Interaction of catalase with montmorillonite homoionic to cations
with dierent hydrophobicity: eect on enzymatic activity and
microbial utilization
L. Calamai
a,*, I. Lozzi
a, G. Stotzky
b, P. Fusi
a, G.G. Ristori
ca
Dipartimento di Scienza del Suolo e Nutrizione della Pianta, UniversitaÁ degli Studi, Piazzale Cascine 28, 50144 Firenze, Italy b
Laboratory of Microbial Ecology, Department of Biology, New York University, New York, NY 10003, USA c
Istituto per la Genesi e l'Ecologia del Suolo (IGES), CNR, Piazzale Cascine 28, 50144, Firenze Italy
Accepted 14 November 1999
Abstract
The exchange sites of montmorillonite (M) were made homoionic to calcium (Ca), hexadecyltrimethylammonium (HDTMA) or pyridinium (PY) cations. The clays were used as adsorbents for the enzyme, catalase (CA). Equilibrium adsorption and binding isotherms (i.e., after washing of the clay±CA complexes after adsorption at equilibrium until no CA was desorbed) were of the L3-type and ®tted the Langmuir equation in the initial, but not in the later, portions of the isotherms. The amounts
adsorbed and bound at the plateau, as well as the anity, were higher for the hydrophobic clays (M±HDTMA and M±PY) as indicated by the Langmuir parameter, Bmax and Keq. In all three systems, there was additional adsorption after the initial
plateau at higher concentrations of CA. In the case of M±Ca±CA this probably resulted from some penetration of CA into the interlayer spaces of the clay, as shown by X-ray diraction analysis. No penetration of the interlayers was observed in the M± HDTMA±CA and M±PY±CA systems. The additional adsorption that occurred after the initial plateau in these systems may have resulted from the formation of multilayers of CA or from a change in the orientation of CA on these clay-organic surfaces, which may also have occurred in the M±Ca±CA system in addition to intercalation. Most of the CA adsorbed at equilibrium was bound on the clays (85±90%). Fourier-transform infrared dierence spectra showed a shift in the Amide I and Amide II frequencies for only M±Ca±CA and M±PY±CA, which was consistent with the hypothesis of a conformational modi®cation of the structure of CA on M±Ca and M±PY. The enzymatic activity of CA adsorbed at equilibrium on the three clays was lower than that of free CA and decreased in the order of M±Ca±CA > M±PY±CA > M±HDTMA±CA. As shown by the values of the overall ®rst-order rate constant,K1, there was a further reduction in activity when CA was bound on the clays, especially on
M±Ca. The pH optimum for the activity of CA remained essentially unchanged when adsorbed or bound on all clays. CA bound on the clay systems, except on M±PY±CA, was poorly utilized in comparison with the free enzyme as a sole source of carbon or nitrogen.72000 Elsevier Science Ltd. All rights reserved.
1. Introduction
Interactions between clay minerals and organic poly-mers of biological interest (e.g., enzymes, other pro-teins, nucleic acids) are important in soil (Stotzky, 1986, 1989; Khanna and Stotzky, 1992; Gallori et al., 1994; Tapp et al., 1994; Vettori et al., 1996, 1999;
Kos-kella and Stotzky, 1997; Alvarez et al., 1998; Tapp and Stotzky, 1998; Khanna et al., 1998). These organic molecules dier in their anity for clay surfaces, which has been related to such physicochemical factors as the types of cations on the clay exchange complex, pH, origin and amount of the layer charge, surface charge density, speci®c surface area, and structure of the molecules (Theng, 1979; Stotzky, 1986; Fusi et al., 1989).
