Environ. Sci. Technol. 1996, 30, 2406-2411
Enhancing Biodegradation of Petroleum Hydrocarbons with Guanidinium Fatty Acids E. C. NELSON,* M. V. WALTER, I. D. BOSSERT,† AND D. G. MARTIN Fuels and Lubricants Technology Department, Texaco Group Inc., P.O. Box 509, Beacon, New York 12540
Bioremediation of hydrocarbon-contaminated soils is an attractive process for treating contaminated soils because it converts contaminants into harmless byproducts at low cost. However, the process is slow and typically requires months to years to reach regulated end points. In laboratory studies, we have been able to improve the process by adding selected guanidinium fatty acids to the contaminated soils. One of these materials, guanidinium cocoate, was synthesized from coconut acid and guanidine carbonate in a facile one-step process. Rates of biodegradation enhancement of nonvolatile hydrocarbons were evaluated using either oxygen and carbon dioxide respirometry in soil slurries or periodic measurements of extractable hydrocarbon residues in unsaturated soil microcosms. Results show rate enhancements in both soil slurries and unsaturated soil microcosms when these systems were treated with 500-1500 ppm of the surfactant. Adding small amounts of CGS to a silty clay soil containing aged lubricant-type hydrocarbons increased rates of hydrocarbon disappearance, mineralization, and oxygen utilization in unsaturated soil and soil slurry systems. Based on these initial investigations, doses of approximately 2 lb of CGS/t of soil appear effective at increasing rates of biodegradation.
Introduction Surface and subsurface releases of petroleum hydrocarbons produce an environment containing an abundant supply of carbon for microbial growth and metabolism. Supplying oxygen to the contaminated area will stimulate microbial biodegradation of the hydrocarbon by indigenous hydrocarbon-degrading microorganisms. Under these conditions, insufficient amounts of assimilable nitrogen or, to a lesser extent, phosphorus are likely to limit the overall rate of biodegradation. A recent review by Alexander summarizes these findings (1). Alternatively, in cases where * To whom correspondence should be addressed; e-mail address:
[email protected]. † Present address: Rutgers University, P.O. Box 909, Engineering Bldg., Room C-258, Brett and Brewster Roads, Piscataway, NJ 088550909.
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there are sufficient amounts of nitrogen and phosphorus and a suitable electron acceptor, bioavailability may limit the rate of biodegradation of the contaminants. Poor water solubility of the contaminant and sorption of the contaminant to regions of the soil particle that are not accessible to microbial activity are potential causes of low biodegradation rates. Enhancing water solubility and increasing mass transfer of the contaminant to the water phase are potential methods for increasing biodegradation rates. Adding surfactants to soils contaminated with hydrophobic contaminants may increase the bioavailability of these materials to hydrocarbon-degrading microorganisms. This potential has been widely recognized, and many investigators have evaluated the effects of adding physical mixtures of surfactants and inorganic nutrients to soils. Inipol EAP 22 is a commercially available example of this approach (2). Although Inipol EAP 22 has been marketed primarily for marine oil spills, Bartha and Abecasis (3) recently described successful applications of this material in soil contaminated with diesel fuel. However, few investigators have described well-controlled studies of the effect of surfactants only on hydrocarbon-contaminated soils. Several recent reports describe well-controlled studies of surfactant effects on enhancing biodegradation of petroleum hydrocarbons in soils. For example, Lindoerfer et. al. (4) demonstrated that rhamnose and trehalose glycolipid biosurfactants will enhance rates of petroleum hydrocarbon biodegradation significantly. They showed that treating crude oil-contaminated soil with a mixture of a glycolipid biosurfactant and a chemical surfactant, e.g., Tween 80, could produce a 3-fold increase in the overall rate of hydrocarbon biodegradation. In these examples, control samples decreased from 10 to 7 wt % (TPH) in 120 days, while biosurfactant/surfactant-treated samples decreased from 10 to 0.9 wt % in the same time. The results demonstrate that adding a surface active compound to a mixed culture of indigenous microorganisms will enhance the rate of biodegradation, albeit at high treatment dosages: 20 lb of biosurfactant/t of soil. Aronstein et al. (5) investigated the surfactant-enhanced desorption and mineralization of phenanthrene and biphenyl in mineral and organic soils. Nonionic alcohol ethoxylates (Alfonic 810-60 and Novel II 1412-56) enhanced mineralization of very low concentrations of these two aromatic substrates (ca. 1 ppm) when 10-100 ppm of the surfactants was added to the soil. However, under somewhat different conditions, Laha and Luthy (6) found that similar alcohol ethoxylates had either no effect or inhibited mineralization of phenanthrene in soil-water systems. Jain et al. (7) evaluated the effect of adding a biosurfactant produced by Pseudomonas aeruginosa UG2 to contaminated soils. They added 10-100 µg of biosurfactant/g of soil. Soils were deliberately contaminated with a mixture of tetradecane (3.2 mg/g), 1-methylnaphthalene (0.8 mg/ g), hexadecene (2.0 mg/g), and pristane (2.0 mg/g). Extraction of soils incubated for 2 months showed that the highest dose of biosurfactant, e.g., 100 µg, was required to provide statistically significant increases in biodegradation rate for all hydrocarbons except 1-methylnaphthalene. Our objective in these investigations was to prepare surfactant/nutrients that might provide a readily assimilable
S0013-936X(95)00903-5 CCC: $12.00
1996 American Chemical Society
source of nitrogen within a water-soluble surfactant. We report that a series of anionic nitrogen-containing compounds will enhance the biodegradation of hydrocarbons that are sorbed to soils (8). In particular, we describe results with the guanidinium salt of coconut acids.
