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The decomposition of

Lolium perenne

in soils exposed to elevated

CO

2

: comparisons of mass loss of litter with soil respiration and

soil microbial biomass

Alwyn Sowerby

a

, Herbert Blum

b

, Tim R.G. Gray

a

, Andrew S. Ball

a,

*

a

Department of Biological Sciences, John Tabor Laboratories, Essex University, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK

b

Department of Plant Sciences, Swiss Federal Institute of Science, Eschikon Research Station, Eschikon, Lindau CH 8315, Switzerland

Received 15 June 1999; received in revised form 10 December 1999; accepted 21 February 2000

Abstract

Two key questions regarding the eects of elevated atmospheric CO2on soil microbial biomass are, (a) will future levels of

elevated CO2 aect the amount of microbial biomass in soil? and (b) how will any observed changes impact on C-¯ux from

soils? These questions were addressed by examining soil microbial biomass, and in situ estimations of soil respiration in grassland soils exposed to free air carbon dioxide enrichment (60 Pa). Corresponding measurements of plant litter mass loss were taken using litter bags, ensuring that ambient litter was decomposed in ambient soil, and elevated CO2 grown litter was decomposed in soils exposed to elevated CO2. Signi®cantly greater levels of microbial biomass (p< 0.05, paired t-test) were detected in soils exposed to elevated CO2(1174.1 compared to 878.9 mg N gÿ1 dry soil for ambient CO2 exposed soils). This corresponded with a signi®cant increase (p< 0.005, paired t-test) in in situ soil respiration from the elevated CO2acclimatised soils (28.7 compared to 20.4 mmol CO2 m2 hÿ1 from soils exposed to ambient CO2). However, when soil respiration was calculated per unit of microbial biomass, no dierences in activity per unit biomass were detected (approx. 0.02mmol CO2 m2 hÿ1 _{unit biomass}ÿ1_{), suggesting that increased soil microbial biomass, rather than increased activity was responsible for the} observed dierences. The mass loss of litter was greater in the elevated CO2acclimatised soils (p< 0.05, ANOVA), even though the initial nutrient ratios of the litter were not signi®cantly dierent.72000 Elsevier Science Ltd. All rights reserved.

Keywords:Decomposition; Elevated CO2; Microbial biomass; Soil; Soil respiration;Lolium perenne

1. Introduction

Resource-balance models predict that plants respond to a higher availability of above-ground resources by increasing the relative allocation to nutrient acquisition (Garnier, 1991). Indeed it has been demonstrated that elevated CO2 favours investment of biomass in roots (and their exudates) relative to that in leaves (Stulen and Den Hertog, 1993), especially when plant growth is nutrient limited. Therefore, the most pronounced

plant responses to elevated atmospheric CO2 concen-tration are probably going to be below-ground (Rogers et al., 1994).

It is not thought that elevated atmospheric CO2will have any direct impacts on the soil community, as the concentration of CO2within the soil atmosphere is suf-®ciently high to not be aected by the predicted increase in atmospheric CO2(O'Neill, 1994). However, there are several indirect eects which may impact on the soil community which could potentially alter rates of carbon turnover in the terrestrial ecosystem. Increased root production could result in an increase in the C allocated below-ground (Norby, 1994). The consequences of increased C input to the soil commu-nity from roots remains unknown. A more

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* Corresponding author. Tel.: 873332; fax: +44-1206-873416.

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ively researched indirect eect of elevated CO2 is that of altered litter quality entering the decomposition pro-cess eecting the rate of decomposition, and therefore, nutrient turnover. A general dilution of nitrogen within plant tissues when grown in elevated CO2 has often been observed. In a meta-analysis of the eects of elevated CO2 on the concentration of nitrogen in over 75 species of plants, Cotrufo et al. found an over-all mean decrease of 14% when plants were grown in elevated CO2 (Cotrufo et al., 1998a, 1998b). The rela-tive composition of other compounds, such as lignin has also been aected by the concentration of atmos-pheric CO2; however, a change in the composition of these compounds has been far less consistent (Penuelas and Estiarte, 1998). However, Norby and Cotrufo (1998) state that there is sucient evidence now to dis-count this potential eect. A more complete review of the hypotheses surrounding the potential direct and indirect impacts of elevated CO2on below-ground pro-cesses can be found in O'Neill (1994).

