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II. Role of Accumulated Soil Enzymes in the Initial Phases of the Decomposition of Organic Residues and of the Transformation of Some Compounds

II. Role of Accumulated Soil Enzymes in the Initial Phases of the Decomposition of Organic Residues and of the Transformation of Some Compounds

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28



s.



KISS, M. DRAGAN-BULARDA,



AND D. R ~ D U L E S C U



saccharolysis had nearly the same intensity in the presence as in the absence of toluene. Proliferation of microorganisms during the 24 hours of

incubation took place in the absence of toluene where the microorganisms

utilized the hydrolytic products as carbon and energy sources and, concomitantly, could synthesize invertase. But the amount of invertase produced

by the proliferating microorganisms was negligible in comparison with the

amount of the preexisting invertase. Therefore, even in incubations lasting

24 hours, most of the soil invertase activity is due to the accumulated invertase already produced by generations of microorganisms, not to the enzyme produced by the proliferating micropopulation of the soil during

incubation.

According to our observations, the accumulated invertase predominates

in the invertase activity of soils during incubations of 48 hours (Kiss el

al., 1971), 14 days (Fig. 2), and 20 days (Kiss et al., 1972).

Voets and Dedeken (1965) and Voets et al. (1965) have compared

invertase activity in soil samples treated with toluene and sterilized by

y-radiation, respectively. In 7-sterilized samples no viable microorganisms



m



Hydrolysis

of sucrose

%



1



Synthesis

of Levan



80



60



60



M



0



-t



+t



+t



FIG.2. Hydrolysis of sucrose and synthesis of levan in soil samples incubated

in the absence ( - t ) or in the presence (+t) of toluene for 14 days. [Redrawn

from S. Kiss, I. Bosica, and P. MCliusz, Stud. Univ. BabepBolyai, Ser. Biol. 7(2),

65-70 ( 1962) .]



ENZYMES ACCUMULATED IN SOIL



29



were detected. However, practically the total invertase activity persisted;

consequently, there was no significant difference between the invertase activity of toluene-treated samples and that of the ,-sterilized samples (Table

I). One can draw the conclusion that the microorganisms, which remained

viable but became nonproliferating in the toluene-treated samples, did not

synthesize new invertase but contributed to the invertase activity of soil

by the amount of invertase they had contained before addition of toluene.

Invertase activity in soil is measurable after 2 hours of incubation

(Kunze and Rickart, 1973). As this incubation time is shorter than the

lag phase of microbial growth in dried and then rewetted soil, the activity

should be ascribed to the accumulated invertase.

Invertase accumulates in soil mostly as an enzyme bound to cell constituents such as disintegrated cells, dead but not disintegrated cells, and

viable but nonproliferating cells, not as an enzyme released from the cells

and then adsorbed (Kiss and Bilint, 1959).

Ukhtomskaya ( 1952) centrifuged suspensions prepared from soil and

buffer solution; the supernatant showed invertase activity. Her paper, however, does not contain a detailed description of the methods used (the same

remark should be made also with respect to Ukhtomskaya’s observation

that the supernatant contains also amylase, protease, peroxidase, and catalase activities). Shcherbakova et al. (1971) extracted an enzyme complex

from a meadow soil that exhibited invertase (and amylase) activity.

2 . Hydrolysis of a-Glucosides, Including Maltose

Toluene-treated soil samples hydrolyze phenyl-a-D-glucoside (Hofmann

and Hoffmann, 1953a) and maltose (Drobnik, 1955; Hofmann and Hoffmann, 1955). Comparative examination of maltase activity in reaction

mixtures incubated in the presence and in the absence of toluene for 7-20

days revealed no significant difference in maltose hydrolysis; the accumulated maltase predominated in the hydrolysis of maltose during incubation

from 7 to 20 days (Kiss et al., 1972).

