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Enzymatic activities in salt affected soils
Zbl. Bakt. II. Abt. 136 (1981),106-116 [Faculty of Agriculture at Shibin EI-Kom, Monoufia University, Egypt]
Enzymatic Activities in Salt Affected Soils M. M. EL-SHINNAWI and S. A. EL-SHIMI With 3 Figures
Summary Activities of dehydrogenase, urease, and phosphatase (nuclease) as affected by the anionic and cationic form of inorganic salts (S04' COa, and CI for Ca, Mg, K, and Na) applied at 15 meg/lOO g soil to alluvial clay and calcareous sandy loam soils, during incubation, were studied. Most of the applied salts stimulated the enzyme activities of soils tested. Carbonates showed the most distinct effect, while chlorides were the least. Nuclease in both soils was activated by all of the applied salts. Dehydrogenase in the calcareous soil was stimulated by all types of salts used. Urease, in the alluvial soil, was promoted by all sulphates and chlorides applied. Urease, in the calcareous soil, was inhibited by chloride salts. In the alluvial soil, sodium as combined with each of the three aniones favoured the enzyme activities; potassium and magnesium showed intermediate figures, while calcium was extremest. In the calcareous soil, potassium in its different forms favoured the enzyme activities; sodium and magnesium showed intermediate results, and calcium gave the least values.
Zusammenfassung In alluvialem Ton sowie kalkhaltigem sandigen Lehm wurde die Wirkung von Ca, Mg, K und Na in Sulfat-, Karbonat- und Chloridform (15 mvalj l Of) g Boden) auf die Aktivitat von Dehydrogenase, Urease und Phosphatase (Nuklease) gepriift. Die meisten Salze stimulierten die Enzymaktivitaten, die Karbonate am deutlichsten, die Chloride am wenigsten. Nuklease wurde bei beiden Boden durch alle Salze aktiviert. Urease wurde im alluvialen Boden durch die Sulfate und Chloride gefordert, im kalkhaltigen sandigen Lehm durch die Chloride inhibiert. Irn alluvialen Boden begiinstigte Na in jeder Salzform die Enzymaktivitaten, Kalium und Magnesium nahmen eine Mittelstellung ein, Kalzium bewirkte die geringsten Stimulierungen. Irn kalkhaltigen Boden fOrderte Kalium in Verbindung mit allen Anionen die Enzymaktivitaten Kalzium hingegen kaum.
The breakdown of the complex organic residues and macromolecules in soil is catalizing by enzymes. Such bio-organic catalysts of proteinaceous nature are produced in soil mainly by microorganisms, and to some extent by roots of the growing plants. Activity of soil enzymes has been found to be dependent on soil characteristics and environmental conditions (SKUJINS 1967). Salts which might occur in/or be applied to soil in various operations could have a considerable influence on the biochemical processes in soil. Reports of GALSTYAN (1964, 1965), showed that dehydrogenase activity was very slight in solonchaks, but high activity of the same enzyme was detected in saline-solonetstic soils. BREMNER and TABATABAI (1973) found that Fe 203 , Mn0 2 , S04 -2, P0 4-3, and CI- all increased the values obtained in the assay of dehydrogenase activity with TTC. Activities of urease and phosphatase in soil were found to be inhibited by carbonates (GALSTYAN 1958, HALSTEAD 1964). On the other hand, ABDEL-MALEK (1973) found a positive correlation between phosphatase activity and the amount of Ca ++, Mg++, and HC0 3 - in soil.
Enzymatic Activities in Salt Affected Soils
107
The present work was carried out in order to clear up the effect of the anionic and cationic form of inorganic salts applied to alluvial fertile and calcareous soils, on the activity of representatives of soil enzymes, namely dehydrogenase (oxidoreductase), urease, and phosphatase (hydrolases). Materials and Methods
Soil samples Fertile alluvial and calcareous - under cultivation - soil samples were collected from the surface layer (0-15 cm) from Banha and Nobaria, respectively. Soil samples were air-dried, ground to pass a 2 mm-sieve, and mixed thoroughly. Pertinent analytical data for these soils (according to the methods of PIPER 1955) are presented in Table 1. Table 1. Characteristics of the soils tested Property
Alluvial
Calcareous
Textural grade Water holding capacity, % pH (1: 2.5 water suspension) Organic carbon, % Total nitrogen, % Total P, % CaCO a, % Total soluble salts, % Soluble ions, meqjlOO g soil HCO a -
Clay 53.60 7.85 1.218 0.120 0.015 2.88 0.110
Sandy loam 36.30 8.43 0.524 0.045 0.007 27.60 0.165
ci-
S04-Ca++ Mg++ K+ Na+
0.46 0.39 0.98 0.39 0.26 0.02 1.16
0.29 0.46 1.47 0.56 0.33 0.67 0.66
Soil treatments Series of sulphates, carbonates, and chlorides for calcium, magnesium, patassium, and sodium were finely ground and mixed with the air dry soil at 15 meq salt per 100 g soil. Treatments - in duplicate - were carried out by using portions of 5 g soil crumbs, placed in large sized (2 X 18 cm) test tubes. The moisture content was adjusted to 60 % of the water holding capacity of the soil. Evaporation losses, during incubation at 30°C, were made up periodically by the addition of distilled water. Variations in pH values of the tested soils, as a result of salt application, appear in Table 2.
Assay of enzyme activities Dehydrogenase was determined colorimetrically for the 2,3,5-triphenyl formazan (TPF) produced from the reduction of 2,3,5-triphenyltetrazolium chloride ('1''1'0), using acetone for extraction (CASIDA et al. 1964). Urease was estimated colorimetrically for the residual urea by P-dimethylaminobenzaldehyde solution (CONRAD 1940, BROADBENT et al. 1958). Phosphatase (nuclease) was determined colorimetrically for the inorganic phosphorus released from nucleic acid added DROBNIKOVA (1961). Sorption of inorganic phosphorus in soil was determined by the method of OLSEN and WATANABE (1957) in order to correct the obtained data of mineralized available phosphorus.
108
M. M. EL-SHINNAWI an d S. A. EL -SBIMI
Table 2. Va ria ti ons in p H val ues of the fertile a ll uv ia l a nd ca lcareous soils t reated with differen t salts
p H value Salt treat ment (15 meq/l OO g s oil) (in 1 : 2.5 soil-water s uspens ion )
Control (no s al t added)
Alluvial
Calcareous
7.85
8.43
S04
Ca Mg K Na
7.53 7.2 0 7.34 7.4 8
8.43 8.00 8.20 8.36
COa
Ca Mg K Na
8.00 8.00 9.1 0 8.92
8.43 8.50 9.00 9.00
Cl
Cu Mg K Na
7.85 7.80 7.85 7.90
8.40 8.40 8. 2 1 8.28
Results and Discussion A. D eh ydrogen a s e 1. Sulphates The application of sulphate salts (at 15 meq/lOO g soil) led to de crease the activity of dehydrogenase in the alluvial soil at time 0 (Fig. lA). Incubation of the moistened soil samples (at 60 % W.H.C.) of the control (unsalted) treatment, however, slightly increased t he enzy me ac tivit y to give a peak on the t hird day, thereafter began to decline. Samples treat ed with MgS0 4 revealed an analogous trend to that of t he control. The other sulpha tes (Ca, K, Na) gave high peaks only on t he seventh day of incubation. Activit y of t he bioorganic cat alyst was promoted by the application of each of CaSO.. and Na 2SO... Potassium sulphate, on t he other hand, inhibited the catalytic reaction of t his soil. Dehydrogenase in thc calcareous soil was stimulated by the applied sulp hat cs at time 0 (F ig. ID). Incubation of the moistened samples showed two definite peaks of dehydrogenase activity for this soil. The first peak on the third day was higher than the second which appeared on t he fifteenth day. All sulphates activated t he dehydrogenase in the calcareous soil, where potassium salt was t he most effective in this regard and followed by those of magnesium, sodiu m, and calcium, respectively. Gene rally, the calcareo us soil show ed somewha t higher dehydrogenase activit y than the fertile alluvial soil. The pat t ern of dehydrogenase activity of the incubated soil sa mples goes along with t he growth curve of micro-organisms when the soil is brough t to favourable conditions of moisture a nd te mperature (SEIFERT 1966). The action of sulphates on decreasing t he pH value of calcareou s soil samples (Table 2) appeared to be favourable for the microbial population and the subsequent biochemical activity (MASHTAKOV et al. 1954 and HOFMANN 1959). It was reported by DIXON and WEBB (1985) that potassium, magnesium, and calcium cations and sulphate anion activate the catalyst dehydrogenase in solution s. The higher dehydrogenase activity shown by the oaloare-
109
Enzymatic Activities in Salt Affected Soils
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Fig. 1. Dehydrogenase activity in the incubated soil samples as affected by the anionic and cationic form of added salts, at 15 meqj l Of) g soil.
