Characteristics of the soil microbial biomass in soils from a long-term field experiment with different levels of C input

Applied Soil Ecology 10 (1998) 37±49

Characteristics of the soil microbial biomass in soils from a long-term ®eld experiment with different levels of C input Ernst Witter, Arno Kanal1,* Department of Soil Sciences, Box 7014, Swedish University of Agricultural Sciences, S-75007 Uppsala, Sweden Received 25 September 1997; accepted 16 February 1998

Abstract Soil samples were taken from a 40 year old ®eld experiment and were chosen so as to obtain soils that mainly differed in the amount rather than quality of past C input. The microbial community of these soils was characterized in terms of its qCO2, the SIR-to-biomass C ratio and its growth and substrate utilization characteristics using glucose as substrate. The microbial substrate utilisation ef®ciency was also studied in relation to the rate of substrate addition. The amount of microbial biomass was closely related to the soil C concentration. The Cmic-to-Corg ratio was, however, not constant but increased with the soil C concentration. Except for the fallow soil, the characteristics of the soil microbial biomass studied differed little between the soils. The microbial community in the fallow soil mainly contrasted from that in the other soils by a lower SIR-to-biomass C ratio and a higher qCO2. It is concluded that differences in the Cmic-to-Corg ratio between the soils was mainly due to differences in the amount of past C input resulting in differences in the quality of soil organic matter, rather than due to intrinsic differences in the microbial ef®ciency of substrate utilization. The microbial substrate utilization ef®ciency measured as the ratio of respired-to-biomass incorporated glucose C decreased with the rate of glucose application. At the same rate of application the ef®ciency was lower in soils with a smaller native biomass than soils with a larger biomass. Compared at a rate of glucose C application of approximately 2 the amount of native biomass C there were only small differences in the microbial substrate utilization between the soils that were not related to the amount of native biomass C. # 1998 Elsevier Science B.V. Keywords: Soil microbial biomass; Soil organic C; C input; qCO2; Cmic-to-Corg ratio;

1. Introduction Agricultural soils that have been under constant management for long enough to be able to assume *Corresponding author. Tel.: +372-7-425075; fax: +372-7425071; e-mail: [email protected] 1 Permanent address: Institute of Soil Science and Agrochemistry, Estonian Agricultural University, Viljandi Road, Eerika, EE2400 Tartu, Estonia 0929-1393/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0929-1393(98)00043-2

14

C-labelled glucose; Substrate utilisation ef®ciency

steady-state conditions are characterised by a constant ratio of biomass C-to-soil organic C, independent of soil C concentrations (Jenkinson and Ladd, 1981; Powlson and Jenkinson, 1981; Anderson and Domsch, 1986, 1989). The Cmic-to-Corg ratio may, however, be modi®ed by factors such as climate (Insam et al., 1989), pH (Witter et al., 1993; Hopkins and Shiel, 1996), crop rotation (Anderson and Domsch, 1989; Insam et al., 1989; Magid et al., 1997) and quality of C input (Anderson and Domsch, 1989), tillage (Magid

38

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

et al., 1997), and soil texture (Kaiser et al., 1992; Sparling, 1992; Hassink, 1994; Franzluebbers et al., 1996) although soil texture is not always found to exert a signi®cant in¯uence on the ratio (Anderson and Domsch, 1989; Insam et al., 1989). In the absence of steady-state conditions the Cmic-to-Corg ratio has been suggested to be an indicator of changes in soil organic matter levels as the soil microbial biomass responds more rapidly to changes in C input than does soil C (Powlson and Jenkinson, 1981; Powlson et al., 1987; Anderson and Domsch, 1989). In a study covering 134 soils from 26 experimental sites Anderson and Domsch (1989) found that the Cmic-to-Corg ratio was constant at about 2.3% in soils under permanent monocultures, but at 2.9% in soils under crop rotation across a wide range of soil C concentrations, a ®nding con®rmed by Insam et al. (1989) in a study of soils from experimental sites across contrasting climatic regions. Anderson et al. suggested that these differences in the Cmic-to-Corg ratio between the monoculture and crop rotation cropping systems could be due to a more ef®cient utilisation of C by the microbial community in soils under crop rotation as suggested by a lower qCO2, qD (Anderson and Domsch, 1990) and a higher af®nity for glucose (Anderson and Gray, 1990) by the soil microorganisms in the latter systems. When effects of the soils' physical and chemical properties on the Cmic-to-Corg ratio can be excluded, differences in this ratio between soils could thus indicate non-steady-state conditions, or differences in the microbial ef®ciency of substrate utilisation. In a previous study of the soils of the Ultuna LongTerm Soil Organic Matter Experiment Witter et al. (1993) found a linear relationship between soil ATP and soil C concentrations. The resolution of the biomass determinations was, however, rather low and the relationship was based on soils which had received organic matter input differing not only in amount but also in quality (Witter, 1996). In this study we choose soils from this experiment ± which at the time of sampling had been under constant management for 40 years ± that mainly differed in the amount of past C input to study the relationship between biomass C and soil C. The biomass was characterised in terms of its qCO2, the SIR-to-biomass C ratio and its growth and substrate utilization characteristics using glucose as substrate.

