Biodegradation of2,4,6-Tribromophenol duringTransport in Fractured ChalkS H A I A R N O N , * , † , ‡ E I L O N A D A R , † , ‡

Z E E V R O N E N , † A L I N E J I D A T , †

A L E X A N D E R Y A K I R E V I C H , † A N DR O N I T N A T I V §

Department of Environmental Hydrology & Microbiology,Zuckerberg Institute for Water Research, J. Blaustein Institutesfor Desert Research, Ben-Gurion University of the Negev,Sede-Boqer 84990, Israel, Department of Geological andEnvironmental Sciences, Ben-Gurion University of the Negev,Beer-Sheva 84105, Israel, and Department of Soil andWater Sciences, The Hebrew University of Jerusalem,Rehovot 76100, Israel

The effect of physicochemical conditions (residence time,oxygen concentrations, and chalk characteristics) onthe biodegradation of 2,4,6-tribromophenol (TBP) duringtransport was investigated in low-permeability fractured-chalk cores. Long-term (∼600 d) biodegradation experimentswere conducted in two cores (∼21 cm diameter, 31 and44 cm long, respectively), intersected by a natural fracture.TBP was used as a model contaminant and as the solecarbon source for aerobic microbial activity. Bacterial isolateswere recovered and identified by both Biolog identificationkit and 16S rDNA sequences from batch enrichmentcultures. One of the strains, with 98% similarity (based onthe 16S rDNA data) to Achromobacter xylosoxidans,was shown to have the ability to degrade TBP in thepresence of chalk. The decrease in TBP concentrationalong the fracture due to biodegradation was not affectedby reducing the residence time from 49 to 8 min. Incontrast, adding oxygen to the water at the inlet andincreasing the flow rates improved TBP removal. Althoughthe matrix pore-size distribution limits microbial activityto the fracture void, the chalk appears to provide an excellentenvironment for biodegradation activity. Approximately90% of TBP removal occurred within 10 cm of the TBPsource, indicating that in-situ bioremediation can be usedto remove organic contaminants in low-permeabilityfractured rocks if nutrient-delivery pathways within theaquifer are secured.

IntroductionBiodegradation of dissolved organic contaminants in ground-water has been studied extensively over the past 20 yr.

However, records of biodegradation processes in low-permeability porous fractured rocks are scarce. Naturalattenuation and in-situ bioremediation in low-permeabilityfractured aquitards appear to constitute an attractive treat-ment scheme for contaminated groundwater, as moreconventional remediation techniques such as pump-and-treat are not feasible. A selection of batch studies havedemonstrated the potential for the biodegradation of syn-thetic organic contaminants in the presence of chalk (1-3).Other studies by Yager et al. (4) and Lenczewski et al. (5),using biochemical and geochemical markers, have demon-strated the biodegradation of various organic contaminantswithin saturated fractured sedimentary rocks under naturalconditions.

Where biodegradation occurs during transport, the phys-icochemical conditions in the aquifer must be considered inaddition to biological factors. Among the former are residencetime, electron acceptor/donor concentrations, and the rock’sphysicochemical characteristics. Whereas these parametersand their impact on biodegradation have been studied inporous media, information on fractured media is scarce inthe literature. For example, Brusseau et al. (6) and Vayenaset al. (7) have shown that increasing the flow rate (i.e.,increasing velocity and decreasing residence time) reducesbiodegradation, probably due to less contact time betweenthe substrate and the microorganisms. This might be crucialin fractured media as the reported velocities are higher thanthose in porous media under the same hydraulic gradients.On the basis of hydraulic and tracer experiments (under bothnatural and forced gradients), typical reported velocities insedimentary fractured media range between 0.01 and 2200m/d (4, 8-11). The influence of physical heterogeneity onmicrobial degradation activity and distribution can also playa major role, as shown by Murphy et al. (12). In their studyon heterogeneous porous media, the physical heterogeneityresulted in uneven oxygen distribution, which influencedbiodegradation activity. Because fast- and slow-flow regions(high and low hydraulic conductivities) are a main charac-teristic of low-permeability fractured rocks, where flow occursmainly in preferential flow paths (e.g., refs 13-15), the masstransport of electron acceptors/donors and other nutrientsis expected to be affected (16, 17). Flow in distinct channelsmay result in small surface area available for biofilmdevelopment and restrict bacterial activity due to limitedsubstrate and nutrient distribution to the low-permeabilityregions.

In addition to the aforementioned physical characteristicsof the flow and the structure of the geological medium, thechemical characteristics of the organic solute are importantwhen biodegradation is being considered. Solubility (18),structure (aliphatic vs aromatic) including functional groupssuch as halogens (19, 20), toxicity (21), and partition (sorption)to the solid phase all affect a compound’s bioavailability andbiodegradability (22, 23).

