Evaluation of methods for measuring microbial biomass C and N and relationships between microbial biomass and soil organic matter particle size classes in West-African soils

PERGAMON

Soil Biology and Biochemistry 31 (1999) 1071±1082

Evaluation of methods for measuring microbial biomass C and N and relationships between microbial biomass and soil organic matter particle size classes in West-African soils B. Vanlauwe a, *, O.C. Nwoke a, N. Sanginga a, R. Merckx b a

b

Soil Microbiology, IITA Ibadan, Nigeria, c/o Lambourn, Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK Laboratory of Soil Fertility and Soil Biology, Faculty of Applied Agricultural Sciences, K. Mercierlaan 92, 3001 Leuven/Heverlee, Belgium Accepted 22 December 1998

Abstract The fumigation±incubation (FI) and the fumigation±extraction (FE) ninhydrin methods for quantifying the microbial biomass pool were tested and the relationships between microbial biomass characteristics and soil organic matter fractions (separated following particle size) investigated for a range of soils representative for the West-African moist savanna zone (13 soils). Three soils from the humid forest zone were also included. Microbial C values calculated using the CO2-C production of the fumigated soils from d 10 to d 20 as control (Bio-C (II)) were better correlated with the ¯ush of ninhydrin reactive N (NRN) during 10 d of fumigation than those calculated with the CO2-C production in the unfumigated soils from d 10 to d 20 as control (Bio-C (I)). The Bio-C (II) values also showed a more consistent range of values (22±210 mg C kg ÿ 1 soil) than the Bio-C (I) values. Using a kC factor of 0.35, which was considered to be more appropriate for our soils than a kC factor of 0.45, kC,NRN could be estimated as 22 and 16, for a 5- and 10-d fumigation, respectively. For all savanna soils, the NRN ¯ush after 5 d of fumigation was closely related to the NRN ¯ush after 10 d, indicating that a 5-d fumigation was sucient provided that the k-values are adapted. Mineral N ¯ushes during incubation and incubation after fumigation were small. Although microbial N values calculated as [NH4+ -N ¯ush of the fumigated soils (0±10 d)]/kN with kN =ÿ 0.014(CO2-C-¯ush-to-NH4+ -N-¯ush during fumigation) + 0.39, which gave values of 14.0±100.7 mg N kg ÿ 1 soil, showed the best relationship with the NRN ¯ush after 10 d of fumigation, microbial N values calculated as [(mineral N ¯ush of the fumigated soils after 10 d) ÿ(mineral N ¯ush of the unfumigated soils after 10 d)]/0.68, which gave values of 3.4±46.2 mg N kg ÿ 1 soil, including 3 values <2 mg N kg ÿ 1 soil and 1 negative value, yielded microbial C-to-N ratios (4.7±16.4) which were better re¯ecting the ratio C-¯ush-to-N-¯ush of fumigated soils (5.2±13.5). Using a kN value of 0.68, kN,NRN could be estimated as 1.5. Between 7 and 17% and between 11 and 28% of the total soil C was part of the soil litter (SL) (organic particles larger than 250 mm) and the particulate organic matter (POM) (organic particles larger than 53 mm), respectively. The CO2-C production of the unfumigated soils was strongly (P < 0.01) related to the SL-C content. Inclusion of the silt and clay content in a linear regression equation increased the R 2 value from 0.70 to 0.91. The Bio-C (II) content showed the best relationship with the C content of the soil particles between 53 and 20 mm and the silt fraction. The NRN ¯ush after 10 d showed the best relationship with the C content of the particles between 250 and 53 mm and between 53 and 20 mm. Maximally 48% of the variation in Bio-C (II) values was explained by the C content of the various fractions, indicating that the present methods do not exclusively measure the active microbial biomass. # 1999 Elsevier Science Ltd. All rights reserved.

1. Introduction * Corresponding author. Fax: 234-2-241-2221; e-mail: b.vanlauwe @cgnet.com

The number of studies in which soil microbial biomass has been measured has been enormous since the

0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 2 1 - 8

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development of a quantitative methodology by Jenkinson and Powlson (1976). Although soils from tropical regions have received some attention (Henrot and Robertson, 1994; Kirchmann and Eklund, 1994; Feigl et al., 1995), soils from the West-African moist savanna zone have not been studied. Although the West-African savanna zone is considered as a vast area where sustainable agriculture can be developed (Pieri, 1992), fertilizers are scarce or too expensive. This implies that any sustainable cropping system must rely partially or completely on the management of organic residues for nutrient supply. A method for reliably estimating microbial biomass C and N values will assist our understanding and aid the design of such cropping systems, because a major part of the di€erent processes transforming those organic amendments (e.g. nutrient supply through mineralization) is microbially-driven. Among the main soil types in the moist savanna zone are Lixisols (25% of the total area), Ferralsols (23%), Arenosols (9%) and Acrisols (7%) (Jagtap, 1995). Several methods have been proposed to measure the microbial biomass pool and the major part of those methods are based on the quanti®cation of a ¯ush of microbially-derived products after fumigation. To decide on the `best' method remains a challenging task as several factors related to methodological diculties, soil characteristics or other considerations, such as analysis time and cost may lead to a di€erent method of choice. We have compared the original fumigation± incubation method (Jenkinson and Powlson, 1976) and its derivatives with the fumigation±extraction ninhydrin-reactive N (NRN) method (Amato and Ladd, 1988). Fumigation±extraction (FE) methods have the advantage that the cumbersome incubation step in the fumigation±incubation (FI) techniques is avoided. Moreover, Vanlauwe et al. (1994) showed that due to the small NRN content of plant residues relative to that of the microbial biomass, this method can potentially be used as early as 4 d after addition of fresh plant materials. Other methods, such as the original fumigation incubation method, usually fail under such conditions. A reduction in fumigation time from 10 to 5 d would make the FE method even more attractive. Soil organic matter (SOM) plays a major role in the supply of nutrients to the growing crop in cropping systems with a minimal input of mineral fertilizer, as is the case in large parts of West-Africa. In the absence of fresh organic matter additions, the SOM pool provides the microbial community with energy and nutrients for maintenance and growth. However, SOM is not a homogeneous pool, but consists of organic components with a wide range of turnover times (Woomer et al., 1994). For a clear understanding of the contribution of SOM to the soil nutrient status, several

