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Soil microbial activity as a biomarker of degradation and

remediation processes

J.A. Pascual*, C. Garcia, T. Hernandez, J.L. Moreno, M. Ros

Department of Soil and Water Conservation and Organic Waste Management, CEBAS-CSIC, P.O. Box 4195, E-30080, Murcia, Spain

Accepted 4 May 2000

Abstract

Several organic matter fractions together with biological and biochemical parameters were measured in a range of intensively farmed soils in SE Spanish Mediterranean region, which had been abandoned (i.e. not used in agriculture) for different periods of time. These soils were compared with adjacent natural soils that had never been used for agriculture. There was a general decline of total organic carbon (TOC), extractable humic substances, water-soluble carbon (WSC) and carbohydrates, microbial biomass and respiration with the time elapsed since abandonment. There was also a decline in plant cover in the abandoned soils. When a degraded soil was amended with municipal solid waste at rates of 6.5 and 26 kg m22as a potential means of remediation, TOC, humic substances, WSC, microbial biomass and respiration rates signi®cantly increased but only at the higher rate of amendment. Plant cover was signi®cantly enhanced by both rates of the amendments and was still present 10 years after the amendment. These data con®rm that agricultural soil abandonment leads to soil degradation and that the addition of urban waste could be a suitable technique with which to restore their quality.q_{2000 Elsevier Science Ltd. All rights reserved.} Keywords: Soil remediation; Organic matter; Dehydrogenase; Hydrolases

1. Introduction

Soil is an important natural resource that needs to be preserved and, if possible, its quality and productive capa-city improved. Doran and Parkin (1994) de®ned soil quality as ªthe capacity to function within an ecosystem and sustain biological productivity, maintain environmental quality and promote plant, animal and human healthº. In natural condi-tions, soils tend towards maintaining an equilibrium between pedogenetic properties and the natural vegetation (Parr and Papendick, 1997).

Soil equilibrium can easily be disturbed, especially by human intervention (e.g. unsuitable agricultural practices). Furthermore, in the Mediterranean region of SE Spain inap-propriate agricultural practices are compounded by adverse environmental and climatic factors (LoÂpez BermuÂdez and Albaladejo, 1990). Soils from semi-arid regions are not resi-lient to the effects of inappropriate land-use and manage-ment, which leads to permanent degradation and loss of productivity. A key factor in degradation of these soils is the loss of natural plant cover, allowing soil water erosion and salinisation processes to occur. This further aggravates

the effects of the semiarid conditions (Garcia et al., 1996) and leads to a loss of soil quality and fertility and the subse-quent abandonment of the land for crop production purposes.

One method of reversing the degradation that is taking abandoned soil back into agricultural production and improving the quality of soils with low organic matter content, involves the addition of municipal solid wastes (Pascual et al., 1998). These materials are rich in carbon and energy sources that increase the soil microbial popula-tion and its activities, and thus reactivate biogeochemical nutrient cycles (Pascual et al., 1997). The organic wastes also increase the soil water-holding capacity, aggregation and improve nutrient status.

Many properties must be used to de®ne soil quality and, once these have been quanti®ed, the most suitable strategies for soil management can be undertaken. Chemical and physical soil parameters such as organic matter, nutrient status, run-off measurements or aggregate content have been used to measure soil quality (Parr and Papendick, 1997). However, these parameters change very slowly, and therefore many years are required to measure signi®cant changes. On the other hand, soil biological and biochemical properties are responsive to small changes that occur in soil, thereby providing immediate and accurate information on

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* Corresponding author. Fax:134-968-266613.

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changes in soil quality. This is because soil microbial activ-ity has a direct in¯uence in a ecosystem stabilactiv-ity and fertilactiv-ity (Smith and Papendick 1993). Microorganisms play a funda-mental role in establishing biogeochemical cycles and are involved in forming the structure of a soil (Harris and Birch, 1989).

In this paper, the changes in soil quality taking place in agricultural soils, in semiarid conditions at different times after abandonment were evaluated by comparison with natural soils exposed to the same climate but not subjected to intensive agriculture and subsequent aban-donment. We also report results obtained from a ®eld site abandoned 20 years previously and amended with the organic fraction of a municipal solid waste (MSW) 10 years previous to this study. To monitor soil quality, organic matter fractions (total organic carbon, humic substances, water-soluble carbon and carbohydrates), microbiological (microbial biomass C, basal respiration) and biochemical (dehydrogenase, phosphatase, b -gluco-sidase, urease and protease activity) properties were measured.