This paper focuses on the effects of residence time andoxygen concentration on the biodegradation of 2,4,6-tri-bromophenol (TBP), a model organic contaminant, in low-permeability porous fractured chalk. Chemical and physicalaspects of the chalk and TBP are discussed in the context ofbiodegradation. These were examined mainly by monitoringbiodegradation in column experiments for ∼600 d, undervarious flow conditions and oxygen concentrations. Althoughthe possible use of natural attenuation or bioremediation toclean up low-permeability fractured rocks has been dem-onstrated by other investigators on a field scale, the variousbiodegradation processes and the factors controlling them

* Corresponding author present address: Department of Civil andEnvironmental Engineering, Northwestern University, 2145 SheridanRd., Evanston, IL 60208-3109; phone: (847)467-4980; fax: (847)491-4011; e-mail: s-arnon@northwestern.edu.

† Department of Environmental Hydrology & Microbiology, Ben-Gurion University of the Negev.

‡ Department of Geological and Environmental Sciences, Ben-Gurion University of the Negev.

§ Department of Soil and Water Sciences, The Hebrew Universityof Jerusalem.

Environ. Sci. Technol. 2005, 39, 748-755

748 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 3, 2005 10.1021/es0491578 CCC: $30.25 2005 American Chemical SocietyPublished on Web 12/21/2004

on the single-fracture scale must be better understood toefficiently use these methods.

Materials and MethodsBiodegradation of TBP was determined by measuring itsdecreases in concentration along with increases in Br-

concentration. The sole source of Br- in the experimentswas TBP dehalogenation, as each mol of dehalogenated TBPreleases 3 mol of Br-. TBP (C6H3Br3O) has a molar weight of330.8 g/mol (21.7% carbon by weight), an aqueous solubilityof 714 mg/L (24), a pKa of 6.25, and a log Kow of 4 (25).

Experimental Apparatus. Two cores, each bisected by asingle fracture, were obtained from chalk bedrock in thenorthern Negev Desert, Israel. The cores’ and the fractures’dimensions appear in Table 1. The cores were saturated undervacuum using degassed artificial groundwater (AGW) withthe following composition: 3700 mg/L Cl-, 1030 mg/L SO4

2-,245 mg/L HCO3

-, 340 mg/L Ca2+, 200 mg/L Mg2+, 2100 mg/LNa+, and 22 mg/L K+ (pH 8 ( 0.2). This composition is similarto that of natural groundwater in the northern Negev’s chalkaquitard (26) and was used to minimize dissolution/precipitation processes.

Figure 1 presents a schematic illustration of the experi-mental apparatus. Each fractured core was wrapped withTeflon tape before it was fixed with epoxy cement (Duralite)inside a PVC casing. Teflon inlet and outlet chambers wereattached to each side of the flow boundaries of the fracturewhile the other two boundaries were sealed with epoxycement to create no-flow boundaries. The chamber’s volumewas relatively large (∼20 mL) to prevent clogging. Fourstainless steel injection ports (1 mm i.d.) were insertedthrough the inlet chamber into the fracture void, 2 cm fromthe inlet boundary. The outlet chamber was internally dividedinto four equal sections, and the flow coming through themwas collected in one vessel using Teflon tubes and a manifold.A pair of piezometers/sampling ports (Teflon tubes, 1 mmi.d.) were inserted approximately 5 cm from the no-flowboundaries, every 10 cm from the inlet toward the outlet ofthe core, and 10 cm apart from each other (i.e., six piezometersfor core 2 and four for core 3). The piezometers were preciselyterminated at the fracture surface, thereby minimizinginterference with flow and transport within the fracture void.The experiments were conducted with the fractures in avertical position, similar to the in-situ fracture orientations(27), while the flow therein was horizontal.

Experiments in cores 2 and 3 were run for 590 and 630d, respectively, and were divided into two phases. Whereasthe conditions prevailing during phase 1 were comparableto those observed in a contaminated chalk aquitard (27, 28),the conditions during phase 2 mimicked an in-situ biore-mediation scenario, characterized by higher fluid velocityand organic load and enhanced oxygen and nutrient supply.The injection procedure during phase 1 (preliminary experi-ments) involved pump 1 (MP3, Gilson), which delivered thesubstrate solution (AGW with 50 mg/L TBP and ∼7.5 mg/Loxygen) at relatively slow flow rates (1.3-7.8 mL/h) into theinlet chamber (Figure 1), while the fluid exited the fractureunder constant hydraulic head. Pump 2 (7554-60, ColeParmer) was used to circulate the inlet-chamber solution, toensure constant concentrations along the boundary. Phase1 lasted 230 and 400 d in cores 2 and 3, respectively. During

this phase, TBP and oxygen concentrations were measuredon a weekly basis. Between days 60 and 120, oxygen couldnot be measured due to a technical problem.

