Environ. Sci. Technol. 1993,27,104-110
material does not imply recognition of, or endorsement by, the National Institute of Standards and Technology, nor does it imply that the materials or equipment used are necessarily the best available for the purpose. Literature Cited Burns, V. R.; Benson, J. D.; Hochhauser, A. M.; Koehl, W. J.; Kreucher, W. M.; Reuter, R. M. SAE Tech. Paper Ser. 1991, No. 912320. Johnson, J. E.; Peterson, F. M. CHEMTECH. 1991, 296. Guidance on Estimating Motor Vehicle Emission Reductions from the Use of Alternative Fuels and Fuel Blends; U S . EPA Report, EPA-AA-TSS-PA-87-4; U.S. EPA, Ann Arbor, MI, 1988. Alcohols and Ethers: A Technical Assessment of their Application as Fuels and Fuel Components; American Petroleum Institute: Washington DC, July 1988; Publ. No. 4261. Keyworth, D. A,; Reid, T. A. Fuel Reformulation 1992,2, 54. Lieder, C. A., Fuels Research, Shell Development Co. private communication, 1992. Calvert, J.; Pitts, J. N., Jr. Photochemistry; Wiley: New York, 1966. Atkinson, R.; Carter, W. P. L. Chem. Rev. 1984, 84, 437. Wallington, T. J.; Atkinson, R.; Winer, A. M.; Pitts, J. N., Jr. J. Phys. Chem. 1986, 90, 5393. Wallington, T. J.; Kurylo, M. J. Znt. J. Chem. Kinet. 1987, 19, 1015. Wallington, T. J.; Dagaut, P.; Liu, R.; Kurylo, M. J. Znt. J. Chem. Kinet. 1988, 20, 541. Wallington, T. J.; Dagaut, P.; Liu, R.; Kurylo, M. J. Environ. Sci. Technol. 1988, 22, 842. Wallington, T. J.; Andino, J. M.; Skewes, L. M.; Siegl, W. 0.;Japar, S. M. Int. J . Chem. Kinet. 1989,21, 933. Japar, S. M.; Wallington, T. J.; Richert, J. F. 0.;Ball, J. C. Znt. J . Chem. Kinet. 1990, 22, 1257.
Wallington, T. J.; Japar, S. M. Enuiron. Sci. Technol. 1991, 25, 410. Wallington, T. J.; Siegl, W. 0.;Liu, R.; Zhang, Z.; Huie, R. E.; Kurylo, M. J. Znt. J. Chem. Kinet. 1990, 24, 1596. Wallington, T. J. Int. J . Chem. Kinet. 1986, 18, 487. Witte, F.; Urbanik, E.; Zetzsch, C. J. Phys. Chem. 1986,90, 3251. Wallington, T. J.; Japar, S. M. J . Atmos. Chem. 1989, 9, 399. Atkinson, R. J . Phys. Chem. Ref. Data 1989, Monograph 1.
Wallington, T. J.; Potts, A. R.; Andino, J. M.; Siegl, W. 0.; Zhang, Z.; Kurylo, M. J.; Huie, R. E. Int. J. Chem. Kinet., submitted. Nelson, L.; Rattigan, 0.; Neavyn, R.; Sidebottom, H.; Treacy, J.; Nielsen, 0. J. Znt. J. Chem. Kinet. 1990,22,1111. Carter, W. P. L.; Atkinson, R. Enuiron. Sci. Technol. 1989, 23, 864. Chang, T. Y.; Rudy, S. J. Atmos. Enuiron. 1990,24, 2421. Hogo, H.; Gery, M. W. User's guide for ercuting OZZPM-4 with CBM-IV or optional mechanism; U.S. Environmental Protection Agency Report, EPA/600/8-88/073b; US.E P A Research Triangle Park, NC, 1988. Japar, S. M.; Wallington, T. J.; Rudy, S. J.; Chang, T. Y. Enuiron. Sci. Technol. 1991, 25, 415. Wallington, T. J.; Dagaut, P.; Liu, R.; Kurylo, M. J. Int. J. Chem. Kinet. 1988,20, 177. Pollack, A. K.; Fieber, J. L.; Noda, A. M.; Lauer, G.; Dunker, A. M.; Schleyer, C. H.; Chock, D. P.; Hertz, M.; Metcalfe, J. E. Air and Waste Management Association, 85th Annual Meeting, Kansas, June 1992; Paper 92-119.04. Dawson, G. A,; Farmer, J. C.; Moyers, J. L. Geophys. Res. Lett. 1980, 7, 725. Norton, R. B. J. Geophys. Res. 1992, 97, 10389, and references therein.
Received f o r review May 1, 1992. Revised manuscript received August 25, 1992. Accepted September 15, 1992.