TABLE I

Enzyme Activities in Normal and y-Irradiated Soil (2’MradY

Treatment



+



Normal soil toluene

7-Irradiated soil

7-Irradiated soil toluene



+



lnvertase



Urease



13.7

12.7

11.6



10.5

12.8

10.0



Acid

Proteolytic

phosphatase enzymes



20.5

20.0

20.4



3.7

4.0

3.9



From J. P. Voets and M. Dedeken, Meded. Landbouwhogesch. Opzoekingssta. Slaat

Gent 30, 2037-2049 (1965).



30



s.



KISS, M.



DRAGAN-BULARDA,



AND D.



RADULESCU



3. Hydrolysis of p-Glucosides, Zncluding Cellobiose

Soils treated with toluene hydrolyze phenyl-p-D-glucoside, arbutin (Hofmann and Hoffmann, 1953a), cellobiose (Hoffmann, 1959), salicin (Hoffmann and Dedeken, 1965), and p-nitrophenyl p-D-glucoside (Hayano,

1973) . In a comparative study of p-glucosidase (cellobiase) activity in

the presence and in the absence of toluene, Galstyan (1965a, 1974) has

found that in reaction mixtures incubated with or without toluene for 24

hours there is no difference in the intensities of arbutin hydrolysis. Consequently, the hydrolytic cleavage of the p-glucoside was catalyzed by the

accumulated p-glucosidase. After prolonged incubation (7-20 days) hydrolysis of cellobiose was due largely to the cellobiase produced by the

proliferating microorganisms rather than to the accumulated cellobiase

(Kiss et al., 1972). Accumulation in soil of some quantity of p-glucosidase

can be deduced also from the observation of Hayano (1973) and Hayano

and Shiojima ( 1974), according to which p-glucosidase activity in soil is

measurable even after incubation for 30-60 minutes.

4 . Hydrolysis of a-Galactosides, Zncluding Melibiose



In soil samples treated with toluene, phenyl-a-D-galactoside (Hofmann

and Hoffmann, 1953a), and melibiose (Hoffmann, 1959) were split enzymatically, a-Galactosidase (melibiase) activities of soil in the presence

and in the absence of toluene were not compared. However, the occurrence

in soil of some accumulated a-galactosidase (melibiase) is expected.



5 . Hydrolysis of p-Galactosides, Including Lactose

Toluene-treated soils are able to split phenyl-p-D-galactoside (Hofmann

and Hoffmann, 1953a) and lactose (Hoffmann, 1959). Comparative examination of soil lactase activity in the presence and in the absence of

toluene has shown that during incubation periods of 7-20 days the accumulated lactase plays a less important role in the hydrolysis of lactose than

the lactase synthesized by the proliferating microorganisms (Kiss et al.,

1972). In a chernozemic soil previously amended with lactose, p-galactosidase activity was measurable even after 2 minutes from the addition of

substrate (0-nitrophenyl p-D-galactoside) . Toluene used at a 2.5-ml dose

per 25 g of soil enhanced the enzyme activity, but higher toluene doses

decreased it. In the soil not amended with lactose, values of enzyme activity

were negligibly low (RySavf and Macura, 1972).

6. Hydrolysis of Starch

Hofmann and Seegerer ( 1951a), Krasil’nikov ( 1952), and Ukhtomskaya (1952) were the first to mention that soils contain amylases, but



ENZYMES ACCUMULATED IN SOIL



31



they did not fully describe the methods used. Drobnik (1955) and Hofmann and Hoffmann (1955) developed and described methods to determine amylase activity in soil. Toluene was used to prevent microbial proliferation. According to Galstyan ( 1965a, 1974), amylolysis in soil

samples incubated in the presence or in the absence of toluene for 24 hours

is of the same intensity. This means that, at least during the first 24 hours,

amylase activity in soil should be attributed to the accumulated amylases.

