Importance of soil water content when estimating soil microbial C, N and P by the fumigation-extraction methods

Soil Bid. Biochem. Vol. 21. No. 2. pp. 245-253. Printed in Great Britain. All rights reserved

1989 Copyright

0038-0717:89 S3.00 + 0.00 C 1989 Pergamon Press plc

IMPORTANCE OF SOIL WATER CONTENT WHEN ESTIMATING SOIL MICROBIAL C, N AND P BY THE FUMIGATION-EXTRACTION METHODS GRAHAM P. SPARLINGand ANDREW W. W~sr N.Z. Soil Bureau. Department of Scientific and Industrial Research, Private Bag, Lower Hutt, New Zealand

(Accepted I October 1988) Summary-The influence of soil water content on the estimation of microbial C. N and P by the fumigation-extraction (FE) method and microbial C and N by the fumigation-incubation (FI) method was investigated using a range of air-dry soils. The estimates of microbial C were compared with those obtained by the substrate-induced respiration (SIR) method. Soils were fumigated overnight with CHCI, when either air-dry. or rewetted to 50% w/w water content immediately before fumigation. The presence of water during fumigation greatly increased the C and N extracted by 0.5 M K,SO, compared with soils fumigated while air-dry. Overnight rewetting of non-fumigated (control) soils decreased extractable-C but the elkct on extractable-N was variable. Rewetting before fumigation also increased CO,-C production and net N-mineralization (during subsequent incubation) compared to soils fumigated while air dry. However, because of high variability the increases were often not significant. The flushes of extractable-C and N (the difference between fumigated and non-fumigated soils) were calculated in three ways. Comparison with the biomass C estimated on air-dry soils by the SIR method suggests the most appropriate way to estimate the flush is: flush = (extractable-C from wetted, fumigated soil) - (extractable-C from air-dry, non-fumigated soil). Estimates of microbial C varied by up to S-fold depending on how the flush was calculated. The release of inorganic P (P,) by fumigation of air-dry soil was generally increased by rewetting. Releases were greatest from rewetted soil fumigated with CHCI, vapour, lower when using liquid CHCI, and usually lowest when air-dry soil was fumigated with CHCI, vapour. On two soils, gradually air-dried in the laboratory, the estimates of microbial C by the FE method were affected by rewetting (to 50% w/w water content) once the soils had dried below 20% w/w water content, and the rewetting effects were highly significant at c 10% w/w water content. To estimate microbial biomass C, N and P of air-dry or moisture-stressed soils when using the FE or FI methods we recommend the soils be rewetted immediately before fumigation.

INTRODUCTION

The importance of soil moisture in regulating microbial activity in soil is well known (Papendick and Campbell, 1981; Harris, 1981; Griffin, 1981). Seasonal fluctuations in soil moisture may be of relevance in the cycling of nutrients through the microorganisms and a considerable proportion of the increased inorganic N and P extracted after airdrying of soils appears to derive from the soil microbial biomass (Marumoto et al., 1982; Sparling et al., 1985; Sparling and Ross, 1988). There is therefore an interest in methods to estimate the microbial biomass of dry soils, or soils under moisture stress. The fumigation-incubation (FI) method, commonly used to estimate soil microbial C and N, was developed for use on moist soils at 50-55% of their water-holding capacity (Jenkinson and Powlson, 1976). The method has since been extended to include rewetted air-dried soils (Shen er al., 1987), but the FI method, when applied to air-dry New Zealand soils, has sometimes given anomalously low or high estimates of microbial C, which showed poor agreement with other methods of estimating microbial C and activity (Sparling et al., 1986). Many factors are known to affect the reliability of the FI method such as: soil pH, calculation of the 245

C-flush, reinoculation, soil moisture content and prior treatment (Jenkinson et al., 1979; Shen et al., 1987; Chapman, 1987; Ross, 1987; West et al.. 1986a). Some of these problems in estimating microbial C and N by the FI method are avoided by the use of the fumigation-extraction (FE) method (Vance el al., 1987; Tate et al., 1988; Sparling and West, 1988a). originally proposed by Brookes er al. (1985b) to estimate soil microbial N. The FE methods use chemical oxidation or digestion stages to estimate the extracted organic C or N which removes many of the uncertainties arising from the IO day microbial mineralisation stage required by the FI method. When applied to moist soils, estimates of microbial C by the FE method have shown good agreement with estimates using the FI method on English soils (Vance et al., 1987). reasonable agreement with New Zealand soils (Tate et al., 1988), and very good agreement with estimates on New Zealand soils using the substrate-induced respiration (SIR) method (Anderson and Domsch, 1978; West and Sparling, 1986; Sparling and West, 1988a). However, estimates of microbial C by the FE method on air-dry soils were anomalously low (West er al., 1988a, b) and showed poor agreement with estimates by the SIR method. These discrepancies warranted further investigation.

