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Microbial growth and sulphur immobilization following the incorporation of plant residues into soil
Biochem. Vol. 25, No. 11,pp. 1567-1573, Printed in Great Britain. All rights reserved
Soil Bid.
1993 Copyright 0
0038-0717/93 $6.00 + 0.00 1993 Pergamon Press Ltd
MICROBIAL GROWTH AND SULPHUR IMMOBILIZATION FOLLOWING THE INCORPORATION OF PLANT RESIDUES INTO SOIL J. WV, A. G. O’DONNELL and J. K. SYERS Department of Agricultural and Environmental Science, Faculty of Agriculture and Biological Sciences, The University, Newcastle upon Tyne NE1 7RU, England (Accepied 12 April 1993)
Summary-The interaction between microbial growth and S immobilization was investigated in an arable soil amended with oil-seed rape (young leaves) and barley straw (1% w/w). Initially, the rape decomposed more rapidly (40 vs 10% by-day 5) and produced a larger microbial biomass (990 pg C g-i soil) than the straw (710 ~a C a-’ soil). The biomass in both of the amended soils then decreased to amounts 3&50% higher than those in the &amended soil by day 35 and was maintained at these levels throughout the 195 day incubation. Most of the rape-S (> 80%) and straw-S (> 60%) added to the soil was released as SOi--S or converted to biomass-S in 5 days. By this time, the amount of S assimilated by the biomass in the rape-amended soil was three times that found using straw. Biomass-S in both soils then decreased but remained twice as high in the rape-amended soil over the period of 15-195 days. The biomass in the straw amended soil had a similar C:S (85-120: 1) to that of the unamended soil but was narrower (40-50: 1) in the rape-amended soil. By day 5, SO,-S in both of the amended soils had increased significantly. The increase in SOi--S in the rape-amended soil was maintained over the 195 day incubation, suggesting that this S was available for plant uptake. However, by day 15, a net immobilization of soil S by the biomass (25% of soil inorganic S) was found using straw. This immobilized S was retained by the biomass throughout the 195 day incubation and was, therefore, unavailable for plant growth. This suggests that the incorporation of plant residues such as straw which contain low amounts of S may decrease the plant availability of soil S.
INTRODUCIION
Plant residues are a major source of organic inputs to soil, accounting for some l.fL5.0 tonnes C (or 2-15 tonnes biomass dry matter) ha-’ annually in arable, grassland and woodland soils (Sanchez et al., 1989; Jenkinson et al., 1992). This plant residue input has a primary role in maintaining soil organic matter content, microbial biomass and activity, and the size of the soil nutrient pool (Sanchez et al., 1989). Soil microorganisms grow rapidly during the decomposition of plant residues (Brookes et al., 1990) and consequently some of the available soil nutrients, particularly N, P and S, major nutrients of both plants and soil microorganisms, may be immobilized. Ckio et al. (1991) have shown that, 7 days after the field incorporation of wheat straw (2% w/w), the amounts of inorganic N in soils both with and without N-amendment, decreased to very low concentrations, whereas those held in the microbial biomass doubled. These data demonstrate a rapid immobilization of soil available N by the microbial biomass following the incorporation of plant residues. However, the interactions between microbial growth and the immobilization and availability of S following the soil incorporation of plant residues remains unknown. We have investigated changes in microbial
biomass, biomass S and inorganic S in an arable soil amended with young leaves of oil-seed rape and with barley straw during an incubation period of 230 days. Our aim was (i) to study the release and subsequent availability of S from plant residues following incorporation into soil and (ii) to determine the relationship between microbial growth and S immobilization in the amended soils. Such studies provide valuable data for both the modelling of S transformations in soil and for the management of crop residues. MATERIALS
AND METHODS
Experimental
The soil (O-20 cm depth) was collected from under permanent arable (winter wheat) at Cockle Park Farm, Northumberland, U.K. This soil was a clay loam (the Wigton Moor series), pH 6.3 and contained 2.8% organic C. The moist sample was dried to 40% of soil water holding capacity (WHC), sieved (< 2 mm) and then conditioned at 100% humidity and 25°C for 10 days. Following conditioning, nine portions (500 g soil o.d.) of moist soil were weighed into 2.5 1.jars. Three of the nine portions were amended with 5 g oil-seed rape (young leaves), and three with 5 g barley straw. The remaining three portions were unamended and used as controls. The chemical properties of the
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amendments are shown in Table 1. Prior to incorporation, plant residues were dried at 35°C and ground (~420 pm). Due to the low N content of straw, the straw-amended soil was supplemented with 100 pg N g-l soil, using 50 ~1 of 143 mM NH&l solution. The unamended soil and the rape-amended soil were treated with distilled water (50 ~1 gg’ soil). Two 100 ml jars containing either 50 ml distilled water or 50 g soda lime (c 420 pm) were placed together with the soil in 2.5 1. jars. Jars were sealed and then kept at 25°C for 230 days. Inorganic-S, biomass-S and biomass-C were measured throughout the incubation following the removal of small samples from each of the jars. The decomposition of plant residues were measured by incubating them with moist soil (1% w/w). Moist soil (50 g on an over-dry basis) was weighed into 100ml jars and amended as outlined above. Each treatment was replicated four times. Soil was then incubated in sealed 1.2 1.jars at 25°C for 210 days. Each jar contained 10 ml free water and a vial containing 25 ml 1 M NaOH to collect the COI-C evolved during incubation. The vial was replaced with fresh NaOH whenever a measurement was made. Analytical procedures CO# evolved from soil was measured by titration of an aliquot (5 ml) of the NaOH using 0.5 M HCI in an autotitrator and measuring the volume of HCl utilized between pH 8.3-3.7 (Jenkinson and Powlson, 1976). Soil SOi--S was measured in 6.5 g samples of moist soil removed during incubation. Soils were weighed into a 50 ml centrifuge tube, 25 ml 0.1 M KH*PO, was then added, and the tube shaken on an end over end shaker for 30 min at 400 rev min-I. The extract was centrifuged at 8000 rev min.-’ for 30min, then filtered through a Whatman No. 42 filter paper. The extract was stored at - 18°C prior to analysis. Soil extracts (4 ml) were mixed with 4 ml HPLC water in a 50ml syringe, and shaken with activated charcoal (ca 0.2 g) for 2 min to remove organic matter. The suspensions were then filtered through a membrane (0.45 pm). SO:--S was measured using ion chromatography (O’Donnell et al., 1992). Microbial biomass-S in soil was measured using a modification of the CHCl,-fumigation-extraction method (Strick and Nakas, 1984). Three portions of Table 1. Some characteristics C contents’
of the plant residues
Total Sb
Hot water soluble SC
Plant residues
%
%
pg S g-’ soil
Rape leaves Barley straw
46.2 45.1
0.724 0.219
2752 925
‘Measured in a carbon analyser (Carbon-Sulphur Determinator, CS-125, LECO). bMeasured by H,Oz digestion method (see Analytical procedures). ‘0.25g plant residue was extracted with 10 ml hot water (100°C) for 1.5 h, made up to 25 ml and analysed
by ion chromatography.