Shcherbakova et al. ( 1970, 1971 ) and Shcherbakova and Galushko

(1971) obtained extracts from a cultivated soil which gave three protein

peaks. One of the peaks exhibited amylase activity. As stated in Section

11, A, 1, Shcherbakova et al. (1971 ) extracted from a meadow soil an

enzyme complex that exhibited both amylase and invertase activities.

7. Hydrolysis of Cellulose

Markus ( 1955) introduced small pieces of cellophane into toluenetreated soil samples. During incubation the weight of cellophane decreased.

This observation was interpreted as evidence of the occurrence of cellulase

in soil. Cellulase activity in toluene-treated soils was also demonstrated

by other authors who used different substrates including cellophane (Kislitsina, 1965, 1968; Kozlov and Kislitsina, 1967), cellulose powder (Rawald,

1968, 1970a,b; Mereshko, 1969; Benefield, 1971) , and carboxymethyl

cellulose (CMC) (Coucke, 1964; Narayanaswami and Veerraju, 1969;

Tomescu, 1970; Bagnyuk and Shchetinskaya, 1971 ;Kislitsina, 1971;Aiyer

and Krishnaswamy, 1971 ; Pancholy and Rice, 1973; Ambroi, 1973b;

Drigan-Bularda and Kiss, 1973; DrBgan-Bularda, 1974). Kong et al.

(1971 ) and Kong and Dommergues (1972) used both cellulose powder

and CMC to determine separately the C, and C, components of the cellulase activity. Merthiolate was substituted for toluene.

Kozlov and Kislitsina (1967) and Kislitsina (1968) compared cellulase

activity in toluene-treated and untreated soils. Cellophane was used as substrate, and the reducing sugars released were analyzed. In the absence of

toluene, smaller amounts of reducing sugars were found indicating that

the proliferating microorganisms consumed a part of the sugars. A similar

comparison was made by Tomescu ( 1970), but she used petroleum ether

instead of toluene, CMC instead of cellophane, and analyzed viscosimetrically the residual CMC. Nearly the same amounts of residual CMC

were found both in the presence and in the absence of the antiseptic. This

shows that in the soil examined (neutral pH; 24-120 hours incubation

time; 28 O or 37OC temperature) the accumulated C, enzyme predominated

in the catalysis of CMC breakdown. Drozdowicz (1971) found C, activity in buffer and water extracts from soils previously composted with



32



s. KISS, M. DRAGAN-BULARDA,



AND D.



RXDULESCU



cellulose powder after 4 weeks. The three organic soils studied by Kong

and Dommergues (1972) showed both C, and C, activities.

In contrast with the positive results concerning occurrence of the C, or

C,plus C, components of cellulase in an accumulated state in soil, some

literature data suggest that cellulase accumulation in soil is not a general

phenomenon.

Hubner (1956-1957) could not demonstrate any cellulase activity in

soils treated with toluene and cellulose. His attempts to extract cellulase

from soils were unsuccessful. Only one out of ten soils studied by Kiss

et al. (1962b) exhibited cellulase activity where powdered cellulose

served as substrate in the presence of toluene, and after incubation the

reducing sugars were analyzed. Seetharaman et al. (1968) treated soil

samples with toluene and CMC and determined the reducing sugar content

after incubation. C, activity was detectable in 11 samples while three

samples showed no C, activity. C, activity was not examined. As mentioned above, Drozdowicz (1971) found C, activity in extracts from soils

previously composted with cellulose powder. These extracts, however, exhibited no C, activity. In addition, both C, and C, were usually lacking

in extracts from noncomposted soils. Dantas and Drozdowicz (1972) incubated soil samples with toluene and with or without cellulose powder.

The differences between the amounts of reducing sugars in cellulose-treated

and untreated samples were negligible.

Further detailed studies are needed for a better understanding of

cellulase accumulation in soil.



8. Hydrolysis of Lichenin

Toluene-treated samples of nine out of ten soils examined were able

to hydrolyze lichenin extracted from Cetraria islandica. This observation

indicates the presence of lichenase in most of the soils examined (Kiss

et al., 1962b).