approaches have been proposed to fractionate SOM by physical techniques based on particle size, particle density, or a combination of both (Stevenson and Elliott, 1989; Christensen, 1992). A successful SOM fractionation scheme should yield fractions having a separate function with respect to the overall contribution of the SOM pool to soil fertility. Vanlauwe et al. (1998) found that fractionating the SOM pool into particle size classes after soil dispersion yielded SOM pools with di€erent abilities to supply N to a growing crop. Our objectives were (i) to test the validity of the fumigation±incubation (FI) and the fumigation±extraction (FE) ninhydrin-reactive N (NRN) method (Amato and Ladd, 1988) for quantifying the soil microbial biomass in a range of soils representative for the West-African moist savanna zone, (ii) to determine the e€ects of a reduction in fumigation time from 10 to 5 d on microbial biomass measurements for the FE method, (iii) to determine the size and selected characteristics of soil organic matter fractions separated following particle size for the same range of soils and (iv) to evaluate relationships between the microbial biomass and soil organic matter fractions. 2. Materials and methods 2.1. Sampling sites and procedure Soil (0±10 cm) was taken from 13 locations (4 in 1994 and 9 in 1995) representing a signi®cant part of the soils of the West-African moist savanna (Table 1). Three soils from the humid forest zone were also included. All soils were taken from land that had been left fallow for a varying number of years. Sites in the savanna zone and the Mbalmayo sites (humid forest) contained grasses and shrubs as fallow vegetation. The Ebolowa soil (humid forest) was taken under Chromolaena odorata fallow. Following the USDA classi®cation (Anderson and Ingram, 1993), the soils are predominantly sandy or sandy loamy, except for the Ebolowa soil (Table 1). Microbial biomass was measured on the fresh, ®eld-moist soils following the fumigation±extraction NRN method with a 5-d fumigation (modi®ed from Amato and Ladd, 1988). A part of the soils was air-dried and stored pending incubation. Another part was oven-dried, sieved to <4 mm and analyzed for organic C (Amato, 1983), Kjeldahl-N, ECEC (IITA, 1982) and pH (H2O) (1:2.5 soil:water ratio). 2.2. Microbial biomass C and N measurements The air-dried soils were conditioned at 34% water holding capacity at 258C for 14 d prior to fumigation

B. Vanlauwe et al. / Soil Biology and Biochemistry 31 (1999) 1071±1082

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Table 1 Selected characteristics of the used soils Site, year of sampling

Abbreviated Country

Coordinates

Agro- Soil type eco(FAO legend) zonea

Soil characteristics organic total C N

C-to-N ECEC

g kg ÿ 1 soil BouakeÂ-94 BouakeÂ-95 Glidji-94 Glidji-95 Niaouli-94 Niaouli-95 Ibadan-94 Ibadan-95 Mbalmayo-94 Mbalmayo-95 FerkeÂ-95 Amoutchou-95 Sarakawa-95 Save-95 Ina-95 Ebolowa-95

B94 B95 G94 G95 N94 N95 I94 I95 M94 M95 F95 A95 Sk95 S95 In95 E95

Ivory Coast ± Togo ± Benin ± Nigeria ± Cameroon ± Ivory Coast Togo Togo Benin Benin Cameroon

6856 0 W 7844 0 N ± 1836 0 E 6815 0 N ± 2810 0 E 6840 0 N ± 3854 0 E 7826 0 N ± 11842 0 E 3830 0 N ± 6855 0 W 9837 0 N 1810 0 E 7822 0 N 18 1 0 E 9837 0 N 2826 0 E 7859 0 N 2844 0 E 9858 0 N 118 8 0 E 2839 0 N

DS ± DS ± DS ± DS ± HF ± SGS DS DS SGS NGS HF

ferric acrisol ± rhodic ferralsol ± rhodic ferralsol ± ferric lixisol ± ferric acrisol ± ferric acrisol haplic arenosol ferric acrisol haplic arenosol ferric lixisol haplic acrisol

6.07 6.57 3.74 3.79 3.24 2.86 7.32 5.51 19.72 11.90 10.83 4.39 4.02 3.75 5.63 19.88

0.49 0.53 0.37 0.37 0.31 0.29 0.73 0.56 1.91 1.23 0.91 0.31 0.31 0.28 0.42 2.16

pH sand silt clay (H2O) g kg ÿ 1 soil

cmol (+) kg ÿ 1 12.4 12.4 10.1 10.2 10.5 9.9 10.0 9.8 10.3 9.7 11.9 14.2 13.0 13.4 13.4 9.2

