates were calculated according to the equations presented in Kirkham and Bartholomew (1954) and Davidson et al. (1991). Net N and C mineralisation were measured in the field in the same plots as gross N mineralisation (total N 1.2– 1.9%). Five samples per plot (total of nine plots) were placed in plastic cylinders similar to those used to measure gross mineralisation and then returned intact for exposure in situ between 14 June and 24 August 1999, and between 6 June and 3 October 2000. The other five samples per plot were taken to the laboratory in the beginning of the field exposure to determine the initial inorganic N and TDN concentration. During the 2000 growing season samples from four plots (total N 1.2, 1.4, 1.8, and 1.9%) were taken on 4 July and 8 August to follow possible seasonal fluctuations in net N mineralisation. Field-exposed cores were taken to the laboratory and the samples were extracted and analysed in the same way as the samples for determination of dissolved N. Net ammonification and nitrification for each period 2 were calculated by subtracting the initial NHþ 4 and NO3 þ 2 concentrations from the final NH4 and NO3 concentrations. Net N mineralisation was calculated as the sum of net ammonification and net nitrification. Negative values for this difference are referred to as net immobilisation (Williams, 1992). The net C mineralisation was calculated as the difference between the post-field exposure and prefield exposure TDC concentrations.
2.5. Statistical analyses Data on seasonal fluctuation and effects of the N gradient and fertilization on dissolved N and C concentrations, ergosterol and Cmic and Nmic were tested with analysis for repeated measures of General Linear Model and linear regression analysis. In the case that the assumption of sphericity was violated (ergosterol), the Greenhouse-Geisser test was used. The N gradient was set as a covariate and fertilisation as a between-subject factor when testing the differences between years, sampling dates and interaction year x date. The average of five subsamples from each plot was used to analyse the mineralisation data. A linear regression model was used to test the influence of the N gradient on net or gross mineralisation, and one-way ANOVA to test the differences between years and fertilisations. All the analyses were made using SPSS 10.0.
3. Results 3.1. Dissolved N and DOC The TDN concentration varied between 0.067 – 0.194 mg g21 DW in the plots. TDN was mainly composed 2 of NH þ 4 and DON (Fig. 2). The extractable NO 3 concentration was usually very low, and often no NO2 3 was detected. The NHþ 4 concentration showed considerable seasonal fluctuation. A significant difference was found between the years in the extractable DOC concentrations (Table 1). A decrease in DOC was observed in July
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Fig. 2. Seasonal fluctuation in 1999 and 2000 in total dissolved N, NHþ 4 , dissolved organic C and N, microbial C and N, ergosterol and microbial C-to-N ratios in the experimental plots ðn ¼ 15Þ:
and August 1999 (Fig. 2), but this was only a temporary change and coincided with a dry period. Concentrations of DON and DOC in the K2SO4 extract decreased significantly along the N gradient, which ranged
from 1.2 to 2.7% N (Fig. 3, Table 1). A negative correlation was found between the N gradient and DON or DOC concentrations (1999: r ¼ 20:667; P , 0:01; r ¼ 20:544; P , 0:05; 2000: r ¼ 20:734; P , 0:01; r ¼ 20:688;
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Table 1 The F-values and significances of differences ðPÞ in the average total dissolved N, NHþ 4 , dissolved organic N and C, microbial C, N and ergosterol concentrations and microbial C-to-N ratio of General Linear Model for repeated measurements between year, sampling dates and interaction year £ date, N gradient and fertilisation ðn ¼ 15Þ Source of variation
Year Dates Year x dates N gradient Fertilisation
df
1 6 6 1 1
F-value TDN
NHþ 4
DON
DOC
Cmic
Nmic
Ergosterol
C-to-N ratio
0.0 2.1 3.3 5.3* 3.5
0.7 1.6 3.8 1.3 5.9*
0.5 1.1 2.1 16.9*** 2.0
29.1*** 2.1 1.9 14.7* 16.2*
4.8* 4.1** 9.0*** 11.6** 1.7
8.9* 4.5** 3.2** 14.8** 2.4
1.1 0.6 0.5 7.0* 0.0
0.1 1.3 0.7 3.3 0.9
P , 0:01). The total peat N concentration had a significant effect on the extractable NHþ 4 concentration at only one sampling: in the beginning of the 1999 growing season when NHþ 4 correlated positively with total N (r ¼ 0:939;
P , 0:001). The total peat N concentration had no significant effect on the extractable DOC-to-DON ratio in this study. Higher NHþ 4 concentrations were found in the non-fertilised plots.
