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Method to measure microbial phosphate in soils
0038-0717/82/040377-09603.00/O Copyright 0 1982 Pergamon Press Ltd
Soil Biol. Biochem. Vol. 14, pp. 377 to 385, 1982 Printed in Great Britain. All rights reserved
METHOD
TO MEASURE MICROBIAL IN SOILS M. J.
HEDLEY*
and J. W. B.
PHOSPHATE
STEWART
Department of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO (Accepred
1 Nouember
1981)
Summary-The amount of phosphate in soil microbial biomass was estimated by adding CHCl, to soil to lyse microbial cells and measuring the proportion of microbial-P released to 0.5 M NaHCOs extracts (pH 8.5). Calculations of total microbial-P were based on the difference between P removed by NaHCOs extraction of ChCI, treated and untreated samples. This method was improved by (1) removing resinextractable P from the soil before lysing microbial cells with CHCIJ, (2) measuring total P rather than inorganic-P in NaHCOs extracts, and (3) prolonging NaHCOs extraction from 30 min to 16 h. The efficiency of extraction of soil microbial-P was determined by measuring the P recovered from two species each of bacteria and fungi grown first in solutions having different P concentrations, then added to soil. The average proportion of microbial-P recovered from a neutral calcareous soil was 37% (KP = 0.37). The KP factor was confirmed by adding 33P-labelled bacteria to a similar soil and measuring the added 33P recovered (38%) by the CHCI,-NaHCO, method.
INTRODUCTION
and organic soil phosphorus observed across environmental gradients (Westin and Buntley, 1967) resulting from prolonged cultivation practices (Haas et al., 1961; Chater and Mattingly, 1980) or with season (Dormaar, 1972; Halm er al., 1972) show that P mineralization and immobilization occurred but provide no information on the rates or mechanisms of transfer. With more accurate methods to evaluate microbial biomass (Babiuk and Paul, 1970; Jenkinson and Powlson, 1976; Paul and Johnson, 1977; van Veen and Paul, 1979), understanding P dynamics in the soil-plant system is limited mainly by lack of analytical methods that identify forms and amounts of various labile inorganic- and organic-P fractions, including P immobilized in microorganisms (Cole et al., 1977; Cosgrove, 1977). Biomass C is measured by making the biomass susceptible to mineralization by fumigation with CHCl,; the subsequent flush of CO2 during incubation gives a measure of soil biomass (Jenkinson, 1976; Anderson and Domsch, 1978a,b). Chloroform fumigation also has been used to measure biomass ATP (Paul and Johnson, 1977) and biomass N (Paul and Voroney, 1980). Cole et al. (1978) used CHCIS fumigation in combination with NaHC03 extraction (Olsen et al., 1954) to remove a portion of the biomass-P. This method was used by Chauhan et al. (1979, 1981) to estimate microbial-P in soils. We report a modification of this technique that more accurately estimates microbial-P. Large net changes in inorganic
Anderson
and
Domsch
(1978a,b)
evaluated
a
CHC13 technique to measure biomass-C by adding known quantities of fungi and bacteria to soil and calculating
the proportion
(Kc) evolved
as COa-C.
* Present address: Soil Science Laboratory, Department of Agricultural Science, Oxford University, Oxford OX1 3PF, England. 377
When this technique is adapted to microbial-P evaluation, consideration must be given to the heterogeneity of soil P compounds, to the diversity of P compounds contained in a microbial cell, and to the ease of extracting these compounds from soil and from soils with different P adsorption capacities. Compounds containing P in bacteria and fungi are made up of 30-50% RNA, 15-20% acid-soluble inorganic- and organic-P compounds consisting of sugar and nucleotide esters and various phosphorylated coenzymes and polyphosphates, < 10% phospholipids, 5510% DNA, and small amounts of inositol phosphates (Alexander, 1977; Caldwell and Black, 1958). The proportion of P in inorganic or organic forms depends on the P concentration in the soil solution, ‘A P in the cell, and age of the cell. In addition, because bacterial and fungal cells differ in cell composition and P content, the proportion of microbial-P measured in a CHCl,-NaHCO, extraction may differ. Among other points that have to be considered during method evaluation are that the CHCl, may affect the extractability of native soil-P, that NaHCO, extraction without CHCI, may release P from microbial cells, and that added microbial biomass could immobilize soil-P during the extraction process (up to the time of CHCl, addition). Finally, an adopted procedure should extract sufficient microbial-P to allow accurate measurement of extra P from lysed microbial cells in extracts which may also contain considerable quantities of soil-P. Anderson and Domsch (1980) using C:P ratios ranging from 7:l to 17:1, estimated that the average quantity of microbial-P in the top 12.5 cm of forest soils is 70 kg ha-‘. Chauhan et al. (1981) found that microbial biomass in incubated soils ranged from 100 to 500 pg biomass-C g- I soil and C:P ratios ranged from 2O:l to 6O:l. Microbial P values in soil, therefore, could range from 1.5 to 50 pg P g- ’ soil. To test extraction procedures and develop methods of measuring microbial-P in soils, we grew two species each of fungi and bacteria in nutrient solutions having
378
M. J. HEDLEY and J. W. B. STEWART
different P concentrations. Quantities of these organisms were added to a soil representative of the Canadian prairie, and the added microbial-P recovered in NaHCO, extracts was measured. MATERIALS
AND METHODS
Soil
The soil (pH 7.4, CEC w 30 m-equiv/lOO g, organic C 2.2%, clay 33%) used in most of our experiments was from the Ap horizon of a well-drained Black Chernozemic soil (Udic Haploboroll) of the Indian Head Association in southern Saskatchewan. A second soil (pH 7.0, CEC _ 30 m-equiv/lOO g, organic C 3.2x, clay 45%) having a larger indigenous microbial biomass and taken from the Ah horizon of a permanent pasture soil of the same association was used where stated. Soils were ground and sieved (< 500 pm), then incubated at 60% field moisture capacity at 24” f 2°C for 21 days before use. Subsamples were oven-dried to determine dry weight. Organisms
TWO species of fungi (Trichoderma harzianum and Fusarium oxysporum) and bacteria (Pseudomonas cepacia and Arthrobacter globtformus), were selected because they are common in soil (Clark and Paul, 1970). Media
Fungi were cultured in potato dextrose broth (PDB, Difco) or the fungal culture medium (FCM) of Burns and Beever (1977). Nutrient broth (NB, Difco) and a standard reference soil solution (RSSC, Herzberg et al., 1978) were used to culture the bacteria. Nutrient agar and potato dextrose agar (Difco) slants were used to maintain stock cultures of the bacteria and fungi, respectively. Phosphate-free media (FCM and RSSC) were prepared for growing bacteria and fungi. Phosphate (KH,P04) solution was sterilized separately, and different volumes were added to FCM and RSSC media in culture flasks to produce a range of P concentrations. Carrier-free radioactive phosphate (33P, -2 mCi in 50 ml) was added to this stock solution to provide uniformly labelled P. Approximate concentrations of the stock solution were added aseptically to each culture flask to provide a 33P-labelled microbial population. Microbial growth and harvesting
Erlenmeyer flasks (250 ml) containing PDB or NB (100 ml) were inoculated from stock slants of fungi or bacteria, respectively. The flasks were incubated for 4 days (fungi) and 2 days (bacteria) at 22°C on an orbital shaker (90 rev min- i). Fungal cells were harvested from the growth media by vacuum filtration onto a cellulose acetate membrane filter (Millipore, 0.45 pm). The harvested cells were washed thoroughly with NaCl solution (8.5 gl-I). Bacterial cells were harvested from the growth media by centrifugation (10,000 rev min- 1 for 20 min. IEC, model B-20 centrifuge) and washed by repeated (4 times) resuspension in NaCl solution and centrifugation. Finally, the washed cells were resuspended in NaCl solution or deionized water. In studies of bacteria and fungi grown in different P
concentrations, the respective inocula were harvested aseptically from 24-h-old cultures of bacteria grown in NB and of fungi grown in FCM. Bacteria and fungi were washed thoroughly with sterile deionized water before being inoculated into the culture flasks containing growth media of different P concentrations. The flasks were incubated (bacteria, 2 days; fungi, 4 days) and harvested as described. Experimental procedure