Published studies that have compared the microbial substrate utilization ef®ciency between soils have differed in their approach. Whereas Chander and Brookes (1991) and Bardgett and Saggar (1994) used a constant rate of substrate addition per weight of soil, Witter and Dahlin (1995) and Dahlin and Witter (1998) adjusted the rate of substrate addition to the size of the native biomass based on ®ndings by Bremer and Kuikman (1994) that the proportion of substrate (glucose) C respired increased with the rate of glucose addition whereas the proportion incorporated into the biomass decreased. Whether or not differences in the substrate utilisation ef®ciency (determined as the ratio of respired to biomass incorporated substrate C) between soils are affected by differences in the rate of substrate addition per unit biomass has, however, so far not been investigated. In our study we therefore also studied the microbial substrate utilisation ef®ciency in relation to the rate of substrate addition in the soils chosen which differed in the size of their native biomass. 2. Materials and methods 2.1. Description of field experiment and soil sampling procedure Samples were taken from The Ultuna Long-Term Soil Organic Matter Experiment. This experiment, situated in central Sweden (608N and 178E), was established in 1956 before which there had been a crop rotation consisting of arable crops and short-term leys (Persson, personal communication). The soil texture is clay loam with 36.5% clay, 41% silt, and 22.5% sand, and has been classi®ed as a Typic Eutrochrept or Eutric Cambisol (Kirchmann et al., 1994). The ®eld experiment has 14 treatments consisting of additions of different N-fertilizers and organic amendments replicated in four blocks. Plots, sized 22 m, are separated from each other by wooden frames sunk to a depth of 0.2 m into the soil. Four soils were selected for this study: (a) bare fallow; (b) cropped without fertilizer N; (c) cropped and fertilized with Ca(NO3)2 at 80 kg N haÿ1 yÿ1, and (d) as (c) but with an additional straw (from barley) input of 4 ton ashfree organic matter haÿ1 yÿ1 applied at double rates every other year. The soils were chosen so as to

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

represent different levels of C input of similar quality (crop residues, or crop residues and straw). The qualitative similarity of the C input is suggested by the similarity in the percentages of crop residue C and the percentage of added straw C mineralized over the period 1956±1991 (Witter, 1996). Only arable crops have been grown since the start of the experiment in 1956, predominantly spring cereals. All treatments have been fertilized with 20 kg P (superphosphate) and 35±38 kg K (KCl) haÿ1 yÿ1. Mineral fertilizers were applied in spring before sowing, and organic amendments in the autumn. The last addition of straw before soil samples were taken was after harvest in October 1995. The ®rst sampling of the soils was carried out at the beginning of the growing season in May 1996 before sowing and the second sampling a few days after harvest of the barley crop in September 1996. Four sub-samples were taken from each plot to a depth of 0.1 m along a diagonal line across the plot and the sub-samples were mixed to form a composite sample for each plot. The ®eld-moist soil was passed without force through a 4 mm screen and thoroughly cleansed of visible organic material. The soil total carbon and nitrogen concentrations were determined by dry combustion on air-dried soil samples. 2.2. Soil microbiological characterization Prior to microbiological analysis the soils were stored at ÿ188C for 3 days. After thawing at 48C, the ®eld moist soils (47±52% WHC in May; 40±43% WHC in September) were adjusted to 65% maximum water holding capacity and pre-incubated at 208C for two weeks before microbiological analysis. Biomass C was determined by the fumigation extraction method (FE) (Vance et al., 1987) with minor modi®cations. Brie¯y, pre-incubated moist soil equivalent to 12.5 g dry weight was extracted for 1 h at room temperature with 50 ml 250 mM K2SO4 and ®ltered through cintered glass funnels. Ethanol-free CHCl3 was used as fumigant and the fumigation time was 24 h. Organic C in the extracts was determined colorimetrically on a TRAACS auto analyzer. Chloroform labile C was converted to biomass C using a kec factor of 0.42 (Wu et al., 1990). Basal respiration was measured by incubating soil equivalent to 50 g dw at 208C in 2000 cm3 airtight jars for 7 days. Respired CO2 was trapped in 5 ml 0.1 M

39

NaOH, the CO2ÿ 3 was precipitated with BaCl2 and the excess OHÿ was titrated with HCl using a phenolphthalein indicator. The SIR response was determined by mixing ®nely ground glucose (2.5 mg gÿ1 dw soil) and talcum powder (100 mg gÿ1 dw soil) with pre-incubated soil equivalent to 5 g dw in 35 ml glass tubes (soil-to-air volume about 1:6). After 2 h incubation at room temperature (about 208C) a 6 ml gas sample was taken from the headspace and analysed for CO2 on a GCIRMS system (Europa Scienti®c, UK). SIR response was expressed as the amount of CO2±C evolved over 2 h after glucose addition after subtraction of the CO2 evolved from the soil after addition of talcum powder only. This subtraction was done in order to base the SIR response on the CO2 evolved in response to the glucose addition only, and differs in that respect from the original method (Anderson and Domsch, 1978). The SIR response was not further converted to biomass C. The speci®c microbial respiration rate (qCO2) was calculated from the basal respiration rate and expressed on basis of biomass C (FE) and SIR response. 2.3. Microbial growth characteristics and substrate utilization efficiency Microbial growth characteristics were derived indirectly from the respiratory response after addition of suf®cient glucose to initiate exponential microbial growth as described in Nordgren (1988) with the exception that the basal respiration rate was subtracted from the respiration data. The microbial substrate utilization ef®ciency was determined in the same experiments by measuring the ratio of respired to biomass incorporated glucose 14 C. For each soil there were three rates of glucose addition ranging from 240 to 880 mg glucose C gÿ1 dw soil with always one rate corresponding to approximately twice the amount of native biomass C in the soil. The uniformly labelled glucose (Amersham, UK) with a speci®c activity of 0.7 kBq mgÿ1 C was added to the soil in a solution containing N, P and K (KNO3 and KH2PO4) in the ratios of C:N:P:K of 15:1:0.3:1.8. The soils, sampled in September, were pre-incubated for 2 weeks at a moisture content adjusted so that addition of the glucose solution (25 mL gÿ1 dw soil) brought the soil up to 65%

