ARTICLE pubs.acs.org/est
Pyrene Biodegradation in an Industrial Soil Exposed to Simulated Rhizodeposition: How Does It Affect Functional Microbial Abundance? Liang Meng† and Yong-Guan Zhu*,†,‡ †
State Key Lab of Regional and Urban Ecology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China ‡ Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 310621, China
bS Supporting Information ABSTRACT: Rhizodeposition is an important biogeochemical process for the phytoremediation of contaminated substrates. This study investigated the effects of various rhizodeposition components from celery (Apium graveolens) on pyrene biodegradation and microbial abundance in a long-term contaminated soil. Batch microcosms simulating in situ contaminated soil were amended with lipophilic extract, water-soluble extract, or debris from celery root to mimic plant rhizodeposition within 70 days. Soil was intermittently analyzed for pyrene concentration and target gene abundance estimated by real-time PCR. Lipophilic extract was the major simulated rhizodeposit enhancing pyrene biodegradation, while water-soluble extract stimulated microbial growth most efficiently. The relative abundance of total polycyclic aromatic hydrocarbon (PAH) degraders was enhanced by lipophilic extract but inhibited by the other two rhizodeposits, indicating that these components exerted different selective pressures on PAH degrader community. Moreover, PAH catabolic pathway may involve in the pollutant detoxification and fatty acid metabolism by microorganisms, which were also affected by rhizodeposition. These results provide insights into plant-microbe interactions responsible for PAH biodegradation and offer opportunities to facilitate PAH phytoremediation in industrial sites.
’ INTRODUCTION Phytoremediation is a promising and cost-effective strategy to clean up PAH contaminated soils.1 The dominant mechanism for this approach is the stimulation of microbial degradation by rhizodeposition (the release of organic compounds from plant roots into soils), the components of which mainly include root exudates, secretions, mucilages, and root turnover from sloughing and cell death, accounting for 8-56% of net plant-fixed carbon annually.2 Several processes, in which rhizodeposition influences PAH biodegradation, have been proposed to involve enhancing microbial growth, initiating PAH cometabolism, increasing PAH bioavailability, and improving microbial habitat.3 All these can create a general effect altering the abundance of PAH-degrading microbes, mainly due to an overall alteration of total microbial population or a selective pressure on catabolic gene expression in the rhizosphere.4 Mixed results from phytoremediation studies have shown that this alteration is highly variable ranging from repression to stimulation, which is attributed to the synergistic and opposing impacts of various rhizodeposits on PAH degrader population.5 Such fundamental complexity and variation make it difficult to evaluate mechanisms of phytoremediation, and thus, it is important to determine how rhizodeposition components specifically influence degradation capacity of soil microbial community. Recently, some studies have addressed this issue by adding root-derived substances as simulated rhizodeposits. Qiu et al.6 demonstrated that some flavonoids, as secondary plant metabolites generated from root exudates, inhibited benzo[a]pyrene r 2010 American Chemical Society
biodegradation, while Da Silva et al.4 showed that phenolic-rich root extracts obviously stimulated phenanthrene mineralization as well as total oxygenase activity. These researchers discussed their results mainly referring to water-soluble exudates as if they represent all rhizodeposition components. However, in fact, there are no sufficient evidence to demonstrate that other components, such as nonaqueous fractions (e.g., lipophilic secretions and sloughed-off cells), contribute less to microbial responses associated with PAH degradation.7 Moreover, the frequent use of artificial root exudates and freshly spiked soils did not exactly conform to actual rhizosphere conditions in contaminated sites.3,8 Thus, further studies are needed to better understand the complex effect of rhizodeposition on the PAH degradation response in real contaminated soils. The aim of the current study was therefore to assess the overall and specific impacts of different rhizodeposition components on pyrene biodegradation potential, expressed as indigenous microbial abundance (mainly for soil bacteria), in an aged PAHcontaminated soil. Specifically, pyrene residues were compared to evaluate efficiencies of components in PAH biodegradation. Total bacterial (16S rDNA) and PAH-ring hydroxylating dioxygenase (PAH-RHDR) gene copies were regularly measured to identify which component(s) had the dominant effect on soil Received: September 1, 2010 Accepted: December 13, 2010 Revised: November 26, 2010 Published: December 31, 2010 1579
dx.doi.org/10.1021/es102995c | Environ. Sci. Technol. 2011, 45, 1579–1585
Environmental Science & Technology microbial abundance, especially for selective enrichment of PAH degraders. We also monitored the expressions of functional genes responsible for pollutant detoxification (GST) and fatty acid metabolism (fadD8) to characterize the likely mechanisms by which rhizodeposition affects PAH catabolic pathways.
’ MATERIALS AND METHODS Chemicals. Standards of pyrene and fatty acids methyl esters were purchased from Sigma-Aldrich (USA) and were chromatographic grade. All other reagents were of analytical grade or better. Soil. The soil from surface layers was collected in a former coke plant in Beijing. Physicochemical properties and 16 U.S. EPA PAH concentrations for this soil are shown in Supporting Information (SI) Table S1. Pyrene was selected as the model PAH, because it was found at a high concentration (1.89 mg kg-1 dry soil) in this soil and frequently applied in PAH phytoremediation studies. Preparation of Rhizodeposition Components. Celery was grown for 6 months in a greenhouse, and fine roots were collected as sources of root extracts and debris due to their predominance in rhizodeposition.4 The freeze-dried roots were ground into powders as whole root debris and extracted with petroleum ether following the methods of AOAC9 to prepare lipophilic extract. After that, the lipid-free roots were spread out for 60 min in a fume hood to ensure solvent volatilization and then extracted in deionized water to collect water-soluble extract according to the method of Qualls10 with a subsequent step described by Nelson and Lym.11 After the above extractions, root residues were oven-dried at 60 °C for 3 days and ground again to obtain extract-free root debris. These products were stored at -20 or 4 °C (for water-soluble extract) until used in biodegradation studies and analyzed for their characterizations. In this study, celery was selected as the model plant because of its high efficiency in PAH removal,12and rhizodeposition components were grouped based on their physicochemical properties (root release patterns). Specifically, water-soluble and lipophilic extract amendments were intended to mimic the release of aqueous and fatty compounds by living roots, respectively, while root debris were added to simulate natural root death and decomposition. Microcosm Setup. Contaminated soils were sieved (
