Plant and Soil 176: 37-49, 1995. © 1995 KluwerAcademicPublishers. Printedin theNetherland,.

Elevated CO2 and temperature effects on soil carbon and nitrogen cycling in ryegrass/white clover turves of an Endoaquept soil

D.J. Ross 1, K.R. Tate 1 and EC.D. Newton 2 1Landcare Research New Zealand, Private Bag 11052, Palmerston North, New Zealand and 2AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand*

Received 2 November 1994. Accepted in revised form 4 April 1995

Key words: C cycling, climate change, elevated CO2, microbial biomass, N mineralization, soil respiration

Abstract

Effects of elevated CO2 (700/.tL L - l ) and a control (350/tL L -I CO2) on the productivity of a 3-year-old rye- grass/white clover pasture, and on soil biochemical properties, were investigated with turves of a Typic Endoaquept soil in growth chambers. Temperature treatments corresponding to average winter, spring, and summer conditions in the field were applied consecutively to all of the turves. An additional treatment, at 700/IL L- i CO2 and a temperature 6°C higher throughout than in the other treatments, was included.

Under the same temperature conditions, overall herbage yields in the '700 #L L- l CO2' treatment were ca. 7% greater than in the control at the end of the 'summer' period. Root mass (to ca 25 cm depth) in the '700 juL L- CO2' treatment was then about 50% greater than in the control, but in the '700 ~L L -1 CO2 + 6°C ' treatment it was 6% lower than in the control. Based on decomposition results, herbage from the '700 ~tL L- l + 6oc , treatment probably contained the highest proportion of readily decomposable components.

Elevated CO2 had no consistent effect on soil total C and N, microbial C and N, or extractable C concentrations in any of the treatments. Under the same temperature conditions, it did, however, enhance soil respiration (CO2-C production) and invertase activity. The effects of elevated CO2 on rates of net N mineralization were less distinct, and the apparent availability of N for the sward was not affected. Under elevated CO2, soil in the higher-temperature treatment had a higher microbial C:N ratio; it also had a greater potential to degrade plant materials.

Data interpretation was complicated by soil spatial variability and the moderately high background levels of organic matter and biochemical properties that are typical of New Zealand pasture soils. More rapid cycling of C under CO2 enrichment is, nevertheless, indicated. Futher long-term experiments are required to determine the overall effect of elevated CO2 on the soil C balance.

Introduction

The concentration of CO 2 in the atmosphere has been increasing since the middle of last century and will continue to do so, based on current industrial and land- management practices (Schneider, 1989). Temperature increases of 3 ° + 1.5°C within the next century are also predicted (Anderson, 1991). Both of these envi- ronmental changes are likely to have significant effects on plant growth and community structure (Anderson, 1991; Newton, 1991; Newton et al., 1994). Their effects on soil C storage and nutrient cycling processes

* FAX no corresponding author: +646355 9230

would probably be indirect, and result from changes in plant productivity, composition and metabolism (Van Veen et al., 1991). Direct effects of increased atmo- spheric CO2 on soil processes are generally consid- ered unlikely because of the high CO2 concentration already present in most soils (Van de Geijn and van Veen, 1993; Van Veen et al., 1991).

Increased inputs, and/or changes in the composi- tion, of plant C can have a complex effect on soil, which is one of the major reservoirs of C on a global scale (Bouwman, 1990). Changes in the size of var- ious C pools, and in rates of cycling of C and other nutrients, are both possible. As yet, there have been

38

few reports of the effects of CO2 enrichment on soil biochemical properties under different plant commu- nities, but appreciable increases in microbial C and N levels were found by Diaz et al. (1993) and in soil protease and xylanase activities by K6rner and Arnone (1992).

We have employed an integrated approach with pasture turves in environmental chambers, in which both CO2 concentrations and temperature differed. Pasture turves were selected because of the importance of grasslands in temperate environments (Emmanuel et al., 1993; Peel, 1986), and their extensive use in New Zealand for grazing by mainly sheep and cat- tle. The pasture used had been established three years previously on a fertile sandy loam and consisted pre- dominantly of ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.). The influence of elevated CO2 and seasonal changes in temperature on species composition and pasture growth in these turves has been reported by Newton et al. (1994). Here we exam- ine the influence of these treatments on soil metabolic processes and pools of total C and N, and microbial C and N which can respond more rapidly than total organic matter to land-management changes (Powlson and Jenkinson, 1981). The relationship between CO2 concentration and temperature differences on the even- tual release of C and N from plant materials to soil was assessed by incubating pasture herbage and roots with soil from the same or different treatment.

Materials and methods

Site properties

The site was located at Flock House, near Bulls, New Zealand, at a latitude of 40 ° 14'S and longtitude of 175 ° 16'E. The mean annual rainfall for the region is 874 mm and mean annual temperature is 12.9°C. The sheep-grazed pasture had been sown three years previously with a ryegrass (Lolium perenne cv. Ellett) and white clover (Trifolium repens cv. Pitau) mixture. The soil was a sandy loam (a Typic Endoaquept) of the Kairanga series (Cowie and Hall, 1965).

