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Published online 27 June 2007
Published in J Environ Qual 36:1137-1144 (2007)
DOI: 10.2134/jeq2006.0304
© 2007 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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Changes in Microbial Biomass Parameters of a Heavy Metal-Contaminated Calcareous Soil during a Field Remediation Experiment

R. Clementea, C. de la Fuentea, R. Moralb and M. P. Bernala,*

a Dep. of Soil and Water Conservation and Organic Waste Management, CEBAS-CSIC, Campus Universitario de Espinardo, PO Box 164, 30100 Espinardo, Murcia, Spain
b Dep. of Agrochemistry and Environment, Miguel Hernandez Univ., EPSO, Ctra Beniel Km 3.2, Orihuela (Alicante), Spain. R. Clemente, current address, School of Biological and Earth Sciences, Liverpool John Moores Univ., Byrom Street, Liverpool L3 3AF, UK

* Corresponding author (pbernal{at}cebas.csic.es)

Received for publication August 1, 2006.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil microbial biomass parameters give useful information about the restoration degree and quality of contaminated soils. These parameters were studied in a field experiment where the effect of two organic amendments on the bioavailability of heavy metals in an agricultural soil and on their accumulation in Beta vulgaris and Beta maritima was assessed. The soil was a calcareous Xeric Torriorthent and the total metal levels were (mg kg–1): 2706 Zn, 3235 Pb, and 39 Cu. The treatments were: fresh cow manure, olive husk, and inorganic fertilizer as a control. Two successive crops (B. vulgaris and B. maritima) were grown on the treated and untreated plots. The soil was sampled before each planting and after each harvest over a 15-mo period. Biomass C and N increased in all plots, especially in the organically amended ones. The ratio CO2–C/biomass C decreased in olive husk and manure-treated plots, in comparison with the control, and also during the experiment, suggesting a beneficial effect of the organic amendments. In olive husk-treated plots a significant increase in the ratio of biomass C/total organic carbon (TOC) with time was observed. This indicated a reduction of heavy metal stress on the microbial population. The amendments showed, in general, a beneficial effect on soil quality and fertility, while microbial biomass parameters were found to be useful indicators of the evolution of the remediation processes.

Abbreviations: DTPA, diethylenetriaminepentaacetic acid • OH, olive husk • OM, organic matter • TOC, total organic carbon


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
SOILS contaminated with heavy metals associated with mining activities usually lack a proper structure and aeration and have low fertility and organic matter (OM) contents that can result in a scarce microbial biomass in these soils (Clemente et al., 2006a), together with poor plant growth potential. Among the different soil remediation techniques, phytoremediation strategies, such as phytostabilization, based on the use of plants with agronomic practices, are being developed for heavy metal-contaminated soils (Salt et al., 1998), as an environmentally friendly technology. The use of organic amendments is a common practice in these reclamation procedures and soil organic matter has been of particular interest in studies of heavy metal retention in soils (Chen, 1996). However, organic materials not only improve soil fertility and increase plant production but also change heavy metal availability (Clemente et al., 2005, 2006a; Walker et al., 2003). The use of certain amendments and/or a plant cover is important for in situ remediation of heavy metal-contaminated soils, since these practices can affect soil chemical properties, as well as the function and structure of the microbial community (Pérez de Mora et al., 2006). Microbial biomass parameters have been widely used for testing soil quality and the degree of restoration in degraded and/or contaminated soils (Clemente et al., 2006a; Pérez de Mora et al., 2005). Soil microbial population and activity have been proposed as useful indicators of soil health (Pankhurst et al., 1995).

It is generally accepted that accumulated heavy metals reduce the amount of soil microbial biomass (Brookes and McGrath, 1984; Chander et al., 1995). However, metal exposure may also lead to the development of metal-tolerant populations (Ellis et al., 2003). Soil microbiological parameters have great potential as early sensitive, effective, and reliable indicators of stresses or perturbations in soils affected by mine wastes (Liao et al., 2005). Therefore, the study of soil microbial function can provide important information when evaluating soil remediation (Pérez de Mora et al., 2006).