40

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

WHC. The rate of basal respiration was measured in the respirometer over 24 h before the glucose was added. Two sets of soil were used, one set containing 40 g dw soil per vessel placed in the respirometer for determination of respired total C and 14 C and for determination of chloroform-labile total C and 14 C 10 d after glucose addition, and one set containing 30 g dw soil per vessel for determination of chloroform-labile total C and 14 C 16 h after the peak respiration rate. The experiment was carried out at 208C. Total respired C was measured by the respirometer and chloroform-labile C was measured as described above for biomass C. 14 C-labelled C in the KOH traps for CO2 in the respirometer and 14 C in the K2SO4 extracts was measured on a WinSpectralTM 1414 scintillation counter (Wallac, Finland) using OptiPhase `HiSafe' scintillation cocktail. In this experiment total C and 14 C rendered extractable by fumigation was not converted to biomass C, but is presented as chloroform-labile carbon. 2.4. Statistical analysis The soil and microbial data are given as arithmetic means of results from four plots. The data were analysed by analysis of variance. 3. Results 3.1. Microbiological and chemical characteristics of the soils Forty years of fallow, of cropping with or without N fertilization and of cropping with N fertilization and straw addition had resulted in soils with chemical characteristics that mainly differed in terms of the soil C concentration, although the C-to-N ratio of the N-fertilized soil was slightly lower than that in the other soils (Table 1). Compared to the soil C concentration of 1.5% at the start of the experiment in 1956 the fallow, the soil without N fertilization and the N fertilized soil showed decreases in the soil C concentration of 35, 23 and 11%, respectively. The N fertilized and straw amended soil showed an increase in soil C of 19%. Both measures of the size of the biomass, i.e. chloroform-labile C and SIR, showed the same ranking of the soils in terms of the amount of biomass

Table 1 Chemical characteristics of the soils sampled in May 1996 Soil

Total C g kgÿ1

Total N g kgÿ1

C-to-N ratio

pH (H2O)

Fallow No N-fertilizer N-fertilized N-fertilized and straw amended

10.1 11.1 13.1 17.7

1.1 1.2 1.5 1.9

9.2 9.3 8.7 9.3

6.2 6.4 6.5 6.6

(Table 2). There were no differences in the amount of biomass C measured by fumigation extraction between the spring and autumn samples,but the SIR responsewas about 12% lower in the autumn samples in all soils (Table 2). There was a close linear relationship between soil C and biomass C (Fig. 1). Because the relationship between soil C and biomass C had a negative intercept, the ratio of biomass C to soil C increased with the soil C concentration. Thus, in the fallow soil biomass C represented 1.2% of soil C, increasing to 2.4% in the N-fertilized‡straw treatment (Table 2). Changes in soil C concentrations between 1956 and 1996 were linearly related to the Cmic-to-Corg ratio measured in 1996 (Fig. 2). The relationship suggests that a ratio of about 2.1% would indicate steady-state conditions in so far that it represents the ratio at which soil C concentrations remained constant over the period 1956±1996. Basal respiration increased with the soil C concentration (Table 3). The ratio of basal respiration to soil C revealed no consistent pattern in the spring samples, but in the autumn samples it increased with the soil C

Fig. 1. Relationship between the soil carbon concentration and biomass C determined by fumigation±extraction.

Fallow Not N-fertilized N-fertilized N-fertilized and straw amended Effect of soil significant at Fallow Not N-fertilized N-fertilized N-fertilized and straw amended Effect of soil significant at

May

a

p<0.002

0.40.1 1.30.1 1.90.2 3.00.2 p<0.0001 0.30.1 1.20.1 1.70.1 2.70.2 p<0.0001

SIR a mg CO2±C gÿ1 soil

CO2±C evolved over 2 h at room temperature after glucose addition.

Effect of sampling time significant at

September

Soil

Sampling time

p<0.005

388 11113 14610 16718 p<0.0001 334 9911 1299 14817 p<0.0001

SIR-to-soil C ratio mg CO2±C gÿ1 Corg

n.s.

11912 16718 2538 44210 p<0.0001 996 17410 2667 41114 p<0.0001

Biomass C mg C gÿ1 soil

n.s.

1.20.1 1.40.1 1.90.1 2.40.1 p<0.0001 1.00.1 1.50.1 2.00.1 2.30.1 p<0.0001

Biomass C-to-soil C ratio %

Table 2 SIR response and soil microbial biomass determined by fumigation extraction of the soils sampled in May and in September

p<0.01

0.0070.001 0.0160.003 0.0150.001 0.0140.001 p<0.0001 0.0070.001 0.0140.001 0.0130.001 0.0130.001 p<0.0001

SIR-to-biomass C ratio mg CO2±C mgÿ1 Cmic

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49 41

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E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

Fig. 2. Relationship between the Cmic-to-Corg ratio determined in 1996 and the change in soil C concentrations between 1956 and 1996. Mean values of 4 replicate blocks.

