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Distribution and characterization of phosphatase and organic phosphorus in soil fractions
_%I/ Bwl. B~~hrrn. Vol. 22. No. 2. pp. 169-174. Pnntcd ,n Great Bntam. All nghts rcxrvcd
1990
0038-07 I7 90 53 00 + 0.00 Press plc
Copyright C I990 Pcrgamon
DISTRIBUTION AND CHARACTERIZATION PHOSPHATASE AND ORGANIC PHOSPHORUS FRACTIONS M. J. ROJO.’ S. G. CARCEDO:
OF IN SOIL
and M. P. MATEOS~
‘Department
of Organic Chemistry. ‘Department of Soil Science and Technology and ‘Department of Biochemistry. Molecular Biology and Physiology, Burgos University College. University of Valladolid. Aptdo 231. 09071 Burgos. Spain (Accepted 25 .4ugusr 1989) Summary-The distribution of organic P (P,). Fe. Al and Ca. and alkaline and acid phosphatase in surface layers of a calciferous alkaline soil and an acidic soil and fractions of these obtained by a granulometric procedure was studied. There was a significant correlation between the P, and Fe or Al contents. The highest rates of P, mineralization (evaluated by means of the organic P,-to-organic C-ratio) were found in the soil units containing less-humified organic matter. These fractions also had the highest amounts
phosphatase activity. Phosphatase activity was concentratedin the larger soil fractions (1OO&lOO~m
dia) and was probably associated with plant debris and the less humitied organic matter. The alkaline soil contained dominantly alkaline phosphatase and the acidic soil acid phosphatase. The relationship between phosphatase activity and pH was essentially the same in soil fractions as in the whole soil. The phosphatase activity of the alkaline soil had a grcatcr thermal stability than that of the acid soil. Enzyme kinetic data give K__,values ranging from 2.0 4.4 IIIM in fractions from the alkaline soil (unfractionatcd soil 4.1 rnti) and frnm 3.6-l 5.4 mM in fractions from the acid soil (unfractionatcd soil 5.7 msr).
and Gonzalez-Carccdo. 19x5). Using this method. WC huvc studied the participation of two important phosphomoncostcrascs in phosphate availability in root-zone soils with high concentrations of organic I’ (P,) and the means through which thcsc enzymes arc stabilized. This study may lead to a bcttcr understanding of the characteristics of soil enzyme immobilization and allow the incorporation of stable enzymatic complexes into soil in order to increase the amount of inorganic phosphate available for plant nutrition.
Soil cnrymcs in the aqueous phase arc gcncrally short-lived bccausc thsy arc inactivated by association with soil particles or colloids or by protcolytic degradation. t lowevcr, soil alkaline and acid phosphatascs (EC 3. I .3. I and 3. I .3.2) and other enzymes can bc immobilized on, or in, soil clays and humatcs, thcrsby constituting a persistent extracellular catalytic activity (Burns, 1986). Soil humic matter is a highly-charged polyclectrolyte which can complex cnzymcs. This incrcascs their resistance to biodegradation and further stabilization can occur by fixation of this complex to clays. A large proportion of total soil P is bound up as ester phosphate in complex organic compounds and, consequently, considerable research has been applied to the characterization of soil phosphatascs and to ways of increasing or supplcmcnting their activity (e.g. Speir and Ross, 1978). Thus, stable artificial humic-phosphatase complexes have been added to soil (Burns and Ladd. 1985). and the USC of a stable phosphatasc seed coating has been cvaluatcd as a means of bringing about localized solubilization of soil organic phosphate (Burns er al.. 1987). Wc have used a granulomctric proccdurc lo scparate soil fractions containing difTerent types of organic matter (incomplctcly transformed animal, plant and microbial debris and polymeric humic fractions). This technique has proved to bc valuable in the location of soil enzyme activities (Perez-Matcos
‘To
whom all correspondence
Soils Samples (O-7 cm) of two soils from under pasture wcrc collected and stored at field moisture content in scaled plastic bags at 2’C. Experimental work was carried out as soon after collection as practicable. Thcsc soils wcrc an Aridic Calcixcroll (pH 8.4; CEC 0. I I cmol kg-‘; organic C (C,) 42.2 g kg-‘; C/N 9.4: sand 26.2%; silt 0.1%; clay 0.21%; CaCO, 63.2%) and an Umbric Dystrochrcpt (pH 4.3; CEC 0.17cmol kg-‘; C, 62.6g kg-‘; C/N 14.9; sand 17.5%; silt 48.6%; clay 13.3%) and have been described by Pcrcz-Matcos and Gonzalez-Carcedo (1985) and Gonzalez-Carcedo er crl. (1983).
Soil enzyme dirtrihutiun The distribution of phosphatascs in soil fractions was determined by the methods of Pcrcz-Matcos and Gonzalez-Carcedo (1985) for other enzymes. Airdried soils were fractionated in three different ways:
should be addressed. 169
(A) sieving at J C without pretreatment. (B) wetsic\~ng after dlsperrion of soil, mixed with distilled water. using reciprocating agttation for 1.5 hat 1.05~. and (C) wet-sie\mg as m B after aqueous dispersion and mechanical dlsruption of the soil by agitation w-ith Nater and 5 agate balls (I cm dia) per 35 g soil for I5 h. Using procedure (A), four soil fractions of with particle-size rcul ttnifs u-ere obtained X00-3OO/~rn (.AF,). 200-100~(m (AF,). 10&50/[m (.AF,) and <50/lrn (AF,). using procedure B four factions of srubi;~ ~ctrirs(BF). and using procedure C four fractions of srrli~turul units (CF) of the same sizes. were obtained. For details see Perez-Mate05 and Gonzalez-Carcedo (198.5). In some assays. fructinns F, and F: were mixed to give fraction F,: (particle-size ~00~100 jirn).
Air-dried soil (~2 mm) (I g). and soil fractions (I 3) were treated with I ml of I I5 mM p-nitrophenyl pll~~sph~Ite (pNP) and 4 ml of modified universal butTcr (MUB) at pH 6.5 (Skujins et trl.. 1962). After agitation. and because of the buffer effect of soil. the pll of thcsc mixtures was 7.1 in the alkaline soil and 5.3 in the acid soil. li~~ubations were carried out at 37 C for i h and then. 0.5 %t CaCII (i ml) ard 0.5 hi NaOf 1 (1 ml) u’cru iiddcd and the mixtures shaken and held at 20 C for 10 min (Tabatubai and Brcmncr, 196Y). The contents were filtcrcd (Whatmun No. 6) and aliquots (I ml) of the filtrates made up to 50 ml with 0.5 M NaOFi. The p-nitrophcnol (pN) produced was dctormincd ~ol~~rim~tricaIly at 410 nm. Assays wcrc c;trricd out in triplicate with two controls (substrata ;&led after incubation). Units of phosphatasc activity were jrmol pN produced g dry soil .’ h ” and specific cnrymc activity v:~Iues with respect lo the organic matter content of samplc~ wcrc calculated in ltmol pN formed g C ‘h ‘.
