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Phosphorus availability in a forest soil determined with a respiratory assay compared to chemical methods
Geoderma 89 Ž1999. 259–271
Phosphorus availability in a forest soil determined with a respiratory assay compared to chemical methods M. Demetz, H. Insam
)
Institut fur ¨ Mikrobiologie, UniÕersity of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria Received 3 April 1998; accepted 14 August 1998
Abstract A forest soil from the Northern Calcareous Alps of Austria was examined with different methods for determining P content and availability. The aim of this study was to compare resin extractable and bicarbonate Žor Olsen. P obtained by a sequential extraction method with bioavailable P as determined with a bioassay based on the kinetics of soil respiration induced by addition of a carbon source and nutrients. With two indices ŽCO 2 evolution within 30 h, and the slope of the growth curves. good agreement between the chemical and respiratory methods was found. The respiratory method appeared to be suitable for estimating the pool of bioavailable P for microorganisms and for a fast qualitative detection of P deficiency. q 1999 Elsevier Science B.V. All rights reserved. Keywords: phosphorus availability; substrate induced respiration; soil microbial biomass; forest; respiration
1. Introduction In several ecosystems of the world, for example, in alpine forests, phosphorus may be limiting for plant production. However, estimates of P availability are often dubious since there is great uncertainty if the measured pools are relevant for plant production. Of the various approaches, some are considered suited to )
Corresponding author. Tel.: q43-512-507-6009; Fax: q43-512-507-2928; E-mail: [email protected] 0016-7061r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 6 - 7 0 6 1 Ž 9 8 . 0 0 0 9 0 - 1
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estimate bioavailable P, such as the extraction with sodium bicarbonate Ž Olsen et al., 1954. or the anion exchange resin method ŽSibbesen, 1978; Schmidt et al., 1997.. However, intensely coloured extracts, precipitations or large amounts of iron and aluminium compounds and organic matter, often limit the validity and reproducibility of these tests Ž Sibbesen, 1978. , especially in acid organic soils. Numerous studies on the respiratory response following glucose amendment to soils have been made, especially since Anderson and Domsch Ž 1978. proposed the substrate induced respiration Ž SIR. method for determining soil microbial biomass. Usually, upon glucose amendment, the respiration rate increases within a few minutes Ž SIR. to a value that is directly proportional to the microbial biomass present. In most cases, after a lag phase of a few hours, the respiration rate further increases. This increase is attributed to microbial growth resulting from the newly available C source Ž glucose. . When the substrate is exhausted, CO 2 evolution starts to decline. However, in some soils glucose addition fails to induce a further increase of CO 2-production after the initial flush. This has been observed for temperate forests Ž Nordgren, 1992; K.H. Domsch, pers. comm.. as well as for tropical forests Ž J. Lodge, pers. comm.. and was attributed to nutrient limitation. Only when nutrients were added with glucose, was the usual increase in respiration found. Nordgren Ž 1992. proposed a bioassay for determination of forest soil N and P availability based on interpreting the respiration curves after amending different amounts of N or P. He argued that if defined amounts of added nutrients would alleviate growth restrictions, it should be possible to obtain figures of endogenous nutrient availability. The aim of the present study was to compare P pools determined with sequential extraction Ž Hedley and Stewart, 1982. with estimates of bioavailable P determined by a bioassay based on respiratory response upon glucose and nutrient addition.
2. Materials and methods 2.1. Soil The soil for this study was a poorly developed mull-rendzina under mixed beech–spruce forest in the Northern Calcareous Alps of Austria Ž 47823X N, 10850X W.. The climate of the study site is typical for the Northern Alps with cold, snowy winters Žmean January temperature - y48C. and cool, rainy summers Ž mean July temperature approx. 168C. . The mean annual temperature and precipitation are 5.78C and 1218 mm, respectively. Soil samples were collected in July and November 1991 and April 1992 from the 5 to 25 cm layer ŽA h-horizon.. Soil was sieved Ž2 mm. to remove larger roots and animals and stored field moist at 48C in polyethylene bags for up to 3
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weeks. Before use, soil samples were equilibrated to room temperature for 3–4 days. Some physical and chemical characteristics are given in Table 1. 2.2. P-extraction A sequential extraction method, described by Hedley and Stewart Ž 1982. and modified by Potter et al. Ž1991. was used. This extraction allows the fractionation of biologically meaningful soil P-pools. Interpretations of the different forms of soil P identified by the sequential fractionation scheme are given in Table 2. All extraction steps were carried out in duplicate. Resin extractable P was measured by the method of Sibbesen Ž1978.. One gram anion exchange resin ŽAmberlite IRA 420, 20–50 mesh. in a nylon mesh bag Ž4 = 4 cm. was placed in a 50 ml Schott bottle that contained 30 ml of distilled water and 1 g of soil d.w. After shaking for 16 h Žend over end. at room temperature, P adsorbed by the resin was displaced by 30 ml of 0.5 M HCl and quantified. Soil was recovered by centrifugation Ž 08C, 8000 = g, 10 min.. One of the duplicate samples was treated with 1 ml CHCl 3 , recapped and shaken intermittently for 1 h; the CHCl 3 was allowed to evaporate overnight in a fume hood. Both samples were then extracted with 0.5 M NaHCO 3 Ž 30 ml, pH 8.5. for 16 h. CHCl 3fumigated and unfumigated samples were sequentially extracted Ž16 h, end over end shaking. with 0.1 M NaOH, sonicated Ž 2 min. and reextracted with 0.1 M
Table 1 Chemical and biological characterization of the soils Sampling time Moisture w%x pH Žwater. pH ŽKCl. C tot w%x Ždry combustion at 9008C. C org w%x Ždry combustion at 6008C. C mic wmg C gy1 d.m.x b Ntot w%x ŽKjeldahl. Nmic wmg N gy1 d.m.x ŽCFEM. a Ptot w%x Žacid digestion. Pmic wmg P gy1 d.m.x ŽCFEM. a K tot w%x Žacid digestion. qCO 2 wmgCO 2 –C gy1 C mic x b Basal respiration wmg CO 2 gy1 d.m. hy1 x b a
July 1991
November 1991
April 1992
63.4 7.4 7.0 27.2 22.8 461 1.25 n.d. 0.046 87.7 0.31 1.57 2.56
67.7 7.5 7.0 29.2 25.1 387 1.36 n.d. 0.033 74.3 0.22 1.88 2.67
60.0 7.6 7.3 24.6 19.1 424 1.00 333.0 0.050 87.1 0.43 1.52 2.14
Brookes et al. Ž1985.. Anderson and Domsch Ž1978.. n.d.—Not determined. C mic , Nmic , Pmic —Microbial biomass C, N and P, respectively.