concentration. In all soils the rate of basal respiration was 2 to 3 times higher in spring than in autumn. Despite the apparently good agreement between the biomass C and SIR estimates (Fig. 3) the ratio of SIRto-biomass C was signi®cantly lower in the fallow soil compared to that in the other soils, between which there were no signi®cant differences (Table 2). The microbial biomass in the fallow soil also differed from the biomass in the other soils in its speci®c microbial respiration rate (qCO2; Table 3). Determined as the ratio of the basal respiration rate to the magnitude of the SIR response the qCO2 was 2±3 times higher in the fallow than that in the other soils in both the spring and autumn samples. In the spring samples the qCO2 in the cropped soil without N fertilization was also higher

Fig. 3. Relationship between biomass C (FE) and the SIR response.

than that in the N fertilized and N fertilized‡straw treatments. Expressed per unit biomass C determined by fumigation extraction, the qCO2 was higher in the spring samples in the fallow and the cropped-withoutfertilizer-N soils compared to the other two soils, whereas in the autumn sampling there were no differences in the qCO2 between soils (Table 3). 3.2. Microbial growth characteristics and substrate utilization efficiency An addition of 880 mg glucose C gÿ1 soil resulted in an exponential increase in the rate of CO2 evolution in all soils (Fig. 4) indicative of exponential microbial growth. Patterns of the rate of CO2 evolution were similar at the lower rates of glucose addition, but with

Table 3 Basal respiration and the specific microbial respiration (qCO2) rate expressed per unit biomass C (qCO2

FE)

and per unit SIR (qCO2

Sampling time

Soil

Basal respiration mg CO2±C gÿ1 soil hÿ1

Basal respiration per unit soil C mg CO2±C gÿ1 Corg hÿ1

qCO2 hÿ1

May

Fallow Not N-fertilized N-fertilized N-fertilized and straw amended Effect of soil significant at: Fallow Not N-Fertilized N-Fertilized N-Fertilized and straw amended Effect of soil significant at: Effect of sampling time significant at:

0.090.01 0.140.01 0.120.01 0.230.01 p<0.0001 0.030.01 0.050.01 0.070.01 0.130.02 p<0.0001 p<0.0001

9.20.4 11.60.2 9.00.8 12.60.7 p<0.0001 3.20.4 3.91.8 5.50.5 7.21.2 p<0.0001 p<0.0001

0.00160.0002 0.00170.002 0.00100.0001 0.00110.0001 p<0.0001 0.00060.0001 0.00050.0002 0.00060.0001 0.00060.0001 N.S. p<0.0001

September

FE

qCO2 hÿ1

SIR) SIR

0.240.02 0.110.01 0.060.01 0.080.01 p<0.0001 0.100.02 0.040.01 0.040.01 0.050.01 p<0.0001 p<0.0001

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

43

Fig. 4. Respiration rates after addition of 880 mg glucose C gÿ1 soil. The rate of basal respiration determined on a separate set of soil samples has been subtracted. Mean respiration curves determined on each soil from 4 replicate blocks.

a shorter exponential phase (results not shown). At the lowest rate of addition (240 mg C gÿ1 soil) an exponential phase was not clearly discernible. Because of the longer exponential phase microbial growth characteristics could be most reliably determined at the highest rate of glucose addition. The initial respiratory response upon substrate addition (in effect the SIR response) increased with the amount of native biomass in the soils, whereas the time between substrate addi-

tion and the time of the peak respiration rate decreased (Table 4). There were no statistically signi®cant differences between the soils in the speci®c microbial growth rate or the lag time (Table 4). The proportion of 14 C glucose respired and the proportion recovered as chloroform-labile (biomass) C was in all soils dependent on the rate of glucose addition. Increasing the rate of glucose addition from 240 to 880 mg C gÿ1 soil increased the proportion

Table 4 Microbial growth characteristics (calculated as described in Nordgren, 1988) determined after addition of 880 mg glucose C gÿ1 soil Soil

Basal respiration a mg CO2±C gÿ1hÿ1

SIR-level b mg CO2±C gÿ1hÿ1

Specific growth rate hÿ1

Lag time h

Time to peak c h

nd

Fallow No N-fertilizer N-fertilized N-fertilized and straw amended Effect of treatment significant at

0.110.07 0.170.04 0.220.07 0.300.08 p<0.05

1.00.4 2.60.1 4.20.5 7.30.8 p<0.0001

0.150.01 0.140.01 0.140.01 0.120.02 n.s.

6.31.7 6.91.3 6.01.7 7.81.5 n.s.

32.00.6 27.31.5 23.30.6 21.30.7 p<0.0001

4 4 3 2

a

Determined over a 5 h period just before glucose addition. The stable rate of respiration 2 to 4 h after glucose addition before the onset of exponential growth. c Time between glucose addition and the peak respiration rate. d Number of replicate soils (representing blocks in the field) used. For some replicate soils the respiration data were omitted due to very uncharacteristic growth curves, probably caused by inadequate mixing of glucose with the soil. b

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E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

Fig. 5. Ratio of respired-to-chloroform-labile 14 C after addition of 240, 550 or 880 mg 14 C[U]-labelled glucose C gÿ1 soil, or added at approximately twice the amount of biomass C in the soil, in relation to the amount of native biomass C in the soil. Mean values of 4 replicate blocks, error bars indicate the standard deviation. See Table 2 for details of native biomass C. Figure A: 16 h after the peak respiration rate, Figure B: 10 d after glucose addition.