246

GRAHAM

P. SPARLING and ANDREW Table

SilC Chyt0-M

Waikanae Camarvon Pomare Horotiu Kaitokc Castlepoint Naike Te Whanga Ekctahuna Egmont Nireaha

I.

W.

WEST

Soil characteristics

TeXttire

%C

%N

PH

USDA soil taxonomy

Fine sandy loam Silt loam Loamy sand Silt loam Silt loam Silt loam Silt loam Clay loam Silt loam Silt loam Black loam Silt loam

1.3 3.7 3.7 4.6 6.0 6.3 6.9 7.0 7.2 8.3 8.8 9.1

0.10 0.40 0.45 0.36 0.68 0.40 0.54 0.55 ND 0.71 0.71 0.53

6.3 6.0 6.2 5.6 6.4 5.8 6.4 5.3 5.4 5.4 2 ;:6

Typic Uditluvcnt Typic Udifluvcnt Mollic Psammaquenf Typic Dystrochrcpt Typic Vitrandept Typic Dytrochrcpt Typic Haplaquoll Typic Haplohumult Udic Ustochrept Aquatic Hapludalf Entic Dystrandept Typic Dystrochrept

ND = not determined.

Where the FI and FE methods have been used to estimate the microbial C of air-dry soils, the soils were usually fumigated while dry and only rewetted for the subsequent incubation or extraction (Shen et al., 1987; Sparling et al., 1986). Moisture and high humidity are known to increase the effectiveness of biocidal treatments such as heat, ethylene-oxide, r-irradiation and sterilants such as ethanol (Sykes, 1969) and it seemed possible that the soil moisture content could affect the biocidal efficiency of CHCI,. We have used liquid CHCI,, rather than vapour, to estimate the microbial P content of soils (Sparling et al.. 1985). Liquid and vapour CHCI, have a similar effect on releasing microbial P from moist soils (Brookes et al., 1982) but in dry soils it was not clear whether organic liquid would have the same effect as rewetting with an aqueous liquid, or whether this would affect the estimates of microbial P. To resolve these various points relating to the estimation of microbial C, N and P of dry soils, a series of experiments was undertaken where soils were fumigated with CHCI,, with or without a rewetting treatment. Some air-dry soils were also tested for NaHCO,-extractable P with the additional treatment of lysis using liquid CHCI,. Finally, the microbial C of two soils was measured during gradual drying under laboratory conditions, to determine the moisture content at which the rewetting effects became significant. METHODS

Soils

Topsoil samples (O-75 mm) were collected between March and May 1987 from a variety of pasture sites in North Island, New Zealand. One hundred cores (25 mm dia) were collected over a IO x IO m area, the surface vegatation was removed and the cores were bulked and sieved c2 mm while field moist. Soils were stored at 25°C for 4-7 days before use. Air-dried samples of the soils were prepared by spreading 200-300 g subsamples on plastic sheet and arranging them in the airflow of a large domestic fan. The subsamples were stirred every l-2 h; the ambient temperature was l8-22’C. The soils were left overnight in the open bench and the drying procedure repeated the next day. All soils were air-dry after 2 days. The dried soils were sealed in glass jars and stored at 25’C for 2-3 days before analyses. Further samples of Castlepoint and Kaitoke soils were col-

lected in October 1987 for the gradual drying experiment. Some characteristics of the soils are shown in Table I. Experimental

Air-dry Waikanae, Carnarvon, Castlepoint and Egmont soils were selected as contrasting soils to compare the effect of rewetting with water on the estimation of microbial C by the FE method. The organic C extracted (triplicate samples) in 0.5 M K2S0, was measured with and without overnight fumigation with CHCI, vapour (Jenkinson and Powlson. 1976) of air-dry soils. and soils rewetted to 20 or 50% w/w water content. The microbial C was also estimated on all the treatments by the SIR method (quadruplicate samples). Seven air-dry soils (Table 4) were selected for further study of the effect of rewetting on the C and N flushes released by fumigation. The soils were tested using the FE method to estimate microbial C and N in 0.5 M K:SO., extracts (triplicate samples), and also by the FI method, estimating microbial C and N from the CO,-C flush and min-N flush during a IO day incubation (triplicate samples). For the FE method unfumigated soil was either rewetted at the same time as the fumigated samples (i.e. at the beginning of the overnight fumigation), or immediately before extraction with 0.5 M K2SOa. The same routine was used for the FI method (i.e. one set of control samples was moistened to 50% w/w water content for overnight rewetting before the IO day incubation). The treatments are summarised in Table 2. The microbial C of all the soils was also estimated using the SIR method (quadruple samples). The effect of rewetting on the estimation of microbial P in air-dry soils was checked with five soils (Table 6). The rewetting and fumigation treatments were the same as the FE method (Table 2) but a further treatment using liquid CHCI, was included. For this treatment 2ml of liquid CHCI, were added to 5 g air-dry soil (triplicate samples) in a 250 ml extraction bottle. The soils were left in the fume cupboard overnight with the bottle caps off to allow the CHCI> to evaporate, and extracted the following day with 0.5 M NaHCOJ (Sparling et al., 1985). Field-moist Kaitoke and Castlepoint soils were air-dried for about 8 h daily in the laboratory for 5 days. During drying the soils were repeatedly handmixed to maintain uniform drying as far as possible. Subsamples were taken daily for analyses and the

