Estimation of soil microbial C by A fumigation-extraction procedure: Influence of soil moisture content

Soil Bid. Biochcm. Vol. 21, No. 6. pp. 761-772. 1989 Printed in Great Britain. All rights reserved

Copyright 0

0038-0717/89 53.00 + 0.00 1989 Pergamon F’resspk

ESTIMATION OF SOIL MICROBIAL C BY A FUMIGATION-EXTRACTION PROCEDURE: INFLUENCE OF SOIL MOISTURE CONTENT D. J. Ross Division of Land and Soil Sciences. Department of Scientific and Industrial Research, Lower Hutt, New Zealand (Accepted 20 Februmy 1989) Summary-The influence of the moisture content of field-moist samples on the estimation of extractable-C Rush, and consequently microbial C. by the fumigation-extraction procedure was determined with five soils from pastures. Three aspects were investigated: (I) moisture content during fumigation; (2) the effect of initial partial drying; and (3) the most suitable extraction procedure for wet, compacted samples. Values of extractable-C Rush were influenced by soil moisture content during fumigation, and were sometimes appreciably lower in drier samples. Maximum values were obtained with soils wetted to ELI50% or more of their water-holding capacity (WHC), corresponding to a water potential of co - 10 kPa or higher. For all field-moist samples, adjustment of soil moisture content immediately before fumigation lo values between 50 and 60% of WHC. or - 5 and - IO kPa. is therefore recommended. Rapid, partial drying at room temperature had no detectable effect on extractable-C flush values, provided the samples were rewelted to ca 50% of WHC immediately before fumigation. For extraction of wet, compacted samples, shaking on a rotary shaker at increased speed, or preferably in an end-over-end shaker, generally gave satisfactory results. Preliminary partial drying of such samples and rewetting to 50% of WHC before fumigation resulted in accurate extractable-C flush values, but could be impractical for routine use.

INTRODUCIION

extractable-C flush values, although it had no effect on microbial C values estimated by a fumigation-incubation procedure. The main objective of this study was to determine whether the moisture content of field-moist samples during fumigation is important in the fumigationextraction procedure and, if so. to determine the moisture content associated with maximum extractable-C flush values. Two other aspects, also, of this procedure were re-examined: (I) the effect, if any, of rapid partial drying of soil (Ross, 1988); and (2) improvement of the extraction procedure for wet, compacted samples (Tate ef al., 1988).

Among the methods now available to estimate soil microbial C, direct extraction procedures have several advantages (Blagodatskiy ef al., 1987; Vance et al., 1987; Jenkinson, 1988; Sparling and West, 1988a.b; Tate et al., 1988). In these procedures, microbial cells are killed by fumigation, or drying (Blagodatskiy ef al., 1987), and the C thereby rendered extractable (the extractable-C flush) is calculated as the difference between the amount extracted from the treated and untreated (control) soil. A conversion factor is subsequently used to estimate microbial C content. In the fumigation-extraction procedure, variations in soil moisture content had no detectable influence on the efficiency of extraction with K*SO, solution (Tate er ul., 1988). An effect of soil moisture content during fumigation has, however, been shown for air-dried samples by Sparling and West (1989). Extractable-C flush values were appreciably higher in samples rewetted immediately before fumigation than in those fumigated at air-dry moisture content. Gradual drying of two soils also indicated that extractable-C flush estimates could be adversely affected when soil water content fell below co IS% w/w (Sparling and West, 1989). These results raised the possibility that the moisture content of field-moist samples during fumigation might also influence extractable-C flush values. This possibility had been suggested by partial-drying experiments (Ross, 1988). Rapid partial drying of soil frequently lowered

MATERIALS AND METHODS Sites and soils

Five soils were examined. Hokio sand occurred under a vegetation of ungrazed grasses and fiatweeds, and the other four soils (Table I) under grazed, introduced pastures (Ross, 1987a). Hokio soil was sampled as turfs (O-3 cm depth). Soil at the other sites was sampled as before (Ross, 1987a), with SO-100 cores (2.5 cm dia. O-5 cm depth) being taken at each sampling time. The times of sampling are indicated in the appropriate tables. The samples were first passed through a 5.6mm mesh and, where indicated, partially dried rapidly with a cold-air fan, with almost continuous mixing of the soil (Ross, 1988). The samples were then further 767

168

D. J. Ross Table I. Some orooerties of the soils Moisture content (%)

Soil Hokio sand Waikanae silt loam Pomare silt loam Castlepoint silt loam Kaitokc silt loam