The properties of smectites homoionic to long-chain
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alkylammonium cations, which are able to change the nature of the clay surface from hydrophilic to hydro-phobic, have been studied, especially in relation to the adsorption of small organic and nonpolar molecules (Boyd and Mortland, 1985, 1986, 1990; Lee et al., 1990; Favre and Lagaly, 1991; Jaynes and Boyd, 1991a, 1991b). These studies have demonstrated that the siloxane surfaces of smectites possess hydrophobic characteristics at sites distant from isomorphous sub-stitution and are able to adsorb such hydrophobic molecules as hydrocarbons. However, the eect of these hydrophobic sites is overshadowed by the pre-ponderance of hydrophilic sites. When inorganic charge-compensating cations on clays are replaced with hydrophobic cations, e.g., trimethylphenylammo-nium (TPMA), hexadecyltrimethylammonium (HDTMA), the hydrophobicity of the clay surfaces increases. Moreover, when the hydrophobic organic cations possess a long alkylammonium tail (e.g., HDTMA), the formation of a ``bulk'' hydrophobic phase is postulated, and the adsorption of small non-polar organics on the clay surfaces may be enhanced (Favre and Lagaly, 1991). Smectites made homoionic to alkylammonium cations are important not only in the adsorption and subsequent deactivation of toxic pollutants in soil, water and industrial wastes (Boyd et al., 1988), but also in the adsorption of macromol-ecules of biological interest, including enzymes, with possible subsequent modi®cation of their activity (Gar-wood et al., 1983; Boyd and Mortland, 1985, 1986, 1990). Depending on the type of organic cation on the clay and the enzyme being studied, the enzymatic ac-tivity of the adsorbed enzyme may be either decreased or enhanced (Boyd and Mortland, 1986).
Our purpose was to determine the eect of dierent hydrophobic and hydrophilic charge-compensating cat-ions, i.e., HDTMA, pyridinium (PY), and calcium (Ca2+), on the equilibrium adsorption and binding and on the enzymatic activity and microbial utilization of bound catalase (CA). These cations were chosen to provide dierent degrees of hydrophobicity (high for HDTMA and low for Ca) of the clay surface.
2. Materials and methods
Montmorillonite (M) from Upton, WY, was obtained from Ward's Natural Science Establishment (Rochester, NY). The <2-mm fraction, separated by sedimentation, was made homoionic to Ca2+, HDTMA+ or PY+. M±Ca was prepared by suspend-ing the clay twice in a volume of 1 M CaCl2 that was
several times the cation-exchange capacity (CEC) of the clay (78 cmol kgÿ1
). M±HDTMA and M±PY were prepared using an amount of the respective chloride salt (0.1 M) that exceeded the CEC by 20%, to avoid
over-saturation of the clay. Excess salt was removed by washing, with centrifugation, the clays with double distilled water (ddH2O) until the AgNO3 test for Clÿ
was negative (six to seven washes at a water-to-clay ratio of 100:1). The clays were stored at 58C and used within 7 d after preparation.
Catalase (EC 1.11.1.6) from beef liver (thymol-free puri®ed powder, type C40; Sigma Co., St Louis, MO) has a molecular mass (Mr) of 225 to 251 kDa and an
isoelectric point (pI) of pH 5.7.
Dierent amounts of CA (0.5±20 mg) dissolved in ddH2O at 228C were added to 10 mg of clay in a ®nal
volume of 10 ml (pH 6.0±6.5), mixed in glass tubes (10-ml capacity) for 1 h on a motorized wheel (50 rev minÿ1
), and the mixture centrifuged at 20,000gfor 1 h. The amount of CA adsorbed at equilibrium was calcu-lated as the dierence between the amount of CA added and the amount present in the equilibrium supernatant, as measured spectrophotometrically at 280 nm. The adsorption data were ®tted to the Lang-muir equation in the Hanes±Woolf linearized form (Schulthess and Dey, 1996): Ce/Cs=C/Bmax+1/(Bmax
Keq), where Ce is the concentration of CA in solution
at equilibrium (mg mlÿ1), C
s is the amount adsorbed
(mg mgÿ1of clay),B
maxis the maximum adsorption
ca-pacity of the clay for CA (mgÿ1 of clay), and K
eq is
the equilibrium constant (ml mgÿ1) for the adsorption reaction (Evans and WennerstroÈm, 1994). A plot of Ce/Cs vs Ce yields an intercept on the ordinate=1/
(Bmax Keq) and a slope=1/Bmax.