The clfcct of ptl was studied by mixing the soil sarupl~‘s with nine solutions of modified universal bufikr (4 mi g ’ ) at ptf vafucs between 3 and I I. Thcrcal’tcr the mixtures were allowed to st:md for 1 h with occasional stirring, the pH was mcasurod again and the cnLymc activi.ty. assayed. The values of the MUB ptl ucrc din~lnlsh~d by the Dystrochrcpt soil and fractions to more acid values whereas the Calci~croll and fractions incrcascd the pH of the six M UB solutions with pH values bctwecn 3 nnd 8 and dccrcased the pH of the other three solutions (see pH of mixtures in Figs 1 and 1). Thermal stability was assayed by measuring cnryme activity of soil samples which had been trcatcd in closed vosscls 31 20, 55. 65. 75. 85. 95 or IO5 C for 21 h. The kinetics of the soil phosphatascs wcrc cvaluatcd by measuring the enzymatic reaction rates at ditfcrcnt substrate concentrations. Five solutions of PNP u’erc used with concentrations of 5; 25; 50; 75 and IO0 msg. Thr values of K,, were dcrivcd from computed Icast-square analysis of Lincwcavcr-Burk plot. \lcasurcmcnts of phosphatase activity for thermal stability and kinetic assays were made at pH IO.27 in
the and pH for
Calcixeroll samples (pH of the added M UB I 1.O) at pH 5.31 in the Dystrochrept samples (MUB 6.5). These pH values correspond to the optima phosphatase activity in the respective soils.
The soil P, content was calculated by a modiiication of the method of Saunders and Williams (1985) from the difference between the contents of total and inorganic orfhophosphate. The total P content was determined by ignition and subsequent extraction with 1 N HSO, and the total inorganic P by extraction of unignited samples with t N H-SO,. P concentrations in all extracts were dete&ined cotorimctrically (Murphy and Riley. 1961). Organic C (C,) and N were measured with a CHN automatic autoanatyser (Carlo Erba. 1106) and Fe. AI and Ca by atomic absorption spectrometry techniques. Catcutations of P,,. C, and elements content in soil fractions (Table I) were made by conversion of absolute results into relative values by taking into account the contribution of each fraction (IV,) to the totat weight of soil (Pcrcz-Matcos and Gonzalez Carccdo. 19x5).
All exporimcntal data were tested by standard deviation and variation cocliicicnt, not exceeding 6% of this cocfficicnt. Two separate variance analyses of
tt;”
C,,’
r*:
pz,C,
57?.3 503.0 47 I) 12.4 YY 71 I ‘3.3 23 X 413 7 Ill 4 Y? I46 ES?,
13.7 14.5 It) Y X3 I I .o
(46) (“iPI C,N fpgg It (x IO $1 .hdU~<‘d-rrerdl wrl 1;s AF, Ai-, A r:I A F, BF, BF: RF, BF. ct:; CF,
CF; CF, f,‘i,thrir
X6 6 X5 ?.Y : 0 32.2 76 X.1 52 I ii.4 7.6 IO.6 65.4
AF, AFL AF, BF, BF, fw, Hf’, :;I CT; CF.
0.4 Y3 10.6 IS.0 Y.1)
11.1
I I.5 16.X IO t l’J.5 15.3 16.5 Y7
&I 6.26 14.0 5.1X 15.3 0.51 13.x 0 ?X 17.0 0.27 13.4 1,X6 23.7 11.57 16.‘) 0.52 17.7 2.X4 13.4 0.x 22.5 0.1’) IX.7 OS1 IX.6 3 3x 13.7
5.4 I?.‘) 2’J.X 21 I 3.0 2.4 4.4 x1.7
Al’ -.
7 6X 6 66 065 II I’J O.IX 1.03 02X 0.30
C.1’
(mga ‘1 7.10 6.05 0.63 O.?? 0.15 0.X! 0.?3 0.35
4 ifs
f,Oft
015 01.8 0 ‘3 S.lX
0.12 OIJ I).IX 4 4.8
X2.‘) 7.0 40 60 32 X 3.2 67 57 3 XI I.? 13.1 77 I
403.5 320.7 37.3 1H.Y 25.1 70x1 I0.Y IS I 3O’J.l 3-i 0 Y.3 IS I 344.7
6.4 62 72 6.7
9.3 3.X IY 2.5 IO Y IS 5 4.Y 30 10.2
iXY3 14.11 I5 7’) iO.hY 11: 1.16 0.72 0.w OY’ OSI h-l3 ?.JY 0 77 0 24 063
IO 5X 5.5‘) 0.72 OX4 If34
.---
7.Y3 h.SII 11.52 If 20 OIZ LY7 061 0.71 2. IO I.?X 0.5Y O.Y? 3.XS
~r.rlr{J~/~?~.~f
us AF,
4.23 3.46 0.43 0 IS 0.0’~ 1.31 0.1x OIJX 1.46 0.15 0.3’) 0.31 2.54
FC’ “-’ .~
0 0 0 0 0 0 0
O?!
0
5.x I.33 0.1’) 027 7.1X
II 0 I) 0 0
Soil phosphatax distribution a rank classification of Kruskal and Wallis (1952) uere performed: one with data of the enzyme distribution and the other with data of the enzyme characterization. For a significance level of 0.05, experimental constants H < 4.95 for the former group of data and H < 5.12 for the latter were obtained for this statistical test. suggesting that the values of independent assays performed in the replicates could not be considered significantly different. RFSL’LTS
ASD
DISCLSSION
Chemical characterization The granulometric procedure we used to fractionate soil phosphatase activity gives soil fractions containing a characteristic distribution of soil organic matter. Real units (AF fractions), specially AF, the fraction formed by the largest aggregates, which include soil units isolated in BF,, BF, and the structural units CF,. contain a mixture of plant debris. less-transformed organic compounds (e.g. pcptides. fatty acids, carbohydrates). polyphenolic humic matter and other kinds of organic hiomolecules. However. the stable (BF fractions) and even more. the structural soil units (CF fractions), scparatc humatcs from fresh organic matter as is indicated hy the C-to-N ratios included in Tahlc I. Thus humic compounds and clay minerals (organomincral complcxcs) constilutc the microapgrcpatcs grouped in the CF, fraction, that has the lowcrt C-to-N ratio (Con~alcz-Carccdo (*I (11.. 1983; Pcrcl-Matcos and Gonlalcz-Cnrccdo. lYX5). A Iarpc proportion of total soil I’ is bound up in organic compounds in thcsc soils: 85.4% in the Calcixcroll soil and 74.3% in the Dystrochrcpt. The distribution of the P,, in the twclvc fractions of both soils (Table I) suggssts an important association bctwccn soil P and humic compounds: soil fractions concentrating clay and humic colloids (AF,. BF,, CF,) showrd the grcatcst P,, concentrations, although the results found in the fractions containing plant debris (AF,. BF,,, CF,,) may indicate the prescncc of P,, associated with non- or partially-humifed organic matter. Esters of inositol, nuclcotidcs and phospholipids, idcntifcd in soil extracts (Hnlslcud and McKerchcr, 1075; Krivonosova and Buscvich, 1981; Stewart and McKerchcr. 1982) have been thought to bc associated with or derived from humic-bound P compounds (c.g. nucleic acids, phospho-proteins, ctc). Sycrs Ed (11. (196’)) also found the grcatcst P, content in soil fractions with the smnllcst particlcsiLc. The P,-to-C,, ratio (or C,-to-P, ratio) has been suggcstcd as an index of P mineralization in soils (c.g. Uriyo and Kcsscba. 1975) and our study showed a high P concentration in the humic organic matter of CF, fractions (Table I ). although in the Dystrochrcpt soil. fresh organic matter prcscnt in thr CF, fraction showed a higher P, concentration. P mineralization appeared to be slower in the calcifcrous soil than in the acidic soil. perhaps due to the prcscncc of calcium in high concentrations (Table I). complexing or insolubilizing P, compounds and protecting them from chemical or biological degradation. Association of P with humatcs through cationic bridges to form organo-metallic P compounds may be a way to
Table
171 2. Phosphatax Aridic
SolIS Real
activity
in the sod fractions’
Calcixcroll
PhA”
Umbric
PhAsp’
PhA”
Dystrochrept Ph.&p’
umts
AF,
90.6
99.3
102.3
1905
AF,
I x4.4
108.7
145.0
x7.
