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Revista de Microbiologia

versão impressa ISSN 0001-3714

Rev. Microbiol. v. 29 n. 3 São Paulo Set. 1998 



Francisco Cleber Sousa Vieira; Ely Nahas *
Departamento de Microbiologia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, SP, Brasil

Submitted: October 19, 1997; Returned to authors for corrections: February 06, 1998;
Approved: July 23, 1998






The enzymatic activity of soil samples stored at temperatures of 5 to -12oC and at room temperature for 0-32 weeks was determined. While alkaline phosphatase and dehydrogenase activity was decreased compared to control in samples stored at low temperatures, acid phosphatase activity showed no significant change.

Key words: Phosphatase, dehydrogenase, soil storage



The enzyme activities have been of interest to the study of many topics in soil microbiology and biochemistry (1). The data of enzyme assays have been connected to many characteristics of soil, including microbial growth and activity as the mineralization of organic P esters (11). Microbial activity can be analyzed on the basis of some parameters that reflect the behavior of soil microorganisms, such as enzyme activity. The form of soil storage for later analysis has also been a source of concern for investigators. Skujins (7) discussed the effect of different forms of soil storage for the determination of enzyme activity and concluded that no rules can be established and that conditions differ for each soil and enzyme to be analyzed. The objective of the present study was to evaluate the effect of soil sample storage at temperatures of -12oC, 5oC and at room temperature (air dried samples) on enzyme activity.

Soil samples from an experimental area of red latossol were collected from 0-20cm depth. The samples were pooled and spread on a tray to remove sticks, leaves and roots. The soil was then air-dried in an oven at 45-50oC for 5 days and sifted through a 2 mm mesh sieve. The results of the chemical and granulometric analyses of the soil are presented in Table 1.


Table 1. Some characteristics of the soil used.

0002i01.gif (10382 bytes)


Soil was stored in field moist condition (70% WHC) for 7 days after the storage period to stabilize the biological and biochemical characteristics. Portions of 500 g were placed in non-sterilized plastic bags closed with string. Samples were used for (a) analysis within a maximum time of 4 h after soil fractionation, or stored at temperatures of (b) 5oC, (c) -12oC, or (d) room temperature. Four samples were removed at different times for the corresponding analyses. Frozen samples were thawed at room temperature for 3 h.

Dehydrogenase activity was determined by the method of Casida (2), using triphenyltetrazolium chloride (TTC) as oxidizing agent. The triphenylformazan formed was extracted with methanol and then measured on a spectrophotometer at a wavelength of 485 nm. One unit of enzyme activity is defined as the amount of enzyme that produces 1.0 µg triphenylformazan per min. Acid and alkaline phosphatase activity was determined by the method of Tabatabai (9) modified by Nahas et al. (4), using p-nitrophenylphosphate as substrate. One unit of enzymatic activity is defined as the amount of enzyme that produces 1.0 µg p-nitrophenol per min. Specific activity is reported as µg triphenylformazan (dehydrogenase) or p-nitrophenol (phosphatases) released per 24 h or 1 h, respectively, per 1 g dry soil.

The data are presented as the means of duplicate analysis of four samples. The study was carried out in a completely randomized design and the data were analyzed statistically using the General Linear Model of the Statistical Analysis System (SAS) software. When a significant F value was detected, the means were compared to control values by the Tukey test, with the level of significance set at 5%.

Data concerning enzymatic activity are summarized in Table 2. Storage of these soil samples at low temperatures did not affect acid phosphatase activity except during the 20th week (5oC) and during the 16th and 20th weeks (-12oC). All the activities of air-dried samples were significantly lower than control and than the values obtained for samples stored at low temperatures. In contrast to acid phosphatase, alkaline phosphatase activity was significantly reduced in stored samples compared to the control. Dehydrogenase activity values were significantly lower than the control, showing that the storage system used decreased dehydrogenase activity (Table 2). Furthermore the activity of samples stored at room temperature was lower than that observed under the other conditions.


Table 2. Effect of temperature and period of storage on enzymatic activities.

0002i02.gif (37755 bytes)

(2) = Tukey, p<0,05
** = significant (p<0,01)
NF = p-nitrophenol
TF = triphenylformazan
(1)  – see text


According to Skujins (7), the enzymatic activity of stored soil samples depends on the enzyme considered. McGarity and Myers (3) obtained significant fluctuations in urease activity over time of storage even during a period as short as 4 days, with no satisfactory explanation. Variations in the responses of enzyme activity were also obtained in this study, probably due to the heterogeneity of the soil used. The enzymes tested in the present study behaved in different manners: while alkaline phosphatase and dehydrogenase activity was decreased compared to control in samples stored at low temperatures, acid phosphatase activity showed no significant change. Except for a few samples, Ross and McNeilly (6) observed increased dehydrogenase activity in samples stored at 4oC and decreased activity at -20oC, with little change at 4oC (5). In contrast, arylsulfatase activity decreased in samples stored at 5oC or at room temperature and was unchanged for as long as 3 months in samples stored at -10oC (10).

Stevenson (8) showed that viable colony counts were reduced in air-dried samples. This result may possibly explain the decreased activity of all enzymes analyzed in the present study, confirming literature data (5, 6, 12). However, other factors may also have an effect since arylsulfatase activity increased in air-dried samples (10).



This work was supported by a grant nº 92/4635-2 from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (EN) and from FAPESP (FCSV).




Atividade microbiana de amostras de latossolo vermelho-escuro armazenadas em diferentes temperaturas

Foi determinada a atividade enzimática de amostras de solo armazenadas às temperaturas de 5, -12oC e ambiente durante 0-32 semanas. A atividade da fosfatase alcalina e da desidrogenase diminuiu nas amostras armazenadas a baixas temperaturas, enquanto que a atividade da fosfatase ácida não variou em relação ao controle.

Palavras-chave: Fosfatase, desidrogenase, armazenagem de solo.




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* Corresponding author. Mailing address: Departamento de Microbiologia, FCAV/UNESP. Rod. Carlos Tonanni, Km 5, CEP 14870-000 Jaboticabal, SP, Brasil; Fax: (+5516)322-4275; E-mail:

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