Biodegradation of Naphthalene from Coal Tar and

Environ. Sci. Technol. 1996, 30, 1282-1291

Biodegradation of Naphthalene from Coal Tar and Heptamethylnonane in Mixed Batch Systems SUBHASIS GHOSHAL,† ANURADHA RAMASWAMI,‡ AND RICHARD G. LUTHY* Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

This research presents the first demonstration of substantial microbial degradation and depletion of naphthalene from coal tar, a multicomponent, aromatic, dense nonaqueous phase liquid (NAPL). The rates and extents of microbial degradation of naphthalene from coal tar and from a two-component NAPL simpler in composition than coal tar were evaluated in gently mixed, NAPL-water batch systems. The rate of degradation of naphthalene, the principal constituent in coal tar, was found to be significantly influenced by the rate of external surface mass transfer from the coal tar. Results show that the rate of mass transfer may control the overall rate of biotransformation in mixed systems where coal tar is present as a globule (=11 mm diameter). Mass transfer is relatively rapid and does not limit biodegradation in slurry systems when coal tar is distributed among a large number of small microporous silica particles (=250 µm diameter). These results were obtained for conditions favorable for biodegradation and provide an indication of the maximum potential rates for microbial degradation of naphthalene from coal tar. The microbial degradation process is dependent on relationships between the NAPL composition and the equilibrium aqueous naphthalene concentration, the naphthalene mass transfer rate between the NAPL and the aqueous phases, and the intrinsic rates of microbial degradation of naphthalene. These relationships have been incorporated in a dissolutiondegradation framework, and the rate-limiting phenomena for the biodegradation process was evaluated using this framework.

Introduction Prior to the widespread use of natural gas, manufactured gas plants (MGPs) supplied gaseous fuel derived from coal, coke, and/or oil. A major byproduct of manufactured gas processes was coal tar, which today is often associated with subsurface contamination at former MGP sites (1). Coal tars are denser-than-water nonaqueous phase liquids

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(DNAPLs), primarily composed of polycyclic aromatic hydrocarbons (PAHs). Naphthalene is often the most abundant PAH compound in such coal tars. This paper discusses the microbial degradation of naphthalene from coal tar NAPL, the source material at contaminated MGP sites. Biotreatment, such as in ex-situ mixed systems, may be a feasible technology for the remediation of coal tarcontaminated material (1); however, factors affecting the biodegradation of PAHs from coal tar NAPL and coal tarcontaminated material need to be evaluated. Although the biodegradation of two- and three-ring PAH compounds from coal tar-contaminated MGP site soils have been investigated, there have been few studies on the biodegradation of PAH compounds from the coal tar NAPL. Microbial degradation of two- and three-ring PAH compounds in natural and laboratory systems has been extensively studied (2, 3). The consensus from various investigations with aqueous suspensions of solid phase PAH compounds has been that bacteria utilize PAHs primarily as dissolved solutes with the rate being dependent on the rate of dissolution of the PAH and not on the amount of the solid phase PAH hydrocarbon present in the system (4-6). Consistent with the findings on the biodegradation of sorbed hydrophobic organic compounds in soil-water systems (7-9), it may also be inferred that it is the aqueous phase organic substrate that is readily available to microorganisms. Thus, in addition to knowledge of biotransformation rates, it is necessary to have an understanding of the physical processes of equilibrium partitioning of solutes and mass transfer of solutes between NAPL and aqueous phases. The work reported here demonstrates the influence of naphthalene mass transfer from coal tar on the microbial degradation of naphthalene. PAH Degradation from Contaminated MGP Site Soils and NAPLs. Studies on biodegradation of PAHs from contaminated MGP site soils (10, 11) have shown unpredictable and incomplete degradation of the PAHs associated with soil, although PAHs present in the aqueous solutions were usually degraded readily. Nakles et al. (12) reported results from laboratory biodegradation studies with contaminated MGP site soils in pan (unmixed) and slurry (mixed) systems. Slurry reactors promoted disaggregation of larger aggregates, while providing no limitations to nutrient or oxygen mass transfer. In tests with soil containing entrapped NAPL, the rate of degradation in slurry reactors was greater than in the pan systems, possibly due to enhanced dissolution of PAHs from the NAPL. Erickson et al. (13) observed no significant loss of PAHs from MGP site contaminated soil (including tar-contaminated soil) in unmixed, unsaturated soil microcosms. They postulated that the failure to observe losses of PAHs was due to the PAHs being unavailable to microorganisms. Morgan et al. (14) have described the aqueous-soluble fraction of PAHs from MGP site soils as being rapidly degradable or ‘readily * Corresponding author telephone: (412) 268-2941; fax: (412) 2687813; e-mail address: [email protected]. † Present address: Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI 48109. ‡ Present address: Environmental Science and Engineering Division, Colorado School of Mines, Golden, CO.

0013-936X/96/0930-1282$12.00/0

 1996 American Chemical Society

available’ and the residual PAHs comprising a ‘resistant fraction’ unavailable to microorganisms. The poor bioavailability of PAH compounds from some soils could be due to diffusional limitations of PAH compounds in micropores and/or slow partitioning of PAHs from NAPLs or solid surfaces to the aqueous phase. The above studies indicate qualitatively that mass transfer limitations may prevent significant biodegradation of PAHs from MGP site contaminated soils. Since the distribution of PAHs in the NAPL phase and within soil particles in those studies was not known and likely varied from case to case, it is difficult to generalize on the processes affecting the biodegradation of PAH compounds from coal tar NAPLs. Moreover, the biodegradation of solutes from complex chemical mixtures such as coal tars may be further complicated by substrate interactions causing unpredictable biodegradation patterns. For example, Alvarez and Vogel (15) have reported both inhibited and enhanced biodegradation of benzene, toluene, and xylene (BTX) in the presence of other BTX compounds, e.g., tolueneenhanced benzene degradation, but p-xylene inhibited its biodegradation. Several laboratory studies have reported on the bioavailability of PAH compounds from two-component NAPLs prepared by dissolving naphthalene or phenanthrene in an insoluble organic liquid such as heptamethylnonane (HMN), diethylhexyl pthalate (DEHP), or hexadecane. It has been demonstrated that microorganisms can utilize naphthalene from HMN or DEHP in NAPL-water systems (16-19). In some of those studies, maximum mineralization rates were reported to be greater than the maximum rates of partitioning measured in abiotic tests. However definite conclusions about the rates and extent of degradation cannot be made from those studies as either the fractions of naphthalene lost from the test systems were not reported (18) or significant fractions of naphthalene were lost from the biometer flasks (17). In this research, naphthalene mineralization from coal tar entrapped in a microporous matrix and from single coal tar globules is evaluated to study the mineralization patterns for significantly different mass transfer conditions in mixed batch systems. The extent of naphthalene degradation from the coal tar and from a two-component NAPL composed of naphthalene and HMN is assessed to provide increased understanding of the biodegradation of PAH solutes from NAPLs.

