Evaluation of methods to estimate the soil microbial biomass and the relationship with soil texture and organic matter

Soil Bid.

Biochem.

Vol. 24,

No. I, pp. 675483,

1992

0038-0717/92

165.00 + 0.00

Copyright 0 1992 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

EVALUATION OF METHODS TO ESTIMATE THE SOIL MICROBIAL BIOMASS AND THE RELATIONSHIP WITH SOIL TEXTURE AND ORGANIC MATTER E.-A. KAISER,‘* T. MUELLER,* R. G. JOERGENSEN,’H. INSAM’ and 0. HEINEMEYER’ ‘Institut fur Bodenbiologie, Bundesforschungsanstalt fur Landwirtschaft, Bundesallee 50, 3300 Braunschweig and %stitut fur Bodenwissenschaften, von-Siebold-Str. 4, 3400 Gottingen, Germany (Accepted 31 January 1992) Summary-Three methods to estimate soil microbial biomass, CHCl, fumigation-incubation (CFI), CHCI, fumigation-extraction (CFE), and substrate-induced respiration (SIR), were compared with each other and with arginine ammonification and basal respiration using soils over a wide range of textural classes and organic matter content. Biomass and activity of the soil microflora were significantly related to clay, soil organic C and especially to total N content. Values for microbial biomass C as estimated by CFI, CFE and SIR were highly correlated and not affected by soil texture. Only the estimation of biomass C by CFE was slightly influenced by soil organic matter including the two peat soils into the regression analysis. While the absolute values for biomass C deviated considerably when estimated by CFI, CFE and SIR, the ranking of the soils was the same for all these methods. These differences were usually not caused by the abiotic soil conditions we examined. The factors to convert the additional amount of organic C extracted after CHCl, fumigation or the substrate-induced respiration to microbial biomass C are discussed.

INTRODUCTION Since introduced by Jenkinson and Powlson (1976) the CHC& fumigation-incubation method (CFE) has been widely used to estimate soil microbial biomass C.

The original method is strictly devised for well-drained agricultural soils with a relatively low content of soil organic matter. Estimations of biomass C by the CFImethod do not appear to be affected by soil texture, probably because the incubation period is long enough to diminish any differences in gas diffusion and CO2 measurements required for the estimation. However, the time for equilibration and gas measurement is much shorter for the substrate-induced respiration method (SIR) than for the CFI-method. Hence, estimates for microbial biomass C may be affected by gas diffusion problems in clay soils, and the abiotic CO2 release from the bicarbonate pool in alkaline soils (Martens, 1987). The most serious influence of soil texture on estimates of microbial biomass C can be expected with the CHCl, fumigation-extraction technique (CFE). Soluble organic matter liberated by fumigation will partition between soil and extractant in a way that may not be the same in soils differing in clay content (Jenkinson, 1988). In the CFE-method for biomass P (Brookes et al., 1982) this is allowed for by measuring the recovery of a spike of PO:-. So far, no comparable correction has been made for C. Available data for biomass C in clay soils measured by CFE are few and contradictory (Joergensen et al., 1989). Arginine ammonification is a substrate*Author for correspondence.

induced activity like SIR and could be expected to relate closely to microbial biomass C (Alef and Kleiner, 1987). However, as for the CFE-method, arginine ammonification is an extraction procedure and so the independence from soil texture may be doubted. The CFI-, CFE-, SIR-procedures and arginine ammonification were compared with each other and with the basal respiration (of the CFI-method) as an independent, easily-available, measure of soil microbial activity. Our comparison was carried out using soils of a wide range of textural classes and soil organic matter content. the aims were (1) to study the interrelations of these methods, (2) to measure the influence of texture and organic matter content on microbial biomass and activity and (3) to test the effect of soil conditions on the procedures to estimate microbial biomass and activity. MATERIALS AND

METHODS

Sampling and storage

Soils were taken as spade samples (O-20 cm) in late April 1988 in the area of Braunschweig and Goettingen, Lower Saxony, Germany. Within 1 day the samples were brought to the laboratory, sieved (~2 mm), moisture-adjusted to 40% of the water holding capacity (WHC) and stored up to 6 weeks at 4°C in aerated polyethylene bags. CHC, fumigation-incubation

(CFI)

After conditioning at 22°C for 1 week, CFI was performed according to Jenkinson and Powlson 615

676

E.-A. KAISER et al.