ous soil could be explained by the findings of CERNA (1969) that the dehydrogenase activity increases as the specific surface of soil particles decreases. It is worth noting that the intensity of enzyme activity of the soil is not usually correlated either with the total number of microbes or with the number of microorganisms in main groups expected to be in such a soil (KOEPF 1954, DROBNIK and SEIFERT 1955, and NOWAK 1964).
2. Carbonates Dehydrogenase of the alluvial soil was activated by the application of Na 2CO s and highly by MgCOa (Fig. IB). Potassium carbonate, however, showed no considerable changes, while calcium carbonate resulted in a slight decrease in the catalytic reaction of the soil. Peaks of the enzyme activity appeared on the 3rd day of incubation for all of the applied carbonates. A second increase, however, was detected on the 15th day. All carbonates markedly stimulated the activity of dehydrogenase in the calcareous soil (Fig. IE). Magnesium carbonate was the most stimulating and was followed, des. cendingly, by sodium, potassium, and calcium carbonates. Two peaks of the catalytic activity appeared within the experimental duration, the first detected on the third day of incubation was higher than the second on the 15th day. Application of carbonates resulted in shifting the pH to higher values in both soils (Table 2); such conditions increased the formation of TPF. Reduction of TTC may be chemically promoted through the alkaline shift that followed the addition of carbonates to soils. 3. Chlorides Inconsiderable changes were shown in the activity of dehydrogenase of the fertile alluvial soil as affected by the addition of chloride salts (Fig. 1C). Calcium and sodium
110
M. M.
E L-SHlN X AWI
an d
S.
A.
EL·SHIMI
chlorides slightly in creased t he en zyme activity, while potassium and magnesium on es decreased it somewhat. P eaks of activit y almost appe a red on t he t hird day for all treatments. Activity of dehydrogenase in t he calcar eou s soil was highly promoted by t he adde d chlorides (Fig. IF). Th e descending order of activation was potassium, sodium, magnesium, and calcium chlorides. Two peaks appeared, a s usual for this soil on the 3rd and the 15th day of incubation. All chlorides showed no effect on t he pH of the alluvial soil (Table 2). Th e pH value of t he calca reous soil, however, was slightly shifted to lower valu es by t he a ddition of eac h of pot assium a nd sodium chlorides (Tabl e 2). The activation of deh ydrogenase show n in t he calca reo us soil following t he addit ion of t he monovalent cati on s may be attributed to t he favourable effect of t he resulted pH on soil mi croflora, The present result s are in acco rdance, to some extent, with t he ea rlier findings of GALSTYAN (1965) a nd KOZLOV a nd MIKHAILOVA (1965) indicati ng t hat t he sa line-so lonetz soils had a high deh ydrogenase acti vity ; a nd of BREl\INER and TABATABAI (1973) which showed t hat S03 -2 and CI- increased t he values obt ained in the assa y of dehydrogen as e activity with TTC. On comparing the an ions as effect ed the activity of dehydrogenase in soil, results showed t hat ca rbonates were t he most enzy me-acti vators in both soils under conside ration , i.e. alluvial clay a nd calca reo us sa ndy loam. Sulphat es a nd chlorides followed , resp ect ively, t he carb onat es in t he alluvial soil, bu t opposite ly in t he calcareous soil. Sodium combined wit h all anions under stud y showe d activati on for deh ydrogen ase in t he fertil e alluvia l soil. Pot as sium, however , resul t ed in a slight inhibition with all a nio ns; wh ':lreas t he effect of t he other cations diff ered stimulating or inhibiting a ccording to the co-anion. Magnesium carbonate was the most stimulatory salt for deh ydrogen ase ac t ivity of t he alluvia l sai l, while calcium carbonate was t he most depressing. The cati on s under consideration activat ed the oxide-reduction cat al ytic reaction in t he calcareou s soil at varying intensities, depending on the complementary ion. Calcium was the least st im ula to ry a mong the different cations with all anion s. Magnesium ca rbonate, likewise, sho wed t he best res ults, and ca lcium chloride ga ve t he least va lues of deh ydr ogen ase ac ti vity in the calcareous soil. Genera lly, deh ydrogenase of t he calca reo us soil resp onded more to t he applied sa lts t ha n t hat of t he alluvial soil. B. U rease 1. Sulphat es
Act ivit y of urease was promot ed by t he application of sulphat e salt s (15 meqJ 100 g) to the alluvial clay soil (F ig. 2 A). Th'3 catalytic reacti on was decreased on the third day of incubation and then began slightly to increase mostly in a lin ear manner. Th e maximum values obtained within the experime ntal duration for t he control as well as for CaS0 4 • K 2S04 , a nd Na 2S04 were at t he beginning, a nd for MgS0 4 on t he t hirtieth day. Magn esium was also t he most st imul a tory cation for t he catalyst urease, followed b y ca lcium, potassium, and sodium, resp ecti vely. Th e calcareous sa ndy loam soil showed, genera lly, similar pat t ern of urease activity to t hat of t he alluvial soil, but at lower valu es. Magn esium and p ot assium sulphates stimu lated t he activity of urease, while calcium a nd sodium ones inhibit ed it (F ig. 2 D ). Max imum valu es for a ll t reatme nts within t he expe ri mental d ura t ion appeared at t ime O. Urease activit y was highest at time 0, follow ed by a depression on the third d ay of incubation a nd again began to show a slight increase . The intensity of these variations differed acoo rding to the soil type and sa lt treatment, i.e . the depression was very
III
Enzymatic Acti vities in Salt Affe ct ed Soils
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F ig. 2. Urease ac t iv ity in t he incubated soil samples as aff ected by the anion ic and ca t io nic forms of added sa lts, at 15 m eq/ l00 g soil.
sha rp in t he calca reo us soil treated with Na 2S04 , and ver y slight in t he alluvial soil trea t ed with MgS0 4 (Figs. 2A a nd D ). Th e highest enzyme activity appea red at t ime 0 could be attributed t o t he initially found urea se released from roots and nonviabl e cells. Maximum prolifer ati on of soil microflora to ok place up to t he t hird da y of in cubation under favourable conditions of moisture a nd te mpe rature (SEIFERT 1966), and subsequently, maximum need for nitrogen to t he reproducing cells would ari se. H ence, ur ease as nit rogeno us compo und is liab le to be decomposed with a higher ra t e t ha n it s accumulation in t his ph ase, when microbial reproduct ion exceeds lysis. The sha rp depression shown in t he calcareo us soil might be referred to its init ially low nitrogen content (Table 1). Th e activation of urea se appeared by application of sulphate sa lts t o t he alluvi al soil might be due to t he role of cations in dimini shing t he adso rpt ion of both t he ur ea substrate and the enzyme on t he cla y part icles which lead s t o increase the hydrolysis pro cess (ZITTLE 1953). In thi s concern , the dival ent s were more effect ive, whereas magnesium sulphate with it s high sol ubilit y (HODGMAN et al. 1955) showed t he best result s. T he shift in p H of t he calca reous soil to lower valu es du e t o t he ap pli cat ion of both MgS0 4 a nd K 2S04 (Ta ble 2) ma y explain t heir suitability to t he en zyme urease. The lower act ivity of urease shown in the calca reous soil in com parison with the alluv ial one could be asc ribe d to eit her t he minor product ion of en zyme or t o soil t exture (Table 1) as CERNA (1966) not ed t hat urease act ivit y in creases with t he in crease in t he spe cific s urface of soi l particles. 2. Carbon at es App licat ion of both CaCOa a nd MgCOa highly accelerated t he hyd rolysis of urea in t he alluvial soil, while Na 2C Oa a nd ~COa di mini shed it (F ig. 2B). Dep ression in t he
112
M. M.
EL-SHINNAWI
and
S.
