Soil microbial biomass C, N and ninhydrin-N in aerobic and anaerobic soils measured by the fumigation-extraction method

Sod Bd. Btochrm. Vol. 23, No. 8. pp. 737-741. 1991 Rntcd I” Great Bntain. All rights reserved

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003843717 91 53 00 +o.oo 1%I991 Pergamon Press plc

SOIL MICROBIAL BIOMASS C, N AND NINHYDRIN-N IN AEROBIC AND ANAEROBIC SOILS MEASURED BY THE FUMIGATION-EXTRACTION METHOD K. INLTI~~SHI.* P. C. BRoonEst Soil Science Department,

AFRC

and

D. S. JESKISWS

institute of Arable Crops Research, Rothamstead Harpenden. Herts AL5 2JQ. U.K. (rlccepprrd IJ Frhruqr

Experimental

Station.

1991)

Summary-The fumigation-extraction method was tested to see if it could be used to measure soil microbial biomass in waterlogged soil. Three Japanese paddy soils were incubated. aerobically or anaerobically (waterlogged). before being fumigated with CHCI, and extracted by 0.5 M KrSO,. CHCI,-fumigation caused large increases in KrSO,-extractable C. N and ninhydrin-N in the aerobic soils, as observed previously. Fumigation caused smaller. but comparable. increases in waterlogged soils. The biomass, measured as K,SO,-extractable C released by CHCI,. declined only slowly. if at all. during an 80 day aerobic incubation. However. under waterlogged conditions KrSO,-extractable C released by CHCI, declined significantly; in 40 days by I I. 22 and 26% in the three soils. Aeration of waterlogged soils for up to I h before extraction did not change the amount of K,SO,-extractable C released by CHCI,. This suggests that 0: does not need IO be rigorously excluded during the sampling or analysis of waterlogged soils. Microbial biomass C (B,) in aerobic soils can provisionally be calculated from the relationship &. = 2.64 EC. where &. is the organic C extracted by 0.5 M K,SO, from fumigated soil, less than extracted from the non-fumigated control.

INTWOIWCTION

Of the various rcccntly-dcvclopcd methods for measuring soil microbial biomass in aerobic soils (e.g. fumigation-incubation, fumigation-extraction, substrate induced respiration. ATP contcnt (see Jcnkinson. 1988 for a rcvicw). the most promising for USC in waterlogged soils appears to bc fumigntionextraction. ATP content is unsuitable, for reasons discussed by lnubushi PI al. (1989) and methods bJsed on aerobic incubation (fumigation-incubation, substrate induced respiration) clearly present dishcultics when applied to anaerobic soils. Inubushi er 01. (1984) did, however, develop a fumigationincubation technique for use in waterlogged soils. In their method. fumigated and unfumigatcd soils were incubated anaerobically for 4 weeks and the increase in NH,-N brought about by fumigation taken as a measure of biomass N. The long incubation period is. however. a disadvantage. The fumigation-extraction technique has already been used to measure biomass in soils containing actively-decomposing substrates. in which the fumigation-incubation method gives unrcliablc results (Ocio and Brookcs, 1990). Tests, not rcportcd hcrc. showed that the ATP content of a watcrloggcd soil, fumigated with CHCI, in the way dcscribcd in this paper. fell to a virtually undctcctablc concentration within 24 h. so that CHCI, is a highly clfcctivc biocidc. cvcn in water-saturated soil. Fumigation-

-

‘Present address: Department of Agricultural Chemistry. Faculty of Bioresources. Mic University. Tsu. Mic 514, Japan. tAuthor for correspondence.

extraction has a special advantage in that it is potentially much more rapid than incubation-based tcchniqucs. WC report exploratory studies. dcsigncd to see if fumigation-extraction has potential as a method for measuring soil microbial biomass in anaerobic soils. ZIATF.WIAIS

ANI) M~T~TIIOIIS

Soils

Soils l-3 were from an unfertilized. a chemically fertilized and an organically manured plot, respcctivcly, of a long-term ficld expcrimcnt on the production of paddy rice at Konosu. Japan. Full details of thcsc soils arc given by lnubushi et ul. (1989).