2. Materials and methods

2.1. Study of degraded, abandoned agricultural soils

Abandoned agricultural soils from the SE Spanish Medi-terranean region, within the province of Murcia were studied. In an area measuring approximately 4 km2, 12 sampling sites were chosen to cover intensively farmed agricultural soils abandoned for different lengths of time. The soils were grouped according to the time elapsed since abandonment (,10, 10±20 and.20 years). The data referring to the dates when the ®elds were abandoned were provided by the landowners. The areas were sampled in May of 1997. All of the soils were clay loams and they were all exposed to the same semi-arid climate (rainfall,250 mm yr21; annual average temperature 178C).

To ascertain how the soils studied differed from others from the same area that had not been subjected to human intervention, a control site supporting natural vegetation typical from Mediterranean soils (principally Quercus rotundifolia) was included.

Three samples were taken from each of the sampling sites: each sample consisted of eight subsamples taken from the top 15 cm of soil. The subsamples were mixed, homogenised, sieved (,2 mm) and stored at 48C until analysed. The main characteristics of the soils are shown in Table 1.

2.2. Long-term soil remediation after the addition of the organic fraction of a municipal solid waste

A soil from an area that had been abandoned for 20 years was amended with the organic fraction of a municipal solid waste (MSW) from Murcia (6.5 and 26 kg m22). The organic matter was incorporated into the top 15 cm using a rotovator. Three plots (one for each treatment and a control) were set up on an east-facing hill slope (10% gradient) with a 40-m2 size. Soil was sampled from each plot 10 years after the amendment. For sampling, eight subsamples were taken randomly from the top 15 cm of soil, mixed and sieved (,2 mm) before analysis. The main characteristics of the soil and MSW are shown in Table 2.

2.2.1. Analytical parameters

The total organic C (TOC) content was determined by oxidation with K2Cr2O7 in a concentrated H2SO4medium

and measurement of the excess dichromate using (NH4)2Fe(SO4)2 (Yeomans and Bremner, 1989). Humic

substances extracted with pH 9.8, 0.1 M sodium pyropho-sphate (solid±liquid ratio 1:10) and water-soluble carbon extracted with distilled water (1:5 solid±liquid ratio) were determined by oxidation with K2Cr2O7and measurement of

absorbance at 590 nm (Sims and Haby, 1971). Soluble carbohydrates from the water extract were determined by the method of Brink et al. (1960).

J.A. Pascual et al. / Soil Biology & Biochemistry 32 (2000) 1877±1883

Table 1

Characteristics of abandoned agricultural soils and natural soils. (WHC: Water holding capacity, EC: electrical conductivity, TOC: total organic carbon, means are indicated^standard deviation)

Time elapsed since abandonment agricultural

,10 years 10±20 years .20 years Natural soil

Texture type Clay loam Clay loam Clay loam Clay loam WHC (%) 37.2^2.1 37.5^1.5 34.6^3.8 46.0^3.0 PH (1:10) 8.58^0.2 8.22^0.31 7.5^0.1 7.76^0.9 EC (dS m21) 0.69^0.05 0.79^0.51 0.83^0.10 0.25^0.10 TOC (g kg21) 11.01^2.33 6.22^0.72 5.30^0.67 20.2^5.81

Table 2

Characteristics of the soil and the municipal solid waste use in the soil remediation experiment

Soil Municipal solid waste

pH (H2O) 7.7 6.8

Electrical conductivity (S m21) 0.78 5.20 Total organic carbon (g kg21) 5.41 300.1 Humic substances (g kg21₎ _1.20 _32.3 Total nitrogen (g kg21₎ _0.41 _13.1 Total phosphorus (g kg21₎ _0.58 _5.6 Total potassium (g kg21₎ _8.10 _3.2

Water holding capacity (%) 34.9 ±

Texture type Clay loam ±

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Microbial biomass C was determined by fumigation± extraction method (Vance et al., 1987), after oxidation with K2Cr2O7, the C content was measured at 590 nm

(Sims and Haby, 1971). Soil respiration was determined using 50 g dry soil, moistened to 65% of its water-holding capacity, placed in hermetically sealed ¯asks and incubated for 30 d at 288C. The CO2 emitted was periodically

collected in 10 ml 0.1 M NaOH and titrated with 0.1 M HCl (Parr and Smith, 1969).

