Environ. Sci. Technol. 2001, 35, 76-83
Cerro Negro Bitumen Degradation by a Consortium of Marine Benthic Microorganisms T H O M A S L . P O T T E R * ,† A N D BRIAN DUVAL‡ USDA-ARS, Southeast Watershed Research Laboratory, Tifton, Georgia 31793, and Association for the Environmental Health of Soils, Amherst, Massachusetts 01004
Cerro Negro bitumen, separated from an Orimulsion sample, was incubated for up to 120 days with sediments collected at a petroleum-impacted site in Tampa Bay, Florida. Biodegradation conditions were optimized by increasing bitumen surface area, continuous agitation on a shaker apparatus, use of a complete growth medium, and maintenance at 37 °C. Aerobic degradation conditions were promoted by maintaining sediment contact with the laboratory atmosphere. Bitumen recovered in solvent extracts when compared to autoclaved controls decreased by up to 40% during the first 56 days. There was no detectable change after this. Molasses addition and use of a culture enriched from the sediments did not change the extent or rate of decrease in bitumen recovery. Chemical fractionation of bitumen control and degraded bitumen showed that aromatic and aliphatic fractions were depleted by ≈50%. Accumulation of polars was observed; however, the apparent increase was relatively small when compared to the mass loss of the other fractions. Selected biomarker ratios were not affected by incubation indicating their utility for fingerprinting the source bitumen in environmental samples. PAH distribution in the aromatic fraction favored the higher alkyl-homologues with the relative degree of alkylation increasing as the mass of bitumen recovered decreased with degradation. The study showed that up to 40% of the bitumen was bioaccessible and that bioremediation may be a treatment option for sediments contaminated with bitumen by an Orimulsion spill.
Introduction The Orinoco heavy oil belt in Eastern Venezuela contains a vast petroleum reserve. Estimates of hydrocarbon resources in the region range as high as 1.2 trillion barrels (1). More than 250 billion barrels are considered recoverable (2). Recently published estimates indicate that this represents nearly 9% of the world’s recoverable petroleum resources (3). Existence of the extensive petroleum deposits in the region was recognized in the early 1920s when the area was explored (2). However, there has been little commercial exploitation or development. The reservoir is comprised almost entirely of extra-heavy crude oil and bitumen with API gravities in the 8° to 10° range. Other properties include relatively high * Corresponding author phone: (912)386-7073; fax: (912)386-7294; e-mail:
[email protected]. † USDA-ARS. ‡Association for the Environmental Health of Soils. 76
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S and V content. Despite these limitations, there is an international consensus that economic potential of these resources to Venezuela and the region is significant (1). Petroleo De Venezuela SA (PDVSA), the state-run oil company of Venezuela, has focused considerable effort on quantifying the Orinoco reserves and developing commercial products. In the late 1980s the company introduced Orimulsion. The product is an emulsion containing 70% bitumen and 30% water stabilized with nonionic surfactant. The principal source of the bitumen is the Cerro Negro field. Orimulsion can be transported via pipelines and tankers. In addition the water does not interfere with bitumen combustion, thus Orimulsion can be used directly in heavyoil fired power generating stations without substantial infrastructure changes (4, 5). A price pegged to coal and offers of long-term contracts has made the product attractive to the electric power industry (1, 2). PDVSA through its subsidiary BITOR has marketed Orimulsion throughout the world. There has been some commercial success. Power plants in Canada, Lithuania, Italy, Barbados, Japan, and Denmark now burn the fuel (4, 5). Nevertheless acceptance has been slow. An application to burn Orimulsion at a 1600 MW power plant in Florida was denied on two occasions and an unfavorable regulatory environment in the United Kingdom (UK) caused an application to burn the fuel at a power plant in Wales to be withdrawn (5). A major concern expressed in public hearings in Florida and the UK was the potential impact of a marine spill in the event of a tanker or pipeline accident (5). In the original Orimulsion formulation the surfactant was a polyethoxylated nonylphenol (6, 7). Surfactants of this type have received widespread international attention because degradation products, which may form under certain environmental conditions, have been shown to behave as weak estrogen mimics (8). Their presence in sewage treatment plant effluents has been associated with estrogenic responses in fish (9). Cause and effect relationships do not appear to have been definitively demonstrated; nevertheless there have been widespread calls to ban these types of surfactants in Europe. BITOR responded