Again. full details arc given by lnubushi et ul. (1989). Briefly. the bulk soils wcrc first sieved ( c 2 mm). then given a conditioning incubation over soda-lime (2 days at 25 C) at 40% of full watcrholding capacity (WHC). Portions of moist soil (containing 2Sg soil on an oven-dry basis) wcrc then incubated aerobically at 50% WHC for up to 80 days. Soils I and 2 wcrc also incubated anaerobically for 40 days and soil 3 for RO days, all at 25 C. The anaerobic incubations wcrc done by placing portions of moist soil (containing log soil on an oven-dry basis) in 60 ml glass ccntrifugc tubes, which were then filled with O!-free water, tightly scnlcd with rubber bungs and incubated at 25 C in the dark. In some casts, anaerobically incubated soils were aerated for up to 60min (after removal of the supernatant water). by bubbling water-saturated air through them.

737

738

K. Table

I.

K,SO,-~atracrable

C. N and

ISBW~I

nmhydrin-reac!rve

et al.

N in sods I-3.

K:SO,txtractable

orgamc C Unfunngawd

Fumrgarcd

after

20.

days aerobtc

or anaerobic K.SO. _

K,SO,sxwaccable total .Ec

Cnfumigated

N

Fumigated

rncubation

- -cxrnctablc

ninhvdrm-N &

Unfumrgatcd

iumrgarcd

.&

Sod No.

tncubation

I 2

3

Pooled

g-1

soil)

(pg

N e-’

sod)

(pg

ninhydnn

N g

’ soil)

Aerobic

49

276

227

51

68

17

0.5

8.3

7.g

Anaerobic

47

227

ISO

I2

31

19

8.2

18.2

10.0

79

103

24

0.8

Il.2

10.3

18

46

28

9.Y

22.9

13.0

Aerobic

44

393

349

Anaerobic

52

337

285

Aerobic Anaerobic

41

703

662

105

135

30

II

15.9

I48

68

526

4%

49

59

40

26 0

43.6

17.6

standard

error ‘20

@g c

days

of mean

6.4

incubatron

after

the conditroning

9.6

II.5

I.5

2.5

2.9

0.22

I .44

Biomass measurements by fumigation-extraction Chloroform fumigation.

The aerobic soils to be fumigated were placed in a desiccator. with a small vial of soda-lime and a beaker container 25mf alcohol-free CHCI,. The desiccator was evacuated for 2 min after the CHCI, had begun to boil vigorously. then sealed. still under vacuum, and incubated for 24 h at 25C in the dark. CHCI, fumigation of the anaerobically incubated soils was done by first siphoning off as much as possible of the su~rnatant wafer, then adding fO~r1 CHCf,g-’ soil and mixing thoroughly on a vortex mixer. The soils were then placed in a desiccator and further fumigated for 24 h, exactly as described above. After fumipafion. the soils wcrc placed in a clean empty desiccator which was evacuated rcpeatcdly until CHCI, could no longer be detected by smell. E=,SO,-exfructicm. In all cases a ratio of I part soil to 4 parts 0.5 M KrSO, (w/v) was used and the mixture shaken for 30 min. For each soil, the unfumigafcd SCI was extracted at the same time that fumigation of the other set commenced, after removal of the supernatant water, as above. Organic C in the fumigated and non-fumigated K,SO, soil extracts was measured by dichromate digestion (Vance er of., 1987). total N by Kjefdahf digestion (Brookes et al., 1985) after treatment with Cr*+ to reduce NO,-N (Prudcn el al.. 1985) and ninhydrin-reactive N colorimetrically (Amato and Ladd. 1988). Fe and Mn in the extracts were determined by ICP spectrometry after diluting I : 10 with 5% v/v HCI. Ammonium-N and NO,-N were measured in the K,SO, extracts by Technicon analysis. If necessary NO,-N was also measured in the siphoned-off supernatant. E,, the amount of C made extractable to 0.5.~ K,SO, by CHCI,, was defined as: EC = [(C extracted by KzSO, from fumigated soil) minus (C extracted from non-fumigated soil)]; Es and ENInsimilarly. All results are the mean of three replicate determinations and are expressed on an oven-dry soil weight basis (105 C, 24 h).