In phase 2, pump 1 was used to deliver AGW (withoutsubstrate), while pump 3 (MP3, Gilson) was used to simul-taneously inject the substrate solution (300 mg/L TBP, 25mg/L K2HPO4 and 3.7 mg/L NH4Cl dissolved in AGW) directlyinto the fracture. The overall flow rate was larger than inphase 1 (8-48 mL/h), while the flow-rate ratio betweenpumps 1 and 3 was kept at 2:1 to avoid substrate backflowinto the inlet chamber. Pump 2 was used to circulate theinlet-chamber solution as in phase 1. In addition, oxygenwas gently bubbled above the inlet chamber into thecirculating AGW. Surplus bubbles were released to theatmosphere through the inlet-chamber piezometer while theAGW, enriched with oxygen (∼20 mg/L without bubbles),was re-circulated into the inlet chamber through its bottom.Phase 2 lasted approximately 200 additional days. TBP, Br-,and oxygen concentrations were measured at the inlet andoutlet of the fractures twice a week and at the piezometersonce a week. The temperature of the solutions was kept at23 ( 2 °C, representing average annual groundwater tem-peratures (unpublished data).

Flow Characterization. To characterize the flow in thefractures, AGW was injected at the inlet of each core, and itseffluent flow rate was measured under different gradients.This experiment was conducted every 1-2 months duringphase 1 (data not shown) and every 10 d during phase 2. Theflow rates were measured gravimetrically at 1-10-minintervals ((0.02 mL) under steady-state conditions and wererepeated 5 times for each gradient. Throughout the entireexperiment, the flow rates were controlled by injection pumpand maintained constant for every test in both cores (Figure1). The hydraulic gradients, ranging from 0.003 to 0.03, weremeasured with a Foxboro differential pressure transducer(IDP10), calibrated for the range of 0-12 mm ((0.05 mm).Under these conditions, the flow rates ranged between 0.1and 50 mL/min.

The flow results were used to calculate the fracturetransmissivity, T (L2/T), based on the cubic law (29):

where 2b is the fracture aperture (L), ν is the kinematicviscosity (L2/T), g is the acceleration of gravity (L/T2), and Ks

is the saturated hydraulic conductivity of the fracture (L/T).The fracture transmissivity was calculated by plotting the(measured) flow rate versus the (set) hydraulic gradients anddividing the linear slope (i.e., fracture conductance) by thefracture width.

Assuming that the cubic law is valid for the range in whicha linear relationship exists between the flow rate and thehydraulic head, the equivalent hydraulic aperture (2bh) canbe calculated from the linear slope obtained by plotting theleft-hand side of eq 2 versus the hydraulic gradients:

where Q is the flow rate (L3/T), W is the width of the fracture(L), ∆h is the hydraulic-head drop along Z (L), and Z is thefracture length (L).

Piezometer Sampling. The six and four piezometers (incores 2 and 3, respectively) were arranged in pairs, penetratinginto the upper and lower halves of the cores (core 3 hadthree active piezometers and one blocked piezometer,probably due to direct contact with the opposite fracturesurface). The sample volume needed for TBP, Br-, and oxygenanalyses was ∼2 mL. On the basis of the equivalent hydraulic

TABLE 1. Core and Fracture Dimensions

sample

corediameter

(cm)

corelength(cm)

fracturelength(cm)

fracturewidth(cm)

initial equivhydraulic

aperture (µm)

core 2 21 44 44 21 495core 3 21 31 31 18 207

T )(2b)3g

12ν) Ks2b (1)

Q12νWg

) (2bh)3∆hZ

(2)

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apertures (determined on the basis of flow experiments andthe cubic law), the initial volume of the fracture void rangedfrom 46 to 12 mL. Thus, extracting 2 mL from each piezometertapping the aforementioned fractures required about one-quarter and one-half (for the six- and three-piezometer setup,respectively) of the total void volume. The volume extractedfor the samples was immediately replenished with waterthrough the inlet chamber. With time, the fracture volumesdecreased due to clogging, resulting in increased relativevolume withdrawn for sampling. The fracture volume wasevaluated by multiplying the length, width, and equivalenthydraulic apertures of the fracture. Because the calculatedvalue of the equivalent hydraulic fracture aperture (eq 2)was used to estimate the fracture volumes, the latter mayhave been erroneous. However, a clear trend of reduction infracture volume was observed.

