Environ. Sci. Technol. 2000, 34, 489-496
Enhanced Degradation of a Model Oil Compound in Soil Using a Liquid Foam-Microbe Formulation MARK B. RIPLEY,† A D R I A N B . H A R R I S O N , * ,† W. BERNARD BETTS,† R. KINSEY DART,‡ AND ASHLEY J. WILSON† Department of Biology, University of York, P.O. Box 373, York YO10 5YW, U.K., and Department of Chemistry, Loughborough University, Loughborough, LE11 3TU, U.K.
This paper describes the development of a protein-based foam formulation and subsequent investigations into its suitability to enhance the degradation of a model hydrocarbon (n-hexadecane) using novel bench-scale soil microcosms. High-density protein-based foam concentrates based upon those developed by the fire-fighting industry were selected for experimental investigation. Using crude protein hydrolysate as a starting material, a foam formulation was developed with properties suitable for bioremediation studies. This formulation incorporated eight hydrocarbondegrading bacteria that had been selected for their ability to degrade hexadecane. In addition to their ability to utilize n-hexadecane, the bacteria were tested for compatibility with the foam formulation and each other. Seven individual Acinetobacter spp. and a Pseudomonas species were selected for use in the consortium based on these criteria. The use of this “bioactive foam” led to enhanced n-hexadecane degradation when compared to controls without foam. Following 7-d incubation, 60% of the n-hexadecane remained in the soil column using a foamed formulation, as compared to 90% with a nonfoamed control. In a subsequent experiment over a 15-d time course, the authors observed significantly greater n-hexadecane degradation in response to oxygenated bioactive foam treatment.
Introduction There are a wide range of bioremediation technologies either in use or proposed for use on oil-contaminated land (1, 2), and these can be divided into two broad groups. In situ techniques treat the contamination at the site of the pollution event, whereas ex situ techniques remove the contamination from the ground and transfer it to another location for treatment. The use of in situ treatment is often preferable in terms of financial considerations, due to the cost of moving large quantities of soil (3). One novel approach to the problem of hydrocarbon contamination of the terrestrial environment is the use of gas-liquid foams to enhance in situ bioremediation. Foams are disperse systems containing at least two distinct phases. * Corresponding author phone: +44 (0)1904 432919; fax: +44 (0)1904 432923; e-mail:
[email protected]. † University of York. ‡ Loughborough University. 10.1021/es9905593 CCC: $19.00 Published on Web 12/22/1999
2000 American Chemical Society
A continuous liquid phase surrounds bubbles of gas and may enclose droplets of a secondary liquid phase or particles of a solid phase. Surfactants are essential for the generation and stabilization of foams, accumulating as a viscoelastic layer at air/water, air/liquid, and liquid/liquid interfaces to maintain the structural integrity of the foam (4). This has an important stabilizing effect by altering surface properties at the interfaces, particularly by lowering the surface tension. The most widespread large-scale application of gas-liquid foams is in fire-fighting, where air is excluded from the combustible material by a thick blanket of foam (5). These fire-fighting foams are supplied as liquid concentrates, which can be diluted on-site to the required strength. The foam is formed from this premix by an aerating device. Several previous studies have been undertaken to investigate the suitability of foams for bioremediation applications. Li et al. (6-8) investigated the degradation of oil using a solid alginate foam carrier inoculated with a marine oil-degrading yeast and nutrients. The foam carrier was prepared from hens’ egg and bovine serum. They observed that a floating alginate carrier could both absorb and hold the oil and that the immobilized nutrients contained in the egg were of use to the microbial population. Using this system, Li et al. found that 61% of the model oil, n-tetradecane, was degraded in 14 days. Stabnikova et al. (9) showed enhanced degradation of crude oil in soil columns using a foamed preparation referred to as Lestan. This contained a hydrocarbondegrading microbial component, a biological surfactant, and a carrier. Eighty-nine percent of the oil was degraded after 35 d of treatment with foamed Lestan (applied at 1-week intervals), which was 43% higher than the untreated controls. A number of different systems have been investigated to treat nonaqueous-phase liquids (NAPLs) in subsurface soils (1012). In these studies, synthetic surfactants were injected directly into soil to mobilize hydrocarbons. The potential use of foams has also been demonstrated for the decontamination of nerve agents (13). In these systems, the detoxification of the nerve agent was carried out by immobilizing the enzyme organophosphorous acid anhydrase within either a fire-fighting or blast-containment foam carrier. The enhanced biodegradation of diesel oil in soil by the addition of protein hydrolysate has been observed previously, in terms of both an increase in the number of degrader bacteria and activity and an elimination of diesel oil (14). In this paper, we describe the development of a gas-liquid foam as a carrier system for the delivery of microbes, nutrients, and oxygen applied as a blanket over hydrocarboncontaminated soil. The subsequent evaluation of the foam for the improvement of degradation rates is also discussed. Due to the large volume of foam that could be required for environmental bioremediation applications, high-density protein-based foam concentrates based on those developed by the fire-fighting industry were selected for experimental investigation. These are relatively inexpensive, easy to apply, durable, and compatible with thickeners; have high elasticity, good temperature stability, a high wetting capacity, and are biodegradable.