b
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Fraction
Treatment
Comments
1 2a
Extraction with an anion exchange resin Extraction with 0.5 M NaHCO 3
2b 3
Chloroform-fumigation followed by extraction with 0.5 M NaHCO 3 Extraction with 0.1 M NaOH
4
Extraction with 0.1 M NaOH, following ultrasonification
5 6
Extraction with 1 N HCl Acid digestion of residual P
Biologically available, soluble inorganic P Labile inorganic and organic P sorbed on soil minerals plus a small amount of microbial P The difference between 2b and 2a represents microbial P Inorganic and organic compounds, held more strongly by chemisorption to Fe and Al components of soil surfaces Inorganic and organic P held on internal surfaces of soil aggregates Removes mainly apatite P Stable organic P forms and relatively insoluble inorganic P compounds
M. Demetz, H. Insam r Geoderma 89 (1999) 259–271
Table 2 Different P fractions obtained by sequential extraction ŽHedley and Stewart, 1982.
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NaOH and finally with 1 N HCl. The soil:extractant ratio was 1:30. To clear the NaOH extracts, the method of Potter et al. Ž1991. was used. After each extraction, the soil was recovered for the subsequent extraction by centrifuging; the supernatant was saved for analyses. A total of 100 mg of the residual material remaining at the end of the extractions were digested with 2 ml concentrated H 2 SO4 and H 2 O 2 at 1708C ŽVogler, 1965. . An aliquot of the original sample Žoven dried and finely ground. also was digested using the H 2 SO4rH 2 O 2 method Ž Vogler, 1965. to determine total P. This estimate should be "5% of the total P calculated as the sum of all P fractions ŽPotter et al., 1991. . 2.3. P determination The ascorbic acidrmolybdate method Ž modified by Vogler, 1965. was applied to measure the orthophosphate concentration in the neutralized extracts and digests. Dark colours in the extracts Ž bicarbonate and NaOH fraction. caused interferences with absorption. Therefore, extracts were acidified to pH 2, cooled to 48C for 1–2 h and then centrifuged Ž 08C, 8000 = g, 10 min.. This procedure resulted in flocculation of humic and fulvic acids, which were removed by centrifugation and produced solutions of sufficient clarity for standard colorimetric determination of orthophosphate Ž Potter et al., 1991. . 2.4. Respiration measurements Moistened soil samples Ž 40–60% of WHC. were amended with 1% glucose and different amounts of P Ž as Na 2 HPO4 . . The concentrations of added P were based on the classification scheme of Kuntze et al. Ž 1981. Ž Table 3. . For the lowest concentration, Na 2 HPO4 was mixed with Talcum as a carrier Ž 1:4. to ensure good distribution in the soil.
Table 3 Classification of P concentrations in soils ŽKuntze et al., 1981. and amounts added to the samples for the respiration assays Classification
Concentration of soil P wmg gy1 x
Qualitative evaluation
P added wmg gy1 x
Na 2 HPO4 wmg gy1 x
A B C D E
- 22 24–44 45–65 67–87 )87
insufficient poor optimum luxury slight surplus surplus surplus
11 30 45 110 150 200 300
50 140 200 500 700 900 1400
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The amended soil samples were connected to a continuously purged measurement device based on IR-gas analysis Ž Heinemeyer et al., 1989. . Carbon dioxide release was measured hourly for 30 h at 228C. 2.5. Analysis of respiration curÕes Three methods were used for the analyses of respiration curves. Ži. ‘slope’-method: From the CO 2 release after addition of glucose and various amounts of P, basal respiration was subtracted. The ‘net’ respiration rate, induced by C and P amendments, was converted into cumulative data. The cumulative curves were presented in diagrams Ž time vs. mg CO 2 ., and the gradient b at the point of inflection of each curve was calculated Ž n s 6.. A linear correlation was calculated between b and the amount of P added. By interpolation, the amount of P available in the sample that had received only glucose was calculated. Žii. ‘area’-method: After subtraction of basal respiration, the area A of the curves above a baseline given by the initial response Ž SIR, hours 1–4. was calculated. By interpolating the regression line of A and the amount of P added, the pool of bioavailable P was calculated. Žiii. ‘Nordgren’-method: The calculations suggested by Nordgren Ž 1992. were applied to the data from this study and the results compared with those of calculation Ži. and Ž ii.. 3. Results and discussion 3.1. P-extraction The sequential extraction method resulted in different amounts of P corresponding to the different P pools in the soil ŽTable 4.. The anion exchange resin
Table 4 P concentrations found in the different fractions Ž ns12. of a sequential extraction Extract
July y1
mg P g Resin NaHCO 3 Microbial P NaOH 1 NaOH 2 HCl Sum Acid digest of residual P a Total P determined a
November
April
4.1"0.2 10.5"0.4 71.6"0.2 22.6"0.3 15.1"0.6 14.2"0.3 138.0 192.0 330.0
4.5"0.4 4.7"0.1 85.9"0.6 22.2"0.6 16.7"0.4 18.6"0.3 152.4 347.6 500.0
d.m."SE
3.4"0.2 10.1"0.2 88.2"0.1 33.4"0.3 19.0"0.8 24.2"0.4 178.2 281.8 460.0
This value was calculated as difference from total P and the sum of the single extractions.