4.0 3.7 1.5 1.4 2.7 1.8 1.9 1.6 3.8 2.1 2.5 1.3 1.3 1.5 1.5 5.1

4.58 4.47 5.88 5.52 4.49 4.67 5.06 5.10 5.98 4.91 5.84 6.28 6.29 6.34 6.82 5.15

74 80 88 91 88 90 80 87 68 72 53 91 90 93 88 40

8 5 6 3 4 2 14 6 20 11 30 4 5 3 6 11

18 15 6 6 8 8 6 7 12 17 17 5 5 4 6 49

a `HF': humid forest zone (length of growing period>270 d); `DS': derived savanna zone (length of growing period between 211 and 270 d); `SGS': Southern Guinea savanna zone (length of growing period between 181 and 210 d); `NGS': Northern Guinea savanna zone (length of growing period 151±180 d) (Jagtap, 1995).

or incubation. All fumigations and incubations were done with 40 g of conditioned soil placed in 100 ml beakers. For the fumigation incubation (FI) method, one triplicate set of soils was fumigated with ethanolfree chloroform for 24 h in the dark, after which the chloroform was evacuated and the soils incubated in 1.5 l jars containing 10 ml of NaOH 1 N in a separate vial. The bottom of the jars was lined with 1 cm of water, the jars were closed air-tight and incubated for 20 d at 258C in the dark. The NaOH was removed after 10 and 20 d and the NaOH solution was titrated with 0.05 N HCl to measure the amount of CO2-C absorbed (Bundy and Bremner, 1972). A second triplicate set of unfumigated soils was incubated in a similar way. Before incubation and after 10 and 20 d of incubation, the soils (40 g wet soil) were extracted with 0.5 M K2SO4 (120 ml). For the fumigation±extraction (FE) method, a third triplicate set of soils was fumigated for 5 d and a fourth set for 10 d at 258C in the dark, after which the soils were extracted in a similar way as described above. The soil extracts of all fumigated and unfumigated soils were analyzed for nitrateN and ammonium-N with an autoanalyzer set-up (IITA, 1982) and for ninhydrin-reactive N (NRN) (Amato and Ladd, 1988).

2.3. Soil organic matter fractionation and analysis The 11 soils collected in 1995 were used for SOM fractionation, following a procedure based on particlesize distribution after soil dispersion. Details are given by Vanlauwe et al. (1998). In summary, 100 g of dry soil was dispersed in 100 ml of a Nahexametaphosphate±Na-carbonate solution through shaking for 16 h on a reciprocal shaker adjusted to 144 rpm. After dispersion, the soil slurry was wetsieved on a wet-sieve shaker to separate the fractions >2 mm, between 250 mm and 2 mm and between 53 and 250 mm. The organic components were separated from the mineral fraction for each of these particle-size classes by careful decantation. The slurry passing through the 53 mm sieve was manually passed through a 20 mm sieve to separate the fraction between 20 and 53 mm. The silt fraction (2 to 20 mm) was separated after four sedimentation cycles at room temperature, while the clay fraction (<2 mm) was collected after four ¯occulation cycles with CaCl2, followed by dialysis in distilled water. The di€erent fractions are referred to as follows: >2 mm mineral: `M2000'; >2 mm organic: `O2000'; 250 mm±2 mm mineral: `M250'; 250 mm to 2 mm organic: `O250'; 53 to 250 mm min-

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eral: `M53'; 53 to 250 mm organic: `O53'; 20 to 53 mm: `MO20'; 2 to 20 mm: `silt' and <2 mm: `clay'. The sum of the O2000 and the O250 material is referred to as `soil litter' (SL), while all organic material larger than 53 mm is referred to as `particulate organic matter' (POM) (Cambardella and Elliott, 1992). The di€erent fractions were analyzed for C and Kjeldahl-N. 2.4. Mathematical and statistical analyses Microbial biomass C values (Bio-C) were calculated following Eqs. (1) and (2) (Jenkinson and Powlson, 1976; Merckx et al., 1985): Bio-C …I † ˆ …F0±10 ÿ UF10±20 †=kC

…1†

Bio-C …II † ˆ …F0±10 ÿ F10±20 †=kC

…2†

with F0±10 the CO2-C production in the fumigated soils during the ®rst 10 d of incubation, UF10±20 the CO2-C produced in the unfumigated soils from d 10 to d 20 and F10±20 the CO2-C produced in the fumigated soils from d 10 to d 20. The kC factor corrects the measured C ¯ush for incomplete die-o€ and mineralization of the microbial cells and a value of 0.45 was used (Sparling and Ross, 1993). Adjustment of the kC factor for low pH soils (Vance et al., 1987) was not necessary as the soil pH was just below 4.5 in only two cases (Table 1). Microbial biomass N values (Bio-N) were calculated following Eqs. (3)±(5) (Shen et al., 1984; Voroney and Paul, 1984): Bio-N …I † ˆ ‰F…Nmin†0±10 ÿ UF…Nmin†0±10 Š=0:68

…3†

‡ Bio-N …II † ˆ ‰F…NH‡ 4 †0±10 ÿ UF…NH 4 †0±10 Š=kN

…4†

Bio-N …III † ˆ ‰F…NH ‡ 4 †0±10 Š=kN

…5†

F(Nmin)0±10 and UF(Nmin)0±10 stand for the total mineral N released in the fumigated and unfumigated soils during the ®rst 10 d of incubation, respectively. F(NH4+ )0±10 and UF(NH4+ )0±10 stand similarly for the ammonium-N released. The kN factor in the Voroney and Paul (1984) equations was adjusted following the CO2-C-¯ush:NH4+ -N-¯ush during fumigation following Eq. (6) (Voroney and Paul, 1984): kN ˆ ÿ0:014  …CO2 -C-flush:NH ‡ 4 -N-flush† ‡ 0:39

…6†

The measurements were subjected to ANOVA (SAS, 1985). Signi®cantly di€erent means were separated following the least signi®cant di€erence (LSD) criterion. Linear and multiple regression analysis was used to investigate relationships between some measured variables (SAS, 1985).