The challenges of using microbial indicators are to identify the best choice among the many techniques available to assess soil quality. Winding et al. (2005) proposed measures of microbial biomass, respiration (rate of aerobic catabolism of soil organic matter by heterotrophs; Cotrufo et al., 1995), N mineralization, and community profiling as techniques to be considered. Brookes (1995) suggested that either the ratio of CO2 production to biomass C (biomass specific respiration rate) or biomass C as a percentage of soil organic C might be better indicators of soil pollution than either microbial activity or biomass measurements alone (Barajas-Aceves et al., 1999). The link between biomass C and total soil organic C may constitute a form of "internal control" in soils of similar type and under similar management (Barajas-Aceves, 2005).

Monitoring soil quality by means of biological indices has been found helpful for the management and sustainability of soils that received sewage sludge application; basal respiration, Biomass C and N, biomass specific respiration, and enzymatic activity increased after the organic residue was added (Fernandes et al., 2005).

We report here a phytoremediation experiment, performed over 15 mo, to determine the usefulness of organic amendments for remediation of a calcareous, metal-contaminated soil in La Unión, Murcia, Spain, by evaluating effects on soil properties and metal availability (Clemente et al., 2007). Mining activity, performed in this area since Roman times, has completely transformed the landscape of the mine site and adjacent land (24.55 km2), due to the accumulation of metalliferous mine wastes (Martínez et al., 2001). Beta vulgaris L. and Beta maritima L. were selected for testing successive changes in metal availability due to the organic amendments (Clemente et al., 2007). The aim of the present work is to study the effects of the organic amendments on microbial biomass parameters, their evolution with time, and the relationships between these parameters, soil properties, and metal bioavailability (soil DTPA extractability and plant uptake) to assess their usefulness as soil remediation indicators. Factor analysis (FA), a multivariable technique adequate for reducing the number of variables and detecting structures in the relationships between variables, was performed with data corresponding to crops of B. vulgaris and B. maritima.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Experimental Design
The experimental site was located in an agricultural area near San Ginés de la Jara (Murcia) (37°38'45'' N, 00°50'50'' W), 3.2 km from the Mar Menor sea, and 2 km north of the nearest old mining site. The soil was a calcareous (15% CaCO3) clay loam, with 39.2% clay, 27.4% silt, and 33.4% sand, classified as Xeric Torriorthent (Natural Resources Conservation Service, 1999). Some properties of the soil are shown in Table 1. Concentrations of Zn (2706 mg kg–1) and Pb (3235 mg kg–1) in this soil greatly exceed statutory EU limits for agricultural soils (300 mg kg–1 of Zn and Pb for soils having pH > 7; Council of the European Communities, 1986). Two organic amendments were tested: cow manure and olive husk. Olive husk or "alperujo" is the waste produced during olive oil extraction by the two-phase centrifugation system (Alburquerque et al., 2004) and its use as an organic amendment for soil reclamation would mean an integrated approach to its disposal, adding value to this by-product (Romero et al., 2005). Heavy metal concentrations in the two residues are well below the limits established for agricultural use of compost and fertilizer products made from wastes in Spain (Cd < 0.7, Cu < 70, Pb < 45, and Zn < 200 mg kg–1; BOE, 2005) (Table 1).


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  		Table 1. Characteristics of the soil and the organic amendments.

 
Three treatments, in a fully randomized design with three replicates per treatment, were set up in experimental plots (6 m2) with 0.5-m distance between plots. The treatments were: mineral fertilizer (a 15 N-15 P2O5–15 K2O commercial inorganic fertilizer was applied at a rate 200 kg ha–1) (internal control), cow manure (51 t ha–1), and olive husk (53 t ha–1). Both organic amendments provided 15 t ha–1 of organic matter.