Suspensions of bacterial cells were pipetted into 40-ml screwcapped polypropylene centrifuge tubes and into identical tubes containing 1 g or 0.5 g of moist soil. Addition aliquots were taken to determine the dry weight of the cells. Moist, whole fungal cells were weighed into polypropylene centrifuge tubes having similar treatments as above; the wet and dry weights of subsamples of each population were determined for calculating the dry weight in each centrifuge tube. Suspensions of 33P-labelled bacteria were added to soil as described above to give an initial specific activity of 0.1 $i g- 1 soil. Adapted chloroform procedure
Chloroform (1 ml) was added to moist soil, microorganisms, or soils amended with microorganisms contained in centrifuge tubes. The centrifuge caps were replaced, and the tubes were shaken by vortex mixing for three lo-set intervals over a period of 30min at 24°C. The caps were removed, and the CHCl, was allowed to evaporate overnight in a fume hood at 24°C. Samples in CHCl,-treated and non-CHCl,-treated tubes were then extracted overnight (16 h) with 0.5 M NaHC03 (30 ml, pH 8.5) at 24°C. After centrifugation (0°C 10,000 rev min- ‘, IEC, Model B-20 centrifuge), the extract was filtered through a membrane filter (Millipore, 0.45 pm). Residue retained by the filter was washed back into the centrifuge tube with deionized water. Inorganic-P (Pi) and total P (PT) concentrations were measured in the NaHCO, extracts and residues, Chloroform treatment of sterilized samples
To investigate whether CHCl, treatment increases the amount of nonmicrobial soil-P extracted in NaHCO, extracts, we compared the quantity of P extracted after CHCl, treatment of sterilized and nonsterilized soil. Two samples of the Indian Head Ah soil were incubated at 60% field moisture capacity (FMC) and 25°C for 10 days. Then one sample was sterilized by fumigation with ethylene oxide, and both were incubated at 60% FMC for 21 days at 25°C in sealed containers. Subsamples of soil from each treatment were extracted with resin for 16 h. Three unsterilized and three sterilized subsamples were treated with CHCl,; three unsterilized subsamples remained untreated. The samples were then placed in a fume hood at 24°C overnight, and all CHC13 evaporated. The samples were then extracted with 30ml 0.5 M NaHCO, for 16 h at 24°C. Extractable-P methods
Resin-extractable P was measured by the method of Amer et al. (1955) as adapted by Sibbesen (1977). To 0.5 g soil, 0.4g anion-exchange resin (Dowex
319
Measurement of biomass-P 1 x 8-50 > 30 mesh) in a Nylon bag was added, plus 30 ml of water, and shaken for 16 h at 24°C. The P adsorbed by the resin was displaced by 25 ml of 0.5 MHCl and analysed. Sodium bicarbonate-P (0.5 MNaHC03, pH 8.5) was measured by the method of Olsen et at. (1954) except that the soil:solution ratio was 1: 30 or 1: 60 and two shaking times, 20 min and 16 h, were used where stated. Phosphate determination
Phosphate (PI) was determined with 1 ml of each extract. A correction was made to account for absorption caused by colored organic matter in the extract. An aliquot of similar size was digested by acidified ammonium ~rsulphate oxidation (Environmental Protection Agency, 1971). The method of Murphy and Riley (1962) was used to measure the orthophosphate concentration in the neutralized extract (Pi) and in the neutralized digest of the extract (P,). Organic-P (P,) was calculated by subtracting Pi and P,. Radioactive-33P assay
Radioactivity (33P) and quantitative (31P) analysis were determined on the same sample. Addition of 0.5-1.0 ml of the 0.5 M HCl extract (resin-P) or the NaHCO, extracts to 14 ml of a complete phase combining cocktail mixture (PCS-II, Amersham) gave a clear, homogeneous fluid that was counted in a Beckman LS 9000 liquid scintillation counter. All counts were corrected to disintegrations min- ‘. RESULTS
Evaluation of lysing agents
Chloroform and two other microbial cell-lysing agents, ethanol and isopropanol, combined with 0.5 M NaHCO,, were compared for efficiency of recovery of P from a pure culture of fungi. All lysing agents gave the same proportional recovery (89%) of fungal-P from pure culture. However, the recovery of fungal-P added to the soil was considerably higher with CHCI, than with ethanol or isopropanol (73% vs 49 and 56%). Uhrasonification of Fusarium sp. mycelia before addition of CHC13 did not increase recovery of microbial-P in subsequent 0.5 M NaHCO, extraction, This suggests that CHCl, effects the complete lysis of fungal cells. Because CHCI, readily evaporates from treated soil, it was the most suitable microbial-lysing agent for this study. Evaporation of the CHCl, also prevents extraction of soil phospholipids, which could produce an overestimate of microbial-P.
E$ect of chloroform on extractability of labile soil-P
To investigate whether CHCl3 treatment may increase extraction of non-microbial soil-P forms, we extracted sterilized and nonsterilized soil samples by NaHC03 with and without CHCI, treatment. Resinextractable Pi increased by 12.1 pgg-’ soil after sterilization with ethylene oxide and subsequent 21-day incubation (Table 1). After the resin extraction, the amount of 0.5 M NaHCO, P, extracted from nonsterilized soil and from CHQ-treated sterile soil was virtually the same, but in the CHCl,-treated nonsterilized soil the 0.5 M NaHC03 Pr increased by 13.0pgg-’ soil. These results illustrate three points. First, ethylene oxide and CHClj treatments do not significantly increase the extractability of 0.5 M NaHC03 soil P,. Second, 0.5 M NaHCOJ alone does not extract significant amounts of indigenous microbial-P. Third, resin extraction after sterilization and incubation is almost as effective in removing P released from the soil microbial population as extrao tion with 0.5 M NaHCO, after CHCl, treatment (12.1 vs 13.0 gg P g- ’ soil). E&et of sorption by soii on recovery of added P in NaHCO, extracts
Preliminary experimentation indicated that age of the fungal culture markedly affects distribution of Pi and P, in the 0.5~ NaHCO, extract. A t-day-old fungal culture had considerably more extractable-p, but the percentage of extractable-P, than-P,, remained similar to those obtained from Cday-old cultures. This suggests that NaHCO~-extractable Pr provides a more reliable measurement from which microbial-P can be estimated. No distinct pattern of change was seen (Table 2) in the amounts of microbial-P, and -P, extracted by 0.5 MN~HCO~ as the cell P content of fungal and bacterial cells increased. However, the sum of these two P values (Pr) increased as the cell-P content inwas creased. Moreover, no useful information obtained by considering the percentage of Pi and P, in NaHC03 soil extracts, compared with that’ obtained in the extract of microbial cells without soil. This is because the rate of hydrolysis of P, to Pi changes once the ceil wall is broken and varies with soil components, cell com~sition, and treatment. Within experimental error, the percentage of P, recovered from both fungi was similar; percentage of P, recovered from both bacteria also was similar. When bacterial and fungal ceils of similar P content were tested, CHC13-NaHCO, extracted fungal-P more effi-
Table 1. Effect of CHC13 treatment on amount of 0.5~ NaHCOJextractable P in sterilized and nonsterilized pasture soils (Ah) preextracted with an anion-exchange resin (Values are the means of three samples; mean deviation < 10%) P fraction
Nonsterilized soil (fig P g- L soil)
Sterilized soil (pg P g- ’ soil)
Resin Pi
40.5
40.5 52.6 CHC13 treatment
0.5 M NaHC03 P, Z fractions
29.7 70.2
42.7 83.2
30.5 83.1
380
M. J. HEIILEY and J. W. B. STEWARI
Table 2. Proportions of total P extracted from bacterial and fungal cells of various P content in the absence of soil (Values are means of three samples: mean deviation < IO”,,)
P content of cells (% dry wt)
0.22 0.28 0.42 0.30 0.90 I.4 0.17 0.40 0.70 0.27 0.55 0.86
“(, of added microbial-P CHCl,Total NaHCO, extracted Residual P,
P,
Pr
Pr
Fusarium oxysporum 13 4 II 79 2 81 74 II 85 Trichoderrna harzianum 60 4 64 71 5 16 69 I5 84 Arthrobacter globiformus 38 8 46 7 43 50 17 41 58 Pseudomonas cepaecu 32 23 55 40 28 68 29 17 46
23 19 15
NaHCO, extraction (Fig. 1). In this figure the amount of microbial-P, added to the soil refers to the amount of P, released to 0.5 M NaHCO, from CHCl,-lysed cells in the absence of soil. Over the range of microbial-P additions (9942 pgg-’ soil), the recovery of added Pi averaged 42 + 5%, whereas the recovery of microbial-P, ranged from 28 to 584; and did not follow a pattern. These results indicate that no simple relationship exists between the recovery of microbial P, from this soil and Pi extracted by NaHCO,. Effect of resin microbial-P
35 24 16 54 50 42 45 32 54
Pi = % of added microbial-P recovered as inorganic P. P, = % of added microbial-P recovered as organic P. P, = Pi + P,.