Experimental details

Turves to a depth of ca. 30 cm were taken along a transect in winter (July 1991) as described by Newton et al. (1994), with the final selection to give six turves per treatment being based on plant composition. Each

J=

2

E

22/16

16/10

10/4,

'Spring' t - - -C ¢ /

'Winter' i ! - - - - - " 0 ~

'Summer' F - - C ¢ .L I

/ , t

I I I I I I I I I I I 20 40 60 80 100 120 140 160 180 200 220

Days of exposure to C02

Fig. 1. Timetable for sampling times and stepped changes in tem- perature applied to the pasture turves growing at 350 or 700 #L L -1 CO2; the timetable was identical for the '700 #L L -1 CO2 + 6°C ' treatment which was at a 6°C higher temperature throughout; - -, acclimation periods; - , measurement periods for pasture growth; C), herbage trimmed; • herbage harvested; • herbage harvested and soil sampled; soil samples (initial) were also taken at day 35 immediately after the winter trimming of the herbage.

turf was contained in a movable steel bin and, after trimming, was ca 1 ×0.5 m and 0.25 m deep.

Details of two of the experimental treatments at 350 #L L - l CO2 ('350') and 700/iL L -1 CO2 ('700') are given by Newton et al. (1994). Turves were exposed consecutively to three temperature regimes that approximated to 'winter' (I0°/4°C day/night), 'spring' (16°/10°C) and 'summer' (22°/16°C) con- ditions at the site. A further treatment ('700 + 6°C'), with temperature in a 700/zL L -1 CO2 room being 6°C higher than in the other two treatments, was also included. The photoperiod used was 12 h at 480/lmol m -2 s -1 photosynthetically active irradiance.

The turves were grown initially for 35 d under the 'winter' regime in the appropriate treatment concen- trations of CO2. Each temperature treatment then last- ed 6 weeks and was separated by a 4-week interval, including a one-week period for adjustment to the next temperature level (Fig. 1). Watering was carried out every 3-4 days, and was designed to avoid soil mois- ture deficits.

Before the experiment commenced, fenitrothion (Verthion, Shell) was applied to control foliage-feeding porina caterpillars (Wiseana spp.). The turves were also sprayed five times during the trial with pimmiphos- methyl/permithrin (Target, ICI) to control aphids (Newton et al., 1994).

Soil sampling and analyses

For routine biochemical measurements, soil was sam- pied at the end of the initial acclimation period and at the end of each 'season' (Fig. 1). Six cores (2.5 cm diam; 0-5 cm depth) of soil were taken around the perimeter of each turf (at least 3 cm in from the edge), with the cores from each turf being pooled to give one replicate sample. Six replicate samples per treatment were thereby obtained. Holes were back-filled with the same Kairanga soil, and the positions marked. At the final harvest, two blocks of soil (ca. 5x5 cm and 5 cm depth) were also taken from each turf for calibration of the fumigation-extraction method for determining microbial C and N, and for herbage and root decompo- sition experiments; the samples from the six replicate turves were here combined to give one pooled sample per CO2 treatment.

All of the soil samples were sieved (<2 mm) and stored moist at 4°C. Sub-samples were air-dried and ground (<250 #m) for total C and N analyses.

Analyses were made in duplicate, except for inver- tase activity and the determinations of kEc- and kEN- factors which were made in triplicate. Results are expressed on the basis of oven-dry (105°C) weight of material. Biochemical measurements generally com- menced within 7 days of sample collection, except for invertase activity which was assayed within 21 days.

Moisture content was determined by drying soil samples overnight at 105° C, and water-holding capac- ity was estimated (WHC) according to Harding and Ross (1964). Total C and N concentrations were deter- mined according to Blakemore et al. (1987).

Extractable-C was measured by shaking 10.0 g soil at 60% of WHC (ca.-5 kPa) with 25 mL 0.5 M K2SO4 in an end-over-end shaker for 30 min. Organic C in the subsequent filtrates was determined by dichromate oxidation in the presence of 70 mg HgO (Jenkinson and Powlson, 1976a; Tate et al., 1988).

CO2-C production was measured in ca. 250 mL biometer flasks (Bartha and Pramer, 1965) with 10.0 g soil at 60% of WHC during 0-14 days at 25°C. Metabolic quotients (qCO2 values; Anderson and Domsch, 1985) were calculated as the average rate of CO2-C production (#g g- l soil h - l ) over 0-14 days per unit of microbial C (mg g-1 soil) present at the start of the incubation.

Microbial C and N were routinely determined by fumigation-extraction (Brookes et al., 1985; Vance et al., 1987), as described by Ross and Sparling (1993).

39

Factors for converting extractable-C flush to micro- bial C, and extractable-N flush to microbial N, were established by calibration against microbial C and N values determined by the fumigation-incubation pro- cedure. The kEc-factors thereby obtained were 0.308 for the '350' CO2 treatment and 0.316 for the '700' treatment; corresponding kEN-factors were 0.299 and 0.326, respectively. Treatment differences were not significant and mean factors, viz. kEc, 0.31 and kEN, 0.31 were taken for routine use. These factors were also used for the '700 + 6°C ' treatment for which specific kEc- and kEN- factors were not determined.