Dissolution-Degradation Model The overall rate of biotransformation of PAH compounds released from NAPLs is influenced by physicochemical phenomena related to the dissolution and mass transfer of PAH compounds from the NAPL phase to the bulk aqueous phase and to biokinetic phenomena related to microbial degradation. A coupled dissolution-degradation model describes the concurrent processes of mass transfer and biodegradation in mixed batch systems. As a working hypothesis, it is assumed that only naphthalene in the aqueous phase is available to the microorganisms. The dynamic change in aqueous phase PAH concentration, C(t) [M/L3], is described by

dC(t) dt

) kla[Ceq(t) - C(t)] - kbioC(t)

(1)

where the first term on the right-hand side represents the

rate of input of PAH to the aqueous phase due to dissolution from the NAPL, and the second term represents the rate of removal of bulk aqueous phase PAH due to biodegradation. For purposes of simplification, the biodegradation rate is expressed as a first-order biokinetic coefficient, kbio [1/T], assuming no substantial change in microbial concentrations once appreciable rates of substrate utilization and biomineralization are observed. In the systems being studied, the rate-limiting step for the mass transfer of solutes from the NAPLs to the aqueous phase is the external mass transfer across the boundary layer in the aqueous phase (20). Thus the dissolution rate is represented by a lumped mass transfer rate coefficient, kla [1/T], and a linear driving force term that represents the departure of the aqueous concentration from the equilibrium concentration, Ceq(t). The lumped mass transfer rate coefficient, kla, is a product of the areaindependent mass transfer coefficient, kl [L/T], and the specific surface area for mass transfer, a [L2/L3]. The aqueous phase concentration of a PAH compound in equilibrium with the NAPL is predicted by PAH Ceq(t) ) X(t) Cpure liquid γNAPL

(2)

where Ceq(t) is the equilibrium aqueous phase concentration of a NAPL-derived PAH compound; X(t) is the mole fraction PAH of the PAH compound in the NAPL at time, t; Cpure liquid is the solubility of the pure subcooled liquid compound in water; and γNAPL is the activity coefficient of the PAH compound in the NAPL. If a significant amount of the NAPL is essentially insoluble, as with coal tar or the twocomponent NAPL, the PAH mole fraction in the NAPL and the equilibrium aqueous phase PAH concentration decreases as cumulatively increasing quantities of the PAH compound are solubilized and then degraded by microbes. The mole fraction of PAH at any time, X(t), is calculated from the mole fraction at t ) 0 and from the fraction of PAH depleted. The equilibrium concentration at any time, Ceq(t), is calculated from eq 2 knowing X(t). The rate of change of aqueous concentration and thus the overall rate of biotransformation of a PAH compound like naphthalene is controlled by the slower of either mass transfer or degradation. When kla > kbio, the maximum rate of mass transfer, [klaCeq(t)] is faster than the maximum rate of microbial degradation [kbioCeq(t)]. Under such conditions, the effective rate of mass depletion is governed by the biokinetic rate constant, kbio. Conversely, when the maximum rate of mass transfer is slower than the maximum rate of biodegradation, the rate of biodegradation is directly related to the mass transfer rate coefficient, kla. The ratio of the biokinetic coefficient to the mass transfer rate coefficient can be used to identify whether biological processes or boundary layer mass transfer phenomena pose a limiting constraint on biotransformation rates. This ratio can be expressed as a dimensionless Damkohler number, Da:

Da ) kbio/kla

(3)

which indicates mass transfer control for values much greater than unity and biokinetic control for values much less than unity. Estimates of the first-order biokinetic rate constants, kbio, for the different systems were obtained by fitting the dissolution-degradation model to the data obtained from mineralization experiments. The objective of applying the dissolution-degradation model to the data obtained from

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the experiments was to obtain approximate values of maximum biodegradation rates for comparison with measured mass transfer rates. The biokinetic rate coefficients were obtained by fitting the model to the data rather than from direct assessment of naphthalene biodegradation in independent tests with aqueous solutions of naphthalene. The rationale was concern that biokinetic rate coefficients obtained from such short-term experiments would not be relevant for long duration tests. Also, there was concern about the complex composition of coal tar on the biokinetic rates. The mineralization of naphthalene was measured in the biodegradation experiments. The rate of naphthalene mass mineralized, normalized to the initial naphthalene mass, dP(t)/dt is given by

dP(t) dt

)

FmkbioC(t)V

(4)

M0

where Fm is the mass of naphthalene mineralized per unit mass of naphthalene degraded, M0 is the mass of naphthalene present in the NAPL at time t ) 0, and [kbioC(t)V] is the mass of naphthalene degraded in time ∆t. The mineralization curve is generated by numerically solving eqs 1, 2, and 4 for P(t) and expressing P(t) as a percent. The initial mass of naphthalene in the coal tar is known from chemical analysis of the coal tar. All tests were started after the naphthalene in the aqueous phase was in equilibrium with the NAPL, and thus the initial conditions [M0,C0, X0, and Ceq(0)] were known. The aqueous concentration of naphthalene for the subsequent time step, C(t + ∆t), was calculated by adding the change in concentration during the time step ∆t to the concentration at the current time step, C(t). The change in aqueous concentration in a time step, ∆C, is obtained by multiplying the right-hand side of eq 1 by the time interval ∆t. The cumulative fraction of naphthalene mineralized, P(t + ∆t ), was also computed in a similar manner, the increment ∆P being computed from eq 4. The mass of naphthalene remaining in the NAPL phase in the next time step, M(t + ∆t), is calculated by accounting for the mass released from the NAPL to the aqueous phase in the time step ∆t and for any mass lost from the system due to volatilization during periodic sampling and oxygen replenishment:

M(t+∆t) ) M(t) - [∆C1V] -

[

]

M0fv∆t tv

(5)

where ∆C1 is the change in naphthalene concentration due to dissolution and is equal to ∆tkla(Ceq(t) - C(t)). fv is the fraction of naphthalene lost from the system during the test (approximately 12%) and tv is the period of active mineralization, during which time losses were found to occur. The assumption of a constant rate of naphthalene loss from the system was based on assessments from sterile controls. The following condition was set for solving the above equations: if ∆C2 > C, where ∆C2 ) kbioC(t)∆t, then ∆C2 ) C - 0 ) C, i.e., only naphthalene in the aqueous phase is bioavailable. The new values of X(t) and Ceq(t) are then recalculated for the current value of M(t). The parameters γNAPL, kla, Fm, fv, and tv being known a priori from independent experiments, the only unknown parameter was the biokinetic rate constant kbio, which was fitted to the data. Optimal values

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TABLE 1

Chemical Composition of Principal Constituents of Baltimore Coal Tar compounds benzene ethylbenzene styrene total xylenes

molecular weight

Volatile Organic Compounds 78 92 106 106

Polycyclic Aromatic Hydrocarbons naphthalene 128 2-methylnaphthalene 142 acenaphthylene 152 acenaphthene 154 fluorene 166 phenanthrene 178 anthracene 178 fluoranthene 202 pyrene 202 228 benz[a]anthracene chrysene 228 252 benzo[b]fluoranthene 252 benzo[k]fluoranthene 252 benzo[a]pyrene 252 dibenz[a,h]anthracene

weight % 0.1 0.34 0.044 0.25 10.0 5.3 0.37 1.3 0.18 0.16 2.0 0.55 0.32 1.0 0.36 0.4 0.16 0.36 0.04

of kbio were obtained using nonlinear least squares regression with a modified Levenberg-Marquardt method (21). The Damkohler number, Da, for each system was calculated from estimated values of kbio and measured values of kla to assess whether mass transfer or biokinetics controlled the overall rate of biotransformation.

Methods Coal Tar. Samples of coal tar were obtained from monitoring wells at MGP sites located at Baltimore, MD, and Stroudsburg, PA. Both coal tars were free flowing brown/ black liquids and were denser than water. The Stroudsburg tar had a kinematic viscosity of 9.94 cSt and a specific gravity of 0.994 (both measured at 30 °C). The kinematic viscosity of the Baltimore tar was 9.5 cSt (at 37 °C) and the specific gravity was 0.99 (at 25 °C). Coal tar samples were characterized by compound class according to ASTM Method D2007. Aromatic compounds accounted for 98% (by weight) of the coal tar from the site in Baltimore. Naphthalene was the most abundant constituent of the Baltimore coal tar, accounting for 10% (by weight) of the of coal tar. The coal tar from Stroudsburg had a different composition with aromatic compounds accounting for 41% of the coal tar by weight, with naphthalene being the most abundant aromatic compound at 2.16% by weight. The average molecular weight of the Baltimore coal tar was 226 g/mol while that for the Stroudsburg tar was 210 g/mol, based on vapor pressure osmometry. Relevant results from the analyses of the Baltimore coal tar is presented in Table 1. The coal tar was analyzed by GC/MS for EPA Priority Pollutant volatiles and PAH compounds as per EPA Methods 8240 and 8270. The composition of the Stroudsburg coal tar has been described elsewhere (22). Preparation of Coal Tar-Coated Microporous Medium. Microporous silica beads (PQ Corporation, Valley Forge, PA, mean diameter 250 µm, average pore diameter ) 140 Å, pore volume 1.2 mL/g) were used as a model microporous medium. The silica beads were conditioned by heating to 800 °C for 2 h in a muffle furnace, whereby surface silica hydrophilic groups were converted to oxides. The beads

were allowed to cool to room temperature and were stored in air-tight containers for future use. Radiolabeled coal tar was prepared by adding 1 µCi [14C]-1-naphthalene (Sigma Chemical Co., St. Louis, MO)/mL coal tar. The radiolabeled naphthalene was dissolved in methanol and between 5 and 10 µL of the solution was added to each mL of coal tar. Approximately 17% of the pore volume of the silica beads was saturated by applying 1 mL of radiolabeled coal tar to 5 g of microporous silica beads. The coal tar-coated silica beads were mixed on end-over-end rotators in Tefloncapped 50-mL centrifuge tubes for approximately 24 h and then allowed to stand for 7 days to provide sufficient time for the coal tar to imbibe into the micropores. The [14C]1-naphthalene added to the coal tar was assumed to be representative of the naphthalene present in the coal tar. Preparation of the Two-Component NAPL. A two-component NAPL was made by dissolving naphthalene crystals in 2,2,4,4,6,8,8-heptamethylnonane (Sigma Chemical Co.), HMN. HMN (specific gravity ) 0.79) has an extremely low solubility in water. HMN was nontoxic to the bacterial culture and was observed not to sustain active growth of the microorganisms. Glass vials containing unlabeled naphthalene and 1 µCi of [14C]-1-naphthalene/mL of HMN were capped with Teflon and tumbled overnight on end-overend rotators to dissolve the naphthalene. Two different naphthalene mass fractions were used in the experiments, one similar to the Stroudsburg coal tar, and the other at 7%. Attempts to add greater amounts of naphthalene in the mixture to achieve a mass fraction of naphthalene at 10% (similar to the Baltimore tar) resulted in the mixture being supersaturated with naphthalene. The mixture comprising unlabeled naphthalene, [14C]-1-naphthalene, and HMN is referred to as the HMN NAPL in this paper. Radiolabeled Measurement Techniques. The activity of 14C-labeled compounds was measured using a Beckman LS 5000TD liquid scintillation counter (LSC) using a correction for quenching and the average of three counts to determine counting efficiency. The activity of 14C in the liquid samples was counted as disintegrations per minute (dpm) such that the 2σ error in dpm was less than 1%. Triplicates of every sample were counted, and the average dpm was recorded after correcting for background activity. Coal tar containing [14C]-1-naphthalene was diluted by a factor of 100 or more with n-butylamine (a solvent that dissolves coal tar readily) to reduce color and opacity. Samples of 200 µL were added to 15 mL of Ultima Gold scintillation liquid (Packard Instrument Co., Meriden, CT) for counting. Aqueous solutions containing 14C-labeled compounds with sample volumes up to 1 mL were added to 10 mL of Optifluor scintillation liquid (Packard Instrument Co.). To reduce errors due to chemiluminescence and static, samples were stored overnight or longer before counting until the random coincidence monitoring (RCM) errors were less than 1%. Equilibrium Aqueous Concentration of Solutes from NAPL. The partitioning of aromatic hydrocarbons from coal tar was measured by both radiolabeled and chromatographic techniques. The equilibrium concentration of naphthalene partitioned from the coal tar into the aqueous phase was determined using 5 g of silica beads coated with 1 mL of radiolabeled coal tar placed in 50-mL Tefloncapped centrifuge tubes filled to minimum headspace with deionized, autoclaved water and rotated for 3 days in endover-end rotators. HgCl2 was added at 200 mg/L to prevent any microbial activity. After being mixed, the tubes were