Soil microbial

biomass measurement and soil moisture

247

Table 2. Soil rewettinn and fumigation treatments Rewet’

FumigatiotP

I.1 I.? 1.3 1.4

No Yes No Yes

No No Yes YCS

Extraction in 0.5 M K:SO,

2.1 2.2 2.3 2.4

No Yes No Yes

No No Yes Yes

Rewctted to 509’0 (w/w) water content and incubated at 25C for IO days

Method

(I)

Fumigation-extraction

(2) Fumigation-incubation

Subsequent treatment

“Rewet’ refers to an overnight rewetting with water, 0.5 ml g-’ air-dry soil. tiFumigation’ refers to overnight fumigation with CHCI, vapour.

soils stored overnight in sealed containers at 25’C. Microbial C was measured on subsamples using the FE and FI methods (triplicate and duplicate samples, respectively), with rewetting treatments as shown in Table 2. The volume of rewetting water was adjusted so that all rewetted treatments were restored to 50% w/w water content. Microbial C was also estimated by the SIR method. Analyses

Fumigations were performed as described by Jenkinson and Powlson (1976) using ethanol-free CHCI,, but sample weights were IOg for the FE method and 5 g for the FI method. Organic C in 0.5 M K:SO, extracts was determined as described by Tate et al. (1988) based on the dichromate oxidation method, with the unused dichromate estimated by back-titration with ferrous ammonium sulphate. Total N in 0.5 M K,SO, extracts was measured on subsamples of the acid-dichromate oxidation mixture, using the indophenol calorimetric method to estimate ammonium-N (Sparling and West, 1988b). CO: evolved during the IO day incubation of the FI method was estimated by gas chromatography (Sparling et al., 1986). The min N-flush from the incubated soils was estimated after IO days by the summation of the nitrate-N and ammonium-N con-

tents in 2 M KCI extracts, measured using standard methods (Blakemore et al., 1987). Inorganic P (Pi) extracted by 0.5 M NaHCO, at pH 8.5 was measured on neutralised decolourised extracts using the molybdenum blue method (Sparling er al., 1985). The SIR method to estimate microbial C was used (West and Sparling. 1986); the rate of CO, respiration (~1 COr g-’ h-i) was converted to microbial C using the relationship: pg microbial C g-i soil = 40.92 (~1 CO2 g-’ h-‘) + 12.9 (West et al., 1986b). Total soil C, N and pH were estimated by standard methods (Blakemore et al.. 1987). Moisture contents were determined after drying soils at 105°C. Moisture contents are calculated on a gravimetric basis and results are expressed on the basis of oven-dry soil.

RESULTS

Fumigation

of rwetred

or air-dry

soil

The amounts of organic C extracted from the rewetted control soil (non-fumigated) generally did not differ significantly from the unwetted soils except for Castlepoint soil where rewetting to 20 or 50% w/w water content caused a decrease in extracted organic C (Table 3). Fumigation much increased extractable organic C,

Table 3. Effect of rewetting air-dry soils to 20 or 50% (w/w) water content and fumigation with CHCI, on the ornanic C extracted bv 0.5 M K,SO.. and microbial C estimated bv the SIR method Extractable organic C (PgCg-‘soil) _

Microbial C estimated by the SIR method _ (PgC g-’ soil)

SoiI

Rewctting treatment

Control

Fumigated

Flush”

Control

Fumigated

CdrlXW”On

Airdry Rewettcd to 20% Rewetted to 50%

I36a’ I28a l44a

327b 447c 417d

l9lc 3l9f 273g

653a 524b 53Sb

8lc 93c 75c

Castlepoint

Airdry Rcwctted to 20% Rewetted to 50%

I72a 113b IlOb

4lOc 537d 535d

238~ 424f 425f

785a 784a 922b

125~ 185~ I39c

h’dikawdc

Airdry Rewettcd to 20% Rewettcd to SO%

I59a I6Oa I67a

298b 39sc 43Sd

I39c 235f 2681

669d 663a 574b

93c lO4.Z 127~

Egmont

Airdry Rewetted to 20% Rcwctted to H)%

317a 318a 309a

S85b 697~ 773d

268e 379f 4641

784a 772a 748a

II6b 7lb 79b

‘For each soil. for either extractable organic C. or microbial C. figures followed by the same letter do not differ significantly at P < 0.05. ‘Flushes calculated by the difference between fumigated soil and the equivalent control soil.