Materials and Methods Foam Generator and Foam Formulation. Equipment for Foam Generation. The system used to generate large quantities of foam consisted of an air cylinder, a liquid reservoir, a mixing chamber, and a dispensing nozzle. Gas (air or oxygen) and protein hydrolysate solution were combined in a column packed with glass beads (diameter 3 and 9 mm) to generate the foam. The foam was directed through a nozzle VOL. 34, NO. 3, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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at the end of the column. A gas pressure of 30 psi (2.06 bar) was used for all experiments. Foam Manufacture. Protein hydrolysate, supplied as a spray-dried powder (Angus Fire Armour Ltd., U.K.), was dissolved in distilled water to provide a range of concentrations (0.25-5.0%), and the samples were sterilized by autoclaving. The resulting solutions (1 L) were then foamed into 25-L vessels using a previously sterilized foam generator. The liquid draining from the foam passed through a small hole in the base of the vessel and was collected in a measuring cylinder. Measurements of the 50% drain time for the different hydrolysate concentrations were made. The 50% drain time was selected as a measurement of foam stability to maintain the standard procedure used in the fire-fighting industry (15). The drainage of liquid from the foam was also considered to be an important factor in bioremediation, providing controlled dispensing of the foam components. All 50% drain time experiments were carried out at 21 °C. Improvements to Foam Stability. To improve the stability of the foam, chemical and viscosity modifications were investigated. Experiments were undertaken to evaluate the effect on foam stability of adding various salts to a 1% w/v protein hydrolysate solution. The following salts were used in the formulation at a concentration of 0.05% w/v: AlCl3‚ 6H2O, NH4Cl, (NH4)2SO4, FeCl3‚6H2O, FeCl2‚4H2O (Sigma, U.K.), FeSO4‚7H2O, MnSO4‚4H2O, MnCl2‚4H2O (BDH, U.K.), MgSO4‚7H2O, and MgCl2‚6H2O. All chemicals were supplied by Fisher, U.K., unless stated. FeCl2‚4H2O was also used in the range of 0.01-0.4% w/v. Foam was generated using a 1% w/v protein hydrolysate solution to which the salt had been added, and the 50% drain time was then measured for each different formulation. The effect on foam stability of three viscosity modifiers [sodium alginate, carboxymethyl cellulose (CMC), and xanthan gum] at a range of concentrations was also investigated. The foam was generated according to the protocol outlined earlier in this section, and the 50% drain time was then measured for each formulation. Oxygen Concentration in Liquid Draining from Foam. The maximum concentration of oxygen that could be dissolved in a 1% protein hydrolysate solution was determined by bubbling either air or oxygen through the solution. Samples of the liquid draining from foam generated (as above) using compressed air or oxygen were collected, and the oxygen concentration was determined using a dissolved oxygen probe (Jenway, U.K.). Microorganisms. Isolation of Hydrocarbon-Degrading Organisms. Samples of activated sludge (Knostrop Sewage Treatment Works, Yorkshire Water plc., Leeds, U.K.) and pressed waste sludge (Linsey Oil Refinery, Immingham, U.K.) were returned to the laboratory at ambient temperature and subsequently stored at 4 °C. Sets of flasks containing 100 mL of sterile Bushnell-Haas broth (BHB) (Difco, U.S.A.) were supplemented with 100 µL of n-hexadecane sterilized by filtration through a 0.2-µm Acrodisc 32 filter (Gelman, U.K.). The two samples of effluent (100 µL or 0.1 g) were used to inoculate sets of 6 flasks each containing n-hexadecane. The flasks were incubated, in an orbital incubator (120 rpm), at 21 °C for 7 d. At the end of the incubation period, the flasks were examined, and if visible signs of growth were present, samples were taken and streaked onto nutrient agar (Oxoid, UK). These plates were then incubated at 21 °C for 24 h. Representative colonies of different morphology were selected and repeatedly subcultured in BHB supplemented with n-hexadecane until pure cultures were obtained; these were then maintained on nutrient agar. Each isolate selected for subsequent use was shown to efficiently utilize n-hexadecane in liquid culture experiments (data not shown). 490