Two Beta species, B. vulgaris L. and B. maritima, were grown in consecutive growing cycles. These species have been used as indicators of soil heavy metal availability (McGrath et al., 2000; Walker et al., 2003); they are also used for human consumption in most of Spain. The risk of heavy metal transfer was evaluated in a previous paper (Clemente et al., 2007). One month after organic amendment addition, Swiss chard (Beta vulgaris L. var. Nomonta) seedlings of uniform size were planted (48 seedlings in each plot) and allowed to grow for 90 d. For the second crop, seeds of B. maritima were collected from a non-polluted site and seeded at a rate of 300 seeds per m2 (1800 seeds per plot) the following year. The plant material was collected after 21 wk; all leaves were sampled, leaving the plants with the flowers for seed production. Plant material was thoroughly washed with distilled water, and the fresh and dry weights (60°C for 1 wk) and heavy metal concentrations were determined.

The soil was sampled four times in each plot, at sowing and sampling of each species, to study the evolution of the soil during the experiment. All soil samples were taken by mixing six subsamples from six sites of each plot at 0- to 20-cm depth. Each soil sample was divided into two fractions, one of which was immediately sieved to <2 mm and stored without drying at <5°C for microbial biomass determination, while the other fraction was air-dried and sieved to <2 mm for chemical analysis. The evolution of soil pH, total organic carbon, and plant-available heavy metal (Zn, Cu, Mn, Fe, Pb, and Cd) concentrations was followed throughout the study.

The timing of the experiment was as follows:

21 Jan. 2003: Addition of soil organic amendments;
5 Mar. 2003: Soil sampling 1, transplanting of B. vulgaris and addition of inorganic fertilizer to control plots;
3 June 2003: Harvesting of B. vulgaris and soil sampling 2;
10 Dec. 2003: Sowing of B. maritima and soil sampling 3;
5 June 2004: Harvesting of B. maritima and soil sampling 4.

Analytical Methods
Soil particle size distribution was assessed by sieving and sedimentation, using the hydrometer method (Gee and Bauder, 1986). The electrical conductivities of the soil and organic waste samples were measured in water suspensions (1:5 and 1:10 (w/v) in soil and organic waste samples, respectively), using a Crison GLP 31 conductivity meter (Crison Instruments, Barcelona, Spain). The pH was determined in saturated soil pastes and in water suspensions 1:10 (w/v) for soil and organic waste samples, respectively, using a Crison GLP 21 pH meter (Crison Instruments, Barcelona, Spain). Soil, organic amendment, and plant pseudo-total heavy metals (Cd, Cu, Fe, Mn, Pb, Zn) were determined by flame atomic absorption spectrometry (AAS) in a UNICAM 969 atomic absorption spectrometer (Thermo Elemental, UK) after nitric-perchloric acid (2:1) digestion in a block digester. The certified "Montana" soil (NIST-SRM 2711) was used to test the digestion method, with a recovery percentage of Cd 101.2, Cu 94.4, Pb 96.7, Zn 94.3, Fe 86.6, and Mn 88.2. Soil-extractable metals (Cd, Cu, Fe, Mn, Pb, Zn) were extracted with 5 mM diethylenetriaminepentaacetic acid (DTPA)/10 mM CaCl2/100 mM triethanolamine at pH 7.3 (Lindsay and Norvell, 1978). The total N and organic C (TOC) concentrations of the soil and organic amendments were measured with a EuroVector automatic microanalyzer (EuroVector, Milan, Italy). The OM content of the soil was determined by multiplying the TOC by 1.72 and the OM of organic amendments by ashing at 430°C. Ammonium nitrogen (NH4+–N) was extracted with 2 M KCl and determined by a colorimetric method based on Berthelot's reaction. Total P was determined colorimetrically as molybdovanadate phosphoric acid in a UV-Visible spectrophotometer, after nitric-perchloric acid (2:1) digestion.

Soil microbial biomass C was determined, after a fumigation-extraction procedure (Vance et al., 1987), in a TOC/Skalar analyzer (Skalar, Breda, The Netherlands) and calculated according to Wu et al. (1990). Ninhydrin N was determined according to Joergensen and Brookes (1990). Biomass N, calculated from the difference between ninhydrin-reactive N in fumigated and non-fumigated soil extracts, is also considered as an indicator of the microbial biomass content in soils (Joergensen and Brookes, 1990). Soil respiration was calculated as the amount of CO2–C emitted during a 10-d incubation. An aliquot of soil (10 g dry wt.) was placed in a 250-mL incubation vessel, the moisture was adjusted to 50% of water-holding capacity, and a vial containing 10 mL 0.1 M NaOH (0.2 M for soil sampling 1) was placed inside the incubation vessel for retention of the evolved CO2. After 10 d the vials were titrated with 0.1 M HCl in an excess of BaCl2. The soils were incubated in triplicate using empty vessels as blanks (triplicates). Chemical analyses were done at least in duplicate and those for microbial biomass determination at least in triplicate.