moist soil (13.0 g) were removed from the jars and exposed to CHC& vapour in a vacuum desiccator for 24 h. Fumigation was carried out in the presence of 50ml free water and at 25°C (Jenkinson and Powlson, 1976). Following fumigation, the soil was transferred to a clean, empty desiccator and the residual CHCl, removed by evacuating for 20 min. Fumigated soils were then extracted with 20ml 10 mM CaCl, (made with HPLC water) by shaking for 60 min at 400 rev min-I. The incubated soils (three portions) were also directly extracted with 20ml CaCl, without prior fumigation. Extracts were then centrifuged, filtered (Whatman No. 42), and stored at - 18°C. After thawing, an aliquot (5 ml) of each extract was transferred to a 10 ml graduated test tube and digested with 1 ml H,O, (AR, 30% v/v) for 5 h in a sand bath (160°C). A further 0.5ml H,O, and 0.2 ml 1 N HCl were then added and digestion continued for an additional 15-20 h. After cooling, the mixture was adjusted to 8 ml by adding HPLC water then filtered through a membrane. SOi--S in the mixture was determined using ion chromatography. Biomass-S was calculated as B, = F,/k,, where F, is the difference between the S extracted from the fumigated soil and the S from the unfumigated soil. The relationship between F, and biomass-S was determined using a conversion factor (k,) of 0.35 (Chapman, 1987; Saggar et al., 1981a). Soil microbial biomass-C was measured by the fumigation-extraction method. Procedures and calculations were as described by Wu et al. (1990) with the exception that 32.5 g incubated soil and 100 ml 0.5 M K,S04 were used. RESULTS AND DISCUSSION
The decomposition of plant residues and growth of soil microbial biomass in the amended soils.
Since the oil-seed rape and the barley straw used in this study were not radiolabelled with 14C, it was impossible to partition between C02-C evolved directly from plant residues and that derived from native soil organic matter. Thus, differences in C02-C evolution between the amended and unamended soils can only be used as an indication of the mineralization of plant residues (Fig. 1). As such, priming effects following the addition of plant residues to soils were not taken into account. Wu et al. (1993) have shown that during the early phase (O-30 days of incubation) of plant residue decomposition, priming effects are small, making it possible to use increases in CO,-C evolution in amended soils as an indication of the mineralization of the substrate during the early phase of decomposition. Figure 1 suggests that rape amendments are decomposed rapidly, with some 40% of rape-C mineralized to CO& within 4 days of incorporation. By day 5, the microbial biomass-C in the rape-amended soil increased by 990 pg C gg’ soil, which was equivalent to 21.4% of the rape-C added (Fig. 2). This rate and
Soil biomass and S immobilization
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SEM
751
50
0
100
150
Incubation
I
200
250
time (days)
Fig. 1. Mineralization of rape and straw amended to soil. The CO,-C evolved from rape and straw was calculated from total CO,-C evolved from the amended soils minus CO,-C evolved the unamended soil.
extent of mineralization rape-C is comparable
and microbial assimilation of to that found using glucose
amendments but is much larger than those of other plant residues such as ryegrass and cereal straw (Kassim et al., 1981; Davenport ef al., 1988; Jawson et al., 1989; Wu et al., 1993). Previous work has suggested that glucose added to soil decomposes completely within 2-3 days, through either mineralization to C02-C or conversion to decomposition products such as biomass, microbial metabolites, or organic matter (Gregorich et al., 1991; Wu et al., 1993). It is likely, therefore, that following the incorporation of rape residues and their rapid decomposition (4-5 days), the rape-C remaining in the soil exists mainly as microbial decomposition products
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(rape-MDP) such as the newly developed biomass, microbial metabolites, or is even incorporated into soil organic matter. Between 4 and 12 days, 13% of the added rape-C (56Opg-’ soil), was mineralized. Nevertheless, the mineralization rate decreased markedly (Fig. 1). This suggests that the supply of carbon and energy from the decomposition of rape to sustain the soil microbial biomass was reduced over this time resulting in a significant decrease in biomass-C (520~~ C g-r soil, Fig. 2). The decrease in biomass-C was equivalent to the amount of rape-C evolved as CO& over 4-12 days. Although unable to partition between CO,-C derived from the rape-C and that from the MDP-C, it is interesting to speculate that the CO*-C evolved during this time resulted
Unamended Straw
1600
(0)
amended
(‘11
Rape amended
n
(01
SEM 600 -
: 0
L ”
I SE
400 -
”
1
I
0
i0
sb
Incubation
1;o
GO
time (days)
Fig. 2. Microbial biomass-C in the amended and unamended soils.
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J. WV et al.
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SEM
Rape amended . Straw
I
amended
Unamended Ol
I
0
50
I
I
100
150
Incubation
I
200
1
250
time (days)
Fig. 3. Microbial biomass-S in the amended and unamended soils.