Effect of Micellar Solubilization on Biodegradation Rates of Hydrocarbons Scott J. Bury and Clarence A. Miller" Department of Chemical Engineering, Rice University, P.O. Box 1892, Houston, Texas 77251
Batch experiments were conducted with a strain of Pseudomonas aeuroginsa and a strain of Ochrobactrum anthropi, both Gram-negative bacteria, growing on aqueous solutions containing straight-chain hydrocarbons solubilized in small micelles (2-4 nm) of nonionic surfactants. Measurements of optical density, a quantity proportional to bacterial cell concentration, and hydrocarbon content were made as a function of time. Since no macroscopic hydrocarbon drops were present and therefore there was no opportunity for the bacteria to attach themselves to oil-water interfaces, the results provided unambiguous confirmation that solubilization greatly enhances rates of hydrocarbon degradation in these systems compared to rates observed with bulk liquid hydrocarbon in the absence of surfactants. Solubilization of n-decane and n-tetradecane in micelles reduced the times required for cell density to double during exponential growth by a factor of -5 for one bacterial strain compared to results obtained for surfactant-free experiments. The improvement was even greater for the other strain. Except for very low hydrocarbon concentrations, the Monod model for a single substrate was able to describe reasonably well the time dependence of both cell growth and hydrocarbon consumption for experiments with n-decane. Introduction Surfactants in Groundwater Cleanup. In recent 404
Environ. Sci. Technol., Vol. 27, No. 1, 1993
years, it has become increasingly clear that organic chemicals have contaminated numerous aquifers. Gasoline from underground storage tanks, petroleum products from refineries, and chlorinated solvents from cleaning processes in various industries are examples of contaminants that threaten groundwater quality. In addition to existing problems, there is always the risk of new contamination from accidental spills and discharges. The current and potential future damage to soils and groundwater resources demands that attention be directed to developing methods for removing contaminants from aquifers. Contamination of an aquifer by a sparingly soluble organic liquid is a complex process. It involves vertical and lateral migration of the liquid through the unsaturated zone, its trapping by capillary forces in both unsaturated and saturated zones as discrete drops or "ganglia", which may extend over several pores and exhibit branched structures, and adsorption of individual compounds on soil particles. Once trapping occurs, mobilization of individual drops or ganglia is usually not possible for the water velocities achievable in practicable pump and treat operations. Similar situations occur in oilfield production after water flooding. Considerable research and development has been carried out on surfactant processes designed to lower the capillary forces drastically and thereby mobilize trapped ganglia (1). A similar application of surfactants
0013-936X/93/0927-0104$04.00/6
0 1992 American Chemical Society
may have possible uses in groundwater and soil cleanup (2-4). Another mechanism by which surfactants can aid in removal of adsorbed or trapped organic liquids is solubilization into micelles-typically aggregates of 50-100 surfactant molecules having diameters of 2-4 nm that provide a less polar and consequently more favorable environment for organic molecules. Solubilization can increase the amount of sparingly soluble hydrocarbons or other organic compounds taken up by aqueous phases by orders of magnitude. Some initial work has been carried out on solubilization as a mechanism for contaminant removal (2-6). It is effective when relatively small amounts of contaminants are present but requires more surfactant than processes based on mobilization when many trapped ganglia exist. Some potential disadvantages, which the process design must avoid or minimize, accompany the potential benefits of using surfactants in remediation processes. Obviously, the surfactant itself must not be a threat to the environment and indeed must be readily biodegradable to levels consistent with applicable regulations ( 4 ) . Such constraints, which are very important for processes in groundwater aquifers, received relatively little attention during the investigation of surfactant processes for petroleum recovery. Moreover, surfactants can, under some conditions, disperse small particles of clay minerals or other solids or interact with petroleum products to form viscous emulsions. Such behavior could lead to pore clogging and subsequent flow reduction (4). The use of surfactants in a remediation process could also affect the biodegradation of contaminants. In some cases, biorestoration is a useful and relatively inexpensive way to reclaim aquifers polluted with organic chemicals. This in situ technique offers the advantage that the bacterial action often converts the contaminant to harmless materials such as carbon dioxide and water rather than simply transferring it to a different phase (7). Evidence exists that surfactants can influence the uptake and consumption of insoluble substrates (8-16). Many microorganisms produce extracellular surface-active compounds, perhaps as a response to the presence of an insoluble substrate (17). The generally accepted purpose of these compounds is to enhance biodegradation. However, the mechanism of enhancement is not entirely clear, as discussed below. Surfactants in Biodegradation of Hydrocarbons. In spite of extensive work on hydrocarbon feedstocks in industrial microbial processes, many questions remain on how bacteria take up sparingly soluble substrates. The three mechanisms traditionally proposed in the literature are as follows: (1)interaction of cells with hydrocarbon dissolved in the aqueous phase; (2) direct contact of cells with hydrocarbon drops considerably larger than the cells; (3) interaction of cells with "solubilized", "pseudosolubilized", or "accommodated" hydrocarbon in entities much smaller than the cells. Note that pseudosolubilization or accommodation need not be into a classical surfactant micelle as described above, but instead into the lipophilic regions of proteins or other polymeric molecules produced and excreted by cells. In any case, the solubilized material dispersed in the small entities is in a thermodynamically stable state. In contrast, the much larger drops of mechanism 2 are thermodynamically unstable with respect to coalescence into a single, large hydrocarbon lens although the presence of surfactant may cause this process to be very slow.