(1976). A moist sample was divided into two portions equivalent to 50 g oven-dry soil. One portion was fumigated with ethanol-free CHCI, for 24 h at 22°C in sealed desiccators containing water and soda-lime. After fumigant removal, the soil was adjusted to 50% WHC and kept for 10 days in 1 litre stoppered glass jars, containing water (10 ml) in the base and M NaOH (20 ml) in a glass vial to trap evolved CP,-C. The portion of non-fumigated soil was treated similarly except for the fumigation step. CO,-C was measured by gas chromatography after acidification of the NaOH solution (Martens, 1985). Soil microbial biomass C by fumigation-incubation (CFI-C,,,) was [(CO, evolved from fumigated soils O-10 days) (CO2 evolved from non-fumigated soils O-10 days)]/ k,, where k, was 0.41 (Anderson and Domsch, 1978). CHCI, fumigation-extraction

(CFE)

CFE was performed according to the method described by Vance et al. (1987). Some modifications were tested to give comparable results to the original procedure. A moist sample was divided into two portions equivalent to 25 g oven-dry soil. One portion was fumigated for 24 h at 25°C with ethanol-free CHCl,. The CHCI, was established with 20 gg 2methyl-2-butene ml-’ (Merck, Darmstadt, Germany), which has proved to give identical results to other forms of ethanol-free CHCl,. Thus, we did not need a purification step and avoid the risks of chemical CHCl, decay. Following fumigant removal, the soil was extracted with 100 ml 0.5 M K,SO, by 45 min over-head shaking (40 rev mm-‘) and filtered through a glass fibre filter (Whatman GF/A). The nonfumigated portion was extracted similarly at the time

No.

I 2 3 4 5 6 7 8 9 IO II I2 13 I4 I5 I6 I7 I8 I9 20 21 22 23 24 25 26 27

Name Speyer FAL Schlag I Timmerlah Cremlingen I Destedt I Dransfeld Flachstoeckheim Cremlingen 2 Salzdahlum Didderse Lengede Zweidorf Salzdahlum 2 Destedt 2 Rosdorf I Soellingen I Gleichen Jerxheim Winnigstedt Bodenstedt Drake&erg Rosdorf 2 Ziegenbreite I Drakenberg 2 Ziegenbreite 2 Soellingen 2 Lauingen

*Arable. tMeadow.

Land use a* a a a a mt a a a a a a a a a a a a a a a a

a m a a m

fumigation commenced. Organic C in the K,SO, soil extracts was measured by an automated u.v.-K&O, oxidation procedure (Wu et al., 1990) using a Dohrman D.C. 80 automatic organic C analyser. for this procedure, 10 ml extract was acidified with 10 ml sodium hexametaphosphate buffer [5% (NaPO,),, pH 2.0 with H,PO,]. Soil microbial biomass C by fumigation-extraction (CFE-C,,,) was [(C extracted from fumigated soils) -(C extracted from nonfumigated soils)]/k,,, where k,, was 0.45 (Wu et al., 1990). Substrate-induced

Arginine ammon$cation

Total N soil

Soil C:N

PH CaCI,

0.69 0.85 0.94

0.057 0.076 0 105

12.1 I I.2 9.0 10.0 9.9 7.9 87 8.8 9.1 17.2 9.5 11.6 9.3 9.3 9.6 10.5 9.9 IO.1 10.8 8.7 7.9 10.9 8.6 6.0 8.3 II.7 31.7

4.5 5.5 7.1 5.9 6.X 7.2 7.0 6.8 7.1 6.7 6.8 7.0 7.0 7.0 7.0 7.3 5.9 7.1 7.1 6.X 6.9 7.2 67 7.1 6.9 7.1 3.7

I .oo I .07 I .08 I .09 I.10 1.15 I.17 I.18 1.40 1.52 I.53 I .73 I .76 I .97 I .98 I .98 2.24 2.35 2.44 2.46 2.61 16.99 21.08

0.100 0.101 0.135 0.124 0.124 0.121 0.067 0.123 0.102 0. I50 0 164 0.160 0.165 0.177 0.195 0. I84 0.227 0.283 0.215 0.283 0.407 0.315 I.451 0.665

(SIR)

(AA)

