The forms of phosphorus in humic and fulvic acids of a toposequence of alpine soils in the northern Caucasus

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Geoderma 80 (1997) 61-73

The forms of phosphorus in humic and fulvic acids of a toposequence of alpine soils in the northern Caucasus M.I. Makarov

a

T.I. Malysheva a L. Haumaier b., H.G. Alt c W. Z e c h h

Soil Science Department, Moscow State University, 119899 Moscow, Russia b Institute of Soil Science and Soil Geography, Unit,ersi~.' of Bayreuth, D-95440 Bayreuth, Germany ~ Laboratoo, of lnorganic Chemisto,, Unil'ersiO' ~fBayreuth, D-95440 Bayreuth, Germany Received 12 February 1997; accepted 7 May 1997

Abstract

Chemical fractionation and 3~p nuclear magnetic resonance (NMR) spectroscopy of humic acids (HA) and fulvic acids (FA) were used to characterize the forms of phosphorus and their changes within a toposequence of alpine soils at the Mt. Malaya Khatipara (Teberda reserve, northwestern Caucasus). Sodium hydroxide extracted 66-82% of total phosphorus from A horizons and 28-51% from B horizons. Organic P amounted to 92-99% of NaOH-extractable P. HA represented the major part of extracted organic P (52-90%). The P species in HA and FA comprised phosphate monoesters (40-86%), phosphate diesters (up to 22%), phosphonates (up to 8%), sugar-diester phosphates (up to 14%), pyrophosphates (up to 11%), polyphosphates (up to 16%), and unknown compounds (up to 6%). Inorganic orthophosphate was found in appreciable proportions only in HA of organic horizons and in FA (up to 16%). The percentages of phosphonates and phosphate diesters were higher in HA, those of sugar diesters and pyrophosphates were higher in FA. Within the toposequence, the contribution of labile P species to total P in HA of surface soil layers increased with increasing thickness of snow cover during winter and correspondingly shorter vegetation periods. The P-species distributions in HA of subsoil horizons were rather similar throughout the toposequence. HA of the surface soils thus seemed to best characterize the P dynamics in these soils. © 1997 Elsevier Science B.V.

Keywords: alpine soils; Caucasus; phosphorus; humic acids; fulvic acids; 3~p-NMR spectroscopy

* Corresponding author. Fax: +49 921 552246; E-mail: [email protected] 0016-7061/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 1 6 - 7 0 6 1 ( 9 7 ) 0 0 0 4 9 - 9

62

M.I. Makarov et al. / Geoderma 80(1997) 61-73

1. Introduction

In numerous investigations, 3~p nuclear magnetic resonance (NMR) spectroscopy has been used to determine the contents and distribution of organic and inorganic P species in alkaline soil extracts (e.g., Tate and Newman, 1982; Adams and Byrne, 1989; Condron et al., 1990; Gil-Sotres et al., 1990; Gressel et al., 1996). Only few studies, however, are dealing with the types and amounts of P species associated with humic substances, i.e., humic acids (HA) and fulvic acids (FA). There are some reports on P species in HA of organic soils (Ogner, 1983; Bedrock et al., 1994a,b). FA have been investigated by 3~p NMR in even less studies (Giusquiani et al., 1994; Guggenberger et al., 1996). Since P in these humic fractions has been suggested to represent a moderately to highly resistant P pool in soils (Bowman and Cole, 1978), knowledge of its chemical nature seems essential for an understanding of its role in the phosphorus cycle. In a recent study (Makarov et al., 1996), we investigated the P-species distributions in HA from A horizons of a series of Caucasian mountain soils. We found that, in the alpine zone, HA contained appreciable proportions of labile P species, indicating limited microbial activity to be responsible for the accumulation of organic P in these soils. The aim of the present study was to investigate in more detail the nature of P in soils along a toposequence of alpine soils in the northwestern Caucasus. We used chemical P fractionation and 3JP-NMR spectroscopy of HA and FA, in order to elucidate if there are characteristic alterations with changes in topography and vegetation. Subsoil horizons (AB and B) were also analyzed to investigate the changes in P species with soil depth. 2. Materials and methods

The soil toposequence investigated is representative of the soil associations at the Mt. Malaia Khatipara (Teberda Reserve, NW-Caucasus, 43028 'N, 41 °45'E), comprising Leptosols and Histosols (Table 1). The sampling sites were located between 2650 and 2750 m a.s.1. (Fig. 1). Mean annual precipitation is about

2750 1

2

stream

265 - . . . . . . . . . . . .