from the mineralization of rape-MDP and reflected the decline in microbial biomass. Between 13-210 days, the mineralization rates of rape-MDP remained small and steady (7 pg C g’ soil day-‘, Fig. 1). Concomitantly, biomass C in the rape-amended soil declined further, to an amount only 35% greater than that in the unamended soil (Fig. 2). This level was then maintained throughout the incubation. In contrast, the microbial biomass C in the unamended soil was subjected to only minor fluctuation throughout the 195 day incubation. The decomposition of barley straw was considerably slower than that of rape, with the maximum mineralization rate only a quarter that of rape (Fig. 1). Unlike the rape, however, the barley straw followed the expected decomposition pattern, decomposing gradually over 60 days. This mineralization of barley straw was, however, biphasic and considerably greater during the early stages (t&12 days) than during the latter period (12-60 days). In the first 60 days, 42.5% of straw-C was mineralized to CO,-C with only an additional 3.8% mineralized during the period 60-210 days. Initially, the biomass-C in this soil increased 3.2-fold following the incorporation of straw (Fig. 2). This increase was significantly smaller than the 3.7-fold increase in biomass-C in the rapeamended soil over the same time (O-S days). The extent of the decline in biomass-C in this soil between 5-35 days was much smaller than that in the rapeamended soil, particularly between 5-15 days (Fig. 2). This pattern of change in biomass-C in the strawamended soil might be due to the gradual decomposition of straw which provided a sustained supply of carbon and energy to maintain high amounts of microbial biomass over an extended period. The rape, on the other hand, whilst supplying more carbon and energy to the biomass, was rapidly decomposed. Thus, the energy supply needed to maintain the biomass-C in the rape-amended soil was available
over a comparatively short time (Fig. 1). These results are in agreement with those of Cochran et al. (1988) and Cogle et al. (1989) who suggested that the labile fractions (e.g. hot-water extractable) of straw were rapidly decomposed during the first 14 days and produced the labile microbial biomass. The resistant fractions (e.g. cellulose and hemicellulose) which were mineralized primarily during the latter stages of incubation, produced the slowly metabolizing microbial biomass which persisted longer. After 60 days, the amount of biomass-C in the straw-amended soil became very similar to that in the rape-amended soil. Similarly, the mineralization rates of the remaining-C of both rape and straw (MDP-C) was reduced but remained throughout the incubation in amounts 3040% higher than that in the unamended soil (Figs 1 and 2). The release and microbial immobilization of plant residue-S
The amounts of biomass-S (Fig. 3) and inorganic-S (Fig. 4) in the unamended soil gradually increased during the 195 or 230 day incubation (from 5.9 to 7.6pg biomass-S and from 16.3 to 23.6pg SO:--S g -’ soil, respectively), due to the mineralization of soil organic matter. A large amount of S was released from rape during the initial rapid decomposition period following amendment and by day 5, the inorganic-S concentrations of the rape-amended soil had increased 3.3fold (from 16.3 to 52.1 c(g S g-’ soil, Fig. 4). The marked increase in the microbial biomass induced by the rapid decomposition of rape resulted in the assimilation of 25 pg S gg’ soil (based on the increase in biomass-S in the rape-amended soil, Fig. 3). Increases in the inorganic-S and biomass-S in the rape-amended soils in the first 5 days accounted for 83.4% of the rape-S added to soil (50% released as inorganic S, 33.4% immobilized in biomass S). This
Soil biomass and S immobilj~tion
and microbial biomass were very different from those of the rape. During the first 5 days following straw amendment, both inorganic-S and biomass-S increased significantly, by 73 and 57%, respectively. These increases were much smaller than those in the rape-amended soil over the same time. The amount of straw-S converted to soil inorganic-S (i.e. the increase in inorganic-S in the straw-amended soil) was equivalent to 42% of the total straw-S added to soil (Fig. 4 and Table 1). The growth of the microbial population during the first 5 days assimilated 20% of the straw-S added to soil. Again, the release and microbial assimilation of straw-S were in accordance with the expected decomposition pattern for straw. At day 5, biomass in the straw-amended soil had a C : S ratio of 130 : I, a value greater than that prior to the amendment (100: 1). It is interesting that between 5 and 15 days, inorganic-S in the strawamended soil dramatically decreased, from 25.6 to 12.1 pg S g-’ soil (Fig. 4). Thus, the amount of inorganic-S in the straw-amended soil was 30% less than that in the unamended soil by day 15. On the other hand, during the same period biomass-S increased by 5.4pg S g-i soil (Fig. 3). Clearly, the S immobilized by the biomass in the straw-amended soil during 5-15 days was from soil available S and included both soil inorganic-S and that converted from straw-S. This immobili~tion of S was not concomitant with an increase in biomass-C (Fig. 2) and thus, the C : S ratio of the biomass in the strawamended soil decreased during this period (from 130 : 1 to 80 : 1). By day 15, the C : S ratio of biomass in the straw-amended soil (80: 1) was similar to that in the unamended soil (87: 1). These changes in biomass-S following the incorporation of straw are very similar to those found using cellulose amendments (Saggar et al., 198lb). Biomass-S in the straw-amended soil decreased by
is in agreement with the extent to which the rape-C was mineralized and with the increase in microbial biomass-C in the first 5 days. It is conceivable that some of the rape-S (17%) could have been transformed into soil organic matter (organic-S) (Fitzgerald and Andrew, 1984; Strickland et al., 1986; Stanko-Golden and Fitzgerald, 1991). As the data show, most (at least 83%) of the rape-S was mineralized in 5 days, with 50% of the S converted to soil inorganic-S and another 33% transformed into soil microbial biomass-S. From day 5 to the end of the incubation, the inorganic-S in the rape-amended soil increased only slightly (Fig. 4). The amount of this increase was similar to that in the unamended soil and was, as in the unamended soil, due to the mineralization of native soil organic-S. These results suggest that the inorganic-S released from the rape-S during the first 5 days was not transformed further throughout the incubation and was available for plant uptake. Changes in biomass-S in the rape-amended soil followed a pattern similar to that of biomass-C (Fig. 3), increasing ca S-fold over the first 5 days. Subsequendy, the biomass-S in the rape-amended soil declined between 6-40 days, from a maximum of 30 to 18 pg S g-i soil. The decline in biomass-S in this soil was not reflected by increased concentrations of inorganic-S over this period and suggests that this biomass-S was transformed into soil organic-S. From day 40 to the end of the incubation (230 days), biomass-S in the rape-amended soil remained in amounts some three times greater than that in the unamended soil. During the 195 day incubation, the C: S ratio of the biomass in the rape-amended soil was 40-50: 1 (calculated from data shown in Figs 1 and 3), a value much narrower than that in the unamended soil (85-100: 1). The effects of straw incorporation on soil organic-S
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1
SEM
Rape amended
45Unamended
0 v
” Straw Ol
I
0
40
I
80 Incubation
I
120
IE t
amended I
180
I
200
time fdrysf
Fig. 4. Inorganic-S (SO,-S) in the amended and unamended soils.
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J. WV et al.