Experimental Section Materials and Methods. Naphthalene, tetradecane, and anhydrous ethyl ether were obtained from Aldrich Chemical Co. Infrared spectra were recorded on a Nicolet 520 FTIR. Samples were taken as thin films between NaCl plates and scanned 10× at a resolution of 4 cm-1. NMR spectra were taken on a Varian UnityPlus-300 spectrometer operating at 299.95 and 75.43 MHz for 1H and 13C, respectively. The probe used a Varian broad band 5-mm probe. 1H spectra were collected with a π/6 pulse width of 4 µs and a 3-s acquisition time at 30 °C. 13C spectra were collected at a π/6 pulse width of 4 µs and with a 533-ms acquisition time and a 3-s relaxation delay at 30 °C. Waltz high-power proton decoupling was used for 13C spectra. Samples were dissolved in perdeuterio DMSO or CDCl3 and are referenced to TMS. Samples for percentage nitrogen analysis were dried in vacuum desiccator against P2O5 for 24 h and analyzed on a Leco CHN-1000 analyzer. Gas chromatographs were run on a Carlo Erba high-temperature GC. Samples were completely dissolved in carbon disulfide and injected onto a 12-m aluminum-clad, methyl silicon column. The column was heated from 40 to 400 °C at a ramp rate of 5 °C/min. For comparison, a standard normal paraffin mixture was run under the same conditions (9). Surface tension and critical micelle concentrations were determined using a Fisher Surface Tensiomat Model 21. Dried samples of the guanidinium cocoate were dissolved in DI water and analyzed in automatic mode with 6.000 cm Pt/Ir ring with an R/r of 53.7488. Surface tension measurements were made at room temperature and are uncorrected. Samples were repeatedly analyzed until the variation between successive measurements was less than 1 dyn/ cm. The critical micelle concentration was determined by a plot of the log C vs γ. Synthesis of Guanidinium Cocoate. Coconut acid (40 kg, Proctor and Gamble C-110) and 60 L of DI water were mixed (150 rpm) and heated to 60 °C in a 450-L stainless steel stirred tank reactor (Chemap). Guanidine carbonate (17.5 kg, Chemie Linz) was dissolved in 64 L of DI water at 60 °C and added to the 450-L reactor in 8 h. The mixture was heated for an additional 24 h and afforded 145.2 kg (25 wt % active) of golden yellow liquid. A sample was lyophilized to give a sticky, pale yellow solid. Dissolution in DI water was facile, producing a clear to slightly cloudy, foaming solution. Chemical analysis of the reaction mixture indicated that the product consisted substantially of the guanidinium salt of coconut acids (10, 11). Percentage nitrogen analysis indicated that 15.3% of the lyophilized mixture was nitrogen. Infrared analysis of a thin film of the reaction mixture revealed major absorbances at 3388, 3148, 2923, 2853, 1670, 1533, and 1408 cm-1. 13C NMR in DMSO showed resonances at 179 and 159 ppm, which are indicative of carboxylate and imine carbons, and resonances at 39.2, 32.3, 29.9-29.5, 26.7, 22.7, and 14.6, consistent with carbon resonances in a mixture of normal aliphatic chains. 1H NMR in DMSO showed resonances consistent with the desired compounds: 7.75 ppm (s, 6 H, guanidinium H), 1.90 ppm (t, 2 H), 1.40 ppm (m, 2 H), 1.23 ppm (m, ∼17 H), 0.78 ppm (t, ∼2.7 H).