3. Results 3.1. Microbial biomass C and N measurements CO2-C production during the ®rst 10 d was higher (but not always signi®cantly) in the fumigated (FI) compared with the unfumigated soils (Table 2). For the unfumigated soils, the amount of CO2 produced was similar for the period 0±10 and 10±20 d, indicating a constant production rate. In 11 of the 16 soils, the fumigated soils produced less CO2 than the unfumigated soils between 10 and 20 d (Table 2). As a result, Bio-C (I) values were often small or even negative, which was not the case for Bio-C (II) values (Table 2). In the fumigated soils, the soil nitrate-N content did not change considerably between 0 and 10 and between 10 and 20 d of incubation, while the ammonium-N content increased signi®cantly for all soils between 0 and 10 d and for more than half of the soils between 10 and 20 d (Table 3). The total mineral N content of the unfumigated soils increased after 20 d of incubation for 11 of the 16 soils, mainly due to an increase of ammonium-N (Table 3). Bio-N (I) values were often small and even negative in one case, resulting in variable microbial C-to-N ratios ranging from ÿ4 to 144 (Table 4). Bio-N (II) values were higher, and resulted in more consistent biomass C-to-N ratios, except for 2 negative values. Bio-N (III) values were always positive and resulted in a rather narrow range of low microbial C-to-N ratios. Ten days of fumigation increased the amount of NRN signi®cantly in all soils, except in the N94 soil (Table 5). The NRN ¯ush after 5 d was highly signi®cantly correlated with the NRN ¯ush after 10 d, after excluding the M94 soil (Fig. 1). This soil produced substantially less NRN after 5 d of fumigation compared to 10 d. The NRN ¯ush after 5 d and 10 d was linearly related to the mineral N ¯ush of the fumigated soils after 10 d, but not to the mineral N release of the unfumigated soils after 10 d of incubation (Fig. 2a and b). The NRN ¯ush of the dried and conditioned soils during 10 d of fumigation was linearly related with the NRN ¯ush during 5 d of fumigation of the fresh soils (Fig. 3). The NRN ¯ush after 5 d (excluding the M94 soil) and 10 d (including all soils) explained most of the variation in Bio-C (II) content (Table 6). The NRN ¯ush after 5 d (excluding the M94 soil) and 10 d (including all soils) explained most of the variation in Bio-N (III) values (Table 6). 3.2. Soil organic matter fractionation Between 55% (Niaouli) and 34% (Mbalmayo) of the total soil C was associated with the clay fraction

B. Vanlauwe et al. / Soil Biology and Biochemistry 31 (1999) 1071±1082

1075

(Fig. 4a). The POM contained between 28% (Glidji) and 11% (BouakeÂ) of the total soil C. Both the size and variability of the C-to-N ratios of the di€erent fractions decreased with particle size (Fig. 4b).

shown). Relationships between the NRN ¯ush after 10 d and the C content of the di€erent fractions were not signi®cant, except for the O53-C and the MO20-C (Table 7).

3.3. Relationships between the size and activity of the microbial biomass and the size of the soil organic matter fractions

4. Discussion

The O250-C content or the SL-C content explained most of the variation in CO2-C production of the unfumigated soils after 10 d (Table 7). Inclusion of silt and clay content in the linear regression equation increased the R 2 value from 0.70 to 0.91 for the SL material (Fig. 5). Coecients of determination of the linear regressions between the mineral N release of the unfumigated soils and the N content of the di€erent SOM fractions were lower, compared with coecients of determination of the linear regressions between CO2 production and C content of the fractions, except for the O2000 material (Table 7). Relationships between the Bio-C (II) values and the C content of the di€erent fractions were not signi®cant, except for the MO20-C and the silt-C (Table 7). Inclusion of more parameters in various multiple regression models did not improve the explanatory value relative to the simple linear regression (results not

4.1. Microbial C and N measurements Assuming (i) that the major part of the NRN components released upon fumigation are derived from the microbial biomass (Carter, 1991) and (ii) that 10 d of fumigation are sucient to reach a plateau for the release of NRN components (Amato and Ladd, 1988; Sparling et al., 1993), it appears that for the range of soils used in this work, the CO2-C production from the fumigated soil between 10 and 20 d of incubation was a better control than the CO2-C production of the unfumigated soil, as the release of NRN during a 10-d fumigation explained a greater proportion of the variation in Bio-C (II) values than in Bio-C (I) values. Sparling and Ross (1993) stated that in some soils, CO2-C production by fumigated soil, once the ¯ush of decomposition has passed, may be markedly less than that by fumigated soils and an unfumigated soil control is then inappropriate. Similar conclusions were

Table 2 CO2-C production of the fumigated and unfumigated soils for the periods between 0±10 and 10±20 d Soil

Microbial biomass Cb

CO2-production fumigated d 0±10

fumigated d 10±20

unfumigated d 0±10

unfumigated d 10±20

mg CO2-C kg ÿ 1 soil BouakeÂ-94 BouakeÂ-95 Glidji-94 Glidji-95 Niaouli-94 Niaouli-95 Ibadan-94 Ibadan-95 Mbalmayo-94 Mbalmayo-95 FerkeÂ-95 Amoutchou-95 Sarakawa-95 Save-95 Ina-95 Ebolowa-95 LSD a

a

(5%)

Bio-C (I)