To minimize mutual effects of adjacent sampling ports,sampling was performed while the outlet valve was shut offand a free surface of water existed only above the inletchamber. Consequently, when samples were extracted, thewater was collected mostly from the space between thepiezometer’s tip and the inlet. However, the exact areaaffected by the sampling procedure could not be estimateddue to variable aperture distribution within the fracture.Sampling consisted of simultaneous extraction from eachpair (upper and lower) of piezometers and advanced fromclose to the inlet chamber toward the outlet chamber. Becausethe samples were withdrawn simultaneously from adjacentzones within the narrow fracture, it was important to confirmthat a sample collected from the area of the upper piezometerwas not withdrawn from the area of the lower piezometerand vice versa. A difference between the two samples wouldindicate that they were collected from different zones. Oxygenserved as a test parameter for the impact of simultaneouslysampling each pair: as long as it was lacking in onepiezometer and measurable in the other (within the samepair), the mutual effect of the simultaneous pumping at eachpair was assumed negligible.

Isolation and Characterization of TBP-Degrading Bac-teria. Water samples from the inlets and outlets of cores 2and 3 were collected 120 d after the core experiments werebegun and were used for the enrichment and isolation ofTBP-degrading bacteria. The sample (10 mL) was inoculatedinto 90 mL of mineral medium (MM) containing TBP (100

mg/L) and yeast extract (50 mg/L). The MM contained saltsand microelements as described by Nejidat et al. (3). Fromour previous studies on TBP-degrading bacteria (19), wefound that the yeast extract supplements some micronu-trients that enable TBP biodegradation. TBP degradation wasconfirmed by HPLC measurements. After two repeat transfers,the culture was streaked onto solidified MM (1.5% agar, Difco,Detroit, MI). Repeated streaking on the same medium servedto purify colonies with varied morphologies. The purity ofthe isolated cultures was tested on R2A agar (Difco). In total,nine isolates were tested for dehalogenation of TBP in thepresence and absence of yeast extract with strain TBPZ (19)used as a positive control. The isolates were further char-acterized using the Biolog system. Three isolates that werepositive for TBP biodegradation and one strain that wasnegative were also characterized based on partial 16S rRNAgene sequence.

Batch Biodegradation Experiments. The potential forTBP biodegradation by bacteria that had been obtained fromthe fractured chalk cores was evaluated independently inbatch experiments. Sterile flasks with 100 mL of MM wereautoclaved (121 °C, 20 min) prior to their use. Triplicates ofthe following treatments, each containing 105 mg/L TBP,were used: (A) noninoculated MM (control), (B) inoculatedMM, (C) inoculated MM with 10 g of crushed chalk, and (D)noninoculated MM with 10 g of crushed chalk (control). Thechalk was sieved (<0.125 mm) and sterilized (autoclaved at121 °C, 20 min) prior to its use. The inoculation was performedusing 0.5 mL of water collected from the outlet of core 2 after120 d of continuous TBP injection (see next section). Theflasks were shaken at 200 rpm, 25 °C, and the concentrationsof TBP and Br- (the degradation product of TBP) weremeasured after 30 d.

DNA Isolation, PCR Amplification, and DNA Sequencing.Genomic DNA from pure cultures was extracted as describedby Ausubel et al. (30). A DNA fragment (323 bp) from the 16SrRNA gene was PCR-amplified using two primers specific fordomain Bacteria (31). The primers were Bac1070f (5′-ATGG-CTGTCGTCAGCT-3′) and universal 1392r (5′-ACGGGCG-GTGTGTAC-3′). PCR amplification (initial denaturation stepof 94 °C for 4 min and 30 cycles of denaturation at 94 °C for1 min, annealing at 53 °C for 1 min, elongation at 72 °C for2 min, and a final elongation step for 5 min) was carried outin 50-µL volumes [10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.1

FIGURE 1. Schematic configuration of the experimental setup for the biodegradation experiments (for the core dimensions see Table 1).

750 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 3, 2005

mM MgCl2, 0.01% gelatin, 200 µM each of four deoxynucle-otide triphosphates, 25 pmol of each primer, 1 µL of templateDNA, and 1 unit of REDTaq DNA polymerase (Sigma, St.Louis, MO)] using a thermocycler (Minicycler, MJ Research,Watertown, MA). A single band of the expected size wasobtained when PCR products were visualized in ethidiumbromide-stained 1.5% agarose gels. DNA bands were carefullyexcised under UV, and DNA was extracted from the gel slicesusing the NucleoSpin Extract kit (Macherey-Nagel, Duren,Germany) and cloned in plasmid pTZ57R using the InsT/APCR Product Cloning Kit (MBI Fermentas, Hanover, MD).DNA sequencing was performed with an ABI Prism 377 DNAsequencer (Perkin-Elmer) at Ben-Gurion University of theNegev (Equipment Center, The Institute for Applied Bio-sciences). Sequences were analyzed using the BLAST (32)similarity search program in order to find the most similaravailable database sequences. The sequences obtained fromthis study were deposited in the GenBank database andassigned the accession numbers: AY189752, AF5408402, andAF394171.