The first mechanism cannot account for the observed rates of growth due to the low solubility of long-chain alkanes (9). However, it can explain many results for aromatics and gaseous hydrocarbons, which have greater solubilities in water (9). Direct contact between cells and macroscopic hydrocarbon drops has been observed in many studies (8, IO), especially those involving yeast (18,19). If hydrocarbon uptake occurs by this mechanism, the surface area available for contact may be a limiting factor for cell growth. Often, formation of emulsions having small drops is a useful device in promoting growth by providing a larger surface area. Direct contact is not limited to liquid droplets. One study found that Mycobacterium cells can colonize phenanthrene crystals (16). The third mechanism gained acceptance during the 1980s although workers had reported this type of interaction in the early 1970s (14,15). Singh and Baruah's group in India has carefully studied this mechanism. They have shown that often a metabolite produced by a microorganism can cause sufficient pseudosolubilization of hydrocarbon to account for the observed growth rates. However, even within the same bacterial genus, e.g., Pseudomonas, the predominant mode of hydrocarbon uptake may vary. A strain they called M1 produced no extracellular metabolite, and direct contact was considered the chief uptake mechanism. However, another strain termed N1 did produce an extracellular material and pseudosolubilization was believed to be dominant (9). There is some ambiguity in previous work with liquid hydrocarbons because it was conducted with both aqueous and oil phases present. Adding surfactants not only facilitates emulsification of the oil, with a resulting increase in interfacial area, but also provides micelles for solubilization. Thus, both mechanisms 2 and 3 are possible. The ability of microorganisms to use hydrocarbons existing solely in solubilized form in a micellar solution without the presence of macroscopic drops of liquid hydrocarbon has not previously been investigated. We present here the results of such a study using a strain of Pseudomonas aeuroginsa, a strain of Ochrobactrum anthropi, pure hydrocarbons, and a single class of synthetic surfactants where the balance between lipophilic and hydrophilic properties can be readily varied. The surfactants used were straight-chain ethoxylated alcohols that are nonionic, nontoxic, readily biodegradable, and indeed widely used in household laundry products and other applications. They have very low critical micelle concentrations (the concentration above which micelles form from individual surfactant molecules dissolved in solution) but are sensitive to temperature. An aqueous solution of nonionic surfactant will separate into two liquid phases with different surfactant concentrations when heated above its cloud point temperature. The degree of ethoxylation strongly affects the cloud point. For example, the cloud points of nearly pure surfactants are 10 "C for C12E4,-30 "C for C12E5,and -50 "C for C12E6(20).By C12E4we mean an ethoxylated alcohol with an alkane chain length of 12 and 4 ethoxyl groups. Since the cloud points of pure C12E4and its commercial counterparts are below ambient temperature, ordinary small micelles do not exist in mixtures of these surfactants with water. Indeed, in this particular case, a dispersion of macroscopic particles of the lamellar liquid crystalline phase exists at room temperature in water (21).The lamellar phase consists of many parallel sheets of surfactant bilayers separated by water layers (21). The present experiments dealt only with the role of solubilization in small micelles and so operated below the
-
Environ. Sci. Technol., Vol. 27, No. 1, 1993
105
cloud point. The weight of hydrocarbon never exceeded half the weight of surfactant and was often much lower. Nevertheless, degradation rates were greatly enhanced compared to those found without surfactants, a confirmation of the importance of solubilization (mechanism 3 above). Experimental Methods Pseudomonas aeuroginsa (ATCC 15528) and Ochrobactrum anthropi (ATCC 21909) were cultured and maintained in nutrient broth (Difco). Glycerol stocks were made and stored at -20 OC. Culture purity was checked periodically using standard microbiological methods. We chose a Pseudomonas strain because Pseudomonads are common soil bacteria, Gram negative, and well-known to use a wide range of substrates, including hydrocarbons. The Ochrobactrum strain was originally identified as a Pseudomonas but has recently been reclassified by the ATCC. This genus was proposed and described by Holmes and co-workers (22). All growth experiments were conducted at 30 "C on a rotary shaker in l-L culture flasks using a mineral salts medium (MSM) (2.42 g of KH2P04,5.6 g of K2HP04,2.0 g of (NH4)2S04, 0.3 g of MgS04.7H20, 0.040 g of CaCl,. 2H20,0.0045 g of MnS04-7H20,100 pg of CuS04.5H20, and 100 pg of FeS04.7H20per liter of ACS reagent grade 1 water). The n-alkanes were purchased from Aldrich and filtered through a 0.22-pm filter before use. The Neodol surfactants were ethoxylated alcohols supplied by Shell Development Co. They were used without further purification. Stocks (10% w/v) of the surfactants in MSM were made and autoclaved separately. We used Neodol (N) 25-9,25-7, and 25-3. The first number in the Neodol name designates the average range of alkyl chain lengths; Le., 25 represents a distribution from C12to CI5. The second number is the average number of ethylene oxide (EO) groups; i.e., 7 represents a distribution around seven EO groups per molecule. The growth experiments consisted of three types: (I) free-phase hydrocarbon, (11)solubilized hydrocarbon, and (111) surfactant only. The type I medium was prepared as follows: Filtered hydrocarbon was added to 200 mL of sterile MSM in a flask. This mixture was shaken for 24 h at 30 "C prior to inoculation. The type I1 medium was prepared by weighing the required amount of hydrocarbon into a sterile, capped tube. The aliquot of sterile 10% surfactant stock was added to this and gently turned for 12 h at 30 "C. The resulting solution was then transferred to the flask containing the remainder of the MSM. In all cases, the result was an optically clear, single-phase, micellar solution. The hydrocarbon was completely solubilized in the micelles. The type I11 medium consisted of surfactant in MSM only. This was also a transparent, single-phase solution. Cell growth was monitored optically using a Brinkman Probe colorimeter at 600 nm. The optical density (OD,) was correlated with dry weight measurements. For the various micellar solutions, OD,, did not differ from that of distilled water. This probe was also used to determine the solubility limits for the surfactants. A 100-mL aliquot of MSM surfactant solution was placed in a beaker and gently stirred via a mechanical stirrer. No air was entrained, and the vortex was minimal. The probe was placed well below the free surface. Hydrocarbon was then introduced in known quantities. The percent transmittance was followed by a strip chart recorder. A return to baseline of the percent transmittance indicated complete solubilization of the hydrocarbon. When the solution re106
Environ. Sci. Technol., Vol. 27, No. 1, 1993
2o
1 0 90/10 and decane 90/10 and tetradecane
-
Q
c
5
0
0
5
10
15
20
25
30
Surfactant Concentration(mg/ml)
Flgure 1. Solubilization density vs surfactant concentration. 90/10 designates a surfactant mixture contalning 90 % Neodol25-9 and 10 % Neodol 25-3.