AF,
69.9
59.3
89.6
149.7
43.9
43.0
52.2
135.5
AF, Stable
I
units
BF,
72.3
67.8
50.6
81.4
BF:
105.3
233. I
1764
113.1
BF,
447
?JJ.8
101.1
lZl.2
BF,
75.5
89.5
J3 z
113.9
CF,
77.0
158.6
61.3
4s3.0
CF, CF,
IO?.5 63. I
87.6 89.0
66.1
CF,
77.6
VI.7
3:. I
Structural
units
‘Phosphalax
activity
of
the
p ml dry soil h -’ Aridic
’
Dystrochrcpi.
Umbric
“Phosphatase
activity
unfracrlonated ‘Specific
phosphdtasc
PhAsp and
in
%
Zl0.Z
unfrwtionated
formed h
I”.1
83.7
soils:
Calcixeroll
with
9x.2
I I .6 pmol
and
respect
to
4.38 pmol
the
actwty
pN
pN g of
’
fhc
samples. activity
in pmol
of the unfracrionaled
IRS 9 U Umbric
pN
soils:
formed
p -’ organic
103.7 U Aridx
C h
‘.
C.kineroll
Dystrochrept.
protect P, from chemical or biochemical attack. Thcrcforc. linear rcgrcssion analysts bctwccn P,, and cations were dcrivcd for all of the soil snmplcs. A signilicant relationship appears bctwccn Fe. Al and P,, in the fractions of the Calcixcroll soil with correlation coeflicicnls. r > 0.X3*** (significant at the P < 0.001 Icvcl). In this soil. the strongest corrclations occurred in the structurd units with cor > O.YY***. This suggests the cfkicnls of participation of Fc and Al, but not C;L. in the association of P compounds with organic matter. Anderson (lY75) and others have outlincd the function of Fc and Al in bonding humic substances with
UMBRIC DYSTROCHREPT
zo-
: + l
’ 456
’
’ 7
8
9
10
11 PH
Fig. I. ElTcct of pH on phosphatase activity in unfractiunated soils (US) and soil real units (AF,,. XKM-IOOIrm; AF,. c50yrm).
172
M. J. ROJO er al
phosphate groups. In contrast, no such relationships were found for the acid Dystrochrept soil. 80
ARiDIC
CALCIXEROL
Enzyme distribution The distribution of phosphate activity in the I2 fractions of both soils is shown in Table 2. This activity was mostty concentrated in the largest size fractions of both soils, both after sieving (AF, . AF?) or after aqueous dispersion and wet sieving (BF,). In the largest size structural units, where most of the fresh less-humiiied organic matter was concentrated (CF: and CF,). the phosphatase activity remained higher, although the humic fractions (CF,) also contained an appreciable amount of activity. The only soil units in which an enzymatic enrichment or puri~cation was observed with respect to the unfractionated samples were the FZ fractions. Plant roots may take up P at a rate greater than indicated by the diffusion coefficient of orf/Iophosphate in soils (Heinrich and Patrick, 1985). This may indicate uptake of fffr~z[~phosphate P derived from organic matter by the action of plant root and associated microbial phosphatases. Plant debris can be obscrvcd, using a low-powered microscope. in the iargcst size fractions (F, and F,). Several authors (e.g. Juma and Tabatabui. 1978) having studied dinirent lcavcs) and soil fractions. plant miltcri;Ils (roots. suggesting ;I plant origin for a part of soil phosphatasc activity. On the other hand, this fraction of structural units CF, (and CF, in the acid soil) had the Iowcst P,,-to-C,, ratio and the greatest phosphatasc activity (compare Tahlcs I anti 2). The highest rate of P mincraii~;ttion (lowest P,,-to-C,, ratios) in the fractions containing fresh organic matter is coincident with the largest rates of activity being found in these soil fractions. Similarly. Tarafdar and Jungk (1987) found a significant corrciation bctwccn the deplction of P,, and phosphatase activity in rhizospherc soils of whsat (r = 0.99) and clover (r = 0.97). Specitic phosphatasc activity vaiucs shown in Table Z may reflect the association of organic matter and enzymes. Enzyme activity is poorly associated with the organic matter of real and stable units. but fresh organic matter accumulated in CF, (and in CF, for the Dystrochrcpt soil) appeared to have a much greater capacity to associate with phosphatase activity than has organic matter in the remaining structural units or unf~~ctionatcd soil. These results agree with those concerned with the distribution of cnryme activity in the soil fractions.
The pal-~Ictivity profiics of phosphatasc in unfractionatcd soil and soil real units (AF,,. AF,) are shown in Fig. I. The differences in pH-optimum vaiucs were not signiticant. Although the presence of many isocnzymcs with different optimal pHs in each sample is suspected. the pH values with maximum activity (cu IO.3 in the Caicixcroii soil and about 5.2 in the Dystrochrcpt) rclatcd to the pH characteristic of each soil sample, suggest the presence of alkaline and acid phosphatase respectively. The incrcascs of activity at pH 5.7 in the Caicixcroil soil may indicate the existence of some acid phosphatasc activity in this soil. although this would have no real significance. Similar results wcrc found for optimum pH values of
60
r; 100
45
6
78
9
10
11
w
z
2
80
E g E
60
UMBAIC DYSTROCHREPT -
us
-
CF
40
5
6
7
8
9
IO
11 PH
Fig. 2. Efkt of pH on phosphatasc Mivity atcd s&is (US) and soil structural units (CF,,. CF,. <5O~tm).
in unfractionZOO@-100 {tm:
phosph~It;I~ activity in the structural units of both soils (Fig. 2). These findings tond to confirm the suggestion of Dick and Tabatabai (1984) that acid phosphatasc dominates in acid soils and alkaline phosphatasc in alkaline soils. although some authors (e.g. Pang and Kolenko, 19X6) have reported neutral phosphutasc in acid woodland soils. These results agree with the conclusion of Frankenbcrgcr and Johanson (1982) to the elTcct that the optimum pH range for phosphatase activity is gcncrally quite narrow. Phosphatase in soil fractions does not usually have its maximum activity at more alkaline pH values than from that of unfractionated soils. suggesting that phosphatase is no more absorbed to colioids in the soil units than in the unfract~onated Table 3. Thrrmul rtltbibty of phosphatwe activity’ in unfraclionatrd sod and sotl frxtions Temperature ( C) 55 Aridk Cul~i.r~roll sod Unfraction;rtcd ~011 Red umtr AF,: AF, AF, Structural units CF,: CF. ufdlrir D,wi?odwrpr sod Unfrsctlonaled sod Real U~IIF AF,: AF, AF, Structural units CF,, CF.