9. Hydrolysis of Laminarin and Fungal Cell Wall p-1,3-Glucan

This problem was studied by Jones and Webley (1968). Fungal cell

walls containing or lacking p-1,3-glucan were incorporated in a kaolinite

paste molded into aggregates that were subsequently incubated on soil. The

developing microorganisms produced p-1,3-glucanase in the aggregates

prepared from cell walls rich in p-1,3-glucan. Glucanase activity in aggregates was assayed in the presence of toluene; laminarin was used as substrate. These experiments were not conducted with the aim to demonstrate

directly the occurrence of p-lY3-glucanasein the soil. It seems probable

that p-1,3-glucanase is produced in soil in localized zones near fungal mate-



ENZYMES ACCUMULATED IN SOIL



33



rial which is being colonized by lytic microorganisms, and p-1,3-glucanase

accumulation should take place in these zones.

10. Hydrolysis of Inulin



Hydrolysis of inulin was found in toluene-treated soil and peat samples

(Hoffmann, 1959; Kiss and PCterfi, 1961a). Control mixtures without

toluene were not prepared. Nevertheless, inulin hydrolysis in the presence

of toluene indicates the occurrence of inulase as an accumulated enzyme

in soil and peat.



1 1 . Hydrolysis of Xylan

Hydrolysis of xylan takes place in toluene-treated soil samples owing

to the occurrence of xylanase in the soil (Sgrensen, 1955). Xylanase

activity measured in ./-irradiated samples was 15-25% lower than that

measured in the toluene-treated samples, indicating that the samples contained accumulated xylanase (SGrensen, 1969).

12. Hydrolysis of Pectin

Hoffmann ( 1959) and Kaiser and Monzon de Asconegui (1971 ) have

demonstrated that hydrolysis of pectin takes place in mixtures consisting

of soil, toluene, buffer, and pectin solutions. Although no comparison was

made between pectinolysis in the presence and in the absence of toluene,

it is clear that the tested soils contained some quantity of pectinolytic enzymes. Monzon de Asconegui and Kaiser (1972) found a parallelism between the activity of pectinolytic enzymes and the development of Azotobacter chroococcum in soil samples amended with pectin. The bacterium

multiplies in the presence of certain degradation products of pectin, such

as galacturonic acid and galactose, which are released by soil pectinolytic

enzymes.

13. Synthesis and Hydrolysis of Levan

Synthesis of levan takes place in reaction mixtures prepared from soil,

toluene, and sucrose solution during a few days of incubation. This was

interpreted as evidence of the occurrence of levansucrase in soil (Kiss,

1961) . Soils also contain levanase: treatment of soil samples with toluene

and a levan solution and incubation of these mixtures result in hydrolytic

cleavage of levan (Kiss et al., 1965). Levan synthesis is more intense in

the absence than in the presence of toluene (Fig. 2). This means that the

accumulated levansucrase plays a less important role than the levansucrase

produced by the proliferating microorganisms (Kiss et al., 1962a, 1972).

Similarly, levanolysis in soils not treated with toluene is due primarily to

the levanase of proliferating microorganisms rather than to the accumulated



34



s.



KISS, M.



DRAGAN-BULARDA,



AND D.



RXDULESCU



enzyme (Kiss ef al., 1965; Drigan-Bularda and Kiss, 1972b). In both the

presence and the absence of toluene, levan synthesis predominates over

levanolysis (Kiss and Dragan-Bularda, 1968a, 1970a). Levansucrase and

levanase activities in soil samples 7-irradiated at minimum sterilizing

dosage did not suffer any changes in comparison with the activities of nonirradiated but toluene-treated samples. At heavier dosages of 7-rays some

reductions occurred in both enzyme activities (M. DrBgan-Bularda and

S. Kiss, unpublished data).



14. Synthesis and Hydrolysis of Dextran

Besides levan, dextran is also synthesized in reaction mixtures consisting

of soil, toluene, and sucrose solution and incubated for several days. This

indicates the presence of dextransucrase in soil (DrBgan-Bularda and

Kiss, 1 9 7 2 ~ )Dextransucrase

.