Statistical Analysis
Using SPSS Version 12.0 software (SPSS Inc., 2003), simple correlations between the different variables were performed. Data were also subjected to ANOVA and differences between means were determined using the Waller–Duncan test. Normality and homogeneity of variances were tested by the Shapiro-Wilk and Levene tests, respectively, before ANOVA. To obtain additional information about the relationships, behavior, and source of contamination, a factor analysis was performed, using Varimax normalized rotation (Kleinbaum et al., 1988). Factor analysis was performed using evaluation of the principal components and retaining only eingenvalues higher than 1 (Kaiser criterion). This technique allows an important reduction in the number of variables and structures the associations between the different variables (Maiz et al., 2000). Factor analysis creates new variables (factors), the number of factors being considerably lower than the number of variables. Factor loadings lower than 0.5 were eliminated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Microbial Biomass Parameters
As expected, the organic amendments led to an increase in the organic-C concentration in the soil with respect to the control, especially in olive husk (OH) plots (Table 2). However, this increase was only significant for OH treatment at the end of the experiment, 15 mo after amendments addition (Table 2). This could be due to the greater organic C content of olive husk compared with cow manure (562 and 412 g kg–1 dw, respectively) and to a lower microbial degradation of olive husk (Clemente et al., 2007; Riffaldi et al., 1997).


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  		Table 2. Microbial biomass parameters in soils at different sampling times (dry matter basis). TOC, total organic carbon.

 
The organic amendments also increased biomass C in the first sampling, 43 d after their addition to the soils, following the order manure > OH > control (Table 2). Throughout the experiment biomass C values increased in OH-treated plots whereas it decreased in manure-treated plots. Both remained significantly higher than in control plots in the last sampling, following the order OH > manure > control. In control plots, where no organic amendment was added, biomass C also increased throughout the experiment, due to soil tillage and plant crops. Biomass N followed the same pattern as biomass C, being significantly higher in manure-treated plots at the beginning of the experiment, while after 15 mo the highest values were found in OH-treated plots (Table 2).

The CO2–C evolved from the soil (measured in a separate incubation experiment) was higher in the organically amended soils than in control plots shortly after the addition of the amendments, although the response was highly variable and not statistically significant (Table 2). In soils from plots receiving the organic amendments, CO2–C evolution rate decreased during the experiment, but was still significantly higher in OH-treated plots after 15 mo (Table 2).

The ratio of CO2–C/biomass C has been proposed as a tool to quantify soil health in heavy metal-contaminated soils (Brookes, 1995). High values generally indicate a high stress on the soil microbial biomass (Barajas-Aceves, 2005). In all the plots this parameter decreased during the experiment (Table 2) showing a clear and significant effect of the remediation procedure in reducing the ratio. Significant reductions with time were found in control and manure-treated plots and a clear, decreasing trend was observed in OH-treated plots (Fig. 1). The values observed were within the range reported by Barajas-Aceves et al. (1999) for Zn-contaminated soils (20–200 mg g–1 d–1), except for the extremely high values found in control plots in March and June 2003 (Table 2).


Figure 1
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  		Fig. 1. Microbial biomass ratios in soils receiving different treatments at different sampling times. Bars with the same letter are not significantly different according to the Waller–Duncan test, at P < 0.05. Groups of bars without letters did not present statistically significant differences (P > 0.05). OH = olive husk; TOC = total organic carbon.