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The bacterial strains were characterized by Gram staining and selected physiological and biochemical tests (16, 17). Gram-negative bacteria were tentatively identified using the API-20-NE test system for nonenteric Gram-negative bacteria (Bio-Merieux, France). Mutual Compatibility of Hydrocarbon-Degrading Bacteria. The isolated organisms were grown overnight in nutrient broth (Oxoid, UK) at 21 °C. Triplicate sets of nutrient agar plates were inoculated with each organism using a rotary plating technique. The plates were then allowed to dry for 30 min. Sterile antibiotic assay disks (Whatman, U.K.) were immersed into a sample of each of the overnight cultures. The disks were blotted on sterile filter paper before placing on the surface of the inoculated agar plates. The plates were inverted prior to incubation at 21 °C for 24 h. At the end of the incubation period, the plates were examined for zones of inhibition around the disks. This method was used to determine if any of the selected organisms produced extracellular compounds that were inhibitory to the other organisms. Effect of Hydrocarbon Degraders on Foam Stability. Cultures of the hydrocarbon-degrading isolates were grown overnight in nutrient broth. Samples of these cultures (25 mL) were placed into 50-mL sterile centrifuge tubes and centrifuged at 6000g for 10 min (High Speed 18, MSE, U.K.). The resulting pellet was resuspended in 25 mL of sterile distilled water. This was added to 100 mL of sterile 10% w/v protein hydrolysate solution in a 500-mL Gibco bottle. Each bottle was shaken for 30 s and allowed to stand. Measurements of the foam collapse were compared to a control bottle containing no bacteria. Compatibility of Foam with Hydrocarbon Degraders. Samples of each isolate were prepared in distilled water as described previously. Triplicate sets of flasks containing 100 mL of protein hydrolysate solution (1% w/v) were prepared. Each flask was inoculated with a 1-mL sample of the bacterial suspension, and the flasks were then incubated at 21 °C for 24 h with shaking. This was repeated for each of the isolates. Samples were removed at 0 and 24 h for bacterial enumeration by plate count. The bacteria were grown on nutrient agar at 21 °C for 24 h prior to counting. The experiment was repeated using protein hydrolysate solution supplemented with 0.05% w/v hydrated ferrous chloride (Sigma, U.K.). Survival of Microbes during the Foaming Process. The foam generator was sterilized to remove any contaminating organisms. This was achieved by dismantling the system and autoclaving at 121 °C, 15 psi for 15 min. The generator was then re-assembled using aseptic procedures. Protein hydrolysate solutions were prepared by dissolving 10.0 g of protein hydrolysate in 900 mL of distilled water. Stock solutions of ferrous chloride were prepared by dissolving 0.5 g (hydrated) of ferrous chloride (Sigma, U.K.) in 50 mL of distilled water. These were autoclaved at 121 °C, 15 psi for 15 min. Suspensions of each of the n-hexadecane-degrading bacteria (approximately 109 mL-1) were prepared in distilled water following centrifugation as described above. From these, 50 mL was aseptically transferred to 900 mL of protein hydrolysate solution. The stock solution of ferrous chloride was added, and then foam was generated into a surfacesterilized vessel. A sample of the foam was immediately removed from the center of the vessel and aseptically transferred into a sterile 500-mL beaker. This sample was placed on ice until the foam collapsed. A sample of the liquid draining from the foam (1 mL) was collected. The number of organisms in the original sample and the drained liquid were estimated by plate count on nutrient agar. Plates were incubated at 21 °C for 24 h. The experiment was repeated for each of the hydrocarbon-degrading bacteria isolated.