Two portions of a moist soil sample (2 g dry wt) were amended with 0.4 ml of 0.25% (w/v) L-arginine solution. After 3 h incubation at 22°C the samples were mixed with 4 ml 2 M KC1 and stirred vigorously for 10 min. After centrifugation for 2-5 min, the clear supernatant was used for NH, determination using an

Soil organic C % (w/w)

I .oo

respiration

After conditioning at 22-C for 1 week, a moist sample equivalent of 50 g oven-dry soil was amended with a powder mixture containing 150mg glucose and 500 mg talcum (Anderson and Domsch, 1978). The CO, production rate was measured hourly, using the method of Heinemeyer et al. (1989) where each sample was continuously purged with air (250ml min’) and evolved CO2 was measured using an IR-detector. Soil microbial biomass C by substrate induced respiration (SIR-C,,,) was calculated from the maximum initial respiratory response, where SIR-C,,, (Irg g ’ soil) was pg CO, g- ’ soil h-’ x 40.04 + 3.75 (Anderson and Domsch, 1978).

Sand %(W/W) 72.0 42.0 6.3 31.5 21.5 8.8 5.5 56. I 15.8 RI.4 3.2 44.8 21.2 29.5 6.X II.1 13.4 8.0 19.2 8.0 12.9 6.8 13.1 3.3 13.1 31.9 76.0

Silt mineral

Clay material

20.9 52.6 x3.5 40.8 61.5 48.4 80.9 34.6 71.3 15.6 83.9 49.5 54.1 54.6 75.8 73. I 60.1 72.2 66.8 75.4 57.6 75.6 48.2 59.9 51.9 51.0 17.3

7.1 5.4 10.2 21.7 17.0 42.8 13.6 9.3 12.9 3.0 12.9 5.7 24.7 15.9 17.4 15.8 26.5 19.8 14.0 16.6 29.5 17.6 38.7 36.8 35.0 Il.1 6.1

Methods to estimate the soil microbial biomass autoanalyser (Technicon). Arginine ammonification @g NH,-N gg’ soil h-‘, Alef and Kleiner, 1986) was NH,-N extracted from soils after 3 h - NH,-N extracted from soils at 0 h. Basal respiration Basal respiration (pg CO,-C g-’ soil 10 days-‘) was calculated from the unfumigated CFI-sample.

peat soils had a far higher soil organic C content. The ratio soil organic C : total N was usually between 6 and 12, only in two sandy soils it was higher, 17 and 31 for soils 17 and 27, respectively. Texture and soil organic C contents are typical for agricultural soils in North-West Germany. A pH around 7.0 was found in most soils, a pH ~4.5 only in two (Table 1). CO,-C

Analytical procedures Soil organic carbon was estimated by dry combustion (C determinator IR 12, Leco) after removing inorganic C by adding HCl (10%) dropwise and subsequent drying at 70°C (Nelson and Sommers, 1982). Total N was determined with an autoanalyser (Technicon) after Kjeldahl digestion (Bremner and Mulvaney, 1982). Sand and silt fractions 220 pm were measured by sieving after treatment with H,O,, silt and clay ~20 pm by a pipette procedure (Schlichting and Blume, 1966). Single and multiple linear regression analyses were performed using the mean of three replicates. All results were expressed on an oven-dry basis (1OYC for ca 24 h).

RESULTS

Soil properties Clay content ranged from 3.0 to 42.8% of mineral material (Table l), silt from 15.6 to 83.9% and sand from 3.2 to 81.4%. Soil organic C content of most soils was between 0.69 and 2.61% dry wt. Only two Table 2. CO&

evolved

from non-fumigated CO,-C evolved

No.

1 2 3 4 5 6 7 8 9 IO II I2 I3 14 I5 I6 17 18 I9 20 21 22 23 24 25 26 27 cvt f % *Not determined.

evolved and organic C extracted

The amount of CO*-C evolved from non-fumigated soils (basal respiration) ranged from 22.9 to 3 11.9 pg CO,-C g-’ soil 10 days-’ (Table 2) and was positively correlated to soil organic C, total N and most markedly to the amount of organic C extracted by 0.5 M K,SO, from non-fumigated soils (Table 4). When the two peat soils were excluded from regression analysis, basal respiration was further positively correlated to soil C: N ratio, pH and clay, but negatively to sand content (Table 4). Basal respiration and the amount of organic C extracted by 0.5 M &SO, from non-fumigated soils comprised a small and variable proportion (0.52% f 0.25 and 0.41% f 0.13, respectively) of soil organic C (Table 3). The quotient basal respiration : soil organic C was positively correlated to soil pH (r = 0.49, n = 27, P I O.Ol), the quotient 0.5 M K,SO, extractable C: soil organic C was positively correlated with soil pH (r = 0.62, n = 26, P I 0.001) and negatively with the soil organic C content (r = -0.56, n = 26, P I 0.01). The other soil properties we analysed did not influence these two ratios significantly.

