Environ. Sci. Techno/. 1995,29,515-521
Reduced Mineralization of Low Concentrations of Phenanthrene Because of Swuesterirg in Nonaqueous-Phase Liquids R E B E C C A A. E F R O Y M S O N t A N D M A R T I N ALEXANDER* Institute for Comparative and Environmental Toxicology and Department of Soil, Crop, and Atmospheric Sciences, Cornel1 University, Ithaca, New York 14853
A nonaqueous-phase liquid (NAPL) may sequester a large fraction of a hydrophobic pollutant away from the aqueous phase. A study was conducted to determine whether the low aqueous concentrations of the compound may be associated with the absence of biodegradation. A phenanthrene-degrading mixed culture did not mineralize phenanthrene when initially dissolved in di-2-ethylhexyl phthalate (DEHP) at concentrations of 0.6-20 pg/mL. Under these conditions, the concentration of phenanthrene in water at equilibrium was less than 1 ng/mL. Such a threshold was not observed when a strain of Pseudomonas or a sample of subsoil was used as the inoculum or when the NAPL added was 2,2,4,4,6,8,8-heptamethylnonane. However, the biodegradation rates by all three populations at the low concentrations of phenanthrene in the NAPLs were slow and far less than expected from the rates at higher concentrations. At high concentrations, the rates of mineralization were higher than the rates of partitioning of phenanthrene to water, whereas mineralization was much slower than partitioning at low concentrations. W e suggest that some NAPLs may sequester hydrophobic compounds away from the aqueous phase to an extent that the concentration falls below the threshold for biodegradation or to a level that results in unexpectedly slow biodegradation.
Introduction Organic chemicals at low concentration often are not degraded by microorganisms. 2,4-Dichlorophenol in a liquid culture of Pseudomonas sp. (I), 2,4-dichlorophenoxyacetate in streamwater (21, dichlorobenzenes in nonsterile soil columns (31, and PCB congeners in freshwater sediments (4) are not metabolized at low concentration. An explanation for the threshold concentrations for biodegradationis the need for more substrate for maintenance energy by the microorganisms than is supplied by diffusive flux of the chemical (9. Moreover, given the importance of microbial acclimation for the biodegradation of contaminants in aquifers (@ or at waste sites, it is possible that microorganisms require a minimum concentration of particular chemicals to degrade them. The sequestering of a hydrophobic compound by nonaqueous-phase liquids (NAPLs) may cause the concentration of the compound in the aqueous phase to fall below the threshold for biodegradation. Indeed, concentrations of organic solutes partitioning from NAPLs to groundwater are often 10timeslower than their solubilities in water (7). Moreover, if the concentration of the solute in the NAPL is low, the concentration in water may be < 1% of the aqueous solubility of the compound (8). Similarly, a hydrophobic phase to which an organic substrate is sorbed may cause the concentration of the substrate in water to fall to levels below the threshold for biodegradation; thus, a strain of Pseudomonas is unable to degrade low concentrations of p-nitrophenol when most of the compound is sorbed to hydrophobic beads (9). Moreover, the inhibition of phenanthrene mineralization by nonionic surfactants was hypothesized to result partlyfrom the sequestering of the substrate from the microorganisms by the micelles (10).
Biodegradation of readily metabolized organic compounds is very slow in the presence of some NAPLs, even whentheorganicliquidsarenot toxic (II,12).Hydrophobic compounds that are largely sequestered by NAPLs have been shown to be degraded slowly (11,121. Indeed, a slow mineralization of phenanthrene has been shown to be associated with di-2-ethylhexylphthalate (DEHP),a NAPL from which the hydrocarbon partitions slowly (8). Therefore, a study was conducted to determine whether a NAPL may sequester a hydrophobic compound to such an extent that no biodegradation occurs.
Materials and Methods Chemicals. Unlabeled phenanthrene and [9-14C]phenanthrene (8.3 mCi/mmol, '98% pure) were purchased from Aldrich Chemical Co. (Milwaukee,WI) and Sigma Chemical Co. (St. Louis, MO),respectively. DEHP, n-hexadecane, and 2,2,4,4,6,8,8-heptamethylnonane were also obtained from Aldrich Chemical Co. The log P of DEHP is 7.9, and its solubility is 0.34 mglL (13). The log P of heptamethylnonane has been estimated to be 10.1 (121, and its solubility is probably much lower than that of DEHP. The NAPLs were chosen because of their low volatility, the inability of the organisms to use them for growth, and their t Present address: Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6036.