FIGURE 1. Schematic of the soil column microcosm used for the bioactive foam treatment of n-hexadecane-contaminated autoclaved soil. Soil Column Microcosms. Collection and Characterization of Soil Samples. Samples of soil were collected from the upper 20 cm of an agricultural site in East Yorkshire, U.K., that was known to be out of cultivation for at least 5 years. The site had no known history of contamination with hydrocarbons. The soil (pH 6.2) was classified as a Blackwood Series of the Typical Sandy Gley Soils (18). All soil samples were sieved (4 mm) to remove any large particles and plant material and were subsequently used without air-drying (19). Waterholding capacity and water content of the soil were determined after Gardner (20). Design of Soil Column Microcosms. Soil column microcosms were designed to allow application of foam to the surface of the soil (Figure 1). The soil columns consisted of two connecting autoclavable plastic tubes (36 mm internal diameter; total length 430 mm), with a Teflon stopper at the base, which contained glass tubing for medium outlet. The columns were opaque to prevent light-induced transformations and the growth of algae. This design allowed easy fitting and removal of the soil-containing section of the column, which, at the end of experiments, could be removed for extraction. The columns could be autoclaved to sterilize. Biodegradation Experiments. Preparation of Microcosms for Experiments. Samples of soil [(500 g (field moist weight)] were taken and placed into plastic bags. These were autoclaved at 121 °C, 15 psi for 30 min. The soil was then incubated at 30 °C for 24 h before the autoclaving process was repeated. When the soil was cool, sufficient n-hexadecane was added to give a final concentration of 0.5% v/w (dry weight) of soil. The bag was shaken vigorously for 5 min to distribute the n-hexadecane evenly. The soil was supplemented with sufficient sterile water to bring the water content up to 40% of the water-holding capacity. The contents of all the plastic
bags were combined, and a sample of the contaminated soil [100 g (dry weight)] was aseptically transferred to the lower section of the presterilized columns. Preparation of Microbial Inoculum. Cultures of the n-hexadecane-degrading bacteria were grown for 48 h in nutrient broth. Samples (100 mL) were then centrifuged (MSE High Speed 18 centrifuge, 6000g for 10 min), and the resulting pellets were resuspended in 100 mL of sterile distilled water. These suspensions were then combined to form the microbial inoculum. This suspension contained approximately equal numbers of each of the bacterial isolates at a concentration of approximately 109 cells mL-1 (as determined by plate counts on nutrient agar). Preparation of Protein Hydrolysate Solutions. Protein hydrolysate solutions were prepared by dissolving 10.0 g of protein hydrolysate powder in either 900 mL (for microbe supplemented solutions) or 950 mL (for sterile solutions) of distilled water. Stock solutions of ferrous chloride were prepared as described earlier. The solutions were autoclaved at 121 °C, 15 psi for 15 min. The two solutions were mixed immediately before use. For the microbe-supplemented protein hydrolysate solution, a sample (50 mL) of the suspension of n-hexadecane-degrading bacteria was added. This provided solutions containing 1% w/v protein hydrolysate and 0.05% w/v ferrous chloride with or without approximately 5 × 107 degraders mL-1 as required. Experimental Setup. For each treatment, soil columns (quadruplicate) were set up containing the equivalent of 100 g (dry weight) of n-hexadecane-contaminated soil. The microcosms were subjected to the following treatment regimes at 48-h intervals for 6 d and incubated at 21 °C: protein hydrolysate solution, aerated foam, and oxygenated foam, each in the presence or absence of the bacterial consortium. Untreated sets of columns were included as controls. Additional columns were sacrificed at the start of the experiment in order to determine the concentration of n-hexadecane and microbial population applied initially. Foam was applied to the soil columns using the foam generator, but the nozzle had been adapted to include a plate at the end to direct foam into the column. Foam was applied until excess was seen to escape from the vent holes immediately above the surface of the soil. These holes were then sealed with electrical tape. For columns that were treated with protein hydrolysate solution, the liquid (15.2 mL) was applied aseptically using an automatic pipet. Repeat treatments were applied to the soil columns at 48-h intervals for 6 d. Samples of soil for analysis were taken on the seventh day. The whole experiment was repeated four times with fresh batches of soil. Three of the columns for each treatment were analyzed for residual n-hexadecane, and the fourth was analyzed for estimation of hydrocarbon-degrader numbers. The results from each of the experiments were combined for statistical analysis using a Student T test. Following the initial soil column experiments, a time course study over a 15-d period was undertaken to measure n-hexadecane degradation in the presence of microbesupplemented foams generated with either air or oxygen. The soil columns were set up as previously described and subjected to foam treatments at 72-h intervals for 12 d at 21 °C. Additional sets of untreated soil columns were set up to serve as controls. Measurement of n-Hexadecane Concentration in Soil Samples. The concentration of n-hexadecane remaining in the columns was determined using a modification of the method of Platen (21). At the end of the experiment, 500 µL of n-tetradecane (Sigma, U.K.) was added to each column as an internal standard. The soil from each of the columns was placed into screw-top Erlenmeyer flasks, and 20 g of anhydrous sodium sulfate (Fisher Scientific, U.K.) was added before mixing. Dichloromethane (HPLC grade, Fisher SciVOL. 