63
75
I
X5
63.7 46.5
9s
Iul
X4 6
73
XS.2 X9.3 94.6
71.6 70.3 X6.4
53.X 51.7 7X.X
3X.7 42.5 55.6
1x.7 32. I 30.5
95.3 92.9
X6.5 73.2
75.4 66.5
55.2 43.9
19.3 24.2
77.6
56. I
JI 3
35.5
29.9
H3.3 RI.X X0.7
62. I 5X.5 6&X
49.x 45.4 so.4
32.9 23.X 37.0
26.9 17.8 30.1
86.9 19.9
69.9 71.4
53.3 65.9
41.9 39 4
41.0 17.7
‘In 96 with resrct to the sctiwty of the untreated samples.
Soil phosphataa ie distribution Table 4. Apparent K_ valucs~of phosphamc in soil fractions Aridic
Calcixeroll
Cmbric
Dystrochrepc
1.12
5.71
Real units AF,, AF, AF,
2.91 ‘.oo 2.55
IS.41 6.88 II.91
S1ructural ““!lS CF,: CF.
4.40 ?.I7
3.62 6.00
Unfractlonatcd
‘In
ITIM
sods
pNP
samples (Perez-Mateos and Gonzalez-Carcedo. 1987). The similarity of optimum pH values afrees with the idea of abiotic phosphatases being associated with materials having lower sorption capacity (e.g. plant residues) in these soils. Only in the CF, fraction (that concentrates humic and clay colloids having a strong sorption capacity) of the Dystrochrept soil does the pH optimum have a higher value than in the unfractionated soil, although the ditTerence is of little significance. The alkaline phosphatase in the carbonatc-containinp soil appears lo have grcuter thermal stability than the acid phosphatasc of the Dystrochrcpt (Table 2). Phosphatase activity shows similar behaviour in unfractionated samplus and soil units when the tcmpcraturc trcatmcnt was higher. although activity in the structural units seems more stahlc than that in unfractionated soils. Higher tcmpcralurcs were rcquircd for enzyme inactivation in the structural soil units Ci-,J, whcrc activity is rhc highcsl (Table 2). th;In in the Cl:, frxlions or in unfraclivnatcd soils. Apparcnl K,,, values of phosphatasc in the ditfcrcnt soil fraclions are shown in Tahlc 4. It has been suggcstod Ihac soil phosphatasc dots not follow the hyperbolical kinetics dcscribcd by the mathcma~ical analysis of Michaelis bccausc the enlymalic rcaclions in soil Lake place at solid-liquid interfaces (Irving and C’osgrovc. 197‘)). tlowevcr, our results indicate that the kinetic bchaviour of phosphatasc in these soils and soil fractions can be analyzed through the Michaclis-Mcntcn equation. with correlation cocfiicients of lines derived from the LincwcaverBurk equation mnging berween O.98Y and 0.99’). The A’,,, values found in these soils are in the same rungs as those found by other authors. e.g. Makboul and Ottow (1979) quote a value of 3.26 rnsl for alkaline phosphalasc. Although the diKcrcnces between K,,, values in both soils do not stem 10 b very significant, the resulls agree with the existence of alkaline and acid phosphatascs. This enzyme-kinetics study suggests that there are different isocnzymcs or ditT’erences in the configuration of the enzyme or acccssibilily of substrate lo the active silt bctwccn soil fractions. If the equilibrium constant of [he rcvcrsiblc formalion of a complex bclwccn substrate and enzyme is assumed lo bc similar to the AMichaclis constant. the alkaline phosphatase would have grcatcr affinity towards PNP in the CF, fraction than in [hc CF,, fraction or unfractionatcd soil. However, the acid phosphatase associated with the plant materials conccntratcd in CF,: would probably bc more accessible to substrate than the enzyme located in the more humic fraclion (CF,) or in the unfractionatcd soil. However. the diKercnces in A;,, values arc probably
173
too small lo consider these interpretations tive conclusions.
as defmi-
Acknowledgemenrs-We thank T. Rey and L. Martinez for some of the analyses. This work was supported in part by grants from Comision Interministerial de Ciencia y Tecnologia (CICYT) and the Acciones lntegradas Programme. REFERENCES Anderson J. P. E. (1975) Other organic phosphorus compounds. In Soil Components. Vol. I. Organic Componenrs (J. E. Gleseking. Ed.). pp. 333-341. Springer-Verlag. New York. Burns R. G. (1956) Interactions of enzymes with soil mineral and organic colloids. In Imerucfion o/Soil Minero/s with .~urrtru~ Orgurncs und Microbes (P. -M. Huang and M. Schnitzer. Eds). no. 429451. Soecial Publication No. 17. Soil Science Society of America. Madison. Burns R. G. and Ladd J. M. (1985) Stability of immobilised phosphatases in soil. Socierx /iv Generul bfrcrobrolog~: Qucrrtcrl~ 12. 17. Burns R. G.. Alstrom S.. Burton C. C. and DartnaIl A. M. (1987) Cyunogenic microbes and phosphatase enzymes in the rhizospherc: properlies and prospcts for manipula1ion. In ~f~rCrr~~krii~~,~.~/,rip &vwe&~ ~ii~roor~tr,li.~“,.~ ;nc/ Pltrn~s in Soil (F. Kunc and V. Vancura. Eds). Academia. Prague and Elscvicr. Amsterdam (in press).. Duck W. A. and Tahat;~hai M. A. (19X4) Kin&c parameters of phosphatascs in SOIIS and organic waste materials. Soil Swnw 137. 7 IS. Frankcnhcrgcr W. T. and Johanson J. B. (1WK!) EIkct of pi I on cnlymc slahilily in soils. .%i/ Bi~~/o~,v ,t Biochemistry I-l, 433 437.
Ilal\tcxl R. L. and McKcrcher R. B. (1075) Biochemistry and cycling of phosphorus. In Soil Biochcwrrsrr~. Vol. 4. (E. A. Paul and A. I>. McLarcn. Edr), pp. 31 63. Xlxccl Dckkcr. New York. Hcinrich P. A. and Patrick J. W. (19X5) Phosphorus acquisition in the soil-root system of Eucu/p/us pihclrrri.~ Sm. seedlings. I. Characlcristics of the soil system. .lusfru/icr,t Jounto/ o/‘ Soil Heseurch 23. 223 -236. Irving G. C. J. and Cosgrove D. J. (1976) The kinetics of soil acid phosphalasc. Soil Biu/qy ,@ Biochentr.~rq~ H. 335 ~340. Juma N. J. and Tabalabai M. A. (1978) Distnbution of phosphomonorslerascs in soils. SoilScicncr 126, 101 -IOX. Krivonosova G. M. and Basevich T. V. (1981) Conlcm and forms of organic phosphutes in the soils of the steppe zone of the Ukraine. Soviet Soil Science 12. 2YO-2Y-l. Kruskal W. H. and Wallis W. A. (1952) Use of ranks in one criterion variance analysis. Journul o/‘Americun Stutisricul Annuls 47. 583 62 I. Makboul H. E. and OIIOW J. C. G. (1979) Alkaline phospharase activity and Michaelis constant in the presence of different clay minerals. Soil Science IZH. I29 -135. Murphy J. and Riley J. P. (1962) A modilicd single solution method for the determination of phosphate in natural uatcrs. Ancr/~rrcu Chimicu Acru 27. 3 I -36. Pang P. C. K. and Kolcnko H. (IYX6) Phosphomonocsrcrasc activity in forest soils. Soil Biology d Biochcnti.~rry 18, 35-N. Perez-Mateos M. and Gonzalez-Carccdo S. (1985) EfTcct of fractionation on location ofcnzymc aclivirics in slruclural units. Biolrqy und Fur/i/i/y q/‘ Soi/.s I, I53- 159. Pcrcz-Mateos M. and Gonzalez-Carcedo S. (I 9X7) Effect of fractionarion on the enzymatic state and behabiour of enzyme aclivitics in difrcrcnt structural soil units. Bio/cjgj rrncl /%rfi/irj~ q/ Soi/.s 4. I5 I - 154.