by offering a reformulated product containing an alternate surfactant (5). In the case of bitumen, there was the perception that it would persist in the environment in the event of spill and that relatively low-cost options such as bioremediation were not suited for treatment of bitumen contaminated sediments. The bitumen’s resistance to biodegradation was inferred from investigations of its chemical composition. A detailed study investigation of Cerro Negro bitumen chemistry was conducted in the 1980s (10). More recently Wang and Fingas (6) characterized the hydrocarbon fraction of bitumen separated from several Orimulsion samples. They observed that the material had high asphaltenes and resin content (>35%) and that the saturates fraction was comprised almost entirely of GC-unresolvable aliphatics. Straight-chain alkanes and other readily degradable substrates, which often dominate the saturates fractions in crude oil, were not detected. They also reported that concentrations of GCresolvable alkylated PAHs were much lower than levels typically found in crude oils and residual fuels. Those PAHs detectable in the bitumen tended toward higher degrees of alkylation. This lead to the conclusion that the bitumen’s composition is similar to residues of highly weathered crude oils and refined petroleum products found in marine and freshwater ecosystems 20 to 30 years after spills (6). Given this it seems likely that the bitumen would be slow to degrade in these 10.1021/es001296b CCC: $20.00
2001 American Chemical Society Published on Web 11/14/2000
environments and that bioremediation of contaminated sediment would be of limited value. However, there are numerous published studies, which have reported that bitumen is bioaccessible and that biodegradation takes place under a wide variety of environmental conditions (11-19). Wyndom and Costerton (11) reported that aliphatic and aromatic fractions in a sample of Athabasca bitumen were degraded by a microbial consortium isolated from surfaces of bitumen deposits in freshwater sediments. Less than 10% of the original mass of both fractions was recovered after 120 days. Incubations were at 16 °C with a complete nutrient medium. Gilbert and Higgens (12) also studied the microbial degradation of Athabasca bitumen. They coated it on filters and placed them in natural seawater. After 40 days at 5 °C, the mass loss of the bitumen was approximately 15%. Comparison of results to a sterile control indicated that microbial metabolism accounted for >50% of the loss. AitLangomazino et al. (17) incubated blown Mexphalte R 90/40 bitumen in batch culture with pure and mixed cultures of yeast and bacteria. After 100 days at 30 °C in the presence of a complete culture medium, 4.4 to 10.8% of the bitumen mass was accounted for by collection of evolved CO2. These investigators also compared the rate and extent of degradation of the blown and distilled Mexphalte samples. The later was enriched with what was reported to be the recalcitrant fraction of the bitumen. Carbon dioxide generation rates indicated that the distilled asphalt sample degradation rate was less than half the blown sample rate. This work emphasized the potential role of chemical composition in controlling bitumen biodegradation. Lapham et al. (19) studied biodegradation of Cerro Negro bitumen separated from an Orimulsion sample. They found that slurries of marine sediments and seawater spiked with bitumen and maintained under aerobic conditions at 25 °C to 30 °C had higher respiration rates than controls. In addition, the stable carbon isotopic composition of the evolved carbon dioxide closely matched the bitumen isotopic signature. Their work showed that the consortium of organisms in the sediment samples had the ability to metabolize bitumen. Nevertheless, the estimated mass of bitumen degraded was only 1 to 3% over 21 to 60 days. It is unknown whether the limited extent of degradation was due to recalcitrance of the bitumen or the conditions of the incubation, e.g. nutrient limitation. The study described in this report was designed to evaluate this question. It was considered a necessary first-step in assessing the potential for bioremediation of sediments, which may be contaminated by bitumen in the event of an Orimulsion spill. Bitumen samples separated from Orimulsion were incubated for 120 days with heavy fuel oil contaminated intertidal sediments collected in Tampa Bay, Florida. Conditions for biodegradation were promoted by maintenance of an optimum growth temperature (37 °C), coating bitumen on 400-mesh glass beads to increase surface area, continuous agitation on a shaker apparatus, use of a complete growth medium, and a preadapted microbial consortium obtained from a sediment enrichment. Contact of the atmosphere in incubation flasks with the laboratory atmosphere likely maintained aerobic degradation conditions. Recovery of bitumen in solvent extracts and changes in chemical composition were compared to autoclaved controls during the course of the incubations.