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44% between 1540 days and then remained at an amount 3040% greater than that in the unamended soil throughout the incubation (Fig. 3). This was only half the biomass-S of the rape-amended soil. However, this difference between inorganic-S in the strawamended soil and that in the unamended soil persisted throughout the incubation. Thus, in the straw-amended soil, the immobilized soil available-S, together with all of the inorganic-S released from the straw during the 195 day incubation, was converted and transformed into soil organic fractions and as such would be no longer available for plant growth. Our results have shown that plant residue-S from both rape (easily-decomposable) and straw (relatively slowly-decomposable) is rapidly released following incorporation. This is presumably because major S-containing components of plant residues such as proteins and amino acids (Stevenson, 1986) are very labile to soil microbial biomass and are decomposed prior to other more resistant fractions such as cellulose. Fitzgerald and Andrew (1984) have shown that methionine8 is rapidly converted to SOi--S and soil organic-S in 2 days following its addition to soil. Strickland and Fitzgerald (1986) have shown that most (70-100%) of the organic-S derived from forest litter can be mineralized or converted into other forms of organic-S after 7 days exposure in soil. Furthermore, our results showed that once S was immobilized by the microbial biomass, this S (e.g. the S represented by the decrease in biomass-S in both of the amended soils, Fig. 3), was directly transformed into the soil organic fraction (no increase in inorganic-S). This suggests that the S immobilized by the biomass and subsequently transformed into soil organic matter is not available for plant until it is remineralized. Differences in the effect of rape and straw incorporation on inorganic-S and microbial biomass S in soil were mainly due to variation in the decomposability and S content of the residues (Table 1). When rape decomposes the rape-S released remains in the inorganic-S pool or is assimilated by the soil microbial biomass. These forms of S increased the size of the soil available-S pool, showing that the incorporation of rape residues into soil can directly provide plant available-S and improve the S-supplying potential of the soil. This is in contrast to the incorporation of barley straw which has a high C: S ratio (206: 1, low S content) and results in a net loss of plant available-S through microbial immobilization and transformation reactions. The incorporation of such plant residues may result in S-deficiency in soil, making it advisable to use S fertilizers during straw incorporation in soils with low available S contents. Acknowledgemenrs-We
are
grateful to the Agricultural and
Food Research Council, U.K. for their financial support and to Dr P. C. Brookes, Soil Science Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, for his assistance in analysing the biomass carbon.
REFJZRRNCRR Brookes P. C., Ocio J. A. and Wu J. (1990) The soil microbial biomass: its measurement, properties and role in soil nitrogen and carbon dynamics following substrate incorporation. Soil Microorganisms 35, 39-5 1. Chapman S. J. (1987) Microbial sulphur in some Scottish soils. Soil Biology d; Biochemistry‘l9, 301-305. Cochran V. L.. Horton K. A. and Cole C. V. (1988) An estimation of microbial death rate and limitation of N or C during wheat straw decomposition. Soil Biology & Biochemistry 20, 293-298. Cogle A. L., SatBgna P. G. and Strong W. M. (1989) Carbon transformations during wheat straw decomposition. Soil Biology & Biochemistry 21, 367-372.
Davenport J. R., Thomas R. L. and Mott S. C. (1988) Carbon mineralization of corn (Zea mays L.) and romegrass (Bromus inermis leyss.) components with an emphasis on the below-ground carbon. Soil Biology & Biochemistry
20, 471476.
Fitzgerald J. W. and Andrew T. L. (1984) Mineralization of methionine sulphur in soil and forest floor layers. Soil Biology & Biochemistry
16, 565-570.
Gregorich E. G., Voroney R. P. and Kachanoski R. G. (1991) Turnover of carbon through the microbial biomass in soils with different textures. Soil Biology & Biochemistry 23, 799-805.
Jawson M. D., Elliott L. F., Papendick R. I. and Campbell G. S. (1989) The decomposition of “C-labelled wheat straw and i5N-labelled microbial material. Soil Biology & Biochemistry
21, 417422.
Jenkinson D. S. and Powlson D. S. (1976) The effects of biocidal treatment on metabolism in soil---I. Fumigation with chloroform. Soil Biology and Biochemistry 8, 167-177.
Jenkinson D. S., Harkness D. D., Vance E. D., Adams D. E. and Harrison A. F. (1992) Calculating net primary production and annual input of organic matter to soil from the amount and radiocarbon content of soil organic matter. Soil Biology & Biochemistry 24, 295-308. Kassim G., Martin J. P. and Haider K. (1981) Incorporation of a wide variety of organic substrate carbon into soil biomass as estimated by the fumigation procedure. Soil Science Society of America Journal 45, 1106-l 122. Ocio J. A., Brookes P. C. and Jenkinson D. S. (1991) Field incorporation of straw and its effects on soil microbial biomass and soil inorganic N. Soil Biology & Biochemistry 23, 171-176.
O’Donnell A. G., He Z. and Syers J. K. (1992) A biphasic extraction procedure for the simultaneous removal of elemental sulphur and sulphate from soils. Journal of the Science of Food and Agriculture
59, 395400.
Saggar S., Bettany J. R. and Stewart J. W. B. (1981a) Measurement of microbial sulfur in soil. Soil Biology & Biochemistry
13, 493498.
Saggar S., Bettany J. R. and Stewart J. W. B. (1981b) Sulphur transformations in relation to carbon and nitrogen in incubated soils. Soil Biology & Biochemistry 13, 499-5 11. Sanchez P. A., Palm C. A., Szott L. T., Cuevas E. and La1 R. (1989) Organic input management in tropical agroecosystems. In Dynamics of Soil Organic Matter in Tropical Ecosystem (D. C. Coleman, J. M. Oades and G. Uehara, Eds), pp. 125-152. NitTAL Project, University of Hawaii Press. Stanko-Golden K. M. and Fitzgerald J. W. (1991) Sulfur transformations and pool sizes in tropical forest soils. Soil Biology & Biochemistry
23, 1053-1058.
Stevenson F. J. (1986) The sulphur cycle. In Cycles in Soil (F. J. Stevenson, Ed.), pp. __ 285-320. Wiley. New York. Strick J. E. and Nakas J. P. (1984) Calibration of a microbial sulfur technique for use in forest soils. Soil Biology & Biochemirtry 16, 289-29 1.
Soil biomass and S immobilization Strickland T. C., Fitzgerald J. W. and Swank W. T. (1986) In situ mobilization of 35S-labelled organic sulphur in litter and soil from a hardwood forest. Soil Biology & Biochemisfry 18, 463-468. Wu J., Brookes P. C. and Jenkinson D. S. (1993) Formation and destruction of microbial biomass during the de-
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composition of glucose and ryegrass in soil. Soil Biology & Biochemistry 25, 1435-1441. Wu J., Joergensen R. G., Pommerening B., Chaussod R. and Brookes P. C. (1990) Measurement of soil microbial biomass by fumigation-extraction-an automated procedure. Soil Biology & Biochemisrry 22, 1167-l 169.