Classification Aquic Typic Typic Typic Tyoic

Udipsamment Udifluvent Dystrochrept Haplaquoll Dvstrochreot

DH

Organic c (%)

Total N (%)

7.4 6.3 5.2 6.4 6.2

0.94 4.2 6.3 6.9 7.8

0.07 0.39 0.41 0.54 0.60

WHC’

-I

45 7s 97 104 115

kPa

Water potential -2 kPa -5 kPa

46 79 100 107 119

43 67 SO I35 100

20 50 58 66 77

’ Determined essentially according to Gasser (1961).

sieved (c 2 mm) and stored at 4°C. Some properties of the soils are given in Table 1. The effect of different extraction conditions on the extractable-C flush values of wet, compacted samples was determined with soils sampled in winter. These samples consisted of remoulded aggregates (lumps) after being forced through the 2 mm mesh. However, the moisture content of Castlepoint soil at that time was unusually low (39% of WHC). Sieved (< 5.6 mm) Castlepoint soil was therefore wetted and mixed with distilled water before further sieving ( < 2 mm) to produce a characteristically smeared and compacted winter sample. This wetted sieved sample (at 55% of WHC) is subsequently referred to as “field-moist” (Table 6).

Anal~~tical methods

Results are expressed on the basis of oven-dry (105’C) weights of material. Analyses were made in triplicate and commenced within 24 h of sieving. The significance of differences between treatments was calculated by analysis of variance and Fisher’s LSD test (Steel and Torrie, 1980). Soil moisture. Soil moisture content was determined by overnight drying at 105°C. WHC was measured according to Gasser (1961) or Harding and Ross (1964); estimates tended to be slightly higher (by an average of ca 3%) by the latter procedure. Soil water potential was measured by tensiometry (Gradwell and Birrell, 1979). Representative values of these properties are shown in Table 1. Soil chemical properties. Organic C and total N contents and pH (1:2.5 in water) were determined according to Blakemore ef al. (1987). Estrac&le-C/?&l. Extractable-C flush was determined essentially by the procedure of Tate er al. (1988). Field-moist soil, partially-dried soil or soil adjusted to various moisture contents by wetting individual, replicate samples (all to give an equivalent weight of 20 g at 60% of WHC) was fumigated with purified CHCI, (Jenkinson and Powlson, 1976b) for 24 h. After removal of the CHCI, and adjustment of the moisture content to 60% of WHC, the soil was extracted with 0.5 M K2S04 (50 ml) by shaking for 30 min. at I IO rev min-’ on an orbital shaker, unless otherwise stated. Unfumigated soil was extracted similarly. The determination of C in the extracts was based on the method used by Jenkinson and Powlson (1976a). and differed from the description given by Tate er al. (1988) only in the inclusion of HgO (70 mg) (Quinn and Salomon. 1964) in the oxidation mixture, unless otherwise stated.

RESULTS

Influence of soil moisture content flush aalues

on extractable-C

Wetting unfumigated, summer samples from their field-moist state (23-30% of WHC) to 50 or 60% of WHC, and standing at room temperature for 24 h had no detectable effect on their extractable-C contents (Table 2). Differences in soil moisture content during fumigation did, however, have a significant effect on extractable-C flush values (i.e. C extracted from fumigated soil minus C extracted from unfumigated soil) (Table 3). Maximum values in the different soils were not directly related to soil moisture content as such. but they were related to the percentage of soil WHC. For example, maximum values of extractableC flush were found at a moisture content of ca 20% in Hokio soil, but at cu 60% in Castlepoint soil; in Castlepoint soil at a moisture content of 26%. the value of extractable-c flush was cu 15% below the maximum. Values of extractable-C flush were greatest at 40% of WHC or higher in Hokio soil, and at ca 50% or higher in the other soils examined. When determined with soil fumigated at field-moisture content (15-30% of WHC), extractable-C flush values were significantly lower than these maximum values, by 22% for Hokio soil and I5 f 1% (SD) for the other soils. In the potassium dichromate-acid digestion mixture for measuring extracted C (Jenkinson and Powlson, 1976a), HgO was added to eliminate interference from halides (Quinn and Salomon, 1964). With three of the soils studied here, the absence of HgO in the digestion mixture had no effect on extractable-C flush values, but in Kaitoke soil values were 2-3% lower in its presence (Table 4). Table 2. Influence of wetting samples to 50 or 60% of WHC. and standing at co 22’C for 24h, on the extractable-C content of unfumigated soil Soil Waikanae Pomare Castlepoint Kaitoke

Moisture (% of WHC)

Extractable-C @cgg-’ soil)

23’ 60 24’ 60 23’ 50 30’ 60

99 97 213 205 84 75 229 220

‘Field-moisture content of samples collected in summer (January. February). For each soil. none of the differences between samples at the different moisture contents was significant.