Bio-C (II)

mg microbial C kg ÿ 1 soil

36.4 59.3 47.8 53.2 37.9 54.9 77.5 78.2 157.0 97.9 153.4 55.8 85.5 61.9 86.1 97.7

26.4 45.0 19.6 26.6 27.2 27.4 48.3 50.2 59.8 53.5 58.7 30.7 36.4 30.2 50.9 42.5

11.9

26.0

35.8 31.2 39.7 47.4 36.8 28.0 70.8 44.3 79.1 71.3 35.8 32.4 35.3 29.1 45.1 56.1

36.2 37.8 36.3 49.7 43.7 48.4 70.3 35.0 88.7 74.5 27.0 45.2 31.5 48.1 36.1 47.1

1 48 26 8 ÿ13 14 16 96 152 52 281 24 120 31 111 113

22 32 63 59 24 61 65 62 216 99 210 56 109 70 78 123

`LSD' refers to `least signi®cant di€erence'. Calculated as [CO2-C production of fumigated soil between 0 and 10 d (F10) minus CO2-C production of the unfumigated soil between 10 and 20 d (UF 20) (Bio-C (I)) or minus CO2-C production of the fumigated soil between 10 and 20 d (F20)]/0.45 (Bio-C (II)). b

a

3.4

`LSD' refers to `least signi®cant di€erence'.

LSDa (5%)

2.4 17.2 42.3 18.3 5.8 13.0 30.5 30.0 105.9 14.0 40.4 13.5 14.0 10.5 14.9 12.9

3.3 19.3 40.2 20.9 6.4 13.4 33.5 24.3 100.7 13.0 37.6 12.6 10.8 5.6 13.5 15.7

2.2 16.9 36.6 18.8 5.7 9.3 28.8 21.9 111.1 11.3 34.2 10.9 9.3 4.7 12.2 14.0

21.2 1.4 0.6 0.4 12.4 2.0 35.0 0.1 27.8 65.9 1.7 0.1 1.1 0.6 1.0 87.8

23.0 5.5 0.3 4.4 16.1 4.6 49.9 4.5 30.9 71.8 2.9 2.4 0.3 2.6 2.3 94.2 3.0

31.1 8.7 4.2 9.0 18.6 6.3 42.5 7.4 38.4 67.9 5.9 7.8 4.8 4.3 2.8 95.5

25.1 8.1 6.0 8.7 15.2 7.9 49.8 11.0 60.3 76.9 20.7 8.2 11.1 9.1 13.2 99.2

33.3 10.8 10.5 12.2 22.0 11.0 44.5 16.3 50.8 74.5 29.5 13.6 13.8 11.4 16.5 103.6

22.7 26.1 19.9 24.7 39.8 43.5 21.4 28.3 20.1 16.0 13.9 18.9 69.2 82.2 25.2 31.3 129.8 129.6 78.1 85.4 36.8 41.7 14.1 16.0 10.2 14.1 10.2 14.3 14.5 18.6 103.3 109.6

4.9

33.5 25.9 46.5 27.3 21.3 19.4 73.0 37.4 144.4 81.9 46.3 21.3 18.8 14.8 17.7 108.4

28.5 27.3 46.1 29.6 21.6 21.3 83.3 35.3 161.0 89.8 58.2 20.7 21.9 14.7 26.7 114.9

35.5 27.7 47.6 31.0 27.7 20.3 73.0 38.2 161.8 85.4 63.7 24.5 23.1 16.1 28.6 117.6

unfumigated unfumigated fumigated fumigated d 10 d 20 d 10 d20

Total mineral N content (mg N kg ÿ 1 soil)

unfumigated unfumigated fumigated fumigated d 0 d 10 d 20 d 10 d 20

Ammonium-N content (mg N kg ÿ 1 soil)

unfumigated unfumigated fumigated fumigated d 0 d 10 d 20 d 10 d 20

BouakeÂ-94 1.4 3.2 BouakeÂ-95 18.6 19.1 Glidji-94 39.2 43.3 Glidji-95 20.9 23.9 Niaouli-94 7.7 9.3 Niaouli-95 11.9 14.3 Ibadan-94 34.3 32.5 Ibadan-95 25.1 26.7 Mbalmayo-94 101.5 98.6 Mbalmayo-95 12.1 13.6 FerkeÂ-95 35.1 38.8 Amoutchou-95 14.0 13.7 Sarakawa-95 9.3 13.8 Save-95 9.6 11.7 Ina-95 12.6 16.4 Ebolowa-95 15.5 15.4

d0

Nitrate-N content (mg N kg ÿ 1 soil)

Table 3 Nitrate-N, ammonium-N and total mineral (min.) N content of in the fumigated and unfumigated soils at 0, 10 and 20 d of incubation (d 0, d 10 and d 20, respectively). Total mineral N values of the fumigated soils incubated for 10 d are not signi®cantly di€erent (P < 0.05) from the total mineral N values of the fumigated soils incubated for 20 d when bold

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Table 4 Microbial biomass N, calculated following Bio-N (I), Bio-N (II) and Bio-N (III), microbial C-to-N ratio and C ¯ush (mg kg ÿ 1 soil) over NH4+ N ¯ush (mg kg ÿ 1 soil) of the fumigated soils (CF-to-NF) for the studied soils Microbial biomass Na Bio-N (I)

Microbial C-to-N ratiob

Bio-N (II)

Bio-N (III)

method for microbial N calculation

mg N kg ÿ 1 soil BouakeÂ-94 BouakeÂ-95 Glidji-94 Glidji-95 Niaouli-94 Niaouli-95 Ibadan-94 Ibadan-95 Mbalmayo-94 Mbalmayo-95 FerkeÂ-95 Amoutchou-95 Sarakawa-95 Save-95 Ina-95 Ebolowa-95