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removed the most labile inorganic P. The resin functions like a plant root with a very high capacity for P uptake; therefore this P fraction may be regarded as the bioavailable fraction Ž Sibbesen, 1978. . It ranged from 0.7% to 1.8% of the total P. The second fraction, obtained by NaHCO 3 extraction, is assumed to contain the labile inorganic and organic P sorbed on soil minerals plus a small amount of microbial P ŽHedley and Stewart, 1982. . NaHCO 3 is a very suitable extractant for calcareous soils with a high amount of CaCO 3 , because the solubility of Ca-phosphates in this fraction depends on the Ca2q concentration in the soil, which is decreased in the presence of NaHCO 3. Thus, the main effect of the NaHCO 3 on acid and neutral soils probably would be through ionic competition of HCO 3-, CO 3- and OH-ions for phosphate adsorbed on the surface of soil particles Ž Olsen et al., 1954. . The lowest concentration was found in the April samples Žthe season with a very high nutrient uptake due to plant growth and increased microbial biomass; Table 1. . The amount of microbial P was estimated by the fumigation–extraction method ŽHedley and Stewart, 1982. ; it was 17–22% of the total P Ž72–88 mg Pmic gy1 d.m... These figures correspond well with those of Diaz-Ravina et al. Ž1993. who found microbial P ranging from 33 to 187 mg P gy1 d.m. depending on soil types and biomass C. C mic and Pmic were positively correlated. The C mic :Pmic-ratio was 5.3, 5.2 and 4.7 for the July, November and April soils, respectively. This relationship shows the important role of soil microorganisms for immobilizing relatively high amounts of P in their biomass ŽMarumoto et al., 1982a,b; Diaz-Ravina et al., 1993. . However, SIR most likely underestimates C mic in acid forest soils, e.g., C mic measured by fumigation–extraction was about twice as high as SIR-C mic in a southern beech forest soil Ž Ross and Tate, 1993.. This may be attributed to a higher fungi-to-bacteria ratio than in the agricultural soils originally used for the calibration of SIR Ž Anderson and Domsch, 1978.. The first extraction with NaOH removed inorganic and organic compounds held more strongly by chemisorption to Fe and Al components of soil surfaces. In the fumigated samples, we found 40% more P than in the unfumigated ones. Therefore, it can be assumed that part of this fraction was biomass P. Thus, presumably some P released from microbial cells after CHCl 3 treatment was adsorbed by soil components ŽHedley and Stewart, 1982.. The second NaOH extraction following ultrasonification yields inorganic and organic P held at internal surfaces of soil aggregates Ž Potter et al., 1991. . The differences between fumigated and unfumigated samples were insignificant in this fraction. The dark colour of both NaOH extracts may be attributed to stable organic phosphate compounds, which reacted with existing humic and fulvic acids and NaOH ŽPsenner et al., 1984. . Finally, after HCl extraction, all Ca- and Mg-phosphates and, if present, apatite, Fe- and Al-phosphates, were removed. A concentration of 14–24 mg P gy1 d.m. was found. The sum of P from fractions
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1–5 gave a percentage of 36–45% of total P in the soil Ža further acid digestion of the residual after HCl extraction was not made. . 3.2. Respiration measurements After glucose addition, soil respiration immediately increased 2.5 fold. Thereafter, the curves of the glucose amended samples ran parallel to the basal respiration curves. When the glucose was exhausted Ž after 24–28 h. , respiration declined. The admixture of N and glucose resulted in the same pattern Ž data not shown., suggesting that some factor other than N or C was limiting. This was not so for the admixture of P and glucose when even a small addition of P Ž 11 mg gy1 . increased soil respiration. CO 2 evolution in this case correlated positively with the amount of added P ŽFig. 1.. With the July sample, P concentrations higher than 2 mg gy1 d.m. did not increase respiration further, which may be due to substrate inhibition or another limiting nutrient Ž Fig. 1. . Foster et al. Ž 1980. also observed this phenomenon in an N limited forest soil, and they attributed the decrease in respiration after addition of N to toxic effects of the added N. The respiration curves of glucose and P amended samples initially had negative slopes Žtype I-curves; Anderson and Domsch, 1978. : there was a slight drop in CO 2 efflux rate for several hours, followed by a constant increase. In the July and April samples, the minimum occurred within 3–4 h; in the November soil the increase began only after 8 h. Such curves often have been found for soils with large amounts of biomass or high organic matter levels Ž Anderson and Domsch, 1978.. Maximum CO 2 release occurred within 18–24 h depending on amount of P added. The more P added, the earlier peaks appeared. The exception were the November samples with respiration maxima after exactly 20 h, regardless of amounts of P added. The rate of decrease was approximately the same for all levels of P. These results indicate that good P supply made possible rapid microbial growth which resulted in earlier exhaustion of glucose than when glucose was added alone ŽStotzky and Norman, 1961. , because the curves with P addition were lower than the glucose curves after 40 h. The highest CO 2 evolution rate was obtained in the November sample with a maximum of 401 mg CO 2 gy1 d.m. The July and April samples showed a respiration maximum of 204 mg CO 2 gy1 d.m. An explanation for the high respiration in the November sample is that in November a large fraction of the microorganisms supposedly were in a dormant state, but optimal temperature Ž 228C. and nutrient conditions in the laboratory stimulated their activity and biomass synthesis. The low biomass and relatively high C and N pools found in the November sample ŽTable 1. support this assumption. 3.3. CurÕe analysis and interpretation Ži. All the data for CO 2 release within 30 h were corrected for basal respiration and, according to Blet-Charaudeau et al. Ž1990. and Amador and
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Fig. 1. Respiration rate Žleft. and cumulative CO 2 evolution Žright. of the July Ža, b., November Žc, d. and April Že, f. samples after addition of 1% glucose and different amounts of P ŽU given in mg Na 2 HPO4 ..
Jones Ž1993. , converted into cumulative data. While only glucose addition resulted in a linear function Ž r - 0.99. , glucose plus P addition resulted in typical sigmoidal growth curves Ž Fig. 1. . The slope of these curves was dependent on the amount of P added. A particularly rapid increase followed by a distinct plateau was obtained with the November sample. Therefore, it can be concluded that microbial biomass in November can be easily stimulated if moisture and nutrient conditions are improved. For each sample, exponential growth rate Žslope b at the point of inflection. was related to the corresponding P amendment in a semilogaritmic diagram resulting in a linear regression
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Fig. 2. Gradient b Žslope gradient in the turning point of cumulative curves. plotted against the amount of P added to the samples. The value y indicates the amount of bioavailable P estimated by interpolation.