The procedure for estimating microbial C by fumigation-incubation (Jenkinson and Powlson, 1976b) is described by Ross and Sparling (1993). It used 20.0 g soil at 60% of WHC, a small (ca. 25 mg) inoculum, a control of fumigated soil incubated Ibr 10-20 days (Ross, 1990), and kc-factor of 0.45 (Jenk- inson, 1988). Microbial N was estimated with 10.0 g soil at 60% of WHC, a small (ca. 15 mg) inoculum, a control of unfumigated soil incubated for 0-14 days, and a kx-factor of 0.57 (Jenkinson, 1988).

Mineral-N (min-N = NH+-N + NO~-N) in 2 M KC1 extracts (1/10, w/v) was determined by autoanalyser procedures (Blakemore et al., 1987). N mineralization was estimated by incubating 10.0 g soil at 60% of WHC for 14 days, with the min-N then being extracted with 100 mL 2 M KC1. Net N mineralization ( ~ rain-N) was the difference between 14-day and 0-day min-N values.

Invertase activity was measured, in the presence of 3 drops toluene, with 1.0 g soil dispersed with a glass rod in 2.0 mL 0.5 M potassium phosphate buffer (pH 5.0) and 2.0 mL aqueous sucrose solution (5% w/v), as described by Ross (1987); incubation was for 24 h at 30°C.

Decomposition of plant materials

Herbage was routinely sampled to a height of 2 cm, as described by Newton et al. (1994), with sub-samples being dried at 95°C for dry weight determinations. One week after the final harvest, futher herbage was cut just above ground level and representative por- tions were separated into grass and clover components. Roots were then also separated from a 10x 10 cm block of soil (0-5 cm depth) from each turf by washing on a 2 mm mesh sieve. Sub-samples of the different plant materials taken at the end of the 'summer' period were freeze-dried for 48 h, with the six replicates from each treatment then being pooled to give one sample per

40

treatment. The pooled samples were ground (< 1 mm) in a Casella mill for subsequent decomposition exper- iments. Total N concentrations were determined in duplicate according to Blakemore et al. (1987), and expressed on an oven-dry (95°C) weight basis.

Decomposition of mixed herbage, grasses, clovers or roots harvested at the end of 'summer' was deter- mined by mixing the plant material from a particu- lar treatment with sieved soil (equivalent to 10.0 g at 60% of WHC) from the identical treatment; the amount of material added was 1.0% on a dry weight basis. After adjustment to the required moisture con- tent, the mixtures were incubated at 25°C for 20 days. Decomposition of mixed herbage or roots from dif- ferent treatments was also examined similarly in soil from a single treatment; (comparisons were not made with plant materials from either the '700' or '700 + 6°C ' treatment in soil from the other treatment). All determinations were made with triplicate samples.

Net mineralization of plant C and N was estimat- ed by subtracting CO2-C or min-N produced by una- mended soil incubated similarly from the soil + plant material values; no account was taken of any possible priming effect. CO2-C production and min-N content were determined as above.

Statistics

The significance of differences between CO2 treat- ments and sampling times was assessed by analysis of variance and Fisher's LSD test (Steel and Torrie, 1980). Significant differences are at p < 0.05, unless stated otherwise. Matched t-tests were used with log- transformed data for estimating errors associated with ratios. Linear relationships between properties were investigated by calculating correlation coefficients.

Results

Yields of plant materials

Herbage yields were not significantly different in the '350' and '700' treatments during 'winter', but were 10-15% greater in the '700' treatment during 'spring' and 'summer' (Table 1). In the '700 + 6°C ' treat- ment, yields were greater than in the other treatments in 'winter' and 'spring' but were smaller in 'summer'. The percentages of clover were similar in all treatments during the first two 'seasons', but were greatest in the '700' and '700 + 6°C ' treatments during 'summer'.

Root mass at the end of the trial was 50% greater in the '700', but 6% lower in the '700 + 6°C ', treatment than in the '350' treatment (P C D Newton, unpubl. data). No data are available for preceding changes dur- ing the different 'seasons'.

Soil properties

Soil moisture content was similar over all treatments and samplings and averaged 38+3 (SD)%.

The average soil pH was 5.84-0.2 at the first sam- plings and 6.2 at the final sampling, and did not differ significantly between treatments.

Mean total C concentrations ranged from 3.8 to 4.3% and total N from 0.34 to 0.38%. They did not differ significantly between the '350' and '700' treat- ments, but were generally lowest at the first three sam- plings in the '700 + 6°C ' treatment (Fig. 2). C:N ratios ranged from 10.8 to 11.9 and did not differ significantly between treatments.

Extractable-C concentrations varied appreciably in the initial samples, but were subsequently indistin- guishable in the '350' and '700' treatments and sig- nificantly higher in the '700 + 6°C ' treatment (Fig. 2).