0013-936)(195/0929-0515$09.00/0
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presumed low toxicity to the bacteria studied. Benzoic acid was purchased from Mallinckrodt (St. Louis MO). Subsoil. A sample of subsurface soil (pH4.6,99.5%sand, '0.05% organic matter) of Typic Haplorthod family was collected above the water table from the C horizon at a depth of 1.0-1.2 m at a site near Tupper Lake in the Adirondack Mountains of New York. Bacteria. Bacteria capable of using phenanthrene as a sole carbon and energy source were isolated as described previously (8). The enrichments were transferred serially to fresh salts solutions containing phenanthrene and then plated on Tryptic soy agar. The characteristicsof Pseudomonas strain R have been reported earlier (8). A phenanthrenedegrading mixed culture was obtained from the subsoil; more than 99% of the colonies of this enrichment had the same morphology. For an experiment in which low concentrations of benzoic acid were used, a phenanthrenemineralizingbacterial isolate, strain M1, was obtained from the enrichment culture. Prior to experiments, the cultures were grown on 50 mg of phenanthrenelL of inorganic salts solution at 30 "C on a rotary shaker operating at approximately 120 rpm. The cultures were used in the late exponential or in the early stationary phase of growth and were first passed through a 40-ym pore-size glass frit to remove the remaining crystals of the hydrocarbon substrate. The cells were collected by centrifugation at about 10400gfor 12 min at 4 "C, and the bacterial pellets were resuspended in buffer and centrifuged two more times to remove soluble substrate and products of biodegradation. The inoculum densitywas between lo3 and lo5 cellslml. Mineralization. The bacteria were added to 50 mL of inorganic salts solution contained in a 250-mL biometer flask. A coarsely porous, aluminum oxide and silicate extractionthimble (Alundum: Norton Scientific,Worcester, MA; inside diameter, 22 mm; height, 70 mm) was placed upright in the flask. The cylindrical thimble confined the substrate-NAPL mixture to a continuous surface area for measurements of mineralization and partitioning. The lowdensity NAPLs did not leak from the coarse pores in the thimble. Large holes at the bottom of the outer wall of the thimble allowed complete exchange of water containing bacteria and phenanthrene inside and out of the thimble. A NAPL-phenanthrene mixture (0.5 mL) was added to the surface of the water within the thimble. The substrate was about 80 000-120 000 dpm of [14Clphenanthreneplus, in some instances, unlabeled phenanthrene. The side arm of the flask contained 2.0 mL of 0.5 M NaOH to trap evolved 14C-labeledand unlabeled COZ. Periodically, the alkali was removed through a cannula and replacedwith fresh NaOH. Four milliliters of Liquiscint scintillation cocktail (National Diagnostics Inc., Atlanta GA) was added to the NaOH that was removed, and radioactivitywas measured with a liquid scintillation counter (ModelLS 7500;Beckman Instruments Inc., Inine CAI. Tests were conducted in duplicate or triplicate. For measurement of phenanthrene mineralization in subsoil, 50 g (dryweight) of subsoil moistened with 5.0 mL of a solution containing 900 mg of KH2P04, 100 mg of K2HP04, and 200 mg of NH4N03/L was added to each biometer flask, the final moisture level being 13.9% (voll wt). DEHP (0.5 mL) containing phenanthrene was then mixed thoroughly with the subsoil. A thimble was not present. 516 m ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 2, 1995
Partitioning. Partitioning of phenanthrene from the NAPL was measured in 50 mL of sterile, aqueous solutions
in 250-mL Erlenmeyer flasks whose shapes were similar to the biometer flasks. Phenanthrene containing 300 0001 400 000 dpm of [14Clphenanthrenewas added in 0.5 mL of a NAPL to the surface of the water phase within thimbles placed in the flasks. At intervals, 1.5 mL of the aqueous solution was removed and added together with 3 mL of scintillation fluid to 7-mL scintillation vials. Before use, the biometer and Erlenmeyer flasks were washed in concentrated sulfuric acid. The aluminum oxide thimbles were cleaned by placing them, in sequence, in methanol, soapy water, a water rinse, concentrated sulfuric acid, and a water rinse. They were then combusted at 500 "C. All flasks were incubated at approximately 21 "C on a rotary shaker (New Brunswick Scientific, New Brunswick NJ) operating at 80-100 rpm. Data Analysis and Calculations. The equilibrium concentration of phenanthrene in the aqueous phase was determined by a simplified two-compartment model: dCldt = k(Ceq- C)
