Environ. Sci. Technol. 2005, 39, 2274-2279
Optimizing Contaminant Desorption and Bioavailability in Dense Slurry Systems. 2. PAH Bioavailability and Rates of Degradation HAN S. KIM AND WALTER J. WEBER, JR.* Department of Chemical Engineering, Energy and Environment Program, The University of Michigan, 4103 Engineering Research Building, Ann Arbor, Michigan, 48109-2099
The effects of mechanical mixing on rates of polycyclic aromatic hydrocarbon (PAH) biodegradation in dense geosorbent slurry (67% solids content, w/w) systems were evaluated using laboratory-scale intermittently mixed batch bioreactors. A PAH-contaminated soil and a phenanthrene-sorbed mineral sorbent (R-Al2O3) were respectively employed as slurry solids in aerobic and anaerobic biodegradation studies. Both slurries exhibited a characteristic behavior of pseudoplastic non-Newtonian fluids, and the impeller revolution rate and its diameter had dramatic impacts on power and torque requirements in their laminar flow mixing. Rates of phenanthrene biodegradation were markedly enhanced by relatively lowlevel auger mixing under both aerobic and anaerobic (denitrifying) conditions. Parameters for empirical models correlating biodegradation rate coefficient (kb) values to the degree of mixing were similar to those for correlations between mass transfer (desorption) rate coefficient (kr) values for rapidly desorbing fractions of soil organic matter and degree of mixing reported in a companion study, supporting a conclusion that performance-efficient and costeffective enhancements of PAH mass transfer (desorption) and its biodegradation processes can be achieved by the introduction of optimal levels of reactor-scale mechanical mixing.
Introduction Soils, buried and lagooned deposits of dredge spoils, industrial sorbents and filtration media, and waste sludges contaminated with polycyclic aromatic hydrocarbons (PAHs) can be found at many industrial and defense-related sites (1, 2). The bioavailability of such PAH contaminants to soil microflora has been studied intensively as a critical factor in the control of both rates and endpoints of bioremediation of contaminated soils and sediments (3). There have been some reports that microbes can utilize sorbed PAHs directly (4-7). In contrast, a number of studies have concluded that bacterial attachment does not occur on the surfaces of solidphase substrates in aqueous systems, that no direct uptake of substrate from the solid phase is evident, that substrate dissolution to the aqueous phase is necessary to make solidassociated hydrophobic organic compounds (HOCs) bioavailable to degrading microbes, and that the mass transfer of PAHs from sorbed or nonaqueous phase liquid phases to * Corresponding author phone: 734-763-2274; fax: 734-936-4391; e-mail:
[email protected]. 2274
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the aqueous phase in which microbial uptake occurs thus controls overall rates of biodegradation (3, 8-14). Despite their relatively high capital and operating costs, and the additional expenses associated with excavation procedures, soil slurry bioreactors have been widely employed as an ex-situ bioremediation technology for soils and sediments contaminated with HOCs (15-19). Slurry-phase treatments can stimulate biodegradation processes by reducing mass transfer resistances and maximizing microbial activity via intermixing of microbes, substrates, and essential nutrients (20). Ex-situ slurry-phase reactor systems overcome many of the inherent issues of gross heterogeneity usually associated with contaminated natural deposits of soils and sediments. Such systems are much more manageable, controllable, predictable, and describable relative to most insitu remediation systems (21). However, quantification of critical mass transfer relationships and associated biodegradation processes has, for the most part, not been adequately addressed in the aforementioned laboratory-scale investigations of slurry-phase bioreactor systems, even though such factors are critical for the optimal design, operation, and scale-up of those processes for field applications. In most cases, ex-situ slurry-phase treatments involve relatively low solids content ranging from 5 to 40% (w/w) because high solids content slurries are very difficult to mix or circulate effectively (22). For instance, Gilcrease et al. (23) investigated the biodegradation of trinitrotoluene in soil slurry reactor systems, reporting that high solids content can reduce mass transfer of hydrophobic substrates because of turbulence damping as a result of high viscosity of slurry fluids. Applications of such technologies, however, in many cases, are limited in in-situ or in-place remediation scenarios due to significantly slow mass transfer of reaction constituents associated with highly compacted solids and almost stagnant fluid environments. Hydrodynamic