Effect of Microbial Polymers on the Sorption and Transport of Phenanthrene in a Low-Carbon Sand Dirk M. Dohse and Leonard W. Lion’ School of Civil and Environmental Engineering, Cornell University, Ithaca, New York 14853
Extracellular polymers of bacterial origin were analyzed for their effect on the sorption behavior of phenanthrene on a low-carbon aquifer sand. Batch experiments indicated that 85% of the polymers tested acted to decrease the distribution coefficient. Column experiments revealed a decrease in the retardation factor of phenanthrene by approximately 40% in the presence of an extracellular polymer produced by a Gram-negative motile rod isolated from a coal tar waste site. This polymer did not, however, influence the mineralization of phenanthrene and was not rapidly degraded by a mixed culture. The combination of the ability of the polymer to influence phenanthrene transport as well as its apparent persistence and lack of a negative effect on phenanthrene degradation suggest that extracellular polymers can act as agents that enhance PAH transport in natural systems.
Introduction The ability to predict the fate of a pollutant compound may involve complex relationships between various processes and phases. The neglect of an important process or phase may lead to erroneous results. For hydrophobic pollutants such as polynuclear aromatic hydrocarbons (PAHs),sorption to solid surfaces is a predominant process in determining their fate in porous media and groundwater (1). Several investigators have observed PAHs in groundwaters and surface waters at higher concentrations and further downgradient than would be predicted by traditional soil/water sorption models (2-51, suggesting some additional transport mechanism. Possible explanations include hydrodynamic processes such as the fingering of flow in the soil or the presence of macropores. Another possible mechanism may be the presence of mobile colloids or dissolved macromolecules that may act as a carrier by sorbing the hydrophobic pollutant and transporting it through the soil matrix (6). Such “facilitated transport” has been observed in column experiments for hydrophobic compounds in the presence of humic materials as well as several exogenic and synthetic colloidal materials (7-10). This research, using batch and column experiments, focused on the role of microbial polymers as potential carriers due to their natural presence in groundwater (11). Surface soils near chemical spills can have elevated
* To whom correspondence should be addressed. 0013-938X/94/0928-0541$04.50/0
0 1994 Amerlcan Chernlcal Society
populations of bacteria (12). These populations may produce extracellular polymers, many of which act as surfactants and emulsifiers (13-16). These polymers may, in turn, act as a carrier to transport pollutants vertically from the vadose zone into the saturated zone during rain storms and horizontally with the groundwater in the saturated zone. Falatko and Novak (17) observed that a mixed bacterial polymer solution increased the solubility of gasoline in batch experiments and reduced its retardation in a sand-filled column. The sorption process for hydrophobic pollutants can be described by a reversible distribution:
where S is the mass of solute sorbed per unit mass of sorbent (mg/g), Kd is the distribution coefficient (mL/g), and C is the aqueous concentration of solute (mg/mL). If organic carbon is assumed to be the primary sorbent, the distribution coefficient can be normalized according to the organic carbon content (1, 18):
where foc is the fraction of organic carbon in the sorbent. The retardation factor, R, is a measure of a pollutant’s movement in porous media and is defined as the ratio of the average velocity of water relative to that of the pollutant. It can be derived from the one-dimensional advection dispersion equation based on the solute/sorbent interaction (19, 20), i.e. (3)
where Pb is the bulk density of the porous media, n is the porosity, and uw and us are the average velocities of the water and solute, respectively. If potential carriers are present in the soil however, the compound is distributed into three, instead of only two, compartments. One must therefore also consider the sorption of the compound to the carrier and the sorption of the carrier to the solid phase. Using the three distribution coefficients, Magee et al. (10) derived a modified expression for the retardation factor in the presence of a carrier that is also subjected to retardation: Envlron. Scl. Technol., Vol. 28, No. 4, 1994 541
where DOM is the carrier concentration, r d m is the distribution coefficient of the pollutant between the aqueous phase and the carrier, Ksd is the distribution coefficient of the pollutant between the aqueous phase and the sorbent, and p d o m is the distribution coefficient of the carrier between the aqueous phase and the sorbent. If one assumes that hydrophobic interactions are involved in the sorption of the carrier to the pollutant and the carrier to the solid phase, then it may be reasonable to expect that the two distribution coefficients involving the carrier will co-vary. A simple assumption consistent with this hypothesis would be to require that the ratio of the carrier distribution coefficients be a constant, r [Le., r = Gm/fldom = constant]. Given this assumption, a plot of R* from eq 4 versus Kimreveals a distinct minimum in the