Soil Handling. All soils used were from a silty clay soil that had been periodically contaminated with lubricanttype oils over a 30-year period. Soils for microcosm experiments were air-dried and sieved to less than no. 4 mesh to remove stones and twigs. Soils for respirometry experiments were air-dried for a minimum of 2 days, ground, sieved to less than no. 30 mesh and riffled to ensure maximum homogeneity between soil samples. Samples were stored at 4 °C until use. Although all soils were obtained from the same site, heterogeneity at the site and multiple sampling events afforded several different batches of soil containing varying amount of extractable hydrocarbon. Therefore, percentage methylene chloride extractable HC is used to identify soils used in the experiments described below. Naphthalene and Tetradecane Treated Soils. The naphthalene and tetradecane were distributed over the soil, by treating 250 g of the control soil with a solution of naphthalene (5 g) and tetradecane (5 g) in ethyl ether (100 mL). After soaking for 10 min, the ethyl ether was immediately evaporated in-vacuo at 30 °C, and the spiked soil was stored in a tightly capped flask at 4 °C to retain the embibed hydrocarbons. The control soil (40 g) was combined with the spiked soil (10 g) to afford a “control + HC” soil containing naphthalene and tetradecane. The mixed soil was not assayed for naphthalene or tetradecane; however, the initial loading could have provided as much as 200 mg each of naphthalene and tetradecane/50 g of soil sample; it is likely that a portion of these biodegradable hydrocarbons volatilized during handling. Microcosm Experiments. Microcosm experiments were conducted using replicate beakers that contained 200 g of soil contaminated with 1.03 wt % extractable hydrocarbon. Two different amounts of guanidinium cocoate (a synonym for guanidinium cocoate is the acronym CGS, representing coconut guanidium surfactant), 173 or 520 mg of CGS/200 g of soil were applied to a series of replicates. Microcosm soils were adjusted to 50% water-holding capacity with DI water and thoroughly mixed with a stainless steel spatula. Microcosms were loosely covered with a plastic cover to retain moisture and stored at room temperature. Throughout the study, soil moisture was periodically adjusted to 50% of water-holding capacity by adding DI water. Periodically, 200-g replicates were air-dried in a laminar flow hood overnight. Duplicate 30-g samples were weighed, mixed with ca. 25 g of anhydrous sodium sulfate and exhaustively extracted with methylene chloride using a Soxhlet extractor. The methylene chloride extracts were concentrated on a rotary evaporator, redissolved in less than 5 mL of methylene chloride, and transferred to tared 10-mL glass vials. The methylene chloride was allowed to evaporate in a hood under an airstream for a minimum of 2 days. The vials containing the residue were weighed, and the percent hydrocarbon was determined. Respirometry. Soil slurry respirometry was carried out in 1-L Erlenmeyer shake flasks or using a Biosciences BI1000 respirometer (Allentown, PA). Shake Flask Respirometry. Soil slurries were made up in a 1-L flask that contained a polypropylene test tube, which had been suspended in the head space of the flask. The tube contained an aqueous KOH solution for trapping out evolved CO2. Soil (30 g) contaminated with 0.95 wt % extractable hydrocarbon was mixed with 300 mL of DI water in the shake flask. The flask was flushed with CO2-free air, sealed, and rotary shaken at 130 rpm and 30 °C. Periodically
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the KOH solution in the CO2 trap was removed, treated with a dilute solution of barium chloride, and titrated with a dilute hydrochloric acid solution as described by Bartha and Pramer (12). Fresh KOH solution was added to the CO2 trap, the flask was flushed with CO2-free air, and rotary shaking was continued. Electrolytic Respirometry. The Bioscience BI-1000 respirometer was operated according to Bioscience’s standard procedures. However, we used a modified threenecked 1225-mL reactor equipped with vacuum-tight glass bearing to permit overhead stirring of the slurry with a glass stir shaft and Teflon paddles (16). Untreated controls were run concurrently with treated samples to account for any changes in soil activity that may have accompanied cold storage of the soils. Initial hydrocarbon concentration was determined using exhaustive Soxhlet extraction with methylene chloride, as described below. Typically, 50 g of soil was added to 1 L of DI water along with any surfactant treatment. A CO2 trap was charged with 5 mL of 45 wt % KOH and suspended in the head space of the reactor. The reactor was tightly sealed and then stirred at ca. 200 rpm in a water bath maintained at 30 °C. The pH of the slurry was evaluated at the start and at the end of the reaction period; pH differences between reactors either at the start or end of the experiment were small (