A.
EL-SHIMI
hydrolysis process was shown on the third day of incubation for all treatments, followed by a, slight linear increase. The maximum values within the experimental duration for all salt treatments appeared at time O. Calcium was the most stimulatory cation for urea hydrolysis in the alluvial soil, while sodium showed the least results. Urease activity in the calcareous soil was increased by the application of each of KaCO a and NaaCO a, respectively, while MgCOa was of inconsiderable effect and CaCOs was inhibitory (Fig. 2E). The same pattern of enzyme activity during incubation time, as in the moistened samples of the alluvial soil, was shown in the calcareous variants. The maximum values within the experimental duration for all salt treatments appeared at time O. Potassium was the most stimulatory cation for urease in the calcareous soil, while calcium showed the least results. The application of carbonates raised the pH values of the tested soils (Table 2). This rise in the pH value was more pronounced in the alluvial soil than in the calcareous one. The unsuitability of potassium and sodium carbonates for urease in the alluvial soil may be referred to the higher alkaline shift of pH value which unfavoured both the ureolytio bacteria and the enzyme urease itself (CECCONI et al, 1974 and ALEXANDER 1977). It is worth noting that the insoluble fine particles of calcium and magnesium carbonates increase the surface area for the catalytic reaction in the alluvial soil and thus increase the activity of urease in accordance with the notes of CERNA (1966). In this connection, ALEXANDER (1977) noted that the presence of such a salt in soil affects the microbial activities even through its insoluble particles to which microorganisms tend to adhere, as in cases of lime and gypsum. The reverse trend appeared in the calcareous soil might be attributed to the native high content of CaCOa and poor properties which lessen the urease activity of such soil, as reported by GALSTYAN (1958). Potassium and sodium carbonates, with their high solubility (HODGMAN et a1. 1955), favoured both the ureolytic microorganisms and urease of the calcareous soil through diminishing the unfavourable action of lime (GALSTYAN 1958). 3. Chlorides Cations combined with chloride very slightly increased the activity of urease in the alluvial soil, where potassium was the most stimulatory, followed descendingly by calcium, magnesium, and sodium (Fig. 2C). The maximum values within the experimental duration for all of the applied chlorides appeared on the thirtieth day. The activity of urease was inhibited by all chlorides applied to the calcareous soil (Fig. 2F). The monovalents, i.e. potassium and sodium, were less inhibitory than the divalents. Calcium was the most depressive cation. The maximum values within the experimental duration for all of the applied chlorides appeared at time O. The presence of cations in the clay soil plays a role in releasing both enzyme protein and substrate adsorbed on the soil particles to soil solution, thus promoting the catalytic reaction (ZITTLE 1953). No evidence in the present results supports the earlier findings of GALSTYAN (1964) in which urease activity was depressed in saline soils. Chlorides appeared to be of inhibiting action on the urease of the calcareous soil which may be due to the specific effect of the anion on both ureolytic bacteria and enzyme in the poor conditions of the calcareous soil. On comparing the anions together how they affect the activity of urease in soil, it is noticed that no convergency could be stated for both soils under consideration. Sulphates were the most enzyme-activators in the alluvial clay soil. Chlorides showed slight increases in the same soil. The divalent cations combined with carbonate highly increased the urease activity, while the monovalents revealed a slight decrease. Calcium and magnesium combined with all anions showed activation for urease in the
113
Enzymatic Activiti es in Salt Affected Soils
alluvial soil, while potassium a nd sodium acted sligh tl y in creasing or decreasing depending up on t he co-anion. Sod ium showed low resul t s in all ca ses. Ca lcium carbonat e was t he most st imulat ory sa lt for urea hydrolysis in t he incubated samples of t he alluvial soil, while sodium carbona te gave t he lea st valu es. Some cations (K , Mg) of both sulphat es and carbonat es slightly stimulated t he urease in t he calcareous sa ndy loam soil, while t he other cati on s (Ca, Na ) were inhibitory. All chlorides inhibit ed t he enzyme of t he same soil. Potassium carbonate was t he most stimulatory salt for t he hydrolysis process in t he calcareou s soil, while calcium chloride ga ve t he least res ult. C. Nu cl ea se
1. Sulpha tes H ydrolysis of nucleic acids in t he moistened sa mples of t he alluvial soil was very slightly stimulated b y applicat ion of Na 2S04 or K 2S04, while MgS0 4 or CaS0 4 showed no notable effect (Fig. 3A). P eaks of activity were shown on the third day of incubation for the different treatments. A second increase was recorded on the fifteenth day. Nucl ease in the calcareou s soil samples was somewhat stimulated by the applied sulphates, where calcium wa s t he best cation, follow ed by magnesium, potassium, and sodium ones, respectively (Fig. 3D). The maximum valu es within the experimental du ra ti on for the control t reatment as well as for MgS0 4 and Na 2S04 , appeared on t he 3rd day, whil e t hose of CaS 0 4 , K 2S04 , and cont rol were on t he 15th day. The stimula tion in t he activity of nu cleic a cid hydrolase caused by t he application of sulp hate salts to both soils tested ma y be attributed to t he specific action of t he a nion on t he contributing microorganism s as well as on t he enzyme itself (DIXON a nd WEBB 1958). In t his regard, HALSTEAD (1964) foun d t hat phosphatas e activity in
-30-
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114
M. M.
EL-SHINNAWI
and
S.
A.
EL-SHIMI
different surface soils was favoured during incubation by CaS0 4 and MgS04 . Order of enzyme activation in the alluvial soil was inversely correlated with the relative replacing power of the cations on the clay surfaces where the order was Ca > Mg > K > Na. Though the situation in the calcareous soil is different. The low activity of the phosphatase in the calcareous soil in the present study agrees with the results of KOTELEV et al. (1960) and EL-EsSAWI (1973). It is noteworthy that DUNARD (1965), KOZLOV (1966), CERNA (1970), ABDEL-MALEK (1973), and EL-EsSAWI (1973) concluded that phosphatase activity is positively correlated with soil texture, organic matter content, and organic phosphorus, Pattern of nuclease activity appeared to be correlated with the microbial growth and expected consistency of microorganisms in such soils. 2. Carbonates Nuclease of the alluvial soil was activated by the applied carbonate salts. Sodium carbonate showed the best results followed by potassium, magnesium, and calcium salts, respectively (Fig. 3B). Peaks of activity were shown on the third day of incubation for all treatments. Hydrolysis of nucleic acids in the calcareous soil was slightly activated by the addition of carbonates, where MgCOa gave the best results followed descendingly by KzCO a, NazCO a, and CaCO a (Fig.3E). Maximum values within the experimental duration for the treatments of MgCOa and NazCO a appeared on the 3rd day, and for KzCO a and CaCO a on the 15th day. The order of activation of the nucleic acids hydrolase in the clay soil caused by carbonate addition is, likewise, inversely correlated with the relative replacing power of the cations on clay surfaces, as shown by sulphates. The rise in pH occurred by the application of carbonate salts to both soils (Table 2) was found to be of a favourable action on the nuclease of the clay soil, whereas such effect was not observed in the calcareous one. In this concern, DROBNIKOVA (1961) found in an arable brown soil having pH 7.9 that as the pH value increases to the alkaline range, the activity of nuclease increases. ABDEL-MALEK (1973) pointed out, in his thesis, that there was a positive correlation between phosphatase activity and the amount of HCO a- in soil. No evidence in our work supports the earlier results of HALSTEAD (1963), who found that phosphatase activity of surface soil samples was inhibited during incubation by CaCO a or MgCOa application. 3. Chlorides Breakdown of nucleic acids was very slightly favoured by addition of chlorides to the alluvial soil. Sodium chloride showed the best results and was followed by potassium, magnesium, and calcium salts, respectively (Fig. 3C). Peaks of activity appeared on the third day of incubation for all treatments. Nuclease of the calcareous soil was promoted by chloride salts, where NaCl was the most promoting, followed descendingly by magnesium, potassium, and calcium chlorides (Fig. 3F). Maximum values within the experimental duration for the treatments of magnesium, sodium, and calcium appeared on the 3rd day, and of potassium on the 15th day. The same order of activation shown by the different cations with sulphates and carbonates was also revealed by chlorides on the nuclease of the alluvial clay soil. HALSTEAD (1964) found that MgClz and CaClz were stimulatory for soil phosphatase. The comparison between the effects of the different anions applied to soils on nuclease activity shows that all of the applied salts stimulated the enzyme in both soils
Enzymatic Activities in Salt Affected Soils
115
tested . I n t he alluvial cla y soil carbonates were t he most enzyme-activa to rs, while sulphates were t he least . I n t he calca reo us sa ndy loam soil, all anions st imulated t he en zyme at about t he same levels. Cations acted at one t re nd as associated with t he different a nions in t he clay soil, where t he monovalent were t he most stimulatory for nuclease. Sodium was t he best while calcium showed t he least result s in all cases of the alluvial soil. In the calcareous soil, cations act ed different ly according to t he complementary an ion; wherea s magnesium and potassium act ed intermediat ely, calciu m and sodiu m acted extremely. Sodium carbo nate WaS t he most stimulatory salt for nuclease in t he alluvial soil, while calcium sulphate ga ve t he least fig ures . Calciu m sulp hate showed t he best resu lts in t he calcareous soil, while calcium ca rbonate was t he least. Ac know l edg ement T h e authors ex press their thanks to Prof. D r . H. H A:Il:DI, Professor of Soil Sc ience, Ain S hams U n iversity, Egypt, for hi s interest in the work and advice.