Microbial C and N levels were initially lower in the '700' than in the '350' treatment, but were indistin- guishable in both treatments in the 'spring' and 'sum- mer' samples (Fig. 2). At the 'winter' sampling, the microbial N concentration was, in contrast to microbial C, higher in the '700' treatment. At most samplings, microbial C concentrations were similar in the '700' and '700 + 6°C ' treatments, whereas microbial N tend- ed to be lower in the '700 + 6°C ' treatment (Fig. 2).

Soil CO2-C production was, at all samplings, high- er in the '700' treatment than in the '350' and '700 + 6°C ' treatments (Fig. 3). Values declined appreciably during the trial.

The values of A Min-N were also higher in the '700' than in the '350' treatment at the first two sam- plings, but were indistinguishable at the last two sam- plings (Fig. 3). Values in the '700 + 6°C ' treatment were, as with CO2-C production, lower than in the '700' treatment at the first three samplings; A min-N values were, however, similar in both treatments at the final sampling.

Invertase activity was significantly higher in the '700' than in the '350' treatment at two samplings, and was consistently higher in the '700' than in the '700 + 6°C ' treatment (Fig. 3).

41

Table 1. Herbage yields and percentage of clover during each 'season'

'Season'

Dry matter yield Percentage of clover ( g m - 2 d - l )

350 z 700 700 + 6 ° C 350 700 700 + 6 ° C

'Winter' 3.7b 3.4b 5.0a 23a 23a 30a

'Spring' 5.2c 5.7b 7.2a 38a 44a 35a

'Summer' 6.1b 7.0a 5.9b 22b 33ab 47a

~CO2 concentration (/zL L - ~ ) during growth. For each property at each 'season', values not marked with the same letter are significantly different (p < 0.05).

150" t a

I:~ 100-

O 50.

0

o ~ 4.0-

~ 2.0-

0

o ~ 0.40- Z 0.20-

0 Initial

Extractable C a

Total C a Total N

'Winter' 'Spring'

z 350 ptL L -1 CO2 700 I.tL L -1 002

--~ 700 ~tL L -1 CO2 +6°C

Microbial C I

~ ~ a ~ ~ ~ I1500/"~5001000 ~

o Microbial N ~ |

200

too z

Io 'Summer' Initial ~inter' 'Spring' 'Summer'

Fig. 2. Total C and N, microbial C and N, and 0.5 M K2504- extractable C contents of soil sampled at the completion of the different 'seasons' from turves grown under different CO2 concentrations. Vertical bars are LSD values (p < 0.05) for sampling time effects; at each sampling time, values not marked with the same letter are significantly different (p < 0.05).

t CO2-C production 1000 b a x ~3~ 750 1 a a a

soo

250 t 0 ;

Z 7st 50 25 0

Initial

B 350 I~L L -1 002 700 ~tL L -1 CO2 700 ~tL L -1 CO2 +6°C

Invertase activity ,5 Min-N t a~ ~ a~ ~ ~ ~ / 3000

~ 1000

~0 'Winter' 'Spdng' 'Summer' Initial 'Winter' 'Spring' 'Summer'

Fig. 3. Soil invertase activity (units: pmol 'glucose' formed g - t soil s - - l ) , and CO2-C and net min-N production (/Xmin-N) during 14 days at 25°C. Vertical bars are LSD values (p < 0.05) for sampling time effects: at each sampling time, values not marked with the same letter are significantly different (p < 0.05).

42

Table 2. Influence of CO2 treatment and sampling time on percentages of microbial C and N in total C and N, and microbial C: microbial N ratios

Sampling Microbial C (%) Microbial N (%) Microbial C

time ~ Total C Total N Microbial N

350 u 700 700+6 ° C = 350 700 700+ 6 ° C 350 700 700+ 6 ° C

Initial 2.7aB 2.5aB 2.5aAB 7.0aA 6.5aABC 6.9aA 4.3aB 4.3aB 4.1aC

'Winter' 3.1aA 3.0aA 2.8aA 6.7abA 8.6aA 6.3bAB 5.5aA 4.1bBC 5.laB

'Spring' 2.3aC 2.2aC 2.4aB 6.3aA 6.5aB 5.9aAB 3.9abB 3.8bC 4.3aC

'Summer ' 2.4aBC 2.3aC 2.4aAB 4.8aB 5.1aC 4.6aB 5.8aA 5.2bA 6.0aA

z Samples taken at the end of the 'seasons' indicated. u CO2 concentration (/zL L -1) during herbage growth. =Temperature throughout the growth periods was 6°C higher than in the other two treatments. For each property at each sampling time, values not marked with the same lower-case letter are significantly different (p < 0.05). For each column, values not marked with the same capital letter are significantly different (p < 0.05).