Previous work looking at the eects of elevated CO2 on the decomposition of litter has generally focused on only one aspect of the decomposition process. For example a number of papers have reported dierences in the mass loss of litter when originally grown in elev-ated CO2 (Cotrufo and Ineson, 1996; Cotrufo et al., 1998a). However, without further investigation into where nutrients lost from the litter are allocated to in the soil community, it is not clear how these dier-ences will impact on nutrient cycling. As yet, no pub-lished work has considered the eects of elevated atmospheric CO2on the decomposition of litter as well as the corresponding soil microbial biomass. Measure-ments of soil microbial biomass alone have also varied, although a trend is observed towards greater microbial biomass under elevated CO2 when estimated by fumi-gation or SIR techniques (Kampichler et al., 1998).

Our objectives with this work were to determine the eects of elevated CO2 on the decomposition of

Lolium perenne litter along with corresponding measurements of soil respiration and microbial bio-mass.

2. Materials and methods

2.1. Site and sampling

The Swiss free air carbon dioxide enrichment (FACE) facility, at Eschikon, Switzerland, examines the eects of elevated CO2on a grassland system. The site consists of six FACE rings, three of which are at ambient levels of CO2, three of which have raised levels of CO2 (60 Pa CO2; 1-min average of 60 Pa2 10% within 92% of the fumigated time, Zanetti et al., 1996). Each of the treatments at the site has undergone

identical crop rotations before the establishment of the FACE experiment, which began in May 1993. The soil in the FACE rings is a fertile, eutric cambisol. Further information regarding the site, including all protocols involved, can be found in Zanetti et al. (1996).

Litter used in this work was harvested from plots sown in monoculture with L. perenne in 1996. Since then the plots have been cut and fertilised eight times a year (NH4NO3; 560 kg N haÿ1yearÿ1). Aerial litter samples were cut during May 1998 and oven dried at 658C.

During July 1998, intact soil cores were taken directly into plastic cylinders (dimensions: 7 cm 20

cm), at random from within plots in each of the FACE rings. Mature L. perenne plants were extracted as well as soil, to produce replica mesocosms for the decomposition of litter. Fig. 1 shows a diagrammatical representation of the intact cores, which were kept within the FACE ring they were extracted from for the duration of the experiment. Twenty-four soil cores were taken in each ring. The soil cores were left for 1 week after extraction to equilibrate before any proto-cols were followed.

2.2. Mass loss of litter in the ®eld

Above-ground litter (0.5±0.8 g, harvested May 1998)

Fig. 1. Diagrammatical representation of the intact Lolium perenne

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was weighed, then placed in mesh bags (mesh size 1

mm, dimensions 5 cm 7 cm). The bags were then

placed into the top layers of the soil in the intact soil cores within the plots in the Swiss FACE rings. Replica bags were removed sequentially throughout the duration of the experiment to measure the mass loss of litter from the litter bags. Litter was decom-posed in the soil it was originally grown in, ensuring that litter grown in elevated CO2 was decomposed in soil acclimatised to elevated CO2. Similarly, litter

orig-inally grown in ambient CO2 was decomposed in

ambient soils.

2.3. Chemical composition of litter

Three individual leaves (taken as the part of the leaf from the base of the stem) from each sample used in the litter bag experiment outlined above, were ground separately by pestle and mortar, then transferred to a foil capsule of a known weight. Percentage compo-sition of carbon and nitrogen, and the carbon to nitro-gen ratio (C:N ratio) of the litter sample was then measured using an automated CHNS/O analyser (Per-kin Elmer).

As well as measurements of mass loss of litter from the litter bags, percentage carbon, nitrogen and the C:N ratio were measured from the remaining litter in the bags, after the extractions of the litter bags from the soil. This was performed on four of the extraction dates.

Polymeric composition of the samples was estimated by sequential extraction and gravimetric analysis. In this case, six leaves were taken from each sample and cut into 1 cm segments before analysis. Full exper-imental protocols are given in Harper and Lynch (1981).

2.4. Soil respiration

Soil respiration measurements were taken in both the morning and afternoon during July and August 1998 using a portable gas monitor (EGM-1, PP Sys-tems) linked to a respiration chamber (diameter 10 cm; soil respiration chamber, PP Systems) at the Swiss FACE site. The grass sward (including roots) was removed from plots (approximate dimensions: 0.5 m

1 m) to avoid the inclusion of root/rhizosphere respir-ation. The root-free soil was then left to equilibrate for a few days until the readings of soil respiration stabil-ised. Measurements were taken at random by placing the respiration chamber over a section of soil for ap-proximately 5 min. Respiratory activity was calculated from the CO2 accumulation rate within the chamber, as described by the manufacturers (Anon, 1990), and expressed asmmol CO2m

2 hÿ1_.

