Hydrolase activities during and after the chloroform fumigation of soil as affected by protease activity

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Abstract

CHCl3 fumigation was used to induce changes in hydrolase activities of a Vertisol under different management (woodland, grassland and arable soils). Measurements were made during the standard 24 h CHCl3 fumigation period used for biomass measurement and after incubation following fumigation. Fumigation of soil was very effective in killing microorganisms as shown by the marked decrease in the ATP content after 4 h of CHCl3 exposure. However, it did not change or slightly decreased β-glucosidase, acid phosphomonoesterase, alkaline phosphomonoesterase and protease as measured by hydrolysis of N-benzoylargininamide, markedly decreased urease activity and increased arylsulphatase activity.

CHCl3 fumigation of soils for 24 h in the presence of several protease inhibitors decreased N-benzoylargininamide hydrolising activities by more than 50% and increased urease and phosphomonoesterase activities, demonstrating that enzymes released during cell lyses can undergo to proteolysis during the CHCl3 fumigation.

During the incubation following fumigation, respiration increased following the typical trend. ATP content also increased in all soils because of the growth of the small microbial population surviving the fumigation. During the incubation following fumigation, β-glucosidase and phosphomonoesterase activities recovered to values not significantly different from those of unfumigated and incubated soil. Arylsulphatase activity decreased in all fumigated soils reaching values not significantly different from those of the unfumigated soils after 24 h. Benzoylargininamide hydrolyzing activities showed only minor changes during the incubation of unfumigated and fumigated soils. Urease activity decreased to low levels in the woodland soil and was not measurable in the grassland and arable soils. N metabolites, derived from the microbial NH4+ immobilization during the incubation following fumigation, may have inhibited the urease synthesis in microbial cells.

Introduction

In the soil environment, active enzymes exist within the microbial cells, adsorbed onto soil colloids, free in soil solution and associated with cell debris (Burns, 1982). Active enzymes, present in the soil solution (free enzymes) or in cell debris, are short-lived and their contribution to the overall enzyme activity is considered to be negligible (Burns, 1982). The measurement of the extra- and intracellular enzyme activity in soil is needed for a better understanding of the response of enzyme activities to changes in agricultural practices, environmental conditions and presence of toxicants (Nannipieri, 1994). Intracellular enzyme activity may be related to microbial activity, while the activity of the soil-stabilised enzymes, protected from thermal and chemical denaturation (Perez-Mateos and Rad, 1989, Speir and Ross, 1990, Nannipieri, 1994) and proteolysis (Nannipieri, 1994), may be insensitive to such changes.

Chemical and physical microbial inhibitors have been used for measuring the contribution of the intracellular enzyme activity to overall soil enzyme activity. These approaches have produced conflicting results (Kiss et al., 1975, Burns, 1978, Ladd, 1985, Frankenberger and Johanson, 1986), possibly due to non-target effects, adsorption onto soil colloids and microbial degradation (Landi et al., 1993, Nannipieri, 1994, Nannipieri et al., 2001). In addition, toluene, a bacteriostatic agent, has been found to increase membrane permeability and thus intracellular enzyme activity (Skujins 1978).

A different approach was followed by McLaren and Pukite (1973) who correlated the number of ureolytic microorganisms to urease activity after microbial growth induced by carbon and nitrogen sources added to soils. Both microbial numbers and urease activity values were those reported by Paulson and Kurtz (1969). The significant correlation plot showed a positive intercept at the zero microbial biomass value, and the intercept value was assumed to be the extracellular enzyme activity. The same approach was followed by Nannipieri et al. (1996) to measure the extracellular phosphomonoesterase activity. They measured ATP content, which is a better measurement of microbial biomass than the plate count, which only reveals 1–10% of the total soil microbial population (Torsvik et al., 1996). Advantages and limitations of this approach, called the Physiological Approach, have been discussed by Nannipieri et al. (2001).

Recently, the CHCl3 fumigation method used for estimating microbial biomass C, has been adapted to assess the intracellular urease and arylsulphatase activities, but not the intracellular activities of 10 other enzymes involved in the C, N, P and S cycling (Klose and Tabatabai, 1999a,Klose and Tabatabai, 1999b, Klose et al., 1999). After the fumigation treatment, both the arylsulphatase and urease activities were significantly higher (more than 50%) than those of the control soils. It was hypothesized that the increased activity was due to the release and measurement of the intracellular enzyme activities, whereas the activity of the unfumigated soils was assumed to be due to the extracellular and soil-stabilised enzymes. However, no data were reported on the behaviour of the urease and arylsulphatase activities during the 24-h fumigation period and no microbial parameters were monitored to determine the efficacy of CHCl3 fumigation in lysing soil microorganisms.

The aims of this research were to study:

  • 1.

    the effect of the CHCl3 fumigation on β-glucosidase, arylsulphatase, urease, protease, acid and alkaline phosphomonoesterase activities in order to assess the validity of CHCl3 fumigation for determining intracellular activities of these enzymes;

  • 2.

    the effects of protease activity released by cell lysis on the persistence of hydrolases released during the 24-h fumigation period;

  • 3.

    the trend of these enzyme activities during the early phases following CHCl3 fumigation and their relation to microbial activity as determined by the CO2 evolution.

The ATP content was used as an index of microbial biomass not only to assess the efficiency of CHCl3 fumigation in lysing microbial cells, but also to follow the growth of surviving microorganisms in the incubation following fumigation.

The three soils differ in their microbial biomass, organic matter contents and enzyme activities (Table 1). Other soil properties (pH, soil texture and clay mineralogy) are similar so that any differences in cell lysis by CHCl3 fumigation among the soils should be due to differences in organic matter content and microbial biomass. It has been reported that the efficiency of the CHCl3 fumigation can be affected by several soil properties (Badalucco et al., 1997).

Section snippets

Soils

The study was conducted on a calcareous soil, Vertic Xerochrept (USDA, 1992) of the Vicarello experimental area located in Tuscany (Central Italy), controlled by the Institute for Soil Study and Protection (Ministry of the Agricultural and Forest Policies). Soil samples were collected under three different permanent management regimes (wheat, grassland and woodland) and they differ only in organic C, microbial biomass content and hydrolase activities (Table 1). Soils were surface (0–10 cm for

The behaviour of ATP and CO2

The ATP content of soils (Fig. 1) decreased in all soils to very low values after 4 h of CHCl3 fumigation, confirming that this treatment is effective in killing microorganisms. The decrease in the ATP content was more rapid than the decline observed by Arnebrant and Schnurer (1990). It has been hypothesised that ATP synthesis is inhibited whereas ATPases remain active during the CHCl3 fumigation (Ciardi et al., 1993). Indeed, both acid and alkaline phosphomonoesterases were active during the

Conclusions

The approach proposed by Klose and Tabatabai (1999a,b) to determine the intracellular enzyme activity after the CHCl3 fumigation of soils presents the problem that hydrolases released after cell lysis are partially degraded by active proteases. Therefore, a complete inhibition of active proteases is required to determine the intracellular enzyme activities using the fumigation method. There may be other drawbacks. For example, denaturation of released enzymes may occur as a result of

Acknowledgements

The authors wish to thank Drs Pagliai, Bazzoffi and Pellegrino of the Institute for Soil Study and Protection of the Minister of the Agricultural and Forest Policy of Florence, for useful information on the soil characteristics of the experimental area where soils were sampled. We wish to thank F. Filindassi for technical assistance.

References (37)

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