ciently than bacterial-P. With bacteria, approximately 50% of the microbial-P was nonextractable, compared with an average of only 22% of fungal-P. The presence of soil reduced recovery of P from both CHCl,-treated fungi and bacteria by almost 50% (Tables 3 and 4). Presumably, some P released from microbial cells was adsorbed by soil components, as some of this was recovered in subsequent extraction in 0.1 M NaOH (Hedley et al., 1982). Addition of clay decreases the extractability of P from mixed microbial tissue (Goring and Bartholomew, 1949). When microbial cells are lysed with CHC13, some of the released P will be sorbed to the soil surface. Recovery of this P in a NaHCO, extraction may be related to the Pi sorption isotherm of a soil if the P, released from the microbial cell is sorbed in a similar manner. To investigate this effect, we compared recovery of added Pi with recovery of microbial-P, by
extraction
on subsequent
Trichoderma harzianum 1.200/;; P (dry wt) 0.47% P (dry wt)
0.5 M NaHCOs
Total
P
A*
Pi P,
B
C
of
Extraction of a soil sample with resin did not decrease the recovery of P from subsequently lysed microbial cells (Table 1). This result was examined further by comparing the results (Table 5) from untreated and resin-pretreated samples subsequently treated with CHCls, then extracted for 30min with 0.5 M NaHCO,. Resin pretreatment has two main advantages (Table 5). First, it reduces the amount of soil Pi extracted by the subsequent NaHC03 extract, as almost all labile Pi is removed by the resin, and P released by CHCl, fumigation then makes up a significant portion of the NaHCO, extract. For example, 0.5 M NAHCO, extracted 17.1 pg P (Table 5) from the chloroformed soil sample, only 20% of which was measurable indigenous microbial-P. In the equivalent resin pretreated sample, measurable microbial P constituted 58% of the NaHCO, P, extracted. Second, the method can be calibrated for use in a particular soil. Changes in resin P provide an estimate of the uptake of labile soil Pi by the added microbial biomass during extraction (Table 5). Because added microbial biomass can take up labile soil Pi during extraction until CHCls is added, values obtained for the apparent recovery of microbial-P from nonchloroform soil were negative (Table 5). As most of the available soil-P is resin extractable, decreases in this value equal microbial-P uptake. Addition of bacterial to the soil (Table 5) reduced resin-extractable P by approximately 12.5 pg P g- 1 soil. Addition of fungi also reduced resin-extractable P but by a lesser amount, about 2.5 pg Pg-’ soil. In the non-resintreated soil, microbial uptake during extraction can not be measured.
Table 3. Effect of CHCI, pretreatment and addition to soil on percentage of total P extracted from fungal cells (Values are means of three samples; mean deviation < 10%)
Extractant
recovery
B o/0 of added
C microbial
Fusarium oxq’sporum 0.62% P (dry wt) A
B
C
P
33 15
49 31
44 I2
63 27
30 20
17 14
48 30
I8 26
48
80
56
90
50
31
78
44
* A = fungi alone. B = fungi alone treated with CHCIs before NaHCOs extraction. C = fungi added to soil and treated with CHCI, before NaHCO,
extraction.
381
Measurement of biomass-P
Table 4. Effect of CHCI:, pretreatment and addition to soil on the percentage of total P extracted from bacterial cells (Values are means of triplicate samples; mean deviation < 10%) Arthrobactergiob~rm~s 1.8% P (dry wt) Extractant
0.5 M NaHC03
Pi P,
Total P
Pseudo~~s 0.8% P (dry wt)
A’
B
C
21 9 30
29 33
20 IS
12 8
62
35
20
cepaeca 2.7% P (dry wt)
C
A
B
c
30 30
23 17
40
54 14
54 7
60
40
40
68
61
B A y0 of added microbial-P
*A = bacteria alone. B = bacteria alone treated with CHCls before NaHCOs extraction. C = bacteria added to soil and treated with CHCls before NaHCO, extraction.
The ~ssibility exists that the indigenous soil microbial population absorbs labile soil-P during extraction. However, microbial-P of incubated soils did not change with time, nor did that of fresh samples from the field. Therefore, this effect will be noticed only when microbial biomass grown in vitro is added to soil or when field soil contains large amounts of undecom~sed plants or plant residues. In resin-treated samples, the 30-min 0.5 MNaHCO, extraction removed 5.3-8.6°~ of the added microbial-P before treatment with CHC13. Thus, the percentage of microbial P measured was that recovered by the CHCl,-NaHCOJ extraction reduced by this amount. Efict of increasing shaking time tion
ofNaHCOs
extrac-
The percentage of soil-P and added microbial-P, especially fungal P, recovered was lower in the CHCI,-treated 30-min NaHCOS extraction than in the CHCl,-treated 16-h NaHCO, extraction (Table
6). This agrees with publish~ data (Bowman et al., 1978}, which indicate that a 16-h 0.5 MN~HCO, extraction removes approximately 20% more labile soil Pi than a 30-min NaHCO, extraction. Similarly, a larger amount of the P released from lysed microbial cells should be recovered by a 16-h extraction. The 16-h 0.5 M NaHCOj extraction recovered 20% more microbial-P from Chili-treated samples than the 30-mm 0.5 M NaHC03 extraction. However, the amount of microbial-P extracted by 0.5 MNaHC03 alone also increased by 8-17x (Table 6). Thus, the 16-h extraction increases measurable recovery of added microbial-P by 3454% over the 30-min extraction (Table 6). The range of measurable microbial-P (P,} recovery of all organisms tested was narrowed to 36.7 f 1.4%. If the indigenous soil microbial population is extracted in a similar manner and consists of 25% bacteria and 75% fungi (Clark and Paul, 1970), then this method measures 37% of microbial-P in the original soil. Using this mean value and data from the Indian Head Ap soil (Table 6), we estimated that this soil contains 18.2 fig P g-i soil of microbial-P. Test of the sequential extraction method with radioactioe phosphate (33P)
Sequential extraction of the Indian Head Ah soil to which 33P-labelled bacteria had been added is an alternative procedure for testing this technique (Table 7). In this case, the amount of added bacterial-P was negligible (0.35 pg “P g- ’ soil) but it had a high specific activity. Adding radioactive bacteria to the soil caused the soil to have an activity of 2.24 x 10’ dis mitt- i g- 1 soil. The increase in percentage recovery of microbial-P between chloroformNaHC03 and NaHCO, extraction was 38.2% (K, = 0.381, which compares favorably with data obtained from adding larger amounts of unlabelled P in fungal and bacterial biomass (Kr = 0.37) (Table 6). Microbial-P method
oA~unt
5
Kl
i5
20
of added P recovered
25
30
35
) pg.g-l soil
Fig. 1. Comparison of the amount of labile P added and P, extracted by a 30-min 0.5 M NaHC03 extraction, The amount of microbial P, added to the soil refers to the amount of P, released to 0.5 M NaHCOo from CHCls-lysed soils in the absence of soil. Symbols: o = Pi, A = Trichodermo sp., Cl = Fusarium sp., + = Pseudomonas sp., 0 = Arthrobocter sp.
We therefore recommend the microbial-P method outlined in Fig. 2 on the basis of our studies. In this method the most biologically available Pi (Amer et al., 1955; Sibbesen, 1977; Bowman et al., 1978) was first removed onto an anion-exchange resin. Labile Pi and P, adsorbed on the soil surface, plus a small amount of microbial-P (Bowman and Cole, 19781,was removed by NaHCO, extraction When a second sample, B, is run concurrently and treated with
14.2
14.2
+
+ _ +
_
+
Pseudomonas
Soil
+
_ +
-
Fusarium + soil
Arthrobacter + soil Pseudomonas + soil
1.3
1.7 1.8 1.2
11.7 il.1
12.0
12.2
ND
ND
ND
17.1
15.8 15.7 17.0
50.8 47.5
39.7
42.9
0.0
0.0
23.4 23.4 0.0 0.0
17.3 17.9
53.4 50.2 41.9 39.7
Microbial P added
12.9
12.5 12.4 13.0
2.5 3.1
2.2
2.0
(b) Resin treated
ND ND
ND ND
30.0
28.3 28.1 30.0
53.3 50.6
41.9
44.9
23.4 23.4
17.3 17.9
53.4 50.2 41.9 39.7
Total microbial P (added + uptake)
(a) Non-resin treated ND ND ND ND
pg P g- ’ soil
Microbial* P uptake
21.4
5.9t 18.4 5.7
8.7 26.8
7.4
26.9?
4.1t 9.8
7.3 20.8t 13.6 17.1
11.3t 22.3
25.4 37.9 27.lt 35.3
NaHCOJ** extractable PT
22.1
33.6
6.0 30.6 5.3
8.6 33.5
1.3
40.8
- 26.9 15.8
- 12.8 29.1
32.2 45.8
41.4
%
Apparent recovery?? of microbial-P
NaHCO,P,(soii + microbe - soil) x loo ” -----~?otal microbial-P
P.
33.3
24.6
24.9
33.5
42.1
41.9
13.6
19.3
Microbial P measured by CHCl, NaHCO,
* Microbial P uptake from time of addition of microbial biomass to soil to CHCI, treatment can be estimated in resin treated soils by the decrease in resin-extractable ** 30-min extraction.
+
-
+
-
Trichoderma + soil
+ soil Soil
+ soil
ND
Arthrobacter
+
-
Fusarium
+ soil
+
+ soil
ND
_
Resinextractable P
Trichoderma
CHC13 treatment
Table 5. Effect of a resin pretreatment and CHCI, treatment on the recovery of microbial P added to soil (Mean of three samples; mean deviations <5%, < lO%t)
P, Pr P, Pr
14.2 1.W 4.1t 7.7 16.9
14.2 5.0 9.8 12.8 23.6
+ 6.1
+ 5.1
+33.s
(b) Fungal species tl 28.2 33.7 45.1 50.4
Chloroformed after resin extraction
(a) Original soil
Change after No chloro- chloroform form
% Microbial-P recovered
Chloroformed after resin extraction
pg P- ’ soil
Chloro- Change formed after No after resin chloro- chloroextraction form form
(41.3 pg P added)
Fusarium sp.