respired and decreased the proportion incorporated into the biomass. The ratio of respired-to-microbially incorporated glucose C therefore increased with the rate of glucose addition. This could be seen when measured 16 h after the time of the peak respiration rate with the exception of the fallow soil receiving 880 mg glucose C gÿ1 soil and when measured 10 d after glucose addition (Fig. 5). The effect was most pronounced 10 d after glucose addition and more pronounced in the 14 C than in the total C data (Tables 5 and 6). Comparing soils at the same rate of glucose addition showed a clear tendency for the ratio of respired to biomass incorporated glucose C to decrease as the amount of native soil biomass increased (Fig. 5), i.e. the biomass in the soils with a smaller amount of native biomass appeared to be

characterised by a lower ef®ciency of substrate utilization. This seems, however, largely to be an artefact caused by the dependency of the substrate utilization ef®ciency on the rate of glucose addition. Comparing the substrate utilization ef®ciency of the soil microbial biomass at a rate of glucose addition adjusted to approximately twice the amount of native biomass C showed only a small decline in the ratio of respiredto-biomass incorporated glucose C with increase in the amount of native biomass, but a slightly higher ratio in the soil with the largest native biomass (Fig. 5). These differences were, however, small compared to the differences caused by comparing substrate utilisation between soils at the same rate of glucose addition per weight of soil in soils with differences in the size of the native biomass.

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

45

Table 5 Change in chloroform-labile (
CL) C, total respired carbon and distribution of 14 C labeled glucose between the chloroform-labile fraction and respired C determined 16 h after the peak respiration rate, and expressed as a percentage of glucose C added Treatment

Fallow Not N-fertilized N-fertilized N-fertilized and straw amended

14

C-labelled glucose added

14

Total C

C-labelled C

mg C gÿ1


CL Respired % of glucose C added

Ratio respired-to-
CL

CL Respired % of glucose C added

Ratio respired-to-CL

240 500 880 240 330 880 240 500 880 240 500 800

142 101 111 161 182 112 291 192 151 333 261 181

2.60.5 2.40.2 2.90.1 1.90.1 2.20.3 4.40.6 1.10.1 1.80.5 3.20.3 0.80.2 1.40.4 2.50.4

121 91 121 191 161 102 261 181 131 291 251 161

1.80.3 2.70.3 2.40.1 1.00.1 1.60.1 3.00.4 0.70.1 1.30.1 2.30.1 0.60.1 0.90.1 1.70.1

376 241 382 312 381 452 313 328 464 264 3510 446

223 251 301 191 261 301 183 241 302 162 221 281

Table 6 Change in chloroform-labile (
CL) C, total respired carbon and distribution of 14 C labeled glucose between the chloroform-labile fraction and respired C determined 10 days after glucose addition, and expressed as a percentage of glucose C added Treatment

Fallow Not N-fertilized N-fertilized N-fertilized and straw amended

14

C-labelled glucose added

14

Total C

C-labelled C

mg C gÿ1


CL Respired % of glucose C added

Ratio respired-to-
CL

CL Respired % of glucose C added

Ratio respired-to-CL

240 500 880 240 330 880 240 500 880 240 500 800

93 61 61 102 112 71 82 81 61 113 82 82

5.50.8 6.20.1 10.40.1 4.61.1 4.80.9 9.61.6 5.91.6 5.21.3 10.41.3 3.91.5 5.20.9 7.31.5

101 91 71 141 141 91 181 141 101 202 171 131

3.10.3 4.20.3 7.60.6 1.90.1 2.50.1 5.60.9 1.40.1 2.50.2 4.70.3 1.20.1 1.90.1 3.30.2

518 342 583 424 523 643 423 4410 626 395 4211 588

4. Discussion 4.1. Size of the microbial biomass in relation to soil organic matter The results of Witter (1996) show that differences in the amounts and concentrations of C between the soils of the Ultuna experiment that we sampled in the current study can be explained by the differences in the amount of C input that these soils received since the start of the experiment in 1956. Increasing amounts of C input thus resulted not only in higher

315 371 502 273 351 472 253 341 462 242 321 421

concentrations of biomass and soil C, but also a higher Cmic-to-Corg ratio. As put forward by Jenkinson and Ladd (1981) and Beck (1984), the ratio of Cmic-to-Corg thus re¯ected quantitative changes in the amounts of soil C. That the ratio increased with increasing amounts of C input suggests that more biomass is supported per unit soil organic C derived from this C input (in this case the input over the period 1956± 1996) than per unit native soil organic C (i.e. C derived from inputs prior to 1956). That the C from the more recent input was more available to soil microorganisms than native soil C is also indicated by the increase

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E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

in the ratio of basal respiration to soil C with increasing amounts of C input. This was seen in the samples taken in September, but not in the samples taken in May. As basal respiration in spring was several times higher than in autumn it is likely that the presence of a large pool of available C, possibly the result of frequent freeze-thaw cycles in late winter and early spring, obscured differences between the soils. Our results are consistent with other observation that an increase in the C input results in an increase in the Cmic-to-Corg ratio (Powlson et al., 1987; Franzluebbers et al., 1994), and conversely that the Cmic-to-Corg ratio decreases when C input decreases (Biederbeck et al., 1984; Sparling, 1992; Houot and Chaussod, 1995; Paterson et al., 1996). It is also consistent with the observation that the Cmic-to-Corg ratio decreases with the soil C concentration with soil depth (Lavahun et al., 1996) which is more pronounced under no tillage than under conventional tillage (Franzluebbers et al., 1994; Costantini et al., 1996; Magid et al., 1997). Paustian et al. (1992) modelled soil organic matter dynamics in some of the soils of the Ultuna experiment between 1956 and 1988. The model predicted changes in the percentage of Corg that is present in the active soil organic matter compartment (conceptually representing the microbial biomass and metabolites). As discussed by the authors these changes agree with the differences in the Cmic-to-Corg ratio between the soils measured by SchnuÈrer et al. (1985) in 1982 and also agree with our data, i.e. the ratio increased with increasing amounts of C input. The model also predicted that the percentage of active C would continue to decline with time in the fallow soil and the soil not receiving N fertilizer. Comparing the data of SchnuÈrer et al. (1985), Witter et al. (1993) and our own data suggest these model predictions appear to have held. In the fallow soil the Cmic-to-Corg ratio decreased over the period 1982±1996 from 1.6%2 in 1982 to 1.4%3 in 1990 and 1.2% in 1996, and from 1.27 to 1.23 and 2 This figure deviates slightly from that given by Paustian et al. (1992). It is calculated from biomass C determinations (autumn 1982) by the fumigation±incubation technique with the use of a control (SchnuÈrer et al., 1985) and soil C determinations on samples from autumn 1983 (Kirchmann et al., 1994). 3 Based on soil ATP determinations which were deemed more reliable than measurements by the fumigation±incubation technique (Witter et al., 1993) and converted to biomass C as shown in Witter (1996).