(1)
where C is the concentration of phenanthrene in the aqueous phase, Ceq is the concentration at equilibrium, k is a partitioning rate constant, and tis time. Although this model was not required for the estimation of Ceq, it was used because the data reported here were obtained in conjunction with a larger study in which the model was needed to estimate partitioning rates (8). In two-compartment models, Ceq is usually not a constant and is determined at each small increment of time by a sorption "isotherm". Ceqis often written as the product of the concentration in the nonaqueous phase ( N U L in the present study) and the equilibrium partition coefficient, which is a constant. In these experiments, however, the change in concentration of phenanthrene in the NAPL was negligible; thus, Ceqwas treated as a constant. The thimbles, which confined the surface areas of the NAPLs, permitted the use of a single value for k. A partition rate constant or mass transfer coefficient is constant if the interfacial area between the two phases is constant. Without the thimble, the NAPLs would have spread during biodegradation. Since C = 0 when t = 0 C=
ceq(I - e-k',
The partitioning data provided measurements of Cfor each time t. Equation 2 was fit to these data, and estimates of Ceqand k were made. According to eq 1, when t = 0, C = 0 and dC/dt = k(C,,). The equilibrium concentration of phenanthrene in water was determined independently for each flask by nonlinear regression using eq 2. The maximum rate of partitioning from the NAPL was the product of the equilibrium concentration and the rate constant. The maximum rates of mineralization were determined either by measurement of the slope of a line or by empirical curve fitting using a fourth-order polynomial equation. The procedure for selecting points to include in the curve fit is described by Efroymson and Alexander (8). The calculation was performed separately for two or three replicate flasks.
Results Studies were undertaken with DEHP and heptamethylnonane as NAPLs and Pseudomonas sp. strain R and the
4
I
T
40
3
30 2
20 1
?i 2 w
DEHP
0
0
0
0
HEPTAMETHYLNONANE
zI
$
10 A
d, z?
40
z?
40t 20
20
l
o
o
o
r
0
0
0
5
10
15
DAYS
FIGURE 1. Mineralization by an enrichment culture (A) or Pseudomonas sp. (B)of 0.6 pg of phenanthrene/ml of DEHP or heptamethylnonane.
mixed culture from subsoil as the microorganisms carrying out the degradation ofphenanthrene. Initially, the substrate (300 ng, was added to 0.5 mL of either DEHP or heptamethylnonane, and the mixture was added to the extraction thimbles in the flasks. Thus, the initial concentration of phenanthrene was O.Gpg/mL of NAPL or 6 ng/mL of water if all of the phenanthrene had been initially in the aqueous phase. Mineralization by the mixed culture of phenanthrene in heptamethylnonane was slow, and the bioactivity on phenanthrene in DEHP was negligible. In 16.6 d, 3.1% and 0.18% of phenanthrene in heptamethylnonane and DEHP, respectively,were mineralized (Figure1A). The latter value may not represent metabolism of phenanthrene because it is less than the level of contamination of the labeled phenanthrene. The initial cell density was 2.1 x lo4 cells/mL of water. In contrast, 55.3%and 9.6%of the hydrocarbon in heptamethylnonane and DEHP, respectively, were mineralized by Pseudomonas sp. strain R in the same time period with an initial density of 1.9 x lo4 cells/ mL (Figure 1B). Mineralization was continuing at the last time point. In the same study, phenanthrene was initiallydissolved in DEHP to give 1000 pg/mL of NAPL;this is equivalent to 10 pg/mL of water if all the phenanthrene initially was in the aqueous phase. For the sake of comparison with this high concentration, the plots of the transformation of 0.6 pg of phenanthrenelmL of DEHP are presented again. Mineralization of this high concentrationby the enrichment culture was both rapid and extensive after an initial acclimation period (Figure 2A). Two plots at 1000 pglmL are shown in the figure because of different lengths of the acclimation phase of the duplicate cultures, but the rates and extents of mineralization were similar. At 0.6 pg of phenanthrenelmL of DEHP, the enrichment did not mineralize the test compound appreciably. Pseudomonas sp. strain R similarlymineralized the higher concentration both rapidly and extensively after an acclimation period, but little phenanthrene present at 0.6pglmL of DEHP was converted to COZby the bacterium (Figure 2B).