Materials and Methods Source of Bitumen. A 5-gallon Orimulsion sample was obtained from a commercial tanker shipment in Dalhousie, NB, Canada by Saybolt, Inc. It was overnight shipped to our laboratory where it was stored under nitrogen in the dark at 20 to 25 °C. Bitumen was recovered from a 500-g subsample by freezing the sample overnight at -20 °C followed by
thawing at room temperature. The water and free-flowing light oil were decanted leaving a mass of highly viscous bitumen. It was repeatedly rinsed with distilled deionized water until the rinse-water was clear. The bitumen was then air-dried and stored at -10 °C. Bitumen Preparation for Incubations. Five grams of bitumen was dissolved in 90 mL of an acetone, hexane, and cyclohexane mixture (4:4:1 v/v/v) and blended with 100-g of Filter Aid 400 (Fisher Scientific) glass beads. The solvents were Optima grade (Fisher Scientific). The composition of the solvent blend was determined by experiments, which showed that it readily dissolved the bitumen. The bitumencoated beads were air-dried for 3 days. Three 10-g subsamples were collected and sequentially extracted by shaking with three 50-mL aliquots of the trisolvent mixture. The solvent was recovered and evaporated to dryness using a rotary thin film evaporator. The mass of bitumen was determined gravimetrically. It was found to be 0.05 g g-1 beads with a coefficient of variation equal to 5%. Source of Sediment. A sediment sample was collected in October 1997 in the intertidal zone of Eleanor Island. It is one of the mangrove islands located in the John’s Pass region of Tampa Bay, Florida, which was oiled by #6 fuel oil after the Barge Bouchard spill in 1993. The sediment was collected to a depth of 15-20 cm at the site designated “4A” by Wetzel (20) using a stainless steel hand trowel. Prior to use, the trowel was washed with a 2-propanol/water mixture (70:30 v/v) and air-dried. The sediment was transferred directly to 1-qt autoclaved glass jars. They were sealed with foil-lined caps and transported to the laboratory in coolers where they were maintained in the dark at 5 °C. The sediment was sequentially extracted with the solvent mixture described above. The weight of the dried residue was 0.1% (wet weight). Analyses described below did not have the potential to separate this background from the bitumen added to the sediments. Sediment Microbial Enrichment. Fifty gram sediment subsamples were transferred to 160-mL serum bottles containing 50 mL of medium. Composition of the medium was based on the basal saltwater medium described by Widdel and Bak (21). Selenite-tungstate, thiamin, vitamin B12, and sodium sulfide solutions, which they recommended for use in enrichments of sulfate-reducing bacteria, were omitted. The bottles were sealed with Teflon-faced silicon rubber septa and agitated at 200 rpm on a reciprocal shaker in a constant temperature room maintained at 37 °C. After 10 days, the serum bottles were removed from the shaker and allowed to settle for 2 h. Twenty milliliters of the supernate were transferred by pipet into 50-mL of fresh medium and fortified with 0.5-g of bitumen coated on glass beads. The bottles were sealed and shaken for an additional two weeks. The culture was then plated on tryptic soy agar (TSA) to enumerate bacteria (22). Approximately 108 cells mL-1 were found. Colonies were picked and examined by light microscopy, gram staining, and catalase testing. The most common bacterial morphotype was motile, 2-3 µm curved rods, aerobic by reaction to catalase, and gram negative. Colonies had white and yellow-green pigmentation. Sediment Incubations. Twenty milliliters of the liquid medium described above was dispensed into 160-mL serum bottles and autoclaved prior to addition of 50-g of sediment. Bitumen coated glass beads (10 g) were then added to the sediment-media slurry. Incubations were made in this manner with the nonamended sediment, sediment plus enrichment culture, sediment plus enrichment culture and 0.5 g molasses, sediment plus 0.5 g molasses. Serum bottles were closed with Teflon-faced rubber septa, wrapped in foil, and incubated on a reciprocal shaker at 37 °C for up to 120 days. Controls were prepared and maintained in the same manner except they were autoclaved after addition of sediment, medium, and bitumen. Given the very low volatile VOL. 35, NO. 1, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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content of the bitumen (10), autoclaving was assumed to have a negligible effect on the chemical composition or physical form of the bitumen. After cooling, bottles were sealed with autoclaved septa. To maintain contact with the laboratory atmosphere during the