(0.44% P dry wt) (49.2 fig P added)
Arthrobacter
+38.9
6.0 1.7 9.5
Change after No chloro- chloroform form
f25.1
sp.
<1 7.1 30.6 19.2 45.5
Pseudomonas
Change after No chloro- chloroform form
+ 35.0
<1 1.7t 5.3 12.7 16.0
(c) Bacterial species
+ 24.6
sp.
Chloroformed after resin extraction
(0.54% P dry wt) (17.0 pg P added)
‘A Microbial-P recovered
Chloroformed after resin extraction
(0.68% P dry wt) (17.0 pg P added)
+ 37.6
+33.3
Change after chloroform
* Added microbial-P = ng P added as a fungi (or bacteria) + amount of resin-l? absorbed by fungi (or bacteria). ** Data is for amount of P removed by both 30-min and 16-h extractions with NaHCO,. Data on later extractions was determined from soil samples that had been extracted with NaHCOs for 16 h.
Resin P, 0.5 M NaHC03 30 mitt 0.5 M NaHCO, 16h
Extractant
No chloroform
Indian Head Ap horizon
Trichoderma sp. (0.37% P dry wt)
Table 6. Amount of P (pg g- ’ soil) extracted from preincubated soil and the percentage recovery of added microbial P* recovered from added fungal and bacterial P with a NaHCO, extractions** (Values are means of three samples; mean deviation ~57~ < lo%?)
M. J.
384 Table 7. soil with error of whereas
HEDLEY
and
J. W. B.
STEWART
Amount of 33P (dis x lo3 mini’ g-I soil) recovered from labelled bacterial additions to CHC13-NaHC03 extraction. Each set of results is the mean of three samples; standard the mean is in parenthesis. Bacterial ‘iP added was negligible at 0.35pg Pg-‘* soil, 33P added had an activity of 223.91 dis x 10e3 min-’ g-i soil at the time of addition 33P Activity in each extract
33P Activity in each extract (dis x lo3 min-’
Extractant Resin 0.5 M NaHCO,
P,
CHCl,-0.5
P,
M NaHCO,
* Percent
of microbial-P
measured
23.1 (1.5) 24.1 (1.5) 106.5 (2.4)
g-i
Average recovery of added microbial P(x)
soil) 26.2 (1.6) 21.2 (0.5) 109.9 (2.5)
by the CHC13 technique
11.0 10.1 48.3
= recovery
in CHCI,-NaHCO,
minus the recovery in NaHCO, (38.29,, K, = 0.36).
CHCl, after resin treatment, the difference in amount of P, removed by NaHCO, extraction of sample B and of a sample A is attributed to P released from microbial cells. However, this extraction removes only part of the microbial-P. Ideally, this method should be calibrated, therefore, for microbial-P recovery from each type of soil studied because proportional recovery of microbial-P differs for more weathered soils. In laboratory experiments with ground incubated moist soils, reproducible results were obtained with samples as small as 0.5 g. Fresh samples of field soils that are not ground require sample sizes of 2-5 g to allow for soil heterogeneity. Soil to solution ratios of the extracting solution should be between I : 30 and 1: 60. The microbial biomass-P technique tested in our studies (Fig. 2) is a means of calculating microbial-P in soils. Measuring P, rather than P, in the NaHCO, extract and extracting the soil for I6 h rather than 30 min are important. Measurable recovery of added microbial-P from the soil was 25534’;, for fungal and 2433% for bacterial-P in a 30-min NaHCO, extraction and 35-39% for fungal-P and 3638”/:, for bacterial-P in a 16-h extraction. Removal of labile Pi by extraction with a resin before extraction with NaHCO, increased the accuracy of measurement of microbial-P. This step is necessary with many soils because of the small size of the microbial-P pool compared with the amount of NaHCO,-extractable P. This step would not be necessary in soils containing low amounts of labile Pi,
Fig.
2.
Flow
chart
of the
microbial-P method. in Pr in extract
such as an acid soil containing high amounts of active aluminum. Resin extraction is important, however, in determining added microbial-P recovered (Kp) from different soils, as changes in resin-extractable P during extraction equals microbial-P uptake. DISCUSSION
The degree of extraction of microbial-P from soils by this method should depend to some extent on the P sorption capacity of the soil. Recovery of added microbial and P, in CHCI,-NaHCO, was measured for a soil with a higher P sorption capacity than the Indian Head soils. Recovery of both bacterial-P and fungal-P was slightly reduced (K, = 0.32) in the higher-P-sorbing soil (Oxbow Bm soil, Hedley et al., 1981). This suggests that, over a range of soils, recovery of microbial-P varies inversely with P sorption capacity. However, as Fig. 1 demonstrates, no simple relationship exists between microbial-P, and Pi extracted in NaHCO,. Over a range of soils we found no direct relationship between P sorption capacity and K, factor (range 0.32-0.47), although K, factors generally were smaller in soils with higher P sorption capacities. Thus, no single K, factor can be used to accurately calculate amounts of microbial-P in a wide range of soils. However, studies of soils with pH values ranging from 6.2 to 8.2 suggest that a K, factor of 0.4 provides a close estimate of microbial-P in soils of this pH range
Estimates of microbial-P B compared with extract
are calculated A.
from the increase
Measurement of biomass-P These results show that use of CHCl, in conjunction with extraction
allows estimation
of microbial-P.
Furthermore,
CHCIa treatment specifically releases with tracers (32P and 33P) this method aliows us to calculate the specific actkty of microbial populations and to determine the dynamics of P transformations in soils. microbic-P.
In conjunction
Acknowledgements-We thank Drs C. V. Cole, S. R. Olsen, and R. A. Bowman for helpful reviews during preparation of this manuscript, published as Journal Paper R 252 of the Saskatchewan Institute of Pedology. Research was supported by the Natural Science and Engineering Research Council of Canada by Western Cooperative Fertilizers Ltd, and by a postdoctoral fellowship (MJH) from the University of Saskatchewan.
REFERENCES ALEXANDER M. (1977) Introduction
CLARK
F. E. and PAUL E. A. (1970) The microflora of grassland. Advonces in Agronomy 22,375435. COLE C. V., INNISG. I. and STEWART J. W. B. (1977) Simulation of phosphorus cycling in semi-arid grasslands. Ecology 58, 115. COLE C.
V., ELLIOTTE. T., HUNT H. W. and COLEMAND. C. (1978) Trophic interactions in soils as they affect energy and nutrient dynamics. V. Phosphorus transformations. Microbial Ecology 4, 381-387.
CO~GROVED. J. (1977) Microbial transformation in the phosphorus cycle. In Advances in Microbial Ecology (M. Alexander, Ed.), pp. 97-134. Plenum Press, New York. D~RMAAR J. (1972) Seasonal patterns of soil organic phosphorus. Canadian Journal ofSoii Science 52, 107-122. ENVIRONMENTAL PROTECTION AGENCY (1971) Methods fir Chemical Analysis of Water and Wastes. Cincinnati, Ohio. GORINGC. A. I. and BARTHOLOMEW W. V. (1949) Microbial products and soil organic matter. II. The effect of clay on the decom~sition and separation of the phosphorus compounds in microorganisms. Proceedings. Soil Science Society of America 14, 152-156.
to Soil Microbiology.
Wiley, New York.
AMERF., BOULDIND. R., BLACK C. A. and DUKE F. R. (1955) Characterization of soil phosphorus by an ion exchange resin adsorption and P3’ equilibration. Plant and Soil 6,391-408. ANDERSONJ. P. E.
and DOMSCHK. H. (1978a) Mineraiization of bacteria and fungi in chloroform-fumigated soils. Siol Biology & Biochemistry 10, 207-213. ANDERSONi’P. E. and D~M~CHK. H. (1978b) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Siology & Biochemistry 10, 215-221. ANDER%JNJ. P. E.
and D~M~CH K. H. (1980) Quantities of plant nutrients in the microbial biomass of selected soils. Soil Science 130, 21 l-216. BABIUK L. A. and PAUL E. A. (1970) The use of fluorescein isothiocyanate in the determination of the bacterial biomass of grassland soils. Canadian Journal of Microbiology 16, 57-62. BOWMAN R. A. and COLE C. V. (1978) Tr~sformations of organic phosphorus substrates in soils evaluated by NaHC03 extraction. Soil Science 125, 49-54. BOWMANR. A., OLSENS. R. and WATANABEF. S. (1978) Greenhouse evaluation of residual phosphate by four phosphorus methods in neutral and calcareous soils. Journal. Soil Science Society of America 42,451-454. BURNS D.