centrifuged, and the activity of 1 mL of the aqueous supernatant was measured after filtering through 0.2-µm PTFE filters by expressing at least 2 mL of the supernatant through the filter to waste. The aqueous concentration of naphthalene was calculated from the radioactivity of unit volume of the aqueous phase, the initial mass of naphthalene in the coal tar, and the radioactivity added to the coal tar. The equilibrium aqueous concentration of naphthalene was determined also by GC analysis of water contacted directly with coal tar NAPL in a closed vial. The equilibrium aqueous phase concentrations of naphthalene measured by both radiolabeled techniques and chromatography were compared with those predicted from eq 2. Radiolabeled techniques were used to measure the equilibrium aqueous concentration of naphthalene from the HMN NAPL. Tests were performed in 22-mL capacity vials fitted with open port caps and Teflon septa. A total of 20 mL of deionized, autoclaved water was contacted with 1.25 mL of the HMN NAPL containing 1 µCi of radiolabeled naphthalene/mL of the NAPL. The vials were put on an orbital shaker and gently mixed so as to not form emulsions, and aqueous samples were taken after 3 and 5 days of mixing by inserting a syringe through the Teflon septa beyond the NAPL layer. Air was expressed through the syringe while the syringe tip was depressed through the NAPL layer into the aqueous phase. Any NAPL in contact with the syringe tip was removed by carefully wiping the tip of the syringe prior to expressing the aqueous sample. The radioactivity in duplicate 0.5-mL samples was measured, and the aqueous phase concentrations were computed as described above. Mass Transfer Experiments. The dissolution of naphthalene from coal tar was studied in gently stirred, flowthrough reactors at different residence times by measuring the 14C activity of the effluent. At the end of each flowthrough test, the equilibrium aqueous phase concentration was measured and compared to that predicted by eq 2 to verify the utility of the equilibrium partitioning relationship in predicting dynamic changes in equilibrium concentration. A description of these experiments have been published elsewhere (20, 23). The intensity of mixing in the flow-through reactors was similar to that for the 250mL biometer flasks mounted on orbital shakers. Batch tests were performed to assess the mass transfer of naphthalene from the HMN NAPL. The tests were performed in 250-mL biometer flasks containing 50 mL of autoclaved, deionized water and 1.25 mL of HMN NAPL confined to a 34 mm i.d. glass tube fused to the inside bottom of the biometer flask as shown in Figure 1. The HMN NAPL-water interfacial area was known by computing the cross-sectional area of the glass vial, which had slots cut at the bottom of the tube to facilitate passage of water through the bottom of the tube. After placing the HMN NAPL in the tube, the glass vial was capped with an aluminum-wrapped stopper to prevent transfer of naphthalene to the headspace of the flask. The aqueous phase in the flask was sampled at frequent intervals, and the aqueous concentration of naphthalene was computed from the radioactivity measured in each sample, as described above. The rate of dissolution of naphthalene from the HMN NAPL in a batch system is given by

dC(t) dt

) kla[Ceq(t) - C(t)]


(6)

9

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FIGURE 1. Schematic of biometer flasks. (A) Coal tar/silica beads system. (B) Coal tar globule system. (C) HMN NAPL system.

The change in the mole fraction of naphthalene in the HMN NAPL during dissolution was negligible and the mass transfer coefficient, kla, was determined by the slope of the plot of the linearized form of the above equation:

ln

(Ceq(0) - C(t)) Ceq(0)

) kla t

(7)

Naphthalene Mineralization Tests. The mineralization of naphthalene from coal tar and the two-component NAPL was measured using radiolabeled techniques. The experiments were carried out under aerobic conditions in 250mL, autoclaved, biometer flasks fitted with a side tube. The side tube and the flask were sealed with neoprene stoppers covered with aluminum foil. Nutrient media (170 mg of KH2PO4, 435 mg of K2HPO4, 850 mg of NH4Cl, 668 mg of Na2HPO4‚7H2O, 22.5 mg of MgSO4‚7H2O, 27.5 mg of CaCl2, and 0.25 mg of FeCl3‚6H2O in 1 L of deionized water) was autoclaved, and 50 mL of this solution was added to each biometer. In mineralization experiments with coal tar-coated silica beads, 5 g of silica beads was coated with 1 mL of coal tar and added to a biometer containing 50 mL of nutrient media. The pH of the slurry was adjusted to approximately 7.2 using a few drops of 2 M NaOH and checked after equilibrating for 2 days, and readjusted if necessary. Tests