174
M. J. RalJ0 ef Oi.
Saunders W. M. H. and Williams E. G. (1955) Observations on the determination of total organic phosphorus in soil. Soil Science 6, 254-267.
Skujins J. J.. Braal L. and McLaren A. D. (1962) Characterization of phosphatase in a terrestrial soil sterilized with an electron beam. Enz~mologiu 25. 125-133. Speir T. W. and Ross D. J. (1978) Soil phosphatase and sulphatase. In Soil Enynes (R. G. Bums, Ed.). pp. 197-250. Academic Press. London. Stewart J. W. B. and McKercher R. B. (1982) Phosphorus cycle. In Experimenrul Microbial Eculog~ (R. G. Burns and J. H. Slater. Eds), pp. 221-238. Blackwell. Oxford.
Sycrs J. K.. Shah R. and Walker T. W. (1969) Fractionation of phosphorus in two alluval soils and particle-size xparates. Soil Science 103. 283-289. Tabatabai M. A. and Bremner J. M. (1969) Use of p-nitrophenyl phosphate for assay of soil phosphatasc activity. Soil Bio1og.r rG Biochemistry
I. 301-307.
Tarafdar J. C. and Jungk A. (1987) Phosphatase activity in the rhizosfere and its relation to the depletion of soil organic phosphorus. Bio/og_v and Fertiliry of Soils 3. 199-204.
Uriyo A. P. and Kesseba A. (1975) Amounts and distribution of organic phosphorus in some profiles in Tanzania. Geoderma 13. 201-210.
1990
0038-07 I7 90 53 00 + 0.00 Press plc
Copyright C I990 Pcrgamon
DISTRIBUTION AND CHARACTERIZATION PHOSPHATASE AND ORGANIC PHOSPHORUS FRACTIONS M. J. ROJO.’ S. G. CARCEDO:
OF IN SOIL
and M. P. MATEOS~
‘Department
of Organic Chemistry. ‘Department of Soil Science and Technology and ‘Department of Biochemistry. Molecular Biology and Physiology, Burgos University College. University of Valladolid. Aptdo 231. 09071 Burgos. Spain (Accepted 25 .4ugusr 1989) Summary-The distribution of organic P (P,). Fe. Al and Ca. and alkaline and acid phosphatase in surface layers of a calciferous alkaline soil and an acidic soil and fractions of these obtained by a granulometric procedure was studied. There was a significant correlation between the P, and Fe or Al contents. The highest rates of P, mineralization (evaluated by means of the organic P,-to-organic C-ratio) were found in the soil units containing less-humified organic matter. These fractions also had the highest amounts
phosphatase activity. Phosphatase activity was concentratedin the larger soil fractions (1OO&lOO~m
dia) and was probably associated with plant debris and the less humitied organic matter. The alkaline soil contained dominantly alkaline phosphatase and the acidic soil acid phosphatase. The relationship between phosphatase activity and pH was essentially the same in soil fractions as in the whole soil. The phosphatase activity of the alkaline soil had a grcatcr thermal stability than that of the acid soil. Enzyme kinetic data give K__,values ranging from 2.0 4.4 IIIM in fractions from the alkaline soil (unfractionatcd soil 4.1 rnti) and frnm 3.6-l 5.4 mM in fractions from the acid soil (unfractionatcd soil 5.7 msr).
and Gonzalez-Carccdo. 19x5). Using this method. WC huvc studied the participation of two important phosphomoncostcrascs in phosphate availability in root-zone soils with high concentrations of organic I’ (P,) and the means through which thcsc enzymes arc stabilized. This study may lead to a bcttcr understanding of the characteristics of soil enzyme immobilization and allow the incorporation of stable enzymatic complexes into soil in order to increase the amount of inorganic phosphate available for plant nutrition.
Soil cnrymcs in the aqueous phase arc gcncrally short-lived bccausc thsy arc inactivated by association with soil particles or colloids or by protcolytic degradation. t lowevcr, soil alkaline and acid phosphatascs (EC 3. I .3. I and 3. I .3.2) and other enzymes can bc immobilized on, or in, soil clays and humatcs, thcrsby constituting a persistent extracellular catalytic activity (Burns, 1986). Soil humic matter is a highly-charged polyclectrolyte which can complex cnzymcs. This incrcascs their resistance to biodegradation and further stabilization can occur by fixation of this complex to clays. A large proportion of total soil P is bound up as ester phosphate in complex organic compounds and, consequently, considerable research has been applied to the characterization of soil phosphatascs and to ways of increasing or supplcmcnting their activity (e.g. Speir and Ross, 1978). Thus, stable artificial humic-phosphatase complexes have been added to soil (Burns and Ladd. 1985). and the USC of a stable phosphatasc seed coating has been cvaluatcd as a means of bringing about localized solubilization of soil organic phosphate (Burns er al.. 1987). Wc have used a granulomctric proccdurc lo scparate soil fractions containing difTerent types of organic matter (incomplctcly transformed animal, plant and microbial debris and polymeric humic fractions). This technique has proved to bc valuable in the location of soil enzyme activities (Perez-Matcos
‘To
whom all correspondence
Soils Samples (O-7 cm) of two soils from under pasture wcrc collected and stored at field moisture content in scaled plastic bags at 2’C. Experimental work was carried out as soon after collection as practicable. Thcsc soils wcrc an Aridic Calcixcroll (pH 8.4; CEC 0. I I cmol kg-‘; organic C (C,) 42.2 g kg-‘; C/N 9.4: sand 26.2%; silt 0.1%; clay 0.21%; CaCO, 63.2%) and an Umbric Dystrochrcpt (pH 4.3; CEC 0.17cmol kg-‘; C, 62.6g kg-‘; C/N 14.9; sand 17.5%; silt 48.6%; clay 13.3%) and have been described by Pcrcz-Matcos and Gonzalez-Carcedo (1985) and Gonzalez-Carcedo er crl. (1983).
Soil enzyme dirtrihutiun The distribution of phosphatascs in soil fractions was determined by the methods of Pcrcz-Matcos and Gonzalez-Carcedo (1985) for other enzymes. Airdried soils were fractionated in three different ways:
should be addressed. 169
(A) sieving at J C without pretreatment. (B) wetsic\~ng after dlsperrion of soil, mixed with distilled water. using reciprocating agttation for 1.5 hat 1.05~. and (C) wet-sie\mg as m B after aqueous dispersion and mechanical dlsruption of the soil by agitation w-ith Nater and 5 agate balls (I cm dia) per 35 g soil for I5 h. Using procedure (A), four soil fractions of with particle-size rcul ttnifs u-ere obtained X00-3OO/~rn (.AF,). 200-100~(m (AF,). 10&50/[m (.AF,) and <50/lrn (AF,). using procedure B four factions of srubi;~ ~ctrirs(BF). and using procedure C four fractions of srrli~turul units (CF) of the same sizes. were obtained. For details see Perez-Mate05 and Gonzalez-Carcedo (198.5). In some assays. fructinns F, and F: were mixed to give fraction F,: (particle-size ~00~100 jirn).