769

Fumigation-extraction and soil moisture Table 3. Influence of soil moisture content during fumigation on values of extractabk-C flush Moisture content during fumigation Soil Hokio

Waikanae

Pomarc

Castlepoint

Kaitoke

(% dry soil)

(% of WHC)

6 12 16 20 23 18 31 41 51 62 22 36 48 60 72 26 36 48 60 72 39 51 67 84 101

Extractable-C flush bg g-’ soil)

IS’ 30 40 50 60 23’ 38 50 63 7s 24’ 38 50 63 1J 23’ 31 41 52 62 30’ 39 53 66 79

98c ll2b 118ab I 24a I2Sa 393c 435b 448ab 464a 464a 659c 728b 758a 166a 774a 708d 782~ 813b 822ab 838a S65b 617a 646a 652a 646a

’ Field-moisture content; sampleswere collected in summer (January, February). except for Hokio (May). For each soil, values not marked with the same letter are significantly different (P < 0.05).

Influence of partial air-drying of soil on extractable-C flush calues

Rapid partial drying of field-moist soil before fumigation generally lowered the values of the extractable-c flush by IO-23% (Table 5); the only exception occurred with a summer sample of Kaitoke soil that was taken immediately after 3 days’ rain (ca 50mm) following a dry spell of several weeks. In contrast, no differences between field-moist and partially-dried samples were observed when the moisture contents of the samples were adjusted to 50% of WHC immediately before fumigation. Influence of shaking conditions on extractable-Cjush values of smeared and compacted samples Because partially-dried samples that had been rewetted to 50% of WHC immediately before fumigation gave a reliable measure of extractable-C flush, Table 4. Influence of the addition of HgO IO the digestion mixture on the measuremcnI of C in esIracIs of fumigated soils

Soil

Moisture content during fumigalion (% of WHC)

Waikanae :r Pomare Castlepoint KaiIokc’

;: 50’ ;:

Extractable-C (pg g-’ soil): mixIure +HgO

dig-lion - HgO 498b 569a 872b 979a 946a 869c 992a

491b S62a 884b 98la 934a 845d 972b

‘The Kaitokc samples were taken at ditferent times. ‘Field-moisture content of samples collected in summer. ‘Samples adjusted IO 50 or 60% of WHC immediately before fumigalion. For each soil, values not marked with the same letrer are significantly ditl’ercnt (P < 0.05).

they were used as the basis of comparison to assess the efficiency of various shaking conditions for determining extractable-C flush in undried, smeared and compacted winter samples. In all except Waikanae soil, extractable-C flush values of the field-moist winter samples were lower with an orbital shaking speed of I10 rev min-’ than with 250 rev min- ’ (Table 6). Dispersion of the soil was less efficient at the lower speed, and lumps of ca 24mm dia remained in some samples after the 30-min extraction. Larger lumps (> 5 mm dia) were also occasionally observed, and their presence resulted in an appreciably lower (by as much as 30%) extractable-C flush value. In all of the field-moist winter samples, values of extractable-C flush were indistinguishable when obtained after orbital shaking at 250 rev min-’ or endover-end shaking at 50 rev min-’ (Table 6). Both of these shaking procedures generally gave satisfactory estimates of extractable-C flush. Compared to values from the more readily dispersed partially-dried samples (rewetted to 50% of WHC). In Waikanae soil however, the flush value of field-moist soil was closest to that of the partially-dried sample (rewetted to 50% of WHC) when end-over-end shaking was used. In the compacted, field-moist winter sample of Pomare soil, none of the extractable-C flush values was as high as those from the partially-dried sample (rewetted to 50% of WHC). In the wetter spring sample taken 3 wk later, the extractable-C flush value of field-moist soil was, however, indistinguishable from that of the partially-dried sample (rewetted to 50% of WHC). The difference between these samples can be attributed to the amount of C extracted from unfumigated soil. At each sampling, the amounts of C extracted from fumigated field-moist soil, and soil that had been partially dried and rewetted before fumigation, were almost identical (data not shown). DISCUSSION