GRAHAM P. SPARLINGand ANDREW Table and

4. Microbial fumigation

C of a range with

CHCI,

on

of air-dry

W.

WEST

soils, and the effects of rewetting

the C or N

extracted

mincralised

over

by 0.5 Y K,SO,:

wth

water

CO:-C

CO,-C N extracted

produced

in 0.5 Y K,SO, (fig g-’

Pomare

Horotui

Castlepoint

Naike

Te Whanga

Eketahuna

Nircaha

‘For

Airdry

day

soil)

and

O-IO

incubarior?

(fig g-’

soil)

N

C

N

9la’

40a

261~1

37a

IlOb

34a

298ab

44b

6lc

37a

255,

Control

Airdry

Fumigated

Rewct

Control

Rewet

Fumigated

I64d

305b

BSb

43b 46b

Airdry

Control

483a

ll3a

933a

62a

Airdry

Fumigated

546b

ll7a

999a

117b

Rewet

Control

303c

63b

907a

Rewet

Fumigated

649d

l7Oc

l072a

Airdry

Control

547a

I2Oa

1045a

IOla

Airdry

Fumigated

693b

I39b

I658b

202b

Rewet

Control

786c

106.Z

7411

95a

Rewet

Fumigated

766d

2OSd

l443b

l8Oc

Airdry

Control

204a

39a

l075a

9%

Airdry

Fumigated

406b

59b

1634b

221b

Rewet

Control

Rewet

Fumigated

Airdry Airdry Rewrt

7oc ll8b

8%

25c

953a

83a

527d

I36d

l985b

346c

Contol

5JOa

Il5a

Fumigated

69Jb

I3Sb

Control

357c

Illa

Rewet

Fumigated

78Jd

19Oc

Airdry

Control

2x4,

60a

Airdry

Fumiguted

417b

72ab

Rewet

Conlrol

19Rc

ReWCl

Fumigatrd

SIYd

IO3b

Airdry

Control

59Xa

79a

I216a

97a

Airdry

Fumigated

67Xb

85a

ll33ab

I56b

RcWcl

Control

4Ohc

l06a

ReWel

Fumigatrd

77Xd

ISIb

920a I542b

Il2a

1414b

227~

639a

89a

l072b

l65b

6l4a

78ab

Illa 250b

761s

949b

958b ll80a

93a l73b

97a l64b

Airdry

Control

?OJa

XXa

l?X?~c

I36a

Airdry

Fumigated

406b

96a

I474ac

23Ub

Rewet

Control

I26ab

IO??b

I54a

Rewrt

Fumigated

15lb

I399c

227b

each soil, and within

each column.

N

min-N

during

C

Treatments

Greytown

and

IO days

C and

Soil

(0.5 ml g-l)

respired

8%~ 527d

figures followed

by the same letter do not differ significantly

at P <0.05. hAll

soils were

rewetted

to 50%

w/w

water

content.

and soils fumigated when rewetted to 20 or 50% W/W water content had significantly greater amounts of extractable organic C (Table 3). The effect of adding different volumes of water was not consistent. For Castlepoint soil there was no difference whether rewetted to 20 or 50% w/w water content. For Camarvon soil more organic C was extracted when wetted to 20 than 50%, whereas for Waikanae and E_mont soils more organic C was extracted when the ~011swere rewetted to 50% w/w water content. The organic C flush, calculated as the difference between the fumigated soil less the conrrol soil at the same moisture content, was greatest when the soils were rewetted. Generally. there was no significant difference in the organic C flush of the soils rewetted to 20 or 50% w/w water content. The SIR method, applied to the air-dry rewetted and fumigated soils, showed that fumigation greatly decreased the SIR response with no significant effect of rewetting (Table 3). The control soils were also affected by the rewetting, but the effects were not consistent, with both increases and decreases in the SIR reponse of the rewetted soils being recorded. In terms of the C flush the volume of water used for rewetting did not appear critical. Consequently

and

reinoculated.

for

the

IO day incubation.

for the remaining experiments, soils were rewetted to 50% w/w water content. Effect of resetting on the estimation of microbial C and N by the FE and FI methods Extractable organic C and N. Rewetting of air-dry soil caused significant changes in the amounts of organic C extracted by the FE method (Table 4). In all soils the trend was the same: overnight rewetting decreased the control values, but greatly increased the organic C extracted from the fumigated soil. The effect of rewetting on extractable N was more variable. Again, the highest amounts were extracted from the rewetted fumigated soil, but the effect of rewetting on the control soils was not consistent, with rewetting causing increases in some soils (Te Whanga, Eketahuna, Nireaha) but decreases in others. In several cases the differences were not significant (Table 4). C and I%’ mineralised during incubation. The amounts of C and N mineralised by the FI method were rather variable and in many instances the rewetted treatments did not differ significantly from the air-dry soils. (Note that “air-dry” refers to the period of overnight fumigation only, all samples were rewetted for the subsequent incubation.) However,