mixing and required power inputs are thus important factors influencing mass transfer of substrates to microorganisms and associated biotransformation reactions. In a companion study (24) we described the underlying logic and operating characteristics of intermittently mixed slurry reactor systems and reported the effects of such mixing with a helical screw-type impeller (auger) on mass transfer (desorption) of sorbed phenanthrene to the aqueous phase. Desorption rates were found to increase rapidly in a range of relatively low-level intermittent mixings, reaching a point of no significant enhancement with further increase of mixing intensity. That work raised logical questions as to how intermittent mechanical mixing would affect rates of overall biodegradation processes for sorbed contaminants in slurry reactor systems. To address those questions, PAH biodegradation experiments were performed using bioactive intermittently mixed batch reactors (IMBRs) in which the same impeller and reactor vessel geometries used in the abiotic IMBRs were employed. Quantitative assessments of the effects of degree of mechanical mixing on rates of PAH biodegradation under aerobic and anaerobic conditions were conducted to evaluate process efficiency by addressing critical operating parameters as well as to evaluate economic benefits that may be applicable for ex-situ and/or in-situ remediation systems. In this regard, dense slurries (67%, w/w) were employed to evaluate conditions that might realistically be anticipated for in-situ operations. The results of the study should prove useful for developing cost-effective, periodic, and low-intensity mixing strategies that can be applied for biotreatment of contaminated soils and sediments. 10.1021/es049564j CCC: $30.25
2005 American Chemical Society Published on Web 02/04/2005
Materials and Methods Materials. Phenanthrene was obtained in analytical grade (purity > 98%) from Sigma-Aldrich (Milwaukee, WI). Methylene chloride, methanol, and acetonitrile (HPLC analysis grade, purity > 99%) were purchased from Fisher Scientific (Chicago, IL). A sandy soil historically contaminated with creosote (organic carbon content of 0.4%, w/w) was collected from the southern Maryland wood treatment (SMWT) site. Physical characteristics and levels of HOCs in the SMWT site soil are provided elsewhere (25). Aluminum oxide (R-Al2O3, spherical powder, particle size 20-50 µm) obtained from Alfa Aesar (Ward Hill, MA) was used as a mineral geosorbent. Culture Media and Microorganisms. Three microbial media were used: (i) a basal salt medium (no carbon); (ii) a Luria-Bertani (LB) medium; and, (iii) a denitrifying mineral medium (no carbon) consisting of 500 mg of KNO3, 4 g of KH2PO4, 2 g of K2HPO4, 2 g of Na2HPO4, 2 g of (NH4)2SO4, 200 mg of MgSO4(7H2O), 10 mg of CaCl2(2H2O), and 1 mg of Fe2SO4(7H2O) and 1 mL of trace mineral nutrient stock solution per liter of Milli-Q water at a pH of 7.1. The trace mineral nutrient stock solution was composed of 0.07 g of ZnCl2, 0.1 g of MnCl2(4H2O), 0.062 g of H3BO3, 0.19 g of CoCl2(6H2O), 0.035 g of CuSO4(5H2O), 0.024 g of NiCl2(6H2O), 0.036 g of Na2MoO4(2H2O), and 5.2 g of EDTA per liter of Milli-Q water. The detailed composition of basal salt medium and LB medium can be found elsewhere (2). All media were sterilized by autoclaving prior to use. The mineral chemicals used in the media were purchased from Sigma-Aldrich, and tryptone and yeast extract were purchased from Difco Laboratories (Detroit, MI). A gram-negative rod, Sphingomonas paucimobilis EPA 505 (EPA505), obtained from P. H. Pritchard (National Environmental Research Institute, Roskilde, Denmark) and a facultative bacterial strain, Pseudomonas stuzeri SAG-R (SAGR), obtained from D. R. Lueking (Michigan Technological University, Houghton, MI) were employed as pure cultures of PAH-degrading bacteria. The former was reported to be isolated from creosote-contaminated soils and capable of metabolizing/cometabolizing PAHs under aerobic condition (26, 27) and the latter was isolated from creosote-contaminated river sediments and capable of degrading PAHs under either aerobic or denitrifying condition (28-30). Both cultures were separately incubated under aerobic conditions at 25 ( 0.5 °C in two 1000-mL Erlenmeyer flasks containing 500 mL of an LB medium and a denitrifying mineral medium supplemented with glycerol (1.5%, w/v), respectively, to increase their biomasses. After 3 days of incubation, the harvested cells were washed to remove residual carbon substrates, and the number of bacterial cells (as CFU) inoculated to each bioreactor was determined by a viable plate count method using LB-agar plates as described elsewhere (2). Phenanthrene Biodegradation in Bioactive IMBRs. Laboratory-scale IMBRs used in our earlier abiotic desorption study (24) were employed as bioactive reactor systems after slight modifications (e.g., no dialysis membrane tubes containing Tenax TA resins in this study). As presented in Table 1, a fixed impeller revolution rate and various total mixing durations were employed to achieve various dimensionless mixing revolution number (NRev ) NθT, defined in ref 24). A relatively low impeller revolution rate was chosen to simulate feasible levels of agitation in field-scale operations. Periods of mixing required for obtaining uniform distributions (i.e., homogenization time) of the solid particles and liquids comprising the SMWT site soil and R-Al2O3 slurries were less than five minutes (data not illustrated). Each aerobic reactor was equipped with a glass tube at the bottom to supply oxygen in the form of air (0.1 mL/min). The SMWT site soil was mixed with a basal salt medium (67% solids content, w/w), and approximately 600 mL of the SMWT site soil slurry was
TABLE 1. Intermittent Auger Mixing Conditions total mixing duration each day (θT, hrs)
intermittent mixing scheme each day
mixing revolution number (NRev, dimensionless)
2 4 6 12 18 24b
2 times, each of 1-hr durationa 2 times, each of 2-hr duration 3 times, each of 2-hr duration 6 times, each of 2-hr duration 6 times, each of 3-hr duration continuous
6936 13 872 20 808 41 616 62 424 83 232
a The same each idle period (no mixing period) between mixing operations (e.g., 2 times of 11 h each, 2 times of 10 h each, 3 times of 6 h each, 6 times of 2 h each, 6 times of 1 h each, and 0 h of idle period from the lowest NRev to the highest NRev, respectively). b Continuously mixed batch reactor (CMBR).
loaded to each aerobic reactor along with EPA 505 inoculums (2 × 106 CFU/mL of aqueous solution). The soil-phase phenanthrene concentrations were monitored at regular intervals over 60 days. The same type of IMBR systems but without glass tubes were used for the anaerobic studies. A denitrifying mineral medium purged with N2 overnight was mixed with R-Al2O3 (67% solids content, w/w). These R-Al2O3 slurries were then spiked with phenanthrene and equilibrated for one month as described in our companion study (24). The final aqueous and solid-phase phenanthrene concentrations at equilibrium were 1000 µg/L and 3.68 ( 0.21 (mean ( standard deviation of triplicate samples) µg/g, respectively. Approximately 600 mL of the phenanthrene-spiked R-Al2O3 slurry was loaded to each anaerobic reactor along with the SAG-R inoculums (5.1 × 106 CFU/mL of aqueous solution). All anaerobic reactors were operated in an aerobic glovebox (Coy Laboratory Incorporation, Grass Lake, MI) filled with 99% N2 and 1% H2 (v/v) to maintain anoxic conditions over the biodegradation period. Concentrations of phenanthrene in the solid phase, and nitrate (NO3-) and nitrite (NO2-) in the aqueous phase, were monitored at regular intervals over 30 days. Redox potentials and dissolved oxygen (DO) levels in the aqueous phase of the R-Al2O3 slurry were also monitored to ensure maintenance of denitrifying conditions. A set of aerobic and anaerobic microbial controls in completely and continuously mixed batch reactors (CMBRs) sterilized by autoclaving for an hour were operated in parallel. Temperature was maintained at 25 ( 0.5 °C during the operation of both aerobic and anaerobic reactors. Collection of slurry samples from reactors and sample treatment (e.g., separation of water and solids from the slurry samples, organic solvent extraction of phenanthrene, etc.) were carefully performed as described in our previous studies (24, 25). Analytical Procedures. Phenanthrene concentrations were analyzed by the HPLC system described elsewhere (2). Concentrations of nitrate and nitrite were also determined by HPLC (Hewlett-Packard, HP series 1050, Palo Alto, CA) equipped with an IonPac AS4A-SC column (125 × 3.2 mm, Dionex, Sunnyvale, CA) and by UV spectrophotometry (Hewlett-Packard, HP series 1050) at a wavelength of 220 nm in a mobile phase (1 mL/min) composed of an ion chromatography eluent (0.191 g/L of Na2CO3, 0.143 g/L of NaHCO3 per liter of Milli-Q water). Apparent viscosity and torque values were obtained as described in our companion study (24). Levels of DO were measured using a DO meter (YSI, model 58, Japan) and redox potential was measured using a pH-redox meter equipped with a redox platinum combination probe (Corning, Big Flats, NY). Nonlinear regression analyses were performed to obtain parameters of models employed in this study by best fits of data using a Kaleidagraph computer program (Synergy Software, Release 3.01, Reading, PA). VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Model Parameters for Fluid Rheology and Power Consumption Power-Reynolds Number Correlation under Laminar Flow Conditions
Power Law Model slurry
power law consistency parameter (K)
power law index (p)
SMWT site soil R-Al2O3
4.23 ( 3.39 ( 0.11
0.37 ( 0.36 ( 0.01
a
0.09a
Mean ( standard error (number of observations ) 50).