incubations, a 23-guage stainless steel needle (Luer-lock design) was inserted through the septum of each bottle. The needle’s hub was packed with autoclaved glass wool. No H2S odor was noted when serum bottles were opened for analysis. If conditions had become anaerobic, H2S production was likely. Sulfate concentration in the medium was approximately 3 mM, the incubation period was long (120 days), and the presence of sulfatereducing bacteria in the sediments was likely. They were collected in shallow intertidal area with little wave energy. Gravimetric Bitumen Analysis. At intervals of 15 to 30 days the sediment in 3 serum bottles from each treatment and one control were solvent extracted, and the bitumen residue was weighed. The extraction involved blending the sediment slurry with 50 to 70 g of 10-60 mesh anhydrous sodium sulfate (Fisher Scientific) followed by sequential (five 40-mL aliquots) extraction with acetone/hexane/cyclohexane (4:4:1, v/v/v). The solvent was transferred to a round-bottom flask and taken to dryness by rotary evaporation. Three replicate 50-g sediment subsamples fortified with bitumen at 10 mg g-1 gave 90-95% recovery with coefficient of variation ) 10%. Bitumen Fractionation. Dried bitumen residue was transferred with three 20-mL aliquots of pentane onto Whatman #2 filter paper in a Buchner funnel. Once on the filter the insoluble solids were washed with an additional 40 mL of pentane. The filter was maintained under vacuum until dry in appearance. The pentane, which was collected in a glass filter flask, was concentrated under N2 to 4 mL and added to the top of an alumina column. Columns were prepared by packing 10 g of neutral alumina (Brockman Activity 1; 60 to 230 mesh) into glass chromatographic columns (8-mm i.d.). They were topped with anhydrous sodium sulfate (1 cm) and pre-eluted with 100 mL of hexane. Separation of the aliphatic, aromatic, and polar compounds was performed by sequential elution with 100-mL volumes of hexane, methylene chloride, and methanol (23). The alumina and Optima grade solvents were obtained from Fisher Scientific (Medford, MA). The three fractions were concentrated by rotary evaporation and taken to dryness under nitrogen gas, and the residue weighed. Each fraction was then redissolved in 4 mL of its separation solvent and stored at -10 °C. GC/FID Analysis. Fractions were analyzed using a HewlettPackard model 5890 Series II gas chromatograph equipped with a flame ionization detector (FID). The oven was fitted with a 30 m × 0.25 mm (i.d.) DB-5 fused silica capillary column (J&W Scientific, Folsom, CA). Ultrahigh purity helium was used as the carrier gas at a head pressure of 100 kPa, with nitrogen as the detector makeup at 30 mL min-1. The oven temperature program was as follows: initial temperature 50 °C (hold 1 min); increase at 20 °C min-1 to 280 °C (hold 27.5 min). Injector and detector temperatures were 280 °C. Automated injections were in the splitless mode. Prior to analysis, each sample was fortified with 100 µg of o-terphenyl (Chem-Serv, West Chester, PA). It served as a retention time marker. GC/MS Analysis. Aliphatic and aromatic fractions isolated from the control and inoculated sediment after 120 days of incubation were analyzed by GC/MS using a Hewlett-Packard 5989 GC/MS system. The GC-column and conditions were identical to those described for the GC-FID analyses with the exception of the oven temperature program. In this case, the initial temperature was 50 °C. It was held for one minute and then increased to 280 °C at 6 °C min-1. The final temperature was held for 21 min. The GC column was directly 78
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FIGURE 1. Cerro Negro bitumen degradation in a laboratory incubation with Tampa Bay sediment inoculated with an enrichment culture. coupled to the ion-source through a heated transfer line maintained at 280 °C. Mass spectral data were obtained in the electron impact mode at 70 eV. The instrument was tuned to meet manufacturer specifications for PFTBA. Prior to analysis, samples were fortified with phenanthrene-d10 (Aldrich, Milwaukee, WI) at 5 µg mL-1. Serial dilutions of a polynuclear aromatic hydrocarbon mixture (QTM PAH mix) purchased from Supelco Inc. (Bellafonte, PA) were used to develop an external calibration curve for the target PAH and their alkyl homologues. Ions used for quantitation were the same as those described by Roques et al. (24). Quantitation was based on relative response to the phenanthrene-d10 base peak (m/z+ ) 188). Biomarker hydrocarbons were identified in the aliphatic fraction by relative retention time and the appearance of the characteristic fragments, m/z+ ) 177, 191, 205, 217, 231, or 253 (6, 25, 26).