mained turbid, the solubilization limit had been reached. The dissolved oxygen concentration was monitored using a YSI Model 58 DO probe. The probe was calibrated against air and corrected for the atmospheric pressure. Measurements indicated that the DO level was maintained at a minimum of 30% of saturation. The disappearance of the n-alkanes during the growth experiments was monitored by GC with a flame ionization detector (FID). The column used was a 15-m SP-1 fused-silica Mega-Bore column with a 2-m guard attached. Results were quantified using an internal standard of n-dodecane (C12). A 0.5-mL aliquot of the sample was added to 0.5 mL of acidified (pH 2) surfactant solution containing solubilized Clz. This step prevented further bacterial oxidation of the hydrocarbon. A 0.1-pL sample was then injected directly onto the column (conditions: injection temperature 200 "C; oven temperature 110 "C for 8 min, then temperature program to 250 "C at 2 "C/ min, hold at 250 "C for 1min; detection temperature 220 "C. The peaks were integrated on a HP-1330A plotting integrator. Results Solubilization Experiments. Solubilization limits of two hydrocarbons were determined for two nonionic surfactants in the aqueous mineral salts medium described above. The experiments were conducted at 30 "C, a temperature well below the surfactant cloud points. As Figure 1 indicates, maximum solubilization was a linear function of surfactant concentration in these systems where values of the critical micelle concentration (cmc) were very small and well below the surfactant concentration range investigated. This linear dependence is consistent with the solubilization literature. We also found the expected greater solubilization capacity of the less hydrophilic surfactant (N25-7) for both hydrocarbons and the greater solubilization of n-decane than of n-tetradecane by both surfactants (23). It is noteworthy that, even in the system with the highest solubilization, the maximum weight of hydrocarbon solubilized was only -60% of the weight of the surfactant in the micelles. Moreover, the amount of hydrocarbon
0.30
1
0.25
IE
0.20
;1
a
6
2 .c I
0
0.05
0.00
0
4
10
5
15
R u n Time (hours)
Flgure 3. Growth and decane consumption data for strain 15528. Conditions: 0.5% Neodol 25-7, pso,= 0.75 mg/mL.
Table I. Specific Growth Rates on Decane in Representative Surfactant Systems
~
0.0
0.1
0.2
0.3
0.4
Solubilization Density (rnglrnl)
Figure 2. Strain 15528 specific growth rates, p, for increasing concentrations of decane soiubilized in 0.5% Neodoi 25-7.
present never exceeded 0.5wt % (5mg/mL) in the surfactant solutions used in the growth experiments discussed below. In many cases it was no greater than 0.1 wt %. Even these values are orders of magnitude above the solubilities of the hydrocarbons in the absence of X mg/mL for n-decane and even surfactants--1 lower for n-tetradecane. Owing to the low hydrocarbon contents, we refer to the surfactant-containing growth media as micellar solutions rather than microemulsions, which typically have much greater solubilization. Growth Experiments. (a) Cell Growth. It is common in batch growth experiments to find a period when the cell density X increases exponentially with a constant growth factor 1.1 (24): dX/dt = PIX (1)
X = Xoexp(11.t)
(2) For exponential growth it is sometimes convenient to refer to the doubling time tD, i.e., the time required for cell density to double: t D = In 2/11. (3)
surfactant concn, %
surfactant
[decane], mg/mL
fi f 0.02
h-'
Strain 15528 0.5
90/10 90/10 90/10 90/10 90/10 90/10 90110 90/10 25-7 25-1 25-7 90/10 25-7 25-1 25-7
0.5 2.0 2.0 2.0 0.5 1.0 2.0 0.25 0.5 1.0 1.0 0.25 0.5 1.0
none
none
0.25 1.00 1.00 2.00 4.00 1.00 1.00 1.00 0.75 0.75 0.75
none none none none 3.5-14
0.29 0.29 0.37 0.37 0.37 0.29 0.33 0.37 0.22 0.26 0.29 0.16 0.12 0.13 0.15 0.05
Strain 21909 90/10 90/10 25-7 25-7 25-7 25-1 90/10
1.0
2.00
0.5 0.5 1.0 2.0 1.0 2.0
1.00
none none
0.16 0.16 0.16 0.15 0.15 0.09 0.09
none
none
8.00
nd"
1.50 1.00 2.00
Not detected. ~
Sometimes an initial period of slower growth, during which the microorganisms adapt to the growth medium, precedes the exponential growth period. The growth rate invariably eventually decreases from the maximum rate as some substrate or nutrient being consumed becomes limiting. During the exponential growth period, the dependence of the growth factor on the concentration S of the limiting substrate, e.g., solubilized hydrocarbon in our experiments, is frequently described by the Monod model (24): 11. = Pm=S/(Ks + S) (4) A key feature of this model is that saturation occurs; i.e., 11. approaches a maximum value pmaxas S increases. K,is called the half-saturation constant. Figure 2 shows the dependence of the growth factor 11. on the solubilization density for several solutions containing 0.5 wt % of N25-7 and various amounts of n-decane below the solubilization limit. Strain 15528 was used.