0.08a
b
R2
reactor geometry parameter (C)
exponential coefficient (a)
R2
0.997 0.992
212.75 ( 129.64 ( 1.99
1.00 ( 1.01 ( 0.02
0.998 0.999
5.89b
0.03b
Mean ( standard error (number of observations ) 10).
FIGURE 1. Power curves for auger mixing of the SMWT site soil and r-Al2O3 slurries.
Results and Discussion Power Consumption. Parameters of the power law model obtained via nonlinear regressions of shear stress data measured as a function of shear rate for the SMWT site soil and R-Al2O3 slurries as performed in our companion study (24) are presented in Table 2. Evidently, both slurries behaved as pseudoplastic non-Newtonian fluids (i.e., p < 1). The SMWT site soil exhibited slightly higher apparent viscosities than the R-Al2O3 slurry over the entire shear rate range tested, which may be attributed to greater heterogeneities in the sizes, shapes, and structures of the natural SMWT site soil particles and/or associated sorbent organic matter (SOM) relative to those of the R-Al2O3 particles (data not illustrated). The correlation obtained between the dimensionless power and Reynolds numbers via dimensional analysis was employed to evaluate power requirements for auger mixing of the dense slurry fluids, as described in our companion study (24): i.e.,
NP ) CNRea
(1)
where C is a characteristic reactor geometry parameter, a is the empirical exponential coefficient, NP is the power number, and NRe is the Reynolds number. Figure 1 presents power numbers as functions of the Reynolds numbers for auger mixing of the SMWT site soil and R-Al2O3 slurries. The power curves for these slurries were similar over a range from laminar to turbulent flow conditions (0.5 < NRe < 104), confirming that system geometry is a primary factor in the correlation. Slight differences between the two curves in terms of power requirements may be attributed to the fact that the torque-mixer system used did not allow precise measurements of torque. Power-Reynolds number correlations analyzed for the two sorbent slurries only under laminar flow conditions (NRe < 20) are presented in Table 2. As observed in the companion study, the a values were close to unity, indicating that the power numbers are inversely proportional to the Reynolds numbers for laminar flow mixing of both slurries. As also described in the companion study, power and torque that relate, respectively, to operating and capital costs for mixing systems (31) were found to increase remarkably with increases in auger revolution rate and its 2276
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FIGURE 2. Phenanthrene biodegradation in aerobic IMBRs (A) and in anaerobic IMBRs (B). Error bars denote standard deviation of triplicate samples. diameter: i.e., power is proportional to the product of N1.99-2.00D2.98-2.99 and torque is proportional to N0.99-1.00D2.98-2.99. In turbulent flow regimes (NRe > 1000), the power numbers were essentially constant regardless of the Reynolds numbers, a situation commonly observed in baffled reactor systems (32). It should be noted that turbulent mixing of viscous nonNewtonian fluids involves high energy dissipation and associated high power consumption (33, 34). PAH Biodegradation under Aerobic and Denitrifying Conditions. Solid-phase phenanthrene concentrations in the SMWT site soil slurry systems monitored during the aerobic biodegradation experiments are illustrated in Figure 2A. Soilphase phenanthrene concentration decreased very slowly in the no-mix control reactor. The variations observed in the lag periods for onset of solid-phase phenanthrene removal likely relate to heterogeneities in contaminated soil samples (e.g., localization of contaminants, uneven distributions of microbial communities, microbial nutrients, and electron acceptors, and the physical and chemical characteristics of the associated SOM). Such spatial heterogeneities can be reduced effectively by mechanical mixing, and the resulting uniform distributions of bio-reaction constituents facilitated initiation of phenanthrene biodegradation, as evidenced by the dependency of lag period on the mixing revolution number (NRev): i.e., the larger the NRev value, the shorter the lag period. Once biodegradation began, rates of phenanthrene removal from the soil phase were enhanced significantly by mechanical mixing. As the NRev value increased, periods for
FIGURE 3. Concentrations of nitrate and nitrite as a function of time during phenanthrene biodegradation under denitrifying conditions in anaerobic reactors. Error bars denote standard deviation of triplicate samples. the removal of more than 95 wt % of extractable phenanthrene (excluding lag periods) became shorter, declining from approximately 21 days for the IMBR with a NRev value of 6936 to 8 days for the CMBR. Note that the principal enhancements of rates of solid-phase phenanthrene biodegradation were achieved at relatively low-level auger mixing. No concentration decrease was observed in the parallel abiotic control reactor over the period studied. Figure 2B presents solid-phase phenanthrene behavior in the R-Al2O3 slurry systems operated under denitrifying conditions. A very gradual decrease of solid-phase phenanthrene concentration was observed in the no-mix