1 O0

200

-

~

distance,

m

300

-, t

400

BBV

500

Fig. 1. Locations of the soil profiles in the landscape ( l - 4 = U m b r i c Histosol; 6 = Fibric Histosol).

Leptosols; 5 = T e r r i c

Umbric Leptosol (bottom)

Terric Histosol (bank of stream)

Fibric Histosol (bank of stream)

5

6

Umbric Leptosnl (lower parts of slope)

Umbric Leptosol (middle parts of slope)

Alpine lichen heath

Umbric Leptosol (ridges, uppermost parts of slope)

H

H'

Alpine swamp

H

(Caret nigra)

(Nardus ~tricta)

Alpine swamp meadow

AB B

Ah

(Sibbaldia semiglabra)

B

Ah AB

B

Ah AB

Ah AB B

Hori zon

Alpine snowbed community

(Geranium gvmnocaulon)

Alpine meadow

(Festuca l'aria)

Alpine meadow

( Festuca orina)

Vegetation type (dominant species)

Soil type (FAO) (position in relief)

4

Profile No.

Table 1 Properties of the Caucasian alpine soils under study

0-10 2(/-30

0-15

0-8 8 16 16-25

0 9 9-23 23 40

0-12 12-22 22-40

0 10 10-20 20-35

Depth (cm)

5.0 5,3

4.8

4.8 4.5 4.9

4.9 4.6 4.9

5.1 4.9 4.8

5.6 5.5 5.6

(H 20)

pH

4.0 4,2

3.8

3.7 4,0 4.3

3.7 4, 1 4,4

3.9 4,0 4.2

4,2 3.7 3.8

(KCI)

293 320

240

29 II 8

41 26 1I

72 15 13

187 160

133

8 3 2

10 5 2

18 7 5

355 375

314

134 39 26

91 60 36

96 68 27

95 52 25

C

17 5 4

Mg

Ca 95 24 14

Total (gkg

Exchangeable (retool kg i) i)

14.2 13.9

13.7

I 1.3 3.9 3.5

8.5 4.9 4.2

9,0 5.3 2,8

10.3 4.9 2.9

N

1.69 1,33

1.80

2,54 1.57 0.94

1.03 0.77

1.22

0.86

1.03

1.35

0.99 0,70

1.07

P

210 282

174

53 25 28

75 58 47

71 66 31

89 53 36

C/P

e~

64

M.L Makaroe et al. / Geoderma 80 (1997) 61-73

1406 mm, and mean annual temperature is - 1.2°C. All soils derived from grey granite and biotite schist (Grishina et al., 1993). Within the toposequence, the snow cover changes from 0 m at the ridge to 5 m and even more at the bottom. Soils are characterized by distinct alpine plant communities. The Leptosols at the wind-exposed ridge position are covered by low-productive alpine leachen heaths. There is no snow cover most of the time, and frost thus deeply penetrates into the soil profile. During summer, the alpine lichen heath is subject to water stress reducing biological activity. Festuca uaria meadow soils at middle slopes with 0.5-1.5 m of snow cover and Geranium gymnocaulon meadow soils at lower slopes with 2 - 4 m of snow cover are characterized by rather high biomass production (Onipchenko, 1990; Grishina et al., 1993). Soils of the alpine snowbed community at the bottom, with more than 5 m of snow cover, are hydromorphic and characterized by a vegetation period of about 2 months. Near streams with water-surplus throughout the vegetation period, Histosols with alpine swamp communities developed. The peat soils of this community are free of snow also about 2 months only. Some properties of the soils are presented in Table 1. The pH was measured in H 2 0 and 1.0 M KCI using a glass electrode at a soil: solution ratio of 1 : 2.5. Organic C was determined by dry combustion, total N by a Kjeldahl procedure, and total P colorimetrically with molybdenum blue after digestion with H2SO 4 :HC104 (20: 1). Exchangeable Ca and Mg were measured in 1.0 M ammonium acetate by atomic absorption (Varian SpectrAA400). HA were extracted with 0.5 M NaOH at soil-to-solution ratios of 1 : 3 (Ah and AB horizons), 1:1.5 (B horizons), or 1:7 (peat horizons). After 1 h of shaking, the suspensions were allowed to stand overnight. Then they were centrifuged for 2 h at 15,000 g. Inorganic P was measured directly in the extracts, and total P was determined after digestion with H2SO 4 : HC104 (20: 1). Organic P was calculated by difference. HA were precipitated by 10% HC1 (pH 1.5), washed with acidified water and dissolved again in 2 0 - 4 0 ml 0.5 M NaOH. Total P in HA and in solution after HA precipitation was determined after digestion with H 2804 and HC104. Total P in FA was calculated by the difference between total P in solution after HA precipitation and inorganic P in the extract. FA were dialyzed, freeze dried, and dissolved in 4 - 5 ml 0.5 M NaOH. HA and FA solutions were concentrated under a stream of N 2 at 40°C until the P concentration was about 0.5 mg m l - ~. One ml D 2 0 was added to 2 ml HA or FA solution for NMR spectroscopy. 31p-NMR spectra were obtained on a Bruker AM 500 NMR spectrometer (11.7 T; 202.5 MHz for 31p). Spectra were acquired without proton decoupling at a temperature of 296 K, using an acquisition time of 0.1 s, a 90 ° pulse, and a relaxation delay of 0.2 s. Chemical shifts were measured relative to external 85% H3PO 4. Spectra were recorded with a line-broadening of 20 Hz. Intensities of signals were determined by integration. Interpretation of spectra was based on

