Water flow drives small scale biogeography of pesticides and bacterial pesticide degraders - A microcosm study using 2,4-D as a model compound

Highlights

• Spatial distribution of 2,4-D and degraders at the mm scale controls the fate of 2,4-D at the cm scale.

• Advection is a key process controlling the accessibility of 2,4-D to bacterial pesticide degraders.

• The formation of non-extractable 2,4-D residues is strongly linked with microbial activity.

Abstract

Complex interactions between biodegradation and mass transfer of organic compounds drive the fate of pesticides in soil ecosystems. We hypothesized that, at the small-scale, co-location of degraders and pollutants in soils may be a prerequisite for efficient biodegradation of these chemicals. In non-co-localized micro-environments, however, diffusive and advective solute transport as well as active transport of microbial degraders towards their corresponding substrate may improve the accessibility of microbial substrates. The objective of this study was to test whether water flow can accelerate microbial pesticide degradation by facilitating the encounter of spatially separated pesticides and bacterial degraders at the millimeter scale.

Combining natural and sterilized soil aggregates, we built soil cores with different spatial localizations of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) and microbial degraders: (i) homogeneous distribution of microorganisms and 2,4-D throughout the soil core, (ii) co-localized microorganisms and 2,4-D in a mm³ soil location, and iii) separated microorganisms and 2,4-D in two mm³ soil locations spaced 1 cm apart.

Following the fate of ¹⁴C labelled 2,4-D (mineralization, extractable and non-extractable residues) as well as the abundance of bacterial 2,4-D degraders harboring the tfdA gene over an incubation period of 24 days, we observed decreased biodegradation of 2,4-D with increasing spatial separation between substrate and bacterial degraders. We found evidence that advection is a key process controlling the accessibility of 2,4-D and pesticide degraders. Advective solute transport induced leaching of about 50% of the initially applied 2,4-D regardless of initial spatial distribution patterns. Simultaneously, advective transport of 2,4-D and bacterial degraders triggered their re-encounter and compensated for the leaching-induced separation of initially co-localized microorganisms and 2,4-D. This resulted in effective biodegradation of 2,4-D, comparable to the homogeneous treatment. Similarly, advective transport processes brought substrate and degraders into contact if both were initially separated. Thus, advection more effectively removed bioaccessibility limitations to pesticide degradation than diffusive transport alone.

These results emphasize the importance of considering spatial microbial ecology as well as biogeophysics at the mm scale to better understand the fate of pesticides at larger scales in soil.

Introduction

The use of pesticides in agriculture has contributed to increased productivity but has also led to contamination of groundwater. Although a fraction of applied pesticides reaches its target, a remaining fraction may persist in soils for longer time periods or be transported to groundwater. The fate of the fraction of pesticides remaining in soil is determined by different chemical, physical, and biological processes such as volatilization, adsorption to soil particles, transport and (biotic and abiotic) degradation depending on the nature of the pesticides and the soil characteristics. These processes can potentially compete with one another, and, consequently, the likelihood of a pesticide reaching groundwater will depend not only on its transfer through the soil but also on other processes of dissipation (Edgehill and Finn, 1983, Alletto et al., 2010). If, for instance, mineralization rates are higher than rates of leaching, the pesticide will disappear from the soil system before reaching the groundwater. Consequently, the mineralization of pesticides by soil microorganisms is a key process for prevention or limitation of potential contamination of waters.

Microorganisms that degrade pesticides are non-randomly distributed in soil (Pallud et al., 2004) and are organized into mm to cm hot spots (Vieublé Gonod et al., 2003, Sjoholm et al., 2010). This heterogeneous spatial distribution of degraders leads to soil volumes with high or low degradation potentials (Dechesne et al., 2010, Poll et al., 2010). Vieublé Gonod et al. (2006a) showed by simulation that the spatial distribution of hot spots may strongly impact 2,4-dichlorophenoxyacetic acid (2,4-D) mineralization. Transport processes that enable the degrading microorganisms to encounter pesticides control biodegradation of pesticides in soil (Banitz et al., 2016, Pagel et al., 2016).