Fig. 3. Dissolved organic N and C, microbial C and N, ergosterol and microbial C-to-N ratio in peat along an N gradient in 1999 and 2000 ðn ¼ 15Þ:
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3.2. Ergosterol, Cmic and Nmic Significant seasonal fluctuation of Cmic and Nmic were observed (Fig. 2, Table 1). There was a significant difference between the two growing seasons, and also interaction between the years and dates. However, no significant variation in the Cmic to Nmic ratio was found (Fig. 2, Table 1). The N gradient significantly affected the ergosterol, Cmic and Nmic, but not the Cmic-to-Nmic ratio (Fig. 3, Table 1). The microbial biomass decreased and ergosterol increased along the N gradient up to a N concentration of over 2%. The correlation between peat total N and the ergosterol or Cmic and Nmic changed to the opposite direction, although
not significantly, when the total N concentration in the peat was over 2% (Fig. 3). Fertilisation did not affect the Cmic, Nmic or ergosterol (Table 1).
3.3. Mineralisation The net N mineralisation rate increased significantly along the N gradient from 1.2 to 1.9% N. This was true for both growing seasons (Fig. 4, Table 2). In 2000 a significant negative correlation was observed between the net C mineralisation rate and peat total N concentration (Fig. 4, Table 2), whereas in 1999 the C mineralisation rates were
Fig. 4. Net C and N mineralisation along N gradient in 1999 and 2000 ðn ¼ 5Þ:
H. Potila, T. Sarjala / Soil Biology & Biochemistry 36 (2004) 1047–1055 Table 2 Coefficients of correlation and significances ðPÞ of linear regression between peat total N concentrations and net N and C mineralisation and gross N mineralisation and immobilisation ðn ¼ 9Þ Year
Net N miner.
Net C miner.
Gross N miner.
Gross N immob.
1999 2000
0.846** 0.828**
NS 20.750*
NS NS
0.721* NS
low and showed no significant correlation with peat N. Fertilisation did not affect N or C mineralisation. The fluctuation in net N mineralisation on four plots with different peat N concentrations in 2000 was measured four times from the beginning of June to October (Fig. 5). In October the cumulative net N mineralisation increased with increasing N concentrations. The dynamics of the mineralisation during the growing season did not show any notable differences between the plots (Fig. 5). The gross N mineralisation rate was greater than immobilisation in both years (Fig. 6). The gross N mineralisation rate in July 2000 was significantly ðP , 0:05Þ higher than that in July 1999 (Fig. 6). The gross N mineralisation did not vary along the N gradient, whereas the gross N immobilisation increased significantly ðP , 0:05Þ along the N gradient in 1999 (Fig. 6, Table 2).
4. Discussion 4.1. Dissolved N The concentrations of dissolved N (mainly NHþ 4 and DON) fluctuated significantly within and between the growing seasons. Seasonal fluctuations in the concentrations of dissolved N in peatland have been reported by Williams (1992) and Williams and Silcock (2000). Their results are only partly in agreement with those of the present study. Owing to the varying weather conditions in different years,
Fig. 5. Cumulative net N mineralisation during the growing season 2000 in peats with four different N concentrations (1.17, 1.42, 1.83, 1.86%) ðn ¼ 5Þ:
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it is not possible to find any consistent patterns in the fluctuation of dissolved N during the growing season. Our study suggests that more active mineralisation of DON by the microbes at higher peat N concentrations, or more efficient uptake of DON by the increasing fungal biomass in the peat along the N gradient, may be the reasons for the negative correlation between extractable DON and total N concentrations in the peat. We observed a positive correlation between the net N mineralisation and N concentration of the peat between 1.2 and 1.9% N. Extractable NHþ 4 did not show any positive correlation with the N gradient except in early June 1999. However, a positive correlation between the net mineralisation and N gradient in the same samples reported here indicates that although more NHþ 4 has been formed at higher peat N concentrations, this has evidently been efficiently utilized by the plants. 4.2. DOC Williams and Silcock (2000) reported significant changes in the concentration of extractable DOC with time, but DOC showed less seasonal variation than DON. They noticed that extractable DOC reached higher values during March and August. In our study the extractable DOC concentrations increased slightly towards autumn, but the seasonal variation was greater than that in DON. Williams and Edwards (1993) postulated that both hydrological factors and the chemical and mineralogical properties of the soil affect DOC concentrations. This is supported by our study, because there was a large temporary decrease in DOC concentrations during July 1999 at the same time as a dry period. Disturbances to fungal mycelia as a result of drying/ wetting events are known to cause mycelial senescence (Pulleman and Tietema, 1999), and this may lead to a mineral N flush. An increase in inorganic N was also observed in our study after the dry period in 1999. According to Gundersen et al. (1998), DOC fluxes are correlated to the amount of litterfall, not to the N status of the sites. In this study the decreasing DOC concentrations along the N gradient may be due to increased consumption of C by the microbes, because N limitation of the microbial community may turn into C limitation as N availability increases (Aber, 1992; Gundersen et al., 1998). This would explain the decline in Nmic concentrations along the N gradient up to a peat N concentration of 2%. Also a decrease in the production of available DOC, when the N concentration in plant litter increases (Aber, 1992), may explain the lower DOC concentrations at higher peat N concentrations. Microbial activity is a key factor affecting N mineralisation and the availability of dissolved N compounds in peat. Lindahl et al. (2002) emphasised in their model that microorganisms, and fungi in particular, play an important role in the transformations of C and nutrients in the soil, as well as the cycling between the atmospheric and plant biomass pool.