5
0
20
10
15
20
DAYS
FIGURE 2. Mineralization by an enrichment from subsoil (A) or Pseudomonas sp. (B) of phenanthrene initially in DEHP at loo0 or 0.6 pg/mL Two plots at loo0pg/mL are shown in panel A because the acclimation phases in duplicate cultures were different.
"
0
5
10
15
20
25
DAYS
FIGURE3. Mineralizationof phenanthreneinitiallydissolved in DEHP (A) or heptamethylnonane (B) by an enrichment from subsoil. The initial concentrations of substrate (in pg/mL) of NAPL are shown in the figure.
Because mineralization by the mixed culture of low concentrations of phenanthrene was slow or did not occur, further experiments were conducted to assess the possible existence of a threshold concentration of phenanthrene for biodegradationby the responsiblemicroorganisms. Both heptamethylnonane and DEHP were used as NAPLs, and the initial bacterial densitywas 3.7 x lo4cells/mL of water. When phenanthrene was dissolved in DEHP to give 6 and 20 pglmL, mineralization by the enrichment was not detected (Figure 3A). On the other hand, 100 pg of phenanthrenelmL of DEHP was extensively mineralized, and almost 50% of the compound was converted to COa. VOL. 29, NO. 2,1995 I ENVIRONMENTAL SCIENCE &TECHNOLOGY
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4
TABLE 1
Partitioning of Phenanthrene from DEHP equilibrium concn initial concn in DEHP kg/mL)
in water (nglmL)
in DEHP (ClglmL)
k(h-l)
10.5 24.5 110
0.319 f 0.034 0.812f 0.071 3.24 j, 0.33
10.5 24.4 109.7
0.137 f 0.029 0.146 f 0.012 0.146 f 0.009
3
.3 E
0
1
CT
w
+
5 z
W
2
Ea
I
c
z
Q
5 2
.
1
-m 5
0.8
1
0 PHENANTHRENE IN DEHP IpglrnL)
FIGURE 5. Isotherm depicting concentrations of phenanthrene in OEHP and water at equilibrium.
0.4
0 0.4
0.3 0.2 0.1 0
1
,
I
I
t
I 1
I
I T a
T
0
10
20
C 30
40
50
HOURS
FIGURE 4. Partitioning to water of phenanthrene initially present at 110 (A), 24.5 (B), or 10.5 (C) pg/mL of DEHP.
In contrast, the mineralization of phenanthrene in heptamethylnonane occurred at 100, 20, or 6 pglmL of the NAPL (Figure 3B). Mineralization was almost linear for extended periods at the three concentrations. The acclimation phase prior to rapid degradation was approximately 9 days at the lowest concentration, and only a short acclimation phase (less than 2 days) was evident at 100 pg/mL. Studies were conducted to determine the rates and extents of partitioning from the NAPL that were associated with concentrations used in tests of mineralization. However, to have adequate sensitivity for determinations of partitioning, at least four times the radioactivity used in mineralization experiments was required. Given the low specific activity of the phenanthrene (8.3 mCilmmol), partitioning tests could not be undertaken at initial concentrations comparable to those used for mineralization. Therefore, partitioning was measured at initial concentrations of 10.5,24.5, and 110 pg of phenanthrene/ 518 1 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 2, 1995
mL of DEHP, and an isotherm of equilibrium concentration in water versus equilibrium concentration in DEHP was plotted. The equilibrium concentration of phenanthrene in water decreased as the initial concentration in the NAPL decreased (Table 1). Less than 1 ng of phenanthrenelmL of water was present at equilibrium when the initial concentration of phenanthrene in the NAPL was 24.5 or 10.5 pglmL. The rate constants were comparable at the three concentrations. The rate and extent of partitioning to water of phenanthrene added to DEHP at three initial concentrations are depicted in Figure 4. The plots represent the fit of the simplified two-compartment model. That model fit the data reasonablywell; however, although few measurements were made after 20 h, the measurements taken at about 50 h suggest that equilibrium may not have been achieved as early as the model estimated. Although the figure depicts the fit of the two-compartment model to the average of the replicate samples, the data in Table 1 were taken from independent fits of the replicate treatments. The partitioning data were used to construct a linear isotherm depicting the relationship between the concentrations of phenanthrene in DEHP and water at equilibrium (Figure 5). The regression line is y = 0.0304~ 0.03330 ( r = 0.99981, where x represents the concentration of phenanthrene in DEHP (inpglmL),yrepresents the concentration ofphenanthrene inwater (innglmL), and risthe correlation coefficient. This equation was used to determine the approximate quantities of phenanthrene in water at equilibrium during the mineralization experiments. At initial phenanthrene concentrations of 0.6, 6, and 20 pglmL of DEHP, the calculated concentrations in water at equilibrium are 0.05, 0.21, and 0.64 nglmL, respectively. Presumably, the y-intercept should be 0. If the slopes from the origin to each point are averaged instead of performing a linear