Air-dried soil (~2 mm) (I g). and soil fractions (I 3) were treated with I ml of I I5 mM p-nitrophenyl pll~~sph~Ite (pNP) and 4 ml of modified universal butTcr (MUB) at pH 6.5 (Skujins et trl.. 1962). After agitation. and because of the buffer effect of soil. the pll of thcsc mixtures was 7.1 in the alkaline soil and 5.3 in the acid soil. li~~ubations were carried out at 37 C for i h and then. 0.5 %t CaCII (i ml) ard 0.5 hi NaOf 1 (1 ml) u’cru iiddcd and the mixtures shaken and held at 20 C for 10 min (Tabatubai and Brcmncr, 196Y). The contents were filtcrcd (Whatmun No. 6) and aliquots (I ml) of the filtrates made up to 50 ml with 0.5 M NaOFi. The p-nitrophcnol (pN) produced was dctormincd ~ol~~rim~tricaIly at 410 nm. Assays wcrc c;trricd out in triplicate with two controls (substrata ;&led after incubation). Units of phosphatasc activity were jrmol pN produced g dry soil .’ h ” and specific cnrymc activity v:~Iues with respect lo the organic matter content of samplc~ wcrc calculated in ltmol pN formed g C ‘h ‘.
The clfcct of ptl was studied by mixing the soil sarupl~‘s with nine solutions of modified universal bufikr (4 mi g ’ ) at ptf vafucs between 3 and I I. Thcrcal’tcr the mixtures were allowed to st:md for 1 h with occasional stirring, the pH was mcasurod again and the cnLymc activi.ty. assayed. The values of the MUB ptl ucrc din~lnlsh~d by the Dystrochrcpt soil and fractions to more acid values whereas the Calci~croll and fractions incrcascd the pH of the six M UB solutions with pH values bctwecn 3 nnd 8 and dccrcased the pH of the other three solutions (see pH of mixtures in Figs 1 and 1). Thermal stability was assayed by measuring cnryme activity of soil samples which had been trcatcd in closed vosscls 31 20, 55. 65. 75. 85. 95 or IO5 C for 21 h. The kinetics of the soil phosphatascs wcrc cvaluatcd by measuring the enzymatic reaction rates at ditfcrcnt substrate concentrations. Five solutions of PNP u’erc used with concentrations of 5; 25; 50; 75 and IO0 msg. Thr values of K,, were dcrivcd from computed Icast-square analysis of Lincwcavcr-Burk plot. \lcasurcmcnts of phosphatase activity for thermal stability and kinetic assays were made at pH IO.27 in
the and pH for
Calcixeroll samples (pH of the added M UB I 1.O) at pH 5.31 in the Dystrochrept samples (MUB 6.5). These pH values correspond to the optima phosphatase activity in the respective soils.
The soil P, content was calculated by a modiiication of the method of Saunders and Williams (1985) from the difference between the contents of total and inorganic orfhophosphate. The total P content was determined by ignition and subsequent extraction with 1 N HSO, and the total inorganic P by extraction of unignited samples with t N H-SO,. P concentrations in all extracts were dete&ined cotorimctrically (Murphy and Riley. 1961). Organic C (C,) and N were measured with a CHN automatic autoanatyser (Carlo Erba. 1106) and Fe. AI and Ca by atomic absorption spectrometry techniques. Catcutations of P,,. C, and elements content in soil fractions (Table I) were made by conversion of absolute results into relative values by taking into account the contribution of each fraction (IV,) to the totat weight of soil (Pcrcz-Matcos and Gonzalez Carccdo. 19x5).
All exporimcntal data were tested by standard deviation and variation cocliicicnt, not exceeding 6% of this cocfficicnt. Two separate variance analyses of
tt;”
C,,’
r*:
pz,C,
57?.3 503.0 47 I) 12.4 YY 71 I ‘3.3 23 X 413 7 Ill 4 Y? I46 ES?,
13.7 14.5 It) Y X3 I I .o
(46) (“iPI C,N fpgg It (x IO $1 .hdU~<‘d-rrerdl wrl 1;s AF, Ai-, A r:I A F, BF, BF: RF, BF. ct:; CF,
CF; CF, f,‘i,thrir
X6 6 X5 ?.Y : 0 32.2 76 X.1 52 I ii.4 7.6 IO.6 65.4
AF, AFL AF, BF, BF, fw, Hf’, :;I CT; CF.
0.4 Y3 10.6 IS.0 Y.1)
11.1
I I.5 16.X IO t l’J.5 15.3 16.5 Y7
&I 6.26 14.0 5.1X 15.3 0.51 13.x 0 ?X 17.0 0.27 13.4 1,X6 23.7 11.57 16.‘) 0.52 17.7 2.X4 13.4 0.x 22.5 0.1’) IX.7 OS1 IX.6 3 3x 13.7
5.4 I?.‘) 2’J.X 21 I 3.0 2.4 4.4 x1.7
Al’ -.
7 6X 6 66 065 II I’J O.IX 1.03 02X 0.30
C.1’
(mga ‘1 7.10 6.05 0.63 O.?? 0.15 0.X! 0.?3 0.35
4 ifs
f,Oft
015 01.8 0 ‘3 S.lX
0.12 OIJ I).IX 4 4.8
X2.‘) 7.0 40 60 32 X 3.2 67 57 3 XI I.? 13.1 77 I
403.5 320.7 37.3 1H.Y 25.1 70x1 I0.Y IS I 3O’J.l 3-i 0 Y.3 IS I 344.7
6.4 62 72 6.7
9.3 3.X IY 2.5 IO Y IS 5 4.Y 30 10.2
iXY3 14.11 I5 7’) iO.hY 11: 1.16 0.72 0.w OY’ OSI h-l3 ?.JY 0 77 0 24 063
IO 5X 5.5‘) 0.72 OX4 If34
.---
7.Y3 h.SII 11.52 If 20 OIZ LY7 061 0.71 2. IO I.?X 0.5Y O.Y? 3.XS
~r.rlr{J~/~?~.~f
us AF,
4.23 3.46 0.43 0 IS 0.0’~ 1.31 0.1x OIJX 1.46 0.15 0.3’) 0.31 2.54
FC’ “-’ .~
0 0 0 0 0 0 0
O?!