Soil microbial biomass measurement and soil moisture

249

Table 5. The C and N released after fumigation of air-dry or rewetted soils with the flush (difference between fumigated and non-fumigated samples) estimated in three ways’ for the direct extraction and incubation methods

Soil

Method of calculation

Flushes of microbial C and N (pgg-’ soil) Fumigal~on~~traction Fumigation-incubation -C

N

C

N

Greyrown (135)”

Dry-dry Rewet-rewet Rewec-dry

19a’ 103b 73c

CO 5la 48a

37a 5Oa 44a

7ab 3a 9b

Pomare (451)

Dry-dry Rewet-rewet Rewet-dry

63a 346b 166c

4a 107b 57c

I6Sa (39a

5% 48b 56a

Horotiu (651)

Dry-dry Rewet-rewet Rewet-dry

146a 380b 219c

194 99b 85~

613a 702a 398a

lbla 85b 79b

Castlepoint (938)

Dry-dry Rewet-rewet Rewet-dry

202a 442b 323c

20a IIIb 96c

.%%d

1032a 9fOa

I26a 263b 251b

Naike (572)

Dry-dry Rewel-rewet Rewetdry

15da 427b 244c

2Oa 79b 75b

622a 653a 494a

I39a i15b 116b

Te Whangd (602)

Dry-dry Rewet-rewet Rewewiry

1331 32lb 23%

I2a 24a 4%

43311 335a 3t@d

76a 80a 84a

Ekerahuna (463)

Dry-dry Rewet-rewel Rrwet-dry

Soil 372b t8Oc

6a 45ab 72b


67a 671

Dry-dry Rewet-rewet Rewet-dry

202a 442b 323c

cd

192a

??d

37?d

63b

117a

NircJha (728)

66a

%d

102a 73a 9la

‘See text for details. “Figures in parentheses show the microbial C of the air-dry soils estimated by the SIR method. ‘For each soil. and within cdch column. finures followed by the same later do not differ signifi~ntly at P < 0.05.

there was a trend for slightly less COz-C to be evolved from the rewetted control treatments during the IO day incubation. The effect of rewetting on the COL-C evolved after fumigation was variable with no significant trend (Table 4). The effect of rewetting on the min-N was also variable and generally there were no significant differences between the air-dry and rewetted controls, nor between the air-dry and rewetted-fumigated soils. Effct of rertetting on the C and N jiushes The flushes of extractable C and N or the C and N mineralised during incubation, were calculated from the amounts extracted from (or mineralised by) the fumigated soil less that extracted from (or mineralised by) the non-fumigated control soil. The ffushes were calculated in three ways. depending upon the rewetting treatments: (i) Fumigated air-dry soil minus air-dry control soil (dry-dry). (ii) Fumigated rewetted soil minus rewetted control soil (rewet-rewet). (iii) Fumigated rewetted soil minus air-dry control soil (rewet-dry). The extractable organic C flushes were significantly affected by the rewetting treatments and method of calculation. In all cases the flush was greatest when calculated using the rewet-rewet method [method (ii)], and least when using the dry-dry method [method (i)] (TabIe 5). The extractable N flush was also affected by the

method of calculation,

in that the dry-dry

method

always gave the lowest N Rush, but the rewet-rewet

method did not always result in the greatest N flush. The CO:-C and min-N flushes were highly variable and, although there were considerable differences between the treatments, the effects were not consistent and usually were not significant (Table 5). Efect of rewetting on the estimation of microbial P Rewetting had no consistent effect on the amounts of inorganic P (P,) extracted from the control soils but increased the P, extracted from fumigated soils (Table 6). Lysis of organisms in air-dry soil with liquid CHCI, usually released more Pi than did vapour fumigation of air-dry soil, but less Pi than did vapour fumigation of rewetted soil. Generally, the greatest Pi flush was obtained by vapour fumigation of rewetted soils corrected for the rewetted control soil, but with Kaitoke soil Iysis of organisms in air-dry soil with liquid CHCI, gave a greater flush. (The flushes for the liquid CHCI, treatment were calculated using the air-dry soil as the control i.e. liquid CHCI, treatment minus air-dry control soil) Estimation of microbial C in gradually -dried soils Rewetting of Kaitoke and Castlepoint soils to 50% w/w water content affected the extractable organic C content and CO?-C respired, even when the soils were not air-dry (Fig. 1). Extractable C contents and COz-C respired from the control soils tended to increase as the soils dried;