Relationships between soil properties 30

No significant treatment effects were apparent in the 2s- percentages of microbial C in total C, or generally of

2 0 microbial N in total N (Table 2). The percentages of ~ ,5 microbial N in total N were, however, lower in the '700 + 6°C ' than in the '700' treatment in the 'winter' ~ lo

"5 samples. ~ s. Microbial C:N ratios were indistinguishable in all

initial samples (Table 2), but were significantly lower ~ o in the '700' than in the '350' treatment at two of the o subsequent samplings (Table 2), and lower in the '700' ~ 25. than in the '700 + 6°C treatment at all subsequent 8 20- samplings. = ~,

15- A l l of the above ratios differed significantly at some 2 sampling times, with the percentage of microbial N in d lO. total N being lowest, and microbial C:N ratios highest, at the final sampling (Table 2). s-

Although the qCO2 values tended to be higher in o

the '700' treatment than in the '350' and '700 + 6°C ' treatments, only one of the differences was significant (Table 3). Differences between sampling times were

Fig. 4. most apparent in the '700 + 6°C ' treatment.

Microbial C and N levels, invertase activity and CO2-C and A min-N values were, in contrast to extractable C, significantly correlated with total C con- centrations and, except for Amin-N, also with total N concentrations (Table 4). Most of these biochemical activities were also significantly correlated with each other; a major exception was extractable C content (Table 4).

Grasses

f Herbage

Clovers

-f i

A 350 p.L L-'r C02

D 70o ld- L -1 CO2

0 700 p.L L°I C02 +6"C

Roots

; , ; ;s 2'o o s ,o ,5 20 25

Incubation period (days)

Net cumulative CO2-C production from decomposition of herbage, grasses, clovers and roots, harvested from a particular treatment at the end of the ' summer ' period, in soil from the identical treatment; the significance of differences between treatments at each incubation period is indicated by * = p<0.05. For simplicity, data for herbage, grasses and clovers are omitted for the '700 + 6°C /zL L -1 CO2' treatment, and root data for the '350/zL L -1 CO2' treatment; values of these particular treatments were intermediate between those of the two treatments shown and, for roots, did not differ significantly (p>0.05) from them.

43

Table 3. Influence of CO2 treatment and sampling time on qCO2 values

qCO2 (tzg CO2-C produced rag- l microbial C h- 1 ) Sampling times ~ 350 v 700 700 + 6 ° C

Initial 2.0aA 2.3aA 2.2aA 'Winter' 1.9aA 2.2aA 2.0aAB 'Spring 1.9abA 2. I aA 1,7bBC 'Summer' 1,6aA 1.9aA 1.5aC

z Samples taken at the end of the 'seasons' indicated. v CO2 concentration (~L L- 1) during herbage growth. At each sampling time, values not marked with the same lower-case letter are significantly different (p < 0.05). For each column, values not marked with the same capital letter are significantly different (p < 0.05)

Table 4. Correlations of soil biochemical properties with total C, total N, and each other

Biochemical property

Correlation coefficient (r)

Total C Total N CO2-C Invertase production activity

Extractable C 0.096 0.205 0.198 0.054 Microbial C 0.710"** 0.297" 0.738"** 0.582*** Microbial N 0,400* * ~ 0.468"** 0.304* * 0.604* * * CO2-C production 0.634*** 0,384*** h000 0,756*** ZX Min-N (0-14 d) 0.270* 0.073 0.594*** 0,258* Invertase activity 0.629*** 0.535**" 0,756*** 1.000

. . . . . . = p < 0,05, 0.01, 0.001; degrees of freedom, 70.

Decomposition of plant materials

Several treatment differences were found when decom- position of mixed herbage or grasses from a particular

treatment was assessed by net CO2-C production in

soil from the identical treatment. Decomposition of herbage during the first 10 days was greater in material

from the '700' than from the '350' treatment (Fig. 4),

and generally in material from the '700 + 6°C ' than from the '350' treatment (data not shown); differences

between the '700' and '700 + 6°C ' treatments were

non-significant (p > 0,05; data not shown). Treatment differences were more marked for the grasses, which were harvested after 7 days re-growth, with decom- position being significantly greater in both the '700'

and '700 + 6°C ' treatments than in the '350' treat- ment throughout the 20-day incubation (Fig. 4, and data not shown). Decomposition of clovers also tend- ed to be greater in the '700' than in the '350' treatment,

although none of the differences was significant (p >

0.05) (Fig. 4, and data not shown). Root decomposition did not differ significantly in the '350' and '700' treat-

ments (data not shown), but it did tend to be greater in the '700' than in the '700 + 6°C ' treatment (Fig. 4).

The influence of soil from one treatment on the

decomposition of herbage from another was not sig-

nificant for the '700' and '350' treatment comparisons

(data not shown), but was pronounced for the '350' and

'700 + 6°C ' treatments. The unamended soils from the '700 + 6°C ' and '350' treatments themselves differed

markedly, with CO2-C production being appreciably greater in the '350' treatment (Fig. 5). Decomposi- tion of herbage from either treatment was, in contrast, greater in soil from the '700 + 6°C ' treatment. When

roots from the different treatments were compared sim- ilarly none of the treatment differences in net CO2-C production was significant (data not shown).