activities in presence and absence of toluene

have not yet been compared. Soils treated with toluene and dextran solution are able to hydrolyze the polysaccharide during incubation. Glucose

is the final hydrolysis product. From these observations one can deduce

that soils contain some quantity of accumulated dextranase (DriganBularda and Kiss, 1972a). In the absence of toluene the proliferating

microorganisms metabolize a large portion of the glucose released from

dextran through the action of the accumulated dextranase and the dextranase recently produced. Toluene prevents utilization of glucose (DriganBularda and Kiss, 1972b) (Fig, 3). Soil samples treated with y-rays at



-t



tt



FIG.3. Decomposition of dextran in soil in the absence (-t) or in the presence

(+t) of toluene. ( 1 ) Dextran solution; (2-8) different soils plus dextran; (9) glucose

solution.



ENZYMES ACCUMULATED IN SOIL



35



minimum sterilizing dose or with toluene showed the same value of dextranase activity. But heavier radiation doses caused a diminution of enzyme

activity (M. DrBgan-Bularda and S. Kiss, unpublished data).



15. Hydrolysis of Native Soil Carbohydrates

In samples of some soils treated with toluene, incubated at 35OC for

2-10 days and then submitted to paper chromatographic analysis, a spot

of glucose appeared. In nonincubated samples and in the heat-sterilized

and incubated soils glucose was not present in detectable amounts. It has

been deduced that glucose was released from native soil carbohydrates by

the action of soil carbohydrases (Kiss and Ptterfi, 1961b; Kiss et al.,

1962b). The reducing sugars present in the extracts of soils incubated with

toluene but without added carbohydrates were produced in part by enzymatic breakdown of soil organic matter (Ross, 1965a).

16. Hydrolysis of Organic Acid Esters, Including Fatty Acid Esters

Soil samples treated with toluene and ethyl butyrate or phenyl acetate

catalyzed the hydrolysis of these esters (Haig, 1955). For the determination of lipase activity in four peats and two muds, Pokorni (1964) used

tributyrin as substrate and titrated the butyric acid liberated from tributyrin

during the 72-hour incubation. Lipase activity, measured in the absence

and in the presence ot toluene, showed only a 15% difference. Consequently, lipase activity in the first 72 hours should be attributed mainly

to the accumulated lipase. Similarly, Pancholy and Lynd (1972, 1973)

found lipase activity in extracts of a loamy sand. Nonfluorescent 4-methylumbelliferone butyrate was used as a substrate which was cleaved by the

lipase to butyric acid and a highly fluorescent compound, 4-methylumbelliferone.

Getzin and Rosefield (1968, 1971 ) have demonstrated that a carboxylesterase takes part in the degradation of the insecticide malathion. The

enzyme which catalyzes the hydrolysis of malathion to its monocarboxylic

acid was extracted from nonirradiated and 7-irradiation sterilized samples

of a clay loam and partially purified. Four to five times more activity was

recovered from nonirradiated soil than from irradiated soil. The enzyme

preparation contained 35 % protein. When this preparation was applied

to two soils, its activity was detected for the duration of the experimental

period (8 weeks). Existence of the malathion esterase as a stable, cell-free

soil enzyme was postulated from evidence based on the persistence and

adsorptive characteristics of the partially purified enzyme in soil. Satyanarayana and Getzin (1973) reported on further purification of the malathion esterase extracted from soil. The purified esterase was resistant to

enzymatic proteolysis. Its digestion with hyaluronidase increased its activity



36



S. KISS, M. DRhGAN-BULARDA, AND D. RXDULESCU



but decreased its stability. This esterase is a carbohydrate-protein complex,

which may account for its stability and persistence in soil as a cell-free

enzyme. Amino acids constituted 65% of the purified complex.