M.1. Makarov et al. / Geoderma 80 (1997) 61-73

65

literature assignments (Newman and Tate, 1980; Hawkes et al., 1984; Condron et al., 1990; Bedrock et al., 1994b).

3. Results and discussion

3.1. Soil P fractionation The soils investigated are characterized by high P accumulation (1.07-2.54 g P kg- ~ in surface layers and 0.70-0.94 g P kg- 1 in B horizons, Table 1) which is directly connected with high humus contents. A maximum in both C and P contents and the narrowest C-to-P ratios were found for the snowbed community soil, probably due to restricted microbial activity under the hydromorphic conditions and due to the short vegetation period. From 66 to 82% of total P in Ah, AB, and H horizons were extracted by 0.5 M NaOH. Inorganic P contents in the alkaline extracts varied from 5 to 110 mg

Table 2 Phosphorus fractions in the Caucasian alpine soils Profile Horizon NaOH-extractableP No.

%Pt inorganic P

organic P

mg/kg %Pe×tr mg/kg %Pextr P in HA

mg/kg

P in FA %Po,e,,,tr a m g / k g

%Po,ext~ "

Ah AB B

68 69 51

29 9 4

4 1 1

695 672 350

96 99 99

550 558 294

79 83 84

145 114 56

21 17 16

Ah AB B

66 68 46

56 5 3

6 I 1

835 700 391

94 99 99

724 632 331

87 90 85

114 68 60

13 I0 15

Ah AB B

82 68 49

27 5 2

3 1 1

969 700 370

97 99 99

787 579 295

81 82 80

182 124 75

19 18 20

Ah AB B

81 80 28

77 69 4

4 5 2

1987 1185 253

96 95 98

1649 1068 184

83 90 73

338 117 69

17 10 27

H

77

110

8

1264

92

840

66

424

34

H H'

67 72

93 77

8 8

1043 880

92 92

543 537

52 61

500 343

48 39

Percentage of extractable organic P.

66

M.L Makarot, et al./ Geoderma 80 (1997) 61-73

P kg-J corresponding to 1-8% of extractable P (Table 2). The surface layers of the hydromorphic soils (profiles 4-6) were characterized by relatively high contents of extractable inorganic P. Extractability of P sharply decreased in B horizons (about 30-50% of total P). Inorganic P amounted to 2 - 4 mg P k g - ~ or 1-2% of extractable P. The low extractability and the small proportions of extractable inorganic P in the B horizons may be due to a relatively low contribution of organic P species to total P and to insignificant weathering of P-containing minerals, respectively. Between 73 and 90% of the alkali-extractable organic P of mineral soil horizons and 52-66% of organic horizons were associated with HA. As FA dominate over HA in alpine meadow soils (Grishina and Makarov, 1987), it is obvious that HA are enriched in P compared to FA.

