The effect of humic acids on biodegradation of polycyclic aromatic hydrocarbons depends on the exposure regime Maria-Carmen Tejeda-Agredano a, b , Philipp Mayer b , Jose-Julio Ortega-Calvo a, * a Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS-CSIC), Apartado 1052, E-41080 Seville, Spain b Department of Environmental Science, Aarhus University, P.O. Box 358, 4000 Roskilde, Denmark article info Article history: Received 23 August 2013 Received in revised form 15 September 2013 Accepted 18 September 2013 Keywords: Biodegradation Pyrene Humic acids Passive dosing Synchronous fluorescence abstract Binding of polycyclic aromatic hydrocarbons (PAHs) to dissolved organic matter (DOM) can reduce the freely dissolved concentration, increase apparent solubility or enhance diffusive mass transfer. To study the effects of DOM on biodegradation, we used phenanthrene and pyrene as model PAHs, soil humic acids as model DOM and a soil Mycobacterium strain as a representative degrader organism. Humic acids enhanced the biodegradation of pyrene when present as solid crystals but not when initially dissolved or provided by partitioning from a polymer. Synchronous fluorescence spectrophotometry, scintillation counting and a microscale diffusion technique were applied in order to determine the kinetics of dissolution and diffusive mass transfer of pyrene. We suggest that humic acids can enhance or inhibit biodegradation as a result of the balance of two opposite effects, namely, solubilization of the chemicals on the one hand and inhibition of cell adhesion to the pollutant source on the other. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous con- taminants with recognized toxicity and carcinogenicity potentials. Biodegradation is one of the most direct pathways for dissipation of these chemicals in polluted soils and sediments. However, the microbial availability of PAHs is often limited by their low water solubility and their high tendency of sorption to the solid surfaces of these environmental compartments, which causes slow biodegradation rates and longer persistence (Juhasz et al., 2010; Posada-Baquero and Ortega-Calvo, 2011). These representative hydrophobic organic contaminants (HOCs) also tend to associate to dissolved macro-molecules (such as humic acids or HA) present in the porewaters. This association will in some exposure scenarios lead to a decrease in the freely dissolved concentrations and then likely to lower biodegradation rates, because degrading bacterial cells usually take up dissolved PAHs by diffusion from the sur- rounding aqueous phase (Thomas et al., 1986; Bosma et al., 1997; Alexander, 1999; Wick et al., 2001; Yang et al., 2009). However, in other situations the presence of dissolved organic matter (DOM) can lead to an enhancement in the biodegradation rate of these HOCs (Ortega-Calvo and Saiz-Jimenez, 1998; Haftka et al., 2008; Smith et al., 2009). Indeed, the addition of DOM (such as HA) is currently considered as a valid biostimulation strategy to speed up bioremediation (Plaza et al., 2009; Yang et al., 2011). Various forms of DOM including humic acids can accelerate the biodegradation of PAHs by increasing the apparent solubility and enhancing the diffusive mass transfer of these contaminants, thus promoting their availability to microorganisms (Smith et al., 2009). The molecular form of HA has been described as a micellar microstructure similar to surfactants, which can incorporate dissolved PAHs (Engebretson and Vonwandruszka, 1994). Despite these studies that show the importance of HA in bioremediation, its exact role in the degra- dation of PAHs by microorganisms remains uncertain. The relative contributions to the biodegradation process by the decreases in freely dissolved concentrations, the enhanced diffusion, and the increases in the total load of pollutant present in the aqueous phase due to enhanced solubilization, still remain unknown. The clarifi- cation of these effects can be relevant for predicting the effects of DOM on biodegradation of PAHs in natural and engineered environments. The present study puts forth a new combination of two analytical techniques, synchronous fluorescence spectrophotom- etry and liquid scintillation counting, to approach this question, by determining the freely dissolved concentration (C free ) and the total concentration in the aqueous phase (C total , where C total ¼ C free þ C bound-HA ) of pyrene in different exposure regimes. The combination of these techniques allowed the determination of * Corresponding author. E-mail address: jjortega@irnase.csic.es (J.J. Ortega-alvo). Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol 0269-7491/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.envpol.2013.09.031 Environmental Pollution 184 (2014) 435e442