3.4 3.9 3.8 1.9 ÿ5.5 3.5 1.7 5.9 46.2 6.6 24.3 6.9 11.6 0.5 11.9 7.8

8.3 9.5 21.6 14.2 ÿ4.3 12.6 ÿ0.3 22.5 91.0 19.2 64.0 19.8 40.2 22.5 37.4 18.5

CF-to-NF

15.1 25.1 20.2 27.5 14.0 22.5 47.0 37.7 100.7 41.4 68.6 27.6 37.1 29.6 41.8 42.2

Meanc SDc

Bio-N (I)

Bio-N (II)

Bio-N (III)

6.5 8.1 16.4 31.1 ÿ4.3 17.2 38.4 10.5 4.7 15.1 8.7 8.1 9.5 143.7 6.6 15.7

2.7 3.4 2.9 4.2 ÿ 5.6 4.9 ÿ205.6 2.8 2.4 5.2 3.3 2.8 2.7 3.1 2.1 6.6

1.5 1.3 3.1 2.2 1.7 2.7 1.4 1.7 2.2 2.4 3.1 2.0 3.0 2.4 1.9 2.9

9.3 8.9 8.9 6.5 13.5 9.5 5.2 7.2 4.8 8.9 8.1 6.9 8.5 7.3 7.1 8.6

10.6 4.4

3.5 1.3

2.2 0.6

8.1 2.0

a Calculated as: Bio-N (I): [total mineral N release day 0±10 in fumigated soil ÿtotal mineral N release d 0±10 unfumigated soil]/0.68 (Shen et al., 1984); Bio-N (II): [NH4+ -N release day 0±10 in fumigated soilÿNH4+ -N release d 0±10 unfumigated soil]/kN; kN =ÿ0.014*(CF-toNF) + 0.39 (Voroney and Paul, 1984); Bio-N (III): [NH4+ -N release d 0±10 in fumigated soil]/kN; kN =ÿ0.014*(CF-to-NF) + 0.39 (Voroney and Paul, 1984). b Microbial C calculated as [F(0±10)ÿF(10±20)]/0.45 was used to calculate all C-to-N ratios. c Bold values were excluded from mean and standard deviation calculations.

Table 5 Ninhydrin-reactive N (NRN) and total mineral N content of unfumigated soils (d 0) and soils fumigated for 5 (d 5) and 10 d (d 10) Ninhydrin-reactive N (mg N kg ÿ 1 soil)

BouakeÂ-94 BouakeÂ-95 Glidji-94 Glidji-95 Niaouli-94 Niaouli-95 Ibadan-94 Ibadan-95 Mbalmayo-94 Mbalmayo-95 FerkeÂ-95 Amoutchou-95 Sarakawa-95 Save-95 Ina-95 Ebolowa-95 LSD (5%)

Total mineral N (mg N kg ÿ 1 soil)

d0

d5

d 10

d0

d5

d 10

23.8 3.6 0.7 0.8 14.9 4.6 37.5 1.2 32.0 74.5 3.2 1.2 2.1 0.8 1.2 86.0

28.0 4.7 2.8 4.7 16.5 6.1 42.9 6.0 34.6 79.3 11.4 4.9 7.5 5.3 7.9 93.7

27.0 6.9 3.8 6.5 16.3 7.6 44.9 7.9 47.0 82.7 14.5 6.4 8.6 12.0 10.1 96.7

22.7 19.9 39.8 21.4 20.1 13.9 69.2 25.2 129.8 78.1 36.8 14.1 10.2 10.2 14.5 103.3

27.5 21.9 37.7 22.2 19.4 14.4 66.6 25.2 135.4 81.5 44.0 15.3 12.5 13.1 16.4 109.7

26.4 22.4 38.8 22.3 20.0 16.9 73.2 27.4 154.6 86.4 44.6 17.0 13.6 17.3 16.9 110.5

2.3

4.7

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B. Vanlauwe et al. / Soil Biology and Biochemistry 31 (1999) 1071±1082

Fig. 1. Relationship between the ninhydrin reactive N ¯ush after 5 and 10 d of fumigation. The encircled symbol was excluded from the regression analysis.

reached by Chaussod and Nicolardot (1982), Merckx et al. (1985) and by Feigl et al. (1995) for Al®sols and Ultisols, but not for Oxisols. Moreover, in our results, only the Bio-C (II) method yielded acceptable values

Fig. 2. Relationships between the NRN ¯ush at 5 and 10 d of fumigation and mineral N release from d 0 to d 10 of the fumigated (FI) (a) and unfumigated (b) soils. The encircled symbols were excluded from the regression analysis.

Fig. 3. Relationship (forced through 0) between the NRN ¯ush (d 0± 5) of fresh soils samples and the NRN ¯ush (d 0±10) of air-dried and conditioned savanna soil samples used in this report (all soils excluding M94, M95 and E95). The encircled symbol was excluded from the regression analysis. The dotted line represents the 1:1 relationship.