 
The ratio of biomass C/TOC has been proposed as a useful index to identify soil pollution by heavy metals (Brookes, 1995) and can be an indicative measure of the changes undergone by soil organic matter (Insam and Merschack, 1997). This ratio was very variable in different treatments at the different samplings, but generally agreed with values reported for contaminated soils (1.2–3.1%, Barajas-Aceves et al., 1999; 1.1–1.8%, Dai et al., 2004) and soils receiving organic amendments (1.4–2.0%, Leita et al., 1999). This ratio was significantly higher in the organic-amended than in control plots and, as observed for other parameters, it was higher for manure-treated plots in March 2003, but higher in OH-treated plots in the last sampling (Table 2). In plots treated with OH a significant increase of this ratio with time was observed (Fig. 1). Chander and Brookes (1993) reported that this ratio ranged from 1.5 to 1.6% in unsludged or uncontaminated sludge-treated soils, and from 0.4 to 0.7% in soils treated with heavy metal-contaminated sludge. Therefore, the combination of these two measurements may provide an ‘internal control’ within soils receiving similar management practices (Barajas-Aceves et al., 1999).

Biomass C/N ratio values were very variable; significant differences were only found (5.37b, 16.07b, and 32.15a for control, OH-, and manure-treated plots, respectively; P < 0.01) in June 2003. This ratio significantly increased with time in control and manure-treated plots mainly at both harvest times (Fig. 1).

Correlations and Statistical Analysis
Simple correlations were calculated for DTPA-extractable soil metal concentrations (Fig. 2), soil pH, TOC, and total N contents, and microbial biomass parameters for all samplings (data not shown). DTPA-extractable metals were measured at all samplings and discussed previously (Clemente et al., 2007). Cow manure did not alter the DTPA-extractable concentrations of metals in the soil compared with the control, whereas olive husk favored solubility of metals in soils mainly due to the reduction of Mn oxides during degradation of phenolic compounds (Clemente et al., 2007). Soil respiration correlated positively to TOC and DTPA Mn and Cu concentrations in soils (r = 0.493, 0.457, 0.340, P < 0.01, 0.01, 0.05, respectively), and correlated negatively to soil pH (r = –0.382, P < 0.05), while biomass C/N ratio positively correlated with DTPA Cu concentrations (r = 0.351, P < 0.05). Soil biomass C correlated with soil TOC (r = 0.523, P < 0.01) and significant positive correlations were found between the different microbial biomass parameters (data not shown). However, no significant correlations were found between DTPA Zn, DPTA Pb, and microbial biomass parameters.


Figure 2
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  		Fig. 2. DTPA-extractable metal concentrations (mg kg–1) in soils at different sampling times. Bars with the same letter are not significantly different according to the Waller–Duncan test, at P < 0.05. Groups of bars without letters did not present statistically significant differences (P > 0.05).

 
In previous work, metal (Mn, Zn, Cu, and Pb) concentrations in plant leaves were significantly intercorrelated, and most of the metal concentrations in plants correlated to their soil DTPA-extractable concentrations (Clemente et al., 2007). Metal concentrations in plants and soil microbial biomass parameters did not show any clear correlation pattern and only isolated positive correlations were found (data not shown). Plant biomass (fresh wt.) of B. vulgaris was negatively correlated with Cu, Mn, Pb, and Zn (r = –0.926, –0.866, –0.938, –0.962, P < 0.001, 0.01, 0.001, 0.001, respectively) concentrations in plant tissue, and DTPA-extractable Cu, Fe, and Mn concentrations in soils from March 2003 (r = –0.823, –0.827, –0.882, P < 0.01). Only the Pb concentration in B. maritima correlated (negatively) to its plant yield (fresh wt.) (r = –0.758, P < 0.05), and a positive correlation was found between plant yield and the ratio of biomass C/TOC (r = 0.732, P < 0.05).

Using data from the B. vulgaris crop, we could identify four factors using factor analysis (Table 3). The first one relates positively plant metal concentrations to DTPA-extractable Mn, Cu, and Fe, TOC, biomass C/N ratio, and negatively to pH and fresh and dry matter production. We called this the toxicity factor as it shows, at lower pH, higher metal availability and plant metal concentrations, and lower plant biomass yield, as well as organic carbon accumulation in soils due to lower mineralization. Factor 2 comprises most of the different microbial biomass parameters determined, so it could be considered as the microbial biomass and activity factor, affected by soil pH. Factor 3 associates DTPA-extractable Cu, Zn, and Pb (metal extractability/solubility factor) and factor 4 relates Fe bioavailability (plant concentration) and CO2–C/biomass C ratio.


