(Anderson and Domsch, 1978). B. Bio-C = COz-Cr/O.41. (Paul and Voroney, 1980). RESULTS. Activity measurements. Oxygen consumption and the ability to ...
Soil Biol. Eiochem. Vol. 17, No. 5, pp. 611-618, 1985 Printed in Great Britain. All rights reserved
0038-0717/85$3.00+ 0.00 Copyright 0 1985Pergamon Press Ltd
MICROBIAL BIOMASS AND ACTIVITY IN AN AGRICULTURAL SOIL WITH DIFFERENT ORGANIC MATTER CONTENTS JOHAN SCIIN~~RBR,MARIANNE CLARHOLM and THOMAS ROSSWALL* Department of Microbiology, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden (Accepted 30 January 1985) Summary-Changes in soil fertility caused by various organic and N-fertilizer amendments were studied in a long-term field trial mostly cropped with cereals. Five treatments were included: (I) fallow, (II) cropping with no C or N addition, (III) cropping with N-fertilization (80 kg ha-’ yr-‘), (IV) cropping with straw incorporation (1800 kg C ha-’ yr-‘) and N-fertilization (80 kg ha-’ yr-‘), and (V) cropping with addition of farmyard manure (80 kg N + 1800 kg C ha-’ yr-‘). The treatments resulted in soil organic matter contents ranging from 4.3% (I) to 5.8% (V). Microbial biomass and activity were determined by chloroform fumigation, direct counting of fun@ (fluorescein diacetate (FDA)-staining and Jones-Mollison agar-film technique) and bacteria (acridine orange staining), most probable number determinations of protozoa, esterase activity (total FDA hydrolysis) and respiration. Both biomass estimates and activity measurements showed a highly significant correlation with soil organic matter. Microbial biomass C ranged from 230 to 600 pg C g-’ dry wt soil, as determined by the fumigation technique, while conversions from direct counts gave a range from 380 to 2260 pg C. Mean hyphal diameters and mean bacterial cell volumes decreased with decreasing soil organic matter content.
INTRODUCTION Management practices, such as tilling and fertilization, affect the quality and quantity of soil organic
matter (SOM) (Voroney et nl., 1981). Simulation modelling has been used to describe the dynamics of SOM under different management conditions. Several models employ various functional divisions of the organic matter, characterized by residence times, rather than the traditional division of organic matter based on chemical extractions (Konova, 1966). The residence times used for different organic matter fractions range from less than a year to more than 1000 years (Paul and Juma, 1981; McGill et al., 1981; van Veen and Paul, 1981; Parton et al., 1983). The SOM components with relatively short residence times consist of microbial biomass and metabolites. Jansson (1958) referred to these components as the “active” SOM fraction. The rapid turnover indicates their importance as potential nutrient sources for plants, especially with regard to nitrogen (N). We studied how soil microorganisms were affected by manure, crop residues and N-fertilizer additions in a field experiment established in 1956. Results on crop yields, SOM contents and soil structure have been reported by Nilsson (1980), Persson (1980) and Eriksson (1980). Data from the same experiment were used by Parton et al. (1983) to develop and evaluate a simulation model for N mineralization and organic matter changes. They divided the SOM into fractions with postulated turnover times of 3, 30 and 1200 years. The model indicated that most of the mineralized N was derived from the fractions with the shortest turnover times (3 and 30 years). The authors did *Present address: Department of Water in Nature and Society, University of Linkeping, S-581 83 Linkdping, Sweden. 611
not, however, have access to any measured values of these fractions. We have determined microbial biomass, which together with microbial metabolites, is equivalent to their active fraction (3 years). Microbial biomass has been recognized both as a transforming agent and as a source and sink for various nutrients. The chloroform fumigation incubation method (CFIM) (Jenkinson and Powlson, 1976) has made possible the quantification of the C, N, P and S contents of the biomass (Jenkinson and Powlson, 1976; Paul and Juma, 1981; Brookes ef al., 1982; Saggar et al., 1981). One of the arguments for the use of the CFIM is the good agreement obtained between biomass determined in this way with that determined from direct counts, but few comparative studies have been published. Accordingly, we also included direct microscopic determinations of bacterial and fungal volumes. Oxygen consumption and the ability to hydrolyse fluorescein diacetate (FDA; Schniirer and Rosswall, 1982) were used to measure overall microbial activity. MATERIAL AND METHOLBS
Site characteristics The site investigated was a 27-year-old field experiment established in 1956 at the Swedish University of Agricultural Sciences, Uppsala, central Sweden. Annual precipitation is 570 mm and mean annual temperature 5.4”C (Rodskjer and Tuvesson, 1975). The soil is a sandy clay loam with a clay content of 35%. At the start of the experiment (1956), the soil organic carbon content was 1.50% and the pH 6.5. The crops have been annuals, generally cereals. All aboveground crop residues have been removed after harvest. Five of the original 15 treatments (Nilsson, 1980) were included in this study:
612
JOHAN SCHN~~RER et al.