Results and Discussion Bitumen recovery expressed as a percent of the total added is shown for sediment inoculated with the enrichment culture and incubated for 120 days in Figure 1. Recovery decreased linearly over the first 56 days. Beyond this, there was no detectable change. The average recovery of the three replicates analyzed on day 56 and day 120 was 61.1% and 61.5%, respectively. Average recoveries in the autoclaved controls ranged from 90 to 98%. Comparison of bitumen recovery in controls to the inoculated sediment indicated that up to 40% of the bitumen was degraded by the indigenous sediment microbial consortium and or the enrichment culture. Hereafter in this discussion material not recovered will be referred to as degraded. As indicated in the description of the sediments, they contained an extractable “background” amounting to 0.1% of the sediment. This included residual hydrocarbons, which were derived from a spill of # 6 fuel oil that had weathered in situ for nearly four years prior to sampling. It is likely that this material was highly resistant to further degradation; however, this is unknown. If 100% of the extractable materials in the sediment were degraded during the incubation, then the bitumen not accounted for in the solvent extracts would have represented 30% of the mass added to the incubation flasks. It follows that between 30 and 40% of the bitumen added to the incubation bottles was not recovered and presumably was degraded. Degradation in this context includes both bitumen mineralization and the possible accumulation of metabolic products, which were not extractable.
TABLE 1. Cerro Negro Bitumen Degradation Rate in Incubations with Tampa Bay Sedimentsa incubation type sediment sediment and enrichment culture sediment and molasses sediment and molasses plus enrichment culture a
rate (µg g-sediment-1 day-1)
r2
-66.4 -57.9 -64.1 -61.9
0.961 0.941 0.852 0.997
Linear regression parameters for the first 56 days of the incubations.
The extent of bitumen degradation, 30 to 40%, was relatively high when compared to results reported by Latham et al. (19). They found that only 1 to 3% of a Cerro Negro bitumen sample was mineralized in seawater-sediment slurries with uncontaminated subtidal marine sediment (J. Chanton, personal communication). Several factors likely explain the greater extent of degradation reported here. This included use of a complete nutrient medium and sediments, which had been contaminated by heavy fuel oil. Addition of the nutrient medium to the sediments likely stimulated the microbial consortium, which had become adapted to fuel oil degradation. Other contributing factors included a higher incubation temperature, 37 °C versus 25 to 30 °C and coating the bitumen on 400 mesh glass beads prior to adding it to the incubation bottles. Use of the beads increased the surface area of the bitumen and they dispersed the sediment during shaking. Enhanced degradation with increased surface area appears to be a general trend for substrates with a high degree of resistance to degradation and low water solubility. For example, Fava (27) reported that coating the polychlorinated biphenyl mixture, Aroclor 1221, on glass beads prior to its incubation in suspension culture with a Psuedomonas sps. increased the rate of aerobic dechlorination. No lag-phase was observed in the degradation experiment described in Figure 1 or in any of the other incubations. This may be due to the length of the sampling interval. Sampling on a shorter time scale at the beginning of the incubation may have allowed identification of a lag-phase. Linear regression parameters for the decrease in bitumen recovery with time are shown for this incubation in Table 1. Over the 56-day period, bitumen loss in the inoculated sediment was approximately 58 µg g-sediment-1 day-1. Similar results were obtained with other incubations. The loss, accounting for up to 40% of the added bitumen, included both mineralization and conversion of neutral compounds to nonextractable metabolites. Given the nature of the extraction conditions, it is expected that the total mass of nonextractable residues was small. The relative distribution of the aliphatic, aromatic, and polar fractions of the bitumen during the incubation is shown in Figure 2 with rates of degradation (or accumulation) over the first 56 days of the incubation presented in Table 2. The aliphatics and aromatics exhibited decreases in the mass added to incubations from 44 to 48% with decay rates ranging from 17.1 to 34.4 µg g-sediment-1 day-1. With the polar fraction, there was an accumulation of approximately 50% of the mass added. The accumulation rate was 5.1 