34, NO. 3, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Effect of different concentrations of protein hydrolysate solution on the 50% drain time of foam. entific, U.K.) (DCM) (150 mL) was added, and the flasks were sealed before being placed on an orbital shaker set at 200 rpm for 30 min to extract the hydrocarbon. The DCM was filtered through a cellulose filter (Whatman No. 1) and then passed through aluminum oxide (Sigma Type WN3 neutral) to remove any polar compounds. The filtered solvent was collected in glass vials. The amount of n-hexadecane was determined by gas-liquid chromatography (GLC), using a Pye Unicam PU 4500 chromatograph fitted with a 30-m 0.2µm film thickness SE54 capillary column. Experimental conditions were as follows: 150-200 °C at 12 °C min-1; injector temperature, 250 °C; detector temperature, 300 °C; carrier gas, helium; split ratio, 80:1; sample size, 1 µL; FID detector. The purity of the n-hexadecane supplied was also checked by GLC using the conditions described above. To determine the extent of n-hexadecane degradation, the areas of individual peaks (from 12 samples) were quantified and normalized to the n-tetradecane peak from each sample. These values were then expressed as the percentage mineralized relative to the amount of the corresponding peak remaining in the sterile abiotic control. Measurement of n-Hexadecane Concentration in Column Drain Liquid. A 500-µL volume of n-tetradecane was added to flasks containing the column drain liquid as an internal standard. The flasks were then extracted twice with 50 mL of DCM in a separating funnel. The solvent layer was passed through alumina (Sigma Type WN3 neutral) to remove any polar compounds. The amount of n-hexadecane was determined by GLC. Measurement of n-Hexadecane-Degrader Numbers. Hexadecane-degrader numbers were determined using a modification of the method of Song and Bartha (22). From each column, 1 g of soil was removed and aseptically transferred to 99 mL of sterile quarter-strength Ringer solution (Oxoid, BR52, U.K.) in a medical flat bottle. This was then shaken vigorously by hand for 10 min. A 5-mL volume of the soil suspension produced was aseptically transferred to 45 mL of sterile quarter-strength Ringer solution to give a 10-3 dilution. Further dilutions were carried out as appropriate. Tubes containing 5 mL of sterile Bushnell-Haas broth with 1 mg L-1 resazurin (Sigma, U.K.) were supplemented with 50 µL of sterile n-hexadecane. Sets of five tubes were inoculated with 1 mL of the appropriate dilution of the soil sample. The inoculated tubes were then incubated at 21 °C for 7 d. The positive tubes (those turning pink or clear due to the reduction of resazurin by microbial oxygen consumption) 492
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TABLE 1. Effect of the Inclusion of Different Salts (0.05% w/v) on the 50% Drain Time of Protein Hydrolysate Foam salt
50% drain time (min)
AlCl3‚6H2O NH4Cl (NH4)2SO4 FeCl3‚6H2O FeCl2‚4H2O
37 100 25 30 93 72
salt
50% drain time (min)
FeSO4‚7H2O MnCl2‚4H2O MnSO4‚4H2O MgCl2‚6H2O MgSO4‚7H2O
94 29 34 36 30
were counted. The MPN values were then obtained by reference to MPN tables.
Results Foam Formulation. Optimization of Formulation. The 50% drain times of foams generated with different concentrations of protein hydrolysate are presented in Figure 2. Using this information together with visual observations of the foam, 1% w/v was selected as an appropriate concentration to use for subsequent experiments. Improvements in Foam Stability. The effects of the inclusion of 0.05% w/v of different metal salts on the 50% drain time of foam are shown in Table 1. Increasing the amount of ferrous chloride in the foam formulation led to an increase in foam stability (Figure 3). Above a concentration of 0.2% w/v, the foam became almost solid and the structure was retained for several days, even when the liquid had drained completely. On the basis of these observations, a concentration of 0.05% w/v was selected. The effect of increasing the amount of thickening agents in the foam formulation was to increase the stability of the foam. However, as the concentration of CMC or xanthan gum was increased foam volume was drastically reduced (data not shown). Sodium alginate proved to be unsuitable due to the formation of a gel upon addition to the foam. Measurement of Oxygen Concentration in Drain Liquid. The amount of oxygen dissolved in protein hydrolysate solutions that had been sparged with air and oxygen was 8.1 and 37.2 mg L-1, respectively. For the foamed preparations, the concentration of oxygen in the liquid draining from the foam generated using air was 5.3 mg L-1, and the liquid draining from foam generated with oxygen was 19.9 mg L-1. Microbial Isolates. Eight n-hexadecane degraders were isolated from the two sites and were tentatively identified,
FIGURE 3. Effect of different concentrations of FeCl2‚4H2O on the 50% drain time of 1% w/v protein hydrolysate foam.