Ž r ) 0.99; Fig. 2.. Bioavailable P was estimated by interpolation of the gradient b from the respiration curve obtained after glucose amendment Žwithout P supply. ŽTable 5.. For the July soil, no interpolation was made, because the chosen P additions were too high. Žii. In the second approach, the area below the respiration curves Ž equivalent to the CO 2 release during 30 h. was calculated as described above. The area was closely related to the amount of P added ŽFig. 3.. Bioavailable P was estimated by interpolation, the results are given in Table 5.
Table 5 Amounts of available P determined with different methods Resin extraction y1
mg P g July November April a
3.42 4.08 4.46
Extraction with 0.5 M Na 2 HCO 3
I ‘slope’
Bioassay II ‘area’
III ‘Nordgren’
10.06 10.47 4.70
n.d. 9.99 7.51
2.85 11.20 2.06
n. d. 140 a r80 a 370r210
d.m.
The left values indicate the ‘total microbially available P’, the values to the right indicate the ‘SIR-corrected microbially available P’ ŽNordgren, 1992.. n.d.—Not determined.
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Fig. 3. The ‘area’ below the respiration curves Žamount of CO 2 evolved during 30 h. plotted against the amount of P added to the samples. The value y indicates the amount of bioavailable P estimated by interpolation.
Žiii. Finally, the approach suggested by Nordgren Ž 1992. was applied to the present data. The results obtained were very different from the results of the other two approaches Ž Table 5.. The ‘total microbially available P’ was 140 and 37 mg P gy1 d.m. and the ‘SIR-corrected microbially available P’ 80 and 210 mg P gy1 d.m. for the November and April soil, respectively. Nordgren Ž 1992. obtained convincing results with an N-limited, acid Ž pH s 4., highly organic forest soil Žmore layer of a podzolic soil. , but his approach was not suited for the soils studied here. In Table 5, the results of the two weakest extractants are compared with the results of the bioassay P determinations. In this experiment, it was assumed that microbially available P corresponds to plant available P. Thus, P in soil is present in several inorganic Ž Pi . and organic ŽPo . forms, but is absorbed by plants almost exclusively as Pi . Complex Po compounds are important sources for plant P that must be mineralized into Pi before plant absorption takes place ŽAnderson, 1980; Yang and Jacobsen, 1990.. The anion exchange resin method is assumed to assess plant available P, because the resin functions like a plant root with a very high capacity for P uptake ŽSibbesen, 1978.. Considering a recovery of only 87–90% of the added P Ždata not shown. , the estimates given in Table 5 may be somewhat low. Hence, it can be supposed that not all of the available P was extracted from the soil. The sodium bicarbonate extraction for P ŽOlsen et al., 1954. is also an accepted method for estimating P availability in soil. It generally removes the labile
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inorganic and organic P, but according to Hedley and Stewart Ž 1982. , some microbial P may also be removed by this extraction. Results therefore overestimate available P. So it can be assumed that the plant available P should lie somewhere between that given by the resin- and the Olsen-P methods. The results of the approaches I and II corresponded to the results obtained with the weak extractants Žion exchange resins and Na 2 HCO 3 . . Approach II resulted in available P concentrations that were slightly lower than the Na 2 HCO 3 extraction, and partly higher than the resin method. The slope method resulted in slightly higher values. With the bioassay approach, the highest bioavailable P was found for the November samples, while no differences were found with resin extracts. Enhanced P availability for the November samples is in accordance with studies of Adams et al. Ž 1989. , who found that most pools of available N and P vary with season, reaching a maximum in the fall. The results suggest that the respiratory approach taken may be useful for determining bioavailable P in forest soils. The results support the findings of Nordgren Ž1992., but suggest other mathematical approaches. If not used for quantitative estimation of bioavailable P Ž which is laborious since P has to be added in different amounts., the respiratory assay can quickly provide qualitative information on P or N limitation for the soil microbiota.
References Adams, M.A., Attiwill, P.M., Polglase, P.J., 1989. Availability of nitrogen and phosphorus in forest soils in northeastern Tasmania. Biol. Fertil. Soils 8, 212–218. Amador, J.A., Jones, R.D., 1993. Nutrient limitations on microbial respiration in peat soils with different total phosphorus content. Soil Biol. Biochem. 25, 793–801. Anderson, G., 1980. Assessing organic phosphorus in soils. In: Khasawneh, F.E., et al. ŽEds.., The Role of Phosphorus in Agriculture. ASA, CSSA and SSSA, Madison, WI, pp. 411–431. Anderson, J.P.E., Domsch, K.H., 1978. A physiological method for the quantitative measurement of microbial biomass in soil. Soil Biol. Biochem. 10, 215–221. Blet-Charaudeau, C., Muller, J., Laudelout, H., 1990. Kinetics of carbon dioxide evolution in relation to microbial biomass and temperature. Soil Sci. Soc. Am. J. 54, 1324–1328. Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation and the release of soil nitrogen, a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842. Diaz-Ravina, M., Acea, M.J., Carballas, T., 1993. Microbial biomass and its contribution to nutrient concentrations in forest soils. Soil Biol. Biochem. 25, 25–31. Foster, N.W., Beauchamp, E.G., Corke, C.T., 1980. Microbial activity in a Pinus banksiana Lamb. forest floor amended with nitrogen and carbon. Can. J. Soil Sci. 60, 199–209. Hedley, M.J., Stewart, J.W.B., 1982. Method to measure microbial phosphate in soils. Soil Biol. Biochem. 14, 377–385. Heinemeyer, O., Insam, H., Kaiser, E.A., Walenzik, G., 1989. Soil microbial biomass and respiration measurements: an automated technique based on infra-red gas analysis. Plant Soil 116, 191–195.