250

GRAHAM

P. SPARLISG

and

ANDREW W. WEST

Table 6. Effecfect of rewetting to 50% w/w water content. fumigation with CHCI, vapour. or lysiswith liquid CHCI, on the amounts of inorganic phosphorus(P,) extracted in 0.5 M NaHCO, from air-dry soils P, extracted in 0.5 I( NaHCO, (rgPg_’ soil) Soil

Treatment

Pomare

Airdry Rewetted Liquid CHCI,

Horotiu

Control

Fumigated

Flushb

5.6b NA

5.9a 8.6b SSa

I .4a 3.0b I .Oa

Airdry Rewctted Liquid CHCI,

18.9a 17.2b NA

l9.6a 21.9b ?I.Zb

0.7a 4.7b 2.3~

Kaitoke

Airdry Rewetted Liquid CHCI,

45.7a 52.lb NA

45.9a 54.2b Sl.Ob

O.Za 2.la J.3b

Castlepoint

Airdry Rewetted Liquid CHCI,

49.9a 49.3a NA

53.Oa 71.Sb 57.sc

3.la 22.2b 7.6c

Egmont

Airdry Rewetted Liquid CHCI,

16.la lS.Ob NA

I7.3a 21.4b 19.3c

I.Za 6.4b 3.2~

4.5a’

NA = Not applicable. ‘For each soil, and for each column. figures followed by the same letter do not differ significantly at P < 0.05. bFlushescalculated by the difference between fumigated soil and the equivalent control soil. For liquid CHCI,. the flush was calculated by subtracting the air-dry control soil

the increases were significant at moisture contents below 20% w/w, and increased sharply as the soils dried below 10% w/w water content. Overnight reco,.c nwr4.d

O-10 ~YS

2800

zoo0 .

2400

tsoa /

I

ii 22w

Fig. I. The amount of organic C (pg g-l) extracted by 0.5 M K:SO, from Kaitoke and Castlepoint soils during gradual drying in the laboratory, and the amounts released after rewetting and/or fumigation with CHCI,; and the amount of CO& respired during subsequent incubation at 50% w’w water content for IO days. 0-O = Non-fumigated air-dry soil; A-A = non-fumigated rewetted soil; 0-0 = fumigated air-dry soils; A-A = fumigated rewetted soil. LSD = least significant difference at P c 0.05.

wetting of the soils before extraction or incubation decreased the extractable C content and COr-C respired on incubation. Fumigation greatly increased extractable-C and CO,-C respired (Fig. I). If the fumigated soils were not rewetted the extractable C content tended to decline as the soils dried, but when the soils were rewetted to 50% w/w water content, extractable C increased significantly, particularly when the soils had dried to ~20% w/w water content. The pattern was slightly different for the C02-C respired after fumigation. The main increases in CO,-C respired from the rewetted soils occurred when the soils had dried below 20% w/w water content, but the effect of overnight rewetting was generally to decrease the CO:-C respired (Fig. 1). High variability meant that many of the changes in CO*-C respiration were not significant for the Castlepoint soil. Flushes of extractable-C and C02-C were calculated using the three methods previously described. The extractable-C was converted to microbial C using an &-factor of 0.35 which is an average value based on Sparling and West (1988a, b). The C02-C flush was converted to microbial C using a mineralisation coefficient, or k-factor, of 0.45 (Jenkinson et al., 1979). These estimates of microbial C were compared with those estimated by the SIR method (Fig. 2). Estimates of the biomass differed greatly depending on which technique and method of calculation was used. For field-moist Kaitoke soil there was initially little difference between the various estimates of microbial C (Fig. 2). However, as the soil dried, the estimates diverged. This was particularly so for the FE method, where at moisture contents below 20% w/w the rewetting treatment had a large effect. Generally, higher estimates of microbial C by the FE and FI methods were obtained by the rewet-rewet method of calculation and estimates by the dry-dry method were much lower. The microbial C content of

Soil

microbial biomass measurement and soil

Fig. 2. The microbial biomass C (pgg-‘) of Kaitoke and Castlepoint soils calculated by the SIR method, the FE and Fl methods with the C-flush calculated by three different ways (see text for details). 0-O = SIR method; A-A = dry-dry method; V-V = rewet-rewet method; R-m = rewet-dry method. LSD = least significant difference at P < 0.05.

Kaitoke soil generally declined on drying as assessed by the SIR and FE methods but was erratic when estimated by the FI method. The estimates of microbial C in Castlepoint soil were very variable. As estimated by the SIR method, there were fluctuations and an overail decline in microbial C on drying, but the FE method showed divergent trends depending on how the C-flush was calculated, particularly when the soil had dried below 20% w/w water content. The microbial C estimated by the FI method was so variable that no consistent trend could be discerned (Fig. 2).