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with coal tar globules were performed with a single globule of 0.7 mL of coal tar immersed in 50 mL of nutrient media. The coal tar globule was formed by carefully expressing coal tar through the tip of a syringe needle immersed in water. Biometers used for the coal tar and HMN NAPL experiments were configured as shown in Figure 1. Biometers were inoculated with 2 mL of an approximately 107 cfu/mL, actively growing, mixed bacterial culture of PAH degrading microorganisms (RET PA101) that have been used in prior studies (24, 25). The microorganisms used for inoculation were grown in the nutrient media supersaturated with naphthalene, the sole carbon source. Standard sterile techniques were followed for inoculation and plate counts. Multiple plate counts were performed on agar plates with naphthalene crystals placed on the lids of inverted plates. The naphthalene from the gas phase was available to the microorganisms plated on the agar. Five milliliters of 2 M NaOH was added to the side arm of the flask to trap CO2 evolved during mineralization. Biometers were continuously shaken in a orbital shaker at approximately 60 rpm. At least duplicates were set up for biometers representing a particular set of experimental conditions. Sterile controls were employed to assess any abiotic mineralization. The control biometers were prepared exactly as for the mineralization tests but were not inoculated and had 200 mg/L mercuric chloride to prevent microbial activity. Radiolabeled CO2 from biomineralization of [14C]-1naphthalene was measured by periodically sampling the NaOH in the biometer side arm and counting the activity of triplicate 0.5-mL NaOH samples. The NaOH was replaced with 5 mL of fresh NaOH, and the headspace of the flask was purged briefly with O2 and then stoppered immediately. After mineralization activity had ceased, additional nutrient and microorganisms were added to the biometers, and mineralization activity was monitored for an additional period to ensure that the mineralization activity had not ceased due to nutrient limitations or lack of viable microorganisms. The percent of naphthalene mineralized at any time was computed from the cumulative fraction of the total activity. Aqueous phase samples were taken at different times from some of the biometers containing coal tar globules or coal tar-coated silica beads for the measurement of naphthalene by GC or HPLC. Supernatant samples from the biometers were filtered through 0.2-µm PTFE filters into 22-mL vials after preconditioning filters by expressing 2 mL of supernatant to waste. Between 2 and 6 mL volumes were collected, and the headspace was filled with a 200 mg/L HgCl2 solution to prevent further biological activity. Mass Balance for 14C. At the conclusion of mass transfer and biodegradation experiments, the radioactivity remaining in the NAPL phases and in the aqueous phases were measured to determine the end points of biodegradation and to evaluate a mass balance for naphthalene in each system. For the coal tar-coated silica systems, the supernatant was pipetted out, weighed samples of the slurry were combusted in a biological material oxidizer, and the 14CO2 from combustion was trapped into vials containing the 14C cocktail (R. J. Harvey Instrument Corp., Hillsdale, NJ). The total activity present in the slurry was calculated from the activity of the combusted samples and the weight of the slurry. The radioactivity remaining in the coal tar globules or the HMN NAPL was determined from 50-µL samples withdrawn directly from the organic phase by syringe. The

TABLE 2

Predicted and Measured Equilibrium Aqueous Naphthalene Concentrations initial naphthalene mass in 1 g of NAPL (mg) Stroudsburg coal tar Baltimore coal tar 2.2% naphthalene in HMN 7.0% naphthalene in HMN

21.6 100 21.6 70

predicted equilibrium naphthalene concnsa (mg/L)

measured equilibrium naphthalene concns (mg/L)

3.76 (γNAPL ) 1) 3.8 18.5 (γNAPL ) 1) 18.7, 19.0c (γNAPL ) 2.35b) 9.6 (γNAPL ) 2.03b)

27.3

a Pure subcooled liquid naphthalene solubility of naphthalene was taken as 104 mg/L (22, 28). b γNAPL values calculated from eq 8. c From GC purge-and-trap analysis.

radioactivity in the supernatant of the biometers was measured also, and a mass balance of naphthalene in each system was performed. Mass balance of 14C from the systems ranged from 82% to 93% with an average of 88%.

Results and Discussion Equilibrium Aqueous Concentration of Solutes from NAPLs. Measured and predicted equilibrium aqueous concentrations of naphthalene from the coal tars are shown in Table 2. The equilibrium aqueous concentrations for the coal tars were predicted using eq 2 with known values of the average molecular weight and the naphthalene mole fraction for each coal tar and a coal tar-phase activity coefficient of unity. Coal tar-phase activity coefficients of unity for two-ring PAHs have been reported (26, 27). The measured equilibrium naphthalene concentrations for both the Baltimore coal tar and the Stroudsburg coal tar matched well with the values predicted by eq 2. The agreement of the measured and predicted equilibrium naphthalene concentrations indicates that the naphthalene in the coal tar NAPL entrapped within the microporous silica beads was in equilibrium with the aqueous phase. The equilibrium concentrations of naphthalene for the HMN NAPLs shown in Table 2 were greater than those observed with the coal tars having similar mole fractions. This was due to activity coefficients being greater than unity for naphthalene in HMN NAPL. For naphthalene dissolved in alkane solvents such as hexane, activity coefficients greater than unity have been reported (28). Equation 2 was used to calculate the activity coefficients from the measured equilibrium concentrations and the respective mole fractions. The equilibrium aqueous concentrations of naphthalene were measured for different mole fractions of naphthalene, and the magnitude of the calculated activity coefficients ranged from 1.97 to 2.98 for mass fractions of naphthalene ranging from 7% to 0.2%. The following empirical expression describes this relationship:

γNAPL ) 1.47 - 0.62 log (XPAH)

(r2 ) 0.98) (8)

The increase in the activity coefficient with decreasing mole fractions of naphthalene is attributed to the NAPL becoming less of an aromatic environment. Such variations in activity coefficients were not observed in the case of coal tar, where the assumption of activity coefficients equal to unity resulted in equilibrium concentration predictions similar