~
These data are taken from the exponential growth phase, which occurred over the first 10 h of the experiments (see Figure 3). It is clear from Figure 2 that 11. does indeed approach a limiting value of -0.25 h-' as hydrocarbon content increases. The value of 1.1 obtained for growth on surfactant alone is only -0.15 h-l and is independent of surfactant concentration over the range investigated (see Table I). As determined by making triplicate runs for virtually all our growth experiments, the uncertainty in the above values of 11. is f0.02 h-l. According to eq 3, the doubling time t D corresponding to the above results is -2.8 h for solubilized n-decane compared to 4.3 h for the hydrocarbon-free surfactant solution. GC data, discussed in a later section, confirmed that the solubilized hydrocarbon was, in fact, oxidized during the former experiments. Note that the Monod equation for a single substrate as given by eq 4 cannot be Environ. Scl. Technol., Vol. 27, No. 1, 1993
107
applicable for very small values of solubilized hydrocarbon content S because p does not approach zero in this limit. Instead, p approaches its value for an oil-free surfactant solution. Of greater interest is the comparison between the growth rate with solubilized n-decane and that with n-decane present but no surfactant. For the latter situation, two experiments were conducted with 3.5 and 14.0 mg/mL liquid n-decane present. Exponential growth was again observed for the first 10 h of the experiment with p in the range of -0.05 h-l. The corresponding time for cell density to double is 13.9 h, -5 times greater than the value given above for n-decane solubilized in surfactant micelles. Since no bulk hydrocarbon drops were present in the micellar solutions and there was therefore no opportunity for the bacteria to attach themselves to oil-water interfaces, these results demonstrate unambiguously that solubilization produces substantial increases in the rate of hydrocarbon degradation in this system. I t is interesting that, when no surfactant was present, accumulation of a viscous material was observed during the experiments with strain 15228. Microscopic examination revealed a rough coating on the hydrocarbon droplets. This material also facilitated dispersion of large hydrocarbon lenses into small drops on swirling the growth medium. No such viscous material was observed during experiments where the hydrocarbon was solubilized in surfactant micelles. The effect of surfactant concentration in this sytem was also investigated. As can be seen from Table I, the maximum growth factor p- increased slightly with increasing surfactant concentration. In other words, cell growth occurred somewhat faster when a fixed amount of hydrocarbon was distributed among a larger number of micelles. The reason for this behavior is not understood though consumption of some surfactant along with hydrocarbon could be involved (see Discussion below). It does not appear to be a mass-transfer effect since the number of micelles is several orders of magnitude greater than the number of cells in all the experiments. Also, the measured growth factors were found to be independent of the rate of shaking of the flasks during growth. Similar experiments were conducted with n-decane and a 90/10 mixture of N25-9 and N25-3. As Table I shows, pwith solubilized hydrocarbon was slightly greater for this somewhat more hydrophilic surfactant but about the same as for N25-7 in the absence hydrocarbon. Again pwith solubilized hydrocarbon present increased slightly with surfactant concentration. The 21909 strain was less effective as a hydrocarbon degrader than the 15528 strain. Indeed, it exhibited negligible growth on n-decane in the absence of surfactant. In contrast, growth did occur with p- equal to -0.16 h-l when n-decane was solubilized in either surfactant. With no hydrocarbon present, pmaxwas -0.09 h-l for both Surfactants (see Table I), again considerably less that with solubilized hydrocarbon. Both strains grew exponentially on solubilized n-tetradecane in the 90/10 surfactant after an initial induction period of -8 h, and both exhibited values of p- of -0.15 h-l. Similar values of p- were found for N25-7 with both strains. GC analysis confirmed that n-tetradecane was oxidized during these experiments. Without surfactant the 21909 strain exhibited negligible growth on this hydrocarbon, while the 15528 strain exhibited very slow growth ( p -0.02 h-l) after a long induction period of -30 h. Thus, the experiments with n-tetradecane, while yielding smaller values of pmarthan those with n-decane, 108 Envlron. Scl. Technol., Vol. 27, No. 1, 1993
confirmed the previous conclusion that solubilization of hydrocarbon in surfactant micelles greatly increased growth compared to that observed with liquid hydrocarbon present but no surfactant. It is interesting that the values of pmm,obtained for the 15528 strain with n-tetradecane solubillzed in both surfactants are about the same as those found for the surfactants alone. In all other cases studied with both n-decane and n-tetradecane, pmaxwas significantly greater when solubilized hydrocarbon was present. Finally, a few experiments used n-octane. Neither strain grew on this hydrocarbon in the absence of surfactant. This result is consistent with previous reports that some microorganisms of the present type have difficulty growing on straight-chain hydrocarbons having chain lengths below 10 when the oil is present as a liquid; i.e., the aqueous phase concentration is at saturation (25). Of course, strains such as Pseudomonas Putida exist which can readily utilize short-chain alkanes. Exponential growth of the 15528 strain did occur with a growth factor of -0.09 h-l when n-octane was solubilized by N25-7. GC analysis confirmed that the n-octane was oxidized. While the value of p obtained is less