References ABDEL-MALEK, S. M.: Enzymatic ac t ivities in so ils as in di cat or s of fertilit y . M. Sc . Thesi s, Faculty of Agriculture, Cairo Univ., Egypt (1973). ALEXANDEIt, 1\1.: I ntroduct ion to So il Microbiology. 2nd ed ., New York 1977. B REMNER, J . 1\1., a nd TABATABAI, 1\1. A. : Effects of some in org an ic su bstan ces on TTC (2,3,5· trip henylt etrazolium chl oride ) assay of d ehydrogenase a ctivity in soils. Soil BioI. Bi och em , 5 (3) (1973) , 385. B ROADBENT, F. E ., H ILL, G. N ., and TYLER, K . B. : Transformations and movement of u rea in soil. Soil. Sci. Soc. Arn er, Proc, 22 (1953), 303. CASIDA, L. E. , KLE IN, D . A., a n d SANTORO, T. : Soil dehydrogenase activity. Soil. Sci. 89 (196 4), 37 1. CECCONI, C. A. , BRUNO, V., and DANIELE, E . : H ydrolysis of urea by t h e so il and by vegetab le in the soil. Georgofili 20 (19 74 ), 177. CERNA, S. : Enzymat ic a ctivity of so il in re lation t o its stru cture. Acta Univ, Carol. - Biologica (65) (1966), 83. - Structura l soil a n d its d ehydrogenase a ct ivit y. Acta Univ. Carol.- Biologica (68) (1969), 279. - Phosphatase activity in st ruct ura l so il. Acta Univ. Ca ro l.-Biologica (69) ( 1970), 455. CONRAD, .J. P. : The n a t u re of the cat a lys t ca u sing t he h y d r oly sis of u rea in soils. Soil. Sci. 50 (1940) , 119 . DIX ON, 1\1. , a n d \VEBB, E . : Enzymes. L ong m a ns, Green & Co., London , N ew York 19 58. DROBNIK, J ., a n d SEIF ERT, J.: The re la tionship of enzymatic inversion in soil to some soil m icrobiological t ests. F o l. Microbiol. 1 ( 1955), 41. DROBNIKOVA, V.: Factors influencing the d etermination of phosphatase in soil. Fo l. Microbiol 6 (196 1), 260 . DUNARD, G.: Enzy m es in soil. Revu e E o, B ioI. Sol. 2 (19 65), 141. EL-EsSAWI, T . M. : Studies on so il enzy m es with special reference to so il phosph at a se (m ethylp a rat h ion hydrolase) . Ph. D. T hesis , F a culty of Ag ri c., Alexan d r ia Univ., Egypt (197 3). GALSTYAN, A. SR. : Fermentative ac t ivity of s om e Armenian s oils. I V. U re ase activity in soi l. D okl. Akad, Nauk Armyan. 26 ( 1958),29. Activit y of en zy mes in sa line soils of t he Ararat lowla n d. D okl. A k ad, Nau k Armyan . 17 (1964), 3. Activ ity of de hydrogena ses in saline-solon etst.ic so il. Dokl, Akad, N auk Armyan. 40 (1965) 4 1. HALSTEAD, R. L. : P h osp h at ase activity of soils as influenced by lim e and other treatments. Canad. J . Soil Sc i. 44 ( 1964) , 137. HODGMAN, C. D., WEAST, R . C., and SELBY, S. M.: H a n db ook of Chemistry and Physi cs. 37th ed., Clevel and 1955. HOFMANN, C.: Dist r ib ution an d or igin of en zy m es in soil. Z. Pfl. Ernahr. Dung . B od en k . 85 (1959), 97.
116
M. M. E L-SHINNAWI an d S. A. E L-SHIMI, Enzymatic Activ ities in Salt Affected Siols
K OEPF, H. : Die bi ologische Ak tivitiit des B od en s u nd ihre e x perimen t e lle K ennzeichnung. Z. p n .Erniihr. Dung. Bodenk, 6. (1954 ), 138. K OTELEV, V. V., MEKHTIEVA, E. A. , and SMIRKOV, V. 1. : Phosphat ase ac t iv it y of s om e so ils a n d r h izospheres of cult iv ated plants. Trud y Pochv, l nst. Molda v ia F il. Akad, N auk 5 (1960), 3. Kozr.ov, K. A ., a n d MIKHAILOVA, E. N.: Dehydrogenase activ ity of som e so ils of Eastern Siberia. P ochvo ved enie 2 (196 5), 58. MASHTAKOV, S. M., K ULAKOVSKAYA, T. N ., a nd GOL'DINA, S. M.: Enzyme a ctivit y and respiration int ensi t y as indices of t he bi ol og ical a ct iv it y of soil. Dokl. Aka d . N auk (1954) , 141. N OWAK, \ V. : Ve rgleichsstudien zwische n dem Mi cro organism engehal t d es B odens und seine r Saccharase-A ktivit at. Pla n t a nd Soil. 20 (196 4), 330. OLSEK, S. R. , a n d \VATANABE, F . S. : A method to d etermi ne a phosphorus adsorptio n maxi m u m of soi ls as meas u re d b y L angmuir isotherm. So il. Sc i. Soc. Am er . Proc. 21 (19 57), 144. PIPER, C. S.: Soi l and P lant Ana lysi s. U n iv. of Ad elaide, Australia , 1955. SEIFIUtT, J. : E cology of soil microbes. II. Act a U n iv. Ca ro l. -Biologica (66) (1966), 139. SKUJINS, J. J. : Enzymes in soil. In: " Soil B iochemistry", Part 1 (Mc LAREN, A. D., a nd PETERSON, G. H., eds .). New York 1967. ZITTLE, C. A.: Adsorption stud ies with en zy mes and other proteins. Enzymol. 1. (1953),319. Authors' a d d r ess : D r . M. M. EL-SHINNAWI and S. A. EL-S HIMI, Faculty of Agriculture, Sh ibin EI-Kom, Monoufia University , Egypt.