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  		Table 3. Factor-rotated matrix for data from soil sampling 1 and the B. vulgaris crop. TOC, total organic carbon.{dagger}

 
Five factors were identified using data from the B. maritima crop (Table 4). Factor 1 associates again plant metal concentrations and DTPA-extractable Mn and Cu with soil respiration, TOC, and biomass C content (toxicity factor: metal availability, C accumulation). Factor 2 positively related biomass C and N with TOC, total N, DTPA Zn and Pb, and negatively with CO2–C/biomass C, showing an unexpected association between microbial biomass content and metal availability. Factor 3 associates fresh and dry weight of the crop with biomass C/TOC ratio, and negatively with CO2–C/biomass C ratio, pH, and Pb concentration in plant tissue, and could be defined as a plant yield factor, affected by pH and related to soil quality ratios. Factors 4 and 5 did not contribute any relevant associations.


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  		Table 4. Factor-rotated matrix for data from soil sampling 3 and the B. maritima crop. TOC, total organic carbon.{dagger}

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Pereira et al. (2006) used soil microbial parameters to evaluate long-term effects of heavy metal contamination on microbial community activity and soil functions and reported that metal content and low pH values negatively affected the biomass and the activity (dehydrogenase activity and potential nitrification) of soil microorganisms. Biomass C values observed in the plots were within the same range as those observed previously in non-polluted soils with organic and/or inorganic fertilization (Leita et al., 1999; Albiach et al., 2000). Similar values were reported (Pérez de Mora et al., 2005; Clemente et al., 2006a) in heavy metal-contaminated soils from an acid mine spillage accident following remediation treatments with organic amendments. Many authors have found that biomass C increases when organic wastes are added to soil, showing long-term effects (Leita et al., 1999).

However, the different composition of the amendments leads to different mineralization patterns in the soil (Bernal et al., 1998; Clemente et al., 2006b), and hence, microbial biomass. Manure contains high concentrations of labile, easily degradable compounds, leading to a rapid increase in microbial biomass in soils shortly after application. In contrast, OH contains less labile molecules and, in addition, its high content of phenolic compounds and fats (Alburquerque et al., 2004) could have led to soil microbial biomass toxicity resulting in a slower degradation/mineralization by soil microorganisms. Olive mill wastes are resistant to degradation by soil microorganisms (Riffaldi et al., 1997). In addition, manure can favor an increased microbial population of the soil, while olive mill wastes have antimicrobial characteristics (Paredes et al., 1987). This accounts for the slower response of the microbial population to olive husk than manure. However, in the last sampling, microbial biomass was significantly higher in OH-treated plots, as a consequence of the gradual degradation of its organic matter. Soil microbial biomass C and biomass C/TOC ratio have been found to be higher in decomposed cow manure than in municipal solid waste compost-treated soils, due to a higher amount of biogenic organic materials like water-soluble organic carbon, carbohydrate, and mineralizable N in the former (Bhattacharyya et al., 2005). The microbial biomass contained in the organic amendments as well as the addition of substrate C was responsible for these results (García-Gil et al., 2000). In control plots an increase in microbial biomass with time was observed as a consequence of inorganic fertilization, plant root residues, and their linked biological activity.

Microorganisms in heavily polluted soils are under stress and are less efficient at C utilization, resulting in more CO2 evolved per unit of substrate (Killham, 1985). Specific respiration rate and biomass as a percentage of soil organic matter were proposed by Barajas-Aceves et al. (1999) as practical indicators of damage to soil biological activity caused by metal contamination. These authors found significant correlations between these linked measurements and total Zn concentration in soils. Control plots showed a higher energetic demand (higher specific respiration) reflecting that soil microbial biomass is under stress conditions as a consequence of soil metal content and needs to expend more energy to survive, resulting in a diversion of energy from biosynthesis (Barajas-Aceves, 2005). Manure produced a greater and more rapid decrease in this ratio when compared to OH-treated plots, although this treatment reached similar values after the first crop had been cultivated. No significant correlations were found between this ratio and DTPA metal extractability in soils and plant metal content, but the CO2–C/biomass C ratio significantly changed with time in both control and manure-treated plots (Fig. 1), this being taken as a symptom of improved soil health. Factor analysis with data from the B. maritima crop agreed with this, as it showed that plant yield in the different plots was linked to this ratio (Table 4).