than that for the surfacttint alone, the results indicate that solubilizing short-chain hydrocarbons in surfactant micelles can, in some cases, increase their biodegradation rates. When the amount of short-chain hydrocarbon solubilized is well below the solubilization limit, as happens here, the concentration of molecularly dksolved hydrocarbon in the aqueous solution is well below its saturation value, a possible explanation for the absence of the inhibiting effect shown by such hydrocarbons for some microorganismsin the absence of surfactant. Further experiments are desirable to determine the generality of these initial results. (b) Hydrocarbon Consumption. The observed increases in cell density discussed above indicated that hydrocarbon degradation occurred during our experiments. Direct analysis of the growth medium by gas chromatography (GC) during the experiments provided quantitative confirmation. If the rate of hydrocarbon consumption (dS/dt) is proportional to the rate of cell growth and if the Monod model applies, one has
where Y (mg of dry cells/mg of hydrocarbon oxidized) is a yield coefficient and eqs 1and 4 have been invoked. For sufficiently large initial values of S, pmmcan be obtained from the exponential portion of the cell growth curve, as discussed above. The yield coefficient Y was determined from a combination of dry cell mass measurements and the GC data. Figure 3 shows an example of the fits obtained for the variation of cell density and hydrocarbon content with time for one experiment with the 15528 strain degrading ndecane solubilized in N25-7. Simulations to determine optimum values of the parameters were performed on a Macintosh IIcx computer running Matlab. The ordinary differential equations were integrated using fourth- and fifth-order RungeKutta formulas. The agreement shown in Figure 3 between the data and the predictions of the model is good. Virtually none of the n-decane remained after -17 h. We verified by separate experiments involving no biodegradation that decane losses from evaporation over this period were negligible; Le., the hydrocarbon must have been oxidized by the microorganisms. For the curves shown in Figure 3, p- = 0.26, Y = 1.1,and K, = 0.1 mg/mL. The last parameter could, one might think, be determined independently from the exponential
3.
2-
0
-
0:
198-
I.
n
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r 0.1
5-
2 ID
2 1-
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2-
: 0.01
0
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0
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tant, the growth factor p would be -0.05 h-l, as indicated in Table I. A simple calculation using eq 5 with the same yield coefficient Y of 1.1 as found for the experiment of Figure 3 and the same initial concentration of bacteria shows that only 10% of the n-decane would be degraded after 17 h in this case where there is no solubilization. In this connection, it is of interest that the n-tetradecane consumption data were not so well described by the Monod model. Moreover, the yield coefficient Y was in the range of 1.6-1.8 and hence considerably greater than the 1.1-1.2 observed for n-decane. The latter are comparable to literature values for hydrocarbon consumption (24,26). In view of the relatively large values of Y in the n-tetradecane experiments and of the significantly lower overall growth rates than for n-decane, it seems likely that surfactant consumption is much more important relative to hydrocarbon consumption in the n-tetradecane system. Further investigation of this matter would require determination of surfactant content as a function of time during the experiments. Goswami and Singh (9) recently reported that pseudosolubilization of n-hexadecane by glycoproteins produced during growth of Pseudomonas strain N1 caused its growth factor p to be a factor of -2 greater than that observed for a similar strain M1 that did not produce molecules capable of incorporating hydrocarbon. They conducted their experiments with bulk n-hexadecane present, in contrast to our procedure, and it is of interest that strain N1 also produced a lipoprotein that facilitated emulsification of the oil. The glycoprotein responsible for pseudosolubilization was rather specific to the particular hydrocarbon present. In our case the surfactant micelles are capable of solubilizing various hydrocarbons though solubilization decreases for longer chain compounds (Figure 1). It is noteworthy that the doubling times reported by Goswami and Singh (9)--5 h for strain N1 with pseudosolubilization and 10 h for strain M1-are of the same order of magnitude as those given above for our systems. While our results indicate significant potential advantages of solubilization, further experiments are needed to determine the effect of surfactant micelles in other systems, especially those containing soil and macroscopic hydrocarbon drops which were not present in our work. Laha and Luthy (27),for instance, recently reported that the presence of micelles of nonionic surfactants similar to ours decreased the rate of degradation of phenanthrene. Soil was present in most of their experiments, but the inhibitory effect of micelles was seen even without soil. It is not clear why this behavior, which is opposite to what we observed in our systems, occurred. In a related study, Guerin and Jones (16) reported that different nonionic surfactants (sorbitol esters with the trade name Tween) enhanced the rate of phenanthrene degradation by a Mycobacterium strain. It appears from their data that the phenanthrene was present both in micelles and as solid crystals. Finally, we note that Miller and Bartha (28) reported that solubilization of long-chain hydrocarbons below their melting points in phospholipid liposomes increased their rate of degradation by a Pseudomonas strain.