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Kuntze, H., Niemann, J., Roeschmann, G., Schwerdtfeger, G., 1981. Bodenkunde. Ulmer, Stuttgart. Marumoto, T., Anderson, J.P.E., Domsch, K.H., 1982a. Decomposition of 14C- and 15 N-labelled microbial cells in soils. Soil Biol. Biochem. 14, 461–467. Marumoto, T., Anderson, J.P.E., Domsch, K.H., 1982b. Mineralization of nutrients from soil microbial biomass. Soil Biol. Biochem. 14, 469–475. Nordgren, A., 1992. A method for determining microbially available N and P in an organic soil. Biol. Fertil. Soils 13, 195–199. Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., 1954. Estimation of available phosphorus in soils by extraction with sodium bicarbonate. Circular 939, US Dept. of Agric., Washington, pp. 1–19. Potter, R.L., Jordan, C.F., Guedes, R.M., Batmanian, G.J., Han, X.G., 1991. Assessment of a phosphorus fractionation method for soils: problems for further investigation. Agric. Ecosyst. Environ. 34, 453–463. Psenner, R., Pucsko, R., Sager, M., 1984. Die Fraktionierung organischer und anorganischer Phosphorverbindungen von Sedimenten. Arch. Hydrobiol. 70, 111–155. Ross, D.J., Tate, K.R., 1993. Microbial C and N in litter and soil of a southern beech Ž Nothofagus. forest: comparison of measurement procedures. Soil Biol. Biochem. 25, 467–475. Schmidt, J.P., Buol, S.W., Kamprath, E.J., 1997. Soil phosphorus dynamics during 17 years of continuous cultivation: a method to estimate long-term P availability. Geoderma 78, 59–70. Sibbesen, E., 1978. An investigation of the anion-exchange resin method for soil phosphate extraction. Plant Soil 50, 305–321. Stotzky, G., Norman, A.G., 1961. Factors limiting microbial activities in soil: I. The level of substrate, nitrogen, and phosphorus. Arch. Mikrobiol. 40, 341–369. Vogler, P., 1965. Probleme der Phosphoranalytik in der Limnologie und ein neues Verfahren zur Bestimmung von gelostem Orthophosphat neben kondensierten Phosphaten und organischen ¨ Phosphorsaureestern. Int. Rev. Hydrobiol. 50, 33–48. ¨ Yang, J.E., Jacobsen, J.S., 1990. Soil inorganic phosphorus fractions and their uptake relationships in calcareous soils. Soil Sci. Soc. Am. J. 54, 1666–1669.
Phosphorus availability in a forest soil determined with a respiratory assay compared to chemical methods M. Demetz, H. Insam
)
Institut fur ¨ Mikrobiologie, UniÕersity of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria Received 3 April 1998; accepted 14 August 1998
Abstract A forest soil from the Northern Calcareous Alps of Austria was examined with different methods for determining P content and availability. The aim of this study was to compare resin extractable and bicarbonate Žor Olsen. P obtained by a sequential extraction method with bioavailable P as determined with a bioassay based on the kinetics of soil respiration induced by addition of a carbon source and nutrients. With two indices ŽCO 2 evolution within 30 h, and the slope of the growth curves. good agreement between the chemical and respiratory methods was found. The respiratory method appeared to be suitable for estimating the pool of bioavailable P for microorganisms and for a fast qualitative detection of P deficiency. q 1999 Elsevier Science B.V. All rights reserved. Keywords: phosphorus availability; substrate induced respiration; soil microbial biomass; forest; respiration
1. Introduction In several ecosystems of the world, for example, in alpine forests, phosphorus may be limiting for plant production. However, estimates of P availability are often dubious since there is great uncertainty if the measured pools are relevant for plant production. Of the various approaches, some are considered suited to )
Corresponding author. Tel.: q43-512-507-6009; Fax: q43-512-507-2928; E-mail: [email protected] 0016-7061r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 6 - 7 0 6 1 Ž 9 8 . 0 0 0 9 0 - 1
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estimate bioavailable P, such as the extraction with sodium bicarbonate Ž Olsen et al., 1954. or the anion exchange resin method ŽSibbesen, 1978; Schmidt et al., 1997.. However, intensely coloured extracts, precipitations or large amounts of iron and aluminium compounds and organic matter, often limit the validity and reproducibility of these tests Ž Sibbesen, 1978. , especially in acid organic soils. Numerous studies on the respiratory response following glucose amendment to soils have been made, especially since Anderson and Domsch Ž 1978. proposed the substrate induced respiration Ž SIR. method for determining soil microbial biomass. Usually, upon glucose amendment, the respiration rate increases within a few minutes Ž SIR. to a value that is directly proportional to the microbial biomass present. In most cases, after a lag phase of a few hours, the respiration rate further increases. This increase is attributed to microbial growth resulting from the newly available C source Ž glucose. . When the substrate is exhausted, CO 2 evolution starts to decline. However, in some soils glucose addition fails to induce a further increase of CO 2-production after the initial flush. This has been observed for temperate forests Ž Nordgren, 1992; K.H. Domsch, pers. comm.. as well as for tropical forests Ž J. Lodge, pers. comm.. and was attributed to nutrient limitation. Only when nutrients were added with glucose, was the usual increase in respiration found. Nordgren Ž 1992. proposed a bioassay for determination of forest soil N and P availability based on interpreting the respiration curves after amending different amounts of N or P. He argued that if defined amounts of added nutrients would alleviate growth restrictions, it should be possible to obtain figures of endogenous nutrient availability. The aim of the present study was to compare P pools determined with sequential extraction Ž Hedley and Stewart, 1982. with estimates of bioavailable P determined by a bioassay based on respiratory response upon glucose and nutrient addition.