and fumigated soils, organic C extracted from non-fumigated induced respiration, arginine ammonification

from:

non-fumigated fumigated soils soils (pg C gg’ soil 10 de’) 22.9 26.8 37.6 34.9 61.9 118.7 108.2 46.2 95.5 38.2 63.2 104.6 60.9 107.4 63.6 89.8 101.8 90.2 87.3 77. I 127.5 123.1 74.5 222.5 127.1 311.9 122.3 3.7

677

31.1 61.7 117.4 135.3 173.7 269.9 197.5 144.7 197.7 100.1 189.8 205.2 230.4 221.9 182.6 222.8 337.1 229.9 242.7 215.1 400.0 284.4 251.3 862. I 400.6 698. I 212.1 3.5

Organic

C extracted

from:

non-fumigated fumigated soils soils (fig C g-’ soil) ND* 29.1 56.3 31.0 39. I 67.5 59.7 43.2 66. I 53.8 45.5 86.0 52.7 67.2 62.9 80.2 37.1 59.5 79.7 70.8 89.7 82.3 73.9 103.3 105.1 443.8 162.5 4.7

ND 67.6 118.7 125.7 148.7 252.2 157.0 135.1 183.7 III.1 157.6 183.1 182.6 209.3 177.4 191.9 243.0 197.2 230.0 213.1 307.7 237.6 262.6 669.2 354.1 894. I 351.1 2.1

and fumigated

Substrate induced respiration (Pl_y~*;-’ g 2.0 3.6 7.2 8.0 il.2 16.0 13.6 8.9 13.4 4.5 10.7 II.2 11.0 15.0 II.0 Il.8 20.7 13.1 14.7 11.2 28.2 lb.2 19.3 49.1 20.4 36.8 8.1 I.5

soils, substrate

Arginine ammonification (pg NHI-N h-’ g-’ soil) 0.87 0.33 0.80 0.4 I 0.13 0.66 0.64 1.28 I.37 0.05 0.03 1.39 ND 0.29 0.07 1.03 1.33 I .24 1.24 1.04 0.97 I .27 I s9 2.71 ND 0.50 I .42 II.4

678

E.-A.

KAISER et

Table 3. Individual conversions factor and the CFI-C,,,: solI organic C ratio for each soil

NO.

E,’ :CFI-C,,,

I

CFI-C,,,: SIRt

NN

2 3 4 5 6 7 8 9 IO II 12 I3 I4 I5 I6 I7 I8 I9 20 21 22 23 24 25 26 27 Mean

10.0 23.6 27.0 30.6 24.4 23.0 16.0 27.0 18.6 33.6 28.9 21.9 37.6 18.6 26.4 27.5 27.1 26.0 25.8 30.0 23.6 24.3 22.4 32.7 32.7 25.6 27.0 25.6

0.45 0.32 0.39 0.40 0.50 0.45 0.38 0.47 0.39 0.36 0.40 0.31 0.51 0.39 0.34 0.36 0.40 0.40 0.42 0.33 0.39 0.44 0.36 0.37 0.48 0.86 0.40

CFI-C,,,: soil organic C % (w/w) 0.29 I .oo 2.07 2.45 2.73 3.45 2.02 2.21 2.27 I.31 2.64 2.08 2.95 1.84 1.90 I .X8 3.26 I .73 1.91 I .70 2.97 I .67 1.77 6.34 2.56

0.55 0.10 2.14

*E, = (C extracted from fumigated soils) - (C extracted from nonfumigated soils. tSlR = substrate-induced respiration (~1 CO, pm’ soil h ‘)_ $Not determined.

al.