(no negative biomass values) within a consistent range. Although the NRN ¯ush after 5 d also explained more of the variation in the Bio-C (II) values than in the Bio-C (I) values, the linear relationship was slightly weaker as compared to the relationship with the NRN ¯ush after 10 d. Although the M94 humid forest soil released only 17% of its d-10 NRN content after 5 d, a 5-d period of fumigation was equally useful for all savanna soils. Of course, in the latter case, an adaptation of the kC,NRN factor is necessary as after 5 d of fumigation, on average 70% of the NRN released after 10 d was measured (Fig. 1). To obtain absolute microbial biomass C values, the measured ¯ush needs to be recalculated with a conversion factor, kC, usually determined by relating the ¯ush of C release with a calibrated reference method. For the range of soils studied no such independently tested reference method is available and as such, it is dicult to make conclusive statements on values for kC. Sparling and Ross (1993) indicated that the kC factor in the fumigation±incubation technique is generally believed to vary between 0.33 and 0.47 and can change depending on soil pH, soil type, composition of the microbial population, age of the microorganisms, time of sampling or soil moisture content. Assuming that a value for kC of 0.45 frequently used in the fumigation incubation method is applicable to the soils studied, the factor which relates the NRN ¯ush to microbial C values (kC,NRN) would equal 17.1 for a 5-d fumigation and 12.3 for a 10-d fumigation (Table 6). These values are less than most other reported values, ranging from 24.3 (Carter, 1991) to 64.8 (Sparling et al., 1993) for a 24-h fumigation and from 21 (Amato and Ladd, 1988) to 29.8 (Sparling et al., 1993) for a 10-d fumigation. Amato and Ladd (1988) obtained a similar factor for a 10-d fumigation period as in this report (11.4) for

B. Vanlauwe et al. / Soil Biology and Biochemistry 31 (1999) 1071±1082

1079

Table 6 Coecients of determination (R 2) and slopes of the linear relationships between Bio-C (I), Bio-C (II), Bio-N (I), Bio-N (II) and Bio-N (III) and the ninhydrin-reactive N (NRN) ¯ushes after 5 and 10 d. The intercepts of the regressions were ®xed at 0 Microbial biomass method (mg kg ÿ 1 soil)

Bio-C (I) Bio-C (II) Bio-N (I) Bio-N (II) Bio-N (III) a *

NRN ¯ush after 5 d (mg kg ÿ 1 soil)

NRN ¯ush after 10 d (mg kg ÿ 1 soil)

R2

slope

excl.a

R2

slope

excl.a

0.69* 0.89* 0.69* 0.77* 0.94*

16.2 17.1 1.5 4.7 7.2

M94 M94 M94 M94 M94

0.70* 0.91* 0.63* 0.79* 0.94*

10.6 12.3 1.5 3.9 5.3

± ± ± ± ±

Excl.: values excluded from the regression analysis. Indicates signi®cant P < 0.001 (n = 16).

soil conditioned for 2 weeks and attributed this to the underestimation of biomass C with the FI method due to relatively large values for CO2 evolved from unfumigated controls. This, however, does not apply here since we used the CO2 production of the fumigated samples as a control for biomass C measurements. However, when using an average kC factor (0.35) (calculated from kC factors for 5 microorganisms, including bacteria and fungi) of a sandy loamy soil with similar pH as most of the soils used in this report

Fig. 4. The total C content (a) and mean C-to-N ratios (b) of the di€erent SOM fractions of the 11 soils collected in 1995. Error bars in (b) are standard deviations.

(Nicolardot et al., 1984), then the kC,NRN becomes 22 and 16 for a 5 and 10-d fumigation period, respectively. For estimating kN values, the situation becomes more complicated because of the wider range of N, compared with C, that can be found in microorganisms (Sparling and Ross, 1993). Microbial N measurements based on the ¯ush of NH4+ -N in the fumigated soils without subtraction of a control seem to be superior to other methods in view of the stronger linear relationships between microbial N and the ¯ush of NRN after 10 d fumigation. This, however, would lead to a discrepancy between the relatively high ratio of C ¯ush-to-N ¯ush of the fumigated soils (CF-to-NF) and the relatively small microbial C-to-N ratios, with microbial N values estimated as Bio-N (III). Microbial N values calculated as Bio-N (I) lead to microbial Cto-N ratios which are much more closely re¯ecting the ratio CF-to-NF. This was also true when no unfumigated control is used to correct for basal N mineralization (results not shown). For the present range of soils, a kN value of 0.68 seems to be closer to reality than often proposed lower values. Based on this, the factor relating the microbial biomass N content with the NRN ¯ush, kN,NRN could be estimated as 1.5 (Table 6). This value is again small when compared with earlier reported values (3.1, Amato and Ladd, 1988; 3.5 to 5.3, Sparling et al., 1993). This is no surprise, as the values of kC,NRN discussed earlier were small compared with values in the literature. The mineral N release of the fumigated soils during the ®rst 10 d of incubation showed a strong linear relationship with the NRN ¯ush after 10 d of fumigation, indicating that the decomposition products are derived from a similar soil organic matter pool, obviously the microbial biomass. Carter (1991) and Henrot and Robertson (1994) similarly found that the release of NRN components from fumigated soils was correlated with the release of mineral N, but the ratio of NRN release-to-mineral N release varied substantially between reports, ranging from 0.5 in this report

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B. Vanlauwe et al. / Soil Biology and Biochemistry 31 (1999) 1071±1082

Table 7 Coecients of determination (R 2) of the linear relationships between (i) the CO2 production of the unfumigated soils from 0 to 10 d and the C content of the di€erent fractions (mg C kg ÿ 1 soil), (ii) between the mineral N release of the unfumigated soils from 0 to 10 d and the N content of the di€erent fractions (mg N kg ÿ 1 soil) and (iii) between the microbial C measured with the FI method as Bio-C(II) and the ninhydrin-reactive N (NRN) ¯ush after 10 d and the C content of the di€erent fractions CO2 production versus fraction-C