1.15%, respectively in the no fertilizer N soil. But even though the model correctly predicted an increase in the percentage of soil that resides in the active fraction in the straw and N fertilized soil, the Cmic-to-Corg ratio in this soil appears to have continued to increase (from 3.2 to 3.8 and 4.4% in 1982, 1990 and 1996, respectively) whereas the model predicted the increase in the ratio to level off 10±15 y after additions were started in 1956 (Paustian et al., 1992). It must, however, be borne in mind that the observed trend in changes in the Cmic-to-Corg ratio over time may be fortuitous. Differences in methodology for biomass C determination between the three studies and unexplained ¯uctuations in soil C concentrations between years (see Kirchmann et al., 1994)) make comparison of this ratio between the studies extremely uncertain. All three studies, however, show that at the time of sampling the Cmic-to-Corg ratio was related to past C inputs as predicted by the model. Also the model developed by Jenkinson and Rayner (1977) predicts that a change to higher amounts of C input results in a transient increase in the Cmic-to-Corg ratio. Powlson and Jenkinson (1981) suggested that an increase in the Cmic-to-Corg ratio may therefore give an early indication of future changes in the amount of soil organic matter. The reason for this is that at steadystate the ratio is independent of the level of C input. Most soils of The Ultuna Long-Term Soil Organic Matter Experiment still show changing soil C concentrations and differences in the Cmic-to-Corg ratio after 40 years of constant management and have therefore not yet reached steady-state conditions. The relationship between the change in soil C concentrations between 1956 and 1996 and the Cmic-toCorg ratio suggests that a ratio of about 2.1% would be associated with steady-state conditions. This can be compared to the Cmic-to-Corg ratio of 2.0% in the unmanured and NPK fertilized plots and of 1.8% in the farmyard manure amended plot of the Broadbalk Continuous Wheat Experiment (Jenkinson and Powlson, 1976) which are close to steady-state conditions after more than 100 years of constant management (Jenkinson and Rayner, 1977). The soils in The Ultuna Long-Term Soil Organic Matter Experiment that are losing C appear to show a continuing decrease in the Cmic-to-Corg ratio in the last 15 years, whereas the soil gaining C shows a further increase in the ratio over this period. Neither are therefore as yet showing a ten-

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

dency to move towards steady-state conditions (i.e. a Cmic-to-Corg ratio of about 2.1%). The fallow soil with virtually zero C input is of course a special case which, theoretically, will not reach steady-state until all C is lost from the soil. With time the soil organic matter remaining can be expected to become increasingly dominated by recalcitrant soil organic matter, reducing its availability to the soil microorganisms. This will lead to further decreases in the Cmic-to-Corg ratio over time, as already seen over the period 1982 to 1996 (see above). The ef®ciency of substrate utilisation may be an additional factor that may affect Cmic-to-Corg ratio. Anderson and Domsch (1989), for example, observed that the Cmic-to-Corg ratio differed between soils (assumed to be near steady-state) that had received NPK fertilizers, straw, farmyard manure or green manure which was thought to be due to qualitative differences in the C input to these soils. Although our data are not conclusive, they do indicate that the biomass in the fallow soil was characterised by a higher respiratory activity (qCO2). Assuming that a higher qCO2 re¯ects a less ef®cient utilisation of C in fallow soils due to the more recalcitrant nature of the soil organic matter (Lavahun et al., 1996), the higher qCO2 in the fallow soil could provide an additional explanation for the low Cmic-toCorg ratio in this soil. Similar observations of a higher qCO2 and lower Cmic-to-Corg ratio have been made in fallow soils (Paterson et al., 1996) and with increasing soil depth (Lavahun et al., 1996). But even though a higher qCO2 may provide an additional explanation for the low Cmic-to-Corg ratio in the fallow soil, there was no tendency for the qCO2 to change in parallel to the Cmic-to-Corg ratio and hence does not explain the differences in the Cmic-to-Corg ratio between the other soils in The Ultuna Long-Term Soil Organic Matter Experiment. With the exception for the fallow soil, characteristics of the soil microbial biomass such as the SIR-tobiomass C ratio, the qCO2, as well as its growth characteristics on glucose and its utilization ef®ciency of this substrate were similar between the soils, with no indication of differences between soils being related to the amount of long term C input to the soil. The differences between the soils in the time between substrate addition and the time of the peak respiration rate are likely to be explained by differences in the initial number of microorganisms in the soil that can