+
TABLE 2
Phenanthrene Partitioning from DEHP and Mineralization rate organisms enrichment culture
Pseudomonas
initial concn of phenanthrene in NAPL bg/mL)
equilibrium concn in water. (nglml)
partitioning* (ng ml-1 h-l)
mineralization (ng ml-l h-l)
0.019 0.19 0.62 3.1 31 0.019 31
0.0027 0.027 0.090
0.000026 f 0.000002 0.00053 f 0.00004 0.0021 f 0.0001 2.0 f 0.2 37 f 7 0.0026 f 0.0008 37 & 7
0.6
6.0 20 100 1000 0.60
1000
0.44 4.4 0.0027
4.4
Equilibrium concentrations (ng/mL of water) were estimated as 0.031 1 x initial concentration of phenanthrene in DEHP @g/mL). Partitioning rates were calculated by kc.., where k was estimated as 0.14 (Table 1). a
regression with the data in Figure 5, the equation is y = 0.0311~;then, 0.6, 6, and 20 pg of phenanthrene/mL of DEHP give equilibrium concentrations of 0.02, 0.19, and 0.62 ng/mL of water, respectively. Mineralization by the enrichment culture was negligible when the equilibrium concentration of phenanthrene in water was 0.62 ng/mL or less (Table 2). Although high rates of phenanthrene partitioning to water (about 0.44or 4.4 ng mL-l h-l) were associated with mineralization rates that were 5-10 times as high, partitioning rates below 0.9 ng mL-l h-l were associated with negligible rates of mineralization. Pseudomonas sp. mineralized phenanthrene initially dissolved in DEHP at 0.60 ng/mL at a rate equivalent to the partitioning rate (Table 2). A study was conducted to ascertain whether the failure of the mixed culture to degrade low concentrations of phenanthrene in the presence of NAPLs resulted from the inability of the microorganisms to grow on or to degrade any organic substrate at low concentrations. The mixed culture and Pseudomonas sp. strain R then were tested for their ability to grow on dissolved organic contaminants in the inorganic salts solution. After a 10-15-h lag period, the population of Pseudomonas sp. strain R increased from 7 x lo3 to about 2.4 x lo5 cellslml, and the mixed culture grew from 1 x lo4 to approximately 1.8 x lo5 cells/mL in 60 h. In both instances, some growth occurred at the expense of organic contaminants in the inorganic salts solution. An experiment was performed to determine whether phenanthrene-degrading microorganisms in the mixed culture could mineralize low concentrations of an organic substrate in the absence of a NAPL. This experiment was intended to ascertain whether the lack of mineralization of phenanthrene in DEHP actually resulted from a threshold in the water phase for the mixed culture. Phenanthrene itself was not tested because of the large percentage that would rapidlyvolatilize at low concentrationswhen a NAPL was not present. The substrate chosen was benzoic acid. The lowest concentration used, 0.3 ng/mL of water, was in the range of concentrations of phenanthrene in water at equilibrium when no microbial activity was previously detected. Because a rare microorganism in the mixed culture incapable of degrading phenanthrene might metabolize benzoic acid, a bacterium that could mineralize both compounds, strain M1, was isolated from the mixed culture. Strain M1 did not have the same threshold as the parent culture, and it mineralized 5% of 6 pg of phenanthrene/mL of DEHP in 44 days. Strain M1 did mineralize 1.0 mg of phenanthrene/mL of DEHP much more rapidly
than the lower concentration. With an initial density of 8.3 x lo3 cellslmL and benzoic acid concentrations of 0.3,3.6, and llOOnglmLofwater,50.3,42.2, and37.6%,respectively, of the substrate were mineralized in 3 d. Thus, the benzoate-utilizing isolate had no detectable threshold, in contrast with the original mixed culture. A study was conducted to determine whether there was a threshold concentration below which phenanthrene dissolved in DEHP was not mineralized in samples of subsoil. Nitrogen and phosphorus were added to the subsoil because biodegradation of phenanthrene in this subsoil is limited by these nutrients (12). In 33.5 d, 56.0 f 5.0,66.0 f3.2, and 6.18 f 1.9% (kl standard deviation) of phenanthrene were mineralized when the initial concentrations of the hydrocarbon in DEHP were 1.7, 15.7, and 100 pg/mL, respectively.