Dehydrogenase activity was determined by the reduction of 2-p-iodo-3-nitrophenyl-5-phenyl tetrazolium chloride to iodonitrophenylformazan by the method of Skujins (1976) as modi®ed by Garcia et al. (1993). Urease and protease (as

N-a-benzoyl-l-argininamide (BAA) protease) activity were

measured following the method of Nannipieri et al. (1980).

Phosphatase and b-glucosidase activities were determined using p-nitrophenyl phosphate disodium (0.115 M) andp -nitrophenyl glucopyranoside (0.05 M), respectively, as substrates (Tabatabai, 1982).

Biomass C, CO2-C emission, dehydrogenase and

hydro-lases activities were determined immediately after sampling, while the other analysis were carried out after storage at 48C for less than 30 d. All assays were carried out by triplicate and data were analysed statistically, using the Statgraph version 4.1 software program.

3. Results and discussion

3.1. Degraded, abandoned agricultural soils

Plant cover is an important soil quality factor (Brockway et al., 1998), mainly due to its contribution towards main-taining a stable biological population in soil by supplying carbon and energy sources from root exudates and plant remains (Balloni and Favilli, 1987). The percentage of plant cover was estimated by a grid-line intersect method. The plant cover supported by the abandoned soils were 5% (,10 years abandonment) and,2% (10±20 and.20 years abandonment). However, the natural soils showed 60% plant cover, supporting natural vegetation typical from the area, mainly Quercus rotundifolia. Natural plant re-estab-lishment could be expected after abandonment of agricul-tural management (Garcia et al., 1997) but it did not occur here due to the low level of organic matter, low microbial activity and extreme climatic conditions (i.e. very long dry periods).

Total organic carbon (TOC) of the degraded soils ranged from 4.40 to 12.90 g kg21(Fig. 1A), with many soils having values,10.00 g kg21. The TOC content of the abandoned soils was below that of the natural soil, since agriculture favours organic matter mineralisation (Tate, 1987), and this may result in dif®culties for plant establishment after soil abandonment. TOC decreased with the time, con®rming the continuing degradation of the soil after abandonment.

The humic carbon content of abandoned soils ranged from 0.67 to 2.33 g kg21, values signi®cantly lower than those of the natural soils (Fig. 1A). The arid climatic condi-tions to which these soils are exposed and the consequent slow rate of humi®cation could be the main factor contribut-ing to these low levels. As with TOC, the lowest values of humic C were in the soils abandoned for the longest times, whereas those abandoned less than 10 years ago had signif-icantly higher humic C contents.

The water-soluble fraction of soil organic matter is of special importance because it is the most degradable, acting as an immediate energy source to the microorganisms (Cook and Allan, 1992). The study of this fraction is also of interest in agricultural soils because it determines the activity of the soils (Janzen et al., 1992). The abandoned soils contained considerably lower levels of water-soluble carbon than did

Fig. 1. Total organic carbon (TOC), humic substance carbon (A), water soluble carbon (mg C kg21 _{soil) and water-soluble carbohydrates (mg} glucose kg21_{) (B) in natural and abandoned agricultural soils at different} times since abandonment. (Error bars denote standard deviation; least signi®cant difference at P#0:05; TOC1:20; humic

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the natural soils, which have retained their cover ofQuercus rotundifolia (Fig. 1B). The water-soluble carbon content declined with time after abandonment, due the continuous degradation of soil quality as consequence of the erosion

processes and the low rainfall (LoÂpez BermuÂdez and Alba-ladejo, 1990). In the abandoned soils, the water-soluble carbohydrates, which represent the most mineralisable frac-tion of the organic matter (De Luca and Keeney, 1993), also had considerably lower levels than in natural soils, but they showed no signi®cant difference with elapsed time (Fig. 1B).