I-bare fallow; Ii-cropped with no additions; III-cropped with 80 kg N haa’ yr-’ as Ca(NO,),; IV-cropped with 80 kg N ha-’ yr’ as Ca(NO,), and 1800 kg C as straw; V-cropped with 80 kg N and 1800 kg C haa’ yrr’ as farmyard manure. Straw and manure were added every second year at twice the rates given above. Additions were made in the autumn of 1981, one year before our sampling. Sampling
Each treatment had four replicates. From each plot, 20 cores (dia = 3.0 cm) were taken down to 20 cm depth at the beginning of November 1982. The cores were mixed and sieved (< 4 mm) to produce four replicate samples from each treatment. Dry weight was determined after drying at 85°C for 20 h. Organic matter contents were determined as loss on ignition (LOI) after combustion at 575°C. Oxygen consumption was measured at 15°C in a modified Gilson respirometer (Rosswall et al., 1977) with 40 g (wet wt) soil samples. Respiration readings were taken every half-hour for at least 3 h. FDA-hydrolytic activity was determined as absorbance at 490nm of a filtrate from a soil suspension incubated with fluorescein diacetate at 24°C (Schni.irer and Rosswall, 1982). Protozoa were enumerated by a modified most probable number technique (Clarholm, 1981). The biomass was calculated assuming a dry weight of 1 ng dry wt animal-’ for amoebae and 0.1 ng dry wt for flagellates. For both groups, a carbon content of 45% of the dry weight was assumed. Bacteria were counted in acridine orange-stained light at 1000 x in fluorescent soil smears magnification (Clarholm and Rosswall, 1980). For biomass estimations, the bacteria were grouped into six size classes with nominal volume values (Table 2). A density of 1.1 g crne3 and a dry weight of 30% were assumed for the biomass calculations (Bakken and Olsen, 1983). A C content of 45% of the dry weight was used for calculating the amounts of C in the biomass. Total hyphal length was estimated by the Jones and Mollison (1948) agar film technique. The soil samples (5 g wet wt) were homogenized for 5 min at 5000 rev min-’ in a Sorvall Omnimixer. The agar film was stained with phenolic aniline blue and mounted in Zeiss immersion oil, 518C. The slides were studied microscope at 1000 x in a phase contrast magnification. Twenty fields of vision were counted for each slide and the lengths of the fungal hyphae were determined by the intersection method (Olson, 1950). Both transparent and blue-stained hyphae as well as melanized (brown) hyphae were counted. FDA-active hyphal lengths were, except for some minor modifications, determined according to Soderstriim (1977). Five grams of soil (wet wt) in 100 ml phosphate buffer (60 mM, pH 7.6) was homogenized for 1 min at 5000 rev min-’ in a Sorvall Omnimixer. This suspension was diluted 250 times and stained with FDA (1Opg ml-‘). Finally 2 mg (wet wt) soil was collected on a membrane filter (Millipore Blackfilter, 0.8 pm). The filters were studied in a Zeiss
fluorescence microscope at 400 x magnification. Thirty fields of vision were counted for each filter, and the fungal length determined by the intersection method (Olson, 1950). Hyphal diameters were determined by measuring 100 individual hyphal fragments from each treatment with a Kellner ocular at 1000 x magnification. The fragments were divided into size classes of 1 pm, and the mean value from each size class was used to calculate the total biovolume. The density of the fungi was assumed to be 1.1 g cm-3, and the dry matter content 21% (Bakken and Olsen, 1983). For calculations of carbon contents, a C content of 45% of dry weight was used. C and N contents of the microbial biomass were determined by the chloroform fumigation technique (Jenkinsson and Powlson, 1976). Fifty grams (wet wt) of sieved (~4 mm) soil was exposed to ethanol-free CHCI, for 20 h. The CHCl, vapours were removed by means of a vacuum pump. An inoculum of 0.5 