Soil Bid.
1993 Copyright 0
0038-0717/93 $6.00 + 0.00 1993 Pergamon Press Ltd
MICROBIAL GROWTH AND SULPHUR IMMOBILIZATION FOLLOWING THE INCORPORATION OF PLANT RESIDUES INTO SOIL J. WV, A. G. O’DONNELL and J. K. SYERS Department of Agricultural and Environmental Science, Faculty of Agriculture and Biological Sciences, The University, Newcastle upon Tyne NE1 7RU, England (Accepied 12 April 1993)
Summary-The interaction between microbial growth and S immobilization was investigated in an arable soil amended with oil-seed rape (young leaves) and barley straw (1% w/w). Initially, the rape decomposed more rapidly (40 vs 10% by-day 5) and produced a larger microbial biomass (990 pg C g-i soil) than the straw (710 ~a C a-’ soil). The biomass in both of the amended soils then decreased to amounts 3&50% higher than those in the &amended soil by day 35 and was maintained at these levels throughout the 195 day incubation. Most of the rape-S (> 80%) and straw-S (> 60%) added to the soil was released as SOi--S or converted to biomass-S in 5 days. By this time, the amount of S assimilated by the biomass in the rape-amended soil was three times that found using straw. Biomass-S in both soils then decreased but remained twice as high in the rape-amended soil over the period of 15-195 days. The biomass in the straw amended soil had a similar C:S (85-120: 1) to that of the unamended soil but was narrower (40-50: 1) in the rape-amended soil. By day 5, SO,-S in both of the amended soils had increased significantly. The increase in SOi--S in the rape-amended soil was maintained over the 195 day incubation, suggesting that this S was available for plant uptake. However, by day 15, a net immobilization of soil S by the biomass (25% of soil inorganic S) was found using straw. This immobilized S was retained by the biomass throughout the 195 day incubation and was, therefore, unavailable for plant growth. This suggests that the incorporation of plant residues such as straw which contain low amounts of S may decrease the plant availability of soil S.
INTRODUCIION
Plant residues are a major source of organic inputs to soil, accounting for some l.fL5.0 tonnes C (or 2-15 tonnes biomass dry matter) ha-’ annually in arable, grassland and woodland soils (Sanchez et al., 1989; Jenkinson et al., 1992). This plant residue input has a primary role in maintaining soil organic matter content, microbial biomass and activity, and the size of the soil nutrient pool (Sanchez et al., 1989). Soil microorganisms grow rapidly during the decomposition of plant residues (Brookes et al., 1990) and consequently some of the available soil nutrients, particularly N, P and S, major nutrients of both plants and soil microorganisms, may be immobilized. Ckio et al. (1991) have shown that, 7 days after the field incorporation of wheat straw (2% w/w), the amounts of inorganic N in soils both with and without N-amendment, decreased to very low concentrations, whereas those held in the microbial biomass doubled. These data demonstrate a rapid immobilization of soil available N by the microbial biomass following the incorporation of plant residues. However, the interactions between microbial growth and the immobilization and availability of S following the soil incorporation of plant residues remains unknown. We have investigated changes in microbial
biomass, biomass S and inorganic S in an arable soil amended with young leaves of oil-seed rape and with barley straw during an incubation period of 230 days. Our aim was (i) to study the release and subsequent availability of S from plant residues following incorporation into soil and (ii) to determine the relationship between microbial growth and S immobilization in the amended soils. Such studies provide valuable data for both the modelling of S transformations in soil and for the management of crop residues. MATERIALS
AND METHODS
Experimental
The soil (O-20 cm depth) was collected from under permanent arable (winter wheat) at Cockle Park Farm, Northumberland, U.K. This soil was a clay loam (the Wigton Moor series), pH 6.3 and contained 2.8% organic C. The moist sample was dried to 40% of soil water holding capacity (WHC), sieved (< 2 mm) and then conditioned at 100% humidity and 25°C for 10 days. Following conditioning, nine portions (500 g soil o.d.) of moist soil were weighed into 2.5 1.jars. Three of the nine portions were amended with 5 g oil-seed rape (young leaves), and three with 5 g barley straw. The remaining three portions were unamended and used as controls. The chemical properties of the
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amendments are shown in Table 1. Prior to incorporation, plant residues were dried at 35°C and ground (~420 pm). Due to the low N content of straw, the straw-amended soil was supplemented with 100 pg N g-l soil, using 50 ~1 of 143 mM NH&l solution. The unamended soil and the rape-amended soil were treated with distilled water (50 ~1 gg’ soil). Two 100 ml jars containing either 50 ml distilled water or 50 g soda lime (c 420 pm) were placed together with the soil in 2.5 1. jars. Jars were sealed and then kept at 25°C for 230 days. Inorganic-S, biomass-S and biomass-C were measured throughout the incubation following the removal of small samples from each of the jars. The decomposition of plant residues were measured by incubating them with moist soil (1% w/w). Moist soil (50 g on an over-dry basis) was weighed into 100ml jars and amended as outlined above. Each treatment was replicated four times. Soil was then incubated in sealed 1.2 1.jars at 25°C for 210 days. Each jar contained 10 ml free water and a vial containing 25 ml 1 M NaOH to collect the COI-C evolved during incubation. The vial was replaced with fresh NaOH whenever a measurement was made. Analytical procedures CO# evolved from soil was measured by titration of an aliquot (5 ml) of the NaOH using 0.5 M HCI in an autotitrator and measuring the volume of HCl utilized between pH 8.3-3.7 (Jenkinson and Powlson, 1976). Soil SOi--S was measured in 6.5 g samples of moist soil removed during incubation. Soils were weighed into a 50 ml centrifuge tube, 25 ml 0.1 M KH*PO, was then added, and the tube shaken on an end over end shaker for 30 min at 400 rev min-I. The extract was centrifuged at 8000 rev min.-’ for 30min, then filtered through a Whatman No. 42 filter paper. The extract was stored at - 18°C prior to analysis. Soil extracts (4 ml) were mixed with 4 ml HPLC water in a 50ml syringe, and shaken with activated charcoal (ca 0.2 g) for 2 min to remove organic matter. The suspensions were then filtered through a membrane (0.45 pm). SO:--S was measured using ion chromatography (O’Donnell et al., 1992). Microbial biomass-S in soil was measured using a modification of the CHCl,-fumigation-extraction method (Strick and Nakas, 1984). Three portions of Table 1. Some characteristics C contents’
of the plant residues
Total Sb
Hot water soluble SC
Plant residues
%
%
pg S g-’ soil
Rape leaves Barley straw
46.2 45.1
0.724 0.219
2752 925
‘Measured in a carbon analyser (Carbon-Sulphur Determinator, CS-125, LECO). bMeasured by H,Oz digestion method (see Analytical procedures). ‘0.25g plant residue was extracted with 10 ml hot water (100°C) for 1.5 h, made up to 25 ml and analysed
by ion chromatography.