Estimation microflora

of biomass and activity of

the soil

Soil microbial biomass C as estimated by the CFI-method ranged from 20.1 to 1560 pg gg ’ dry soil (Table 2) and comprised an extremely variable proportion of soil organic C from 0.1 to 6.3% (Table 3). The lowest CFI-C,,:soil organic C proportion was found in the acidic peat soil, the highest in a grassland soil on clay. The quotient of CFI-C,,: soil organic C was positively linear related to the clay content (r = 0.66, n = 27, P I 0.001) and negatively to the sand content (r = -0.55, n = 27, P I 0.01) and to the soil C:N ratio (r = -0.57, n = 27, P
Table 4. Correlation coefficients of basal respiration, arginine ammonification and biomass estimates with different solI propertIes n Basal resp.t Arg. ammon. SIR-C,,, CFI-C,,, CFE-C,,,

251 27 23~ 25 25f 27 251: ::, 26

Soil organic C

Total N

Soil C:N

0.5 M K,SO, extractable C

pH CaCI,

0.66*** 0.58** 0.60** 0.1 I 0.70*** 0.25 0.68*** 0.22 0.66*** 0.42*

0.79*** 0.84*** 0.70”’ 0.12 0.88*** 0.58** 0.87*** 0.53” 0.87’” 0.69***

-0.54” -0.01 -0.43* -0.03 -0.62** -0.31 -0.56” -0.29 -0.56** -0.11

0.76*** 0.84*‘* 0.61** 0.05 0.64” 0.53*. 0.59** 0.46* 0.61** 0.63”’

0.50’ 0.24 0.18 0.00 0.35 0.35 0.30 0.31 0.21 0.10

Sand

Silt

-0.53** -0.19 -0.24 -0.15 -0.50’ -0.40* -0.44. -0.37 -0.40 -0.19

0.28 0.08 0.05 -0.01 0.18 0.17 0.13 0.12 0.02 -0.06

Clay 0.60” 0.2x 0.41 0.37 0.69*** 0.56” 0.66** 0.57” 0.69*** 0.48’

‘P < 0.05; l*P s 0.01; ***p 5 0.001. &C evolution of non-fumigated soils (CFI). iWithout the two peat soils.

Table 5. Correlation of CFI-C,,, with basal respiration, arginine ammonification and other biomass estimates, increase in I’ when soil wooerties are included in multi& linear resression models

Basal respt Arg. ammon. SIR-C,,, CFE-C,,,

n

r (%) CFI-C,,,

255 27 235 25 259 27

70.0”’ 60.5*** 45.3’ 27.6’ 92X*** 93.3***

24§ 26

97.2’*’ 92.l***

Increase in r2 (%)

____ Soil organic C

Total N

1.6 7.5 8.41 n.i.$ n.1. n.i. n.i.

IO.91 4.6. 30.1’” 20.5** n.i. n.i. n.i.

5.2*‘*

*P < 0.05; **p < 0.01; l**P 5 0.001. tC&-C evolution of non-fumigated soils (CFI). fNo increase. #Without the two peat soils

4.3***

Soil C:N 0.4 6.7’ 5.8 4.1 n.i. n.i.

0.2 3.1***

0.5 M K,SO, extr. C

pH CaCI,

Sand

Silt

Clay

n.i. 10.2” 3.3 18.3f n.i. n.i. n.i.

0.7 n.i. 0.7 6.8 n.i. n.i. n.i.

2.9 3.5 5.6 4.9 n.i. n.i. n.i.

n.i. n.1. n.i. n.i. n.i. n.i. n.i.

2.9 I2.6** 14.3**+ 11.5’ n.i. n.1.

1.5’

1.4

0.7

3.6”.

0.2 0.7

Methods to estimate the soil microbial biomass

n z g

2000

-

1800

-

679

60 r-30y-43.7

rz-0.98

50

1600-

B Y 1400 J p 1200 ,p, 1000 z .g

800

p

600

% L %

400 200

u:l!I

0

0

SIR

CFE

WI

Fig. 1. Notched box-and-whisker plot for the microbial biomass estimations with the CHCI, fumigation-incubation method (CFI), CHCI, fu~gation~xtraction method (CFE) and the substrate induced respiration method (SIR).