44

Table 5. Total N concentration of, and net min-N production (on decomposition in soil from the same treatment) from, herbage, grasses, clover, and roots harvested at the end of the 'summer' period

Plant Total N (%) material

Net min-N production (0-20 days; mg g- 1 plant dry wt)

350 z 700 700 + 6 ° C 350 700 700 + 6 ° C

Herbage 3.9 3.5 4.3 14.2a 9.1b 15.1a Grasses u 4.0 4.1 4.0 15.9a 16.4a 14.8b Clovers u 4.4 4.3 4.1 18.8a 18.7a 13.0b Roots 1.7 1.9 2.0 -0.1b -0.1b 1.9a

c02 concentration (#L L- l) during growth. UGrasses and clovers were harvested from sward re-growth one week after the harvesting of the mixed herbage. For each plant material, values of net min-N production not marked with the same letter are significantly different (p < 0.05); (total N was determined in only single analyses and could not be statistically compared).

The total N concentrations of the mixed herbage, and grasses and clovers harvested one week later, (Table 5) were typical of those for high-producing pas- tures (Metson, 1972). Net N mineralization of these plant materials was broadly related to total N concen- trations (r, 0.871; p < 0.01). In both the grasses and clovers net N mineralization was lowest in the '700 + 6°C ' treatment (Table 5). The total N concentrations of the roots were comparatively low, and net N miner- alization over 20 days was negligible or absent.

Discussion

Our experimental system differed in some respects from a naturally occurring pasture, with soil moisture levels being adequate throughout and not fluctuating at the different 'seasons'. Temperatures in the sward and soil would also have been more uniform than in the field. Light may have limited growth at the higher tem- peratures (Newton et al., 1994), although single leaf photosynthesis was strongly stimulated under elevated CO2 (Newton et al., 1995). In addition, the herbage- harvesting regime, with all material removed, could have placed a greater demand on soil nutrient supply. In spite of these limitations, and the absence of grazing animals, our use of large turves is considered appro- priate for studying the effects of elevated CO2 on plant and soil processes in a well-established pasture.

In a recently established mixed sward on an arti- ficial medium, elevated CO2 markedly stimulated the growth of ryegrass and white clover (Hardacre et al.,

1986). In contrast, elevated CO2 had a comparative- ly small effect on plant yields in our older pasture communities, and then mainly at the higher tempera- tures. Yields in the '700 + 6°C ' treatment were essen- tially one 'season' out of phase, and comparable to those obtained by the '700' treatment in the following 'spring' or 'summer' . The overall cumulative increase in yield of about 7% under elevated CO2 is unlikely to have been restricted by N availability, with the N contents of component grasses being similar in both the '350' and '700' treatments at the end of the trial. Larigauderie et al. (1988) also found similar N lev- els in mature grass foliage after growth in enriched CO2, although N levels in young leaves were high- er in the elevated CO2 treatment. The proportions of component plant species did change throughout the trial, with clovers being favoured, and ryegrass growth depressed, under elevated CO2 (Newton et al., 1994).

Soil properties

Storage of the sieved soil for a few days at 4°C before the commencement of some of the biochemical mea- surements would probably have had only a negligible effect on the values obtained. The original field values of these biochemical properties were not determined and may have differed from those found with the ini- tial samples, which could have been influenced to some extent by growth of the turves for 5 weeks under the 'winter ' conditions (Fig. I). However, these initial

"O 0

o & o o

N "5 E

30-

1o

E E o

"6 3o.

60O

8 T 400

200

(c) Herbage (700 IlL L -1 CO2 +6°C)

(b) Herbage (350 I.tL L -1 C02)

' ('a) sob '

Incubation period (days)

Fig. 5. CO2-C production from unamended soil and net cumulative CO2-C production from decomposition in soil from either the '350 #L L - l CO2 or '700/zL L - l CO2 + 6°C ' treatment of herbage harvested at the end of the 'summer' period; • - • soil from the' 350' ~L L - l CO2 treatment, ~ - ~ soil from the '700/zL L -1 + 6°C ' treatment; (a) soil, (b) herbage harvested from the '350/zL L - 1 CO2 treatment, and (e) herbage harvested from the '700/~L L - [ CO2 + 6 ° C' treatment; the significance of differences between incubations in the different soils at each incubation period is indicated by * = p < 0.05.

results did act as a basis for assessing ensuing changes during the subsequent periods of growth.

The size of the increases in dry matter produc- tion suggests that few major changes in soil proper- ties might be expected over the three 'seasons' of this trial. The detection of possible changes would have also been hindered by the high background levels of

45

the soil biochemical properties and by soil variabil- ity. The intensity of sampling, to reduce variability, was restricted by the size of the turves and the oth- er concurrent measurements that were made (Clark et al., 1995; Newton et al., 1994; Yeates and Orchard, 1993). With the sampling procedure adopted, coeffi- cients of variation (%) for the different properties of the replicate turves of each treatment at each sampling times had the following averages: total C, 5; total N, 4; extractable C, 7; microbial C, 9; microbial N, 12; CO2- C production and A min-N, 9; invertase activity, 13. This spatial variability was inherent in the turves, and not the result of the CO2 treatments, with the average coefficients of variation at the 'initial' sampling being at least as high as those over the whole trial period.