Influence of soil moisture flush values

content

on extractable-C

The importance of soil moisture content in the fumigation-extraction procedure for measuring microbial biomass C has been clearly demonstrated for field-moist samples in this study, and for air-dried samples by Sparling and West (1989). Because the extractable-C values of unfumigated, field-moist soil were not lowered by overnight wetting of the samples, the effects of soil wetting immediately before fumigation can be attributed solely to the amounts of C extracted from fumigated soil. In air-dried soils, in contrast, the amounts of extractable-C decreased when unfumigated samples were rewetted and allowed to stand overnight (Sparling and West, 1989). When soil moisture contents were low or moderate during fumigation, extractable-C flush values were often appreciably lower than those obtained with wetted samples. These results have implications for the determination of factors for converting extractable-C flush to microbial C values (Vance et al., 1987; Sparling and West. 1988a.b; Tate et al.. 1988). for drying experiments (West et al., 1988) and for the measurement of microbial C in soils from different environments or seasons.

770

D. J. Ross Table 5. lnlluace

of partial drying and adjustment of soil water content before fumiaation on values of extractable-C flush Extractable-C flush (pg g-’ soil): moisture content during fumigation

Soil

Treatment

Name Waikanae

FM’

Pomare

:; PD FM PD FM’ PD FM PD

Castlepoint Kaitokc

Moisture content (X of WHC) 32 22 37 25 30 17 48 26 39 2s

I

Unadjusted

Adjusted to 50% of WHC 419a 4l4a 594a 603a 735a 724a 764a 759a 679a 667a

38gb 331c 590a 533b 6glb 525c 733a 765a 634b WC

‘FM, geld-moist; PD. partially dried. *Sample collected in summer (February), immediately afier 3 days of rain (cd 50 mm) following a dry period of several weeks. All other samples were collected in autumn (March-May). For each soil at each sampling time, values not marked with the same letter are significantly different (P < 0.05).

Although the optimum moisture content (expressed as a percentage of WHC) can vary with different soils (Table 3), and to some extent in the same soil at different sampling times, e.g. Kaitoke soil (Table 5) all soils gave maximum extractable-C values when fumigated at co 50% of WHC or higher. In terms of soil-water potential, - 10 kPa would here correspond to ca 50% of WHC or higher (based on measurements with previous samples of these soils; F. J. Cook, personal communication) and -5 kPa (determined with the present samples) to cu 60% of WHC. Less efficient enzymic hydrolysis of cell components, as suggested by Spariing and West (1989) for air-dried samples, could be mainly responsible for the lower amounts of C extracted from field-moist samples fumigated at suboptimum moisture contents. The involvement of hydrolytic enzymes in increasing the extractability of microbial N has been indicated by the fumigation experiments of Brookes et al. (1985a.b) and Amato and Ladd (1988). Similar hydrolysis of other cell components appears likely. Support for this hypothesis is provided by experiments in which partial air-drying often lowered extractable-C flush, but not CO& flush values (Ross, 1988). To bc estimated in the fumigation-

extraction procedure, components from killed cells would either have to be initially soluble or rendered soluble through enzymic activity during fumigation. For this, an adequate moisture content would be required. In the fumigationincubation procedure, such initially insoluble components would be metabolized during incubation of the soil, which is adjusted to a standardized WHC ufkr fumigation; the actual moisture content during fumigation would therefore not be critical (Ross, 1988). Differences in rates of soil dispersion during extraction may also have contributed to the differences in extractable-C values of samples fumigated at various moisture contents (degree of dispersion was roughly assessed by assuming an inverse relationship with speed of filtration). Dispersion was frequently greatest with the wetted fumigated samples, and could thereby have resulted in greater extractability of cell-derived components held within fine particles. The dispersion of a soil during shaking may also be affected by soil texture. However, no conclusions on the possible influence of moisture-texture relationships on extractable-C values can be drawn from this study, in which only a sand and four silt loam soils were examined.