g (wet wt) soil was mixed into both unfumigated (control) and fumigated samples. The water content was adjusted to 0.25 g H,O gg’ dry wt soil by addition of distilled water. The sample bottles (300 ml with butyl rubber membranes) were held at 24°C for 10 days. The CO> produced after 3, 6 and 10 days was determined in a Carlo Erba model 230 GC equipped with a hot-wire detector. The column was a 5 m-long Porapak QS (mesh SO-loo), 6mmdia. The oven temperature was 60°C the detector temperature 150°C and the flow of the carrier gas, He, was 30 ml min-‘. After the CO, measurements on days 3 and 6, the bottles were evacuated three times with a vacuum pump, which was enough to decrease the CO, concentration to ambient air concentration (0.034J. Inorganic N was determined in soil extracts (2 M KCl. 1: 5 w/v) before fumigation. and in fumigated and unfumigated samples after 10 days of incubation. Ammonium-N was determined by the indophenol blue method (Runge, 1971). and nitrate by flow injection analysis (Gine et al., 1980). The microbial biomass N-contents were calculated by dividing the amounts of NH:-N accumulated in the fumigated samples by a k, of 0.4 (see Discussion). The C contents of the biomass were calculated in two ways.
A. Bio-C = (CO,-Cr - COz-Cc)/O.41 (Anderson and Domsch, 1978). B. Bio-C = COz-Cr/O.41 (Paul and Voroney, 1980). RESULTS
Activity
measurements
Oxygen consumption
and the ability to hydrolyse FDA generally increased with soil organic-matter content (Table 1). For both activities the straw + N treatment (5.55% soil organic matter) gave higher values than the farmyard manure treatment (5.75%). The good correlation between the two activity measurements was noted earlier in a sampling with depth in the soil profile (Schniirer and Rosswall, 1982). Organism
determinations
Total hyphal lengths and bacterial numbers were
Microbial
biomass
and soil organic
613
matter
II : No addition
LO 30
0
1
IU:+N
@j
L
6
8
20
10
10
2
L
H: Straw+N
301
20
00
2
LO
6
8’0
2
LO
L
6
8
Y: Manure 20 10 l-l
-0
2
i
Hyphal diameter (pm)
Fig. 1. Contributions of hyphae of different diameter classes to the total fungal length in the five soil treatments. Calculated as percentage
8
z d +I
2
$
,o rj
”
d +I 6
of the total
length
in each treatment.
of the same magnitude as in other agricultural soils (Baath and Soderstrom, 1980; Domsch et al., 1979; Paul and Johnson, 1977). The lengths of FDA-active hyphae as well as total hyphal lengths were greater in the treatments with higher organic-matter contents (Table 1). The amounts of FDA active hyphae were around 2% of total hyphal lengths. This is in accordance with the 24% active fungal biomass reported by Soderstrijm (197913). Bacterial numbers also increased with organic matter content (Table 1). Bacteria were twice as numerous in soils with C amendments as compared with fallowed soil (Table 1). In the cropped but unfertilized soil, bacterial numbers were 17% higher compared with the fallow soil, while the treatment with only fertilizer additions had 40% higher numbers. Mean hyphal diameters decreased with decreasing SOM content and had a range of 1.62.7 pm (Table 1). The size distribution is shown in Fig. 1. Thinner hyphae dominated in the fallow where only 33% had a diameter exceeding 2 pm, while in the straw + N treatment 66% were larger than 2pm. B&%th and Sijderstriim (1979) determined mean hyphal diameters in horizons from seven different forest soils. They found thinner hyphae in the mineral horizons and an overall range of mean diameters from 1.3 to 3.0 pm. The mean bacterial volume was highest in the straw + N treatment (Table 1). The fallow treatment had the highest percentage (93%) of the smallest size class (small cocci), and the straw + N treatment the lowest, 71% (Table 2). The average bacterial cell in the fallow was only half the size (0.4 pm’) of that in
614
JOHAN
SCHNORER
et al.