385
J. W. and BEF/ER R. E. (1977) Kinetic characterization of the two phosphate uptake systems in the fungus Neurospora crassa. Journal of Bacteriology 132, 511-519. CALDWELLA. G. and BLACKC. A. (1958) Inositol hexaphosphate. 2. Synthesis by soil microorganisms. Proceedings. Soil Science Society of America 22, 293-296. CHATERM. and MATTINGLYG. E. G. (1980) Changes in organic phosphorus content of soils from iong~ontinu~ experiments at Rothamsted and S~mundham. Rothamsted Report for 1979, Part 2, pp. 41-61. CHAUHANB. S., STEWARTJ. W. B. and PAUL E. A. (1979) Effect of carbon additions on soil labile inorganic, organic and microbially held phosphate. Canadian Journal of Soil Science 59, 387-396. CHAUHANB. S., STEWART1. W. B. and PAUL E. A. (1981) Effect of labile inorganic phosphate status and organic carbon additions on the microbiaI uptake of phosphorus in soils. Canadian Journal of Soil Science 61, 373-385.
HAAS H. J., GRUNESD. L. and RE~CHMAN G. A. (1961) Phosphorus changes in Great Plains soils as influenced bv crouoinn and manure aonlications. Proceedinas. Soil * Siience Society of America ii, 21.5-218. HALM8. J., STEWARTJ. W. B. and HALSTEADR. L. (1972) The phosphorus cycle in a native grassland ecosystem. In Isotopes and Radiation in Soil-Plant Relationshios Ineluding Forestry, pp. 571-586. SM151/7, IAEA, Vienna. HEDLEYM. J.. STEWARTJ. W. B. and CHAUHAN B. S. (1982) Changes in labile inorganic and organic soil phos: phorus fractions induced by cultivation practices or regular carbon additions. Journal. Soil Science Society of America (in press). HERZBERGM. A., KLEIN D. A. and COLEMAND. C. (1978) Trophic interactions in soils as they affect energy and nutrient dynamics. II. Physical responses of selected rhizosphere bacteria. Microbial Ecology 4, 351-359. JENKIN~~ND. S. and POWL~~ND. S. (1976) The effects of biocidal treatments on metabolism in soil. I. Fumigation with chloroform. Soil Biology d; Biochemistry 8,167-177. MURPHYJ. and RILEYJ. P. (1962) A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27, 31-36. OLSEN S. R., COLE C. V., WATANABE F. S. and DEAN L. A. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. USDA Circular 939. PAUL E. A. and JOHNSONR. L. (1977) Microscopic counting and adenosine 5’-triphosphate measurement in determining microbial growth in soils. Applied Environmental Microbiology 34, 263-269.
PAUL E. A. and VORONEYR. P. (1980) Nutrient and energy flows through soil microbial biomass. In Contemporary Microbial Ecology (D. C. Ellwood et al., Eds), pp. 215-237. Academic Press, London. SIBBE~ENE. (1977) A simple ion-exchange procedure for extracting plant-available elements from soil. Plant and Soil 46,665669. VAN
VEEN J. A. and PAUL E. A. (1979) Conversion of biovolume measurement of soil organisms grown under various moisture tensions to biomass and their nutrient content. Applied Enuironmental Microbiology 37, 686692.
WESTINF. C. and BUNTLEYG. J. (1967) Soil phosphorus in South Dakota. III. Phosphorus fractions in some Borolls and Ustolls. Proceedings. Soil Science Society of America 31, 521-528.
Soil Biol. Biochem. Vol. 14, pp. 377 to 385, 1982 Printed in Great Britain. All rights reserved
METHOD
TO MEASURE MICROBIAL IN SOILS M. J.
HEDLEY*
and J. W. B.
PHOSPHATE
STEWART
Department of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO (Accepred
1 Nouember
1981)
Summary-The amount of phosphate in soil microbial biomass was estimated by adding CHCl, to soil to lyse microbial cells and measuring the proportion of microbial-P released to 0.5 M NaHCOs extracts (pH 8.5). Calculations of total microbial-P were based on the difference between P removed by NaHCOs extraction of ChCI, treated and untreated samples. This method was improved by (1) removing resinextractable P from the soil before lysing microbial cells with CHCIJ, (2) measuring total P rather than inorganic-P in NaHCOs extracts, and (3) prolonging NaHCOs extraction from 30 min to 16 h. The efficiency of extraction of soil microbial-P was determined by measuring the P recovered from two species each of bacteria and fungi grown first in solutions having different P concentrations, then added to soil. The average proportion of microbial-P recovered from a neutral calcareous soil was 37% (KP = 0.37). The KP factor was confirmed by adding 33P-labelled bacteria to a similar soil and measuring the added 33P recovered (38%) by the CHCI,-NaHCO, method.
INTRODUCTION
and organic soil phosphorus observed across environmental gradients (Westin and Buntley, 1967) resulting from prolonged cultivation practices (Haas et al., 1961; Chater and Mattingly, 1980) or with season (Dormaar, 1972; Halm er al., 1972) show that P mineralization and immobilization occurred but provide no information on the rates or mechanisms of transfer. With more accurate methods to evaluate microbial biomass (Babiuk and Paul, 1970; Jenkinson and Powlson, 1976; Paul and Johnson, 1977; van Veen and Paul, 1979), understanding P dynamics in the soil-plant system is limited mainly by lack of analytical methods that identify forms and amounts of various labile inorganic- and organic-P fractions, including P immobilized in microorganisms (Cole et al., 1977; Cosgrove, 1977). Biomass C is measured by making the biomass susceptible to mineralization by fumigation with CHCl,; the subsequent flush of CO2 during incubation gives a measure of soil biomass (Jenkinson, 1976; Anderson and Domsch, 1978a,b). Chloroform fumigation also has been used to measure biomass ATP (Paul and Johnson, 1977) and biomass N (Paul and Voroney, 1980). Cole et al. (1978) used CHCIS fumigation in combination with NaHC03 extraction (Olsen et al., 1954) to remove a portion of the biomass-P. This method was used by Chauhan et al. (1979, 1981) to estimate microbial-P in soils. We report a modification of this technique that more accurately estimates microbial-P. Large net changes in inorganic
Anderson
and
Domsch
(1978a,b)
evaluated
a
CHC13 technique to measure biomass-C by adding known quantities of fungi and bacteria to soil and calculating
the proportion
(Kc) evolved
as COa-C.
* Present address: Soil Science Laboratory, Department of Agricultural Science, Oxford University, Oxford OX1 3PF, England. 377
When this technique is adapted to microbial-P evaluation, consideration must be given to the heterogeneity of soil P compounds, to the diversity of P compounds contained in a microbial cell, and to the ease of extracting these compounds from soil and from soils with different P adsorption capacities. Compounds containing P in bacteria and fungi are made up of 30-50% RNA, 15-20% acid-soluble inorganic- and organic-P compounds consisting of sugar and nucleotide esters and various phosphorylated coenzymes and polyphosphates, < 10% phospholipids, 5510% DNA, and small amounts of inositol phosphates (Alexander, 1977; Caldwell and Black, 1958). The proportion of P in inorganic or organic forms depends on the P concentration in the soil solution, ‘A P in the cell, and age of the cell. In addition, because bacterial and fungal cells differ in cell composition and P content, the proportion of microbial-P measured in a CHCl,-NaHCO, extraction may differ. Among other points that have to be considered during method evaluation are that the CHCl, may affect the extractability of native soil-P, that NaHCO, extraction without CHCI, may release P from microbial cells, and that added microbial biomass could immobilize soil-P during the extraction process (up to the time of CHCl, addition). Finally, an adopted procedure should extract sufficient microbial-P to allow accurate measurement of extra P from lysed microbial cells in extracts which may also contain considerable quantities of soil-P. Anderson and Domsch (1980) using C:P ratios ranging from 7:l to 17:1, estimated that the average quantity of microbial-P in the top 12.5 cm of forest soils is 70 kg ha-‘. Chauhan et al. (1981) found that microbial biomass in incubated soils ranged from 100 to 500 pg biomass-C g- I soil and C:P ratios ranged from 2O:l to 6O:l. Microbial P values in soil, therefore, could range from 1.5 to 50 pg P g- ’ soil. To test extraction procedures and develop methods of measuring microbial-P in soils, we grew two species each of fungi and bacteria in nutrient solutions having
378
M. J. HEDLEY and J. W. B. STEWART
different P concentrations. Quantities of these organisms were added to a soil representative of the Canadian prairie, and the added microbial-P recovered in NaHCO, extracts was measured. MATERIALS
AND METHODS
Soil
The soil (pH 7.4, CEC w 30 m-equiv/lOO g, organic C 2.2%, clay 33%) used in most of our experiments was from the Ap horizon of a well-drained Black Chernozemic soil (Udic Haploboroll) of the Indian Head Association in southern Saskatchewan. A second soil (pH 7.0, CEC _ 30 m-equiv/lOO g, organic C 3.2x, clay 45%) having a larger indigenous microbial biomass and taken from the Ah horizon of a permanent pasture soil of the same association was used where stated. Soils were ground and sieved (< 500 pm), then incubated at 60% field moisture capacity at 24” f 2°C for 21 days before use. Subsamples were oven-dried to determine dry weight. Organisms
TWO species of fungi (Trichoderma harzianum and Fusarium oxysporum) and bacteria (Pseudomonas cepacia and Arthrobacter globtformus), were selected because they are common in soil (Clark and Paul, 1970). Media
Fungi were cultured in potato dextrose broth (PDB, Difco) or the fungal culture medium (FCM) of Burns and Beever (1977). Nutrient broth (NB, Difco) and a standard reference soil solution (RSSC, Herzberg et al., 1978) were used to culture the bacteria. Nutrient agar and potato dextrose agar (Difco) slants were used to maintain stock cultures of the bacteria and fungi, respectively. Phosphate-free media (FCM and RSSC) were prepared for growing bacteria and fungi. Phosphate (KH,P04) solution was sterilized separately, and different volumes were added to FCM and RSSC media in culture flasks to produce a range of P concentrations. Carrier-free radioactive phosphate (33P, -2 mCi in 50 ml) was added to this stock solution to provide uniformly labelled P. Approximate concentrations of the stock solution were added aseptically to each culture flask to provide a 33P-labelled microbial population. Microbial growth and harvesting