moist soil (13.0 g) were removed from the jars and exposed to CHC& vapour in a vacuum desiccator for 24 h. Fumigation was carried out in the presence of 50ml free water and at 25°C (Jenkinson and Powlson, 1976). Following fumigation, the soil was transferred to a clean, empty desiccator and the residual CHCl, removed by evacuating for 20 min. Fumigated soils were then extracted with 20ml 10 mM CaCl, (made with HPLC water) by shaking for 60 min at 400 rev min-I. The incubated soils (three portions) were also directly extracted with 20ml CaCl, without prior fumigation. Extracts were then centrifuged, filtered (Whatman No. 42), and stored at - 18°C. After thawing, an aliquot (5 ml) of each extract was transferred to a 10 ml graduated test tube and digested with 1 ml H,O, (AR, 30% v/v) for 5 h in a sand bath (160°C). A further 0.5ml H,O, and 0.2 ml 1 N HCl were then added and digestion continued for an additional 15-20 h. After cooling, the mixture was adjusted to 8 ml by adding HPLC water then filtered through a membrane. SOi--S in the mixture was determined using ion chromatography. Biomass-S was calculated as B, = F,/k,, where F, is the difference between the S extracted from the fumigated soil and the S from the unfumigated soil. The relationship between F, and biomass-S was determined using a conversion factor (k,) of 0.35 (Chapman, 1987; Saggar et al., 1981a). Soil microbial biomass-C was measured by the fumigation-extraction method. Procedures and calculations were as described by Wu et al. (1990) with the exception that 32.5 g incubated soil and 100 ml 0.5 M K,S04 were used. RESULTS AND DISCUSSION
The decomposition of plant residues and growth of soil microbial biomass in the amended soils.
Since the oil-seed rape and the barley straw used in this study were not radiolabelled with 14C, it was impossible to partition between C02-C evolved directly from plant residues and that derived from native soil organic matter. Thus, differences in C02-C evolution between the amended and unamended soils can only be used as an indication of the mineralization of plant residues (Fig. 1). As such, priming effects following the addition of plant residues to soils were not taken into account. Wu et al. (1993) have shown that during the early phase (O-30 days of incubation) of plant residue decomposition, priming effects are small, making it possible to use increases in CO,-C evolution in amended soils as an indication of the mineralization of the substrate during the early phase of decomposition. Figure 1 suggests that rape amendments are decomposed rapidly, with some 40% of rape-C mineralized to CO& within 4 days of incorporation. By day 5, the microbial biomass-C in the rape-amended soil increased by 990 pg C gg’ soil, which was equivalent to 21.4% of the rape-C added (Fig. 2). This rate and
Soil biomass and S immobilization
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SEM
751
50
0
100
150
Incubation
I
200
250
time (days)
Fig. 1. Mineralization of rape and straw amended to soil. The CO,-C evolved from rape and straw was calculated from total CO,-C evolved from the amended soils minus CO,-C evolved the unamended soil.
extent of mineralization rape-C is comparable
and microbial assimilation of to that found using glucose
amendments but is much larger than those of other plant residues such as ryegrass and cereal straw (Kassim et al., 1981; Davenport ef al., 1988; Jawson et al., 1989; Wu et al., 1993). Previous work has suggested that glucose added to soil decomposes completely within 2-3 days, through either mineralization to C02-C or conversion to decomposition products such as biomass, microbial metabolites, or organic matter (Gregorich et al., 1991; Wu et al., 1993). It is likely, therefore, that following the incorporation of rape residues and their rapid decomposition (4-5 days), the rape-C remaining in the soil exists mainly as microbial decomposition products
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(rape-MDP) such as the newly developed biomass, microbial metabolites, or is even incorporated into soil organic matter. Between 4 and 12 days, 13% of the added rape-C (56Opg-’ soil), was mineralized. Nevertheless, the mineralization rate decreased markedly (Fig. 1). This suggests that the supply of carbon and energy from the decomposition of rape to sustain the soil microbial biomass was reduced over this time resulting in a significant decrease in biomass-C (520~~ C g-r soil, Fig. 2). The decrease in biomass-C was equivalent to the amount of rape-C evolved as CO& over 4-12 days. Although unable to partition between CO,-C derived from the rape-C and that from the MDP-C, it is interesting to speculate that the CO*-C evolved during this time resulted
Unamended Straw
1600
(0)
amended
(‘11
Rape amended
n
(01
SEM 600 -
: 0
L ”
I SE
400 -
”
1
I
0
i0
sb
Incubation
1;o
GO
time (days)
Fig. 2. Microbial biomass-C in the amended and unamended soils.
2;;
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SEM
Rape amended . Straw
I
amended
Unamended Ol
I
0
50
I
I
100
150
Incubation
I
200
1
250
time (days)
Fig. 3. Microbial biomass-S in the amended and unamended soils.