TABLE 2. Survival of n-hexadecane-Degrading Isolates in Protein Hydrolysate Solution over a 24-h Period viable bacterial population (×106 ( SEM) protein solution + ferrous chloride
protein solution isolate
0h
24 h
0h
24 h
Acinetobacter johnsonii Acinetobacter junii Pseudomonas aeruginosa Acinetobacter haemolyticus Acinetobacter sp. Acinetobacter baumannii Acinetobacter haemolyticus Acinetobacter haemolyticus
6.80 ( 1.00 19.90 ( 0.17 67.00 ( 1.53 5.37 ( 0.03 2.18 ( 0.11 22.90 ( 0.30 42.00 ( 2.08 4.97 ( 0.14
13.90 ( 2.05 65.00 ( 2.65 90.00 ( 4.16 61.00 ( 4.73 17.30 ( 1.80 24.10 ( 2.87 103.00 ( 4.91 32.90 ( 2.74
5.90 ( 0.36 17.4 ( 0.76 51.0 ( 5.65 7.77 ( 1.18 4.67 ( 0.32 6.70 ( 0.17 37.3 ( 0.33 10.10 ( 1.42
20.40 ( 1.31 28.80 ( 4.05 30.40 ( 5.46 46.50 ( 1.84 8.23 ( 1.68 18.00 ( 4.13 40.10 ( 7.59 36.30 ( 1.88
based on biochemical tests and staining, as Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter baumanni, Pseudomonas aeruginosa, three strains of Acinetobacter haemolyticus, and an Acinetobacter species. None of the organisms isolated produced any compounds that were inhibitory to any of the other organisms used in the consortium. Work carried out by the authors (unpublished data) suggested that some organisms do have a deleterious effect on foam stability. However, the organisms selected here for further investigation showed no effect on the stability of the foam. The selected bacteria were not killed by the foam formulation. Indeed, a limited amount of growth was observed for all the organisms selected. The results of this are presented in Table 2. Soil Microcosm Experiments. The results of the 7-d experiments using soil column microcosms clearly demonstrated that the inclusion of both microbes and oxygen in the foam formulation enhanced the removal of n-hexadecane from the soil (Figure 4). The pattern of n-hexadecane removal was consistent for all four experiments, although there was some variation between experiments due to the use of fresh batches of soil for each run. The removal of n-hexadecane was matched by increases in the numbers of n-hexadecanedegrading bacteria present in the soil column (Figure 5). The process of autoclaving the soil twice at 121 °C, 15 psi for 30 min, was not totally effective at killing all the hydrocarbondegrading bacteria present in the nonsterile soil. In no case was n-hexadecane detected in the liquid draining from the soil columns. The n-tetradecane added as an internal
standard was recovered by the extraction process, confirming that the method was reliable. The results of the time course experiment conducted over a 15-d period of n-hexadecane degradation are shown in Figure 6. After 6 d, the rate and extent of n-hexadecane was markedly increased by treatment with oxygen-supplemented bioactive foam. At the end of the study (15 d), 32.9% of the n-hexadecane was degraded in the aerated foam treated columns, and 51.3% was degraded in the oxygenated foamtreated columns. These data provide evidence that oxygensupplemented bioactive foam significantly enhances the removal of n-hexadecane from the soil columns over that using aerated bioactive foam treatment.
