Factor analysis of the relationships between several physico-chemical and microbiological characteristics of some Belgian agricultural soils

Soil Bid. Biochem. Vol. ?I, No. I. pp. 53-58. 1989 Printed in Great Britain. All rights reserved

Copyright 0

0038-0717;89 53.00 + 0.00 1989 Pergamon Press plc

FACTOR ANALYSIS OF THE RELATIONSHIPS BETWEEN SEVERAL PHYSICO-CHEMICAL AND MICROBIOLOGICAL CHARACTERISTICS OF SOME BELGIAN AGRICULTURAL SOILS X. VEKEMAXS, B. GDDDEN and M. J. F’ENNINCKX Laboratoire de Microbiologic, Groupe d’Ecologie. Microbienne et Appliqued, Universitt Libre de Bruxelles. c/o CERIA. Av. E. Gryson I, 1070 Bruxelles, Belgium

(Accepted 29 June 1988)

Summary-Twenty Belgian agricultural soils, 16 of which had been organically cultivated, were examined for their biochemical and microbiological properties. In spite of the very different nature of the characteristics studied. close relationships were found between soil respiration and glucose mineralization rates, biomass-C as measured by the fumigation-incubation method, and several enzyme activities, namely FDA-hydrolase, alkaline phosphatase and dehydrogenase..Correlation coefficients between urease activity and other biological measurements were always found to be endowed with a negative sign. Moreover, factor analysis of the data showed that some physico-chemical characteristics such as soil organic C. total N. clay content and CEC were closely related to most of the biological measurements, while pH and sand content were not. Two procedures for the determination of dehydrogenase activity and two methods of calculation of biomass-C were also compared.

MATERIALS AND METHODS

INTRODCCTION

Soils

Most studies of the effects of soil management have been mainly concerned with the modifications of physico-chemical and microbiological properties induced by soil alteration. In this context, enzyme activities, respiration rates and production of biochemicals (e.g. ATP) have been considered as possible indexes of soil microbial activity (Beck, 1984; Verstraete and Voets, 1977). Recent methodological advances. for example the fumigation-incubation technique (Jenkinson and Ladd, 1981) or the initial respiratory response method (Domsch et af., 1979), have, however, opened new prospects in this field. Numerous methods are now available for the soil scientist, but only few detailed studies (Chaussod er al., 1986; Sarathchandra er al.. 1984) have considered the possible relationships between the results obtained by the different techniques mentioned above. Factor analysis, used by these investigators, has been shown as a powerful tool for analysing such data. Our work, carried out on 20 Belgian agricultural soils, was intended to illustrate this approach. Sixteen organically-cultivated soils (Cacek, 1984) were included in our study. As far as we are aware, only few investigations have considered such soils (Elmholt and Kjiiller, 1987). In order to extend the range of soils investigated, we included four samples used for other cropping systems. We looked for the pattern of relationships among several biological characteristics as soil enzyme activities, respiration and glucose and classical mineralization rates, biomass-C physico-chemical properties of the soils investigated.

Surface (0-20cm) samples of Belgian agricultural soils were collected in March and April 1986 (Table I). Soils l-16 were managed according to the USDA definition of “organic farming” system (Cacek, 1984) and had received heavy annual application of composted farmyard manure. Soils 17-20 came from non “organic farming” systems selected to obtain a wide range in physico-chemical soil properties.

Analytical methods

Most analyses were made on fresh samples stored at 4C for no longer than 2 weeks. The determinations of organic-C, clay and sand content, pH and cation exchange capacity were made on air dried sieved soils ( < 2 mm). All results were expressed in terms of g-’ dry wt of soil. Physico-chemical charucteristics. Particle size distribution was determined by sieving (sand fraction > 50 pm) and by the pipette method (silt and clay fractions) following peroxide and NH, treatment. Chemical analyses included the determination of soil pH (stiff soil-water paste), cation exchange capacity (CEC) at pH 7 (ammonium-acetate), organic-C (dry combustion) and total N (Kjeldahl). Biomass-C. Soil (50 g) was fumigated for 24 h with water-washed chloroform and incubated for I4 days, without reinoculation, at 28’C in 0.5 I. flasks (Chaussod and Nicolardot, 1982). 53

X. VEKEMANSer

al.