Chemical Analysis. TBP was analyzed using high-performance liquid chromatography (HPLC; Kontron). Analy-sis error was estimated at (2%, and a concentration of 0.2mg/L was considered to be the lower detection limit.

Oxygen was measured with a flow-through microelectrode(Microelectrodes, Inc., model 16-730). Analysis error wasestimated at (0.2 mg/L.

Bromide concentrations were measured using a modifiedversion of method 4500-Br- B (33) with a Hitachi U-2000spectrophotometer. The procedure was scaled down to asample size of 5 mL. This modification was necessary tomeasure samples collected from within the fracture void.More details regarding these analyses can be found in Arnon(34).

Results and DiscussionBatch Biodegradation Experiments. Following 30 d ofincubation, TBP biodegradation was evident only in thepresence of chalk (Table 2, treatment C). In the noninoculatedflasks (treatments A and D) as well as in the chalk-freeinoculated flask (treatment B), neither the appearance ofBr- nor the disappearance of TBP were detected. Superiormicroorganism activity in the presence of chalk had beenpreviously shown by Nejidat et al. (3) in his study on thebiodegradation of TBP by Achromobacter piechaudii, strainTBPZ, although the exact reason for this remains unclear. Intreatment C, calculations of mass balance between Br-

concentration and the concentration expected from a sto-ichiometric release from 105 mg/L TBP (72% Br-) indicatedthat full dehalogenation had occurred. The results from thisexperiment demonstrate the potential for TBP biodegradationin the presence of chalk, as will be discussed later. Br- wasused as an indicator for TBP biodegradation because, basedon the metabolic pathway for trihalophenols described byPadilla et al. (35), our HPLC analysis of the culture supernatantdid not reveal accumulation of the expected quinones orquinol.

Characterization of TBP-Degrading Bacteria. Charac-terization of the different isolates obtained from the coreeffluents suggested that all of them are related to theAlcaligenes-Achromobacter family, known to degrade halo-genated compounds (Table 3). Although the phenotypicobservation (including Biolog identification) suggested con-siderable differences among the isolates, analysis of theirpartial 16S rRNA gene sequence revealed limited diversityamong them. Some of the closely related (98% similar)sequences were of bacteria that are involved in the biodeg-radation of organic compounds. For example, isolate 3 wassimilar to a sequence from an uncultured bacterium foundwhere the natural attenuation of trichloroethylene in frac-tured shale bedrock had been observed (5). Furthermore,Ronen et al. (19) had previously found TBP-degradingAchromobacter piechaudii in soils overlying the chalkformations where the cores had been obtained. Nevertheless,analysis of the 16S rDNA sequences of that strain and thecurrent isolates showed that they are not closely related.

Biodegradation of TBP during Transport. The analysisof biodegradation during transport in the fractured chalk isbased on the results obtained during phase 2 of theexperiments. This is because during phase 1, TBP was partiallydegraded in the inlet chamber, imposing uncontrolled TBPand oxygen concentrations at the inlet boundary. Duringphase 2, TBP was injected directly into the fracture toovercome the unintended partial biodegradation that hadoccurred during phase 1, while AGW was introduced via theinlet chamber (Figure 1). Due to this injection procedureand consequent mixing between the TBP solution and theAGW, the exact inlet concentrations could not be determined.However, assuming full mixing of AGW with the TBP solution(with known flow rate and TBP solution concentration), anestimate of 95 ( 3 mg TBP/L was obtained. This was theexpected TBP concentration at the outlet if no attenuationoccurred during its transport (Figure 2). This value was alsoused as the inlet concentration considered for the modeling(the results of which are presented in Figure 2).

TBP was considerably attenuated in the fractured cores(i.e., a large difference was observed between the expectedand measured concentrations at the fracture outlet) duringphase 2, as shown in Figure 2 (the results for phase 1 arepresented in the Supporting Information, Supplement 1).TBP loss to the atmosphere was considered insignificant, asindicated by the constant TBP concentrations in the feedingflasks (measured on a weekly basis, while new solutions werereplaced approximately every 3 weeks). Chemical degradationappeared to have little, if any, impact on TBP attenuation,as batch experiments showed no TBP degradation in flaskswith or without sterile chalk (Table 2). In addition, TBP wasfound to only slightly adsorb to chalk (distribution coefficient,Km ) 0.1 mL/g), as determined in batch adsorption experi-ments at pH 7.6 after equilibrium had been reached (>24 h)(25). Conversely, diffusion has been shown to play a majorrole in solute transport within fractured porous rocks (36)and specifically in fractured chalk from the study area(17). In a preliminary experiment during which TBP andBr- (as a nonreactive tracer) were injected into a sterilefracture at flow rates of 1.3 mL/h, we found an effectivediffusion coefficient (De) of TBP in chalk of 6.75 × 10-7 (cm2/s). After 2 months of continuous injection (with a biocide,sharomix-mci 30 mg/L, to prevent microbial activity), 30%of the injected mass was retained in the fractured core dueto diffusion and adsorption (34). Given the low distributioncoefficient of TBP (0.1 mL/g), it may be concluded thatdiffusion, and to a lesser extent adsorption, are the non-biological causes responsible for TBP attenuation in thefractured chalk.