the three soils, compared to the amounts in the corresponding aerobic soils. In contrast, EN and E,,, both increased. suggesting that the biomasses in the waterlogged soils actually increased their N concentrations. This is consistent with a change in the composition of the biomass (Takai et al., 1956): certain bacteria, with narrow C:N ratios, tending to prosper under anaerobic conditions. at the expense of the fungi. which usually have wider C: N ratios than bacteria. There were also some changes in total amounts of C. N and ninhydrin N extracted from tho nonfumigated soils due to watcriogging. Anaerobically incubated soil contained a little more cxtractablc organic C than the corresponding acrobicafly incubated soil. but markedly less total N. by it factor of 4 in soil 2. This WIS bccausc the substantial quantities of nitrate initially present were dcnitri~~d during anaerobic incubation (Table 2). Soils incubated acrobicafly for 20 days contained very little NH,-N (co.4 pg N g-i soil: Tabfc 2) or ninhydrin-reactive N (< I.1 pg N g-’ soil: Table I). Soils incubated anaerobically contained much more of both, and the two forms of N were present in similar quantities. Thus soif I incu~ted anaerobically for 20 days contained 7.4 pg NH,-N g-* soil (Table 2) and 8.2 118 ninhydrin-N (Table 1); the values for soil 2 were 8.8 and 9.9 pg; for soil 3, 25.9 and 26kg, respectively. Since NH,-N is more than 95% as effective as leucine-N in the ninhydrin reaction under our experimental conditions (Joergensen and Brookes, 1990). nearly ati the ninhydr~n-reactjve material extracted from our waterlogged (but unfumigatcd) soils must have been present as NH,-N. In the aerobic incubations the ratio EC: E, ranged from 14 to 22 and E,: EN,,, from 29 to 45 (Table 3). Anaerobic incubation caused these ratios to decrease by about a factor of two in soil 3, and rather less in Table

2. K,SO,-exlraclablc after

20 dayr

inorganic aerobic

or

N

in sods

AND

DISCUSSION

Eflecrs of f~~~igu~ion on ~~SO~-e.~~ruet~b~e C, N und ninh.wirin-N in aerobic and tvaferlogge’d sds Fumigation increased K:SO,-extractable C. N and ninhydrin-N. whether the soils had been incubated aerobically or anaerobically (Table 1). Anaerobic incubation caused EC to decline by about 20-30% in

Soil No.

I

Incubation

incubarion

NO,-N .~___.

NH&-N

2

~UKNK

43. I

2.1

63.6

Aerobic Anaerobic

29.6

SH.7

before

20 days NO,-N

‘roil) 0.1

3.1

Acrobrc Anaerobic

3

After

Aerobic Anaerobic

1-3.

anaerobic

lnirial NH,-N RESULTS

1.46

incubauon.

49.4

7.4

6.3

0.2

17.0

H.X

6.7

0.4

103.2

25.9

0.9

and

Biomass measurements Table 3. Rams of 4:

&., .

4: EYlnand

in waterlogged soils

739

Eu : E.,,.in soils

sod

:Cu.. Eru: Et... 2.2

No.

Incubation

I

Aerobic Anaerobic

13.5 9.5

29. I 18.0

1.9

2

Aerobic AnaerobIc

14.5 10.3

33.9 22.0

2.3 2.1

3

Acroblc Anaerobic

21.7 II.5

44.7 26.0

2.1 2.3

G:E.

E,

soils I and 2. It should be noted that the E,: EN ratios of the aerobically incubated soils in Table 2 are much wider (14-X) than those collated from the literature by Jenkinson (1988) from similar measurements on I04 aerobic soils. Our three soils had all carried paddy rice for many years and it may be that the C: N ratio of the biomass in such soils, when aerobically incubated. is considerably greater than in welldrained soils. In contrast, the ratio EN: EN,” was much more constant (range 1.9-2.3) between the soils. irrespective of whether they had been incubated aerobically or anaerobically (Table 3). This ratio was very similar to that reported by Joergensen and Brookes (1990) for I2 aerobically incubated soils, when their biomass N values are recalculated as EN. It must be pointed out that the total N values (i.c. organic plus inorganic N) in Table 1 are subject to large analytical errors, since the soils contained so much inorganic N (Table 2). All three soils initially contained two to three times as much inorganic N as was subscqucntly rcleascd by fumigation as organic and inorganic N.

Thcrc was little consistent change in E, in any of the soils during aerobic incubation (Fig. I). This is cntircly consistent with the findings of lnubushi er ul. (1989) that ATP in these soils remained at fairly constant concentrations throughout the go-day aerobic incubation. There are ample results in the literature suggesting that ATP is a reliable measure of the amount of biomass in moist aerobic soils (see Jcnkinson. 1988). so that both ways of measuring biomass concur in suggesting that there was little change during the aerobic incubation. In centrist. Ec declined when the soils were incubated anaerobically. In soil I it fell by 22%. in soil 2 by I I % and in soil 3 by 26%. all over 40 days. In soil 3 it fell by about 40% during an 80 day incubation (Fig. I). However. this fall was much less than the fall in ATP measured in the same experimcnt (Inubushi er al., 1989). Thus, ATP in soils I and 2 fell by about 50% during 40 days anaerobic incubation and by about 60% in soil 3 during 80 days anaerobic incubation. Taken at their face value, ATP measurements thus indicated a large decrease in biomass during anaerobic incubation, E, measuremcnts indicated rclativcly small declines. The reason for this discrepancy is that. while the total pool of adcninc nucleotides (AT = ATP + ADP + AMP) declined relatively slowly during anaerobiosis (like E,). ATP as a percentage of A, declined very much faster (Inubushi et al., 1989). Thus ATP is not a reliable indicator of the amount of biomass present under anerobic conditions.