Neither total C nor N concentrations changed con- sistently in the elevated COo treatment. The slightly lower values in the '700 + 6°C ' treatment at the first three samplings could have resulted from inital differ- ences between turves. The problem of identifying any changes in these two properties can be illustrated with total C data expressed on an area basis. Over the 217 days of the trial, yields of roots (to ca. 25 cm depth) were 1268 kg ha- 1 greater in the '700' than in the '350' treatment (Newton et al., 1994). These yields would be equivalent to about 510 kg C ha -1, respectively (assuming a root C content of 40%). If total soil C is taken to be 4.0% (Fig. 2), with a coefficient of varia- tion of 5.0%, and dry bulk density is ca 1.0 g cm -3, the C content of soil to 0-5 cm depth could vary by 1000 kg ha- ~. Such variability is obviously greater than any likely extra input of C to the soil from root growth under elevated CO2. Kong et al. (1993) also found no measurable changes in total C and N concentrations in soil supporting different yields of clover for several years. In other systems, variable effects of elevated CO2 on soil total C concentrations have been report- ed. Increases were found by Kuikman et al. (1991) and Lekkerkerk et al. (1990), and decreases by K6rner and Amone (1992). Van de Geijn and van Veen (1993) considered that changes would be gradual and, as in our study, difficult to detect.

Within the constraints of the trial, there is also no conclusive evidence that levels of microbial C or N responded to elevated CO2. They were initially great- est in the '350' treatment, but similar in the '350' and '700' treatments at the end of 'spring' and 'summer'. To some extent, the variations in microbial biomass could have resulted from the similar variations in total organic matter (Table 4); (we cannot account, how- ever, for the comparatively high microbial N value in

46

the 'winter' '700' treatment). Our results consequently differ from those of Diaz et al. (1993) with systems of mixed herbaceous plants or acidic grasslands, and Zak et al. (1993) with soil under poplar. Soil organic mat- ter contents did, however, differ appreciably between these systems, with only 0.2% total C in the soil of Zak et al. (1993); microbial C was also there compar- atively low, at 63/~g g- l ; (absolute values were not given by Diaz et al., 1993). Variability, and the very much higher background levels in our samples, may, as with total C and N, have obscured any CO2-induced increases in microbial C and N. It is also likely that biological populations and activity were greater and more varied in our topsoil than in the subsoil of Zak et al. (1993). Newly formed microbial biomass could then have turned over more rapidly in our system and resulted in only small, if any, net changes in biomass levels.

There are, on the other hand, indications that com- ponent microorganisms did change in response to ele- vated CO2. Although microbial C:N ratios were lower in the '700' than in the '350' treatment at two sam- piing times, and could suggest a greater proportion of bacteria (Anderson and Domsch, 1980) under elevated CO2, differences between treatments were generally too small to provide conclusive evidence. However, support for this concept is provided by the soil nema- tode populations which were enriched in bacterial feed- ers in the elevated-CO2 treatment (Yeates and Orchard, 1993). These microbial changes were partly dependent on temperature, with microbial C:N ratios in the '700 + 6°C ' treatment not differing significantly from those in the '350' treatment.

Any increase in plant productivity under elevated CO2 would eventually result in the addition of extra C to the soil through decomposing litter, roots and/or rhi- zodeposition (Kuikman et al., 1991; Lekkerkerk et al., 1990; Van Veen et al., 1991). Increased C metabolism and CO2-C release in soil could, therefore, be expect- ed, especially where levels of total and microbial C remained unchanged (Lamborg et al., 1984). In agree- ment with this postulate, rates of CO2 production were significantly higher throughout in the '700' treatment than in the control soil. Rates of cellulose breakdown were also enhanced in the '700' treatment (Yeates and Orchard, 1993). Increased rates of CO2 production, in response to elevated CO2, were also found by K6rner and Arnone (1992) and Zak et al. (1993), particularly in rhizosphere soil. The enhancement of CO2 production in our turves cannot be attributed directly to the levels of extractable C, which differed significantly between

the '700' and '350' treatments only at the initial sam- pling.

All CO2-C production rates in soil declined in the 'spring' and 'summer' samples. In accord with this inverse relationship with temperature, values were also consistently lower in the '700 + 6°C ' than in the '700' treatment. These CO2-C production values were measured under standardized conditions and would be dependent on substrate availability, as well as micro- bial activity, at the time of sampling. A larger pool of readily metabolized substrates in the '700' than in the '350' treatment is, therefore, suggested. In the '700 + 6°C ' treatment this labile pool may already have been metabolised appreciably before the samples were taken. Greater metabolic activity in situ might also account for the root mass being lowest in the '700 + 6°C ' treatment at the final harvest.