The total amount of TBP degraded within the fracturewas estimated by comparing the experimental breakthrough

TABLE 2. Observed Dehalogenation of TBP Following 30 d ofIncubation

(A)nonin-

oculatedMM

(B)inoculated

MM

(C)inoculatedMM with

chalk

(D)noninoculated

MM withchalk

TBP disappearance - - +a -Br- formation - - +b -

a TBP completely disappeared from the solution. b Complete deha-logenation was observed, based on mass-balance calculations (eachmol of TBP releases 3 mol of Br-).

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curve (BTC) and the BTC predicted by a mathematical modeldescribing solute transport through a single fracture formedbetween two parallel, porous plates (37). Contaminanttransport in such a fracture is controlled by advective anddispersive fluxes, diffusion from the fracture into the porousmatrix, and sorption onto the solid phase within the fracture-matrix system. No chemical decay or microbiological deg-radation was accounted for in these calculations (more detailsregarding the mathematical formulations are included in theSupporting Information, Supplement 2). As an analyticalsolution is unavailable for the case of temporally variableflow velocity and inlet concentration, a numerical solution(38) was used for the simulations. Comparison of theexperimental BTC (affected by diffusion into the matrix,adsorption, and biodegradation) with the theoretical BTC(affected by only diffusion and adsorption) enabled anestimation of biodegraded TBP.

In addition to the expected (no attenuation) and measuredTBP concentrations at the outlet of core 2, Figure 2 illustratesthree simulated BTCs based on the parameters presented inTable 4. The values of the parameters measured directly inthe fractured chalk (Km, θ, Fb) were the same for all threesimulations. The tortuosity (τ) was obtained by inversemodeling during a 2-month-long tracer experiment con-ducted at a flow rate of 1.3 mL/h in a sterile fractured chalkcore with Br- as the nonreactive tracer (34). The values ofRL and 2b in the three simulations were also identical, as itwas shown by Arnon (34) that, in the long term (>50 d),these parameters have only a minor effect (<1 mg/L) onsolute concentrations.

Whereas the parameters used in simulation 1 wereobtained experimentally (Table 4), simulations 2 and 3differed by (1 order of magnitude in the De value with respectto that of simulation 1, representing two extreme casesconsidered to be the limits of our parameter estimation.

If biodegradation is negligible, the simulated concentra-tions should be close to the experimentally measuredconcentrations at the outlet. Following this logic, the largedifferences between the simulations and the experimentallymeasured TBP concentrations leave no doubt regarding thesubstantial amount of biodegraded TBP. The comparisonbetween the biodegraded TBP and the measured Br- basedon the stoichiometric release of Br- suggests that fulldehalogenation occurred (34).

After ∼100 d of operation in phase 2 (day 440), thepredicted outlet concentrations suggested that diffusion and

TABLE 3. Characterization of the Bacterial Isolates Using Biolog Identification and 16S DNA

TBPdebrominationmicroscopic

characterizationcolonycharacteristics

isolate color border morphologyGramstain

withyeast

extract

withoutyeast

extractBiolog

ID (similarity)

blast results(similarity) and

GenBank Accession No.

1 yellow regular Bacillus negative + - Sphingomonas paucimobilis(0.227)

Achromobacter xylosoxidans(0.98) AY189752

3 white regular short Bacillus negative + - Alcaligenes denitrificans(0.225)

uncultured bacterium clone I2(0.98) AF5408402

5 beige regular Bacillus (longand fat)

negative + - Alcaligenes denitrificans(0.820)

Blackwater bioreactor bacteriumBW6 (0.98) AF394171

TBPZ white regular Bacillus negative + - Alcaligenes denitrificans(0.55)

Achromobacter piechaudii(100) AF237784

FIGURE 2. Measured and simulated TBP concentrations during its transport and degradation in core 2 during phase 2.