0
5.x I.33 0.1’) 027 7.1X
II 0 I) 0 0
Soil phosphatax distribution a rank classification of Kruskal and Wallis (1952) uere performed: one with data of the enzyme distribution and the other with data of the enzyme characterization. For a significance level of 0.05, experimental constants H < 4.95 for the former group of data and H < 5.12 for the latter were obtained for this statistical test. suggesting that the values of independent assays performed in the replicates could not be considered significantly different. RFSL’LTS
ASD
DISCLSSION
Chemical characterization The granulometric procedure we used to fractionate soil phosphatase activity gives soil fractions containing a characteristic distribution of soil organic matter. Real units (AF fractions), specially AF, the fraction formed by the largest aggregates, which include soil units isolated in BF,, BF, and the structural units CF,. contain a mixture of plant debris. less-transformed organic compounds (e.g. pcptides. fatty acids, carbohydrates). polyphenolic humic matter and other kinds of organic hiomolecules. However. the stable (BF fractions) and even more. the structural soil units (CF fractions), scparatc humatcs from fresh organic matter as is indicated hy the C-to-N ratios included in Tahlc I. Thus humic compounds and clay minerals (organomincral complcxcs) constilutc the microapgrcpatcs grouped in the CF, fraction, that has the lowcrt C-to-N ratio (Con~alcz-Carccdo (*I (11.. 1983; Pcrcl-Matcos and Gonlalcz-Cnrccdo. lYX5). A Iarpc proportion of total soil I’ is bound up in organic compounds in thcsc soils: 85.4% in the Calcixcroll soil and 74.3% in the Dystrochrcpt. The distribution of the P,, in the twclvc fractions of both soils (Table I) suggssts an important association bctwccn soil P and humic compounds: soil fractions concentrating clay and humic colloids (AF,. BF,, CF,) showrd the grcatcst P,, concentrations, although the results found in the fractions containing plant debris (AF,. BF,,, CF,,) may indicate the prescncc of P,, associated with non- or partially-humifed organic matter. Esters of inositol, nuclcotidcs and phospholipids, idcntifcd in soil extracts (Hnlslcud and McKerchcr, 1075; Krivonosova and Buscvich, 1981; Stewart and McKerchcr. 1982) have been thought to bc associated with or derived from humic-bound P compounds (c.g. nucleic acids, phospho-proteins, ctc). Sycrs Ed (11. (196’)) also found the grcatcst P, content in soil fractions with the smnllcst particlcsiLc. The P,-to-C,, ratio (or C,-to-P, ratio) has been suggcstcd as an index of P mineralization in soils (c.g. Uriyo and Kcsscba. 1975) and our study showed a high P concentration in the humic organic matter of CF, fractions (Table I ). although in the Dystrochrcpt soil. fresh organic matter prcscnt in thr CF, fraction showed a higher P, concentration. P mineralization appeared to be slower in the calcifcrous soil than in the acidic soil. perhaps due to the prcscncc of calcium in high concentrations (Table I). complexing or insolubilizing P, compounds and protecting them from chemical or biological degradation. Association of P with humatcs through cationic bridges to form organo-metallic P compounds may be a way to
Table
171 2. Phosphatax Aridic
SolIS Real
activity
in the sod fractions’
Calcixcroll
PhA”
Umbric
PhAsp’
PhA”
Dystrochrept Ph.&p’
umts
AF,
90.6
99.3
102.3
1905
AF,
I x4.4
108.7
145.0
x7.
AF,
69.9
59.3
89.6
149.7
43.9
43.0
52.2
135.5
AF, Stable
I
units
BF,
72.3
67.8
50.6
81.4
BF:
105.3
233. I
1764
113.1
BF,
447
?JJ.8
101.1
lZl.2
BF,
75.5
89.5
J3 z
113.9
CF,
77.0
158.6
61.3
4s3.0
CF, CF,
IO?.5 63. I
87.6 89.0
66.1
CF,
77.6
VI.7
3:. I
Structural
units
‘Phosphalax
activity
of
the
p ml dry soil h -’ Aridic
’
Dystrochrcpi.
Umbric
“Phosphatase
activity
unfracrlonated ‘Specific
phosphdtasc
PhAsp and
in
%
Zl0.Z
unfrwtionated
formed h
I”.1
83.7
soils:
Calcixeroll
with
9x.2
I I .6 pmol
and
respect
to
4.38 pmol
the
actwty
pN
pN g of
’
fhc
samples. activity
in pmol
of the unfracrionaled
IRS 9 U Umbric
pN
soils:
formed
p -’ organic
103.7 U Aridx
C h
‘.
C.kineroll
Dystrochrept.
protect P, from chemical or biochemical attack. Thcrcforc. linear rcgrcssion analysts bctwccn P,, and cations were dcrivcd for all of the soil snmplcs. A signilicant relationship appears bctwccn Fe. Al and P,, in the fractions of the Calcixcroll soil with correlation coeflicicnls. r > 0.X3*** (significant at the P < 0.001 Icvcl). In this soil. the strongest corrclations occurred in the structurd units with cor > O.YY***. This suggests the cfkicnls of participation of Fc and Al, but not C;L. in the association of P compounds with organic matter. Anderson (lY75) and others have outlincd the function of Fc and Al in bonding humic substances with
UMBRIC DYSTROCHREPT
zo-
: + l
’ 456
’
’ 7
8
9
10
11 PH
Fig. I. ElTcct of pH on phosphatase activity in unfractiunated soils (US) and soil real units (AF,,. XKM-IOOIrm; AF,. c50yrm).
172
M. J. ROJO er al
phosphate groups. In contrast, no such relationships were found for the acid Dystrochrept soil. 80
ARiDIC
CALCIXEROL
Enzyme distribution The distribution of phosphate activity in the I2 fractions of both soils is shown in Table 2. This activity was mostty concentrated in the largest size fractions of both soils, both after sieving (AF, . AF?) or after aqueous dispersion and wet sieving (BF,). In the largest size structural units, where most of the fresh less-humiiied organic matter was concentrated (CF: and CF,). the phosphatase activity remained higher, although the humic fractions (CF,) also contained an appreciable amount of activity. The only soil units in which an enzymatic enrichment or puri~cation was observed with respect to the unfractionated samples were the FZ fractions. Plant roots may take up P at a rate greater than indicated by the diffusion coefficient of orf/Iophosphate in soils (Heinrich and Patrick, 1985). This may indicate uptake of fffr~z[~phosphate P derived from organic matter by the action of plant root and associated microbial phosphatases. Plant debris can be obscrvcd, using a low-powered microscope. in the iargcst size fractions (F, and F,). Several authors (e.g. Juma and Tabatabui. 1978) having studied dinirent lcavcs) and soil fractions. plant miltcri;Ils (roots. suggesting ;I plant origin for a part of soil phosphatasc activity. On the other hand, this fraction of structural units CF, (and CF, in the acid soil) had the Iowcst P,,-to-C,, ratio and the greatest phosphatasc activity (compare Tahlcs I anti 2). The highest rate of P mincraii~;ttion (lowest P,,-to-C,, ratios) in the fractions containing fresh organic matter is coincident with the largest rates of activity being found in these soil fractions. Similarly. Tarafdar and Jungk (1987) found a significant corrciation bctwccn the deplction of P,, and phosphatase activity in rhizospherc soils of whsat (r = 0.99) and clover (r = 0.97). Specitic phosphatasc activity vaiucs shown in Table Z may reflect the association of organic matter and enzymes. Enzyme activity is poorly associated with the organic matter of real and stable units. but fresh organic matter accumulated in CF, (and in CF, for the Dystrochrcpt soil) appeared to have a much greater capacity to associate with phosphatase activity than has organic matter in the remaining structural units or unf~~ctionatcd soil. These results agree with those concerned with the distribution of cnryme activity in the soil fractions.