Table I. Some physico-chemical properties of soils used Soil numbe?

I 2 3 4 5 6 7 8 9 IO II I2 I3 I4 I5 16 I7 I8 I9 20

Organic-C (Oh)

Total N f%/o)

PH

CEC (mequiv 100 g-‘)

Clay (%)

Sand P0)

3.9 5.2 3.3 4.1 1.9 4.2 2.3 5.4 1.7 2.7 I.5 1.5 I.4 2.1 1.9 4. I 1.4 0.9 0.9 0.7

0.25 0.39 0.29 0.32 0.17 0.22 0.25 0.24 0.15 0.18 0.12 0.09 0.13 0.21 0.16 0.37 0.09 0.07 0.10 0.08

6.2 6.1 5.6 5.1 6.6 6.0 6.2 7.7 6.4 6.6 6.2 5.4 6.9 6.2 5.8 5.1 6.1 5.5 6.8 7.0

25.2 23.7 23.0 33.0 21.2 16.9 29.0 19.4 23.0 23.9 12.6 Il.2 15.7 18.1 17.5 22.0 6.6 15.6 10.8 13.5

15 24 23 22 13 I6 19 8 15 I4 II 9 I5 I5 I4 I3 I4 I4 IO I3

17 I4 I? 33 74 6 41 I6 17 I2 27 49 I2 I8 23 20 13 I3 2s 16

‘All soils were silt loams except 7 and I2 which were loams (USDA-textural

Two different correction factors were used to calculate the microbial flush; (A) CO,-C evolved by unfumigated soil, 7-14days; and (B) C02-C evolved by fumigated soil, 7-14 days. Biomass-C was calculated using a k factor of 0.42 (Chaussod ef al., 1986). CO, euofufion. The amounts of CO, evolved by unfumigated soils between 7-14days at 28C (Chaussod et ul., 1986) were used as respiration values. Glucose mineralization acticity. The initial rates of CO2 production in the presence of glucose were determined by radiorespirometry (Mayaudon, 1971). Soil (2 g) was held for 30 min at 30-C in a IOml scintillation vial after the addition of 3.7 MBq of “C-uniformly-labelled glucose (specific activity 148 MBq mmol-‘) and 0.1 ml of 4 mw glucose solution. The “COL evolved was trapped and measured as described by Legrain and Stalon (1976). FDA-hydrolase. The assay was based on the method of Schniirer and Rosswall (1982). Fifteen ml of 60 mM, pH 7.6, phosphate buffer, and 0.4 ml Fluorescein-diacetate (FDA) solution (0.2% in acetone) were added to 5 g soil in 250 ml capacity vials; the controls containing no substrate. After incubation for 45 min at 30’C in an oscillating shaker (280 rev min-‘), the vials were placed in ice and 40 ml acetone were added. Absorbance was determined at 490 nm after centrifugation at lO,OOOg for IO min. Fluorescein standards were prepared in order to express the activity as pmol fluorescein released g-’ soil h-l. Alkaline phosphatoses. The method of Tabatabai (1982) was used but a 10rn.~ p-nitrophenyl phosphate solution was used. This substrate concentration was about 5 times the mean K,,, (Michaelis constant) determined on our soils (Malcolm, 1983). Dehydrogenuse. Two methods were used (see Discussion). Dehydrogenase - TTC. method, using This 2.3,5-triphenyltetrazolium chloride (TTC) as substrate, was adapted from Domsch ef al. (1979). Three ml of a Tris buffer plus TTC (0.5 M, pH 7.6, I % TTC) were added to 3 g soil and the reaction mixture

class).