Malathion is susceptible also to chemical degradation due to both alkaline hydrolysis and adsorption by the soil particles. But its microbial

breakdown predominated over its chemical degradation in each of the

three soils studied by Walker and Stojanovic (1973).



17. Oxidation of Glucose

Ross ( 1968) found slight amounts of gluconic and 2-ketogluconic acids

in reaction mixtures prepared from soils and glucose solution when incubated for a maximum of 16 hours at 37OC. In reaction mixtures containing

toluene, the acids were also detectable even after 24-28 hours of incubation

and their amounts were larger than in the absence of toluene. These results

suggest the occurrence of glucose oxidase and gluconate dehydrogenase

as accumulated enzymes in soil. Utilization of glucose, however, by glucose

oxidase and gluconate dehydrogenase is of minor importance. Thus, in mixtures containing toluene and about 150 pmoles of glucose per gram of

dry soil incubated at 37OC for 24 hours, only 0.8% of the glucose was

oxidized.



18. Oxidation of Ascorbic Acid

Galstyan and Marukyan ( 1973) treated heat-sterilized and unheated soil

samples with ascorbic acid and incubated them at 3OoC for 1 hour. Oxidation of ascorbic acid to dehydroascorbic acid took place in each sample

but at a higher rate, in general, in the unheated than in the sterilized soil.

These observations were interpreted as evidence of the occurrence in soil

of both ascorbate oxidase and ascorbic acid-oxidizing thermostable, inorganic catalysts. In some soils the enzyme predominates over inorganic catalysts in oxidation of ascorbic acid, and in others the oxidation is due primarily or exclusively to inorganic catalysts.

19. Oxidation of Phenols

According to Galstyan (1958), extracts prepared from soils contained

heat-labile factors capable of oxidizing pyrogallol and producing purpurogallin in easily detectable amounts during incubation (30 minutes). The

oxidation took place in the presence of H,O, (peroxidase activity) or 0,

(polyphenol oxidase activity). The soil extracts can be replaced by intact

soils (Galstyan and Tatevosyan, 1964; Shatsman and Kalikina, 1972;

Galstyan, 1974). Kozlov ( 1964) used catechol to detect peroxidase and

polyphenol oxidase activities in soils. Incubation lasted 2 minutes. Kuprevich and Shcherbakova ( 1965, 1966, 1971 ) assayed polyphenol oxidase



37



ENZYMES ACCUMULATED IN SOIL



activity in soil samples treated with pyrogallol, catechol, hydroquinone,

tyrosine, or phenylalanine and incubated for 1 hour. The highest rate of

0, uptake was noted upon addition of pyrogallol and the lowest upon addition of phenylalanine (the only nonphenolic compound studied). Ross and

McNeilly ( 1973) demonstrated the presence of polyphenol-oxidizing enzymes in litter and soil under hard beech (Nothofagus truncata) forest,

by using catechol and phloroglucinol as substrates and measuring the 0,

uptake for 90-1 20 minutes. Resorcinol-oxidizing activity was not detected.

Mayaudon et al. (1973a) found that neutral, filter-sterilized (cell-free)

extracts from fresh samples of forest, meadow, and cultivated soils decarboxylated DL-3,4-dihydroxyphenylalanine ( DL-DOPA), DL-tyrosine, DLtryptophan, and DL-phenylalanine with relative decarboxylation rates of

100, 30, 10, and 0, respectively. Each amino acid was 14C-labeled at carbon atom 1. In order to obtain more information about this decarboxylation, Mayaudon et al. (1973b) submitted the soil extracts to a purification

procedure for removing the humic materials, The purified preparation had

the properties of a- and p-diphenol oxidases. Thus, it oxidized D-catechin,

p-cresol, catechol, DL-DOPA, and p-quinol with relative 0, absorption

rates of 298, 251, 156, 100, and 20, respectively. Decarboxylation of the

amino acids mentioned above was preceded by their enzymatic oxidation.