The slightly higher qCO2 values in the '700' than in the '350' treatment are also suggestive of a greater sub- strate supply under elevated CO2~ The lower qCO2 val- ues in the '700 + 6°C ' than in the '700' treatment may, as with CO2-C production, be indicative of greater substrate metabolism before the measurements were made.

The influence of elevated CO2 on soil N mineral- ization was, as in other systems, less clearly defined. Increased net min-N production has been found in some elevated-CO2 systems (Billes et al., 1993; Zak et al., 1993), and a greater release of N from 'native' organic matter is also considered possible (Zak et al., 1993). Griffiths (1993), on the other hand, concluded that increased exudation of soluble C from growing roots did not enhance the mineralization of soil organ- ic N. Although the pool of mineralizable N may have increased in our '700' treatment under 'winter' condi- tions, it appears to have been readily metabolised in the warmer 'seasons' and then been essentially the same in the '700' and '350' treatments. Again, evidence for a temperature-related breakdown of this pool is provided by the '700 + 6 °' treatment, in which Amin-N values were generally significantly lower than in the '700' treatment. Although a stimulatory effect of elevated CO2 on N pools and metabolism cannot be unequiv- ocally established, our/~ min-N values, and herbage total N contents, strongly suggest that increased CO2 had no adverse effects on N availability for the growing sward.

The increased soil invertase activity found in the '700' treatment could be indicative of a greater input of plant-derived invertase than in the '350' treatment, and/or greater synthesis by soil organisms in response

47

to the greater C input (Ross et al., 1982). The declines in invertase activity observed with increasing 'season- al' temperatures, and in the '700 + 6°C ' treatment, suggest that an appreciable proportion of this enzyme would have been relatively unprotected in this panic- ular soil and not stabilised by organic colloids or clays (Burns, 1982).

Decornposition of plant materials

Under field conditions, the input to the soil of above- ground plant materials would be predominantly from litter and from herbage trampled by grazing animals. For the decomposition experiments, the collection of litter was impractical and cut herbage was used to indi- cate possible changes induced by the CO2 treatments. Because between-treatment comparisons were of pri- mary interest, we did not investigate other factors that could affect decomposition rates (Jenkinson, 1981), and have assumed that any grinding (Bremer et al., 1991; Jensen 1994) or priming (Azam et al., 1993; Jenkinson, 1981; Mary et al., 1993) effects were simi- lar for all treatments.

When determined in soil from the identical treat- ment, rates of C mineralization of mixed herbage, grasses and clovers were initially significantly high- er in the '700' than '350' treatment and are in accord with the more rapid release of CO2-C from unamended soil in the '700' treatment. The C mineralization pat- terns of the mixed herbage from these two treatments did not, however, differ significantly when incubated in the one soil and suggest that the composition of the herbage did not differ markedly. On the other hand, the significantly greater rate of C mineralization found in herbage from the '700 + 6°C ' than from the '350' treatment (Fig. 5) may indicate a greater percentage of non-structural carbohydrates in plants under this ele- vated CO2 treatment (K6rner and Arnone, 1992; Yelle et al., 1989).

Overall, results do not imply that the additional plant material produced with elevated CO2 would have no long-term effects on soil C levels; these would also depend on the long-term rates of decomposition, for which we have no data. Moreover, the much lower decomposition rates of roots (Fig. 4), which were of mixed age and species, were similar in the '700' and '350' treatments.

Relationships between plant chemical composition and the net release of min-N on degradation can be complex, being directly proportional to total N in some materials (Constantinides and Fownes, 1994) but not

in others (Fox et al., 1990; Palm and Sanchez, 1991). In our above-ground materials, net N mineralization was closely related to total N content and, during 20 days, similar in the grass and clover components. Val- ues in the '350' and '700' treatments were somewhat lower for the mixed herbage, probably because it con- tained more mature material than did the grass and clovers, which were harvested from the younger sub- sequent re-growth. Further work is required to assess factors influencing net N mineralization in the root material.

Conclusions

The levels of soil organic matter and biochemical prop- erties in our turves were typical of those found in pro- ductive New Zealand pastures; however, their compar- atively high values, and appreciable spatial variability, made it difficult to determine whether elevated CO2 had any effect on some properties.

Under the same temperature conditions, yields of herbage increased slightly, and roots to a greater extent, under elevated CO2; the growth of clover was then also stimulated. Under elevated CO2, soil respiratory activity was enhanced; no change in N availability was indicated, and levels of total C and N and microbial C and N also showed no consistent change.

In the elevated-CO2 treatment at the 6°C higher temperature, more rapid metabolism of labile C and N in the turves before the soil was sampled was indicat- ed.

Longer growth periods, and/or the use of soils with a lower organic matter content, are now required to establish more clearly the possible effects of elevated CO2 on levels of total C and the more labile organic components.

Acknowledgements

We thank Colin C Bell for maintaining the growth chambers and turves, Elaine M Glasgow for herbage dissections, Charles W Feltham for total C and N analy- ses, Matthew D Taylor for autoanalyser measurements of ammonium-N and nitrate-N, David J McQueen for statistical assistance, and two anonymous referees for constructive comments.

48

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