2.5. Microbial biomass

Immediately after each measurement of soil respir-ation, soil samples were taken using a soil corer (30

cm 3 cm) and placed into sealed plastic bags.

Samples were temporarily stored at 48C before the esti-mation of microbial biomass (1 h maximum time before fumigation). On each occasion, three replicates were taken from each of the plots (in each of the FACE rings), giving a total number of nine replicates for each of the treatments. Microbial biomass-N was determined in the samples taken above. First the whole core taken by the soil corer (30 cm3 cm) was

mixed then two sub-samples (10 g) of the soil were used in a fumigation-extraction method of measuring microbial biomass, as outlined in Rowell (1995).

2.6. Statistical analysis

Mass loss of litter from litter bags was analysed using two way analysis of variance (ANOVA). Dier-ences between the chemical composition of the elev-ated CO2 and ambient grown litter were identi®ed using t-tests.

In the statistical analysis FACE rings were paired (i.e. C1 with F1, C2 with F2 and C3 with F3), as analysis of the soil in the FACE rings in a previous report showed dierences in basic soil chemistry (Nitschelm, 1996).

Fig. 2. Mass loss of litter from litter bags containing litter cut from swards in ambient and elevated CO2 (60 Pa CO2) rings. Ambient

grown litter was decomposed in ambient soil, elevated CO2 grown

litter in elevated CO2-acclimatised soil. Error bars show the standard

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3. Results

3.1. Chemical composition of harvested litter

The percentage carbon and nitrogen content, and therefore, the C:N ratio of L. perenne litter harvested in May 1996 from plants grown in elevated and ambi-ent CO2 was not statistically dierent (C:N 13:1 and 15:1 for ambient and elevated CO2-derived litters, re-spectively, Table 1). The percentage composition of the hemi-cellulose and lignin content was also not signi®-cantly dierent between ambient and elevated CO2 grown litters (Table 1). However, dierences were observed in the soluble and cellulose/ash fractions of the litter (Table 1).

A comparison of the mass loss of L. perenne litter from litter bags showed statistically signi®cant dier-ences (Fig. 2; two way ANOVA,F= 17.53, p< 0.01). The litter originally grown in the elevated CO2 was found to have consistently greater rates of decompo-sition than the litter grown and decomposed in the ambient soils. This pattern was seen throughout the decomposition period, showing that the length of time during which the litter was in the soil did not change the eect of the elevated CO2on the decomposition of the litter (Two way ANOVA, F= 0.167, not signi®-cant).

3.3. Chemical composition of decomposing litter

Analysis of decomposing litter showed a decline in the C:N ratios of the litter until ratios levelled out at approximately 10:1 (Fig. 3). The decline in the C:N ratio of the elevated CO2grown litter was slower than the ambient litter (taking approximately 20 and 30 days, respectively). The dierences observed in the loss of carbon and nitrogen from the litter were mainly seen between day 5 and 25 in the soil. Interestingly, this does not correspond to the dierences observed in

mass loss between the two litter types (Fig. 2), where signi®cant dierences were mainly observed in the ®rst 5 days and then later on after approximately 35 days.

Microbial biomass was measured during 3 days in July and August 1998. Comparing ambient and elev-ated CO2 rings for dierences in microbial biomass showed elevated CO2-acclimatised soils to have a sig-ni®cantly greater biomass than ambient soils (Paired

t-test p < 0.05; elevated CO2 exposed soil

mean=1174.072129.0 mg N gÿ1

soil, ambient CO2 exposed soil mean=878.94 276.58 mg N gÿ1

soil). Readings of microbial biomass did dier between the dates; ranging from 641.9 to 1151.9 mg N gÿ1 _{soil in}

the control soil samples, and from 959.7 to 1785.9 mg N gÿ1 _{soil in the elevated CO}

2soils. However, the

el-Table 1

Chemical analysis of litter grown in ambient and elevated CO2FACE rings in Switzerlanda

Ambient grown litter Elevated CO2grown litter Signi®cance level

% Carbon 45.28 45.72 n.s.

% Nitrogen 3.53 3.29 n.s.

C:N 13.2:1 14.7:1 n.s.

Soluble fraction 35.38 39.94

Hemicellulose 27.97 29.77 n.s.

Cellulose and ash 32.43 26.88

Lignin 4.22 3.41 n.s.

Lignin:N 1.2:1 1:1 Not applicable

a_{Signi®cance is reported on t-tests,}_p_{< 0.05,}_p_{< 0.01,}_p_{< 0.005.}

Fig. 3. C:N ratio of litter remaining in the litter bags decomposing in ambient and elevated CO2 (60 Pa CO2) acclimatised soils at the

Swiss FACE site. Ambient grown litter was decomposed in ambient soil, elevated CO2 grown litter in elevated CO2-acclimatised soil.