retardation factor (Figure 1A). At low r d m values (and correspondingly low f l d o m values), the carrier does not sorb the pollutant and, hence, does not influence its transport. Under these conditions R* remains nearly constant (see Figure 1A for KOm < 2 X lo3). At intermediate p d o m and r d m values, the carrier sorbs the pollutant but remains sufficiently mobile ( p d o m is less than p d ) so that the pollutant retardation factor, R*, is reduced. However, at high p d o m and K:m values (see Figure l a for Kim > lo6), the carrier sorbs more strongly to the solid phase than does the pollutant (K"dom is greater than K"d) and, hence, increases the calculated value of R*. As illustrated in Figure lA, the magnitude of the effect of the carrier upon R* depends upon the magnitude of the value that is assumed for the ratio, r of p d o m and Kim. [Note, all calculations for Figure 1assume a porous medium and solute comparable to the aquifer sand and phenanthrene employed in this research; therefore, p d and pb/n were held constant.] Figure 1B illustrates the effect of the carrier concentration at a fixed I(Odm/pdom ratio. As expected, increased carrier concentration results in higher pollutant mobility. A sensitivity analysis of the model by Magee et al. (10) revealed that the pollutant distribution coefficients, p d and Kim,as well as the carrier concentration were the most important model parameters. The objective of this research was to test the hypothesis that dissolved extracellular polymers of bacterial origin can enhance the transport of hydrophobic pollutants in porous media, Batch sorption isotherms were used to screen candidate polymers for their effects on pollutant sorption, and column transport experiments were used to confirm pollutant mobility in the presence of a selected polymer. Altering the phase distribution of a pollutant may also alter its rate of mineralization by soil bacteria. Therefore, the effect of the presence of the selected polymer on pollutant mineralization was considered as was the susceptibility of the polymer to bacterial attack. Experimental Methods Phenanthrene is a polynuclear aromatic hydrocarbon (PAH) and was used as a model pollutant because of its hydrophobic nature. The aqueous solubility of phenanthrene is reported to be 1.29 mg/L, and its octanol-water partition coefficient is 3700 ( I ) . Radiolabeled phenan542
Environ. Scl. Technol., Vol. 28. No. 4, 1994
mg TOCiL
DOM.100
500 mg TOC/L '
s
0'
r=50,000 102
103
10'
1o5
106
1
Figure 1. Effect of the distribution coefficient between the carrier and the solid phase on the retardationfactor of a hydrophobic pollutant as (A) a function of the ratio of the carrier distributlon coefficient with the pollutantto the carrler distribution coefficientwith the stationary phase, r = $m/phm and (B) as a function of carrier concentration. (Ail calculations assume the sorbent and sorbate combination are comparableto phenanthreneand an aquifer sand, $ = 12.9 and pb/n = 4.38.)
threne (9J4C, 13.1 pCi/pmol, Sigma Chemical Co., St. Louis, MO) was tested for purity by high-performance liquid chromatography and determined to be free of chemical and radiochemical impurities. For batch and column experiments, the phenanthrene crystals were placed in a saturator similar to that developed by Burris and MacIntyre (21)and dissolved in 18.9 MWcm distilleddeionized water containing 5 mM Cas04 and 0.02 % NaN3. Cas04 was added as a background electrolyte, and NaN3 was added as a biological inhibitor. The aquifer sand used in all experiments was obtained from a quarry in Newfield, NY. The organic carbon content of the sand was determined to be 0.049 f .012% (n = 7) using the WalkleyBlack method (22). A size analysis of the sand has been previously reported by Magee et al. (10)and revealed that 47.2% and 47.6% of the sand were in the fine (0.1-0.25 mm) and medium (0.25-0.5 mm) size ranges, respectively. Extraction of Polymers. Bacterial isolates were obtained primarily from a coal tar waste site and a laboratory culture collection (C. Thomas and W. Ghiorse; Section of Microbiology, Cornel1 University). Other cultures were obtained from the American Type Culture Collection (ATCC) and the Department of Energy Subsurface Microbial Culture Collection (D. Balkwill, Florida
Table 1, Summary of Isolates and Their Sources depth
Gram source or reaction catalase oxidase reference
bacterium
(m)
Pseudomonas A100 9701A-2‘ 9701A-4 9701M 9702A-2 9702M-4 9703A-1 9703111-5 9704M-1 9706M-2 9707M-3 9709A-3 9711A-2 9711A-4 9712M-3 9714A-4 B649‘ B693 Pseudomonas cepacia 249-100 Pseudomonas fluoresceme
surfacea 1.8 1.8 1.8 4.0 4.0 11 11 12 4.8 7.0 9.4 5.8 5.8 8.1 9.1 259 259 NAd
+ ++ + ++ + + + + +-
NA
-
+ + + +
-
-
-
+ ++ + + ++ + + + + ++ +
+ + + + + + +-
+ ++ +
23 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 49 49 50 50
Rhizosphere isolate from experimental soybean plants cultured in Honeoye silt loam obtained from the Aurora research farm of Cornel1 University, Aurora, NY. Strains designated 97x2 were isolated from an Electric Power Research Institute (EPRI) manufactured gas plant site in the midwestern United States. Strains desginatedB6xx were from the Department of Energy (DOE) field site at Savannah River Plant site P24; Aiken, SC (DOE Subsurface Microbiology Collection at Florida State University). NA = not applicable. e ATCC 13524 (American Type Culture Collection, Rockville, MD).