The amount of CA bound was determined by sub-tracting the amount of CA in the equilibrium super-natant plus the amounts desorbed after washings with ddH2O (usually four washes, as no CA was detected
after two washes; lower level of detection was 10 mg mlÿ1
) from the initial amount added. Each washing was performed by resuspending and shaking the clay± CA complex (pellet recovered after centrifugation) in 10 ml of ddH2O for 1 h, centrifuging, and
spectropho-tometrically determining the amount of CA desorbed. X-ray diractograms of bound clay±CA complexes were obtained on oriented samples, air-dried at room temperature and after heating at 1508C, as described by Fusi et al. (1989). A Philips 1410 diractometer with Co Ka radiation was used. Fourier-transform in-frared (FT-IR) analyses (Perkin Elmer 1710 FT-IR spectrometer) were done by air-drying a few drops of suspensions of bound clay±CA complexes or pure clays or of a solution of pure CA on ZnS (Irtran) win-dows. The spectra were recorded between 2000 and 1200 cmÿ1
spectra of the pure clays from those of the correspond-ing clay±CA complexes.
The enzymatic activity of CA was measured at 208C and pH 7 (optimum for the activity of CA) by adding 5 ml of a H2O2solution (at six concentrations ranging
from 0.06 to 1.2%, to yield 3.94±81.7 mM, respect-ively) to CA, either free or adsorbed or bound on the clays, in glass ¯asks (200 ml) containing 45 ml of phos-phate buer (150 mM KH2PO4 and Na2HPO4,
31.5:68.5 ratio). The reaction was stopped after 0.5, 1, 2, 4, 8 or 16 min by adding 40 ml of H2SO4 (25%)
containing Ti(SO4)2 (0.6%) (Thomas and Chamberlin,
1980). The yellow complex that formed with unreacted H2O2 was measured at 410 nm, and the amount of
H2O2 decomposed was calculated as the dierence
between the amounts added and remaining. To obtain comparable amounts of decomposed H2O2, 10, 50 and
100 mg of free, adsorbed, and bound CA, respectively, were used, as the enzymatic activity decreased in the order of free CA > adsorbed CA > bound CA. Paral-lel analyses were performed using comparable amounts of the clays only, to estimate the contribution of the clays to the decomposition of H2O2. No detectable
de-composition of H2O2by the clays alone was observed
at the clay-to-H2O2ratios used.
CA is partially inactivated by contact with high con-centration of H2O2, e.g., in the range of the
Michae-lis±Menten constant (Km=1.1 M) where
pseudo-saturating kinetics are obtained unless the initial vel-ocity is measured within a few seconds after mixing (Ogura, 1955), which is incompatible with obtaining a uniform mixture of H2O2 and suspensions of
clay-bound CA. Therefore, an interval of 1 min and initial concentrations that would not cause decomposition of CA (3.94 to 81.7 mM H2O2) were used. At these
con-centrations, CA exhibits ®rst-order kinetics, and, there-fore, the kinetic parameter calculated was the overall ®rst-order rate constant, K1 (in Mÿ1 sÿ1), for the
equation, V=K1x[H2O2]x[CA], where V= velocity of
decomposition of H2O2 (in M sÿ 1
), and [H2O2] and
[CA] are the concentrations of H2O2and CA (both in
M), respectively. A plot of V over [H2O2]x[CA] yields
a straight line with slope=K1
The enzymatic activity of free and clay-bound CA was also determined at pH 4±10 using 9.5 mM H2O2
and the following buers (150 mM): citrate, pH 4±5; KH2PO4/Na2HPO4, pH 5±8; and borate, pH 8±10.
The activity of CA is dependent on pH, but it is not in¯uenced by the type or concentration of buer (Ogura, 1955). Because H2O2 can decompose
chemi-cally at alkaline pH, parallel analyses were done in the buer solutions alone, to estimate the amounts of H2O2decomposed chemically. There was no detectable
chemical decomposition of H2O2at any pH.