Enzymatic Activities in Salt Affected Soils M. M. EL-SHINNAWI and S. A. EL-SHIMI With 3 Figures
Summary Activities of dehydrogenase, urease, and phosphatase (nuclease) as affected by the anionic and cationic form of inorganic salts (S04' COa, and CI for Ca, Mg, K, and Na) applied at 15 meg/lOO g soil to alluvial clay and calcareous sandy loam soils, during incubation, were studied. Most of the applied salts stimulated the enzyme activities of soils tested. Carbonates showed the most distinct effect, while chlorides were the least. Nuclease in both soils was activated by all of the applied salts. Dehydrogenase in the calcareous soil was stimulated by all types of salts used. Urease, in the alluvial soil, was promoted by all sulphates and chlorides applied. Urease, in the calcareous soil, was inhibited by chloride salts. In the alluvial soil, sodium as combined with each of the three aniones favoured the enzyme activities; potassium and magnesium showed intermediate figures, while calcium was extremest. In the calcareous soil, potassium in its different forms favoured the enzyme activities; sodium and magnesium showed intermediate results, and calcium gave the least values.
Zusammenfassung In alluvialem Ton sowie kalkhaltigem sandigen Lehm wurde die Wirkung von Ca, Mg, K und Na in Sulfat-, Karbonat- und Chloridform (15 mvalj l Of) g Boden) auf die Aktivitat von Dehydrogenase, Urease und Phosphatase (Nuklease) gepriift. Die meisten Salze stimulierten die Enzymaktivitaten, die Karbonate am deutlichsten, die Chloride am wenigsten. Nuklease wurde bei beiden Boden durch alle Salze aktiviert. Urease wurde im alluvialen Boden durch die Sulfate und Chloride gefordert, im kalkhaltigen sandigen Lehm durch die Chloride inhibiert. Irn alluvialen Boden begiinstigte Na in jeder Salzform die Enzymaktivitaten, Kalium und Magnesium nahmen eine Mittelstellung ein, Kalzium bewirkte die geringsten Stimulierungen. Irn kalkhaltigen Boden fOrderte Kalium in Verbindung mit allen Anionen die Enzymaktivitaten Kalzium hingegen kaum.
The breakdown of the complex organic residues and macromolecules in soil is catalizing by enzymes. Such bio-organic catalysts of proteinaceous nature are produced in soil mainly by microorganisms, and to some extent by roots of the growing plants. Activity of soil enzymes has been found to be dependent on soil characteristics and environmental conditions (SKUJINS 1967). Salts which might occur in/or be applied to soil in various operations could have a considerable influence on the biochemical processes in soil. Reports of GALSTYAN (1964, 1965), showed that dehydrogenase activity was very slight in solonchaks, but high activity of the same enzyme was detected in saline-solonetstic soils. BREMNER and TABATABAI (1973) found that Fe 203 , Mn0 2 , S04 -2, P0 4-3, and CI- all increased the values obtained in the assay of dehydrogenase activity with TTC. Activities of urease and phosphatase in soil were found to be inhibited by carbonates (GALSTYAN 1958, HALSTEAD 1964). On the other hand, ABDEL-MALEK (1973) found a positive correlation between phosphatase activity and the amount of Ca ++, Mg++, and HC0 3 - in soil.
Enzymatic Activities in Salt Affected Soils
107
The present work was carried out in order to clear up the effect of the anionic and cationic form of inorganic salts applied to alluvial fertile and calcareous soils, on the activity of representatives of soil enzymes, namely dehydrogenase (oxidoreductase), urease, and phosphatase (hydrolases). Materials and Methods
Soil samples Fertile alluvial and calcareous - under cultivation - soil samples were collected from the surface layer (0-15 cm) from Banha and Nobaria, respectively. Soil samples were air-dried, ground to pass a 2 mm-sieve, and mixed thoroughly. Pertinent analytical data for these soils (according to the methods of PIPER 1955) are presented in Table 1. Table 1. Characteristics of the soils tested Property
Alluvial
Calcareous
Textural grade Water holding capacity, % pH (1: 2.5 water suspension) Organic carbon, % Total nitrogen, % Total P, % CaCO a, % Total soluble salts, % Soluble ions, meqjlOO g soil HCO a -
Clay 53.60 7.85 1.218 0.120 0.015 2.88 0.110
Sandy loam 36.30 8.43 0.524 0.045 0.007 27.60 0.165
ci-
S04-Ca++ Mg++ K+ Na+
0.46 0.39 0.98 0.39 0.26 0.02 1.16
0.29 0.46 1.47 0.56 0.33 0.67 0.66
Soil treatments Series of sulphates, carbonates, and chlorides for calcium, magnesium, patassium, and sodium were finely ground and mixed with the air dry soil at 15 meq salt per 100 g soil. Treatments - in duplicate - were carried out by using portions of 5 g soil crumbs, placed in large sized (2 X 18 cm) test tubes. The moisture content was adjusted to 60 % of the water holding capacity of the soil. Evaporation losses, during incubation at 30°C, were made up periodically by the addition of distilled water. Variations in pH values of the tested soils, as a result of salt application, appear in Table 2.
Assay of enzyme activities Dehydrogenase was determined colorimetrically for the 2,3,5-triphenyl formazan (TPF) produced from the reduction of 2,3,5-triphenyltetrazolium chloride ('1''1'0), using acetone for extraction (CASIDA et al. 1964). Urease was estimated colorimetrically for the residual urea by P-dimethylaminobenzaldehyde solution (CONRAD 1940, BROADBENT et al. 1958). Phosphatase (nuclease) was determined colorimetrically for the inorganic phosphorus released from nucleic acid added DROBNIKOVA (1961). Sorption of inorganic phosphorus in soil was determined by the method of OLSEN and WATANABE (1957) in order to correct the obtained data of mineralized available phosphorus.
108
M. M. EL-SHINNAWI an d S. A. EL -SBIMI
Table 2. Va ria ti ons in p H val ues of the fertile a ll uv ia l a nd ca lcareous soils t reated with differen t salts
p H value Salt treat ment (15 meq/l OO g s oil) (in 1 : 2.5 soil-water s uspens ion )
Control (no s al t added)
Alluvial
Calcareous
7.85
8.43
S04
Ca Mg K Na
7.53 7.2 0 7.34 7.4 8
8.43 8.00 8.20 8.36
COa
Ca Mg K Na
8.00 8.00 9.1 0 8.92
8.43 8.50 9.00 9.00
Cl
Cu Mg K Na
7.85 7.80 7.85 7.90
8.40 8.40 8. 2 1 8.28
Results and Discussion A. D eh ydrogen a s e 1. Sulphates The application of sulphate salts (at 15 meq/lOO g soil) led to de crease the activity of dehydrogenase in the alluvial soil at time 0 (Fig. lA). Incubation of the moistened soil samples (at 60 % W.H.C.) of the control (unsalted) treatment, however, slightly increased t he enzy me ac tivit y to give a peak on the t hird day, thereafter began to decline. Samples treat ed with MgS0 4 revealed an analogous trend to that of t he control. The other sulpha tes (Ca, K, Na) gave high peaks only on t he seventh day of incubation. Activit y of t he bioorganic cat alyst was promoted by the application of each of CaSO.. and Na 2SO... Potassium sulphate, on t he other hand, inhibited the catalytic reaction of t his soil. Dehydrogenase in thc calcareous soil was stimulated by the applied sulp hat cs at time 0 (F ig. ID). Incubation of the moistened samples showed two definite peaks of dehydrogenase activity for this soil. The first peak on the third day was higher than the second which appeared on t he fifteenth day. All sulphates activated t he dehydrogenase in the calcareous soil, where potassium salt was t he most effective in this regard and followed by those of magnesium, sodiu m, and calcium, respectively. Gene rally, the calcareo us soil show ed somewha t higher dehydrogenase activit y than the fertile alluvial soil. The pat t ern of dehydrogenase activity of the incubated soil sa mples goes along with t he growth curve of micro-organisms when the soil is brough t to favourable conditions of moisture a nd te mperature (SEIFERT 1966). The action of sulphates on decreasing t he pH value of calcareou s soil samples (Table 2) appeared to be favourable for the microbial population and the subsequent biochemical activity (MASHTAKOV et al. 1954 and HOFMANN 1959). It was reported by DIXON and WEBB (1985) that potassium, magnesium, and calcium cations and sulphate anion activate the catalyst dehydrogenase in solution s. The higher dehydrogenase activity shown by the oaloare-
109
Enzymatic Activities in Salt Affected Soils
ALL UVIAL SOIL 2000~
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/600 s,
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~
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~
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Fig. 1. Dehydrogenase activity in the incubated soil samples as affected by the anionic and cationic form of added salts, at 15 meqj l Of) g soil.
ous soil could be explained by the findings of CERNA (1969) that the dehydrogenase activity increases as the specific surface of soil particles decreases. It is worth noting that the intensity of enzyme activity of the soil is not usually correlated either with the total number of microbes or with the number of microorganisms in main groups expected to be in such a soil (KOEPF 1954, DROBNIK and SEIFERT 1955, and NOWAK 1964).