)

Festuca varia

)

Geranium gymnocau/on

22' snowbed. community

I[ ~ I1 Jl

1~

I I

A' PinernSW~lmp o

Alpine

I

20

I

I

10 PPM 0

I

-10

swamp

I

-20

Fig. 2. 3~P-NMR spectra of humic acids extracted from Ah and H horizons of alpine soils with different types of vegetation.

M.L Makarov et al. / Geoderma 80 (1997) 61-73

67

3.2. Phosphorus-31 N M R spectroscopy

Phosphorus-31 NMR spectra of HA and FA extracted from surface soils are shown in Figs. 2 and 3. Resonances in the range of 19.0-20.0 ppm have been assigned to phosphonates, the sharp resonance at about 6.0 ppm to inorganic orthophosphate, signals between 6.0 and 3.0 ppm to orthophosphate monoesters, and those around 0 ppm to orthophosphate diesters (Hawkes et al., 1984). Signals in the resonance region between monoesters and diesters (3.0-0.5 ppm) have been attributed to teichoic acids (Condron et al., 1990) or to nucleic acids (Bedrock et al., 1994b). Both types of compounds represent sugar-diester phosphates. We therefore used this term for resonances in that region. For the small signals at about - 1 . 5 ppm, no assignments have been given for soil extracts so far. At high pH, acyl phosphates show resonances in that region (Vogel and Bridget, 1983). Acetyl phosphate added to an alkaline solution of

j

Alpine lihhath

Festuca varla

Geranium gymnocaulon

(3) Alpine snowbed community

(4)

Alpine swamp meadow (5)

Alpine swamp

I

20

I

10

I

PPM

0

I

-10

I

-20

Fig. 3. 31P-NMR spectra of fulvic acids extracted from Ah and H horizons of alpine soils with

different types of vegetation.

68

M.I. MakaroL~et al. / Geoderma 80 (1997) 61-73

humic acid, however, rapidly hydrolyzed to orthophosphate (Haumaier, unpublished data). The resonances at - 1.5 ppm thus remain unidentified. The range of - 4 . 0 to - 5 . 0 ppm has been attributed to pyrophosphate and to polyphosphate end groups, and that between - 1 9 and - 2 1 ppm to polyphosphates (Hawkes et al., 1984; Bedrock et al., 1994b). The contribution of ATP (Bedrock et al., 1994b) to these resonances can be neglected because of the absence of a signal for P~ around - 10 ppm. The P-species distributions in HA and FA are shown in Tables 3 and 4. The presence of inorganic phosphate species in HA and FA solutions can be explained by slow hydrolysis of organic P or, more probably, by the release of orthophosphate associated with humic materials. Humic and fulvic-acid metal complexes are able to bind appreciable amounts of phosphate (Levesque and Schnitzer, 1967; Gerke and Hermann, 1992). In our experiments, up to 10% of total P in HA was released as orthophosphate during reprecipitation of freshly prepared HA. Phosphate monoesters were the dominant P species in HA (Fig. 2, Table 3). This is in agreement with the generally held view that monoesters are the organic P species most resistant to mineralization. The lowest proportions were found in HA of the surface layers of hydromorphic soils (profiles 4-6). Accordingly, these soils contained the highest proportions of the more labile species, i.e., phosphate diesters (resonances at 0 ppm) and phosphonates (resonances at 19 ppm, Fig. 2). Restricted microbial activity due to the cool and moist environment seems to be the cause for relatively high levels of labile organic P species. Tate and Newman (1982) found significant correlations between annual precipitation and proportions of these labile P species in alkali extracts of tussock grassland soils. The rise in phosphate-diester percentage, when going down the slope from the ridge (profile 1) to the bottom (profile 4), was accompanied by a comparable rise in the percentage of polyphosphates (Table 3, Fig. 2). Polyphosphates are short-lived intermediates in the soil-P cycle (Pepper et al., 1976), and they can be synthesized by microorganisms under conditions of nutrient imbalance (Ghonsikar and Miller, 1973). From profile 1 to profile 4, pH and contents of exchangeable Ca and Mg decreased whereas P contents increased (Table 1). This may be indicative of some nutrient imbalance which could have led to the accumulation of labile polyphosphates, particularly in the Ah horizon of profile 4. The percentages of inorganic orthophosphate were rather high for HA from peat samples (Table 3, Fig. 2). Similar proportions of orthophosphate have been found by Bedrock et al. (1994b) in HA from a blanket peat in Great Britain. These authors reported even higher proportions of orthophosphate in HA from fertilized peat and in HA from an agricultural mineral soil. In our study, the peat samples had the highest percentages of NaOH-extractable inorganic P (Table 2). This may indicate that the distribution of P species in HA mainly is controlled