2. Materials and methods 2.1. Soil The soil for this study was a poorly developed mull-rendzina under mixed beech–spruce forest in the Northern Calcareous Alps of Austria Ž 47823X N, 10850X W.. The climate of the study site is typical for the Northern Alps with cold, snowy winters Žmean January temperature - y48C. and cool, rainy summers Ž mean July temperature approx. 168C. . The mean annual temperature and precipitation are 5.78C and 1218 mm, respectively. Soil samples were collected in July and November 1991 and April 1992 from the 5 to 25 cm layer ŽA h-horizon.. Soil was sieved Ž2 mm. to remove larger roots and animals and stored field moist at 48C in polyethylene bags for up to 3
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weeks. Before use, soil samples were equilibrated to room temperature for 3–4 days. Some physical and chemical characteristics are given in Table 1. 2.2. P-extraction A sequential extraction method, described by Hedley and Stewart Ž 1982. and modified by Potter et al. Ž1991. was used. This extraction allows the fractionation of biologically meaningful soil P-pools. Interpretations of the different forms of soil P identified by the sequential fractionation scheme are given in Table 2. All extraction steps were carried out in duplicate. Resin extractable P was measured by the method of Sibbesen Ž1978.. One gram anion exchange resin ŽAmberlite IRA 420, 20–50 mesh. in a nylon mesh bag Ž4 = 4 cm. was placed in a 50 ml Schott bottle that contained 30 ml of distilled water and 1 g of soil d.w. After shaking for 16 h Žend over end. at room temperature, P adsorbed by the resin was displaced by 30 ml of 0.5 M HCl and quantified. Soil was recovered by centrifugation Ž 08C, 8000 = g, 10 min.. One of the duplicate samples was treated with 1 ml CHCl 3 , recapped and shaken intermittently for 1 h; the CHCl 3 was allowed to evaporate overnight in a fume hood. Both samples were then extracted with 0.5 M NaHCO 3 Ž 30 ml, pH 8.5. for 16 h. CHCl 3fumigated and unfumigated samples were sequentially extracted Ž16 h, end over end shaking. with 0.1 M NaOH, sonicated Ž 2 min. and reextracted with 0.1 M
Table 1 Chemical and biological characterization of the soils Sampling time Moisture w%x pH Žwater. pH ŽKCl. C tot w%x Ždry combustion at 9008C. C org w%x Ždry combustion at 6008C. C mic wmg C gy1 d.m.x b Ntot w%x ŽKjeldahl. Nmic wmg N gy1 d.m.x ŽCFEM. a Ptot w%x Žacid digestion. Pmic wmg P gy1 d.m.x ŽCFEM. a K tot w%x Žacid digestion. qCO 2 wmgCO 2 –C gy1 C mic x b Basal respiration wmg CO 2 gy1 d.m. hy1 x b a
July 1991
November 1991
April 1992
63.4 7.4 7.0 27.2 22.8 461 1.25 n.d. 0.046 87.7 0.31 1.57 2.56
67.7 7.5 7.0 29.2 25.1 387 1.36 n.d. 0.033 74.3 0.22 1.88 2.67
60.0 7.6 7.3 24.6 19.1 424 1.00 333.0 0.050 87.1 0.43 1.52 2.14
Brookes et al. Ž1985.. Anderson and Domsch Ž1978.. n.d.—Not determined. C mic , Nmic , Pmic —Microbial biomass C, N and P, respectively.
b
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Fraction
Treatment
Comments
1 2a
Extraction with an anion exchange resin Extraction with 0.5 M NaHCO 3
2b 3
Chloroform-fumigation followed by extraction with 0.5 M NaHCO 3 Extraction with 0.1 M NaOH
4
Extraction with 0.1 M NaOH, following ultrasonification
5 6
Extraction with 1 N HCl Acid digestion of residual P
Biologically available, soluble inorganic P Labile inorganic and organic P sorbed on soil minerals plus a small amount of microbial P The difference between 2b and 2a represents microbial P Inorganic and organic compounds, held more strongly by chemisorption to Fe and Al components of soil surfaces Inorganic and organic P held on internal surfaces of soil aggregates Removes mainly apatite P Stable organic P forms and relatively insoluble inorganic P compounds
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Table 2 Different P fractions obtained by sequential extraction ŽHedley and Stewart, 1982.
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NaOH and finally with 1 N HCl. The soil:extractant ratio was 1:30. To clear the NaOH extracts, the method of Potter et al. Ž1991. was used. After each extraction, the soil was recovered for the subsequent extraction by centrifuging; the supernatant was saved for analyses. A total of 100 mg of the residual material remaining at the end of the extractions were digested with 2 ml concentrated H 2 SO4 and H 2 O 2 at 1708C ŽVogler, 1965. . An aliquot of the original sample Žoven dried and finely ground. also was digested using the H 2 SO4rH 2 O 2 method Ž Vogler, 1965. to determine total P. This estimate should be "5% of the total P calculated as the sum of all P fractions ŽPotter et al., 1991. . 2.3. P determination The ascorbic acidrmolybdate method Ž modified by Vogler, 1965. was applied to measure the orthophosphate concentration in the neutralized extracts and digests. Dark colours in the extracts Ž bicarbonate and NaOH fraction. caused interferences with absorption. Therefore, extracts were acidified to pH 2, cooled to 48C for 1–2 h and then centrifuged Ž 08C, 8000 = g, 10 min.. This procedure resulted in flocculation of humic and fulvic acids, which were removed by centrifugation and produced solutions of sufficient clarity for standard colorimetric determination of orthophosphate Ž Potter et al., 1991. . 2.4. Respiration measurements Moistened soil samples Ž 40–60% of WHC. were amended with 1% glucose and different amounts of P Ž as Na 2 HPO4 . . The concentrations of added P were based on the classification scheme of Kuntze et al. Ž 1981. Ž Table 3. . For the lowest concentration, Na 2 HPO4 was mixed with Talcum as a carrier Ž 1:4. to ensure good distribution in the soil.