from the mineralization of rape-MDP and reflected the decline in microbial biomass. Between 13-210 days, the mineralization rates of rape-MDP remained small and steady (7 pg C g’ soil day-‘, Fig. 1). Concomitantly, biomass C in the rape-amended soil declined further, to an amount only 35% greater than that in the unamended soil (Fig. 2). This level was then maintained throughout the incubation. In contrast, the microbial biomass C in the unamended soil was subjected to only minor fluctuation throughout the 195 day incubation. The decomposition of barley straw was considerably slower than that of rape, with the maximum mineralization rate only a quarter that of rape (Fig. 1). Unlike the rape, however, the barley straw followed the expected decomposition pattern, decomposing gradually over 60 days. This mineralization of barley straw was, however, biphasic and considerably greater during the early stages (t&12 days) than during the latter period (12-60 days). In the first 60 days, 42.5% of straw-C was mineralized to CO,-C with only an additional 3.8% mineralized during the period 60-210 days. Initially, the biomass-C in this soil increased 3.2-fold following the incorporation of straw (Fig. 2). This increase was significantly smaller than the 3.7-fold increase in biomass-C in the rapeamended soil over the same time (O-S days). The extent of the decline in biomass-C in this soil between 5-35 days was much smaller than that in the rapeamended soil, particularly between 5-15 days (Fig. 2). This pattern of change in biomass-C in the strawamended soil might be due to the gradual decomposition of straw which provided a sustained supply of carbon and energy to maintain high amounts of microbial biomass over an extended period. The rape, on the other hand, whilst supplying more carbon and energy to the biomass, was rapidly decomposed. Thus, the energy supply needed to maintain the biomass-C in the rape-amended soil was available
over a comparatively short time (Fig. 1). These results are in agreement with those of Cochran et al. (1988) and Cogle et al. (1989) who suggested that the labile fractions (e.g. hot-water extractable) of straw were rapidly decomposed during the first 14 days and produced the labile microbial biomass. The resistant fractions (e.g. cellulose and hemicellulose) which were mineralized primarily during the latter stages of incubation, produced the slowly metabolizing microbial biomass which persisted longer. After 60 days, the amount of biomass-C in the straw-amended soil became very similar to that in the rape-amended soil. Similarly, the mineralization rates of the remaining-C of both rape and straw (MDP-C) was reduced but remained throughout the incubation in amounts 3040% higher than that in the unamended soil (Figs 1 and 2). The release and microbial immobilization of plant residue-S
The amounts of biomass-S (Fig. 3) and inorganic-S (Fig. 4) in the unamended soil gradually increased during the 195 or 230 day incubation (from 5.9 to 7.6pg biomass-S and from 16.3 to 23.6pg SO:--S g -’ soil, respectively), due to the mineralization of soil organic matter. A large amount of S was released from rape during the initial rapid decomposition period following amendment and by day 5, the inorganic-S concentrations of the rape-amended soil had increased 3.3fold (from 16.3 to 52.1 c(g S g-’ soil, Fig. 4). The marked increase in the microbial biomass induced by the rapid decomposition of rape resulted in the assimilation of 25 pg S gg’ soil (based on the increase in biomass-S in the rape-amended soil, Fig. 3). Increases in the inorganic-S and biomass-S in the rape-amended soils in the first 5 days accounted for 83.4% of the rape-S added to soil (50% released as inorganic S, 33.4% immobilized in biomass S). This
Soil biomass and S immobilj~tion
and microbial biomass were very different from those of the rape. During the first 5 days following straw amendment, both inorganic-S and biomass-S increased significantly, by 73 and 57%, respectively. These increases were much smaller than those in the rape-amended soil over the same time. The amount of straw-S converted to soil inorganic-S (i.e. the increase in inorganic-S in the straw-amended soil) was equivalent to 42% of the total straw-S added to soil (Fig. 4 and Table 1). The growth of the microbial population during the first 5 days assimilated 20% of the straw-S added to soil. Again, the release and microbial assimilation of straw-S were in accordance with the expected decomposition pattern for straw. At day 5, biomass in the straw-amended soil had a C : S ratio of 130 : I, a value greater than that prior to the amendment (100: 1). It is interesting that between 5 and 15 days, inorganic-S in the strawamended soil dramatically decreased, from 25.6 to 12.1 pg S g-’ soil (Fig. 4). Thus, the amount of inorganic-S in the straw-amended soil was 30% less than that in the unamended soil by day 15. On the other hand, during the same period biomass-S increased by 5.4pg S g-i soil (Fig. 3). Clearly, the S immobilized by the biomass in the straw-amended soil during 5-15 days was from soil available S and included both soil inorganic-S and that converted from straw-S. This immobili~tion of S was not concomitant with an increase in biomass-C (Fig. 2) and thus, the C : S ratio of the biomass in the strawamended soil decreased during this period (from 130 : 1 to 80 : 1). By day 15, the C : S ratio of biomass in the straw-amended soil (80: 1) was similar to that in the unamended soil (87: 1). These changes in biomass-S following the incorporation of straw are very similar to those found using cellulose amendments (Saggar et al., 198lb). Biomass-S in the straw-amended soil decreased by
is in agreement with the extent to which the rape-C was mineralized and with the increase in microbial biomass-C in the first 5 days. It is conceivable that some of the rape-S (17%) could have been transformed into soil organic matter (organic-S) (Fitzgerald and Andrew, 1984; Strickland et al., 1986; Stanko-Golden and Fitzgerald, 1991). As the data show, most (at least 83%) of the rape-S was mineralized in 5 days, with 50% of the S converted to soil inorganic-S and another 33% transformed into soil microbial biomass-S. From day 5 to the end of the incubation, the inorganic-S in the rape-amended soil increased only slightly (Fig. 4). The amount of this increase was similar to that in the unamended soil and was, as in the unamended soil, due to the mineralization of native soil organic-S. These results suggest that the inorganic-S released from the rape-S during the first 5 days was not transformed further throughout the incubation and was available for plant uptake. Changes in biomass-S in the rape-amended soil followed a pattern similar to that of biomass-C (Fig. 3), increasing ca S-fold over the first 5 days. Subsequendy, the biomass-S in the rape-amended soil declined between 6-40 days, from a maximum of 30 to 18 pg S g-i soil. The decline in biomass-S in this soil was not reflected by increased concentrations of inorganic-S over this period and suggests that this biomass-S was transformed into soil organic-S. From day 40 to the end of the incubation (230 days), biomass-S in the rape-amended soil remained in amounts some three times greater than that in the unamended soil. During the 195 day incubation, the C: S ratio of the biomass in the rape-amended soil was 40-50: 1 (calculated from data shown in Figs 1 and 3), a value much narrower than that in the unamended soil (85-100: 1). The effects of straw incorporation on soil organic-S
75
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1
SEM
Rape amended
45Unamended
0 v
” Straw Ol
I
0
40
I
80 Incubation
I
120
IE t
amended I
180
I
200
time fdrysf
Fig. 4. Inorganic-S (SO,-S) in the amended and unamended soils.
1
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J. WV et al.