2. Carbonates Dehydrogenase of the alluvial soil was activated by the application of Na 2CO s and highly by MgCOa (Fig. IB). Potassium carbonate, however, showed no considerable changes, while calcium carbonate resulted in a slight decrease in the catalytic reaction of the soil. Peaks of the enzyme activity appeared on the 3rd day of incubation for all of the applied carbonates. A second increase, however, was detected on the 15th day. All carbonates markedly stimulated the activity of dehydrogenase in the calcareous soil (Fig. IE). Magnesium carbonate was the most stimulating and was followed, des. cendingly, by sodium, potassium, and calcium carbonates. Two peaks of the catalytic activity appeared within the experimental duration, the first detected on the third day of incubation was higher than the second on the 15th day. Application of carbonates resulted in shifting the pH to higher values in both soils (Table 2); such conditions increased the formation of TPF. Reduction of TTC may be chemically promoted through the alkaline shift that followed the addition of carbonates to soils. 3. Chlorides Inconsiderable changes were shown in the activity of dehydrogenase of the fertile alluvial soil as affected by the addition of chloride salts (Fig. 1C). Calcium and sodium
110
M. M.
E L-SHlN X AWI
an d
S.
A.
EL·SHIMI
chlorides slightly in creased t he en zyme activity, while potassium and magnesium on es decreased it somewhat. P eaks of activit y almost appe a red on t he t hird day for all treatments. Activity of dehydrogenase in t he calcar eou s soil was highly promoted by t he adde d chlorides (Fig. IF). Th e descending order of activation was potassium, sodium, magnesium, and calcium chlorides. Two peaks appeared, a s usual for this soil on the 3rd and the 15th day of incubation. All chlorides showed no effect on t he pH of the alluvial soil (Table 2). Th e pH value of t he calca reous soil, however, was slightly shifted to lower valu es by t he a ddition of eac h of pot assium a nd sodium chlorides (Tabl e 2). The activation of deh ydrogenase show n in t he calca reo us soil following t he addit ion of t he monovalent cati on s may be attributed to t he favourable effect of t he resulted pH on soil mi croflora, The present result s are in acco rdance, to some extent, with t he ea rlier findings of GALSTYAN (1965) a nd KOZLOV a nd MIKHAILOVA (1965) indicati ng t hat t he sa line-so lonetz soils had a high deh ydrogenase acti vity ; a nd of BREl\INER and TABATABAI (1973) which showed t hat S03 -2 and CI- increased t he values obt ained in the assa y of dehydrogen as e activity with TTC. On comparing the an ions as effect ed the activity of dehydrogenase in soil, results showed t hat ca rbonates were t he most enzy me-acti vators in both soils under conside ration , i.e. alluvial clay a nd calca reo us sa ndy loam. Sulphat es a nd chlorides followed , resp ect ively, t he carb onat es in t he alluvial soil, bu t opposite ly in t he calcareous soil. Sodium combined wit h all anions under stud y showe d activati on for deh ydrogen ase in t he fertil e alluvia l soil. Pot as sium, however , resul t ed in a slight inhibition with all a nio ns; wh ':lreas t he effect of t he other cations diff ered stimulating or inhibiting a ccording to the co-anion. Magnesium carbonate was the most stimulatory salt for deh ydrogen ase ac t ivity of t he alluvia l sai l, while calcium carbonate was t he most depressing. The cati on s under consideration activat ed the oxide-reduction cat al ytic reaction in t he calcareou s soil at varying intensities, depending on the complementary ion. Calcium was the least st im ula to ry a mong the different cations with all anion s. Magnesium ca rbonate, likewise, sho wed t he best res ults, and ca lcium chloride ga ve t he least va lues of deh ydr ogen ase ac ti vity in the calcareous soil. Genera lly, deh ydrogenase of t he calca reo us soil resp onded more to t he applied sa lts t ha n t hat of t he alluvial soil. B. U rease 1. Sulphat es
Act ivit y of urease was promot ed by t he application of sulphat e salt s (15 meqJ 100 g) to the alluvial clay soil (F ig. 2 A). Th'3 catalytic reacti on was decreased on the third day of incubation and then began slightly to increase mostly in a lin ear manner. Th e maximum values obtained within the experime ntal duration for t he control as well as for CaS0 4 • K 2S04 , a nd Na 2S04 were at t he beginning, a nd for MgS0 4 on t he t hirtieth day. Magn esium was also t he most st imul a tory cation for t he catalyst urease, followed b y ca lcium, potassium, and sodium, resp ecti vely. Th e calcareous sa ndy loam soil showed, genera lly, similar pat t ern of urease activity to t hat of t he alluvial soil, but at lower valu es. Magn esium and p ot assium sulphates stimu lated t he activity of urease, while calcium a nd sodium ones inhibit ed it (F ig. 2 D ). Max imum valu es for a ll t reatme nts within t he expe ri mental d ura t ion appeared at t ime O. Urease activit y was highest at time 0, follow ed by a depression on the third d ay of incubation a nd again began to show a slight increase . The intensity of these variations differed acoo rding to the soil type and sa lt treatment, i.e . the depression was very
III
Enzymatic Acti vities in Salt Affe ct ed Soils
-- --
A LV 'tAl.. SOIL
00 ~
....
~
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0
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e
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. -
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r
_
.
_
.
~
0200 -,--,-
r~
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~
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I
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00
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r
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r
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Contro/
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i
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F ig. 2. Urease ac t iv ity in t he incubated soil samples as aff ected by the anion ic and ca t io nic forms of added sa lts, at 15 m eq/ l00 g soil.
sha rp in t he calca reo us soil treated with Na 2S04 , and ver y slight in t he alluvial soil trea t ed with MgS0 4 (Figs. 2A a nd D ). Th e highest enzyme activity appea red at t ime 0 could be attributed t o t he initially found urea se released from roots and nonviabl e cells. Maximum prolifer ati on of soil microflora to ok place up to t he t hird da y of in cubation under favourable conditions of moisture a nd te mpe rature (SEIFERT 1966), and subsequently, maximum need for nitrogen to t he reproducing cells would ari se. H ence, ur ease as nit rogeno us compo und is liab le to be decomposed with a higher ra t e t ha n it s accumulation in t his ph ase, when microbial reproduct ion exceeds lysis. The sha rp depression shown in t he calcareo us soil might be referred to its init ially low nitrogen content (Table 1). Th e activation of urea se appeared by application of sulphate sa lts t o t he alluvi al soil might be due to t he role of cations in dimini shing t he adso rpt ion of both t he ur ea substrate and the enzyme on t he cla y part icles which lead s t o increase the hydrolysis pro cess (ZITTLE 1953). In thi s concern , the dival ent s were more effect ive, whereas magnesium sulphate with it s high sol ubilit y (HODGMAN et al. 1955) showed t he best result s. T he shift in p H of t he calca reous soil to lower valu es du e t o t he ap pli cat ion of both MgS0 4 a nd K 2S04 (Ta ble 2) ma y explain t heir suitability to t he en zyme urease. The lower act ivity of urease shown in the calca reous soil in com parison with the alluv ial one could be asc ribe d to eit her t he minor product ion of en zyme or t o soil t exture (Table 1) as CERNA (1966) not ed t hat urease act ivit y in creases with t he in crease in t he spe cific s urface of soi l particles. 2. Carbon at es App licat ion of both CaCOa a nd MgCOa highly accelerated t he hyd rolysis of urea in t he alluvial soil, while Na 2C Oa a nd ~COa di mini shed it (F ig. 2B). Dep ression in t he
112
M. M.
EL-SHINNAWI
and
S.