Neither the variation of the conversion factor for SIR nor the &-factor of the CFE-method were significantly affected by any of the soil properties we examined (Table 5). The linear relationship between CFI-C,, and arginine ammonification was much weaker, but still significant (Table 5). Stepwise multiple linear regression analysis was used to test whether chemical and physical soil properties improved the correlations between the estimates of microbial biomass and activity (Table 5). No effect of soif properties was found on the correlation between CFI-C,,, and SIR-C,,,. A weak but significant effect on the correlation between CFI-C,,, and CFE-C,,, was found. The correlation of CFI-C,i, with the basal respiration was significantly increased by inclusion of organic matter related properties and clay content into a multiple regression model (Table 5). When the two peat soils were excluded, only total N content improved the correlation of basal respiration to CFI-C,i,. Similar results were observed for the correlation of CFI-Ctic with arginine ammonification, however, at a much lower level of significance.

~'9.96 f0.88 x r2= 0.92

0

200

400

Microbial

600 biomass

800

1000

1200

1400

1600

ICFI) [pg Cmleg-lso113

Fig. 2. Relationship between microbial biomass C estimated by the CHCIJ fumigation-extraction method (CFE-C,& and by the CHCI, fumigation-incubation method (CFI-C,,).

I 200

I 400

Microbial

1 600 biomass

I 800

I 1000

fCFIt

I 1200

I 1400

Cpg Cmic g-’

I 1600

soil3

Fig. 3. ReIationship between substrate-indu~ ~ximum irkal respiration rates (SIR) and microbial biomass C estimated by the CHCI, fumigation-incubation method (CFI-C,,&

Inclusion of total N content significantly improved the correlation between CFI-C,;, and a&nine ammonifi~tion. DISCUSSION

Soil microjora in relation to soil properties

For most of the soils included in this study, microbial biomass and activity were reiated to several of the soil chemical and physical properties (Table 4). In some cases, the two peat soils were exceptions. A positive relationship between microbial biomass C and soil organic C has been reported (e.g. Anderson and Domsch, 1989). Here, biomass and activities were correlated with total N at a higher level of significance than with soil organic C. This stresses the importance of N availability for microbial metabolism, while soil organic C solely reflects the size of the organic matter pool. Insam et al. (1991) also found a close relationship between SIR-C,, and the soil nutritional status. While the biomass C:soil organic ratio increased with increasing crop yield, the metabolic quotient (basal respiration: biomass C) decreased. Among others, Merckx et al. (1985) and Van Veen et al. (1985) found positive relationships between microbial biomass C and clay content. Our correlation between biomass C and clay content is not extremely strong because there are many other factors effective, such as climate, vegetation and topography (Insam et al., 1989). Not only microbial biomass C content, but also the biomass C : soil organic C ratio were found to increase with clay content (Soerensen, 1983), indicating an increasing C availability and a more efficient substrate utilization. The negative relationship of all microbial properties with the sand content was in most cases not significant. The effects of sand on organic matter turnover and microbial performance seems to be more complex and indirect than those of clay. In accordance, Sparling (1981)

680

E.-A. KAISER et al.

and Veremans et al. (1989) did not find correlations between microbial biomass C and soil texture for sandy soils. Relationships at a low level of significance were found between pH and the soil microbial properties. In contrast, Wolters and Joergensen (1991) found strong effects of neutral acidification on microbial performance in forest soils. However, most of our arable soils were from a relatively small range around neutrality, as a result of Ca-fertilizer application. Also, in contrast to Wolters and Joergensen (1991) the amount for 0.5 M K,SO, extractable C was significantly correlated to microbial biomass C and particularly strongly with basal respiration. This suggests a relationship between the fractions of readily mineralizable C and easily extractable C, as it is often expected (e.g. Stanford et al., 1975; Katz et al., 1985). However, this relationship may also be disputed since soils usually are C limited. Under normal conditions it seems very unlikely that the soil solution or aqueous soil extracts would contain high amounts of mineralizable material. Basal respiration has been found to be highly correlated with microbial biomass C as has been observed by others (Van de Warf and Verstraete, 1987; Veremans et al., 1989). Here it was found that basal respiration is affected by several soil properties, especially the total N content. Furthermore, nutritional status and population structure of the microflora, as well as the amount of available C sources had marked effects on the respiration rate. In the arginine ammonification assay, glucose induced respiration is replaced by arginine desamination resulting in NH,-liberation. In contrast to Alef and Kleiner (1987) or Suttner and Kleiner (1988) our correlation of CFI-C,,, to arginine ammonification is relatively poor, although significant. It is evident that abiotic factors, such as clay and again total N content, significantly affect arginine ammonification. A reason could be NH,-retention by clay minerals hampering the extraction or NH,-uptake by microorganisms in soils with a low N content. Effect of soil properties on the estimation of biomass C The values for microbial biomass C as estimated by CFI, CFE and SIR are highly correlated and not affected by soil texture. The inclusion of clay content into the regression model did not improve the relationship of CFI with CFE and SIR, although problems with high clay contents were anticipated for the CFE-method (Jenkinson, 1988). Only CFE-C,,, was slightly influenced by soil organic matter when the two peat soils were included into the regression analysis. Although the absolute value of microbial biomass C varied, the ranking was the same for the CFI, CFE and SIR methods (Table 3). If greater precision is needed to quantify the microbial C pool, preferentially two independent methods, e.g. SIR and CFE, should be used. It is an important result that the differences between the methods are not caused