*
State University). One isolate from a soil root zone was also used (23). Sources of the isolates are indicated in Table 1. The isolates were grown in a 5 g/L glucosegrowth medium (24) until early stationary growth. To obtain a 1%-labeled polymer for polymer mineralization experiments, [14C]glucose(uniformly labeled, specific activity = 9.1 pCi/pmol, ICN Biomedicals; Irvine, CA) was added to the growth medium. The procedure for extraction of extracellular polymers was adapted from techniques used by Corpe (25). An extracellular polymer was separated from the cell mass by centrifugation at 8000g and precipitated by mixing solutions containing a polymer with equal volumes of acetone. Cell-bound polymers were extracted according to a modified version of the procedure described by Brown and Lester (26). A 2% solution of Na4EDTA was added to the cell mass after separation of the extracellular polymers as described above and refrigerated for 30 min. The sample was then centrifuged at 12000g for 50 min at 5 “C. The resulting supernatant contained the cell-bound polymers that were solublized by EDTA treatment. After centrifugation, the concentrated polymer solutions were placed in 8000 molecular weight cutoff tubing and dialyzed against distilleddeionized water to remove excess solvents and low molecular weight impurities. The purified polymer was then freeze-dried. Prior to use, the polymer was redissolved in the CaSO4NaN3 electrolyte solution at a concentration of 100 mg TOC/L. Polymer that was difficult to dissolve was sonicated and/or placed on a rotary shaker until the dissolution was complete. Samples were then filtered through an ll-pm filter (Whatman No. 1) to eliminate any large flocculant particles. HPLC analysis for polymer molecular weight was carried out using a Hewlett-Packard 1090 HPLC on a BioSep-
SEC-S4000 column (Phenomenex, Torrance, CA) using a mobile phase of 50 mM PO4 buffered to pH 6.0. UV analysis of the eluate was determined by a diode array detector at wavelengths of 195,210, and 275 nm. Infrared spectra of polymers was determined using KBr pellets and a Perkin-Elmer Model 683 spectrophotometer. Isotherm Experiments. Batch isotherms were used to determine the sorption of phenanthrene onto the sand in both the presence and the absence of polymer and to determine the distribution of polymer between the aqueous phase and sand. All experiments were run in the dark at 25 “C. The fluorescence quenching method described by Gauthier et al. (27)was used to determine the distribution between phenanthrene and the polymer. Flame-sealed borosilicate glass ampules were used to provide an all-glass enclosure for the batch isotherm experiments since phenanthrene has been shown to significantly sorb to Teflon seals (28). One milliliter of [l4C1phenanthrenefrom the saturator (activity 0.011-0.016 pCi/mL) was added to ampules filled with varying amounts of sand and CaS04-NaN3 electrolyte solution and immediately flame-sealed. Sand concentrations ranged from 1 to 4 g/10 mL of solution with a typical initial [l4C1phenanthrene concentration of 100 pg/L. Samples were equilibrated for 24 h, with agitation provided by a rotary tumbler. A 24-h equilibration period was determined to be sufficient to give aqueous concentrations that were stable over a time interval of 10 days (29). Aqueous concentrations were then measured by liquid scintillation after centrifugation to remove suspended matter. All isotherm data presented are based on the linearized form of the mass balance equation:
where C is the concentration, V is the volume, M is the mass of sorbent, and the subscripts o and s refer to controls (no sorbent) and samples (with sorbent), respectively. Equation 5 was used to determine the distribution coefficient instead of eq 1 due to the lack of interdependence of the regression variables. The linearity of data plotted in the form of eq 5 and the presence of low concentrations of TOC in sand blanks were taken as evidence of negligible solids effects on the phenanthrene distribution coefficient. To determine the distribution coefficient between the polymer and the sand, rdom, a similar procedure was used. Polymer dissolved in electrolyte solution was added to 10 mL of Teflon-capped test tubes and equilibrated for 24 h on a rotating mixer. Samples were subsequently centrifuged, and the supernatant was measured for total organic carbon. Dissolved polymer results were corrected for the amount of organic carbon released from the sand as measured from control samples without polymer. To determine the distribution coefficient between phenanthrene and the polymer, KOrn, the fluoreeence quenching method described by Gauthier et al. (27) was used. The fluorescence of phenanthrene was measured using a quartz cuvette and excitation/emission wavelengths of 288 and 364 nm, respectively. The data were corrected for dilution effects, fluorescence of polymer alone, and the “inner filter effect” (27). Environ. Scl. Technol., Vol. 28, No. 4, 1994 543
10
Table 2. Summary of Column Experiment Parameters
experiment
8“ v b
De
Pbd
ne
-
pulse polymer widthf concng
phenanthrene 5.0 8.0 3.72 1.77 0.37 8.41 0 phenanthrene 5.0 6.6 2.39 1.69 0.37 8.48 0 phenanthrene/polymer 5.0 7.2 3.34 1.68 0.35 8.85 93.9 phenanthrene/polymer 5.0 7.0 3.26 1.73 0.37 8.40 103.2 polymer 5.0 7.2 3.34 1.68 0.35 2.80 93.9 a Bulk flow rate (mL/h). Pore water velocity (cm/h). c Dispersion coefficient determined from nonlinear least-squares fit of chloride BTC (cm2/h). d Sand bulk density (g/mL). e Porosity of sand in column (mL/mL). f Pulse duration for phenanthrene in pore volumes (O(pore volumes) = [time (h)][bulk flow rate (mL/h)l/[porosity X empty column volume (mL)]). 8 In mg of organic carbon/L.
v)
> v)
61
y
u.0
0.1
=
0.90
+
19.99x
/
?0
> 0
0
~-
Column Experiments. Miscible displacement experiments were performed to determine the retardation of phenanthrene in porous media and to quantify the effect of polymer on the transport of phenanthrene. Conditions for column experiments are summarized in Table 2. A detailed description of column methodology is provided by Dohse (24). Briefly, an all-glasscolumn assembly (25-mm i.d., 5-cm length) was used comparable to that described by Lion et al. (28). Two continuous syringe pumps were used (Pharmacia Model LHB P-500): one to deliver the phenanthrene pulse and the second to deliver the mobile phase. In experiments for phenanthrene in the presence of polymer, the polymer was added to both the pulse input and the mobile phase used to elute the pulse. The pump delivering phenanthrene was presaturated by circulating a phenanthrene/electrolyte solution until the effluent concentration remained constant. Stainless steel tubing was used wherever possible to limit the sorption of phenanthrene onto the experimental apparatus. The retardation factor was estimated from the data using the first temporal moment of the phenanthrene breakthrough curve (BTC), i.e.
where C/C, is the relative concentration, 8 is the pore volume, and 8, is the pulse width. Mineralization Experiments. Mineralization experiments on phenanthrene and polymer were performed using 155-mLserum bottles containing a 500-pL glass vial filled with 0.5 M NaOH to trap 14C02evolved from the mineralization of [14CIphenanthrene or 14C-labeledpolymer. Sand (1g) and an inorganic salt growth medium (30) (5 mL) were added to bottles that were autoclaved prior to the addition of [Wlphenanthrene and the bacterial inoculum. Samples without a sorbent were also analyzed. [l4C1Phenanthrenedissolved in a 5050 mixture of methanol and deionized water was added aseptically to the serum bottle, which was immediately crimp capped with a Teflon septum and allowed to equilibrate with the sand, when present, for 24 h before inoculating. The total initial [Wlphenanthrene concentration was ZO.8 mg/L. The diluted concentration of methanol was sufficiently low (