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Fig. 6. Gross mineralisation rates and immobilisation along N gradient in July 1999 and 2000 ðn ¼ 5Þ:
4.3. Microbial biomass and activity Microbial biomass values showed both seasonal fluctuations and differences between the years. In the 1999 the microbial biomass decreased from June to a minimum in July, and increased again to the highest value in October. The dry period in July 1999 probably had an adverse effect on the microbial population in the peat, causing a lower gross N mineralisation rate in July 1999 than in 2000. Lund and Goksøyr (1980) reported that disruption of the microbial community, for example as a result of a drying and rewetting event, induced a 20-fold increase in the numbers of culturable bacteria. Furthermore, a 3-fold increase in net N mineralisation in corresponding conditions has been reported by Pulleman and Tietema (1999). Wardle (1998) noted that the temporal variability of Cmic in forest ecosystems was most closely related to the soil N concentration. In our study the Cmic and Nmic decreased along the N gradient up to the value of 2% N, which was also reflected in the extractable DOC and DON concentrations. In our study decreasing decomposition, measured as C mineralisation, along the N gradient followed
the reduction in microbial biomass. The rate of C mineralisation was much less or even turned to immobilisation in 1999 compared to 2000. Possible changes in litter quality along the N gradient may explain the decreasing decomposition because, among other factors, the N or C-toN ratios of litter (Koenig and Cochran, 1994; Scheffer et al., 2001) have been shown to control decomposition. Mycorrhizal fungi, that have access to recent photosynthates from the plant, will not be limited by the availability of organic C in the soil. The concurrent decrease in microbial biomass and increase in fungal biomass up to a peat N concentration of 2% may indicate a competitive advantage for fungi in the microbial community. When the peat total N concentration was more than 2% there was a shift in the opposite direction in the correlations between the N gradient and ergosterol concentration, as well as between the N and Cmic and Nmic. This indicates a change in the balance in the microbial community that was less favourable for the fungi. Also Arnebrant (1994) recognised that the growth of the extramatrical mycelium of an unidentified ectomycorrhizal fungus was totally inhibited once it was exposed to a peat total N concentration of
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2 mg g21, and growth of the extramatrical mycelium decreased at a peat total N concentration above 1 mg g21. Increase in fungal biomass in Scots pine roots along an increasing N gradient up to 2% peat N concentration in drained peatland was reported by Sarjala and Kaunisto (2000). Wallenda et al. (1996) have hypothesized that ectomycorrhizal fungi may become C limited at higher N availabilities, because of the lower amounts of fungusspecific sugars.
5. Conclusions Our study revealed that production and availability of dissolved N forms and microbial biomass in peat in a drained peatland varies considerably during the growing season, but without a consistent pattern. Dissolved inorganic N fluctuated much more than the relatively stable fraction of DON. The total N content of peat affected the dissolved N, especially DON, DOC and microbial biomass and activity, but NHþ 4 in the 0 –10 cm peat layer did not show any correlation. With a higher peat N concentration (. 2%) the fungal biomass declined with a simultaneous increase in microbial biomass C and N. According to our results, the relative importance of bacteria and fungi in the C and N fluxes in drained peatland may vary depending on the peat N concentration.
Acknowledgements We are grateful to Ms Eeva Pihlajaviita, Mr Kari Honka and Ms Helena Railo for technical assistance, and to John Derome and Professor Rauni Stro¨mmer for critical reading of the manuscript. This work was supported by Ministry of Agriculture and Forestry in Finland.
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