A.
EL-SHIMI
hydrolysis process was shown on the third day of incubation for all treatments, followed by a, slight linear increase. The maximum values within the experimental duration for all salt treatments appeared at time O. Calcium was the most stimulatory cation for urea hydrolysis in the alluvial soil, while sodium showed the least results. Urease activity in the calcareous soil was increased by the application of each of KaCO a and NaaCO a, respectively, while MgCOa was of inconsiderable effect and CaCOs was inhibitory (Fig. 2E). The same pattern of enzyme activity during incubation time, as in the moistened samples of the alluvial soil, was shown in the calcareous variants. The maximum values within the experimental duration for all salt treatments appeared at time O. Potassium was the most stimulatory cation for urease in the calcareous soil, while calcium showed the least results. The application of carbonates raised the pH values of the tested soils (Table 2). This rise in the pH value was more pronounced in the alluvial soil than in the calcareous one. The unsuitability of potassium and sodium carbonates for urease in the alluvial soil may be referred to the higher alkaline shift of pH value which unfavoured both the ureolytio bacteria and the enzyme urease itself (CECCONI et al, 1974 and ALEXANDER 1977). It is worth noting that the insoluble fine particles of calcium and magnesium carbonates increase the surface area for the catalytic reaction in the alluvial soil and thus increase the activity of urease in accordance with the notes of CERNA (1966). In this connection, ALEXANDER (1977) noted that the presence of such a salt in soil affects the microbial activities even through its insoluble particles to which microorganisms tend to adhere, as in cases of lime and gypsum. The reverse trend appeared in the calcareous soil might be attributed to the native high content of CaCOa and poor properties which lessen the urease activity of such soil, as reported by GALSTYAN (1958). Potassium and sodium carbonates, with their high solubility (HODGMAN et a1. 1955), favoured both the ureolytic microorganisms and urease of the calcareous soil through diminishing the unfavourable action of lime (GALSTYAN 1958). 3. Chlorides Cations combined with chloride very slightly increased the activity of urease in the alluvial soil, where potassium was the most stimulatory, followed descendingly by calcium, magnesium, and sodium (Fig. 2C). The maximum values within the experimental duration for all of the applied chlorides appeared on the thirtieth day. The activity of urease was inhibited by all chlorides applied to the calcareous soil (Fig. 2F). The monovalents, i.e. potassium and sodium, were less inhibitory than the divalents. Calcium was the most depressive cation. The maximum values within the experimental duration for all of the applied chlorides appeared at time O. The presence of cations in the clay soil plays a role in releasing both enzyme protein and substrate adsorbed on the soil particles to soil solution, thus promoting the catalytic reaction (ZITTLE 1953). No evidence in the present results supports the earlier findings of GALSTYAN (1964) in which urease activity was depressed in saline soils. Chlorides appeared to be of inhibiting action on the urease of the calcareous soil which may be due to the specific effect of the anion on both ureolytic bacteria and enzyme in the poor conditions of the calcareous soil. On comparing the anions together how they affect the activity of urease in soil, it is noticed that no convergency could be stated for both soils under consideration. Sulphates were the most enzyme-activators in the alluvial clay soil. Chlorides showed slight increases in the same soil. The divalent cations combined with carbonate highly increased the urease activity, while the monovalents revealed a slight decrease. Calcium and magnesium combined with all anions showed activation for urease in the
113
Enzymatic Activiti es in Salt Affected Soils
alluvial soil, while potassium a nd sodium acted sligh tl y in creasing or decreasing depending up on t he co-anion. Sod ium showed low resul t s in all ca ses. Ca lcium carbonat e was t he most st imulat ory sa lt for urea hydrolysis in t he incubated samples of t he alluvial soil, while sodium carbona te gave t he lea st valu es. Some cations (K , Mg) of both sulphat es and carbonat es slightly stimulated t he urease in t he calcareous sa ndy loam soil, while t he other cati on s (Ca, Na ) were inhibitory. All chlorides inhibit ed t he enzyme of t he same soil. Potassium carbonate was t he most stimulatory salt for t he hydrolysis process in t he calcareou s soil, while calcium chloride ga ve t he least res ult. C. Nu cl ea se
1. Sulpha tes H ydrolysis of nucleic acids in t he moistened sa mples of t he alluvial soil was very slightly stimulated b y applicat ion of Na 2S04 or K 2S04, while MgS0 4 or CaS0 4 showed no notable effect (Fig. 3A). P eaks of activity were shown on the third day of incubation for the different treatments. A second increase was recorded on the fifteenth day. Nucl ease in the calcareou s soil samples was somewhat stimulated by the applied sulphates, where calcium wa s t he best cation, follow ed by magnesium, potassium, and sodium ones, respectively (Fig. 3D). The maximum valu es within the experimental du ra ti on for the control t reatment as well as for MgS0 4 and Na 2S04 , appeared on t he 3rd day, whil e t hose of CaS 0 4 , K 2S04 , and cont rol were on t he 15th day. The stimula tion in t he activity of nu cleic a cid hydrolase caused by t he application of sulp hate salts to both soils tested ma y be attributed to t he specific action of t he a nion on t he contributing microorganism s as well as on t he enzyme itself (DIXON a nd WEBB 1958). In t his regard, HALSTEAD (1964) foun d t hat phosphatas e activity in
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114
M. M.
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and
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EL-SHIMI
different surface soils was favoured during incubation by CaS0 4 and MgS04 . Order of enzyme activation in the alluvial soil was inversely correlated with the relative replacing power of the cations on the clay surfaces where the order was Ca > Mg > K > Na. Though the situation in the calcareous soil is different. The low activity of the phosphatase in the calcareous soil in the present study agrees with the results of KOTELEV et al. (1960) and EL-EsSAWI (1973). It is noteworthy that DUNARD (1965), KOZLOV (1966), CERNA (1970), ABDEL-MALEK (1973), and EL-EsSAWI (1973) concluded that phosphatase activity is positively correlated with soil texture, organic matter content, and organic phosphorus, Pattern of nuclease activity appeared to be correlated with the microbial growth and expected consistency of microorganisms in such soils. 2. Carbonates Nuclease of the alluvial soil was activated by the applied carbonate salts. Sodium carbonate showed the best results followed by potassium, magnesium, and calcium salts, respectively (Fig. 3B). Peaks of activity were shown on the third day of incubation for all treatments. Hydrolysis of nucleic acids in the calcareous soil was slightly activated by the addition of carbonates, where MgCOa gave the best results followed descendingly by KzCO a, NazCO a, and CaCO a (Fig.3E). Maximum values within the experimental duration for the treatments of MgCOa and NazCO a appeared on the 3rd day, and for KzCO a and CaCO a on the 15th day. The order of activation of the nucleic acids hydrolase in the clay soil caused by carbonate addition is, likewise, inversely correlated with the relative replacing power of the cations on clay surfaces, as shown by sulphates. The rise in pH occurred by the application of carbonate salts to both soils (Table 2) was found to be of a favourable action on the nuclease of the clay soil, whereas such effect was not observed in the calcareous one. In this concern, DROBNIKOVA (1961) found in an arable brown soil having pH 7.9 that as the pH value increases to the alkaline range, the activity of nuclease increases. ABDEL-MALEK (1973) pointed out, in his thesis, that there was a positive correlation between phosphatase activity and the amount of HCO a- in soil. No evidence in our work supports the earlier results of HALSTEAD (1963), who found that phosphatase activity of surface soil samples was inhibited during incubation by CaCO a or MgCOa application. 3. Chlorides Breakdown of nucleic acids was very slightly favoured by addition of chlorides to the alluvial soil. Sodium chloride showed the best results and was followed by potassium, magnesium, and calcium salts, respectively (Fig. 3C). Peaks of activity appeared on the third day of incubation for all treatments. Nuclease of the calcareous soil was promoted by chloride salts, where NaCl was the most promoting, followed descendingly by magnesium, potassium, and calcium chlorides (Fig. 3F). Maximum values within the experimental duration for the treatments of magnesium, sodium, and calcium appeared on the 3rd day, and of potassium on the 15th day. The same order of activation shown by the different cations with sulphates and carbonates was also revealed by chlorides on the nuclease of the alluvial clay soil. HALSTEAD (1964) found that MgClz and CaClz were stimulatory for soil phosphatase. The comparison between the effects of the different anions applied to soils on nuclease activity shows that all of the applied salts stimulated the enzyme in both soils
Enzymatic Activities in Salt Affected Soils
115
tested . I n t he alluvial cla y soil carbonates were t he most enzyme-activa to rs, while sulphates were t he least . I n t he calca reo us sa ndy loam soil, all anions st imulated t he en zyme at about t he same levels. Cations acted at one t re nd as associated with t he different a nions in t he clay soil, where t he monovalent were t he most stimulatory for nuclease. Sodium was t he best while calcium showed t he least result s in all cases of the alluvial soil. In the calcareous soil, cations act ed different ly according to t he complementary an ion; wherea s magnesium and potassium act ed intermediat ely, calciu m and sodiu m acted extremely. Sodium carbo nate WaS t he most stimulatory salt for nuclease in t he alluvial soil, while calcium sulphate ga ve t he least fig ures . Calciu m sulp hate showed t he best resu lts in t he calcareous soil, while calcium ca rbonate was t he least. Ac know l edg ement T h e authors ex press their thanks to Prof. D r . H. H A:Il:DI, Professor of Soil Sc ience, Ain S hams U n iversity, Egypt, for hi s interest in the work and advice.
