Influence of Low Oxygen Tensions and Sorption to Sediment Black Carbon on Biodegradation of Pyrene

Sorption to sediment black carbon (BC) may limit the aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) in resuspension events and intact sediment beds. We examined this hypothesis experimentally under conditions that were realistic in terms of oxygen concentrations and BC content. A new method, based on synchronous fluorescence observations of ¹⁴C-pyrene, was developed for continuously measuring the uptake of dissolved pyrene by Mycobacterium gilvum VM552, a representative degrader of PAHs. The effect of oxygen and pyrene concentrations on pyrene uptake followed Michaelis-Menten kinetics, resulting in a dissolved oxygen half-saturation constant (K_om) of 14.1 μM and a dissolved pyrene half-saturation constant (K_pm) of 6 nM. The fluorescence of ¹⁴C-pyrene in air-saturated suspensions of sediments and induced cells followed time courses that reflected simultaneous desorption and biodegradation of pyrene, ultimately causing a quasi-steady-state concentration of dissolved pyrene balancing desorptive inputs and biodegradation removals. The increasing concentrations of ¹⁴CO₂ in these suspensions, as determined with liquid scintillation, evidenced the strong impact of sorption to BC-rich sediments on the biodegradation rate. Using the best-fit parameter values, we integrated oxygen and sorption effects and showed that oxygen tensions far below saturation levels in water are sufficient to enable significant decreases in the steady-state concentrations of aqueous-phase pyrene. These findings may be relevant for bioaccumulation scenarios that consider the effect of sediment resuspension events on exposure to water column and sediment pore water, as well as the direct uptake of PAHs from sediments.The aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) constitutes one of the main processes for dissipation of these toxic compounds from polluted soils and sediments. The oxygen dependence of this process has long sustained the belief that the anaerobic conditions usually found in environments such as sediments in estuaries and ports are the main cause of a long persistence of PAH pollution. However, recent findings have demonstrated that microorganisms can also use other electron acceptors, such as nitrate and sulfate, to oxidize PAHs in sediments (30, 33). Less attention has been given to aerobic biodegradation operating at low oxygen tensions. This process may also be important at the interface between anoxic sediments and the overlying waters. Resuspension of PAH-polluted anoxic sediments can result in the exposure of sediment particles to low concentrations of oxygen in the immediately overlying water column, thereby promoting the aerobic biodegradation of PAHs (22) under conditions in which they can be desorbed and taken up by competent microorganisms. The resulting decreases in the aqueous-phase concentration (and in the associated chemical activity) caused by oxygen-limited aerobic biodegradation may be relevant for bioaccumulation scenarios that consider exposure to the water column and sediment pore water, in addition to the direct uptake from sediments (23). Despite its significance, the capacity for prediction of aerobic biodegradation rates of PAHs at low oxygen tensions is still very limited. Whereas the oxygen dependence of fast biodegradation of PAHs in soils and sediments is a well-known phenomenon (5, 17), studies reporting precise measurements of the dissolved oxygen half-saturation constant (K_om) for biodegradation of PAHs—a key modeling parameter—are very scarce (7, 21). The only available estimation for a K_om value of a high-molecular-weight (HMW) PAH (5.9 μM) was provided for pyrene on the basis of growth rates on pyrene of a Mycobacterium strain in a fermenter (7). However, the pyrene concentration chosen (500 mg/liter)—well above the level of its aqueous solubility (0.13 mg/liter) known to support bacterial growth (34)—was not representative of those concentrations present in the environment.Besides the oxygen concentration, another factor that may control the biodegradation of sedimentary PAHs is their bioavailability. Due to their partitioning into sorbents, such chemicals exhibit only weak chemical activity gradients that promote their uptake and transformation by active microbial cells. Hence, the biodegradation rates are likely far below those corresponding to maximum rates, and they may reflect nonlinear biochemical dependencies. Also, these low rates may be due to the lower chemical activity of PAHs causing the microbial acquisition of the aqueous-phase chemicals to become a bottleneck for the biodegradation process (31). Examples of conflicts of bioavailability with biodegradation can be found when PAHs are predominantly sorbed onto solid aggregates (12) and dissolved in non-aqueous-phase liquids (28). Sorption is especially important in sediments. During recent years, the traditional, one-phase organic carbon (OC) partitioning model has been expanded for PAHs and other hydrophobic pollutants to include uptake both into OC and onto the ubiquitous, solid-phase products of incomplete combustion, collectively called black carbon (BC). Therefore, adsorption to BC and absorption to OC would occur in parallel during the sorption process (1, 2, 15). The new model has been useful in understanding field observations of the PAH solid-water distribution coefficient (K_d), which have evidenced a higher sorption capacity than would have been expected on the basis of OC content only (25, 26). Several studies have shown that strong sorption of PAHs to BC may also significantly limit biodegradation. For example, Ghosh et al. showed that 16 U.S. Environmental Protection Agency (USEPA) PAHs associated with carbonaceous coal-derived material present in harbor sediments exhibited negligible biodegradation rates in aerobic sediment slurries, whereas similar conditions led to significant losses (up to 75% after 2 months) of PAHs present in semisolid coal tar pitch (10). Little or no biodegradation was also observed for 3- to 6-ringed PAHs associated with BC-rich street dust added to soils to simulate diffuse pollution (18) and with naphthalene sorbed to granular activated carbon, a material similar to BC in its physicochemical characteristics, in suspensions of two different bacterial species with dissimilar modes of acquisition of the sorbed compound (14). Finally, Rhodes et al. examined the effect of BC on bioavailability of phenanthrene in soils (32). They found that the addition of BC to soils caused a significant decrease both in the total extent of mineralization and in extractability by the use of cyclodextrin solutions (32). Despite these advances in the field, it is still uncertain whether sorption to BC causes the sequestration of PAHs or whether their microbial assimilation is still possible, although at a very low rate. This gap in knowledge is a major limitation in predicting the fate of these chemicals in many contaminated sediments, making it difficult to achieve a proper perception of the risks posed by resuspensions in overlying waters and bioturbated sediment beds.We considered that sorption to sediment BC may limit the aerobic biodegradation of aqueous-phase PAHs such as pyrene and examined this hypothesis experimentally under conditions that were realistic in terms of concentrations of oxygen and suspended-solids typical for sediment resuspension events. For this aim, we developed a new method, based on synchronous fluorescence observations of ¹⁴C-pyrene, for both measuring the rates of uptake of dissolved pyrene at low oxygen concentrations by a representative PAH-degrading bacterial strain and simultaneously assessing the appearance of ¹⁴CO₂. The method also allowed us to characterize the evolution of aqueous-phase pyrene during biodegradation in initially equilibrated suspensions of sediment with a known content in black carbon. The information obtained experimentally was integrated in model calculations of the evolution of aqueous pyrene concentrations in sediment suspensions. To our knowledge, this is the first report connecting these two major factors—oxygen limitation and sorption to BC—in the biodegradation of HMW PAHs.