M.I. Makaror et al. / Geoderma 80 (1997) 61-73

c-

O

"6

0 e~

t~

0

©

u

g

0

e-

,<

e-

.u © e-

.==.

,-"

c-

oo

o

"V.

0 0 ~Z o

69

Ah

Ah

H

H

4

5

6

3

B

3

10 4

All AB

2

15

16

9

8

12

Ah

1

Inorganic orthophosphate

Horizon

Profile. No.

0

0

0

0

0

0 0

0

Phosphonates

54

59

67

68

71

61 73

53

M0noesters

9

7

4

8

13

11 14

14

Sugar-diester phosphates

Table 4 Phosphorus-species distribution (%) in fulvic acids (FA) of the Caucasian alpine soils

7

6

1

6

7

8 5

5

Diesters

0

0

0

5

6

3 4

3

Unknown compounds

11

7

3

4

7 0 0

I0

Pyrophosphates

4

5

16

1

0 0 0

3

Polyphosphates

M.I. Makarov et al. / Geoderma 80 (1997) 61-73

71

by the proportions of the respective P species in the total alkaline extract from which HA are precipitated, and that a part of P is only in loose association with HA. Consequently, this part of P would not necessarily be 'highly resistant'. Phosphate monoesters were preponderant also in FA (Fig. 3, Table 4), but diester proportions generally were lower than in HA and phosphonates were absent. Proportions of inorganic orthophosphate, pyrophosphates, and sugar-diester phosphates were higher than in HA, and they decreased from profile 1 to profile 4. The FA of the snowbed community soil (profile 4) were characterized by an extraordinary high proportion of polyphosphates which was even higher than that of the HA. The FA of peat soils differed from the corresponding HA mainly by the absence of phosphonates and lower percentages of diesters. The other features of the spectra were rather similar. Greater proportions of inorganic phosphates and sugar-diester phosphates in FA than in HA were also found for a Colombian Oxisol by Guggenberger et al. (1996). Possibly, the distribution of organic P species between FA and HA is controlled to a certain extent by their hydrophilic/hydrophobic properties. Accordingly, the more hydrophilic sugar phosphates would be relatively enriched in the FA fraction. The ability to form metal bridges to inorganic P species can be expected to be higher for FA because of higher contents of carboxyl groups. The lower total P content of FA would result in higher proportions of inorganic P. Monoester proportions in HA generally increased with soil depth whereas the contributions of other organic species and of condensed inorganic phosphates decreased (Table 3, Fig. 4). Inorganic orthophosphate and unknown compounds showed no consistent trend. The increase of monoesters was thus mainly due to lower proportions of labile P species. This is in agreement with the view that the organic matter of subsurface horizons is more degraded and thus contains less labile components. HA

FA

Ah .,_..k.._,-,,j

Ah

AB j

AB

B l 20

B I 10

PPM

I 0

I -10

1 -20

I 20

I 10

PPM

I 0

I -10

I -20

Fig. 4. 31P-NMR spectra of humic and fulvic acids extracted from horizons of profile 2.

72

M.L Makarov et al. / Geoderma 80 (1997) 61-73

Except for the disappearance of pyrophosphate and a decrease of orthophoshate resonances, spectra of FA showed little alterations with depth (Fig. 4). Considering the low contribution of FA-P to total extractable P (Table 2), the changes with depth in P-species distribution seem to be best represented by the HA fractions.

4. Conclusions Most of the alkali-extractable P was associated with the HA fractions. The P-species distributions in HA of surface soils showed distinct alterations with changes in topography and vegetation. Increasing thickness of snow cover during winter, correspondingly shorter vegetation periods, and increasing water-surplus at the lower elevations led to increasing proportions of inorganic and labile organic P species. This indicates reduced P mineralization under conditions unfavourable for microbial activity, but also limited P uptake by the plants. In the subsurface horizons, the differences in P-species distributions levelled off. No clear tendencies were observed for the FA fractions. Thus, the HA fractions of the surface soils seem to best characterize the P dynamics in these alpine soils.