Discussion The concentration of a hydrophobic compound in water in proximity to a NAPL may be too low for biodegradation by some microorganisms to occur. On the other hand, other organisms may not have a demonstrable threshold and may transform low concentrations of a chemical in water in equilibrium with the NAPL. Thus, the mixed culture from subsoil did not mineralize phenanthrene initially present in DEHP at concentrations of 20 pg/mL or less. However, the mixed culture mineralized the substrate at low concentrations in heptamethylnonane, and Pseudomonas sp. strain R and the indigenous microbial community of subsoil degraded phenanthrene at low concentrations in DEHP. Moreover, an isolate (strain M1) from the mixed culture utilized benzoic acid in water even at concentrations of 0.3 ng/mL. In all instances, the rate of mineralization was low. The existence of a threshold concentration belowwhich little or no biodegradation occurs is not unexpected. A model has been formulated that predicts that a threshold will exist when the rate that a microorganism uses carbon to supply the energy for its maintenance equals the rate of diffusion of that compound to the surface of the cell (5). Experimental data have been reported for various bacteria giving threshold concentrations for glucose and quinoline of2-18 ng/mL (12-14). However, those dataare for solutes in water alone and not for a NAPL-water mixture. In the latter case, a hydrophobic molecule, such as phenanthrene, would chiefly be in the organic liquid whether or not equilibrium with water was achieved. Sorption to a hydrophobic surface has previously been shown to reduce the concentration of p-nitrophenol to levels below the VOL. 29, NO. 2, 1995 /ENVIRONMENTAL SCIENCE &TECHNOLOGY
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threshold for biodegradation (9). In the present instance, the partitioning of phenanthrene present at a concentration of 24.5pg or less per mLof DEHP would give, at equilibrium, less than 1.0 ng/mL of water, a value below the thresholds for the bacteria cited above. For the mixed culture, a threshold for mineralization probably exists between 0.62 and 3.1 ng/mL of water. Moreover, the rate of partitioning of phenanthrene from DEHP rather than the equilibrium concentration may have been too low for the enrichment to be maintained. Partitioning rates of 0.090 ng mL-’ h-l or less were associated with negligible mineralization. Indeed, low dilution rates of substrates in chemostats have been shown to cause cell starvation ( 1 7). The mineralization by the enrichment of phenanthrene initially dissolved in heptamethylnonane at 0.6-20 pglmL but not in DEHP is consistent with differences in partitioning. Phenanthrene partitions more rapidly and to a greater extent from heptamethylnonane to water than from DEHP. The rate of biodegradation of organic substrates at low concentrations is usually directly related to the substrate concentration. This is characteristic of Monod kinetics of bacterial growth and metabolism at substrate levels appreciably below the Ksvalue (181,and this direct relationship has been observed with a wide range of concentrations of substrates in natural waters (19). The data herein are not presented as amountbut rather as percent of phenanthrene mineralized as a function of time. If the amount degraded is a direct function of the substrate concentration, then the percent degraded after a period of time should be the same at several substrate concentrations. Clearly, the partitioning rate or the rate at which a compound enters a biodegradable phase is also important. Under many conditions in this study, including the mineralization of phenanthrene in heptamethylnonane by the mixed culture or phenanthrene in DEHP by Pseudomonas sp. or the subsoil community, the percentage of phenanthrene mineralized as a function of time declined with decreasing concentrations of the substrate