Microbial degraders can reach their target substrate by active as well as passive transport processes: microbial growth, motile dispersion or passive transport (Dechesne et al., 2014, Kim et al., 2015). Pallud et al. (2004) showed that after 2,4-D addition, the soil volume occupied by degraders could increase from 1% to 50% and the abundance of 2,4-D degraders from 10² to 10⁶ degraders per g of soil due to microbial growth. Ingham et al. (2011) showed that Paenibacillus was able to swarm at rates up to 10.8 mm h⁻¹ on agar; however, bacterial motility on agar surfaces is higher than in soil due to soil structure and water content (Wang and Or, 2010). The dispersion of motile bacteria depends on intrinsic characteristics of the microorganisms (Gannon et al., 1991) and the matrix potential of the soil (Dechesne et al., 2010). Zvyagintsev (1962) observed that relatively few soil bacteria (<1%) are motile, suggesting that bacterial movement in soil is limited or relies on passive mechanisms (Parke et al., 1986, Bahme and Schroth, 1987, Kemp et al., 1992, Hekman et al., 1994, Dechesne et al., 2010). Transport of bacteria by diffusion is typically negligible (Yates and Yates, 1990) and becomes only relevant in water-saturated soils (Dechesne et al., 2010). Instead, advective transport of bacteria along macropores, earthworm burrows, cracks, and fractures acts as a more effective mechanism of bacterial dispersal in soil (Breitenbeck et al., 1988, McCarthy and Zachara, 1989, Abu-Bashour et al., 1994, Or et al., 2007). Fungal hyphae may act entirely as physical vectors for bacterial transport, enabling bacteria to cross air-filled gaps in soil and thus gain access to otherwise inaccessible contaminants - a mechanism referred to as the “fungal highway” (Ellegaard-Jensen et al., 2014). Transport of bacteria via macroorganisms may also occur by feeding, burrowing and cast deposition (e.g. earthworms) or by translocation of attached bacteria with root growth (Daane et al., 1996, Vos et al., 2013). However, Madsen and Alexander (1982) showed 100-fold higher bacterial dispersal by percolating water (advection) in comparison to translocation of bacteria by earthworms and growing plant roots.

The encounter of pesticides and microbial degraders is additionally determined by diffusive and advective transport of dissolved and colloid-attached pesticides. Up to a few percent of a surface applied pesticide may be vertically leached in soil through advective flow, particularly during rainstorm events occurring just after pesticide application (Flury, 1996, Kladivko et al., 2001) unless it has been rapidly adsorbed or degraded. One consequence of advective flow is that pesticides may bypass a large part of the soil matrix. Compared with advective flow, diffusive flow leads to a slower migration of pesticide through soil, resulting in a longer contact time of the pesticide with soil particles and microorganisms. Diffusion is important for transport over small distances while advective transport occurs at larger scales (Howard, 1991, Or et al., 2007).

Water, therefore, is one of the main factors controlling biodegradation of pesticides in soil: water is relevant as transport agent at high water potentials and controls both the diffusion and the metabolic activity of the microorganisms at lower potentials. Better understanding of how transport of pesticides and microorganisms is controlled by water dynamics, and of accessibility of pesticides to degraders under heterogeneous soil conditions, is needed. Some studies have investigated the relationships between transport pathways and microbial activity in static or dynamic water conditions (Estrella et al., 1993, Kelsey and Alexander, 1995, Shaw and Burns, 1998, Ellegaard-Jensen et al., 2014). Pinheiro et al. (2015), who studied the impact of heterogeneity in diffusive conditions at the microbial habitat scale found that diffusion was not sufficient to establish contact between microorganisms and pesticides. Babey et al. (2017) showed that when microorganisms and 2,4-D were initially separated by 2.6 cm, 2,4-D mineralization was reduced by a factor of 55 near saturation (−1 kPa) as compared to when they were co-localized and by a factor of 1100 at the wilting point (−1585 kPa). Transport by advection may be more effective than diffusion to facilitate contact (Pinheiro et al., 2015, Babey et al., 2017).

The objective of this work was to understand the impact of water flow on the fate of the pesticide 2,4-D and its degraders, taking into account different initial spatial distributions of the pesticide and soil microorganisms in soil. We have chosen 2,4 D as a model systemic herbicide which is one of the oldest and most widely available herbicides in the world used as a weedkiller on cereal crops, pastures, and orchards (currently, over 1500 herbicide products contain 2,4-D as an active ingredient). 2,4-D is very mobile with low adsorptive capacities and is relatively rapidly degraded and mineralized in soils with half-lives of a few days (Ou, 1984, Smith and Aubin, 1991, Vieublé Gonod et al., 2006b). 2,4-D is mainly degraded by specific microorganisms for which it is a source of C and energy (Fournier, 1980). In addition, the metabolic pathways for the degradation of the compound as well as genes encoding 2,4-D catabolism are well-known.

We hypothesized that co-location of 2,4-D and its degraders is a prerequisite for efficient 2,4-D biodegradation. Diffusive and advective solute transport as well as active transport of microbial degraders towards their corresponding substrate may, however, improve accessibility of microbial substrates in non-co-localized micro-environments. This paper reports the use of repacked soil cores to study pesticide fate at the millimeter scale under irrigation events leading to both diffusive and advective transport.