Acknowledgements This work was supported by research grants awarded to M.I. Makarov by the European Environmental Research Organisation (EERO) and by the German Academic Exchange Office (DAAD).

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M.L Makarot, et al. / Geoderma 80 (1997) 61-73

73

Gerke, J., Hermann, R., 1992. Adsorption of orthophosphate to humic-Fe-complexes and to amorphous Fe-oxide. Z. Pflanzenern~ihr. Bodenkd. 155, 233-236. Ghonsikar, C.P., Miller, R.H., 1973. Soil inorganic polyphosphates of microbial origin. Plant Soil 38, 651-655. Gil-Sotres, F,, Zech, W., Alt, H.G,, 1990. Characterization of phosphorus fractions in surface horizons of soils from Galicia (N.W. Spain) by 31p NMR spectroscopy. Soil Biol. Biochem. 22, 75-79. Giusquiani, P.L., Gigliotti, G., Businelli, D., Macchioni, A., 1994. Spectroscopic comparison between humic and fulvic acids from urban waste compost and soil. In: N. Senesi and T.M. Miano (Eds.), Humic Substances in the Global Environment and Implications on Human Health. Elsevier, Amsterdam, pp. 1303-1310. Gressel, N., McColl, J.G., Preston, C.M., Newman, R.H., Powers, R.F., 1996. Linkages between phosphorus transformations and carbon decomposition in a forest soil. Biogeochemistry 33, 97-123. Grishina, L.A., Makarov, M.I., 1987. Soils of alpine lichen heaths. In: Biogeocoenoses of Alpine Lichen Heaths. Moscow, pp. 9-19 (in Russian). Grishina, L.A., Onipchenko, V.G., Makarov, M.I., Vanyasin, V.A., 1993. Changes in properties of mountain-meadow alpine soils of the northwestern Caucasus under different ecological conditions. Eurasian Soil Sci. 25, 1-12. Guggenberger, G., Haumaier, L., Thomas, R.J., Zech, W., 1996. Assessing the organic phosphorus status of an Oxisol under tropical pastures following native savanna using 31p NMR spectroscopy. Biol. Fertil. Soils 23, 332-339. Hawkes, G.E., Powlson, D.C., Randall, E.W., Tate, K.R., 1984. A ~P nuclear magnetic resonance study of the phosphorus species in alkali extracts of soils from long-term field experiments. J. Soil Sci. 35, 35-45. Levesque, M., Schnitzer, M., 1967. Organo-metallic interactions in soils, 6. Preparation and properties of fulvic acid-metal phosphates. Soil Sci. 103, 183-190. Makarov, M.I., Guggenberger, G., Zech, W., Alt, H.G., 1996. Organic phosphorus species in humic acids of mountain soils along a toposequence in the Northern Caucasus. Z. Pflanzenern~ihr. Bodenkd. 159, 467-470. Newman, R.H., Tate, K.R., 1980. Soil phosphorus characterisation by 3tp nuclear magnetic resonance. Commun. Soil Sci. Plant Anal. 11,835-842. Ogner, G., 1983. 3tP-NMR spectra of humic acids: a comparison of four different raw humus types in Norway. Geoderma 29, 215-219. Onipchenko, V.G., 1990. Phytomass of alpine communities in the North-Western Caucasus. Proceeding of Moscow Environment Society (MOIP), Set. Biology, 95, pp. 52-62 (in Russian). Pepper, I.L., Miller, R.H., Ghonsikar, C.P., 1976. Microbial inorganic polyphosphates: factors influencing their accumulation in soil. Soil Sci. Soc. Am, J. 40, 872-875. Tate, K.R., Newman, R.H., 1982. Phosphorus fractions of a climosequence of soils in New Zealand tussock grassland. Soil Biol. Biochem. 14, 191-196. Vogel, H.J., Bridger, W.A., 1983. Phosphorus-31 nuclear magnetic resonance pH titration studies of the phosphoproteins tropomyosin and glycogen phosphorylase-a. Can. J. Biochem. Cell Biol. 61,363-369.