was held for I8 h at 30’C in tubes closed with rubber stoppers. After this, triphenylformazan (TPF) produced was extracted by shaking in the presence of 20ml methanol. Absorbance was determined at 480 nm after centrifugation at 30,OOOg for 5 min. Because the activity was not linear during the reaction period, we expressed the results as nmol TPF released g-’ soil I8 h-‘. Dehvdrocenose-INT. The substrate used was 2-p-iddophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride ([NT) (Trevors. 1984). The orocedure was the same ‘as‘ for TTC except that’ the substrate concentration was 0.7% INT in Tris buffer and the exposure time was 4 h. The activity was linear with the time during the reaction. The results were expressed as nmol INF released g-’ soil h-‘. Ureuse. Enzymatic extract of the soil samples were prepared as follows: IS ml 0.5% Triton X-100 were added to log soil; the suspension was shaken for IO min then centrifugated at 30,OOOgfor IO min and the supernatant used directly for enzyme determination after filtration on Whatman No. 3 paper. Statistical analysis. The data were analyzed using an SPSS (Nie et al., 1975) factor analysis which is designed to reduce the number of variables that need to be considered to a small number of indices called factors. It is assumed that each original variable can be expressed as a linear combination of these factors, plus a residual term that reflects the extent to which the variable is independent of the other variables. This residual term is derived from the squared multiple correlation between the variable and the rest of the variables in the set, using an iteration method. Plotting the original variables in a two- (or more) dimensional factor space gives an illustration of the pattern of relationships among the variables. Physic0 -chemical properties of the soils

A broad range of organic-C (0.7-5.4%). total N (0.06-0.39%) and CEC (6.6-33.0 mequiv 100 g-’ soil) values were observed for the soils surveyed (Table \ I). Most of the soils were acidic or nearly

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56

neutral (pH,, 5.1-6.9) except for soil 8 which was slightly alkaline @H 7.7).

Table 4. The contribution of each inferred factor’ lo

the rocal variance of the system % of Variance

Cumulative % 57.5 69.9

8

s7.5 12.4 1.9 5.9 5.6 3.8 2.8 I.4

9 IO II 12 13 14

0.9 0.3 0.1 0.1 0. I

Factor

Biomass-C

Biomass-C ranged from 81 to 932 cg Cg-’ soil according to the fumigation-incubation technique (Table 2). A range of 108-972pgCg-’ soil was obtained with the Chaussod and Nicolardot procedure (1982). These values indicate that the microbial biomass comprised between about O&2.9% of the organic-C in the soils.

2 3 4 6

Respirometry CO2 production in soils without glucose addition ranged from 5.3-62.9 fig C g-’ soil day-’ (Table 2). The specific respiration rate (flux of C-CO, unit-’ biomass-C; Chaussod et al., 1986) calculated from these values ranged from 0.03 to 0.08 day-‘. The initial rates of CO1 production in the presence of glucose ranged from 0. I to 4.7 nmol CO2 produced g -’ soil min-‘. Soil en:yme acrivities

Alkaline phosphatase, FDA-hydrolase and urease activities ranged respectively from 0.1 to 6.8, 0.08 to 0.6 and 4.5 to 12.9 pmol product released g-’ soil h-’ (Table 2). Dehydrogenase activity ranged from 5 to 100 units according to the TT’C method (see Materials and Methods) and from 14 to 81 nmol INF released g-’ soil h-’ according to the INT procedure. Correlabon analysis

Table 3 shows the simple linear correlation matrix obtained among the different soil characteristics. In general, close relationships (r = 0.47*-0.95***) were found among soil microbiological properties, except for urease and alkaline phosphatase. Soil biomass values were highly correlated (P < 0.001) with all microbiological parameters but urease. In addition some significant positive correlations (P < O.OS0.001) were found between soil organic-c. total N, clay content, CEC and the various microbiological parameters.