DISCUSSIOS

Effect of retsetting on the release of C, N and P from fumigated air -dry soils Rewetting of air-dry soils before fumigation markedly affected the amounts of C and N extracted by 0.5 M KZS04 and to a lesser extent, the amounts of CO:-C and N mineralised during incubation. Depending on how the flushes of C or N were calculated. the estimates of microbial C or N could vary greatly. The main effect of rewetting was to greatly increase the 0.5 M KzSO, extractable-C and N of the fumigated soils. When the non-fumigated (control) soils were rewetted at the same time as the fumigated soils (i.e. an overnight rewetting) the extractable-C content was slightly decreased. This Iatter effect can be explained by the overnight mineralisation of organic C

moisture

251

and loss as CO? from therewetted control soil. This resulted in a lower extractable-C content when analysed the next day and a lower amount of CO?-C respired on incubation, although this latter effect was much less marked. We are unsure why greater amounts of C and N were extracted from soils fumigated when rewetted rather than fumigated while dry, but the effect does not seem to relate to the biocidal efficiency of CHCI, fumigation. For exampie, the SIR response after fumigation was suppressed by a similar amount for both rewetted and air-dried soils, and in many instances rewetting had no significant effect on the C and N flushes estimated by the FI method. The large effect of rewetting on the microbial biomass estimated by the FE method rather than the FI method suggested that the presence of water during fumigation may influence the e.~tractabif~t~ of soil microbial C, N and P. On fumigation. microbia1 C, N and P are thought to be released primarily as organic compounds, yet the main component of the P Rush in 0.5 M NaHCO, extracts is Pi because soil phosphatases rapidly transform the organic P to P, (Brookes et al., 1982; Sparling et al., 1986). Soil enzymes continue to function in the presence of CHCI,, but their activity is iikety to be much reduced if the soil is dry, because of the restricted mobility and poor contact between enzyme and substrate. A mobility effect could explain why the treatment of air-dry soil with liquid CHCI, rather than vapour CHCI, resulted in larger Pi flushes. Both liquid and vapour CHCI, effectively Iyse microbial cells (Brookes er al., 1982) but in air-dry soils, the transformation of organic P to P, may have been more rapid in the presence of liquid CHCI, because of better contact between enzymes and substrates. Soit enzymes are less important for the mineralisation. of microbial C and N by the FE method because released organic C and N are oxidised or digested chemically. Nonetheless, considerable involvement of soil enzyme processes in the extractability of microbial N in 0.5 M K,SO, was inferred by Brookes et al. (1985a). After fumigation of moist soil for 5 days the extractable-N was greatly increased compared to fumigation for 1 day. The biocidal effect of CHCI, is completed by 1 day, hence the greater amount of N extracted was due to increased solubility rather than increased biocidal action. The involvement of soil enzymes in this process was shown by a decrease in extractable-N of fumigated soils if the fumigation temperature was 6O’C or above. Brookes et al. (1985b) suggested that, at the higher soil enzymes were deactivated, tem~ratures, resulting in lower extractability of the released microbial N. We provisionally suggest that, in air-dry soils, reduced enzyme activity can limit the extractability of both microbial C and N released by fumigation. Consequently, air-dry soils should be rewetted before fumigation for greater extractability of these compounds in 0.5 M KZS04. For the limited number of soils we have tested. the degree of wetting did not appear crucial, provided adequate moisture was restored. We used a standard amount of 0.5mlg-’ air-dry soil, but soils with differing characteristics may require different amounts of water and rewetting

‘52

GRAHAM

P.

SPARLIXG and ANDREW

to D-55% water-holding capacity may be preferable (Jenkinson and Powlson. 1976; Shen et al., 1987). Calculation of microbial C, N and P in air-dry soils