Table 3 Classification of P concentrations in soils ŽKuntze et al., 1981. and amounts added to the samples for the respiration assays Classification
Concentration of soil P wmg gy1 x
Qualitative evaluation
P added wmg gy1 x
Na 2 HPO4 wmg gy1 x
A B C D E
- 22 24–44 45–65 67–87 )87
insufficient poor optimum luxury slight surplus surplus surplus
11 30 45 110 150 200 300
50 140 200 500 700 900 1400
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The amended soil samples were connected to a continuously purged measurement device based on IR-gas analysis Ž Heinemeyer et al., 1989. . Carbon dioxide release was measured hourly for 30 h at 228C. 2.5. Analysis of respiration curÕes Three methods were used for the analyses of respiration curves. Ži. ‘slope’-method: From the CO 2 release after addition of glucose and various amounts of P, basal respiration was subtracted. The ‘net’ respiration rate, induced by C and P amendments, was converted into cumulative data. The cumulative curves were presented in diagrams Ž time vs. mg CO 2 ., and the gradient b at the point of inflection of each curve was calculated Ž n s 6.. A linear correlation was calculated between b and the amount of P added. By interpolation, the amount of P available in the sample that had received only glucose was calculated. Žii. ‘area’-method: After subtraction of basal respiration, the area A of the curves above a baseline given by the initial response Ž SIR, hours 1–4. was calculated. By interpolating the regression line of A and the amount of P added, the pool of bioavailable P was calculated. Žiii. ‘Nordgren’-method: The calculations suggested by Nordgren Ž 1992. were applied to the data from this study and the results compared with those of calculation Ži. and Ž ii.. 3. Results and discussion 3.1. P-extraction The sequential extraction method resulted in different amounts of P corresponding to the different P pools in the soil ŽTable 4.. The anion exchange resin
Table 4 P concentrations found in the different fractions Ž ns12. of a sequential extraction Extract
July y1
mg P g Resin NaHCO 3 Microbial P NaOH 1 NaOH 2 HCl Sum Acid digest of residual P a Total P determined a
November
April
4.1"0.2 10.5"0.4 71.6"0.2 22.6"0.3 15.1"0.6 14.2"0.3 138.0 192.0 330.0
4.5"0.4 4.7"0.1 85.9"0.6 22.2"0.6 16.7"0.4 18.6"0.3 152.4 347.6 500.0
d.m."SE
3.4"0.2 10.1"0.2 88.2"0.1 33.4"0.3 19.0"0.8 24.2"0.4 178.2 281.8 460.0
This value was calculated as difference from total P and the sum of the single extractions.
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removed the most labile inorganic P. The resin functions like a plant root with a very high capacity for P uptake; therefore this P fraction may be regarded as the bioavailable fraction Ž Sibbesen, 1978. . It ranged from 0.7% to 1.8% of the total P. The second fraction, obtained by NaHCO 3 extraction, is assumed to contain the labile inorganic and organic P sorbed on soil minerals plus a small amount of microbial P ŽHedley and Stewart, 1982. . NaHCO 3 is a very suitable extractant for calcareous soils with a high amount of CaCO 3 , because the solubility of Ca-phosphates in this fraction depends on the Ca2q concentration in the soil, which is decreased in the presence of NaHCO 3. Thus, the main effect of the NaHCO 3 on acid and neutral soils probably would be through ionic competition of HCO 3-, CO 3- and OH-ions for phosphate adsorbed on the surface of soil particles Ž Olsen et al., 1954. . The lowest concentration was found in the April samples Žthe season with a very high nutrient uptake due to plant growth and increased microbial biomass; Table 1. . The amount of microbial P was estimated by the fumigation–extraction method ŽHedley and Stewart, 1982. ; it was 17–22% of the total P Ž72–88 mg Pmic gy1 d.m... These figures correspond well with those of Diaz-Ravina et al. Ž1993. who found microbial P ranging from 33 to 187 mg P gy1 d.m. depending on soil types and biomass C. C mic and Pmic were positively correlated. The C mic :Pmic-ratio was 5.3, 5.2 and 4.7 for the July, November and April soils, respectively. This relationship shows the important role of soil microorganisms for immobilizing relatively high amounts of P in their biomass ŽMarumoto et al., 1982a,b; Diaz-Ravina et al., 1993. . However, SIR most likely underestimates C mic in acid forest soils, e.g., C mic measured by fumigation–extraction was about twice as high as SIR-C mic in a southern beech forest soil Ž Ross and Tate, 1993.. This may be attributed to a higher fungi-to-bacteria ratio than in the agricultural soils originally used for the calibration of SIR Ž Anderson and Domsch, 1978.. The first extraction with NaOH removed inorganic and organic compounds held more strongly by chemisorption to Fe and Al components of soil surfaces. In the fumigated samples, we found 40% more P than in the unfumigated ones. Therefore, it can be assumed that part of this fraction was biomass P. Thus, presumably some P released from microbial cells after CHCl 3 treatment was adsorbed by soil components ŽHedley and Stewart, 1982.. The second NaOH extraction following ultrasonification yields inorganic and organic P held at internal surfaces of soil aggregates Ž Potter et al., 1991. . The differences between fumigated and unfumigated samples were insignificant in this fraction. The dark colour of both NaOH extracts may be attributed to stable organic phosphate compounds, which reacted with existing humic and fulvic acids and NaOH ŽPsenner et al., 1984. . Finally, after HCl extraction, all Ca- and Mg-phosphates and, if present, apatite, Fe- and Al-phosphates, were removed. A concentration of 14–24 mg P gy1 d.m. was found. The sum of P from fractions
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1–5 gave a percentage of 36–45% of total P in the soil Ža further acid digestion of the residual after HCl extraction was not made. . 3.2. Respiration measurements After glucose addition, soil respiration immediately increased 2.5 fold. Thereafter, the curves of the glucose amended samples ran parallel to the basal respiration curves. When the glucose was exhausted Ž after 24–28 h. , respiration declined. The admixture of N and glucose resulted in the same pattern Ž data not shown., suggesting that some factor other than N or C was limiting. This was not so for the admixture of P and glucose when even a small addition of P Ž 11 mg gy1 . increased soil respiration. CO 2 evolution in this case correlated positively with the amount of added P ŽFig. 1.. With the July sample, P concentrations higher than 2 mg gy1 d.m. did not increase respiration further, which may be due to substrate inhibition or another limiting nutrient Ž Fig. 1. . Foster et al. Ž 1980. also observed this phenomenon in an N limited forest soil, and they attributed the decrease in respiration after addition of N to toxic effects of the added N. The respiration curves of glucose and P amended samples initially had negative slopes Žtype I-curves; Anderson and Domsch, 1978. : there was a slight drop in CO 2 efflux rate for several hours, followed by a constant increase. In the July and April samples, the minimum occurred within 3–4 h; in the November soil the increase began only after 8 h. Such curves often have been found for soils with large amounts of biomass or high organic matter levels Ž Anderson and Domsch, 1978.. Maximum CO 2 release occurred within 18–24 h depending on amount of P added. The more P added, the earlier peaks appeared. The exception were the November samples with respiration maxima after exactly 20 h, regardless of amounts of P added. The rate of decrease was approximately the same for all levels of P. These results indicate that good P supply made possible rapid microbial growth which resulted in earlier exhaustion of glucose than when glucose was added alone ŽStotzky and Norman, 1961. , because the curves with P addition were lower than the glucose curves after 40 h. The highest CO 2 evolution rate was obtained in the November sample with a maximum of 401 mg CO 2 gy1 d.m. The July and April samples showed a respiration maximum of 204 mg CO 2 gy1 d.m. An explanation for the high respiration in the November sample is that in November a large fraction of the microorganisms supposedly were in a dormant state, but optimal temperature Ž 228C. and nutrient conditions in the laboratory stimulated their activity and biomass synthesis. The low biomass and relatively high C and N pools found in the November sample ŽTable 1. support this assumption. 3.3. CurÕe analysis and interpretation Ži. All the data for CO 2 release within 30 h were corrected for basal respiration and, according to Blet-Charaudeau et al. Ž1990. and Amador and
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Fig. 1. Respiration rate Žleft. and cumulative CO 2 evolution Žright. of the July Ža, b., November Žc, d. and April Že, f. samples after addition of 1% glucose and different amounts of P ŽU given in mg Na 2 HPO4 ..