The pal-~Ictivity profiics of phosphatasc in unfractionatcd soil and soil real units (AF,,. AF,) are shown in Fig. I. The differences in pH-optimum vaiucs were not signiticant. Although the presence of many isocnzymcs with different optimal pHs in each sample is suspected. the pH values with maximum activity (cu IO.3 in the Caicixcroii soil and about 5.2 in the Dystrochrcpt) rclatcd to the pH characteristic of each soil sample, suggest the presence of alkaline and acid phosphatase respectively. The incrcascs of activity at pH 5.7 in the Caicixcroil soil may indicate the existence of some acid phosphatasc activity in this soil. although this would have no real significance. Similar results wcrc found for optimum pH values of
60
r; 100
45
6
78
9
10
11
w
z
2
80
E g E
60
UMBAIC DYSTROCHREPT -
us
-
CF
40
5
6
7
8
9
IO
11 PH
Fig. 2. Efkt of pH on phosphatasc Mivity atcd s&is (US) and soil structural units (CF,,. CF,. <5O~tm).
in unfractionZOO@-100 {tm:
phosph~It;I~ activity in the structural units of both soils (Fig. 2). These findings tond to confirm the suggestion of Dick and Tabatabai (1984) that acid phosphatasc dominates in acid soils and alkaline phosphatasc in alkaline soils. although some authors (e.g. Pang and Kolenko, 19X6) have reported neutral phosphutasc in acid woodland soils. These results agree with the conclusion of Frankenbcrgcr and Johanson (1982) to the elTcct that the optimum pH range for phosphatase activity is gcncrally quite narrow. Phosphatase in soil fractions does not usually have its maximum activity at more alkaline pH values than from that of unfractionated soils. suggesting that phosphatase is no more absorbed to colioids in the soil units than in the unfract~onated Table 3. Thrrmul rtltbibty of phosphatwe activity’ in unfraclionatrd sod and sotl frxtions Temperature ( C) 55 Aridk Cul~i.r~roll sod Unfraction;rtcd ~011 Red umtr AF,: AF, AF, Structural units CF,: CF. ufdlrir D,wi?odwrpr sod Unfrsctlonaled sod Real U~IIF AF,: AF, AF, Structural units CF,, CF.
63
75
I
X5
63.7 46.5
9s
Iul
X4 6
73
XS.2 X9.3 94.6
71.6 70.3 X6.4
53.X 51.7 7X.X
3X.7 42.5 55.6
1x.7 32. I 30.5
95.3 92.9
X6.5 73.2
75.4 66.5
55.2 43.9
19.3 24.2
77.6
56. I
JI 3
35.5
29.9
H3.3 RI.X X0.7
62. I 5X.5 6&X
49.x 45.4 so.4
32.9 23.X 37.0
26.9 17.8 30.1
86.9 19.9
69.9 71.4
53.3 65.9
41.9 39 4
41.0 17.7
‘In 96 with resrct to the sctiwty of the untreated samples.
Soil phosphataa ie distribution Table 4. Apparent K_ valucs~of phosphamc in soil fractions Aridic
Calcixeroll
Cmbric
Dystrochrepc
1.12
5.71
Real units AF,, AF, AF,
2.91 ‘.oo 2.55
IS.41 6.88 II.91
S1ructural ““!lS CF,: CF.
4.40 ?.I7
3.62 6.00
Unfractlonatcd
‘In
ITIM
sods
pNP
samples (Perez-Mateos and Gonzalez-Carcedo. 1987). The similarity of optimum pH values afrees with the idea of abiotic phosphatases being associated with materials having lower sorption capacity (e.g. plant residues) in these soils. Only in the CF, fraction (that concentrates humic and clay colloids having a strong sorption capacity) of the Dystrochrept soil does the pH optimum have a higher value than in the unfractionated soil, although the ditTerence is of little significance. The alkaline phosphatase in the carbonatc-containinp soil appears lo have grcuter thermal stability than the acid phosphatasc of the Dystrochrcpt (Table 2). Phosphatase activity shows similar behaviour in unfractionated samplus and soil units when the tcmpcraturc trcatmcnt was higher. although activity in the structural units seems more stahlc than that in unfractionated soils. Higher tcmpcralurcs were rcquircd for enzyme inactivation in the structural soil units Ci-,J, whcrc activity is rhc highcsl (Table 2). th;In in the Cl:, frxlions or in unfraclivnatcd soils. Apparcnl K,,, values of phosphatasc in the ditfcrcnt soil fraclions are shown in Tahlc 4. It has been suggcstod Ihac soil phosphatasc dots not follow the hyperbolical kinetics dcscribcd by the mathcma~ical analysis of Michaelis bccausc the enlymalic rcaclions in soil Lake place at solid-liquid interfaces (Irving and C’osgrovc. 197‘)). tlowevcr, our results indicate that the kinetic bchaviour of phosphatasc in these soils and soil fractions can be analyzed through the Michaclis-Mcntcn equation. with correlation cocfiicients of lines derived from the LincwcaverBurk equation mnging berween O.98Y and 0.99’). The A’,,, values found in these soils are in the same rungs as those found by other authors. e.g. Makboul and Ottow (1979) quote a value of 3.26 rnsl for alkaline phosphalasc. Although the diKcrcnces between K,,, values in both soils do not stem 10 b very significant, the resulls agree with the existence of alkaline and acid phosphatascs. This enzyme-kinetics study suggests that there are different isocnzymcs or ditT’erences in the configuration of the enzyme or acccssibilily of substrate lo the active silt bctwccn soil fractions. If the equilibrium constant of [he rcvcrsiblc formalion of a complex bclwccn substrate and enzyme is assumed lo bc similar to the AMichaclis constant. the alkaline phosphatase would have grcatcr affinity towards PNP in the CF, fraction than in [hc CF,, fraction or unfractionatcd soil. However, the acid phosphatase associated with the plant materials conccntratcd in CF,: would probably bc more accessible to substrate than the enzyme located in the more humic fraclion (CF,) or in the unfractionatcd soil. However. the diKercnces in A;,, values arc probably
173
too small lo consider these interpretations tive conclusions.
as defmi-
Acknowledgemenrs-We thank T. Rey and L. Martinez for some of the analyses. This work was supported in part by grants from Comision Interministerial de Ciencia y Tecnologia (CICYT) and the Acciones lntegradas Programme. REFERENCES Anderson J. P. E. (1975) Other organic phosphorus compounds. In Soil Components. Vol. I. Organic Componenrs (J. E. Gleseking. Ed.). pp. 333-341. Springer-Verlag. New York. Burns R. G. (1956) Interactions of enzymes with soil mineral and organic colloids. In Imerucfion o/Soil Minero/s with .~urrtru~ Orgurncs und Microbes (P. -M. Huang and M. Schnitzer. Eds). no. 429451. Soecial Publication No. 17. Soil Science Society of America. Madison. Burns R. G. and Ladd J. M. (1985) Stability of immobilised phosphatases in soil. Socierx /iv Generul bfrcrobrolog~: Qucrrtcrl~ 12. 17. Burns R. G.. Alstrom S.. Burton C. C. and DartnaIl A. M. (1987) Cyunogenic microbes and phosphatase enzymes in the rhizospherc: properlies and prospcts for manipula1ion. In ~f~rCrr~~krii~~,~.~/,rip &vwe&~ ~ii~roor~tr,li.~“,.~ ;nc/ Pltrn~s in Soil (F. Kunc and V. Vancura. Eds). Academia. Prague and Elscvicr. Amsterdam (in press).. Duck W. A. and Tahat;~hai M. A. (19X4) Kin&c parameters of phosphatascs in SOIIS and organic waste materials. Soil Swnw 137. 7 IS. Frankcnhcrgcr W. T. and Johanson J. B. (1WK!) EIkct of pi I on cnlymc slahilily in soils. .%i/ Bi~~/o~,v ,t Biochemistry I-l, 433 437.
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