Erlenmeyer flasks (250 ml) containing PDB or NB (100 ml) were inoculated from stock slants of fungi or bacteria, respectively. The flasks were incubated for 4 days (fungi) and 2 days (bacteria) at 22°C on an orbital shaker (90 rev min- i). Fungal cells were harvested from the growth media by vacuum filtration onto a cellulose acetate membrane filter (Millipore, 0.45 pm). The harvested cells were washed thoroughly with NaCl solution (8.5 gl-I). Bacterial cells were harvested from the growth media by centrifugation (10,000 rev min- 1 for 20 min. IEC, model B-20 centrifuge) and washed by repeated (4 times) resuspension in NaCl solution and centrifugation. Finally, the washed cells were resuspended in NaCl solution or deionized water. In studies of bacteria and fungi grown in different P
concentrations, the respective inocula were harvested aseptically from 24-h-old cultures of bacteria grown in NB and of fungi grown in FCM. Bacteria and fungi were washed thoroughly with sterile deionized water before being inoculated into the culture flasks containing growth media of different P concentrations. The flasks were incubated (bacteria, 2 days; fungi, 4 days) and harvested as described. Experimental procedure
Suspensions of bacterial cells were pipetted into 40-ml screwcapped polypropylene centrifuge tubes and into identical tubes containing 1 g or 0.5 g of moist soil. Addition aliquots were taken to determine the dry weight of the cells. Moist, whole fungal cells were weighed into polypropylene centrifuge tubes having similar treatments as above; the wet and dry weights of subsamples of each population were determined for calculating the dry weight in each centrifuge tube. Suspensions of 33P-labelled bacteria were added to soil as described above to give an initial specific activity of 0.1 $i g- 1 soil. Adapted chloroform procedure
Chloroform (1 ml) was added to moist soil, microorganisms, or soils amended with microorganisms contained in centrifuge tubes. The centrifuge caps were replaced, and the tubes were shaken by vortex mixing for three lo-set intervals over a period of 30min at 24°C. The caps were removed, and the CHCl, was allowed to evaporate overnight in a fume hood at 24°C. Samples in CHCl,-treated and non-CHCl,-treated tubes were then extracted overnight (16 h) with 0.5 M NaHC03 (30 ml, pH 8.5) at 24°C. After centrifugation (0°C 10,000 rev min- ‘, IEC, Model B-20 centrifuge), the extract was filtered through a membrane filter (Millipore, 0.45 pm). Residue retained by the filter was washed back into the centrifuge tube with deionized water. Inorganic-P (Pi) and total P (PT) concentrations were measured in the NaHCO, extracts and residues, Chloroform treatment of sterilized samples
To investigate whether CHCl, treatment increases the amount of nonmicrobial soil-P extracted in NaHCO, extracts, we compared the quantity of P extracted after CHCl, treatment of sterilized and nonsterilized soil. Two samples of the Indian Head Ah soil were incubated at 60% field moisture capacity (FMC) and 25°C for 10 days. Then one sample was sterilized by fumigation with ethylene oxide, and both were incubated at 60% FMC for 21 days at 25°C in sealed containers. Subsamples of soil from each treatment were extracted with resin for 16 h. Three unsterilized and three sterilized subsamples were treated with CHCl,; three unsterilized subsamples remained untreated. The samples were then placed in a fume hood at 24°C overnight, and all CHC13 evaporated. The samples were then extracted with 30ml 0.5 M NaHCO, for 16 h at 24°C. Extractable-P methods
Resin-extractable P was measured by the method of Amer et al. (1955) as adapted by Sibbesen (1977). To 0.5 g soil, 0.4g anion-exchange resin (Dowex
319
Measurement of biomass-P 1 x 8-50 > 30 mesh) in a Nylon bag was added, plus 30 ml of water, and shaken for 16 h at 24°C. The P adsorbed by the resin was displaced by 25 ml of 0.5 MHCl and analysed. Sodium bicarbonate-P (0.5 MNaHC03, pH 8.5) was measured by the method of Olsen et at. (1954) except that the soil:solution ratio was 1: 30 or 1: 60 and two shaking times, 20 min and 16 h, were used where stated. Phosphate determination
Phosphate (PI) was determined with 1 ml of each extract. A correction was made to account for absorption caused by colored organic matter in the extract. An aliquot of similar size was digested by acidified ammonium ~rsulphate oxidation (Environmental Protection Agency, 1971). The method of Murphy and Riley (1962) was used to measure the orthophosphate concentration in the neutralized extract (Pi) and in the neutralized digest of the extract (P,). Organic-P (P,) was calculated by subtracting Pi and P,. Radioactive-33P assay
Radioactivity (33P) and quantitative (31P) analysis were determined on the same sample. Addition of 0.5-1.0 ml of the 0.5 M HCl extract (resin-P) or the NaHCO, extracts to 14 ml of a complete phase combining cocktail mixture (PCS-II, Amersham) gave a clear, homogeneous fluid that was counted in a Beckman LS 9000 liquid scintillation counter. All counts were corrected to disintegrations min- ‘. RESULTS
Evaluation of lysing agents
Chloroform and two other microbial cell-lysing agents, ethanol and isopropanol, combined with 0.5 M NaHCO,, were compared for efficiency of recovery of P from a pure culture of fungi. All lysing agents gave the same proportional recovery (89%) of fungal-P from pure culture. However, the recovery of fungal-P added to the soil was considerably higher with CHCI, than with ethanol or isopropanol (73% vs 49 and 56%). Uhrasonification of Fusarium sp. mycelia before addition of CHC13 did not increase recovery of microbial-P in subsequent 0.5 M NaHCO, extraction, This suggests that CHCl, effects the complete lysis of fungal cells. Because CHCI, readily evaporates from treated soil, it was the most suitable microbial-lysing agent for this study. Evaporation of the CHCl, also prevents extraction of soil phospholipids, which could produce an overestimate of microbial-P.
E$ect of chloroform on extractability of labile soil-P
To investigate whether CHCl3 treatment may increase extraction of non-microbial soil-P forms, we extracted sterilized and nonsterilized soil samples by NaHC03 with and without CHCI, treatment. Resinextractable Pi increased by 12.1 pgg-’ soil after sterilization with ethylene oxide and subsequent 21-day incubation (Table 1). After the resin extraction, the amount of 0.5 M NaHCO, P, extracted from nonsterilized soil and from CHQ-treated sterile soil was virtually the same, but in the CHCl,-treated nonsterilized soil the 0.5 M NaHC03 Pr increased by 13.0pgg-’ soil. These results illustrate three points. First, ethylene oxide and CHClj treatments do not significantly increase the extractability of 0.5 M NaHC03 soil P,. Second, 0.5 M NaHCOJ alone does not extract significant amounts of indigenous microbial-P. Third, resin extraction after sterilization and incubation is almost as effective in removing P released from the soil microbial population as extrao tion with 0.5 M NaHCO, after CHCl, treatment (12.1 vs 13.0 gg P g- ’ soil). E&et of sorption by soii on recovery of added P in NaHCO, extracts
Preliminary experimentation indicated that age of the fungal culture markedly affects distribution of Pi and P, in the 0.5~ NaHCO, extract. A t-day-old fungal culture had considerably more extractable-p, but the percentage of extractable-P, than-P,, remained similar to those obtained from Cday-old cultures. This suggests that NaHCO~-extractable Pr provides a more reliable measurement from which microbial-P can be estimated. No distinct pattern of change was seen (Table 2) in the amounts of microbial-P, and -P, extracted by 0.5 MN~HCO~ as the cell P content of fungal and bacterial cells increased. However, the sum of these two P values (Pr) increased as the cell-P content inwas creased. Moreover, no useful information obtained by considering the percentage of Pi and P, in NaHC03 soil extracts, compared with that’ obtained in the extract of microbial cells without soil. This is because the rate of hydrolysis of P, to Pi changes once the ceil wall is broken and varies with soil components, cell com~sition, and treatment. Within experimental error, the percentage of P, recovered from both fungi was similar; percentage of P, recovered from both bacteria also was similar. When bacterial and fungal ceils of similar P content were tested, CHC13-NaHCO, extracted fungal-P more effi-
Table 1. Effect of CHC13 treatment on amount of 0.5~ NaHCOJextractable P in sterilized and nonsterilized pasture soils (Ah) preextracted with an anion-exchange resin (Values are the means of three samples; mean deviation < 10%) P fraction
Nonsterilized soil (fig P g- L soil)
Sterilized soil (pg P g- ’ soil)
Resin Pi
40.5
40.5 52.6 CHC13 treatment
0.5 M NaHC03 P, Z fractions
29.7 70.2
42.7 83.2
30.5 83.1
380
M. J. HEIILEY and J. W. B. STEWARI
Table 2. Proportions of total P extracted from bacterial and fungal cells of various P content in the absence of soil (Values are means of three samples: mean deviation < IO”,,)
P content of cells (% dry wt)
0.22 0.28 0.42 0.30 0.90 I.4 0.17 0.40 0.70 0.27 0.55 0.86
“(, of added microbial-P CHCl,Total NaHCO, extracted Residual P,
P,
Pr
Pr
Fusarium oxysporum 13 4 II 79 2 81 74 II 85 Trichoderrna harzianum 60 4 64 71 5 16 69 I5 84 Arthrobacter globiformus 38 8 46 7 43 50 17 41 58 Pseudomonas cepaecu 32 23 55 40 28 68 29 17 46
23 19 15
NaHCO, extraction (Fig. 1). In this figure the amount of microbial-P, added to the soil refers to the amount of P, released to 0.5 M NaHCO, from CHCl,-lysed cells in the absence of soil. Over the range of microbial-P additions (9942 pgg-’ soil), the recovery of added Pi averaged 42 + 5%, whereas the recovery of microbial-P, ranged from 28 to 584; and did not follow a pattern. These results indicate that no simple relationship exists between the recovery of microbial P, from this soil and Pi extracted by NaHCO,. Effect of resin microbial-P
35 24 16 54 50 42 45 32 54
Pi = % of added microbial-P recovered as inorganic P. P, = % of added microbial-P recovered as organic P. P, = Pi + P,.