TABLE 4. Physical and Chemical Parameters Used for TBPTransport Simulations

parameter simulation 1 simulation 2 simulation 3

effective diffusion coeff,De (cm2/s)

6.75 × 10-7 a 6.75 × 10-8 6.75 × 10-6

fracture aperture, 2b (µm) 500a 500 500dispersivity, RL (cm) 1a 1 1tortuosity, τ 0.27a 0.27 0.27matrix porosity, θ 0.28a 0.28 0.28distribution coeff,

Km (mL/g)0.1b 0.1 0.1

flow velocity, u (cm/h) varied with flow rate and aperture

a Arnon (34). b Wefer-Roehl et al. (25).

752 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 3, 2005

adsorption had become negligible for all three simulationsand thus, all of the TBP attenuation could be attributed tobiodegradation. Biodegradation activity within the fracturedchalk cores is assumed to be limited to the fracture voidbecause the size of more than 99% of the pores is smallerthan 0.4 µm (39), whereas the average bacterial size is 1 µm.Indeed, no bacteria were detected in the chalk samplescollected ∼3 mm perpendicular to the fracture surface aftertermination of the experiment (determined by vigorousmixing of a buffer solution and chalk and plate-counting onR2A agar). The relatively low TBP adsorption to the secondaryminerals formed on the fracture surface (Km ) 0.05 mL/g;25) may account for some of the reduction in its bioavail-ability, and consequently for some of the limitations to thebiodegradation. However, extensive TBP diffusion into thematrix was by far the major limiting process with respect toits biodegradation, as discussed earlier in the text.

The cumulative mass of removed TBP is presented inFigure 3, along with the accompanying reduction in relativetransmissivity (T/T0, where T0 is the fracture transmissivitybefore the beginning of the experiments). The amount ofbiodegraded TBP was calculated, following Figure 2, as theconcentration difference between the experimental BTC andthat predicted by simulation 1. This is a conservativeestimation, because for both BTCs (experimental andtheoretical) the TBP masses absorbed by the porous matrixwere taken as equal. However, when biodegradation occurs,the TBP concentrations in the fracture and its gradients acrossthe matrix are smaller than those accounted for in thesimulations (where biodegradation is ignored), resulting instronger matrix diffusion of TBP and lower theoreticalconcentrations at the outlet. Thus, the above estimate ofbiodegraded TBP should be considered the lower theoreticallimit of the actual biodegraded mass.

A link between the rate of TBP removal and the reductionin transmissivity is clearly seen, especially after day 420, whenthe relative transmissivity dropped drastically (g90%) fol-lowing a sharp increase in TBP mass removal (Figure 3).Although TBP concentrations were similar in cores 2 and 3(inlet and outlet, respectively), its removal was enhanced incore 2 because the flow rate in that core was ∼2.5-fold larger(Table 5 after day 420). The relative reduction in thetransmissivity of core 3 was similar to that in core 2 (Figure3), despite the lower TBP removal, probably because its initialaperture was much smaller than that of core 2 (Table 1).Moreover, the average reduction in the equivalent hydraulicaperture per TBP mass consumption was similar for cores2 and 3: 28.53 and 25.48 mg of TBP per 1 µm reduction,respectively.

TBP removal improved after day 420 as a result ofincreased nutrient and oxygen supply and the increased flowrates (Table 5), as discussed in the next section. Thetransmissivity reduction was due to clogging by bacteria andextracellular polymeric substances (EPS), as visualized in SEM

micrographs (34). This further supports the notion thatbacteria biodegraded TBP and used its carbon to producebiomass.

Effect of Residence Time on TBP Biodegradation. Theincreasing flow rates during phase 2 (Table 5), withoutdeteriorating TBP biodegradation (Figure 2), was the majorcause for the significant increase in mass removal (Figure 3).In addition to the experimentally imposed changes in theflow rates, residence time decreased as a result of fractureclogging and the use of constant flow rate, as displayed inTable 6, when clogging became the dominant process (day420 onward).

Despite the decrease in residence time (increasing flowrates until day 420 and clogging after day 420), the amountof biodegraded TBP fluctuated between 20 and 60 mg/L withno clear trend. This observation suggests no correlationbetween TBP biodegradation and residence time, at leastwithin the range of the calculated residence times (8-49min), and differs from previous studies reporting a dete-rioration of biodegradation with decreasing residence time(e.g., refs 7 and 40). It is expected, however, that if theresidence time were further reduced, biodegradation ef-ficiency would eventually deteriorate.

The calculated residence times of 8 and 49 min (cores 3and 2, respectively) translate to average velocities of 56 and13 m/d, respectively. It should be noted that the hydraulicgradients generating these velocities were comparable to thevalues usually observed in fractured aquitards (8-10, 27).