A significant time-dependant increase in biomass C/TOC ratio was observed in plots treated with OH, reflecting changes in mineralization processes as the toxicity of this amendment decreased with the degradation of its phenolic compounds (Fig. 1). Basal respiration and microbial coefficients (biomass C/TOC) have been reported to be significantly negatively correlated with soil Cu and As content, while a significant, positive correlation was reported between Cd concentration in soil and metabolic quotient (qCO2) (Shukurov et al., 2006). Li et al. (2005) reported that metal stress resulted in relatively low ratios of microbial biomass C/TOC and in inhibition of both the microbial metabolic quotient and C mineralization rate, eventually leading to increases in soil organic C, which are in agreement with the results reported above. Liao et al. (2005) found both microbial biomass and basal respiration to be negatively affected by elevated heavy metals levels and that biomass C/TOC and qCO2 were closely correlated to heavy metal stress.

Joergensen et al. (1995) suggested that high microbial biomass C/N ratios are caused by increased fungal to microbial biomass ratios. Dai et al. (2004) suggested that soil microbial community composition varied along concentration gradients, with fungi being predominant in polluted soils and bacteria in the uncontaminated soils, in terms of the values observed for biomass C/N ratio. The ratio of biomass C/N was found to be part of the factor 1 (identified as ‘metal availability and toxicity’ factor) from the factor analysis performed with data from the B. vulgaris crop (Table 3), indicating an effect of metal toxicity on soil microbial composition. This parameter showed a rapid and significant increase in manure-treated plots and later in control and OH-treated plots, which may reflect the changes in microbial community structure in these soils. Chen et al. (2006) found negative correlations between bacterial diversity and NH4NO3–extractable Cu. Dai et al. (2004) found that organic C and N and biomass C and N were positively correlated with the metal content of the soils. These authors also reported that the ratios of biomass C/N were highly and positively correlated with soil metal content, indicating variation in the microbial community in relation to contamination, while microbial activities (specific respiration, etc.) were negatively correlated with the heavy metal content of the soils.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Microbial biomass parameters reflected mineralization patterns of the different amendments and showed that soil receiving these amendments experienced an increase in soil quality and fertility, even in planted, non-amended soil receiving inorganic fertilizer. Shortly after addition of the amendments to the soil, the extractable metals were related to soil pH, both affecting the variation of the microbial composition. DTPA-extractable metal concentrations are not directly related to the microbial biomass and its activity, as the behavior of the microorganisms differed according to the organic waste added. Fifteen months after amendment addition, the extractability of the metals in soils, plant metal accumulation, and plant biomass production, in addition to soil pH, were related to soil microbial biomass ratios.

Although the addition of organic amendments to heavy metal-contaminated calcareous soils can improve soil fertility, the response of the microbial population depends on the amendment used. Manure helps to reduce the stress symptoms enabling the soil microbial biomass to increase quickly. This amendment is more appropriate for creating suitable conditions for phytoremediation of metal-polluted soils than olive husk. Microbial biomass parameters are suitable to follow the progress of the recovery processes, complementing metal extractability in soil and plant metal accumulation measurements.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. D.J. Walker for his scientific comments and the language revision of the manuscript. This work was financed by the Spanish Ministry of Science and Technology (REN2001-1113-C02-02) and CSIC (Intramural Especial. Ref.: 200440E047).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 





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The SCI Journals Agronomy Journal Crop Science
Journal of Natural Resources
and Life Sciences Education		 Vadose Zone Journal
Soil Science Society of America Journal Journal of Plant Registrations The Plant Genome