47

utilize glucose for growth, as there were no statistically signi®cant differences between the soils in the speci®c microbial growth rate or the lag time. Differences in the time between substrate addition and the time of the peak respiration have therefore probably no physiological basis. Differences in the Cmic-to-Corg ratio between the soils were therefore not related to any of the characteristics of the soil microbial biomass determined in this study. The biomass of the fallow soil mainly differed in that its SIR response was only half that of the biomass in the other soils. Apparently soil microorganisms depend on input of fresh organic matter in order to maintain the same level of respiratory response to substrate addition, which would con®rm the suggestion by Hassink (1993) that smaller amounts of available carbon coincide with a reduced activity of the biomass and a lower SIR response of the biomass. 4.2. Microbial utilization of 14 C glucose in relation to the rate of glucose addition Bremer and Kuikman (1994) showed that the proportion of glucose C mineralized increased with the rate of addition but only up to rates of addition of 300 mg glucose C gÿ1 soil, while the proportion of glucose C that was recovered in the chloroform-labile (microbially incorporated) fraction decreased in parallel, even at rates of substrate addition of up to 2300 mg glucose C gÿ1 soil. The ratio of respired to biomass incorporated glucose C thus increased with the rate of glucose addition, most markedly at the lower rates of addition. Surprisingly, however, the proportion of glucose-C respired or incorporated into the biomass differed little between the two soils studied, even though the size of the native microbial biomass in these soils differed by 50%. Based on four soils mainly differing in their content of soil organic matter our results, however, clearly show that ratio of respired to biomass incorporated glucose C at three rates of glucose addition (240, 500 and 880 mg C gÿ1 soil) decreased as the size of the native biomass of the soil increased. This is entirely consistent with the observation in our experiment that with an increase in the rate of glucose addition the ratio of respired to biomass incorporated glucose C increased. That the decrease in this ratio with increasing size of the native biomass is not due to innate differences in the sub-

48

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49

strate utilization ef®ciency of the native biomass in these soils is seen when comparing the ratio of respired to biomass incorporated C at rates of glucose addition corresponding to approximately twice the size of the native biomass in these soils. At these rates of glucose addition differences in the substrate utilization ef®ciency between the soils remain, but these appear unrelated to the size of native biomass. These results put into question in how far the higher ratio of respired to biomass incorporated glucose C found in some metal contaminated soils (Chander and Brookes, 1991; Bardgett and Saggar, 1994) may have been an artefact caused by the fact that these authors applied the same amount of glucose to soils differing in the size of their native biomass. It must, however, be pointed out that the rates of glucose addition used by these authors exceeded the highest rate of addition in our experiment, and that the effect of the rate of addition on the ratio of respired-to-biomass incorporated glucose C appears to decrease with increasing rates of glucose addition. Acknowledgements The Department of Soil Sciences, Division for Soil Fertility is thanked for making available soils from The Ultuna Long-Term Soil Organic Matter Experiment. A. Kanal was supported by a grant from the Swedish University of Agricultural Sciences. References Anderson, J.P.E., Domsch, K.H., 1978. A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology and Biochemistry 10, 215±221. Anderson, T.-H., Domsch, K.H., 1986. Carbon link between microbial biomass and soil organic matter. In: Mergusar, F., Gantar, M. (Eds.), Perspectives in Microbial Ecology. Proceedings of the Fourth International Symposium on Microbial Ecology. Slovene Society for Microbial Ecology, pp. 467±471. Anderson, T.-H., Domsch, K.H., 1989. Ratio of microbial biomass carbon to total organic carbon in arable soils. Soil Biology and Biochemistry 21, 471±479. Anderson, T.-H., Domsch, K.H., 1990. Application of ecophysiological quotients (qCO2 and qD) on microbial biomasses from soils of different cropping histories. Soil Biology and Biochemistry 22, 251±255. Anderson, T.-H., Gray, T.R.G., 1990. Soil microbial carbon uptake characteristics in relation to soil management. FEMS Microbiol. Ecol. 74, 11±20.