77.8 83.7 89.3 93.1 95.9 97.3 98.4 99.3 99.6 99.8 99.9 100.0

I.1

‘Factors computed by factor analysis with iteration.

methodologies used. Modifications of the usual procedures (see Materials and Methods) were made here for alkaline phosphatase, FDA-hydrolase and dehydrogenase-INT in order to satisfy the conditions for correct enzymatic assays as discussed by Malcolm (1983). No comparisons with the literature could thus be made for these results. However, the values obtained for biomass-C, dehydrogenase-TTC, soil respiration and glucose mineralization of the organically-cultivated soils were generally higher than reported for cropping systems not receiving large amounts of organic materials, but were also lower than those for grassland soils (Beck, 1984; Chaussod et al., 1986; Domsch et al., 1979; Jenkinson and Ladd, 1981; Sarathchandra et al., 1984; Shen et al., 1987).

Soil enzymes have been widely used as convenient indicators of microbial activity (Tabatabai. 1982). However these measures are not necessarily directly correlated with the metabolically-active microflora (Burns, 1982). We observed close correlations between all of the enzyme activities, except for urease.

Factor analysis

Factor analysis was introduced to explain the observed relationships among soils characteristics in terms of simpler relationships. The process leads in principle to a smaller number of hypothetical variables (or factors) representing most of the data variation. In this study, Factor I alone accounted for 57% of the total variance (Table 4). This result was consistent with a close correlation between the characteristics studied. The factor loadings for each of the I5 soil characteristics were plotted against Factors 1 and 2 (accounting both for 70% of the total variance) in Fig. I. All characteristics except urease, pH and sand content were located in a small area on the plot, showing a common source of variation. DlSCtiSSlON

Comparison between the results obtained by different authors working in the field of soil biochemistry, is often difficult because of the different

Factor

1

Fig. I. Plot of the loadings from the first two factors. AP = alkaline phosphatase; BA = biomass-C by method A; BB = biomass-C by method B; Cl = clay content; CEC = cation exchange capacity; CO, = CO, production; DI = dehydrogenase-INT; DT = dehydrogenase-TfC; mineralization; GI = glucose FDA = FDA-hydrolase: OC = organic-C; pH = soil pH; Sa = sand content: TN = total N; Ur = urease.

Factor analysis of microbiological characteristics Similar results were reported by Beck (1984). Moreover factor analysis has shown that the enzyme activities, considered as a group, are strongly correlated with other microbial activity indexes such as soil respiration, glucose mineralization and biomassC. This points out the potential of enzymes as indicators of metabolic activity of microorganisms, in soils treated with large amount of organic matter. FDA-hydrolysis activity has been proposed as a measure of total microbial activity in soil and litter (Schniirer and Rosswall, 1982). This method estimates a set of different hydrolases (esterases, lipases and proteases) produced by the soil microflora (Nicolardot et al., 1982). The close correlations observed between FDA-hydrolysis activity, other soil enzymes, glucose mineralization and microbial biomass show the potential of this method for agricultural soils. We observed only a low but mostly significant correlation (r = 0.28-0.69***) between alkaline phosphatases and the other biological properties. This contrasts with the results of Frankenberger and Dick (1983) who observed that, in the case of non cultivated soils, alkaline phosphatase was closely related with the relative activity and mass of the microbial populations. The difference is not necessarily explained by the type of the soils compared but could result from the more limited range of pH of the soils investigated by these authors (pH 7.07-7.42). Nannipieri et al. (1979) pointed out that a pH lower than 6.0 can destabilize the fraction of enzyme present in the medium. Six of our soils were in fact characterized by a pH lower than 6.0 and presented low alkaline phosphatase activity (Table 1). Closer correlations (r = 0.59**-0.89***) were observed when these samples were discarded, which supports this hypothesis. Dehydrogenase-TTC activity has been frequently used in soil microbiological studies (Domsch et al., 1979). However this method presents two major disadvantages: (I) a lack of sensitivity implicating either the use of I624h incubations (Domsch er al., 1979) or the addition of electron donors (Casida, 1977) (2) the activity is not linear throughout the entire incubation. A method adapted from Trevors (1984)

I).