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evated CO2 soils consistently held greater microbial biomass.

Measurements of soil respiration showed that ambi-ent soil on average produced 20.4 mmol CO2 m

2 hÿ1_,

elevated CO2-acclimatised soil produced on average 28.7mmol CO2m2hÿ1. This shows signi®cantly greater levels of soil respiration from elevated CO2 -acclimat-ised soils than from ambient soils (paired t-test; p< 0.005), when compared on individual sample dates.

However, when soil respiration was calculated per unit of microbial biomass, ambient soils produced on average 0.02 mmol CO2 hÿ1 unit biomass-Nÿ1, (stan-dard error; 3.910ÿ3), with elevated CO2-acclimatised soils producing on average 0.03 mmol CO2 hÿ1 unit biomass-Nÿ1

(standard error; 4.910ÿ3). These values

showed no statistically signi®cant dierences in CO2 production per unit of microbial biomass between ambient and elevated CO2 acclimatised soil samples (paired t-test).

4. Discussion

4.1. Chemical composition of litter

The chemical analysis of the litter used in the mass loss work showed no signi®cant dierences in carbon and nitrogen content, although a trend towards greater C:N ratios were observed in elevated CO2 grown lit-ters. Early predictions, based on green leaf chemistry, estimated that elevated atmospheric CO2would reduce nitrogen in senesced plant material (O'Neill and Norby, 1996). However, this has not been realised, as these initial predictions did not consider the resorption of leaf constituents before senescence (Norby and Cotrufo, 1998), or dierences in plant chemistry when growth is restricted by pots, etc. (O'Neill and Norby, 1996). It is generally agreed that the C:N ratio of litter is not greater when plants are grown in elevated CO2 (Walker and Steen, 1997). The results from our work con®rm these ®ndings.

The majority of published results have focused on the decomposition of litter originally grown in elev-ated CO2, in ambient soils. Previous research has pro-vided variable results. When litter grown in elevated CO2 is decomposed in soil not exposed previously to elevated CO2 either no dierence in mass loss was observed (Kemp et al., 1994; O'Neill and Norby, 1996), or a decline in the mass loss of litter was seen

(Cotrufo et al., 1994; Cotrufo and Ineson, 1995; Cotrufo and Ineson, 1996; Cotrufo et al., 1998b).

Although no direct eects of elevated CO2 are expected on the soil community, indirect eects may become increasingly important and could potentially amplify to the extent of altering soil community struc-ture and functions. In contrast to the majority of papers, our data shows increased rates of mass loss when litter was grown and decomposed in soils exposed to elevated CO2. Other work reporting the de-composition of litter originally grown in elevated CO2 in soil exposed to elevated CO2 has not shown many signi®cant dierences (Hirschel et al., 1997; Gahrooee, 1998). However, looking at the data presented by Gah-rooee (1998), for one of the species monitored,Quercus cerris, a clear elevated CO2 eect was observed with the soil exposed to elevated levels of CO2. Here soils exposed to elevated CO2 showed a consistent decline in the mass loss of litter when compared to soils

exposed to ambient conditions. This eect was

observed for both the ambient grown litter and the el-evated CO2grown litter (Gahrooee, 1998).

4.3. Chemical composition of decomposing litter

Although the litters had similar C:N ratios when entering the soil, almost as soon as the decompo-sition process began, dierences arose in the C:N ratio of the litters until the C:N ratio of the litter reached approximately 10:1, suggesting that the in-itial decomposition of the litter did not occur in the same manner. Whether this was the response of an altered decomposer community, or some other factor is not clear. Dierences seen in the mass loss of the litter continued throughout the decomposition period. However, after the initial 20±30 days, con-sistent signi®cant dierences were not seen in the C:N ratio of the dierent litters as they decom-posed. This suggests that the C:N ratio is not a good indicator of the nitrogen or carbon dynamics of decaying plant material, which is veri®ed by Wagener and Schimel (1998), who studied the tem-poral and spatial decomposition of litter on a Alas-kan birch forest ¯oor.

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In our study elevated CO2exposed soil samples had consistently greater microbial biomass than ambient soil samples. Several other published results have observed increased microbial biomass in elevated CO2 acclimatised soils (Diaz et al., 1993; Zak et al., 1993; Schenk et al., 1995). Other published results have not shown an increase in the soil microbial biomass with elevated CO2 (Kampichler et al., 1998; Runion et al., 1994). However, the results presented in Kampichler et al. (1998), are from containers within a controlled en-vironment exposed to elevated CO2, and in Runion et al. (1994), microbial biomass was estimated using di-lution plate counts, a method notoriously biased when considering the microbial community in soil.