Effect of Oxygen on Biodegradation of TBP. TBP wasnot fully degraded within the fracture (Figure 2) while theoutlet oxygen concentrations rarely exceeded 1 mg/L, whichfor obligatory aerobic bacteria implies that anoxic conditionsdeveloped along the fracture (41). During phase 1, the inletoxygen concentrations ranged between 2 and 5 mg/L,averaging 3.6 mg/L; during phase 2, the oxygen concentra-tions in the inlet chamber and in the nutrient solution werekept at 23 ( 2 and 7.5 ( 0.5 mg/L, respectively. Figure 4illustrates the rate of TBP removal along with the rate ofoxygen consumption. When the entire data set is examined

FIGURE 3. Cumulative removed TBP mass and the changes in relativetransmissivity.

TABLE 5. Flow Rates during the Biodegradation Experiments:Phases 1 and 2

flow rate(mL/h) core 2 (d) core 3 (d)

1.3 0-240a 0-345 (not active from 345 to 400)a

3 240-335a

7.8 335-390b 400-421b

12 390-420b

20 421-590b

48 420-520b

a Phase 1. b Phase 2.

TABLE 6. Residence Time during the BiodegradationExperiments during Phase 2

core day

equivhydraulicaperture,2b (µm)a

relativetransmissivity

(T/To)b

fracturevolume(mL)c

flowrate

(mL/h)

residencetime

(min)d

core 2 420 425 0.64 39.3 48 49.1core 2 520 262 0.1 24.2 48 30.3core 3 420 182 0.66 10.2 20 30.46core 3 590 50 0.017 2.8 20 8.37

a Calculated using the cubic law. b Calculated from the flow experi-ments (see Materials and Methods). c The fracture volume was calcu-lated by multiplying the length, width, and equivalent hydraulicapertures of the fracture. d The residence time was calculated as themeasured flow rate divided by the fracture volume.

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(phases 1 and 2), the increase in oxygen consumption uponTBP biodegradation is clearly seen. Nevertheless, duringphase 1, no link was found between the biodegraded TBPand the amount of oxygen consumed. Conversely, duringphase 2, a relationship between TBP biodegradation andoxygen consumption was evident, though the relatively weakcorrelation (R2 ∼ 0.6) suggests that other factors must alsobe affecting biodegradation. The poor correlation betweenoxygen consumption and TBP removal can also be explainedby the presence of other microorganisms which are incapableof degrading TBP but do use up some of the oxygen. Thecrucial role of oxygen for biodegradation was also apparentaround day 380 (Figure 2), when a short failure in oxygensupply caused an immediate decline in the amount ofdegraded substrate, while the recovery to previous levels tookabout 1 week.

A comparison of the data from phases 1 and 2 clearlyshows that increasing available oxygen increases the amountof substrate consumption. Though it was difficult to increasethe inlet oxygen concentration, a further increase in TBPconsumption was achieved by increasing the flow rate underthe same oxygen and substrate concentrations at the inlet(phase 2).

Spatial Distribution of Biodegradation Activity alongthe Fracture. Figure 5 presents Br- and oxygen concentra-tions along the fracture intersecting core 2, as measured insamples collected from the piezometers (which served assampling ports). The oxygen along the fracture was rapidlydepleted (∼80%) within the first 11 cm (Figure 5a). Br-

concentrations mirrored those of oxygen as most of it wasformed close to the fracture inlet (Figure 5b), while along therest of the fracture, Br- remained fairly constant or slightlydecreased. Though temporal changes in the oxygen and Br-

concentrations are evident, the general concentration trendalong the fracture remained similar throughout phase 2 ofthe experiments. For both oxygen and Br- concentrations,the overall scatter of the data between the two piezometerslocated at the same distance from the inlet (illustrated by theheight of the error bars) increased with time, reflecting thedynamic nature of the biodegradation process and thevariable conditions that developed in different regions withinthe fracture following flow-path redistribution due to clogging(34). The dynamic conditions within the microbial com-munity could have also contributed to these temporalvariations, but that subject is beyond the scope of this paper.

Oxygen consumption and Br- formation within the first11 cm of the fracture suggest that most of the biodegradationoccurs within this region. The temporal changes in biodeg-radation (Figures 2 and 5, mainly after day 420) suggest thatsteady-state biodegradation (i.e., constant inlet and outletconcentrations) was never achieved. This was probably dueto clogging, which became a major controlling factor as theexperiments progressed while maintaining constant hy-draulic and chemical conditions at the fracture boundaries.

AcknowledgmentsWe thank Zoe Grabinar and Camille Vainstein for theireditorial assistance. We also thank three anonymous review-ers for their comments and suggestions on this work. Thisresearch was supported in part by a grant from the IsraelScience Foundation (251/98).

Supporting Information AvailableA discussion of the biodegradation of TBP during transportand the mathematical model of solute transport. This materialis available free of charge via the Internet at http://pubs.acs.org.

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Received for review June 4, 2004. Revised manuscript re-ceived October 25, 2004. Accepted November 1, 2004.

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