Discussion The protein hydrolysate selected proved to be an excellent starting material for the development of a bioactive foam formulation. The foams considered in this study possessed an expansion ratio of 20:1, a parameter that was fixed by the foam-generating apparatus. Increases in the concentration of hydrolysate led to improvements in foam stability, although diminishing returns were observed as more hydrolysate was added. A 1% w/v hydrolysate solution was selected for further investigation. This was based principally on observations of the quality of the foam formed and the perceived need to limit the amount of nitrogen (in the form of protein) added to soil. In the field, the addition of excess hydrolysate could lead to excess nitrogen leaching into groundwater. The lifetime of the foam generated using this concentration was deemed by the authors to be insufficient for bioremediation studies. For this reason, alternative ways of improving foam VOL. 34, NO. 3, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. n-Hexadecane remaining in soil columns after 7 d, following different treatment regimes at 21 °C. Results are expressed as % n-hexadecane added at start of experiment. All data are means ( SEM.
FIGURE 5. MPN n-hexadecane degrading organisms isolated from soil columns after 7 d following different treatment strategies at 21 °C. stability were investigated. The use of viscosity modifiers, while retarding drainage, had a deleterious effect on foam expansion ratio and were difficult to generate. Sodium alginate was shown to be unsuitable due to the formation of a gel upon addition to the foam. Further work by the authors (unpublished data) indicated that this was due to a reaction of the alginate with calcium present in the protein hydrolysate. The inclusion of viscosity modifiers in the foam formulation to prevent drainage caused two further problems. First, the viscosity of the solution draining from the foam was so great that oxygen transfer was inhibited, resulting in anoxic conditions. Second, the use of these compounds increased the carbon content of the formulation. This could prevent or delay hydrocarbon degradation by microbes favoring these foam-derived compounds as an alternative carbon source or could limit growth by nitrogen limitation. The inclusion of metal salts into the formulation at relatively low concentrations had a marked effect on both foam quality and retardation of drainage from the foam. Ferrous chloride solution at 0.05% w/v was selected as a stabilizing agent based upon the improvements in foam 494
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stability and the fact that ferrous chloride has been accepted previously for use in foams used in agriculture. A number of bacteria capable of degrading n-hexadecane were isolated from both sample source sites. In addition to the ability to degrade n-hexadecane, they were tested for their compatibility with the foam formulation and with each other. Eight organisms were selected for further tests based on these criteria. The predominant genus of n-hexadecanedegrading bacteria was Acinetobacter, which accounted for seven out of the eight organisms selected. Other organisms screened for their compatibility with the foam resulted in a reduction in foam stability and were not used further. The degradation of a suitable simple hydrocarbon in a model ecosystem was investigated to allow an initial laboratory appraisal of the effectiveness of the bioactive foam technique prior to any field trials. The choice of hydrocarbon was governed by five major factors: low chemical complexity, high degradability, low toxicity, high stability in the environment (principally low volatility), and easy analytical methods. A single compound, n-hexadecane, was selected as it has a relatively simple aliphatic hydrocarbon structure, is readily degradable (23-
FIGURE 6. n-Hexadecane remaining (% original; mean three replicates ( SEM) over time from soil columns incubated at 21 °C following various bioactive foam treatments. Symbols: (() untreated control; (2) aerated foam with microorganisms; (9) oxygenated foam with microorganisms. 25), and is stable under the experimental conditions used. Several studies have been carried out using n-hexadecane as a model substrate for hydrocarbon degradation in soil (26, 27). The selection of bacteria chosen for subsequent experiments in microcosms was therefore governed principally by their ability to degrade n-hexadecane. In preliminary experiments using soil columns, the foam was generated separately and then applied to the surface of the microcosm. This led to changes in foam structure. When attempts were made to dispense foam into vessels, difficulties were encountered due to air being trapped below the foam. Venting the air through escape holes in the final microcosm design prevented this. The small size of the microcosm employed was originally believed to be limited by edge effects that would reduce foam stability, i.e., the relatively larger surface area inside a small diameter tube would accentuate the foam collapse. However, experiments conducted with different sized vessels showed that this was not the case (unpublished data). The authors therefore decided to proceed using small microcosms. While not representing a true model of the soil, these microcosms did allow for superior control of parameters such as temperature and the use of a greater number of replicates. The results of the soil column experiments showed clearly that the use of a protein hydrolysate solution, supplemented with bacteria and delivered as a foam (i.e., a bioactive foam), led to enhanced n-hexadecane degradation when compared to controls without foam. This was true with either air or oxygen in the formulation. Statistical analysis showed that the bioactive foam had a significant effect on n-hexadecane removal at the 99% confidence level when compared to both the untreated control and the nonfoamed protein hydrolysate with (or without) microbes in the formulation. However, no significant difference (at the 90% confidence level) in n-hexadecane degradation was observed with bioactive foam generated with oxygen when compared to bioactive foam generated with air. This was unexpected, as in all repeats of the experiment the residual level of n-hexadecane was less in experiments using oxygen. The lack of a significant difference was probably due to the relatively short period over which the experiment was run. To confirm this, a further experiment was carried out over an extended period of time. The results of the 15-d time course experiment indicated that oxygenated foam significantly enhanced the biodegradation of n-hexadecane when compared to aerated foam.