Various methods to calculate the flush of C, N or P following fumigation were used, which gave widely varying estimates of the microbial biomass. Our main interest was to estimate the microbial biomass at the time of sampling rather than after incubation. The question arises as to the most appropriate method of calculating the flush and allowing for the rewetting effect. When estimating microbial C, N or P content in dry soils by the FE method we favour the calculation: microbial biomass C, N or P = [(extracts from rewetted. fumigated soil) - (extracts from airdry control soil)]/E-factor. The basis for this preference is that: (i) greater amounts of C. N and P were extracted from rewetted, fumigated soils suggesting a better recovery of microbial C, N or P; (ii) the use of air-dry. non-fumigated soil avoids the overnight loss of C and possible immobilisation of N that occurs if rewetted (control) soil is used; (iii) the microbial C content, calculated by the above method, and using an &-factor of 0.35 derived for moist soils. showed the closest agreement with the microbial C estimated by the SIR method when applied to air-dry soils. For this study the &-factor to convert the organic C flush of air-dry soil (rewet-dry method) to microbial C (estimated by the SIR method) was 0.41 + 0.06, which is similar to values calculated for moist soils (Sparling and West, 1988a, b). The equivalent factors, estimated using the air-dry soils or the rewetted soils for both fumigated and control treatments, were 0.21 + 0.05 and 0.66 + 0.12, respectively. This strongly infers that the C flushes estimated using only the dry soils [method (i)] were too low, while those estimated using only the rewetted soils [method (ii)] were too high. Microbial C content estimated by the FI method was more variable and often differed markedly from that estimated by the SIR method. We do not consider the FI method to be reliable on air-dry soils, but if using this method we suggest that a rewetting treatment should be included for the fumigated soils, and that the CO:-C flush be calculated as suggested for the FE technique. Shen et al. (1987) used the FI method to estimate the microbial C content of air-dry English soils. They did not investigate the effects of overnight rewetting on the CO,-C flush, but stressed the importance of correcting the CO:-C respired from fumigated soil with that respired by an appropriate control treatment. We endorse their view that it is always essential to correct for the CO:-C respired by the rewetted control because of the very much higher levels of decomposable organic C after airdrying which contribute to the respiration flush after rewetting. This reasoning also applies to the calculation of the organic C flush by the FE method. Microbial dearh on resetting of soils

The rewetting of air-dry soils causes a rapid increase in water potential and considerable osmotic stress for the soil organisms (Harris, 1981). Kieft er

W. WEST

al. (1987) rewetted moisture-stressed

soils with KCI solutions of different osmotic potential to investigate whether the osmotic shock of rewetting dry soils was killing an otherwise viable biomass. Using a modified FI technique, and including a correction factor for the inhibitory effect of KC1 on decomposition and respiration, they concluded that during a drying and rewetting cycle, a greater proportion of organisms could be killed during the rapid rewetting than during the slower drying process. This effect could be important in the determination of microbial biomass if some of the non-fumigated (control) soil was killed during a rewetting procedure. Some of the effects noted in our experiments could have been influenced by the osmotic potential of the rewetting solutions. For the rewetted treatments, water was added (0.5 ml g-‘) to the air-dry soils. For the “air-dry” treatments the water was added only a few seconds before extraction with the 0.5 M KzSOJ (to make the extraction conditions equivalent) so for the FE method the air-dry soils were effectively rewetted with 0.5 M K,SO, which will cause less osmotic stress than wetting with water and, according to Kieft ef al. (1987). should cause less microbial death. The present experiments were not designed to check the effect of the extracting solutions on microbial viability but in a further series of experiments (Sparling and West, 1989) we remoistened air-dry soils with various glucose-NaCI solutions and measured the SIR response over the next 2 h. The SIR response was maximum when the soils were rewetted with solutions of high osmotic potential (low salt concentrations) and was markedly decreased by low osmotic potentials. which is contrary to expectation if rewetting caused osmotic stress and microbial death. Clearly further work is required to clarify these points. but part of the difference between the conclusions of Kieft ef al. (1987) and ours, may be in the use of two different techniques (FI method and SIR, respectively) to estimate viable microbial biomass. Reverting effects on gradually-drying soils

Previous studies on the changes in microbial indices during the gradual drying of soils, and our present results, suggest the modified SIR method (West and Sparling, 1986) can give reasonable estimate of microbial C over a wide range of moisture contents (Sparling et nl.. 1986; West et al., 1988a. b). On the two soils tested here the FE method underestimated microbial C content when the water content approached 20% w/w and the rewetting effect became highly significant at water contents below 10% (w/w). To obtain reliable estimates of microbial C and N by the FE method, for soils with similar characteristics to those tested we recommend rewetting to 50% (w/w) before fumigation. We do not consider the FI method to be reliable on drier soils, but if used, recommend that the soils also be rewetted for fumigation. West et al. (1988a, b) measured the microbial C and N of gradually-dried soil by the FE method, but no rewetting treatment was used. Our present findings explain why at low moisture contents (c 10% w/w) these estimates of microbial C and N by the FE method were anomalously low. Estimates by Sparling et al. (1985, 1987) on the microbial con-

Soil microbial biomass measurement and soil moisture

tribution to the extractable P content of soils after air-drying are not affected by the present findings. Microbial P was estimated by treatment of moist soil with liquid CHCI~, and the microbial P of dry soils estimated from the change in microbial C (measured by the SIR method). Liquid or vapout treatment with CHCI, appear equally effective in releasing microbial P from moist soils. Consequently. the conclusions reached regarding the large contributions from microbial P to the extractable P contents of air-dry soils remain valid. AcknawIedgcmnf

-We

thank C. W. Feltham for technical

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