The authors suggested the following pathway of DOPA degradation:

DOPA



-



>SO2



$402



phenylalanine-3,4-quinone

2-carboxy-2,3-dihydroindole-5,6-quinone

(dopachrome) +

COz



-



+ 5.6-dihydroxyindole YAoz indole-5,6-quinone



+



a blue-violet product conventionally termed melanin



20. Decomposition of Diethylstilbestrol



Diethylstilbestrol (DES) is a very active synthetic estrogen. It is administered to young animals to increase their growth rate. DES is excreted

with the feces and urine. Thus, the hormone reaches the soil either directly

in pastures or through the use of stable manure. If DES is not rapidly

decomposed in the soil, this may result in its uptake by plants, which can

thus present a hazard to man and animals. For the study of the DES decomposition 'T-labeled DES was used in nonsterilized, y-radiation-sterilized, and autoclaved soil samples, and during the incubation (4 and 60

days) ' T O , was assayed. It has been found that 14C0, was produced not

only in the nonsterile soil, but also in the 7-sterilized soil. From the observations made with the 7-sterilized soil, it is evident that soil contains DESdecomposing enzyme(s) . Contribution of the accumulated enzyme( s) to

'TO, production from DES during the 60 days of incubation represents

two-thirds of the total 14C0, evolution in nonsterile soil (Gregers-Hansen,



38



s.



KISS, M.



DRAGAN-BULARDA,



AND D.



RXDULESCU



1964). The nature of the DES-decomposing soil enzyme(s) was not

studied.



21. Decomposition of Sodium Dioctylsulfosuccinate

This compound is a synthetic anionic detergent. Rotini (1959a) and

Rotini and Galoppini (1967) added the detergent to heat-sterilized,

toluene-treated, and untreated samples of a garden soil. The mixtures were

incubated at 25OC for 1-16 days. Detergent concentration decreased to

a lesser extent in the sterilized than in the toluene-treated samples. The

highest decrease occurred in the untreated soil. These observations indicate

that decomposition of the dioctylsulfosuccinate added to soil is due not

only to proliferating microorganisms, but also to accumulated soil enzyme( s) and to thermostable, inorganic catalyst(s) . Products and pathway

of decomposition, and the nature of the detergent-decomposing soil enzyme(s) were not studied.



B. NITROGEN

CYCLE

I . Hydrolysis of Urea

Rotini ( 1 9 3 5 ~ )compared urease activity in soil samples treated and

not treated with toluene and observed that toluene did not diminish, but

on the contrary increased, urease activity in soil. Similar results were obtained by Tabatabai and Bremner (1972). McLaren et al. (1957) worked

with sterilized and native, nonsterile soil samples. Sterilization was carried

out by either autoclaving or irradiation with an electron beam. Sterilization

resulted in destruction of all microorganisms including bacterial spores.

After sterilization, the soil samples were treated with a sterile urea solution,

then incubated in the absence of toluene and analyzed to determine urease

activity. In the autoclaved soil ureolysis did not take place owing to heat

inactivation of the enzymes. The electron-sterilized soil retained its urease

activity. In addition, in irradiated soil ureolysis was faster than in native

soil. Microbial proliferation occurred in the absence of toluene and, concomitantly, urease could be synthesized, but the amount of synthesized

urease was negligible in comparison with the amount of accumulated

urease.

Increase of urease activity following treatment with toluene or electron

beam can be attributed to an increase of permeability for urea and reaction

products in the viable microbial cells, which became nonproliferating in

the presence of toluene, to the increase of permeability in radiation-killed

cells, and also to the lytic effect of toluene on some microorganisms and

to the autolysis of the radiation-killed cells. Urease as an endoenzyme will



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II. Role of Accumulated Soil Enzymes in the Initial Phases of the Decomposition of Organic Residues and of the Transformation of Some Compounds

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