to measured concentrations even after significant fractions of naphthalene had been depleted from the tars (23). Mass Transfer Rate Coefficients. Mass transfer experiments with coal tar-coated microporous silica beads and single coal tar globules were performed in small, flowthrough reactors (20). For the coal tar-coated silica beads used, the pore diffusion rates were much larger than the particle surface external mass transfer rate. Lumped mass transfer rate coefficients for naphthalene (kla) were derived from the flow-through dissolution experiments and were approximately equal to 3550/day for the Stroudsburg tar and 4550/day for the Baltimore tar. In experiments where the dissolution of naphthalene was studied from coal tarcoated silica beads, the measured mass transfer coefficients were found to decrease with time, and this is believed to be a result of an interfacial ‘aging’ phenomenon (20, 23). The mass transfer rate coefficients were observed to decrease to a minimum of about 500/day after aging for 7-30 days. Mass transfer rates from single 0.7-mL coal tar globules (11 mm diameter when approximated as a sphere) were significantly slower than the silica beads, and kla values of 2/day and 1.6/day for naphthalene were determined for the Stroudsburg and Baltimore coal tars, respectively. For the coal tar globules, the mass transfer coefficients did not vary with time. Mass transfer experiments with the HMN NAPL were performed in the retrofitted biometers with the HMN NAPL confined in the glass vial. The data for aqueous concentration of naphthalene resulting from the dissolution from HMN NAPL confined in a 34 mm diameter glass vial were fitted to eq 7, and a value of kla equal to 26/day was obtained. Although the NAPL-water interfacial area for the coal tar globule was about 1.7 times larger than that for the HMN NAPL, the lumped naphthalene mass transfer rate coefficients (kla) for the coal tar globules were significantly less than for the HMN NAPL. This indicates that the areaindependent mass transfer coefficient, kl (L/T), for naphthalene is greater for the HMN NAPL. This may be a combined result of the effects of orbital mixing on mass transfer coefficients for a globule versus a flat interface, differences in NAPL-phase mixing due the density differences in the organic phases, or differences in diffusional resistances in the coal tar and the HMN NAPL phase. Naphthalene Mineralization Profiles. The percent naphthalene mineralized from the coal tar-coated silica beads are shown in Figure 2a,b. Rapid mineralization of naphthalene in the first 10 days was followed by an extended period of slow mineralization. Variations of an overall maximum of 10% in the extents of mineralization for a particular tar were observed between tests, as seen in Figure 2. The differences between tests were probably due to small variations in the inocula. Variations for duplicate samples within an individual test was less than 5%. The naphthalene mineralization profiles from experiments with single 0.7-mL coal tar globules are shown in Figure 3. These profiles are significantly different from those obtained with the coal tar-coated silica beads. Mineralization occurred at a slow rate over approximately 120 days. The mineralization of naphthalene was measured over a period of 150 days, and approximately 70% of the naphthalene in the coal tar globules was mineralized over this period. The differences in the mineralization profiles of the Stroudsburg coal tar and the Baltimore coal tar are attributable to differences in the composition of the two tars and to microbial kinetics.


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a

FIGURE 4. Mineralization profiles for naphthalene from HMN NAPLs. Biometers were fitted with 34 mm diameter vials.

b

FIGURE 2. Mineralization profiles for naphthalene from coal tar imbibed in silica beads. Panels a and b are the results of two independent experiments.

FIGURE 3. Mineralization profiles for naphthalene from single coal tar globules.

As shown in Figure 4, the naphthalene mineralization profiles from the HMN NAPL were similar to those obtained from the coal tar-coated silica bead system. Mineralization was rapid in the first 20 days and was followed by a period of slow mineralization. About 60-70% of the naphthalene present in the NAPL phase was mineralized. The mineralization patterns were similar for HMN NAPL containing

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2.2% and 7% naphthalene as well as for the Stroudsburg and Baltimore coal tars coated onto silica beads. The similarity in the mineralization profiles do not necessarily imply similar rates of biodegradation. The rate of biodegradation is influenced by the relative magnitudes of the mass transfer rate coefficients, the biokinetic rate constants, and the initial mass of naphthalene in the NAPL. The similarity of the naphthalene mineralization profiles obtained from biodegradation of the two coal tars coated onto microporous silica and from naphthalene mineralized from the HMN NAPL suggests that naphthalene mineralization was not affected significantly by the large number of solutes in coal tar that may be present in the aqueous phase. The extent of mineralization of naphthalene from the two coal tars and from the HMN NAPL range from 50 to 70% and is relatively independent of large differences in the initial naphthalene mole fraction in the NAPL. In similar tests with HMN, Efroymson and Alexander (17) and OrtegaCalvo and Alexander (18) reported that the extent of naphthalene mineralization ranged from 20 to 30%. Significant losses may account for the extents of mineralization in those studies being much smaller than the results presented in Figure 4. The pH in the aqueous phase in the biometers decreased slightly from an initial value of 7.2 to approximately 6.5 at the end of the experiment. Biofilms were observed to grow on the coal tar globules but were not observed to attach to the HMN phase. With all of the above systems, plating aqueous phase samples on naphthalene plates resulted in colonies visually similar to those observed for the inocula, suggesting no major changes in the microbial population. End Points of Biodegradation for NAPL Phase Naphthalene. When significant mineralization activity was not observed for a length of time even after the biometers were supplemented with additional nutrients and microorganisms, the NAPL and the aqueous phases were sampled to determine the percent distribution of 14C in these phases. The 14C in the aqueous phase represents the fraction of naphthalene in solution as well as the fraction of naphthalene that goes to creating suspended biomass and metabolic end products. Table 3 lists the percent distribution of 14C at the end of the mineralization tests for the biometers containing the coal tar globules and the HMN NAPL. Initially in these tests, the NAPL phase contained the entire amount of naphthalene. For the Baltimore tar,

TABLE 3

Percent 14C Distribution at the Conclusion of Biomineralization Testsa globule (Stroudsburg coal tar) globule (Baltimore coal tar) two-component NAPL (2.2% naphthalene) two-component NAPL (7% naphthalene) a

remaining in NAPL

mineralized

remaining in aqueous phase

lost

7 6 0.31-0.33 0.3-0.6

70 73 63.9-70.0 53.2-67.6

16 9 21.8-22.1 17.3-31.1

7 12 7.84-13.6 14.9-15.9

Average (single) values given when the variations are less than 5% in duplicate tests.