Diaz et al. (1993) hypothesised that the extra carbon resulting from elevated levels of atmospheric CO2, will be allocated to the microbial biomass in soil and will bring about carbon and nutrient accumulation in soil organic matter. With this hypothesis, increased carbon inputs will stay within the soil system and not be returned to the atmosphere immediately. If this was to occur, increasing levels of CO2 within the atmosphere may be ameliorated by increased carbon storage in soil organic matter. From the measurement of microbial biomass, our ®ndings, in part, support this theory.

Overall, the CO2 eux from the bulk soil at the Swiss FACE site was greater in the rings exposed to el-evated CO2. Other published work examining the CO2 eux from soil shows somewhat variable results. CO2 eux was greater in soils planted with Ponderosa pine (Vose et al., 1997), temperate zone shrubland (Ball et al., 2000), Scots pine seedlings (Janssens et al., 1998) and Douglas ®r seedlings (Lin et al., 1999). However, CO2 eux from a tidal marsh ecosystem showed no eects of elevated CO2 (Ball and Drake, 1997). Pre-vious measurements of CO2 eux at the Swiss FACE site showed lower levels of CO2 eux from soils exposed to elevated CO2 (Ineson et al., 1998). How-ever, readings of gas exchange were limited only to one control ring and one fumigated ring.

5. Conclusions

Zak et al. (1993) hypothesised that the result of increased carbon inputs from the atmosphere to the soil under elevated concentrations of CO2, may increase rates of turnover and mineralisation of carbon within the soil system. Theoretically, carbon entering the soil will be transformed rapidly and returned to the atmosphere, giving at best a neutral eect on rates

of CO2accumulation in the atmosphere, and at worse potentially result in a positive feedback.

Our results do show an increase in overall rates of soil respiration from soils exposed to elevated CO2. However, this was the result of an increase in mi-crobial biomass rather than due to an increase in ac-tivity of individuals. Our results from the measurement of mass loss of litter, and the nutritional quality of that litter as it decomposes, suggest that there may be a dierence between the decomposer communities in the soils in the dierent CO2 treatment rings at the Swiss FACE site. Whether the observed increase in mi-crobial biomass and soil respiration in the FACE rings is driven by altered or increased litter input is not com-pletely clear as other factors, such as the water content of the soil, have been shown to eect microbial bio-mass and soil respiration. However, various environ-mental parameters have been measured alongside microbial biomass and soil respiration at the Swiss site with very few dierences being observed between elev-ated CO2 exposed soils and ambient soils (data not shown). This would seem to suggest that any dier-ences observed in this work were the result of the plants response to elevated CO2, be it either through a change in the input of litter to the soil or through altered rooting strategies or exudates.

Ineson et al. (1998) showed greatly accelerated rates of N2O production from the soils at the Swiss FACE site. They speculated that increased available soil-C had fuelled the increased rates of denitri®cation. The rate of mass loss of litter from the litter bags in the soil at the Swiss FACE site was also rapid, when com-paring rates with other published work (e.g. Kemp et al., 1994; Cotrufo and Ineson, 1996; Hirschel et al., 1997). Clearly a dierence is being observed in soil exposed to elevated CO2 at the Swiss FACE site. In corroboration with some of the results published in Ineson et al. (1998), our results seem to be suggesting increased rates of C and N cycling within the soils exposed to elevated CO2in the Swiss FACE site.

Future work on the eects of elevated CO2 on the decomposition of litter and the turnover of carbon within soil needs more precise measurements of the rate of C and N cycling within the system. Our results presented here also show there may be dierences in the process of decomposition in soils exposed to elev-ated CO2. The use of stable isotopes such as 15N and

13_C_{, will become invaluable in this, and have already}

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atmospheric CO2bringing about dierences in the soil community? (2) Is elevated CO2, or any elevated CO2 -derived eect, changing the diversity of individuals within the decomposer community, as well as the population size of the microbial component of the community? Clearly long-term integrated research will be needed to answer some of these questions, so as precise as possible predictions can be made on how el-evated CO2 will impact on the turnover of carbon in the terrestrial system.

Acknowledgements

The award of a NERC studentship to Alwyn Sowerby is gratefully acknowledged. Technical assist-ance provided by Tania Cresswell-Maynard in UK and Werner Wild in Switzerland is also appreciated.

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