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44% between 1540 days and then remained at an amount 3040% greater than that in the unamended soil throughout the incubation (Fig. 3). This was only half the biomass-S of the rape-amended soil. However, this difference between inorganic-S in the strawamended soil and that in the unamended soil persisted throughout the incubation. Thus, in the straw-amended soil, the immobilized soil available-S, together with all of the inorganic-S released from the straw during the 195 day incubation, was converted and transformed into soil organic fractions and as such would be no longer available for plant growth. Our results have shown that plant residue-S from both rape (easily-decomposable) and straw (relatively slowly-decomposable) is rapidly released following incorporation. This is presumably because major S-containing components of plant residues such as proteins and amino acids (Stevenson, 1986) are very labile to soil microbial biomass and are decomposed prior to other more resistant fractions such as cellulose. Fitzgerald and Andrew (1984) have shown that methionine8 is rapidly converted to SOi--S and soil organic-S in 2 days following its addition to soil. Strickland and Fitzgerald (1986) have shown that most (70-100%) of the organic-S derived from forest litter can be mineralized or converted into other forms of organic-S after 7 days exposure in soil. Furthermore, our results showed that once S was immobilized by the microbial biomass, this S (e.g. the S represented by the decrease in biomass-S in both of the amended soils, Fig. 3), was directly transformed into the soil organic fraction (no increase in inorganic-S). This suggests that the S immobilized by the biomass and subsequently transformed into soil organic matter is not available for plant until it is remineralized. Differences in the effect of rape and straw incorporation on inorganic-S and microbial biomass S in soil were mainly due to variation in the decomposability and S content of the residues (Table 1). When rape decomposes the rape-S released remains in the inorganic-S pool or is assimilated by the soil microbial biomass. These forms of S increased the size of the soil available-S pool, showing that the incorporation of rape residues into soil can directly provide plant available-S and improve the S-supplying potential of the soil. This is in contrast to the incorporation of barley straw which has a high C: S ratio (206: 1, low S content) and results in a net loss of plant available-S through microbial immobilization and transformation reactions. The incorporation of such plant residues may result in S-deficiency in soil, making it advisable to use S fertilizers during straw incorporation in soils with low available S contents. Acknowledgemenrs-We
are
grateful to the Agricultural and
Food Research Council, U.K. for their financial support and to Dr P. C. Brookes, Soil Science Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, for his assistance in analysing the biomass carbon.
REFJZRRNCRR Brookes P. C., Ocio J. A. and Wu J. (1990) The soil microbial biomass: its measurement, properties and role in soil nitrogen and carbon dynamics following substrate incorporation. Soil Microorganisms 35, 39-5 1. Chapman S. J. (1987) Microbial sulphur in some Scottish soils. Soil Biology d; Biochemistry‘l9, 301-305. Cochran V. L.. Horton K. A. and Cole C. V. (1988) An estimation of microbial death rate and limitation of N or C during wheat straw decomposition. Soil Biology & Biochemistry 20, 293-298. Cogle A. L., SatBgna P. G. and Strong W. M. (1989) Carbon transformations during wheat straw decomposition. Soil Biology & Biochemistry 21, 367-372.
Davenport J. R., Thomas R. L. and Mott S. C. (1988) Carbon mineralization of corn (Zea mays L.) and romegrass (Bromus inermis leyss.) components with an emphasis on the below-ground carbon. Soil Biology & Biochemistry
20, 471476.
Fitzgerald J. W. and Andrew T. L. (1984) Mineralization of methionine sulphur in soil and forest floor layers. Soil Biology & Biochemistry
16, 565-570.
Gregorich E. G., Voroney R. P. and Kachanoski R. G. (1991) Turnover of carbon through the microbial biomass in soils with different textures. Soil Biology & Biochemistry 23, 799-805.
Jawson M. D., Elliott L. F., Papendick R. I. and Campbell G. S. (1989) The decomposition of “C-labelled wheat straw and i5N-labelled microbial material. Soil Biology & Biochemistry
21, 417422.
Jenkinson D. S. and Powlson D. S. (1976) The effects of biocidal treatment on metabolism in soil---I. Fumigation with chloroform. Soil Biology and Biochemistry 8, 167-177.
Jenkinson D. S., Harkness D. D., Vance E. D., Adams D. E. and Harrison A. F. (1992) Calculating net primary production and annual input of organic matter to soil from the amount and radiocarbon content of soil organic matter. Soil Biology & Biochemistry 24, 295-308. Kassim G., Martin J. P. and Haider K. (1981) Incorporation of a wide variety of organic substrate carbon into soil biomass as estimated by the fumigation procedure. Soil Science Society of America Journal 45, 1106-l 122. Ocio J. A., Brookes P. C. and Jenkinson D. S. (1991) Field incorporation of straw and its effects on soil microbial biomass and soil inorganic N. Soil Biology & Biochemistry 23, 171-176.
O’Donnell A. G., He Z. and Syers J. K. (1992) A biphasic extraction procedure for the simultaneous removal of elemental sulphur and sulphate from soils. Journal of the Science of Food and Agriculture
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Saggar S., Bettany J. R. and Stewart J. W. B. (1981a) Measurement of microbial sulfur in soil. Soil Biology & Biochemistry
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Saggar S., Bettany J. R. and Stewart J. W. B. (1981b) Sulphur transformations in relation to carbon and nitrogen in incubated soils. Soil Biology & Biochemistry 13, 499-5 11. Sanchez P. A., Palm C. A., Szott L. T., Cuevas E. and La1 R. (1989) Organic input management in tropical agroecosystems. In Dynamics of Soil Organic Matter in Tropical Ecosystem (D. C. Coleman, J. M. Oades and G. Uehara, Eds), pp. 125-152. NitTAL Project, University of Hawaii Press. Stanko-Golden K. M. and Fitzgerald J. W. (1991) Sulfur transformations and pool sizes in tropical forest soils. Soil Biology & Biochemistry
23, 1053-1058.
Stevenson F. J. (1986) The sulphur cycle. In Cycles in Soil (F. J. Stevenson, Ed.), pp. __ 285-320. Wiley. New York. Strick J. E. and Nakas J. P. (1984) Calibration of a microbial sulfur technique for use in forest soils. Soil Biology & Biochemirtry 16, 289-29 1.
Soil biomass and S immobilization Strickland T. C., Fitzgerald J. W. and Swank W. T. (1986) In situ mobilization of 35S-labelled organic sulphur in litter and soil from a hardwood forest. Soil Biology & Biochemisfry 18, 463-468. Wu J., Brookes P. C. and Jenkinson D. S. (1993) Formation and destruction of microbial biomass during the de-
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composition of glucose and ryegrass in soil. Soil Biology & Biochemistry 25, 1435-1441. Wu J., Joergensen R. G., Pommerening B., Chaussod R. and Brookes P. C. (1990) Measurement of soil microbial biomass by fumigation-extraction-an automated procedure. Soil Biology & Biochemisrry 22, 1167-l 169.