Since the oxidation of n-alkanes by bacteria is an oxygendependent reaction, catalyzed by monoxygenases and dioxygenases, it was hypothesized that the use of oxygenated bioactive foams would further enhance biodegradation rates by maintaining oxygen availability and transfer at the contaminated site. Dissolved oxygen levels in the liquid draining from aerated and oxygenated foams were found to be 5.3 and 19.9 mg L-1, respectively, supporting the hypothesis that biodegradation was enhanced by improved oxygen transfer. A limited amount of degradation occurred when the protein hydrolysate (nonfoamed) was added as a solution. This is thought to be due to the effects of relatively rapid addition of the solution. This could have led to two problems: (i) the nutrient content of the protein hydrolysate was not available for the hydrocarbon degrading organisms due to its rapid migration through the soil column, and (ii) soil compaction caused by draining of the liquid through the column may have caused a reduction in oxygen transfer. Some removal of n-hexadecane (although not statistically significant at the 90% confidence level) was seen with the addition of sterile foam formulations. This was found to be due to the growth of soil organisms that were not killed by the autoclaving process. The results of the MPN counts confirmed this, showing a substantial number of n-hexadecane-degrading bacteria present in columns to which only apparently sterile additions had been made. Further evidence for this was obtained when n-hexadecane-degrading, Grampositive, spore-forming bacteria were isolated from autoclaved soil. Several studies (28) have indicated that it is very difficult to achieve sterilization of soil without substantial damage to its structure. Methods using metabolic poisons such as cyanide or mercuric chloride were not appropriate for these experiments, as subsequent additions of microbes were to be carried out. Despite problems with sterilizing soil, the principal aim of the experiments was achieved, showing that a foamed protein hydrolysate can enhance the degradative ability of hydrocarbon-degrading bacteria. A very valuable property of aqueous foams is their ability to immobilize (contain or trap) all three states of matter: gases, liquids, and solids. This is possible because the aqueous component of the foam exists as thin sheets or lamellae, compressed between adjacent bubbles of gas, which provide a dynamic trapping surface. Oil spills could be blanketed VOL. 34, NO. 3, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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using a long-persistence, slow-draining aqueous-based foam mixed with microbes (and/or enzymes), nutrients, and oxygen. This would help to keep the microbes in contact with the oil as well as providing rate-limiting nutrients and oxygen. The foam could also act to protect organisms from harsh environmental conditions such as extremes of temperature and to provide a treatment technique in situations where aqueous bioremediation systems would be either wasteful or difficult to apply, e.g., on steep slopes or surfaces with numerous hollows or crevices. The treatment of spills of oil on (or close to) the surface will probably be the most amenable to treatment with bioactive foam. Treatment of oil contamination of the vadose zone is also a possibility. If the bioactive foam investigated here was to be injected into soil under pressure, it may be possible to force both oxygen and nutrients into the soil. The lamella of the individual foam bubbles could act as traps pushing the oxygen and nutrients through the soil (29). The technology described in this paper could have applications in the treatment of hydrocarbon-based (and other) pollutants in a range of scenarios. It is anticipated that bioactive foam will now be tested in the field at larger scale and appraised for use in environmental bioremediation.
Acknowledgments The authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council that funded this project under the Realizing Our Potential Scheme (Grant 87/SYS04727, Mechanisms for enhanced oil biodegradation in a liquid foam-microbe system), and an earmarked studentship for Mark Ripley. We also thank Angus Fire Armour Ltd., U.K., for supplying protein hydrolysate; Yorkshire Water plc, Knostrop Sewage Treatment Works, Leeds, U. K., and Linsey Oil Refinery, Immingham, U.K., for environmental samples; Bob Palmer, Cranfield University, for identifying soil types; Dr Brendan Keely, Department of Chemistry, University of York for GLC facilities; and Nutrasweet Kelco for supplying xanthan gum and sodium alginate samples.
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Received for review May 17, 1999. Revised manuscript received October 12, 1999. Accepted November 5, 1999. ES9905593