in the NAPLs and in water at equilibrium. This lack of proportionality between rate and concentration has been observed before (19) and probably reflects concentrations slightly above the threshold for biodegradation. The lack of association between partitioning and mineralization rates also supports the likelihood of the existence of a threshold. Precedents exist for a pure culture to have no demonstrable threshold for a particular test compound, as observed here for the isolate that metabolized benzoate even at 0.3 ng/mL. Although this bacterium was isolated from a mixed culture that could not metabolize phenanthrene at 0.3 ng/mL, the data are not unexpected since the threshold concentrations for the biodegradation of two compounds sometimes differ (20). Moreover, the presence of one degradable substrate can eliminate the threshold for another. For example, a strain of Pseudomonas can mineralize low concentrations of methylene chloride in the presence of acetate but not in its absence (21),and the addition of arabinose permitted Salmonella typhimurium to grow on glucose at concentrations below the observed threshold (15). Presumably strain M1 was able to grow on contaminating organic carbon in the inorganic salts solution, as shown here for Pseudomonas sp. strain R and the mixed culture from which strain M1 was obtained. Benzoate, but not phenanthrene, that was apparently transformed at levels below the threshold was transformed concomitant with that growth. A difference in the ability 520
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of Pseudomonas sp. and the mixed culture to use organic contaminants in the inorganic salts solution has not been shown to account for the ability of the pseudomonad to degrade phenanthrene at low concentrations. The experiments with benzoate and the contaminating carbon in the inorganic salts solution did not confirm that the failure of the mixed culture to mineralize low concentrations of phenanthrene in DEHP resulted from a threshold in the aqueous phase, but they did highlight the dependence of the phenomenon of thresholds on the substrate, NAPL, and cultural conditions. These findings have a number of implications for biodegradation of pollutants present in NAPLs in soils, aquifers, and waters. A compound with a high NAPLwater partition coefficient may persist because the concentration in the water phase is below the threshold for the growth of a small population of microorganismspotentially able to metabolize that compound. Alternatively,the rate of degradation may be slower than expected from rates observed at higher concentrations. Although the cessation of biodegradation because of bacterial thresholds usually occurs when little substrate remains, in polluted sites containing NAPLs, large quantities of a biodegradable compound may persist if it has ahigh NAPL-water partition coefficient. On the other hand, if the potentially active microorganisms are able to utilize other carbon sources in the aqueous phase, the soil, or the NAPL or if the microorganisms can attach to the NAPL and degrade the more concentrated substrate there (221, a compound whose partitioning coefficient should give subthreshold concentrations in the water phase might still be transformed.
This work was supported by the Cornell University Center for Advanced Technology in Biotechnology and by a grant from the National Institute of Environmental Health Sciences (ES05950).
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(17) Brock, T.D.;Madigan, M. T. Biology of Microorganisms, 6th e d Prentice Hak Englewood Cliffs, NJ, 1991;p 316. (18) Simkins, S.; Alexander, M. Appl. Environ. Microbiol. 1984, 47, 1299-1306. (19) Rubin, H.E.;Subba-Rao, R. V.; Alexander, M. Appl. Environ. Microbiol. 1982, 43, 1133-1138. (20) Martin,P.;MacLeod, R. A. Appl. Environ. Microbiol. 1984, 47, 1017-1022. (21) LaPat-Polasko, L. T.;McCarty, P. L.; Zehnder, A. J. B. Appl. Environ. Microbiol. 1984, 47, 825-830.
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Received for review July 19, 1994. Revised manuscript received November 11, 1994. Accepted November 15, 1994."
ES940446D @
Abstract published in AdvanceACSAbstracts, December 15,1994.
VOL. 29, NO. 2, 1995 I ENVIRONMENTAL SCIENCE &TECHNOLOGY
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