ciently than bacterial-P. With bacteria, approximately 50% of the microbial-P was nonextractable, compared with an average of only 22% of fungal-P. The presence of soil reduced recovery of P from both CHCl,-treated fungi and bacteria by almost 50% (Tables 3 and 4). Presumably, some P released from microbial cells was adsorbed by soil components, as some of this was recovered in subsequent extraction in 0.1 M NaOH (Hedley et al., 1982). Addition of clay decreases the extractability of P from mixed microbial tissue (Goring and Bartholomew, 1949). When microbial cells are lysed with CHC13, some of the released P will be sorbed to the soil surface. Recovery of this P in a NaHCO, extraction may be related to the Pi sorption isotherm of a soil if the P, released from the microbial cell is sorbed in a similar manner. To investigate this effect, we compared recovery of added Pi with recovery of microbial-P, by
extraction
on subsequent
Trichoderma harzianum 1.200/;; P (dry wt) 0.47% P (dry wt)
0.5 M NaHCOs
Total
P
A*
Pi P,
B
C
of
Extraction of a soil sample with resin did not decrease the recovery of P from subsequently lysed microbial cells (Table 1). This result was examined further by comparing the results (Table 5) from untreated and resin-pretreated samples subsequently treated with CHCls, then extracted for 30min with 0.5 M NaHCO,. Resin pretreatment has two main advantages (Table 5). First, it reduces the amount of soil Pi extracted by the subsequent NaHC03 extract, as almost all labile Pi is removed by the resin, and P released by CHCl, fumigation then makes up a significant portion of the NaHCO, extract. For example, 0.5 M NAHCO, extracted 17.1 pg P (Table 5) from the chloroformed soil sample, only 20% of which was measurable indigenous microbial-P. In the equivalent resin pretreated sample, measurable microbial P constituted 58% of the NaHCO, P, extracted. Second, the method can be calibrated for use in a particular soil. Changes in resin P provide an estimate of the uptake of labile soil Pi by the added microbial biomass during extraction (Table 5). Because added microbial biomass can take up labile soil Pi during extraction until CHCls is added, values obtained for the apparent recovery of microbial-P from nonchloroform soil were negative (Table 5). As most of the available soil-P is resin extractable, decreases in this value equal microbial-P uptake. Addition of bacterial to the soil (Table 5) reduced resin-extractable P by approximately 12.5 pg P g- 1 soil. Addition of fungi also reduced resin-extractable P but by a lesser amount, about 2.5 pg Pg-’ soil. In the non-resintreated soil, microbial uptake during extraction can not be measured.
Table 3. Effect of CHCI, pretreatment and addition to soil on percentage of total P extracted from fungal cells (Values are means of three samples; mean deviation < 10%)
Extractant
recovery
B o/0 of added
C microbial
Fusarium oxq’sporum 0.62% P (dry wt) A
B
C
P
33 15
49 31
44 I2
63 27
30 20
17 14
48 30
I8 26
48
80
56
90
50
31
78
44
* A = fungi alone. B = fungi alone treated with CHCIs before NaHCOs extraction. C = fungi added to soil and treated with CHCI, before NaHCO,
extraction.
381
Measurement of biomass-P
Table 4. Effect of CHCI:, pretreatment and addition to soil on the percentage of total P extracted from bacterial cells (Values are means of triplicate samples; mean deviation < 10%) Arthrobactergiob~rm~s 1.8% P (dry wt) Extractant
0.5 M NaHC03
Pi P,
Total P
Pseudo~~s 0.8% P (dry wt)
A’
B
C
21 9 30
29 33
20 IS
12 8
62
35
20
cepaeca 2.7% P (dry wt)
C
A
B
c
30 30
23 17
40
54 14
54 7
60
40
40
68
61
B A y0 of added microbial-P
*A = bacteria alone. B = bacteria alone treated with CHCls before NaHCOs extraction. C = bacteria added to soil and treated with CHCls before NaHCO, extraction.
The ~ssibility exists that the indigenous soil microbial population absorbs labile soil-P during extraction. However, microbial-P of incubated soils did not change with time, nor did that of fresh samples from the field. Therefore, this effect will be noticed only when microbial biomass grown in vitro is added to soil or when field soil contains large amounts of undecom~sed plants or plant residues. In resin-treated samples, the 30-min 0.5 MNaHCO, extraction removed 5.3-8.6°~ of the added microbial-P before treatment with CHC13. Thus, the percentage of microbial P measured was that recovered by the CHCl,-NaHCOJ extraction reduced by this amount. Efict of increasing shaking time tion
ofNaHCOs
extrac-
The percentage of soil-P and added microbial-P, especially fungal P, recovered was lower in the CHCI,-treated 30-min NaHCOS extraction than in the CHCl,-treated 16-h NaHCO, extraction (Table
6). This agrees with publish~ data (Bowman et al., 1978}, which indicate that a 16-h 0.5 MN~HCO, extraction removes approximately 20% more labile soil Pi than a 30-min NaHCO, extraction. Similarly, a larger amount of the P released from lysed microbial cells should be recovered by a 16-h extraction. The 16-h 0.5 M NaHCOj extraction recovered 20% more microbial-P from Chili-treated samples than the 30-mm 0.5 M NaHC03 extraction. However, the amount of microbial-P extracted by 0.5 MNaHC03 alone also increased by 8-17x (Table 6). Thus, the 16-h extraction increases measurable recovery of added microbial-P by 3454% over the 30-min extraction (Table 6). The range of measurable microbial-P (P,} recovery of all organisms tested was narrowed to 36.7 f 1.4%. If the indigenous soil microbial population is extracted in a similar manner and consists of 25% bacteria and 75% fungi (Clark and Paul, 1970), then this method measures 37% of microbial-P in the original soil. Using this mean value and data from the Indian Head Ap soil (Table 6), we estimated that this soil contains 18.2 fig P g-i soil of microbial-P. Test of the sequential extraction method with radioactioe phosphate (33P)
Sequential extraction of the Indian Head Ah soil to which 33P-labelled bacteria had been added is an alternative procedure for testing this technique (Table 7). In this case, the amount of added bacterial-P was negligible (0.35 pg “P g- ’ soil) but it had a high specific activity. Adding radioactive bacteria to the soil caused the soil to have an activity of 2.24 x 10’ dis mitt- i g- 1 soil. The increase in percentage recovery of microbial-P between chloroformNaHC03 and NaHCO, extraction was 38.2% (K, = 0.381, which compares favorably with data obtained from adding larger amounts of unlabelled P in fungal and bacterial biomass (Kr = 0.37) (Table 6). Microbial-P method
oA~unt
5
Kl
i5
20
of added P recovered
25
30
35
) pg.g-l soil
Fig. 1. Comparison of the amount of labile P added and P, extracted by a 30-min 0.5 M NaHC03 extraction, The amount of microbial P, added to the soil refers to the amount of P, released to 0.5 M NaHCOo from CHCls-lysed soils in the absence of soil. Symbols: o = Pi, A = Trichodermo sp., Cl = Fusarium sp., + = Pseudomonas sp., 0 = Arthrobocter sp.