The utilization of CA, either free or bound on the dierent clay systems, as a sole source of carbon or
nitrogen by a mixed community of microbes was eval-uated by measuring, by FT-IR, CO2 evolution during
incubation of CA with a microbial inoculum derived from soil. Free or bound CA (2 mg) suspended in 0.5 ml of ddH2O was mixed with 1 ml of modi®ed
Davis' minimal medium that did not contain nitrogen (Dmin) (3concentrated and sterilized) (Gerhard et al.,
1981) and 0.2 ml of soil inoculum (Si) (10 g of a
non-sterile and unfertilized garden soil suspended in 25 ml of tap water, ®ltered through a paper towel, centri-fuged at 10,000 g, washed 3 times in ddH2O, and
resuspended in ddH2O, to a ®nal optical density at
500 nm of 0.8 to 1) in 100-ml Erlenmeyer ¯asks and adjusted to a ®nal volume of 3 ml with sterile ddH2O.
The ¯asks were sealed with air-tight rubber stoppers and maintained at 258C in the dark. CO2 evolution
was measured after 3 and 7 d by injecting 2 ml of air from the head-space of each ¯ask with a syringe into a micro gas cell (optical path=10 cm, total
volume=8 ml), which was ¯ushed extensively with N2
before each analysis by FT-IR. Preliminary exper-iments showed that the best FT-IR scanning con-ditions for quantitative determination of CO2 were
two scans at a resolution of 32 cmÿ1 and peak inte-gration of the spectra at absorbances between 2440 and 2100 cmÿ1. The areas of the integrated peaks were compared with a calibration curve constructed with pure CO2.
Free or bound clay±CA was incubated with: (i) Dmin+Si; (ii) Dmin+Si+glucose; (iii) Dmin
+-Si+NH4NO3; or (iv) Dmin+Si+glucose+NH4NO3
(with the amounts of glucose and NH4NO3 being
equivalent to the carbon and nitrogen contents of 2 mg of CA). Control experiments with (i), (ii), (iii), and iv either alone or with the homoionic clay systems (i.e., without CA) or with only HTDMA chloride or PY chloride (in amounts equivalent to those present on the clays), were done to evaluate the inhibition by or utilization of the organic cations, free or bound on the clay, by soil microorganisms.
3. Results and discussion
The shapes of the binding isotherms were similar to those of the equilibrium adsorption isotherms, and 85± 90% of CA adsorbed at equilibrium was bound (Fig. 1). Both isotherms were of the L3-type, according
to the classi®cation system of Giles et al. (1960), and showed a good ®t to the Langmuir equation at low concentrations of CA (Table 1), suggesting the for-mation of a monolayer of CA on the external surface of the clays. This was con®rmed by X-ray diraction analyses of the clay±CA complexes, which showed no dierences, neither after drying at room temperature, nor at 1508C, in the basal d001 spacing from that of
the pure clays at these lower concentrations of protein (Fig. 2). The amount of CA adsorbed at equilibrium on M±Ca at the initial plateau (Bmax), and the value
of the equilibrium constant (Keq), calculated with the
Langmuir equation, were lower than those with M± HTDMA and M±PY (Table 1), indicating that the adsorption of CA diered with the degree of hydro-phobicity of the clay surface. More adsorption at equi-librium was expected on M±HDTMA than on M±PY, as M±HDTMA was more hydrophobic. However, higher BmaxandKeqvalues were obtained with M±PY,
probably because of its greater external surface area (Table 1).
The isotherms did not ®t the Langmuir equation over the entire range of concentrations of CA at equili-brium. The additional adsorption and binding of CA on M±Ca after the initial plateau was probably the result of the penetration of the protein into the inter-layer spaces of the clay, as the X-ray diractograms after drying at room temperature (Fig. 2) and after heating at 1508C (not shown) showed an expansion of the interlayer spaces with the higher amounts of CA bound. In contrast, the additional adsorption and binding of CA on M±HDTMA and M±PY after the plateau, albeit not as great as on M±Ca, cannot be explained by an intercalation by CA, as the d001
spa-cings of M±HDTMA±CA and M±PY±CA remained unchanged regardless of the amount of CA bound. The second rise in the adsorption and binding iso-therms in the M±HDTMA±CA and M±PY±CA sys-tems may have resulted from the formation of multilayers of CA on the external surfaces of the clay or from changes in the orientation of CA on the sur-faces. The formation of multilayers and changes in orientation may have also occurred in the M±Ca±CA system, in addition to the intercalation of CA.
