Dynamics of soil microbial populations involved in 2,4-D biodegradation revealed by FAME-based Stable Isotope Probing

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Abstract

Stable Isotope Probing (SIP) is a powerful tool for analysing the fate of pesticides in soil. Together with FAME (Fatty Acid Methyl Esters), it can help identify biodegradation pathways and recycling into the microbial biomass. The fate of ring-labelled 13C-2,4-dichlorophenoxyacetic acid or 2,4-D (C2,4-D) was determined in soil during a 6-month incubation. The distribution of 13C among the microbial biomass, the CO2 respired, the water, methanol and dichloromethane soluble fractions, and the residual non-extracted bulk soil was measured. Molecular analyses were carried out on the lipid and the non-extractable fractions. After 8 days, about half of the initial amount of C2,4-D was mineralised; the other half remained in soil as non-extractable residues (NER). C2,4-D continued to be mineralised, suggesting that NER were still bioavailable. Analysis of C2,4-D-enriched FAME contained in the lipid fraction suggested that a succession of microbial populations was involved in 2,4-D biodegradation. This is possibly due to the change of 2,4-D availability. The C2,4-D yield coefficient and degrader diversity evolved during the incubation, providing corroboratory evidence that different physiological groups were active during the incubation. The 13C-labelled microbial community was always less diverse than the total community, even at the end of the incubation, suggesting that the cross-feeding community is also a specific part of the total community. This work shows that molecular analysis of 13C-labelled pesticides is a useful tool for understanding both chemical and biological aspects of their fate in soil.

Introduction

In order to reduce the environmental impact of pesticides it is essential to understand the mechanisms affecting their sorption and degradation in soil (Barriuso, 1994). In many cases, degradation in soil follows more complicated kinetics than first-order kinetics from which a half-life is deduced (Soulas, 1982). This simplification is generally used because of the difficulty in taking into account the interactions among soil microbial populations (Soulas, 1993). Furthermore, sorption over long time scales, which results in the formation of non-extractable residues (NER), modifies the availability of pesticides (Gevao et al., 2000). The formation of NER, resulting from the irreversible adsorption, the chemical stabilisation and/or the physical sequestration of pesticides and their metabolites in the organo-mineral matrix is a major impediment to their complete degradation by microorganisms (Barriuso et al., 2008). A better understanding of how microbial community activity is related to the formation and degradation of bound substrates may contribute to a more precise prediction of the long-term fate of pesticides in soil. Whilst a number of studies have been carried out to determine whether the structure of microbial populations responsible for pesticide degradation changes as function of application rate (Macur et al., 2007), no study has dealt with the dynamics of degrader populations over the long term. It is known that the formation of NER reduces the availability of a pesticide and therefore its mineralisation. However, degrader community structure may change in response to the reduction in pesticide bioavailability with possible consequences on microbial activity. The effects of such changes on the long-term fate of pesticides have not been dealt within the literature.

The characterisation of both pesticide residues and microbial degrader populations is necessary for a better understanding of how pesticide availability and degrader community structure interact. The availability of pesticides in soil is generally assessed by sequential extraction with an inverse polarity gradient (Mordaunt et al., 2005). Among the molecular tools with which microbial communities in soil can be characterised, lipid biomarkers analysed as Fatty Acid Methyl Esters (FAME) after saponification or transmethylation have been used successfully (Zelles, 1999a). FAME-Stable Isotope Probing (SIP) is a powerful tool for targeting active microbial populations in environmental samples (Boschker et al., 1998). Although RNA-SIP or DNA-SIP is more appropriate than FAME-SIP for taxonomic studies, they are less sensitive because they require high levels of isotopic enrichment of the biomass in order to separate labelled from unlabelled nucleic acids (MacGregor et al., 2006, Timms-Wilson et al., 2006, Neufeld et al., 2008). The fate of compounds that are present in small quantities or are poorly labelled cannot be studied using these approaches. For monitoring pesticides in soil over the long term (defined here as beyond the point when NER are the dominant pesticide forms), sensitivity is likely to be an important issue, especially as the 13C enrichment of the target biomass can be diluted by the consumption of other C sources and because cross-feeding can lead to the labelling of other communities (Gallagher et al., 2005). Under these circumstances, the high sensitivity of FAME-SIP is very useful, particularly when specific identities of microorganisms are not sought. As the precise identification of the microorganisms involved in the degradation was not a primary objective of this study, FAME-SIP was the preferred method. Furthermore, the extraction procedure for lipid biomarkers (Bligh and Dyer, 1959) being similar to that for measuring pesticide potential availability (Mordaunt et al., 2005), both using methanol and dichloromethane, the FAME extraction procedure was adapted to allow the determination of 13C-enriched pesticide residues and 13C-enriched FAME in the same sample.

The aim of this study was to identify the links, if any, among pesticide bioavailability, mineralisation and the structure of degrader populations during a 6-month incubation. The model chemical used was 2,4-dichlorophenoxyacetic acid (2,4-D), one of the most applied herbicides worldwide. This compound is known to be both very degradable (Voos and Groffman, 1997) and likely to form NER (Benoit et al., 1998). By using FAME-SIP, it was possible to assess the 13C-2,4-D degrader populations' dynamics and compare it to that of the whole microbial biomass. The hypothesis was that the diversity of 2,4-D degraders would be lowest during the first 2 weeks (Cupples and Sims, 2007) when the pesticide was still readily available. It was then expected that the diversity of the populations involved would increase due to the dilution of the labelling during microbial turnover and that it would approach that of the overall population.

Section snippets

Soil samples

In order to ensure the validity of the labelling approach, the same pesticide concentration and the same soil were used as in a recent 14C-2,4-D experiment (Vieuble-Gonod et al., 2003) allowing a comparison of 13C and 14C results. Samples were obtained from the ploughed layer (0–30 cm) of a cultivated Luvisol from an INRA's experimental site in Versailles, France. The soil is a silt loam (30% sand, 53% silt and 17% clay), with 13.8 g kg−1 of total organic carbon (TOC), 1.35 g kg−1 of nitrogen,

Results

For every fraction analysed (CO2, different extracts, soil microbial biomass), differences in δ13C values between 2,4-D treated and control samples were significant (P < 0.001) but the quantity of C was similar (data not shown). GC-FID analyses showed that FAME profiles were similar in treated and control samples (data not shown) and GC-IRMS analyses revealed that 25 FAME were significantly 13C enriched in the treated soil compared to those in the control soil (P < 0.001). Those results allowed

Methodological considerations

Stable isotope probing has been used to study microbial involvement in the transformation of numerous molecules, including methane, acetate, cellulose and xenobiotics, in environmental samples (Neufeld et al., 2008 and references therein). Here, the use of 13C labelling to follow the fate of 2,4-D in soil gave a similar estimation of mineralisation kinetics, incorporation into the biomass, extractability and NER formation to that obtained with traditional 14C labelling techniques (Vieuble-Gonod

Conclusion

Using FAME-SIP, we detected C2,4-D in the microbial biomass for the duration of the incubation, long after the peak of degradation activity. This would not have been readily achieved using nucleic acid-based SIP because of the relatively poor sensitivity. The dynamics of the 13C-FAME profile over a 6-month period suggested that the biodegradation of labelled 2,4-D involved a succession of microbial communities. We confirmed that proteobacteria are the principal degraders of 2,4-D between 3 and

Acknowledgements

The study was supported by the French National Research Program ECODYN of the French National Institute for Earth Sciences and Astronomy (INSU). The authors thank Nicolas Péchot and Valérie Pouteau for their help with experiments as well as Claire Chenu and two anonymous referees for reviewing the manuscript.

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