µg g-sediment-1 day-1. This included both bitumen metabolites and “natural” products produced by sediment microorganisms. Overall the accumulation of polars was relatively minor process. Their mass represented 1.0 implied the opposite. Based on this classification, the stearanes (217), demethylhopanes (177), methylhopanes (205), and tricyclic terpanes (191) were the most labile biomarker families. RCs ranged from 0.6 to 0.9. The tetracyclic-terpanes (191) and hopanes (191) were intermediate in stability (RC ) 1.0). C-ring-monoaromatics and triaromatics were most resistant with RCs equal to 1.2 and 2.0, respectively. Munoz et al. (28) observed a similar trend in biomarker transformation in crude oil contaminated mangrove soil. They reported that stearanes degraded most rapidly, that triaromatics were “refractory”, and that there was no degradation of C-ring-monoaromatics. The apparent stability of the aromatic biomarkers is an indication of their potential for fingerprinting highly weathered petroleum residues and evaluating the extent of biodegradation of environmental samples. In degraded and control bitumen samples, a close match in peak profiles of the two families of compounds was observed. This is shown in Figure 5 for peaks in the m/z+ ) 253 series (C-ring monoaromatics). Given their resistance to degradation, it appears that one or more of these compounds could serve as a conserved marker for biodegradation studies. The data indicated that they were more highly conserved than the hopanes. A prominent member of the hopane family, 17R(H),21β(H)-hopane, was proposed as a conserved internal marker for estimating the biodegradation of crude oil (29) and has been used for this purpose in several investigations (29-31). To evaluate the quantitative contribution of the biomarkers, the concentration of the cyclic-terpanes, the most
FIGURE 5. “C-ring-monoaromatics” distribution in control and degraded Cerro Negro bitumen residues. abundant of the biomarker groups, was estimated. This was done using full-scan MS data for the seven most intense peaks in the m/z+ ) 191 ion current chromatogram. For each spectrum, the fractional contribution of m/z+ ) 191 to the total ion current in a background subtracted apex scan was computed. This yielded an average of 8.4% with the coefficient of variation ) 24%. Dividing the summed m/z+ ) 191 ion current/TIC ratio by this value yielded a value for the percent cyclic-terpanes composition. It was 4.2% in the control and 5.0% in the degraded residue. These calculations show that although these compounds have diagnostic significance their quantitative contribution to the aliphatic fraction is low. This was also the case for the other biomarker classes whose summed ion current was 2 to 10 times less than the cyclic-terpanes. As indicated in Table 2, the rate of degradation of the aromatic fraction was nearly twice the aliphatic fraction. However the overall extent of degradation of the two fractions was similar. The total decrease in the aliphatics was 44% and the aromatics 48%. Wyndom and Costerton (11) in their study of Athabasca bitumen degradation reported similar results, i.e., approximately equal degradation of both fractions. AitLangomazino et al. (15) described reduction of 40% for the aliphatic and 25% for the aromatic fractions of a Mexphalte bitumen sample in batch culture. These data indicate that the extent of degradation of aromatic and aliphatic fractions in bitumen varies with the source of the bitumen. Similar behavior has been reported for crude oil (34). Compounds specifically identified in analyses of the aromatic fractions included alkylated naphthalenes, flourenes, phenanthrenes, dibenzothiphenes, pyrenes, and chrysenes. Figure 6 shows that a general feature of their distribution was higher concentrations of the higher alkyl homologues in each PAH series. Wang and Fingas (6) observed the same pattern in a sample of unweathered Orimulsion bitumen and noted that the pattern is characteristic of highly weathered crude oils. In the unweathered bitumen, it is probably the result of paleo-weathering (35). Comparison of the control and degraded bitumen PAH concentrations profiles revealed depletion of C0,C1, C2, and C3 homologues in the naphthalene series and the C0 and C1 homologues in the phenanthrene, fluorene and dibenzothiophene series (Figure 6). The trend in susceptibility of each PAH homologous series, specifically detected in the bitumen, was evaluated by normalizing summed concentration data of each series on the sum of the alkyl-chrysenes concentration (Table 5). This
FIGURE 6. “Alkylated PAH” distribution in control and degraded Cerro Negro bitumen.