TABLE 4

Estimates of kbio and Damkohler Number NAPL Stroudsburg coal tar Baltimore coal tar two-component NAPL (2.2% naphthalene) two-component NAPL (7% naphthalene) a Estimated from data presented in Figure 2a. ∼kla.

b

test system

Fm

kla (day-1) (measured)

kbio (day-1) (estimated)

Da

rate-controlling phenomena

silica beads globule silica beads globule 34-mm vial 34-mm vial

0.83 0.83 0.89 0.89 0.76 0.63

3550 1.6 4550 2.0 26.1 26.1

34.1,a 26.1b c 11.9,a 14.7b c 8.8 4.7

∼0.008 16-21 ∼0.003 6-7 0.3 0.2

biokinetics mass transfer biokinetics mass transfer biokinetics biokinetics

Estimated from data presented in Figure 2b. c kla < kbio, thus apparent biodegradation rate constant

only about 6% of the naphthalene initially present in the coal tar globule remained in the coal tar phase after the extended period of biomineralization; approximately, 73% of the naphthalene was mineralized and converted to CO2. In this test, the aqueous phase contained microbial mass, metabolic end products of naphthalene degradation, and a small mass of naphthalene, all of which accounted for 9% of the 14C initially present in the NAPL phase. The fraction of naphthalene in the aqueous phase was insignificant (less than 0.06% based on estimates from eq 2), as the mole fraction of naphthalene remaining in the coal tar was very small. These results indicate that a very large fraction of the naphthalene may be released from an accessible coal tar NAPL in the presence of a continuous sink such as microorganisms. However, the coal tars used in this study were free-flowing tars, and the biodegradation of PAHs from more viscous or solid tar may be significantly different from that observed in this study. As shown in Table 3, between 7 and 16% of the 14C was lost from the various biometers during the tests. It is likely that naphthalene may have been lost while transferring and handling the naphthalene HMN or the coal tar at the beginning of the experiments, or while the system was opened for sampling and purging with O2. Calculations suggest the major loss was probably due to volatilization during periodic purging with O2. For the HMN NAPL, only 0.3-0.6% of the naphthalene initially present in the NAPL remained in that phase after mineralization had ceased. This indicates a significant depletion of the mass of naphthalene from the NAPL phase and suggests that under ideal biodegradation conditions naphthalene may be essentially entirely eliminated from a NAPL. The fraction of naphthalene depleted from the coal tar globule was substantial as well. It is unclear why a small fraction of naphthalene remained in the coal tar phase. Possible reasons could be that the mineralization of naphthalene had become imperceptibly slow at the point where the experiment had been terminated or that the microorganisms were utilizing other aromatic compounds as substrates after the concentration of naphthalene had become very small.

The mass of naphthalene mineralized per unit mass of naphthalene degraded, Fm, was computed from the results for the HMN NAPL systems and from the coal tar globule systems. The total amount of naphthalene degraded was determined by the difference of the initial naphthalene mass and the amounts of naphthalene lost and remaining in the NAPL phase at the end of the test. For the HMN NAPL systems, Fm values ranged from 0.63 to 0.79, with an average value of 0.73. For these systems, the Fm calculated from the individual systems was used in the estimation of kbio. Fm values of 0.83 and 0.89 were calculated from the systems employing single Stroudsburg and Baltimore tar globules, respectively. For estimating kbio for the systems with coal tar-coated silica beads, the Fm values were assumed to be same as for the coal tar globules. The Fm values are summarized in Table 4. Rate-Controlling Phenomena. Values of kbio were determined by fitting the dissolution-degradation model equations to the mineralization data. For the HMN NAPL, γNAPL for each X(t) was recalculated from eq 8. In all cases, only data for the initial part of the mineralization curves were used to be consistent with the assumptions of constant microbial concentration. The mass of naphthalene lost from each system, fv, was determined to be about 12% from the mass balance analysis for 14C. It was assumed that the time over which these losses were significant varied from about 7 to 30 days, during which time the mineralization was rapid. This was the time to the break in the mineralization profiles after 50-70% mineralization occurred. Lag phases of duration upto 2 days occurred in the experiments with coal tar and silica beads, and the time and percent mineralization axes were adjusted to exclude the lag phase. The modified data set was used for parameter estimation. The estimated values of kbio for each system is presented in Table 4, and the residual sum of squares for each estimate is presented in Table 5. The rate-controlling phenomena for the different NAPLwater systems were identified by comparing the magnitudes of the mass transfer rate coefficient (kla) and the biokinetic rate constant (kbio). The estimated values of kbio for the systems with coal tar-coated silica beads ranged from 12


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TABLE 5

Residual Sum of Squares for Estimates of kbio

NAPL

no. of data residual kbio (day-1) points sum of test system (estimated) usedc squaresd

Stroudsburg coal tar

silica beads

Baltimore coal tar

silica beads

two-component NAPL 34-mm vial (2.2% naphthalene) two-component NAPL 34-mm vial (7% naphthalene)

34.1a 26.1b 11.9a 14.7b 8.8

3 3 3 3 3

0.052 0.205 0.037 0.011 0.009

4.7

3

0.125

a

Estimated from data presented in Figure 2a. b Estimated from data presented in Figure 2b. c Data points from initial period of mineralization excluding any lag phase. d Residuals calculated as relative residuals ([Pobs - Ppred]/Pobs).

to 34/day, which is much smaller than the mass transfer rate coefficients of approximately 4000/day measured in the flow-through tests. The resulting Damkohler numbers are in the range of 0.003-0.008 and being much less than unity provide evidence that the overall rate of naphthalene biotransformation with the coal tar-coated silica particles is controlled by the intrinsic biokinetics of the microorganisms. For the case of coal tar globules, the observed rates of mass transfer were slower than the biokinetic rates. The measured values of kla were 1.6 and 2/day, which were less than the kbio values of 12-34/day obtained in the tests with the coal tar-coated silica beads. The resulting Damkohler numbers for these systems were in the range of 6-21 and being greater than unity suggest that the overall rate of biodegradation is limited by the mass transfer rates. Under mass transfer limited conditions, naphthalene released from the coal tar globule is degraded readily by the microorganisms, causing the aqueous concentrations of naphthalene in the biometer to be much less than equilibrium. Measurements of the aqueous phase naphthalene concentration were made at day 50 during the tests to assess the aqueous naphthalene concentrations. In the biometers containing the Stroudsburg tar globule, aqueous naphthalene concentrations were less than a detection limit of 0.1 mg/L. In the biometers containing the Baltimore tar, aqueous naphthalene concentrations were approximately 3 mg/L, which was significantly smaller than an estimated equilibrium aqueous naphthalene concentration of 12 mg/L at that time. These observations with both Stroudsburg and Baltimore coal tar globules further indicate mass transfer limitations. The rate of biomineralization of naphthalene from the HMN NAPL was controlled by the intrinsic biokinetics of the microorganisms. The mass transfer coefficient of 26/ day was greater than the estimated biokinetic rate constants of 4.7 and 8.8/day. These estimated biokinetic rate constants are smaller than those obtained from the coal tar systems. The equilibrium aqueous concentrations of naphthalene from the HMN NAPL are greater than those from coal tar, and although the biokinetic rate constants are smaller, the initial rates of mineralization given by eq 4 do not differ significantly when first-order kinetics are assumed. For all the above cases, the biokinetic rate constants vary from one system to another but are all in

