Real-Time, in Situ Monitoring of Bioactive Zone Dynamics in Heterogeneous Systems JONATHAN G. DORN, MARK L. BRUSSEAU, AND RAINA M. MAIER* Department of Soil, Water and Environmental Science, The University of Arizona, 429 Shantz Building #38, Tucson, Arizona 85721 Successful implementation of in situ bioremediation is contingent upon understanding how physicochemical and microbial factors affect the formation and dynamics of microbially active regions known as bioactive zones (BAZs). This study demonstrates how a novel fiber optic detection system can be used to test hypotheses concerning real- time, in situ BAZ formation and dynamics. This study focuses on naphthalene transport in saturated porous media containing defined physicochemical and microbial heterogeneities. Biological activity was measured using a lux reporter bacterium, Pseudomonas putida RB1353, that bioluminesces during naphthalene catabolism. Results show that the presence of defined heterogeneities drives the development of BAZs at material-property interfaces where the confluence of naphthalene, dissolved oxygen, and sufficient microbial density is optimal. Thus, despite successful transport of P. putida RB1353 into a sterile low-permeability region containing substrate, BAZ formation in this region was limited by local physicochemical conditions (e.g., naphthalene and dissolved oxygen bioavailability). In another instance, transport of P. putida RB1353 occurred against advective flow, resulting in BAZ formation upgradient of inoculated regions. Defined systems such as this can be used as a basis for predicting localization of activity in complex subsurface systems. Introduction The utility of in situ bioremediation (1, 2) is limited by our lack of understanding of how coupled physicochemical and biological factors influence bioactive zone (BAZ) formation and dynamics. Bioactive zones are regions where the microbial community is sufficiently active to catabolize bioavailable substrates, and successful in situ bioremediation is contingent upon the formation and maintenance of BAZs (3-6). Additional research concerning how coupled phys- icochemical and biological processes influence BAZ forma- tion and dynamics in porous media is still necessary to improve (1) our understanding of how transformation-related nonideality, such as non-steady-state microbial activity, affects contaminant transport and fate and (2) our ability to predict the efficacy of in situ bioremediation in dynamic, heterogeneous environments. Several field- and laboratory-scale studies have been performed to characterize BAZ dynamics in porous media. Brusseau et al. (7) examined the impact of residence time, substrate concentration, and electron acceptor concentration during benzoate biodegradation in hydrocarbon-contami- nated aquifer material. Biodegradation was greatest for longer residence times without limiting substrate or electron acceptor concentrations. Murphy et al. (8) conducted flow- cell experiments to determine the influence of physical heterogeneity on microbial degradation of benzoate. The results indicate that benzoate biodegradation was controlled by the spatial and temporal variations in nutrient flux generated by nonuniform flow within the heterogeneous media. Yarwood et al. (9) monitored bacterial growth dynamics in the presence of glucose under unsaturated flow conditions in a 2-D flow-cell. The temporal and spatial bacterial colonization pattern was found to be dependent upon oxygen availability. Devlin et al. (10) examined field- scale bioactive zone development in response to sequenced bioremediation of mixed contaminants in groundwater and found that nutrient injection stimulated BAZ formation and enhanced bioremediation over the natural attenuation control. Dyer et al. (11) examined the reduction of 1,2- dichloroethane at field-scale in response to injection of methanol, ammonium chloride, and sodium chloride. BAZ formation was limited by inadequate mixing of the carbon substrate within the test zone and clogging of the recharge wells. Odencrantz et al. (3) investigated BAZ formation in response to electron acceptor availability at laboratory-scale and concluded that BAZ size and location were controllable through location-specific electron-acceptor injection. Keijzer et al. (5) developed an analytical approximation, based on contaminant removal rates and BAZs, to characterize in situ aquifer bioremediation performance. This study found that a decrease in electron acceptor concentration or a decrease in flow rate produced a smaller, more efficient BAZ that enhanced in situ bioremediation performance. Additional studies are required to further enhance our understanding of BAZ formation and dynamics, especially for substrates, such as naphthalene, that exhibit limited bioavailability. To address this demand, our laboratory developed a novel, fiber optic detection system that employs a lux bioreporter organism to monitor real-time, in situ microbial activity in porous media at laboratory-scale (12, 13). The fiber optic detection system was recently employed to investigate the dynamics of in situ bacterial activity in response to changing salicylate and electron-acceptor [e.g., dissolved oxygen (DO)] concentrations. The results indicated that the location and size of the BAZs were correlated with salicylate and DO bioavailability (14). The objective of this study is to use the fiber optic detection system to study real-time, in situ BAZ dynamics during naphthalene transport in saturated porous media containing defined physical, chemical, and microbial heterogeneities. Two scenarios are examined. The first represents a situation where a toxic contaminant has sterilized a high-permeability matrix but an adjacent low-permeability matrix still contains a degrading population. The contaminant is present only in the high-permeability zone. The second scenario represents a situation where subsurface injection with a degrading population has successfully inoculated a high-permeability zone but has had varying success with the adjacent low- permeability zone. In this case, the contaminant is located in the low-permeability zone. Experimental Materials Chemicals and Porous Media. Naphthalene (C10H8; EM Science, Gibbstown, NJ) was used as a model polycyclic aromatic hydrocarbon (PAH) because it has a relatively large * Corresponding author phone: (520) 621-7231; fax: (520) 621- 1647; e-mail: rmaier@ag.arizona.edu. Environ. Sci. Technol. 2005, 39, 8898-8905 8898 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 22, 2005 10.1021/es0508626 CCC: $30.25  2005 American Chemical Society Published on Web 10/11/2005