Bardgett, R.D., Saggar, S., 1994. Effects of heavy metal contamination on the short-term decomposition of labelled [14 C] glucose in a pasture soil. Soil Biology and Biochemistry 26, 727±733. Beck, T., 1984. Mikrobiologische und Biochemische Charakterisierung landwirtschaftlich genuÈtzter Boden. II. Mitteilung. Beziehung zum Humushaushalt. Zeitschrift FuÈr PflanzenernaÈhrung Und Bodenkunde 147, 467±475. Biederbeck, V.O., Campbell, C.A., Zentner, R.P., 1984. Effect of crop rotation and fertilization on some biological properties of a loam in Southwestern Saskatchewan. Canadian Journal of Soil Science 64, 355±367. Bremer, E., Kuikman, P., 1994. Microbial utilization of 14 C‰UŠ glucose in soil is affected by the amount and timing of glucose additions. Soil Biology and Biochemistry 26, 511±517. Chander, K., Brookes, P.C., 1991. Microbial biomass dynamics during the decomposition of glucose and maize in metalcontaminated and non-contaminated soils. Soil Biology and Biochemistry 23, 917±925. Costantini, A., Cosentino, D., Segat, A., 1996. Influence of tillage systems on biological properties of a Typic Argiudoll soil under continuous maize in central Argentina. Soil and Tillage Research 38, 265±271. Dahlin, S., Witter, E., 1998. Can the low microbial biomass C-toorganic C ratios in an acid and a metal contaminated soil be explained by differences in growth characteristics, substrate utilisation efficiency or maintenance requirements? Soil Biol. Biochem., in Press. Franzluebbers, A.J., Haney, R.L., Hons, F.M., Zuberer, D.A., 1996. Active fractions of organic matter in soils with different texture. Soil Biology and Biochemistry 28, 1367±1372. Franzluebbers, A.J., Hons, F.M., Zuberer, D.A., 1994. Seasonal changes in soil microbial biomass and mineralizable C and N in wheat management systems. Soil Biology and Biochemistry 26, 1469±1475. Hassink, J., 1993. Relationship between the amount and the activity of the microbial biomass in Dutch grassland soils: comparison of the fumigation±incubation method and the substrate-induced respiration method. Soil Biology and Biochemistry 25, 533± 538. Hassink, J., 1994. Effect of soil texture on the size of the microbial biomass and on the amount of C and N mineralized per unit of microbial biomass in Dutch grassland soils. Soil Biology and Biochemistry 26, 1573±1581. Hopkins, D.W., Shiel, R.S., 1996. Size and activity of soil microbial communities in long-term experimental grassland plots treated with manure and inorganic fertilizers. Biology and Fertility of Soils 22, 66±70. Houot, S., Chaussod, R., 1995. Impact of agricultural practices on the size and activity of the microbial biomass in a long-term field experiment. Biology and Fertility of Soils 19, 309±316. Insam, H., Parkinson, D., Domsch, K.H., 1989. Influence of macroclimate on soil microbial biomass. Soil Biology and Biochemistry 21, 211±221. Jenkinson, D.S., Ladd, J.N., 1981. Microbial biomass in soil: measurement and turnover. In: Bollag, J.M., Stotsky, G. (Eds.), Soil Biochemistry 5. Marcel Dekker, New York, pp. 415±471.

E. Witter, A. Kanal / Applied Soil Ecology 10 (1998) 37±49 Jenkinson, D.S., Powlson, D.S., 1976. The effect of biocidal treatment on metabolism in soil. V. A method for measuring soil biomass. Soil Biology and Biochemistry 8, 209±213. Jenkinson, D.S., Rayner, J.H., 1977. The turnover of soil organic matter in some of the Rothamsted Classical Experiments. Soil Science 123, 298±305. Kaiser, E.A., Mueller, T., Joergensen, R.G., Insam, H., Heinemeyer, O., 1992. Evaluation of methods to estimate the soil microbial biomass and the relationship with soil texture and organic matter. Soil Biology and Biochemistry 24, 675± 683. Kirchmann, H., Persson, J., Carlgren, K., 1994. The Ultuna longterm soil organic matter experiment, 1956±1991. Swedish University of Agricultural Sciences, Uppsala. Lavahun, M.F.E., Joergensen, R.G., Meyer, B., 1996. Activity and biomass of soil microorganisms at different depths. Biology and Fertility of Soils 23, 38±42. Magid, J., Jensen, L.S., Mueller, T., Nielsen, N.E., 1997. Sizedensity fractionation for in situ measurements of rape straw decomposition ± An alternative to the litterbag approach?. Soil Biology and Biochemistry 29, 1125±1133. Nordgren, A., 1988. Apparatus for the continuous, long-term monitoring of soil respiration rate in large number of samples. Soil Biology and Biochemistry 20, 955±957. Paterson, E., Rattray, E.A., Killham, K., 1996. Effect of elevated atmospheric CO2 concentration on C-partitioning and rhizosphere C-flow for three plant species. Soil Biology and Biochemistry 28, 195±201. Paustian, K., Parton, W.J., Persson, J., 1992. Modeling soil organic matter in organic-amended and nitrogen- fertilized long-term plots. Soil Science Society Of America Journal 56, 476±488.

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Powlson, D.S., Brookes, P.C., Christensen, B.T., 1987. Measurement of soil microbial biomass provides an early indication of changes in total soil organic matter due to straw incorporation. Soil Biology and Biochemistry 19, 159±164. Powlson, D.S., Jenkinson, D.S., 1981. A comparison of the organic matter, biomass, adenosine triphosphate and mineralizablenitrogen contents of ploughed and direct-drilled soils. Journal of Agricultural Science, Cambridge 97, 713±721. SchnuÈrer, J., Clarholm, M., Rosswall, T., 1985. Microbial biomass and activity in an agricultural soil with different organic matter contents. Soil Biology and Biochemistry 17, 611±618. Sparling, G.P., 1992. Ratio of microbial biomass carbon to soil organic carbon as a sensitive indicator of changes in soil organic matter. Australian Journal of Soil Research 30, 195±207. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass carbon. Soil Biology and Biochemistry 19, 703±707. Witter, E., 1996. Soil C-balance in a long term field experiment in relation to the size of the microbial biomass. Biology and Fertility of Soils 23, 33±37. Witter, E., Dahlin, S., 1995. Microbial utilisation of 14 C‰UŠ-labelled straw and 13 C‰UŠ-labelled glucose in soils of contrasting pH and metal status. Soil Biology and Biochemistry 27, 1507±1516. Witter, E., MaÊrtensson, A.M., Garcia, F.V., 1993. Size of the soil microbial biomass in a long-term field experiment as affected by different N-fertilizers and organic manures. Soil Biology and Biochemistry 25, 659±669. Wu, J., Joergensen, R.G., Pommerening, B., Brookes, P.C., 1990. Measurement of soil microbial biomass C by fumigation extraction ± an automated procedure. Soil Biology and Biochemistry 22, 1167±1169.