by the influence of abiotic soil properties, except for CFI-C,,, in the two very acid soils (Nos 1 and 27) which is obviously too low. It is a well established phenomenon that the common CFI-procedure gives unreliable results in acid soils (Jenkinson, 1988). In all the other cases, a single cause for the differences in the conversion of biomass C-estimates by CFI, CFE and SIR could not be detected among the parameters tested here. The soil chemical and physical properties examined in this paper did not affect the two fumigation procedures, nor the glucose-induced maximum initial response. Consequently, CFI, but especially CFE and SIR could be used over a wide range of soils in contrast to methods which use different kinds of heat energy to kill and measure the soil biomass, such as heat extraction with salt solutions (e.g. Jenkinson, 1968) or CO,-evolution after micro-waving (Speir et al., 1986) or drying-rewetting (Blagodatskiy et al., 1987). The adsorption capacity of different soils for heat energy varies enormously, depending on organic matter and clay content. Estimation

of biomass C by the CFE-method

The estimation of microbial biomass C by the two fumigation methods is based on the difference of fumigated minus non-fumigated soils. The accuracy of the estimation is affected by the size of this difference in relation to the values from nonfumigated samples, i.e. the quotient of fumigated: nonfumigated soils. The quotient of extractable C from fumigated : non-fumigated soils was on average 3.2 (2.01-6.55). In the two peat soils the ratio was relatively small. Consequently, in none of our soils is the estimation of biomass C seriously affected by high amounts of extractable soil organic matter. A similar quotient was found for the CO* evolved from fumigated:non-fumigated soils, with a mean value of 2.8 (range from 1.83 to 3.88, excluding the two acidic soils Nos 1 and 27). The additional amount of organic C extracted by 0.5 M K,SO, after CHCl, fumigation must be converted to microbial biomass C using a proportionality factor (ICE,-) which corrects incomplete release and extraction of microbial C. This conversion is the most crucial point of all methods, because fluctuations of the proportionality factor are the biggest source of error. We used a k,,-factor of 0.45 which was obtained empirically by calibration against the CFImethod (Wu et al., 1990). Since the CFE-method yielded the lowest biomass values (Fig. l), however not significant, a k,,-factor of 0.41 should be used which could be obtained by regression analysis and by averaging the individual &-factors (E,: CFI-C,,,, Table 3). The automated u.v.-KzS,Os oxidation procedure (Wu et al., 1990) we used to measure organic C in the K,SO, soil extracts give ca 19% higher values than oxidation procedures by refluxing with acid dichromate reagents. Thus kac-factors of 0.45 and 0.41 for u.v.-K&O, oxidation correspond to k,,-

Methods to estimate the soil microbial biomass

681

factors for dichromate oxidation of 0.38 and 0.34, respectively. these two are similar to conversion factors for dichromate oxidation obtained by others using different calibration procedures, which range from 0.33 (Ross, 1990) to 0.40 (Sparhng et al., 1990). Considering all available data on &,-factors, we do not propose a reassessment of the factor given by Wu et al. (1990) at the moment (Fig. 2). Estimation of microbial biomass C by CFE was slightly influenced by soil organic matter in the two peat soils. Consequently, this method should be used cautiously in highly organic soils, despite encouraging results by us and Sparling et al. (1990).

The factor proposed by Anderson and Domsch (1978) to convert the maximum initial respiratory response to biomass C needs to be discussed in view of the data presented here (Figs 1 and 3). Still, we refrain from recommending a new calibration factor for general use. However, whenever biomass is being calculated from SIR, one has to critically evaluate which factor should be employed. In our soils measured with continuous aeration, regression analysis of SIR and CFI-C,ic gave the linear retationship: SIR-C,,, (,ug gg ’ soil) = ~1 CO, gg ’ soil h--’ x 30 (the intercept was not significantly different from 0).