Table 6. Influence of shaking conditions on values of extractable-C flush of field-moist and partially-dried winter samples Shaking conditions Speed (rev min-‘)

Extractable-C flush (cg g-’ soil): soil Pomare Winter Spring

Treatment

Shaker

Castlepoint

Kaitoke

Field-moist’ Field-moist Field-moist Partiallydried’ Partially-dried (50% of WHC)’ Partially-dried (50% of WHC)’

Orbital Orbital End-over-end Orbital

II0 250 50 II0

34Oc 344c 34gbc 31ld

473d 525c 524c 548bc

ND ND 576a ND

635~ 704ab 700ab 68lb

59lb 65la 654a 577b

Orbital

II0

365a

58la

ND

725a

629a

361ab

566a

566ab

720a

653a

End-over-end

’ Field-moisture contents ranged from

50

Waikanae

54-60% of WHC, with those for the winter (I9 August) and early spring (6 September) samples of Pomare soil being 53 and 57%. respectively; all of these sieved samples were smeared and compacted. *Moisture contents of the partially-dried samples. consisting of discrete soil particles, ranged from 3l-39% of WHC. ‘Moisture content adjusted to 50% of WHC immediately before fumigation. ND = not determined. For each soil at each sampling time, values not marked with the same letter are significantly different (P < 0.05).

Fumigation-extraction

A factor that can be eliiinated in these wetting is interference from CHCI, decomposition during the fumigation period. According to Vance et d (1987), the presence of HgO in the oxidation mixture to measure the C extracted is necessary, because pure CHCI, decomposes rapidly to phosgene and HCI. However, the absence of any effect of HgO in the digests of Waikanae and Pomare soils that had been fumigated at rather low field-moisture contents or at 60% of WHC (Table 4) strongly suggests that decomposition during fumigation was here absent or negligible, even in the samples that had been wetted to 60% of WHC. effects

For discrete soil “aggregates” (< 2 mm), shaking with 0.5 M &SO, for 30 min at 110 rev min-’ on a rotary shaker, or at 50 rev min-’ on an end-over-end shaker (Sparling and West, 1988a) appeared equally satisfactory for the extraction of C from fumigated and unfumigated soil (Tate er al., 1988). Problems had, however, been encountered with wet samples that gave compacted lumps after being forced through a 2mm mesh; the efficacy of alternative shaking speeds and extraction times or initial partial drying had therefore been examined (Ross, 1988). Appropriate procedures for estimating extractableC flush in such wet, compacted samples can now be proposed. An accurate estimate appeared to result when the samples were partially dried and re-sieved ( -=z2 mm) and subsequently rewetted to 50% of WHC immediately before fumigation. Partial drying may, however, be impractical when handling large numbers of samples, because constant attention is needed to prevent localized over-drying of the soil (Ross, 1988). Direct fumigation and extraction of undried soil is therefore required. No extraction procedure was entirely satisfactory, but end-over-end shaking generally appeared slightly more suitable than rotary shaking at an increased speed (2SOrev min-I). Although a rotary shaking speed of I10 rev min-’ (Tate et al., 1988) was reasonably satisfactory for some wet compacted samples, e.g. Waikanae and a Southdown soil [a Typic Dystrochrept (Ross, 1987b); data not shown], it was unsuitable for other soils and cannot be recommended as a general procedure. The dispersibility of the samples during extraction appeared to be the most critical factor affecting these results. Even with the higher shaking speed, or end-over-end shaking, dispersion of soil lumps was sometimes incomplete. Incomplete or slow dispersion of soil would probably account for the low extractable-C contents and occasionally variable results observed with wet, compacted samples (Tate et al., 1988) and for the differences found here between the two unfumigat~, compacted, undried samples of Pomare soil taken in August and September. The fact that wet, compacted samples can give satisfactory extractable-C values under modified extraction conditions shows that CHCI, is as effective a fumigant for very wet soil as for drier soil. Problems that had been encountered in the fumigationincubation procedure with wet, compacted samples, which often gave unrealistically low biomass-C values (Ross ef d. 1985; Ross, 1987a), can therefore be

771

and soil moisture

attributed mainly to impeded ~ne~li~tion of killed organisms. In these particular samples, the possible inability of CHCll to kill nearly a11of the organisms (Jenkinson and Powlson, 1980; Adams and Laughlin, 1981) appears less likely and, at most, of only minor importance. General recommendations Although unfumigated samples can be satisfactorily extracted at field-moisture content, control of the moisture content of fumigated samples during fumigation has been found to be essential for maximum extractable-C flush values. Based on these results, a water potential of -5 to - IO kPa (ca 5040% of WHC), adjusted immediately before fumigation, appears to be optimum. For wet, compacted sampla, extraction on a rotary shaker at 250 rev min-‘, or preferably an end-over-end shaker at 50 rev min-’ is recommended; the use of large soil lumps (> ca 5 mm) should, however, be avoided. Acknow/edgemenrs--I thank Charles W. Feltham for the chemical analyses, Freeman 1. Cook and Linton J. Palmer for determining soil-water potentials, and Graham P. Sparling for valuable discussions. REFERENCES

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