The frequencies of the Amide I and Amide II bands of free CA and of the clay±CA complexes in the FT-IR spectra are shown in Table 2. The Amide I band of the M±Ca±CA and M±PY±CA complexes shifted to a higher frequency (1655 vs 1650 cmÿ1
for free CA), whereas the Amide II band shifted to a lower fre-quency (1536 and 1534, respectively, vs 1545 cmÿ1
for free CA). These dierences were signi®cant, as the fre-quencies were the average of 128 repeated scans. The
Table 1
Values ofBmax andKeq, regression coecients for ®tting adsorption data to the Langmuir equation, and speci®c surface areas of and water
retention by the clay systems
System Bmaxa2SEMb(mmol gÿ1clay) Keqc2SEM (mMÿ1) r2d Surface areae(m2gÿ1clay) H2O retentionf(ml gÿ1clay)
M±HDTMA±CA 1.420.07 17.822.9 0.997 9.9 7.54
M±PY±CA 1.720.11 25.024.1 0.998 18.0 6.17
M±Ca±Ca 0.620.04 3.120.9 0.993 27.5 4.04
a
Bmax=Maximum adsorption at plateau calculated by the Langmuir equation; see text for details. b
SEM=Standard error of means.
c
Keq=Anity coecient for adsorption of Ca on the surfaces; see text for details. d
Regression coecient for ®tting adsorption data to the Langmuir equation at the ®rst plateau.
e
External surface area measured by N2adsorption at 77 K. f
changes may have resulted from a distortion of the structure of CA caused by its binding on these clays. Shifts of the Amide I band (C1O stretching band) to higher wavenumbers and of the Amide II bands (NH bending) to lower wavenumbers are diagnostic of an increase in random or less-ordered portions of second-ary structure (D'Esposito and Koenig, 1978). No dierences were observed in the Amide I and II bands of M±HDTMA±CA in comparison with free CA, indi-cating that binding of CA on this clay did not cause any signi®cant perturbation of the structure of the pro-tein. These observations were consistent with the hy-pothesis of a weaker binding of CA by hydrophobic interactions on M±HDTMA than by hydrophilic inter-actions on M±Ca, as also suggested by Garwood et al. (1983) for glucose oxidase and by Boyd and Mortland (1985, 1986, 1990) for urease.
The values for the kinetic parameter, K1, of the
ac-tivity of free, adsorbed and bound CA on the dierent
clays are reported in Table 3. The value for free CA was higher than the values reported in the literature for CA from beef liver (Ogura, 1955; Nichols and Schombaum, 1963), but it was consistent with the K1
calculated from information provided by Sigma Co. for type C40 CA. These dierences in the K1 for free
CA probably resulted from dierences in the prep-aration and purity of the enzyme. When CA was adsorbed at equilibrium on M±Ca, the value for K1
was reduced by 80% (Table 3), whereas the reduction was 90% for M±PY±CA and 95% for M±HDTMA± CA. The value of K1 for CA bound on M±Ca was
reduced by more than two order of magnitude (99.3%) in comparison with free CA. This dierence between the activities of CA adsorbed at equilibrium and bound suggests that dierent pools of CA with dier-ent enzymatic activity existed in the equilibrium com-plex of the hydrophilic M±Ca±CA system, i.e., tightly bound CA, loosely bound CA, and CA dissolved in the water retained by the clay pellet. For M±HDTMA and M±PY, the dierences in K1 values between
bound and adsorbed CA were small.