Dehydrogenase-INr’ activity (nmol INF g-lsol I h-l) Fig. 2. Regression analysis of dehydrogenase-INT dehydrogenase-TTC activities.

vs

1000

57

r

0

500

1000

Biomass-C by method E (vg c 9-f soi I) Fig. 3. Regression analysis of biomass-C estimated method B vs biomass-C estimated by method A.

by

using INT in place of TTC and an incubation time of less than 4 h has proved to be very sensitive and time linear (X. Vekemans, unpublished observations). Trevors (1984) reported diverging results for both methods. We have found a close correlation (0.75***) between these methods, but Fig. 2 shows that the results were not similar at the level of individual soils. Soil urease activity is a controversial method of estimating microbial activity. Indeed this enzyme can be associated with the soil organic matter while retaining its activity (Burns et al., 1972). We found almost negative and non-significant correlations between urease and other soil microbial activities. This contrasts with other results (Frankenberger and Dick, 1983; Sarathchandra et al., 1984) who found low but significant positive correlation between urease and other properties for various soils. These discrepancies can not be explained. Soil respiration and glucose mineralization activity, estimated here by radiorespirometry, are thought to be valid indicators of the biological activity of microorganisms in their natural habitats (Mayaudon. 1971). These methods were used here as reference in the evaluation of more indirect estimations of the soil microbial activity such as soil enzyme activities. Biomass-C, calculated by two different procedures in this work, is highly correlated (r = 0.96***) with Factor I representing 58% of the total variance, as shown by the factor analysis (Fig. I). In spite of the high correlation coefficient (r = 0.95***), it was found, however, that the results obtained by the two methods used for calculation of biomass-C were not the same for individual soils (Fig. 3). This could be related to the different nature of the correction factor for non-microbial C mineralization, used in the two methods. Method A used the respiration of a nonfumigated soil during the 7-14day period as the correction factor (Jenkinson and Ladd. 1981). whereas method B used the respiration of a fumigated soil after the decomposition flush during the same period (Chaussod and Nicolardot, 1982). The relationship between correction factor B and A is given by the equation:

B = 0.8 1 A + I I .7 (r = 0.873***).

X.

58

VEKEMA~J et al.

This equation is close to that obtained by Shen et al. (1987). The discrepancies between the two methods correspond to different amounts of respiration in non-Fumigated soils compared to fumigated soils after the flush. This difference could be explained to some extent by the selection of a particular microflora in the fumigated samples (Chaussod and Nicolardot, 1982; Shen er al., 1987). From the different soil physico-chemical properties studied in this work, soil organic C, total N, clay content and CEC were found to be correlated with all the microbiological properties but urease, as reported by Chaussod et al. (1986) and Frankenberger and Dick (1983). This is well illustrated by the factorial analysis (Fig. I) and supports the general concept of a clay-organic complexes-microflora link in soil systems (Verstraete and Voets. 1977). Finaily, it must be noted that in spite of the close relationships that can be observed among soil biological properties, specific interactions between individual soils and biological indicators can introduce misleading results (Pancholy and Rice, 1973; Vance et af., 1987). We conclude with the necessity of using several complementary methods in order to evaluate the biological activity of soils. In this respect, the use of factor analysis might provide a rational basis for treating these data. Ackno,r/rdRemenrs-We sincerely thank B. DupontSaussoy and E. Delmotte for technical assistance. We also thank Professors J. Herbauts and W. Gruber for their help in the determination of some phys~co-chern~~l properties.

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fumigation-incubation

method in

Chaussod R., Nicolardot B., Catroux G. and Chretien J. strongly acid soils. Soil-Biology & Biochemistry 19, (1986) Relations entre les caracteristiques physico697-701. chimiques et microbioiogiques de quelques sois cultives. Verstraete W. and Voets J.-P. (1977) Soil microbial and Scienre du Sol 2, 213-226. biochemical characteristics in relation to soil management and fertility. Soil Biology & Biochemisrry 9, 253-258. Domsch K. H., Beck Th.. Anderson J. P.. Soderstrom B.,