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the range of reported first-order naphthalene biokinetic rate constants of 1-25/day reported for soil slurry systems (8, 29). In this study, the degradation of naphthalene has been evaluated in gently mixed batch reactors under favorable conditions of oxygen and nutrient availability, and thus the rates of biodegradation represent maximum possible rates achievable for the microorganisms used. The equilibrium aqueous naphthalene concentration and the mass transfer rate coefficient together affect the aqueous naphthalene concentration and in turn the biodegradation rate for the NAPL systems.

Acknowledgments This study was supported by an industrial sponsor and by Texaco Inc. Research and Development, Beacon, NY. Mr. Ian MacFarlane of EA Engineering, Science and Technology, Sparks, MD, provided us the results of the physical and chemical characterization of the Baltimore coal tar and the results of the chromatographic analysis of the aqueous samples.

Literature Cited (1) Luthy, R. G.; Dzombak, D. A.; Peters, C. A.; Ramaswami, A.; Roy, S. B.; Nakles, D.; Nott, B. R. Environ. Sci. Technol. 1994, 28 (3), 266A. (2) Cerniglia, C. E. Adv. Appl. Microbiol. 1984, 30, 31. (3) Heitkamp, M. A.; Freeman, J. P.; Miller, D. W.; Cerniglia, C. E. Appl. Environ. Microbiol. 1992, 54 (10), 2556. (4) Woodzinski, R. S.; Bertolini, D. Appl. Environ. Microbiol. 1972, 23 (6), 1077. (5) Woodzinski, R. S.; Coyle, J. E. Appl. Environ. Microbiol. 1974, 27 (6), 1081. (6) Stucki, G.; Alexander, M. Appl. Appl. Environ. Microbiol. 1987, 53 (2), 292. (7) Ogram, A. V.; Jessup, R. E.; Ou, L. T.; Rao, P. S. C. Appl. Environ. Microbiol. 1985, 49 (3), 582. (8) Mihelcic, J. R.; Luthy, R. G. Environ. Sci. Technol. 1991, 25 (1), 169. (9) Rijnaarts, H. H. M.; Bachmann, A.; Jumelet, J. C.; Zehnder, A. J. B. Environ. Sci. Technol. 1990, 24 (6), 1349. (10) Smith, J. R.; Weightman, R. L. Co-Treatment of Manufactured Gas Plant Site Groundwaters with Municipal Wastewaters. Final Topical Report to the Gas Research Institute, Chicago, IL, 1988; Contract 5086-254-1350. (11) Sherman, D. F.; Loehr, R. C.; Neuhauser, E. F. Proceedings of the International Conference on Physicochemical and Biological Detoxification of Hazardous Waste; Atlantic City, NJ; Technomic Publishers: Lancaster, PA, 1988; pp 1035-1045. (12) Nakles, D. V.; Linz, D. G.; Murarka, I. P. Bioremediation of MGP Soils: Limitations and Potentials. Presented at EPRI Technology Transfer Seminar on Management of Manufactured Gas Plant Sites, Atlanta, GA, Apr 1991. (13) Erickson, D. C.; Loehr, R. C.; Neuhauser, E. F. Water Res. 1993, 27 (5), 911. (14) Morgan, D. J.; Battaglia, A.; Hall, B. J.; Vernieri, L. A.; Cushney, M. A. Technical Report GRI-92/0499. Gas Research Institute: Chicago, IL, 1992. (15) Alvarez, P. J. J.; Vogel, T. M. Appl. Environ. Microbiol. 1991, 57 (10), 2981. (16) Woodzinski, R. S.; Larocca, D. Appl. Environ. Microbiol. 1977, 33 (3), 660. (17) Efroymson, R. A.; Alexander, M. Appl. Environ. Microbiol. 1991, 57 (5), 1441. (18) Ortega-Calvo, J.; Alexander, M. Appl. Environ. Microbiol. 1994, 60 (7), 2643. (19) Efroymson, R. A.; Alexander, M. Environ. Sci. Technol. 1994, 28 (6), 1172. (20) Ramaswami, A. Ph.D. Dissertation, Carnegie Mellon University, 1994. (21) IMSL. International Math Software Library Manual; IMSL, Inc.: Houston, TX, 1987; Vol. 3. (22) Peters, C. A.; Luthy, R. G. Environ. Sci. Technol. 1993, 27 (13), 2831. (23) Luthy, R. G.; Ramaswami, A.; Ghoshal, S.; Merkel, W. Environ. Sci. Technol. 1993, 27 (13), 2914. (24) Laha, S.; Luthy, R. G. Biotechnol. Bioeng. 1992, 40 (11), 1367.

(25) Liu, Z.; Jacobson, A. M.; Luthy, R. G. Appl. Environ. Microbiol. 1995, 61 (1), 145. (26) Lee, L. S.; Rao, P. S. C.; Okuda, I. Environ. Sci. Technol. 1992, 26 (11), 2110. (27) Lane, W. F.; Loehr, R. C. Environ. Sci. Technol. 1992, 26 (5), 983. (28) Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. Environmental Organic Chemistry; John Wiley and Sons: New York, 1992; p 128.

(29) Guerin, W. F.; Boyd, S. A. Appl. Environ. Microbiol. 1992, 58 (4), 1142.

Received for review July 10, 1995. Revised manuscript received November 10, 1995. Accepted December 14, 1995.X ES950494D X

Abstract published in Advance ACS Abstracts, March 1, 1996.


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Biodegradation of Naphthalene from Coal Tar and

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