i 35
Run Time (HOURS)
Flgure 4. Growth and decane consumption data for strain 21909. Conditlons: 0.5% Neodoi 25-7, pso, = 1.5 mg/mL.
portions of the cell growth curves for various initial hydrocarbon contents S. From available data of this type we concluded that 0.1 was the right order of magnitude for K,, but a precise value could not be obtained. The reason is that, as indicated above, eq 4 cannot be correct for small S when surfactant is present because p eventually approaches its value for pure surfactant as S continues to decrease. Improving the fit between the curves and data in Figure 3 would require additional information on the rate of consumption of surfactant when solubilized hydrocarbon is present and an extension of eq 4 to include the effects of both hydrocarbon and surfactant concentrations. A similar plot for the 21909 strain growing on n-decane solubilized in N25-7 is given in Figure 4. For the curves shown, pmsx= 0.16, Y = 1.2, and K, = 0.15 mg/mL. As might be expected, the values of Y and K, are not very different from those found above for the 15528 strain. The agreement between the calculated curves and the experimental data is reasonable. Discussion The data presented above demonstrate clearly that when hydrocarbon is solubilized in small micelles of synthetic surfactants, its rate of biodegradation can substantially increase. The ability of the Monod model, which was developed for soluble substrates, to fit the cell growth and hydrocarbon consumption data for n-decane suggests that, at least in these situations where individual micelles are much smaller than the bacteria and typically contain no more than 10-100 hydrocarbon molecules, the main effect is an effective increase in the solubility of the hydrocarbon in the aqueous phase. Further support for this explanation could be obtained by using a different type of surfactant, a study we are currently conducting. The variations observed in the growth factor p with changes in surfactant concentration and average surfactant EO number (see Table I) could simply reflect differences in surfactant degradation occurring in parallel with, though at a slower rate than, that of the hydrocarbon. It is important to recognize, however, that, whatever the situation regarding surfactant degradation, hydrocarbon degradation is greatly enhanced as a result of solubilization in surfactant micelles. Consider, for example, the experiment of Figure 3 where virtually all the n-decane was degraded after 17 h, according to the GC data shown. If instead one started with the same amount of n-decane but no surfac-
Summary Experiments were conducted to assess the effect of solubilization in small nonionic surfactant micelles on the biodegradation of straight-chain alkanes in the absence of a bulk hydrocarbon phase, e.g., macroscopic hydrocarbon drops. Solubilization of n-decane and n-tetradecane in such micelles reduced the times required for cell density to double during exponential growth of one bacterial strain to about 15-20% of the values obtained for surfactant-free Environ. Sci. Technol., Vol. 27, No. 1, 1993
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Yoshida, F.; Yamane, T.; Nakamoto, K.-I. Biotechnol. Bioeng. 1973,15, 257-270. Guerin, W. F.; Jones, G. E. Appl. Environ. Microbiol. 1988, 54, 937-944. Duvnjak, Z.; Kosaric, N. Biotechnol. Lett. 1985,7,793-796. McLee, A. G.; Davies, S. L. Can. J. Microbiol. 1972, 18, 315-319. Gutierrez, J. R.; Erickson, L. E. Biotechnol. Bioeng. 1977, 29, 1331-1349. Miller, C. A.; Neogi, P. Interfacial Phenomena: Equilibrium and Dynamic Effects;Marcel Dekker: New York, 1985; p 354. Mitchell, D. J.; Tiddy, G. J. T.; Waring, L.; Bostock, T.; McDonald, M. P. J. Chem. Soc., Faraday Trans. 1 1983, 79, 975-1000. Holmes, B.; Popoff, M.; Kiredjian, M.; Kersters, K. Int. J. Syst. Bacteriol. 1988, 38, 406-416. Mackay, R. A. In Nonionic Surfactants: Physical Chemistry; Schick, M. J., Ed.; Marcel Dekker: New York, 1987; p 1135. Bailey, J. E.; Ollis, D. F. Biochemical Engineering Fundamentals, 2nd ed.; McGraw-Hilk New York, 1986; p 984. Drozd, J. W. In Carbon Substrates in Biotechnology; Stowell, J. D., e t al., Eds.; IRL Press Limited: Oxford, England, 1987; p 206. Siktya, B. Methods in Industrial Microbiology; Ellis Horwood Ltd.: Chichester, England, 1983; p 420. Laha, S.; Luthy, R. G. Environ. Sei. Technol. 1991, 25, 1920-1930. Miller, R. M.; Bartha, R. Appl. Environ. Microbiol. 1989, 55, 269-274.
