Water Air Soil Pollut (2012) 223:3385–3392 DOI 10.1007/s11270-012-1117-5
Assessment of s-Triazine Catabolic Potential in Soil Bacterial Isolates Applying atz Genes as Functional Biomarkers Carmen Fajardo & Maria Ludovica Saccà & Alicia Gibello & María José Martinez-Iñigo & Mar Nande & Carmen Lobo & Margarita Martin
Received: 22 November 2011 / Accepted: 14 February 2012 / Published online: 29 February 2012 # Springer Science+Business Media B.V. 2012
Abstract Fluorescence in situ hybridization (FISH) technique and qPCR analyses, targeting atz genes, were applied to detect the presence of simazinedegrading bacteria in an agricultural soil with a history of herbicide application. atzB-targeted bacteria detected by FISH represented 5% of total soil bacteria with potential capability to metabolize the herbicide. The soil natural attenuation capacity was confirmed in soil microcosms by measuring simazine degradation. Moreover, four bacterial strains were isolated from the soil and identified as Acinetobacter lwoffii, Pseudomonas putida, Rhizobium sp. and Pseudomonas sp. C. Fajardo (*) : A. Gibello : M. Nande : M. Martin Universidad Complutense, Avenida Puerta de Hierro s/n, 28040 Madrid, Spain e-mail:
[email protected] M. L. Saccà Campus de Excelencia Internacional de Moncloa, Edificio del Real Jardín Botánico Alfonso XIII, Ciudad Universitaria, 28040 Madrid, Spain
The isolates were able to grow using different s-triazine compounds and related metabolites as the sole carbon source. Growth parameters in presence of simazine were calculated using the Gompertz model. Rhizobium sp. showed the highest simazine degradation (71.2%) and mineralization (38.7%) rates, whereas the lowest values were found to A. lwoffii—50.4% of degradation and 22.4% of mineralization. Results from qPCR analyses of atzA, atzB and atzC genes revealed their presence in Rhizobium sp. and A. lwoffii, being atzB and atzC the most abundant functional genes. Rhizobium sp. showed a higher amount of the three biomarkers compared to A. lwoffii: the atzA, atzB and atzC gene copy number per microlitre were, respectively, 101, 102 and 103-fold higher in the former. Therefore the proposed molecular approaches based on the use of atz genes as biomarkers can be considered as useful tools to evaluate the presence and potential capability of degrading-s-triazines soil microorganisms. Keywords Triazines . atz genes . FISH . qPCR . Biomarker . Biodegradation
M. J. Martinez-Iñigo Universidad Carlos III, Calle Madrid, 126, 28903 Madrid, Spain
1 Introduction
C. Lobo Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA), Finca “El Encín” Km 38,2 A-II Apdo 127, 28800 Madrid, Spain
s-Triazine compounds form a common group of herbicides used for the control of a wide variety of broadleaf weeds in agricultural soils. Atrazine was the first triazine ring herbicide produced and extensively
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applied to corn cultivation (EPA 2003). Following the prohibition of atrazine in the EU in 2004, it was replaced by other triazines such as simazine, terbuthylazine, terbutryn, ametryn, cyanazine and prometryn. Simazine (2-chloro-4,6-bis(ethylamine)-s-triazine) has been used in agriculture, forestry and non-crops soils in many countries for more than 40 years (Dinamarca et al. 2007), and together with two chlorinated metabolites (deethylatrazine and deisopropyl atrazine), simazine has shown estrogenic properties with several cell lines (Sanderson et al. 2001). Currently, simazine has also been banned in EU member states (2007) although it is commonly used in South America for the control of annual weeds in fruit plantations, vineyards and in forestry (Flores et al. 2009). Moreover simazine and related s-triazines may persist in the environment for many years depending on climatic and edaphic factors (e.g. temperature, soil moisture, organic carbon content), and on the degradation activity of the soil native microbiota (Gunasekara et al. 2007). Biodegradation of s-triazines has been extensively studied, although there are few reports on soil microorganisms with high capacity to mineralize the simazine triazine ring (Iwasaki et al. 2007; Sánchez et al. 2005; Santiago-Mora et al. 2005). According to the triazine biodegradation pathways described for different bacteria (Martínez et al. 2001; Topp et al. 2000a), dechlorination and deamination reactions are previously required for triazine ring cleavage. The atrazine chlorohydrolase (AtzA) found in Pseudomonas sp. strain ADP and other Gram-negative bacteria (Mandelbaum et al. 1995; Seffernick et al. 2007), and the s-triazine hydrolase described in Rhodoccocus corallinus and other Grampositive bacteria (Topp et al. 2000b) are the enzymes involved in dechlorinating simazine. Less diversity is found in the dealkylaminase activity of soil microorganisms. AtzB hydroxyatrazine Nethylaminohydrolase is an amidohydrolase superfamily enzyme, which is involved in the degradation of multiple s-triazine compounds (Seffernick et al. 2007). atzC is the gene encoding the enzyme responsible for the hydrolytic deamidation of N-isopropylammelide to cyanuric acid and isopropylamine (Ralebitso et al. 2002; Wackett et al. 2002). Most strains that mineralize s-triazine harbour atzB gene (Iwasaki et al. 2007; Santiago-Mora et al. 2005), the essential gene for bacterial growth on triazines. However important gaps remain in the knowledge of how widely distributed other s-triazine-degrading
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genes are among different bacterial species and whether or not certain genes may be present only in certain species. Moreover, it is required to understand the effect of these genes copy number in the degradation capability of s-triazine natural degrading bacteria. The advent of molecular techniques, such as functional gene-targeted fluorescence in situ hybridization (FISH) and PCR amplification, improved our capability to analyse the microbial structure of organisms in native populations without the need for cultivation (Fajardo et al. 2011; Martín et al. 2008). In studies on the natural attenuation capacity of soils contaminated with s-triazine herbicides, the monitoring of functional genes encoding one of the key enzymes associated with this process is considered to be an attractive approach to determine the soil remediation potential. Particularly, a specific sequence of atzB gene has been revealed to serve as powerful probe for FISH detection of bacterial strains capable of degrading simazine (Martín et al. 2008; MartínezIñigo et al. 2010). The aim of this study is to apply the atz genes as functional biomarkers to detect the simazine degradation potential capacity of soil microbial agents. In an attempt to relate the atz genes copy number with the herbicide catabolism, soil bacterial strains were isolated and characterized metabolically. qPCR analyses targeting atzA, atzB and atzC genes were applied to characterize genetically the isolates. Therefore, this approach would contribute to monitor the occurrence of simazine-degrading organisms and the effectiveness of remediation strategies.
2 Materials and Methods 2.1 Soil Sample Bulk soil sample was collected from the surface layer A (0–22 cm depth) of a loam Calcic Haploxeraf (USDA), with a long herbicide exposure history, located in Alcalá de Henares (Madrid, Spain). The main geopedological and chemical characteristics of the soil are described in Table 1. 2.2 Chemicals Simazine, atrazine, 2-hydroxysimazine, deethylatrazine, isopropylamine, ethylamine and [U-ring 14C]
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Table 1 The main characteristics of the soil
0.5 ml min−1. Simazine was monitored at 214 nm. The injection volume was 10 μl, and simazine was identified by coelution with standard in HPLC analysis.
Depth (cm), 0–30
Soil
Total sand (%)
49.0
Total silt (%)
21.2
Clay (%)
29.8
Texture class USDA (1994)
Sandy clay loam
pH (H2O)
7.9–9.1
Organic matter (%)
0.63±0.04
EC (dS cm−1)
0.11±0.02
Ca carbonates (%)
5.6±0.3
Total nitrogen (%)
0.05±0.01
P (mg kg−1 soil)
21±0.9
Ca (mg kg−1 soil)
4,787±540
Mg (mg kg−1 soil)
146±24
Na (mg kg−1 soil)
42±4
K (mg kg−1 soil)
355±74
−1
Cu (mg kg
soil)
53±5.6
Zn (mg kg−1 soil)
44±1
Cd (mg kg−1 soil)
0.90±0.03
Pb (mg kg−1 soil)
13±0.3
Hg (mg kg−1 soil)