control reactor, while solid-phase phenanthrene concentrations decreased to levels below 5% of their initial values in less than 10 days in the anaerobic CMBR. Similar to behaviors observed for the aerobic IMBRs, phenanthrene biodegradation rates under denitrifying conditions were enhanced markedly by relatively low-level auger mixing, while further enhancement became marginal as the NRev value increased. Solid-phase phenanthrene concentrations were essentially constant in the parallel abiotic control reactor over the period of observation. No lag time was observed in any of the anaerobic bioreactors, indicating that the inoculated SAG-R appears to have readily been adapted to denitrifying environments. McNally et al. (30) reported that SAG-R pre-grown on glycerol did not require an adaptation period for phenanthrene degradation under denitrifying conditions because glycerol appears not to promote severe catabolite repression of PAH biodegradation. Redox potential values varied from -25 to 95 mV, and DO concentrations were at nondetectable levels ( 41 616). The empirical model developed in our companion study (24) was employed to evaluate the dependency of biodegradation rate coefficients on degree of mixing: i.e.,
kb ) kb,R + βbNRevc
(3)
where kb,R (day-1) is the value of kb under quiescent conditions (no mixing), βb (day-1) is the mixing sensitivity parameter representing impacts of mixing intensity on biodegradation rates, and c is an empirical exponential coefficient. As presented in Table 3, the evident nonlinear relationships between kb and NRev values (i.e., c < 1) under both aerobic and anaerobic conditions again demonstrate that auger mixing at low levels can be highly effective. The mixing revolution number can be increased by increasing either impeller revolution rate or total mixing duration (i.e., NRev ) NθT). High revolution rates, however, create high shear stresses, which may in turn decrease microbial activity via inhibition of adenosine triphosphate generation and uptake of electron acceptors, thus reducing specific growth rates or damping microbial cell membranes (39-41). Increases of overall mixing time, on the other hand, usually entail higher operating costs (31). In view of the strong dependency of power and torque on mixing revolution number (i.e., auger revolution rate) discussed earlier, it can be concluded that low-level auger mixing of dense slurries is much more performance-efficient and cost-effective for the design and operation of slurry-phase biodegradation processes than high-level mixing. Nonlinear correlations between degree of mixing and phenanthrene mass transfer (desorption) rates were observed in our companion study of abiotic IMBRs (24), demonstrating that low-level mixing enhances rates of release of sorbed contaminants associated with rapidly desorbing fractions of SOM, while only marginal further increases in rates of release were observed at much higher levels of mixing. Such trends were most evident in slurries comprising geosorbents containing rubbery or soft-carbon organic matrixes (e.g., cellulose and Chelsea soil). Parameters of the empirical model used to describe rapid desorption rate dependence on degree of mixing were very similar in magnitude to those determined for the bioactive IMBRs. This similarity suggests that the enhancement of phenanthrene biodegradation observed is similarly attributable to the introduction of optimal levels of reactor-scale mechanical mixing. In summary, IMBR systems can effectively address limitations associated with dense slurry biochemical transformation processes by compressing the temporal and spatial scales of reactor-scale mass transport processes to more closely match those of the reaction-scale microbial processes involved. Reactor-scale process analyses such as those performed here with respect to mechanical mixing and PAH biodegradation can be used to optimize slurry-phase treatment systems in terms of maximizing process efficiency and cost-effectiveness. The IMBR strategy described may in many instances function to obviate the need for excavation of 2278
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We thank Dr. Parmely (Hap) Pritchard (National Environmental Research Institute, Roskilde, Denmark) and Dr. Donald R. Lueking (Michigan Technological University, Houghton, MI) for providing cultures of the EPA 505 and SAG-R used in this study, and Dr. Jixin Tang of our Energy and Environment research group for his helpful data review and associated discussions. Funding for this research was provided by the Strategic Environmental Research and Development Program through a grant from the Army Corps of Engineers Waterways Experiments Station to the Great Lakes and Mid-Atlantic Center (GLMAC) for Hazardous Substance Research, operating under U.S. Environmental Protection Agency baseline Grant R-825539. Baseline support of the activities of GLMAC was also provided by the State of Michigan Department of Environmental Quality.
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Received for review March 19, 2004. Revised manuscript received December 10, 2004. Accepted December 21, 2004. ES049564J
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