experiments. It also greatly increased the rate of hydrocarbon consumption. For a second strain, significant degradation of the same two hydrocarbons occurred in the presence of the surfactant, but degradation was negligible in ita absence. L i t e r a t u r e Cited Miller, C. A.; Qutubuddin, S. In Interfacial Phenomena in Apolar Media; Eicke, H. F., Parfitt, G. D., Eds.; Marcel Dekker: New York, 1987; Chapter 4. Edwards, D. A.; Luthy, R. G.; Liu, Z. Environ. Sei. Technol. 1991,25, 127-133. Jafvert, C. T.; Heath, J. K. Environ. Sei. Technol. 1991, 25, 1031-1044. Abdul, A. S.; Gibson, T. L.; Rai, D. N. Ground Water 1992, 30, 219-231. Fountain, J. C. In Transport and Remediation of Subsurface Contaminants; Sabatini, D. A., Knox, R. C., Eds.; ACS Symp. Ser. 1992, No. 491, Chapter 15. Kile, D. E.; Chiou, C. T. Environ. Sei. Technol. 1990,24, 205-208. Lee, M. D.; et al. CRC Crit. Rev. Environ. Control 1988, 18, 29-86. Erikson, L. E.; Nakahara, T. R o c . Biochem. 1975,10,9-13. Goswami, P.; Singh, H. D. Biotechnol. Bioeng. 1991,37, 1-11. Kennedy, R. S.; Finnerty, W. R.; Sudarsanan, K.; Young, R. A. Arch. Microbiol. 1975, 102, 75-83. Roy, P. K.; Singh, H. D.; Bhagat, S. D.; Baruah, J. N. Biotechnol. Bioeng. 1979,21, 955-974. Reddy, P. G.; Singh, H. D.; Roy, P. K.; Baruah, J. N. Biotechnol. Bioeng. 1982, 24, 1241-1269. Reddy, P. G.; Singh, H. D.; Pathak, M. G.; Bhagat, S. D.; Baruah, J. N. Biotechnol. Bioeng. 1983, 25, 387-401. Whitworth, D. A.; Moo-Young, M.; Viswanatha, T. Biotechnol. Bioeng. 1973, 15, 649-675.
Received for review May 11,1992. Revised manuscript received September 2, 1992. Accepted September 9,1992. The Texas Advanced Technology Program generously supported this research. Amoco Gorp. provided fellowship support for S.J.B.
Peroxyacyl Nitrates at Southern California Mountain Forest Locations Danlel Grosjean,* Edwln L. Wllllams, 11, and Erlc Grosjean DGA Inc., 4526 Telephone Road, Suite 205, Ventura, Callfornla 93003
Ambient levels of the peroxyacyl nitrates [RC(O)OONOz]PAN (R = CH,) and PPN (R = C2HJ have been measured at two southern California mountain forest locations impacted by urban photochemical smog. The highest levels recorded were 22 ppb for PAN and 4.3 ppb for PPN; 24-h averages were 2-10 ppb for PAN and 0.25-1.7 ppb for PPN. PPN and PAN were highly correlated, with PPN/PAN ratios of 0.28 (August-October 1989), 0.18 (August-September 1990), and 0.14 and 0.19 (August-September 1991).Diurnal and seasonal variations in PAN/ozone, PPN ozone, and PPN/PAN ratios are discussed in terms of ormation and removal processes for PAN and PPN. The relative contribution of olefins, aromatics, carbonyls, and paraffins to the observed PPN PAN ratios has been estimated. Other peroxyacyl nitrates measured included PnBN (R = n-C3H7),which was frequently observed at one location at levels of 0.2-0.4 ppb. With a detection limit of 0.2 ppb, the isoprene oxidation product MPAN [R = CH2 = C(CH,)] was only observed in a few instances at one location but was observed more frequently at the other at levels of 0.3-1.2 ppb.
f/
Introduction
Photochemical oxidants that may adversely impact vegetation include ozone and peroxyacyl nitrates [RC(O)OONO,]. The two most abundant peroxyacyl nitrates are peroxyacetyl nitrate (PAN, R = CH,) and peroxy110 Environ. Sci. Technol., Vol. 27,
No. 1, 1993
propionyl nitrate (PPN, R = CzH,). While the adverse effecta of ozone on forests and crops are well documented, less information is available regarding the phytotoxic properties of peroxyacyl nitrates (1-3). The phytotoxicity of PAN has been demonstrated in several studies (1-3), and there is limited evidence suggesting that PPN and higher molecular weight peroxyacyl nitrates may be even more phytotoxic than PAN (1). In addition, ambient levels of peroxyacyl nitrates, while required to obtain a realistic assessment of photochemical oxidant damage to vegetation, have been seldom measured at locations where observations of severe oxidant damage have been recorded. In a recent study, we carried out detailed measurements of the ambient levels of peroxyacyl nitrates at a southern California mountain forest location (4). This study included the first set of quantitative measurements of PPN in ambient air and described the diurnal and seasonal variations of PAN and PPN along with those of ozone at a mountain forest site severely impacted by photochemical air pollution ( 5 , 6 ) . In this study, we have measured the ambient levels of the two most abundant peroxyacyl nitrates, PAN and PPN, during the 1990 and 1991 smog seasons. These measurements were carried out at two southern California mountain forest locations including the one surveyed in 1989 as part of our earlier study (4). The two C,-substituted peroxyacyl nitrates, namely, peroxy-n-butyryl nitrate (PnBN, R = n-C,H,) and peroxy-
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