Table 2. Size dist~bution of bacteria I% of total> Cocci
Rods
Nominal volume (flm’)
0.3
-..-___ 1.4
4.0
0.8
2.4
-.___ 10
Treatment: I (fallow) II (no addition) III (+N) IV (straw + N) V (manure)
93.2 81.7 84.8 71.9 18.7
3.0 11.0 7.0 17.3 14.4
0 0 0 0.6 1.1
2.6 4.0 2.8 1.4 1.2
1.2 3.2 4.9 8.3 3.5
0 0 0.5 0.4 1.1
the straw + N treatment (0.8 @m3). Cells of the two largest size classes were only found in the soils with C additions. The observation of variation in hyphal diameter and bacterial cell size with SOM contents shows the importance of size determinations for subsequent conversions of lengths and number to biomass. Flagellate numbers were 3 and 10 times larger in treatments with straw and manure additions, respectively, as compared with treatments I-III (Table 1). The large number of flagellates recorded in treatment V, the o@y measurement with a higher value for V than for IV, had a large standard error. It was a result of three values being lower than for IV and one very high. The straw + N treatment contained twice as many amoebae as the manure treatment, and three times more than the N treatment. The fallowed and unamended soils contained less than 2% of the numbers of amoebae observed in the soils with additions. The high numbers of naked amoebae found in treatment IV as compared with the other treatment indicates a high bacterial production
-
_ n 761 824 1023 1447 1303
in this cropping system. Due to differences in cell size, each amoeba represents about 10 times higher bacterial production as compared with a flagellate. Carbon and nitrogen in biomass
Carbon mineralization after fumigation was positively correlated with organic matter content (r = 0.83, P < 0.001). This was also found for unfumigated samples (r = 0.79, P < 0.001) (Table 3). There were low rates of N mineralization during incubation in the unfumigated samples (Table 3). In the fumigated samples, NOT-N contents had generally decreased during incubation, but there was a more than 50 times increase, in the amounts of NH:-N. This accumulation of NEIL-N was strongly correlated to soil organic-matter contents (r = 0.85, P < 0.001).
Biomass C was calculated from direct counts and from the amounts of CO,-C evolved from the fumigated sample with or without subtraction of CO,-C from the unfumigated sample (Table 4). Conversion from direct counts gave the highest values. All three
Table 3. Mineralization of carbon and nitrogen in fumigated and unfumigated samples during 10 days of incubation. Results are given as ~a C or ua N P-’ drv wt soil (mean values f SE, n = 4)
Treatment I (fallow) II (no addition) III (+N) IV (straw + N) v (manure) Cokelation with loss nn ienition
***p
C mineralized during -___-incubation -.___ -__-_ .I_Before incubation NOT-N NH,+-N Unfnmieated Fomizated 30.3 k 45.6 f 50.5 * 118.9 k 100.9 *
1.6 6.6 12.0 3.8
95.5 * 4.0 129.5 k 4.2 164.6 + 3.5 250.2 + 7.3 241.6 k 3.0
r = 0.79***
I = 0.83*“*
I .6
7.6+ 1.1 5.0 * 0.5 6.4 & 0.6 5.2 + 0.7 9.7 * 0.8
0.4iO.l 0.4 f 0.1 0.5 rt 0.1 0.8kO.l 0.8 + 0.1
Mineral N in soil -.___-_--.After incubation Fumigated Unfumigated NOT-N NH,+-N NO; -N NH:-N 7.3 IO.8 5.4 * 0.3 7-l f0.6 5.8 f 1.4 9.9 It 0.3
1.1 fO.1 1.OkO.l 1.OkO.O 1.0+0.2 0.9fO.i
6.2 i 3.9 f 5.4 * 5.2 * 7.5 +
Table 4. Calculated values for C and N contents of microbial biomass. The combined biomass of bacteria, fungi and protozoa is given under “direct counts”. Biomass C (method A and B) and N by the fumigation method is calculated as described in Materials and Methods. All values are given as UPC or up, N a-’ drv wt (mean values + SE, n = 4) Biomass C Direct counts
Treatment
***p