Mineral N release versus fraction-N

Bio-C (II) versus fraction-C

NRN ¯ush after 10 d versus fraction-C

O2000 O250 O53 MO20 Silt Clay

0.456* 0.701** 0.503* 0.461* 0.478* 0.252

0.627** 0.420* 0.328 0.249 0.310 0.255

0.342 0.217 0.351 0.482* 0.406* 0.235

0.228 0.258 0.376* 0.396* 0.344 0.236

Soil litter POM Total soil

0.697** 0.607** 0.417*

0.467* 0.386* 0.286

0.247 0.302 0.290

0.267 0.325 0.293

*

and

**

indicate signi®cant P < 0.05 and P < 0.01 (n = 11).

to 0.9 or 1.4 (Henrot and Robertson, 1994). This was caused by di€erences between soils in enzyme activity during fumigation (release of NRN components) or in microbial activity during incubation after fumigation (release of mineral N). Although further speculation on exact mechanisms may not be useful in view of the lack of detailed soil data in the latter publication, this observation merits further attention. 4.2. Relationships between microbial biomass, soil organic matter fractions and soil C and N mineralization The C contents of the di€erent savanna soils are small, ranging from 0.3 to 1.1% and ®t closely to the classical relationship between total soil C and clay content for West African soils, reported by Jones (1973)

Fig. 5. Relationship between the estimated (via multiple linear regression) and the measured CO2-C production of the 11 unfumigated soils collected in 1995. The dotted line represents the 1:1 relationship.

(%C = 0.236 + 0.0289 (% clay), R 2 = 0.876). Between 7 and 17% and between 11 and 28% of the total soil C is part of the SL and POM material, respectively. The proportion of total soil C in the POM material is large compared with the range determined for a set of Danish arable soils by Christensen (1985) (5±10%), but small compared with the range determined by Cambardella and Elliott (1992) for a set of Mollisols (18±39%). Due to the small clay contents of the used savanna topsoils, there is little protection of C by its association with clay particles, which could result in a relatively larger amount of C in the larger organic matter fractions. Not only the total soil organic C content (2.86±19.88 g C kg ÿ 1 soil), but also the Bio-C (II) values of the savanna soils were small (22±216 mg C kg ÿ 1 soil). The conditioning time must have been long enough to almost completely restore the original microbial biomass values as NRN ¯ushes measured on fresh soils were similar to the NRN ¯ushes determined on the air-dried, conditioned soils (Fig. 3). Although total soil C values were similar to the values reported by Kirchmann and Eklund (1994) for Zimbabwean sandy savanna soils (4.6±12.4 g C kg ÿ 1 soil for the top 5 cm of soil), the proportion of total C in the microbial biomass is much smaller in our report (0.5±2.5%) compared with the former (5.5±9.3%). This large discrepancy between the Zimbabwean and West-African savanna soils in the fraction of the total soil C incorporated in microbial tissue may be caused by a di€erent amount of easily decomposable substrates, di€erences in climatic conditions and periods of stress for the microbial community, di€erences in soil architecture and related contacts between microorganisms and substrates or di€erences in the methodology used.

B. Vanlauwe et al. / Soil Biology and Biochemistry 31 (1999) 1071±1082

Most of the CO2-C produced by unfumigated soils appeared to be derived from the fractions larger than 53 mm or the particulate organic matter (POM) and especially from the O250-C. The silt, but especially the clay material seemed to reduce the potential release of CO2-C from the SL fraction, as over 90% of the variation in CO2-C production could be explained by a multiple linear regression equation including the SL-C pool and the silt and clay content. Merckx et al. (1985) has already demonstrated that clay minerals may act as an adsorption sink for root-derived organic products in soils, but particles in the SL fraction are larger than soluble organic components released in the soil solution by plant roots. It appears that clay and silt particles may also reduce the mineralization potential of large organic particles by coating their surface and thus reducing accessibility to microbial biomass and enzymes, by entrapping the particles in aggregates, by physically protecting secondary decomposition products, or by other mechanisms enhancing physical protection. Franzluebbers and Arshad (1997) also hypothesized that clay may play an important role in sequestering POM-C by enhancing the physical protection of POM within macroaggregates. Mineral N release in the soil solution was less clearly related to the N content of distinct soil organic matter fractions which is not surprising in view of the small amounts of soil N mineralized after 10 d (maximally 7 mg kg ÿ 1 soil). Although relationships between the N content of the O2000 and SL material were most signi®cant, it is unlikely that a substantial part of the mineral N is derived from fractions with a C-to-N ratio of 36 (217) (O2000) or 24 (27) (SL). A longer incubation period is surely required to make more conclusive statements on relationships between net N mineralization and SOM fractions. The O53, MO20 and silt fractions explained most of the variation in Bio-C (II) contents and NRN ¯ushes after 10 d. However, even the strongest relationships explained only a low proportion of the variation (<50%). This may not be surprising as the microbial community may show di€erent levels of activity (Gregorich et al., 1991). Although the microorganisms metabolizing the SL material are likely to be more active than the overall microbial community, the inactive part of the biomass is also quanti®ed with fumigation techniques. Van Gestel et al. (1996) showed that microorganisms were associated with all particle size classes. To further mask any relationships between microbial biomass and SOM fractions, the e€ectiveness of the chloroform treatment may be di€erent for microorganisms located di€erently in the soil fabric and having di€erent degrees of activity.

1081

Acknowledgements The authors gratefully acknowledge the Belgian Administration for Development Cooperation (ABOS) who sponsored this work in the framework on the collaborative project between KU Leuven and IITA on ``Process-based studies of soil organic matter dynamics in relation to the sustainability of agricultural systems in the tropics''.

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