We therefore recommend the microbial-P method outlined in Fig. 2 on the basis of our studies. In this method the most biologically available Pi (Amer et al., 1955; Sibbesen, 1977; Bowman et al., 1978) was first removed onto an anion-exchange resin. Labile Pi and P, adsorbed on the soil surface, plus a small amount of microbial-P (Bowman and Cole, 19781,was removed by NaHCO, extraction When a second sample, B, is run concurrently and treated with
14.2
14.2
+
+ _ +
_
+
Pseudomonas
Soil
+
_ +
-
Fusarium + soil
Arthrobacter + soil Pseudomonas + soil
1.3
1.7 1.8 1.2
11.7 il.1
12.0
12.2
ND
ND
ND
17.1
15.8 15.7 17.0
50.8 47.5
39.7
42.9
0.0
0.0
23.4 23.4 0.0 0.0
17.3 17.9
53.4 50.2 41.9 39.7
Microbial P added
12.9
12.5 12.4 13.0
2.5 3.1
2.2
2.0
(b) Resin treated
ND ND
ND ND
30.0
28.3 28.1 30.0
53.3 50.6
41.9
44.9
23.4 23.4
17.3 17.9
53.4 50.2 41.9 39.7
Total microbial P (added + uptake)
(a) Non-resin treated ND ND ND ND
pg P g- ’ soil
Microbial* P uptake
21.4
5.9t 18.4 5.7
8.7 26.8
7.4
26.9?
4.1t 9.8
7.3 20.8t 13.6 17.1
11.3t 22.3
25.4 37.9 27.lt 35.3
NaHCOJ** extractable PT
22.1
33.6
6.0 30.6 5.3
8.6 33.5
1.3
40.8
- 26.9 15.8
- 12.8 29.1
32.2 45.8
41.4
%
Apparent recovery?? of microbial-P
NaHCO,P,(soii + microbe - soil) x loo ” -----~?otal microbial-P
P.
33.3
24.6
24.9
33.5
42.1
41.9
13.6
19.3
Microbial P measured by CHCl, NaHCO,
* Microbial P uptake from time of addition of microbial biomass to soil to CHCI, treatment can be estimated in resin treated soils by the decrease in resin-extractable ** 30-min extraction.
+
-
+
-
Trichoderma + soil
+ soil Soil
+ soil
ND
Arthrobacter
+
-
Fusarium
+ soil
+
+ soil
ND
_
Resinextractable P
Trichoderma
CHC13 treatment
Table 5. Effect of a resin pretreatment and CHCI, treatment on the recovery of microbial P added to soil (Mean of three samples; mean deviations <5%, < lO%t)
P, Pr P, Pr
14.2 1.W 4.1t 7.7 16.9
14.2 5.0 9.8 12.8 23.6
+ 6.1
+ 5.1
+33.s
(b) Fungal species tl 28.2 33.7 45.1 50.4
Chloroformed after resin extraction
(a) Original soil
Change after No chloro- chloroform form
% Microbial-P recovered
Chloroformed after resin extraction
pg P- ’ soil
Chloro- Change formed after No after resin chloro- chloroextraction form form
(41.3 pg P added)
Fusarium sp.
(0.44% P dry wt) (49.2 fig P added)
Arthrobacter
+38.9
6.0 1.7 9.5
Change after No chloro- chloroform form
f25.1
sp.
<1 7.1 30.6 19.2 45.5
Pseudomonas
Change after No chloro- chloroform form
+ 35.0
<1 1.7t 5.3 12.7 16.0
(c) Bacterial species
+ 24.6
sp.
Chloroformed after resin extraction
(0.54% P dry wt) (17.0 pg P added)
‘A Microbial-P recovered
Chloroformed after resin extraction
(0.68% P dry wt) (17.0 pg P added)
+ 37.6
+33.3
Change after chloroform
* Added microbial-P = ng P added as a fungi (or bacteria) + amount of resin-l? absorbed by fungi (or bacteria). ** Data is for amount of P removed by both 30-min and 16-h extractions with NaHCO,. Data on later extractions was determined from soil samples that had been extracted with NaHCOs for 16 h.
Resin P, 0.5 M NaHC03 30 mitt 0.5 M NaHCO, 16h
Extractant
No chloroform
Indian Head Ap horizon
Trichoderma sp. (0.37% P dry wt)
Table 6. Amount of P (pg g- ’ soil) extracted from preincubated soil and the percentage recovery of added microbial P* recovered from added fungal and bacterial P with a NaHCO, extractions** (Values are means of three samples; mean deviation ~57~ < lo%?)
M. J.
384 Table 7. soil with error of whereas
HEDLEY
and
J. W. B.
STEWART
Amount of 33P (dis x lo3 mini’ g-I soil) recovered from labelled bacterial additions to CHC13-NaHC03 extraction. Each set of results is the mean of three samples; standard the mean is in parenthesis. Bacterial ‘iP added was negligible at 0.35pg Pg-‘* soil, 33P added had an activity of 223.91 dis x 10e3 min-’ g-i soil at the time of addition 33P Activity in each extract
33P Activity in each extract (dis x lo3 min-’
Extractant Resin 0.5 M NaHCO,
P,
CHCl,-0.5
P,
M NaHCO,
* Percent
of microbial-P
measured
23.1 (1.5) 24.1 (1.5) 106.5 (2.4)
g-i
Average recovery of added microbial P(x)
soil) 26.2 (1.6) 21.2 (0.5) 109.9 (2.5)
by the CHC13 technique
11.0 10.1 48.3
= recovery
in CHCI,-NaHCO,
minus the recovery in NaHCO, (38.29,, K, = 0.36).
CHCl, after resin treatment, the difference in amount of P, removed by NaHCO, extraction of sample B and of a sample A is attributed to P released from microbial cells. However, this extraction removes only part of the microbial-P. Ideally, this method should be calibrated, therefore, for microbial-P recovery from each type of soil studied because proportional recovery of microbial-P differs for more weathered soils. In laboratory experiments with ground incubated moist soils, reproducible results were obtained with samples as small as 0.5 g. Fresh samples of field soils that are not ground require sample sizes of 2-5 g to allow for soil heterogeneity. Soil to solution ratios of the extracting solution should be between I : 30 and 1: 60. The microbial biomass-P technique tested in our studies (Fig. 2) is a means of calculating microbial-P in soils. Measuring P, rather than P, in the NaHCO, extract and extracting the soil for I6 h rather than 30 min are important. Measurable recovery of added microbial-P from the soil was 25534’;, for fungal and 2433% for bacterial-P in a 30-min NaHCO, extraction and 35-39% for fungal-P and 3638”/:, for bacterial-P in a 16-h extraction. Removal of labile Pi by extraction with a resin before extraction with NaHCO, increased the accuracy of measurement of microbial-P. This step is necessary with many soils because of the small size of the microbial-P pool compared with the amount of NaHCO,-extractable P. This step would not be necessary in soils containing low amounts of labile Pi,
Fig.
2.
Flow
chart
of the
microbial-P method. in Pr in extract
such as an acid soil containing high amounts of active aluminum. Resin extraction is important, however, in determining added microbial-P recovered (Kp) from different soils, as changes in resin-extractable P during extraction equals microbial-P uptake. DISCUSSION
The degree of extraction of microbial-P from soils by this method should depend to some extent on the P sorption capacity of the soil. Recovery of added microbial and P, in CHCI,-NaHCO, was measured for a soil with a higher P sorption capacity than the Indian Head soils. Recovery of both bacterial-P and fungal-P was slightly reduced (K, = 0.32) in the higher-P-sorbing soil (Oxbow Bm soil, Hedley et al., 1981). This suggests that, over a range of soils, recovery of microbial-P varies inversely with P sorption capacity. However, as Fig. 1 demonstrates, no simple relationship exists between microbial-P, and Pi extracted in NaHCO,. Over a range of soils we found no direct relationship between P sorption capacity and K, factor (range 0.32-0.47), although K, factors generally were smaller in soils with higher P sorption capacities. Thus, no single K, factor can be used to accurately calculate amounts of microbial-P in a wide range of soils. However, studies of soils with pH values ranging from 6.2 to 8.2 suggest that a K, factor of 0.4 provides a close estimate of microbial-P in soils of this pH range
Estimates of microbial-P B compared with extract
are calculated A.
from the increase
Measurement of biomass-P These results show that use of CHCl, in conjunction with extraction
allows estimation
of microbial-P.
Furthermore,
CHCIa treatment specifically releases with tracers (32P and 33P) this method aliows us to calculate the specific actkty of microbial populations and to determine the dynamics of P transformations in soils. microbic-P.
In conjunction
Acknowledgements-We thank Drs C. V. Cole, S. R. Olsen, and R. A. Bowman for helpful reviews during preparation of this manuscript, published as Journal Paper R 252 of the Saskatchewan Institute of Pedology. Research was supported by the Natural Science and Engineering Research Council of Canada by Western Cooperative Fertilizers Ltd, and by a postdoctoral fellowship (MJH) from the University of Saskatchewan.
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CLARK
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