Throughout this study, the time elapsed since soil aban-donment seems to be an important factor in¯uencing organic matter fractions. After intensive agriculture, soils are often exhausted (Tate, 1987), and if they are abandoned without any subsequent treatment, they may be subjected to erosion (Garcia et al., 1996), due to their low capacity to recover and to establish a natural plant cover.

Microbial biomass C can be considered to be a more sensitive indicator of soil quality than organic matter or TOC, since it responds more rapidly and to a greater extent to changes (e.g. degradation; Ross et al., 1982; Powlson et al., 1987). The microbial biomass C detected in the aban-doned soils varied greatly but it declined when agricultural soils were abandoned and decreased with time elapsed (Fig. 2A), presumably as a consequence of the loss of the capacity to protect soils against the erosion processes.

Basal respiration is a good indicator as soil microbial activity (Anderson, 1982) The basal respiration in all the abandoned soils showed signi®cantly lower values than in natural soils (Fig. 2B). The lowest values for this parameter were in the soils abandoned for the longest times.

Dehydrogenase activity has been proposed as a measure of microbial activity in soil (Garcia et al., 1993), although some authors have criticised this approach (Nannipieri et al., 1990; Beyer et al., 1992) because the enzyme is affected by numerous factors (soil type, pH, etc). Dehydrogenase activ-ity is involved in the initial breakdown of soil organic matter (Bolton et al., 1985). We found that the abandoned soils, which showed low values for other measures of microbial activity (e.g. biomass carbon content, basal respiration rate), also display the lowest dehydrogenase activity (Table 3). In general, the lowest values for dehydrogenase activity were in the soils abandoned for the longest time, whereas those abandoned less than 10 years had signi®cantlyP#0:05

higher contents. The decrease in activity with the passing time may be due to progressive erosion of the abandoned

Fig. 2. Microbial biomass carbon (A) and basal respiration (B) in natural and abandoned agricultural soils at different time since abandonment. (Error bars denote standard deviation; least signi®cant difference atP#

0:05;microbial biomass carbon56.0, basal respiration3.2).

Table 3

Dehydrogenase and hydrolase enzyme activities in natural soil and abandoned agricultural soils with different time elapsing since abandonment. (INTF: iodonitrotetrazolium formazane; BAA:N-a-benzoyl-l-argininamide; PNP:p-nitrophenol. Means are indicated^standard deviation; LSD: least signi®cant differences atP#0:05

Elapsed time Dehydrogenase (mg INTF g21)

Urease

(mmol NH3g21h21)

Protease BAA (mmol NH3g21h21)

Phosphatase (mmol PNP g21h21)

b-Glucosidase (mmol PNP g21h21)

,10 years 50.0^6.2 1.38^0.61 0.63^0.12 44.8^9.1 45.1^12.0

10±20 years 16.2^5.1 0.63^0.63 0.52^0.20 23.7^8.1 28.2^11.2

.20 years 16.8^4.7 0.75^0.23 0.34^0.12 30.9^8.2 21.5^10.1

Natural soil 61.2^4.6 1.40^0.35 1.60^0.46 127.0^22.0 105.1^20.1

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soils as a consequence of the low levels of plant cover (Brockway et al., 1998) and the low levels of organic matter (Pascual et al., 1999).

The study of different hydrolase enzyme activities is important since they indicate the potential of a soil to carry out speci®c biochemical reactions, and are important in maintaining soil fertility (Burns, 1982). Urease and protease (that hydrolysesN-a-benzoyl-l-argininamide) act

in the hydrolysis of organic to inorganic nitrogen, the former using urea-type substrates and the latter simple peptidic substrates. Phosphatases catalyse the hydrolysis of organic phosphorus compounds to phosphates. b-Glucosidase hydrolyseb-glucosides in soil or in decomposing plant resi-dues (Hayano and Tubaki, 1985). The overall levels of hydrolytic enzymes detected in the abandoned soils were low compared with the soil of the same area which have not suffered human intervention (Table 3). The existence of plant cover and the subsequent losses of carbon due to mineralisation is again shown to be the key to the enzymatic activities were reduced. Once again, the longest abandoned soil showed the lowest enzymatic activities.