-I-a x-x-x

SOll 3 =

\

OLI 0

’

10

X

X

’

I

I

20

40

80

Incubation

time

( day,)

Fig. I. Changes in EC during aciobic ( x ) or anaerobic (0) incubation of soils l-3. Standard errors of means shown.

Eflects of aeration soils

on C extracted from

waterlogged

The aim of this experiment was to set how sensitive the fumigation-extraction measurements were to brief periods of aeration prior to fumigation and extraction. Once an anaerobic field soil is sampled, it is difficult to prevent 0, penetrating the sample and producing partial aerobiosis during the period before analysis, however brief. Table 4 shows that there were few, if any, consistent differences in K,SO,extractable C in unfumigated or fumigated samples of soils 2 or 3 after 20 days anaerobic incubation, whether or not the soils had been aerated before extraction. Biomass C measurements on waterlogged soils by the fumigation-extraction method should therefore not be sensitive to brief exposure to 0,. unlike ATP measurements.

Table 4. Organic C in K,SO,-extracts or soils firs1 incubawd irnacrobically for 20 days a~ 25 C and then aerated bcrore extraction

Soil No. 2

3

Aeration lime (min) 0

K,SO,-cxwactabk C Fumigalcd Unfumigatcd hR

c

R

’ Soil)

IO 60

39 44 35

333 343 342

294 299 307

0 IO 60

60 53 48

520 532 546

460 479 498

Pooled standard error of mean

8.9

12.7

IS.5

740

K.

fNU8CsHI et uf.

Table 5. Soluble Fe and Mn in 0.5 H K,SO, cxtrac~s of samples of soil 3 that had been Incubated aerobIcally or anacroblcally. and then extracted. either directly or after fumigatmn vith CHCI, Incubation condttzonr

Davs of rncubacton

Aeration trmc (mini

Fe Treatment

(pg g

Mn

’ soil)

Aerobic

xl

n a* “a

Unfumigared Fumigated


Anaerobic

20

0 0

Unfumigatcd Fumigated

530 163

137 139

AnaerobIc

IO IO

Unfumigated Fumigated

376 2

133 I32

Anaerobic

60 60

Unfumiga~ed Fur&&d

7 3

127 134

Anaerobic

0 0

Unfumipated Fumigated

I450 toso

128 I33

0.3 2.6

*Not applicable.

Deierminnkm soils

of C in K,SO,

extracts qf waterfogged

Extracts of anaerobically-incubated soils contain much more Fe and Mn than extracts of aerobically incubated soils (Table 5). However. even a brief exposure to 0: before extraction rcduccd the amount of Fc extracted. Extractable Fe also fell during CHCI, fumigation. but Icss markedly. Detcrminatjon of organic C in soil extracts by the dichromatc oxidation method. as in our work, is of course affcctcd by the presence of any iron present as Fe?+. Howcvcr. the quantities present wcrc insutiicicnt to make more than a small ditfcrcncc to organic C as measured by dichromato oxidation. Consider the worst case in Table 5. and assume that at1 the extracted iron was present as Fe”. The extract of soil 3 that had been incubated anacrobially for 40 days contained 1450 jig Fe g ’ soil. equivalent in reducing capacity to 78/1g C. After fumigation the extract contained IO50 erg Fe g’ ’ soil, equivalent to 56/1g C. Thus EC would have been ovcrcstimatcd by 22 j;g C, or by 4.6%. This probtcm of changes in iron contents during fumigation only arises if orpanic C is determined by dichromate oxidation. If organic C is measured in extracts by modern automatic oxidation procedures based on measurement of the CO: formed (Wu PI ul., 1990). changes in ferrous iron should not interfere. Cuiculrttiort of soil microbid bicimuss frum ftmtigctriott -e.rlrocrivn meusuremettts in ftttuerobic soils