Calibration of the SIR-method

Choice of methods to estimate microbial biomass C

In spite of the high correlation between the two methods, the absolute values for SIR-C,, were significantly higher (25%) than for CFI-C,,, (Fig. 1). 20% higher values for SIR than for CFI have also been reported by Sparling and Eiland (1983). The data imply that per unit biomass C more CO,release from glucose utilization was measured than in the original work of Anderson and Domsch (1978). For their initial calibration of the SIRmethod, they used a variation of the CFI-method, where CO, evolution was continuously measured for 10 days and only that measurement period was used for calculating CFI-C,,, during which the fumigated sample showed higher respiration than the non-fumigated one. This may have caused an overestimation of CFI-C,,,. Other reasons for the discrepancy between SIR- and CFI-C,,, may be a different age structure of the microbial community, another fungal: bacterial ratio or a different availability of degradable C compounds. In contrast to the original publication, our soils were sampled solely in spring when baseline conditions are most likely to prevail (Anderson and Domsch, 1986). We found the largest differences between SIR- and CFI-C,, in the soils with the lowest microbial biomass C content. This is in accordance with the findings of Ocio and Brookes (1990) who suggested that a disproportionally large part of the microbial biomass was active in decomposing glucose in soils with little biomass. A wide variety of methodological alterations in the SIR-assay has been employed, among them headspace sampling and gas chromatography for CO, (Sparling and West, 1990), 0, measurement with the Sapromat system (Beck, 1984) and analysis systems with discontinuous (Anderson and Domsch, 1978) and continuous aeration (Heinemeyer et aI., 1989). Static systems have the disadvantage of chang ing CO, gradients during the course of incubation, and CO, retention in the soil solution which may cause underestimations, unless they are corrected for (Sparling and West, 1990). Discontinuous aeration may cause overestimations in neutral or alkaline soils (Martens, 1987). The Sapromat system does not have these limitations, however, its sensitivity is low.

The CFI-method was the first of the new methods to estimate biomass C of soil microorganisms and was considered a breakthrough in soil ecology (Jenkinson and Powlson, 1976). However, methodological problems (choice of control) and the time of incubation (at least 10 days) limits the use of CFI, especially if routine analysis of many samples is required. In contrast to the CFI-method, the CFE-technique can be used over the whole range of soil pH (Vance et al., 1987). It is also applicable to soils that have recently received substrate (Ocio and Brookes, 1990), to water-logged soils (Inubushi et al., 1991) and to soils supersaturated with K,SO, solution (Widmer ef af., 1989; Meuller et al., 1991). Nevertheless, smearing and compaction of wet soils must also be avoided in this method, e.g. if soils are sieved. Especially clay soils are sensitive to this kind of damage (Ross, 1987). In contrast to CFIand SIR-method, which need an optimum water content between 40 and 60% WHC, the CFE-method is only affected by dryness. It is recommended that dry soils (below - 10 to -5 kPA water potential) should be rewetted to ensure that the fumigation is fully effective (Sparling et al., 1990). In the 0.5 M K,SO, extracts of the CFE-method, not only C can be measured but also NH, and total N (Brookes et al., 1985), ninhydrin N (Joergensen and Brookes, 1990) and carbohydrates (Joergensen et al., 1990). As the CFI-method, the CFE-method is not affected by high clay content, but should be used cautiously in highly organic soils. Major problems arise in soils with extremely low biomass C content when the ratio of extractable C from fumigated: non-fumigated soils gets closer and the error of the two measurements accumulates. Problems of the SIR-method are mainly the fluctuations of the glucose mineralization caused by changes in the ~pulation structure. However, the SIR-method gives biomass data within 6 h, requires no toxic chemical and has a very low coefficient of variation (Table. 2). therefore this method is also suitable for routine measurements of soils containing small amounts of microbial biomass C.

E.-A. KAISERet al.

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Acknowledgements-We would like to thank Volkmar Walters for support and Ingrid Ostermeyer for analysis of the particle size distribution. We also thank Sabine Schintzei, Andrea Oehns-Rittgerott and Angelika Gonser for expert technical assistance.

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