3.2. Long-term soil remediation after the addition of the organic fraction of a municipal solid waste

As mentioned above, plant cover is an important factor in soil quality (Brockway et al., 1998), therefore it was measured 10 years after soil amendment with the organic fraction of a municipal solid waste (MSW). The percentage of plant cover on the MSW amended sites increased in the following order: control plot unamended soil (3% plant cover)_,plot treated with the low dose (25%)_,plot treated with the high dose (60%). The amendment with organic matter from MSW had been suf®cient to re-establish a considerable plant cover that could protect soils against erosion.

Ten years after the amendment, the TOC content in the amended soils was higher than in the unamended soil (two times with the low dose and four times with the high dose) (Fig. 3A). According to Pascual et al. (1997), approximately half of the organic matter added as MSW is mineralised in the ®rst 12 months, thus the increase in organic C after 10 years was mainly due to the presence of plant cover and the resulting root exudates and plant residues. Humic substances and water-soluble compounds also showed higher values in the amended soil than in the

Fig. 3. Total organic carbon (TOC) (A), humic substance carbon (B) and water-soluble carbon (C) in the amended and unamended soils. (Error bars denote standard deviation; least signi®cant difference atP#0:05;TOC

0:8;humic substance carbon140, water-soluble carbon30).

Table 4

Dehydrogenase and hydrolase enzymes activities of abandoned soil 10 years after organic amendment. (INTF: iodonitrotetrazolium formazane; BAA:N-a -benzoyl-l-argininamide; PNP:p-nitrophenol. Means are indicated^standard deviation; LSD: least signi®cant differences atP#0:05

Dehydrogenase (mg INTF g21)

Urease

(mmol NH3g21h21)

Protease BAA (mmol NH3g21h21)

Phosphatase (mmol PNP g21h21)

b-Glucosidase (mmol PNP g21h21)

Low dose 13^3.1 1.60^0.25 0.51^0.22 80.0^20.0 200.0^20.6

High dose 42^3.2 3.23^0.33 1.32^0.23 180.0^30.0 370.0^20.2

Control 8.5^2.2 1.00^0.26 0.35^0.22 40.0^15.0 25.0^12.1

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control (Fig. 3B and C). The addition of MSW to soil abandoned after intensive agricultural use could be a strategy to preserve and improve long-term soil quality, due to improvement in the soil structure and the devel-opment of plant cover.

Soil amendment with MSW gave rise to signi®cantly P#0:05higher microbial biomass C and basal

respira-tion values than found in the control soil (Fig. 4A). This improvement could also be attributed to the plant cover developed as consequence of the initial organic amendment. In short-term laboratory incubations without vegetation, microbial biomass C and basal respiration decreased with length of incubation time, because of the decline in the easily biodegradable carbon (Pascual et al., 1997). The addi-tion of MSW under ®eld condiaddi-tions stimulated the develop-ment of a plant cover, which acted as a carbon and nutrient source and which, in principle, maintained the levels of organic matter, microbial biomass and therefore basal respiration.

Ten years after the amendment, dehydrogenase activity in the amended soil was signi®cantlyP#0:05 higher than

that in the control soil (and signi®cantly higher at the higher than lower dose, Table 4). This con®rms that the active

microbial biomass is mainly supported by plant root exudates and residues through out this period.

Amendment with organic matter had a positive effect on the activity of the different hydrolases studied, probably due to the higher microbial biomass (Garcia et al., 1994; Pascual et al., 1998). This suggests that 10 years after the amendments, the biochemical cycles of N (urease and protease-BAA activity), P (phosphatase activity) and carbon (b-glucosidase activity) could have been reactivated, thus improving the fertility of the amended soil (Ladd, 1985).

The abandonment of Mediterranean soils after intensive agricultural practices causes a loss of soil quality and the longer the soils are left, the more degraded they become. The main reasons for this decline is probably a combination of the low levels of organic matter, nutrients and microbial activity. Addition of organic waste after abandoning agri-cultural use of these soils could be a good strategy to preserve and improve soil quality for future use. The organic amendments not only increase the organic matter, but also lead to an increase in natural vegetation capable of main-taining high microbial biomass. Knowledge of soil biologi-cal and biochemibiologi-cal status has helped in the diagnosis of the capacity for the soil to be regenerated.

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