Jones Ž1993. , converted into cumulative data. While only glucose addition resulted in a linear function Ž r - 0.99. , glucose plus P addition resulted in typical sigmoidal growth curves Ž Fig. 1. . The slope of these curves was dependent on the amount of P added. A particularly rapid increase followed by a distinct plateau was obtained with the November sample. Therefore, it can be concluded that microbial biomass in November can be easily stimulated if moisture and nutrient conditions are improved. For each sample, exponential growth rate Žslope b at the point of inflection. was related to the corresponding P amendment in a semilogaritmic diagram resulting in a linear regression
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Fig. 2. Gradient b Žslope gradient in the turning point of cumulative curves. plotted against the amount of P added to the samples. The value y indicates the amount of bioavailable P estimated by interpolation.
Ž r ) 0.99; Fig. 2.. Bioavailable P was estimated by interpolation of the gradient b from the respiration curve obtained after glucose amendment Žwithout P supply. ŽTable 5.. For the July soil, no interpolation was made, because the chosen P additions were too high. Žii. In the second approach, the area below the respiration curves Ž equivalent to the CO 2 release during 30 h. was calculated as described above. The area was closely related to the amount of P added ŽFig. 3.. Bioavailable P was estimated by interpolation, the results are given in Table 5.
Table 5 Amounts of available P determined with different methods Resin extraction y1
mg P g July November April a
3.42 4.08 4.46
Extraction with 0.5 M Na 2 HCO 3
I ‘slope’
Bioassay II ‘area’
III ‘Nordgren’
10.06 10.47 4.70
n.d. 9.99 7.51
2.85 11.20 2.06
n. d. 140 a r80 a 370r210
d.m.
The left values indicate the ‘total microbially available P’, the values to the right indicate the ‘SIR-corrected microbially available P’ ŽNordgren, 1992.. n.d.—Not determined.
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Fig. 3. The ‘area’ below the respiration curves Žamount of CO 2 evolved during 30 h. plotted against the amount of P added to the samples. The value y indicates the amount of bioavailable P estimated by interpolation.
Žiii. Finally, the approach suggested by Nordgren Ž 1992. was applied to the present data. The results obtained were very different from the results of the other two approaches Ž Table 5.. The ‘total microbially available P’ was 140 and 37 mg P gy1 d.m. and the ‘SIR-corrected microbially available P’ 80 and 210 mg P gy1 d.m. for the November and April soil, respectively. Nordgren Ž 1992. obtained convincing results with an N-limited, acid Ž pH s 4., highly organic forest soil Žmore layer of a podzolic soil. , but his approach was not suited for the soils studied here. In Table 5, the results of the two weakest extractants are compared with the results of the bioassay P determinations. In this experiment, it was assumed that microbially available P corresponds to plant available P. Thus, P in soil is present in several inorganic Ž Pi . and organic ŽPo . forms, but is absorbed by plants almost exclusively as Pi . Complex Po compounds are important sources for plant P that must be mineralized into Pi before plant absorption takes place ŽAnderson, 1980; Yang and Jacobsen, 1990.. The anion exchange resin method is assumed to assess plant available P, because the resin functions like a plant root with a very high capacity for P uptake ŽSibbesen, 1978.. Considering a recovery of only 87–90% of the added P Ždata not shown. , the estimates given in Table 5 may be somewhat low. Hence, it can be supposed that not all of the available P was extracted from the soil. The sodium bicarbonate extraction for P ŽOlsen et al., 1954. is also an accepted method for estimating P availability in soil. It generally removes the labile
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inorganic and organic P, but according to Hedley and Stewart Ž 1982. , some microbial P may also be removed by this extraction. Results therefore overestimate available P. So it can be assumed that the plant available P should lie somewhere between that given by the resin- and the Olsen-P methods. The results of the approaches I and II corresponded to the results obtained with the weak extractants Žion exchange resins and Na 2 HCO 3 . . Approach II resulted in available P concentrations that were slightly lower than the Na 2 HCO 3 extraction, and partly higher than the resin method. The slope method resulted in slightly higher values. With the bioassay approach, the highest bioavailable P was found for the November samples, while no differences were found with resin extracts. Enhanced P availability for the November samples is in accordance with studies of Adams et al. Ž 1989. , who found that most pools of available N and P vary with season, reaching a maximum in the fall. The results suggest that the respiratory approach taken may be useful for determining bioavailable P in forest soils. The results support the findings of Nordgren Ž1992., but suggest other mathematical approaches. If not used for quantitative estimation of bioavailable P Ž which is laborious since P has to be added in different amounts., the respiratory assay can quickly provide qualitative information on P or N limitation for the soil microbiota.
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