Them can be little doubt that the C, N and ninhydrin-reactive N rctcascd by the fumigation of anaerobic soils comes from the lysis of killed microorganisms, just as with aerobic soils. Certainty. the very similar E,: E,,, ratios of the N released by CHCI, in the anaerobi~lly-incubated and aerobically-incubated soils (Table 3) suggest that the sdmc soil N fraction is being extracted in both casts. Rather mom indirect evidcncc that fumigation-extraction can be used as a measure of soil microbial biomass under anaerobic conditions comes from the observations of lnubushi LPItrl. (1989) made at the same time as the measurements in Fig t were done Thus. when anaerobically incubated soils 2 and 3 were aerated for I h after 20 days anaerobic incubation, ATP increased to 82 and 84% of initial ATP vatucs in the aerobically incubated soils. suggesting a

decline in total biomass of about U-20% during the 20 day anaerobic incubation (tnubushi ef al., 1989. This is very close to the measured decline in EC during anaerobic incubation of these soils during the same period (Fig. I). suggesting that both ATP, following a period of aeration. and fumigationextraction arc measuring the same fraction (presumably microbial biomass) in thcsc anaerobic&y incubated soils. In aerobic soils, biomass C (Be) is rektted to Ec by the relationship: & = 2.64 EC. if organic C is dctcrmincd by dichromatc digestion (Vance ct ul.. 1987) or from: B c’ = 2.22 &. if organic C is determined by automated u.v.-persutphatc oxidation (Wu et d.. 1990). Whether or not the same relationships hold for anaerobic soils is an opt question. Until proved othcrwisc. the best that can be done is to assume that they do, and use the same equations for calculating fIc under anaerobic and aerobic conditions. With N the situation is cvcn less satisfactory, as there is evidence (Table 3) that the C: N ratio of the biomass is less after anaerobic than aerobic incubation. The relationship B, = 2.22 EN (Jenkinson, 1988) should not be used on anaerobic soils until m-evaluated for these conditions. Although much more needs to be done to evaluate fumigation-extraction fully as a tcchniquc for measuring biomass in waterlogged soils, the exploratory studies presented here do suggest that it could be useful under such conditions. Ackno~/e~~en~cn/.r-K. lnubushi thanks the British Council for a scholarship and the Faculty of Agriculture. The University of Tokyo. for study leave.

REFERENCFS

Amato M. and Ladd J. N. (1988) Assay for microbial biomass based on ninhydrin-rcactivc nitrogen in extracts of fumigated soils. Soil Biology d Biochemistry 20, 107-l 14. Brookes P. C., Landman A.. Pruden G. and Jenkinson D. S. (1985) Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method for measuring microbial biomass nitrogen in soil. Soil Biukogy & Biorhcnri.srr~ 17, 837-842. lnubushi K., Wada H. and Takai Y. (1984) Determination of microbial biomass nitrogen in submerged soil. Suil Scicwcr md Plunl Nurritiott 30, 455-459.

Biomass measurements in waterlogged soils lnubushi K.. Brooka P. C. and Jenkinson D. S. (1989) Adenosine 5’-triphosphatc and adcnylatc energy charge in waterlogged soil. Soil Bio1ogy & Biochemistry 21. 733-739. Jenkinson D. S. (1988) The determination of microbial biomass carbon and nitrogen in soil. In A&unces in Nitrogen C.vcling in Agrinrlrurai Ecosysrems (J. R. Wilson, Ed.), pp. 368-386. CAB. Wallingford. Jocrgenxn R. G. and Brookcs P. C. (1990) Ninhydrinreactive nitrogen measurements of microbial biomass in 0.5 M Kr SO, soil extracts. Soil Biology & Biochemisrry 22, 1023-1028. Ocio J. A. and Brookes P. C. (1990) An evaluation of methods for measuring the microbial biomass in soils following recent additions of wheat straw and the charac-

741

terisation of the biomass that develops. Soil Biology & Biochemistry

22, 685-694.

Prudcn G.. Powlson D. S. and Jenkinson D. S. (1985) Reduction of nitrate prior to Kjcldahl digestion. Journal of the Science of Food and Agriculture

36, 7 l-73.

Takai Y.. Koyama T. and Kamura T. (1956) Microbial metabolism in reduction processesof paddy soils. Part I. Soil and Plant Food 2, 63-66.

Vance E. D., Brookcs P. C. and Jenkinson D. S. (1987) An extraction method for measuring soil microbial biomass. Soil Biology & Biochemistry 19. 703-707. Wu J.. Joergenscn R. G.. Pommerening B.. Chaussod R. and Brookes P. C. (1990) Measurement of soil microbial biomass C by fumigation-extraction-an automated procedure. Soil Biology & Biochemisrr.v 22. 1167-l 170.