To evaluate the contribution to the enzymatic ac-tivity of the unadsorbed CA dissolved in the water retained by the clay pellets after the initial centrifu-gation for the determination of the amount of CA adsorbed at equilibrium, the pellets were weighed after drying at 1058C for 12 h. The amount of water retained by the hydrophobic systems was greater than by M±Ca±CA (Table 1), probably because the rapid
Fig. 2. X-ray diraction spectra of bound M±HDTMA±CA, M±PY±CA, and M±Ca±CA complexes after air-drying at room temperature. The amounts of CA bound are indicated. Similar results were obtained when the complexes were dried at 1508C.
Table 2
Infrared frequencies (cmÿ1) of CA free or bound on M±Ca, M±
HDTMA, or M±PYa
Form of CA Amide I Amide II
Free CA 1650 1545
M±HDTMA±CA 1650 1544
M±PY±CA 1655 1536
M±Ca±CA 1655 1534
a
¯occulation that occurred in the hydrophobic systems resulted in a random orientation of the quasi-crystals and in the formation of large pores. In the hydrophilic M±Ca system, the quasi-crystals remained dispersed and a close-packing occurred during centrifugation, which resulted in small pores. The amounts of CA dis-solved in the water within the pores created by the arrangement of the particles after centrifugation prob-ably contributed to the apparent activity of adsorbed CA, but this eect should have been more evident in the M±PY±Ca and M±HDTMA systems, as more water was retained by these systems than by M±Ca± CA. As large dierences between the enzymatic ac-tivity of adsorbed vs bound CA were observed only with M±Ca, larger structural modi®cations of CA or dierences in steric hindrance in the binding of H2O2
may have occurred with bound CA than with CA adsorbed at equilibrium on M±Ca.
The optimal pH for the activity of CA was not aected by the adsorption or binding of CA on the dierent clays, regardless of the type of charge-com-pensating cation (Fig. 3). Garwood et al. (1983) reported that the optimal pH for the activity of free and ``immobilyzed'' glucose oxidase was the same for hydrophobic and ionic modes of binding. The optimal pH for the activity of several enzymes has been reported to shift to higher values after adsorption of the enzymes on clays homoionic to inorganic cations, presumably as a result of the acidic character of the clay surface (Hattori, 1973; Theng, 1979). Although these authors did not dierentiate between adsorption at equilibrium and binding, the interaction of these enzymes with clays homoionic to inorganic cations was presumably mainly via ionic interactions, whereas our present study, the adsorption and binding of CA occurred in unbuered systems at pH values above the pI of CA, suggesting that hydrogen bonding between CA and M±Ca was primarily involved (Stotzky, 1986; Fusi et al., 1989).
Soil microorganisms utilized free CA and glucose as sole C sources to the same extent, as the amounts of CO2evolved almost doubled when an equal amount of
C as glucose was added to free CA (Fig. 4, experiment (iii) vs experiment (i)). The N contained in CA appeared to be adequate for growth of the microbes, as supplementation with NH4NO3 did not increase
CO2 evolution (Fig. 4, experiment (ii) vs experiment
(i)). Also, in the CA+glucose and CA+gluco-se+NH4NO3experiments (Fig. 4, experiments (iii) and
(iv)) no signi®cant dierences in CO2 evolution were
observed. There were no signi®cant dierences in CO2
evolution between the control (M±HDTMA) and M± HDTMA±CA alone (Fig. 4, experiments (i) and (ii)). This was not primarily the result of an inhibition of microbial growth, as the addition of glucose, with or without NH4NO3, to M±HDTMA or M±HDTMA± Table 3
Kinetic parameter,K1, after 1 min for the activity of CA free, adsorbed or bound on M±Ca, M±HDTMA, or M±PY
Form of CA K1(Mÿ
1
sÿ1
) ra Residual CA activityb(%) Reduction in CA activityc(%)
Free CA 6.46108 0.97 100 0
M±HDTMA±CA
Absorbed 3.84107 0.98 5.9 94.0
Bound 2.20107 0.99 3.4 96.6
M±PY±CA
Adsorbed 6.43107 0.97 9.9 90.0
Bound 5.53107 0.99 8.6 91.4
M±Ca±CA
Adsorbed 1.23108 0.95 19.0 80.9
Bound 4.21106 0.92 0.6 99.3
aRegression coecient for the equation,V=K
1[E][S]. See text for details.
bK
1(adsorbed or bound clay±CA complex)/K1(free CA)100.
c100
ÿK1(adsorbed or bound clay±CA complex)/K1(free CA)100.