TABLE 5. PAH Homolog Concentration in the Control and Degraded Bitumen Residue Following a 120-Day Incubation sum concn (µg g-1)
ratio to sum of alkyl-chrysenes
control degraded control degraded C0 to C4-naphthalenes 342 C0 to C3-phenanthrenes 531 C0 to C3-dibenzothiophenes 742 C0 to C3-fluorenes 335 C0 to C3-pyrenes 197 C0 to C3-chrysenes 90.9 total-PAH 2240
235 542 802 293 461 222 2550
3.8 5.8 8.2 3.2 2.2 1.0 25
1.1 2.4 3.6 1.5 2.1 1.0 11
PAH family is reportedly more recalcitrant than most of the others and has been used in the same manner in studies of PAH degradation in soil and sediment (26). The alkyl-pyrenes ratio in the control and degraded bitumen was nearly equal indicating that the stability of this PAH series was similar to the alkyl-chrysenes. All other ratios decreased by 2 to 3 times when the control and degraded samples were compared. The decrease reflects increased susceptibility to biodegradation of these PAH. Results were in general agreement with PAH data reported by Wang and Fingas (26) for residues of heavy fuel oil of which had weathered in intertidal sediments for 20 to 30 years. Table 5 data also show that during the incubations the overall decrease in these PAH was approximately 60%. This was indicated by comparison of total-PAH/alkyl-chrysenes ratios. However, it should be noted the PAH detected in the GC/MS analyses represented only 0.2 to 0.3% of the aromatic fractions. Structural assignments for the bulk of the aromatics (>99%) which were degraded and which remained in the degraded residue were beyond the limits of the GC/MS analyses. In sum, the composition of the bulk of the hydrocarbons which was degraded or which accumulated in the degraded residues remains uncertain. This includes both the aliphatic and aromatics fractions. They were likely similar to the UCM described in other petroleum products, in particular highly weathered environmental residues of spilled oil. Killops and Al-Juboori (32) and Gough and Rowland (33) used a variety of techniques to characterize the UCM in highly weathered crude oil. They reported that cyclic and aromatic structures with long branched alkyl side chains were prominent. This included a wide-variety of “T-branched” alkane structures and aromatics with long (up to C17) alkyl side chains. Results obtained with other sediment-bitumen incubations are shown in Figure 7. Linear regression parameters VOL. 35, NO. 1, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Conditions had been optimized to promote biodegradation. The data have indicated that consortium of bitumendegrading organisms had developed on the sediments and that bioremediation has potential as a treatment option for sediments contaminated by an Orimulsion spill.
Acknowledgments
FIGURE 7. Cerro Negro bitumen degradation in laboratory incubations with Tampa Bay sediment.
Financial support was provided by the Florida Power & Lights Company (FPL) through a grant made by Association of the Environmental Health of Soils (AEHS) to the University of Massachusetts. Ted van Vleet and Dana Wetzel, Department of Marine Science, University of South Florida made collection of sediment samples possible. Paul Kostecki, School of Public Health, University of Massachusetts, Jeff Chanton and Lita Proctor, Department of Oceanography, Florida State University, and Jerry Kirk and John Gnecco, Florida Power & Lights Company, provided helpful discussions during the course of the study. The work was performed in laboratory facilities provided by the University of Massachusetts Department of Food Science.
Literature Cited
FIGURE 8. Cerro Negro bitumen degradation by an enrichment culture isolated from Tampa Bay sediments. for the rate of decrease in bitumen recovery are presented in Table 2. Results were remarkably similar to those described for the sediment plus enrichment culture (Figure 1; Table 1). There was up to 40% reduction in bitumen recovery after 56 days with little decrease observed thereafter. The molasses was added with the expectation that it may stimulate utilization of the bitumen hydrocarbons. When it was added as a supplemental carbon source to incubations of crude oil in seawater, respiration was stimulated along with n-alkane metabolism (36). No effect was seen in the bitumen incubations. This may due to the abundance of cometabolizable carbon in the sediment and the lack of n-alkanes in the bitumen. One aspect of the data was that in the sediment only incubation, the highest rate of decrease in bitumen recovery was observed (Table 2). It was significantly (P ) 0.05) greater than when the enrichment culture was added in an incubation (Figure 1; Table 1). A question about the viability of the enrichment culture arose from this result. The culture was tested in a 38-day incubation in which all factors were held constant except that no sediment was added to the serum bottles. Results are summarized in Figure 8. Culture viability was demonstrated by a rapid and linear decrease in the bitumen percent recovery. It was 5.2 mg day-1. In the sediment-based incubations, the decrease was 2.9 to 3.3 mg day-1. The lower rate of degradation observed in the later incubation may be attributed to “protective” action of the sediments. They likely adsorbed a portion of the bitumen thereby decreasing its bioavailability. Taken together, results of this study show that a substantial portion of the bitumen, 30 to 40%, was metabolically available to a consortium of marine benthic microorganisms. This was based on direct comparison of recoveries in solvent extracts of autoclaved controls and sediment incubations. 82
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Received for review May 24, 2000. Revised manuscript received September 25, 2000. Accepted September 29, 2000. ES001296B
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