References ABDEL-MALEK, S. M.: Enzymatic ac t ivities in so ils as in di cat or s of fertilit y . M. Sc . Thesi s, Faculty of Agriculture, Cairo Univ., Egypt (1973). ALEXANDEIt, 1\1.: I ntroduct ion to So il Microbiology. 2nd ed ., New York 1977. B REMNER, J . 1\1., a nd TABATABAI, 1\1. A. : Effects of some in org an ic su bstan ces on TTC (2,3,5· trip henylt etrazolium chl oride ) assay of d ehydrogenase a ctivity in soils. Soil BioI. Bi och em , 5 (3) (1973) , 385. B ROADBENT, F. E ., H ILL, G. N ., and TYLER, K . B. : Transformations and movement of u rea in soil. Soil. Sci. Soc. Arn er, Proc, 22 (1953), 303. CASIDA, L. E. , KLE IN, D . A., a n d SANTORO, T. : Soil dehydrogenase activity. Soil. Sci. 89 (196 4), 37 1. CECCONI, C. A. , BRUNO, V., and DANIELE, E . : H ydrolysis of urea by t h e so il and by vegetab le in the soil. Georgofili 20 (19 74 ), 177. CERNA, S. : Enzymat ic a ctivity of so il in re lation t o its stru cture. Acta Univ, Carol. - Biologica (65) (1966), 83. - Structura l soil a n d its d ehydrogenase a ct ivit y. Acta Univ. Carol.- Biologica (68) (1969), 279. - Phosphatase activity in st ruct ura l so il. Acta Univ. Ca ro l.-Biologica (69) ( 1970), 455. CONRAD, .J. P. : The n a t u re of the cat a lys t ca u sing t he h y d r oly sis of u rea in soils. Soil. Sci. 50 (1940) , 119 . DIX ON, 1\1. , a n d \VEBB, E . : Enzymes. L ong m a ns, Green & Co., London , N ew York 19 58. DROBNIK, J ., a n d SEIF ERT, J.: The re la tionship of enzymatic inversion in soil to some soil m icrobiological t ests. F o l. Microbiol. 1 ( 1955), 41. DROBNIKOVA, V.: Factors influencing the d etermination of phosphatase in soil. Fo l. Microbiol 6 (196 1), 260 . DUNARD, G.: Enzy m es in soil. Revu e E o, B ioI. Sol. 2 (19 65), 141. EL-EsSAWI, T . M. : Studies on so il enzy m es with special reference to so il phosph at a se (m ethylp a rat h ion hydrolase) . Ph. D. T hesis , F a culty of Ag ri c., Alexan d r ia Univ., Egypt (197 3). GALSTYAN, A. SR. : Fermentative ac t ivity of s om e Armenian s oils. I V. U re ase activity in soi l. D okl. Akad, Nauk Armyan. 26 ( 1958),29. Activit y of en zy mes in sa line soils of t he Ararat lowla n d. D okl. A k ad, Nau k Armyan . 17 (1964), 3. Activ ity of de hydrogena ses in saline-solon etst.ic so il. Dokl, Akad, N auk Armyan. 40 (1965) 4 1. HALSTEAD, R. L. : P h osp h at ase activity of soils as influenced by lim e and other treatments. Canad. J . Soil Sc i. 44 ( 1964) , 137. HODGMAN, C. D., WEAST, R . C., and SELBY, S. M.: H a n db ook of Chemistry and Physi cs. 37th ed., Clevel and 1955. HOFMANN, C.: Dist r ib ution an d or igin of en zy m es in soil. Z. Pfl. Ernahr. Dung . B od en k . 85 (1959), 97.
116
M. M. E L-SHINNAWI an d S. A. E L-SHIMI, Enzymatic Activ ities in Salt Affected Siols
K OEPF, H. : Die bi ologische Ak tivitiit des B od en s u nd ihre e x perimen t e lle K ennzeichnung. Z. p n .Erniihr. Dung. Bodenk, 6. (1954 ), 138. K OTELEV, V. V., MEKHTIEVA, E. A. , and SMIRKOV, V. 1. : Phosphat ase ac t iv it y of s om e so ils a n d r h izospheres of cult iv ated plants. Trud y Pochv, l nst. Molda v ia F il. Akad, N auk 5 (1960), 3. Kozr.ov, K. A ., a n d MIKHAILOVA, E. N.: Dehydrogenase activ ity of som e so ils of Eastern Siberia. P ochvo ved enie 2 (196 5), 58. MASHTAKOV, S. M., K ULAKOVSKAYA, T. N ., a nd GOL'DINA, S. M.: Enzyme a ctivit y and respiration int ensi t y as indices of t he bi ol og ical a ct iv it y of soil. Dokl. Aka d . N auk (1954) , 141. N OWAK, \ V. : Ve rgleichsstudien zwische n dem Mi cro organism engehal t d es B odens und seine r Saccharase-A ktivit at. Pla n t a nd Soil. 20 (196 4), 330. OLSEK, S. R. , a n d \VATANABE, F . S. : A method to d etermi ne a phosphorus adsorptio n maxi m u m of soi ls as meas u re d b y L angmuir isotherm. So il. Sc i. Soc. Am er . Proc. 21 (19 57), 144. PIPER, C. S.: Soi l and P lant Ana lysi s. U n iv. of Ad elaide, Australia , 1955. SEIFIUtT, J. : E cology of soil microbes. II. Act a U n iv. Ca ro l. -Biologica (66) (1966), 139. SKUJINS, J. J. : Enzymes in soil. In: " Soil B iochemistry", Part 1 (Mc LAREN, A. D., a nd PETERSON, G. H., eds .). New York 1967. ZITTLE, C. A.: Adsorption stud ies with en zy mes and other proteins. Enzymol. 1. (1953),319. Authors' a d d r ess : D r . M. M. EL-SHINNAWI and S. A. EL-S HIMI, Faculty of Agriculture, Sh ibin EI-Kom, Monoufia University , Egypt.