CA (Fig. 4, experiments (iii) and (iv)) resulted in sig-ni®cant CO2 evolution but, rather, to a reduced
bioa-vailability of the bound CA. As expected for a quaternary ammonium salt such as HDTMA, essen-tially complete inhibition of microbial growth occurred with free HTDMA chloride in all treatments (Fig. 4, experiments (i), (ii), (iii), and (iv)), but this inhibition did not occur when HTDMA was present as the charge-compensating cation on M.
In contrast, CA bound on M±PY was utilized by microbes as a sole source of C and N to the same extent as free CA, showing that the binding on M±PY did not aect the bioavailability of CA. The control treatments with only M±PY and PY chloride in amounts corresponding to those present in M±PY showed neither inhibition of microbial growth nor ap-preciable utilization of the PY as a source of C, although it was utilized as a source of N, con®rming that the CO2evolution observed with M±PY±CA was
the result of the utilization of bound CA.
When CA was bound on M±Ca, CO2evolution was
reduced by about 90% in comparison with free CA. As also observed with M±HDTMA±CA and M±PY± CA, the addition of glucose, without or with NH4NO3
(experiments (iii) and (iv)), resulted in increased respir-ation, con®rming that the reduced CO2evolution from
M±Ca±CA was the result of reduced utilization of bound CA and not of inhibition of microbial growth. The results on microbial utilization indicate that CA is poorly available as source of nutrients when bound on M both by hydrophobic or ionic interactions. The M± PY±CA bound represent a unique case. When present
on the clay as a charge-compensating cation, PY pre-sented a rapid ¯occulation of the clay suspension, i.e., the diuse double layer surrounding the clay colloidal particles was suppressed. Nevertheless, the anity for nonpolar organic molecules, or ``organophilicity'', was limited, as no hydrophobic tail was present. According to Tanford (1991) the ability of organic amphiphylic molecules, such as fatty acids, alcohols, amides, etc., to form micelles in bulk solution increases threefold for each CH2 group added to the alkyl chain
(``Traube's rule''). For these reasons, the interactions of CA with M±PY probably involved both weak hydrogen bonding (as demonstrated by the shift in the Amide I and Amide II bands observed by FT-IR) and limited hydrophobic eect, which rendered CA avail-able for the microbes.
4. Conclusions
Both hydrophobic and hydrophilic modes of inter-action of montmorillonite with CA resulted in high amounts of CA being adsorbed, in a reduction but not complete elimination of the enzymatic activity, and in reduced bioavailability of CA. However, the anity of the hydrophobic surfaces for CA was higher and the reduction in enzymatic activity less than that observed on M±Ca, where ionic and hydrogen-bonding inter-actions are assumed, indicating that CA can probably persist and function in soil when bound in clay-humic complexes having a hydrophobic character. The spec-troscopic data of CA bound on M±HDTMA and M±
Fig. 4. Evolution of CO2from dierent systems inoculated with a microbial community from soil after a 7 d incubation at 258C (mean2
PY indicated that the structural modi®cations, detected by FT±IR, may not be related to changes in the enzy-matic activity or bioavailability of CA, and that more information on the eect of clay on the active center of CA is needed. For these reasons, further investi-gation on the structure of the active center of CA bound on clay systems is needed.
Acknowledgements
This work was supported by the Italian Consiglio Nazionale delle Ricerche (CNR) within a cooperative program between the Dipartimento di Scienza del Suolo e Nutrizione della Pianta UniversitaÁ di Firenze, Italy, and the Laboratory of Microbial Ecology, Department of Biology, at New York University. We thank Mr Fabrizio Filindassi for his assistance in the preparation of the graphs.
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