Aerobic Biodegradation of Hopanes and Other Biomarkers by Crude

Oct 5, 2002 - The degradation of petroleum biomarkers was examined using mixed cultures of microorganisms enriched from surface soils at four differen...
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Environ. Sci. Technol. 2002, 36, 4585-4592

Aerobic Biodegradation of Hopanes and Other Biomarkers by Crude Oil-Degrading Enrichment Cultures R O B E R T O F R O N T E R A - S U A U , †,| F . D A N I E L B O S T , †,# THOMAS J. MCDONALD,‡ AND P A M E L A J . M O R R I S * ,†,§ Department of Microbiology and Immunology and Marine Biomedicine and Environmental Sciences, 221 Fort Johnson Road, Medical University of South Carolina, Charleston, South Carolina 29412, and B&B Laboratories, 1902 Pinon Drive, College Station, Texas 77845

The degradation of petroleum biomarkers was examined using mixed cultures of microorganisms enriched from surface soils at four different hydrocarbon-contaminated sites. These cultures degraded C30 17R(H),21β(H)-hopane and the C31-C34 extended hopanes in Bonny Light crude oil after 21 days of incubation at 30 °C. The C35 extended hopanes were conserved, and no 25-norhopanes were detected during the incubation. Denaturing gradient gel electrophoresis (DGGE) analysis of the enrichment cultures demonstrated distinct microbial community profiles. Additional studies with the LC culture demonstrated a consistent biomarker degradation pattern after growth on three crude oils: a Nigerian Bonny Light crude, a Venezuelan crude oil, and Alaskan North Slope 521. The onset of biomarker degradation was observed between days 14 and 21 but only at 30 °C and at oil concentrations below 6 mg/mL. The biomarker profiles following degradation by these enrichment cultures are similar to numerous field observations and may represent the dominant biodegradation pattern found in many hydrocarbon-contaminated aerobic surface environments.

Introduction While microorganisms that degrade crude oil are ubiquitous in the environment, monitoring crude oil degradation under field conditions remains a challenge. A common approach is the use of biomarker compounds as internal standards to estimate crude oil biodegradation. Microbial transformation of crude oils in reservoirs, during storage, or in surface environments results in the alteration of many biomarker parameters used by petroleum geochemists (1, 2). The study of these alterations has led geochemists to develop ranking systems that measure the extent of crude oil biodegradation based on the transformation of biomarkers (3-5). * Corresponding author phone: (843)762-5533; fax: (843)7625535; e-mail: [email protected]. † Department of Microbiology & Immunology, Medical University of South Carolina. ‡ B&B Laboratories. § Marine Biomedicine & Environmental Sciences, Medical University of South Carolina. | Present address: Biology Department, SPIRE Program, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599. # Present address: Biotechnology Center for Agriculture and the Environment, Rutgers University, New Brunswick, NJ 08901-8520. 10.1021/es025894x CCC: $22.00 Published on Web 10/05/2002

 2002 American Chemical Society

To monitor crude oil degradation in the field, ratios of n-alkanes to pristane and phytane have been used (6). However, pristane and phytane can be degraded, limiting their use to the early stages of biodegradation (7-9). Biomarkers more resistant to degradation (i.e., steranes, triterpanes) can also be used to assess microbial transformation of crude oils (1). Hopanes, a series of pentacyclic triterpanes derived from bacterial lipid precursors, appear as ubiquitous and abundant components of the aliphatic fraction of crude oils (10, 11). This series includes C30 17R(H),21β(H)-hopane and the C31-C35 homohopanes that occur as 22S and 22R epimers based on the asymmetric center at C-22 (12). C30 17R(H),21β(H)-Hopane has been successfully used as a conserved biomarker to assess crude oil biodegradation in the field (13, 14). However, C30 17R(H),21β(H)hopane degradation has been observed during landfarm bioremediation of a soil contaminated with refinery byproducts (15). Hopane degradation under laboratory conditions has often been unsuccessful, possibly due to short incubation times, use of pure cultures, the absence of hopane-degrading bacteria, or inadequate growth conditions (4, 16-18). However, degradation of C30 17R(H),21(H)β-hopane and the homohopane series with preferential degradation of the higher molecular weight homologues (C35 > C34> C33> C32> C31> C30) has been reported (19, 20). Other studies, using crude oil and no other added carbon source, have demonstrated degradation of C30 17R(H),21β(Η)-hopane with preferential degradation of the lower molecular weight homohopanes (21, 22). Laboratory studies of biomarker degradation are hindered by the lack of radiolabeled substrates. However, Tritz and colleagues (23) synthesized tritium-labeled hopane and observed hop-17(21)-ene as the only product of hopane oxidation by cholesterol-induced Arthrobacter simplex ATCC 13260. Studies of microbial cultures capable of biomarker degradation will further the understanding of the environmental fate of hopanes and other biomarkers as well as give insight into the interpretation and use of biomarker-based geochemical and biodegradation parameters. In this study, the specificity and pattern of biomarker degradation in different crude oil-degrading microbial enrichment cultures was determined. Additional studies focused on one of these cultures, the LC culture, and the effect of culture parameters (i.e., time, temperature, oil concentration) and oil composition on the pattern of biomarker degradation and microbial community structure.

Experimental Section Enrichment Cultures. Cultures were enriched from five hydrocarbon-impacted soils using Bonny Light crude oil or Alaskan North Slope 521 crude oil (Table 1). The dichloromethane-extractable fraction of each soil was determined after Soxhlet extraction with dichloromethane/acetone (9:1, vol:vol) for 24 h. Enrichment cultures were initiated by adding 1 g of soil and 25 mL of basic salts/trace metal media (BMTM) (24) to a 125 mL Erlenmeyer flask with Teflon-lined screw caps. Each flask was amended with 2 mg/mL crude oil unless otherwise noted, incubated at 30 °C and 200 rpm, and maintained by monthly 4% inoculum transfers. Three monthly transfers occurred prior to any analysis. Experimental Design. The LC culture was used to examine the effect of oil composition on the specificity of biomarker degradation. The LC culture was transferred into flasks containing Bonny Light crude oil (BLC), Alaskan North Slope VOL. 36, NO. 21, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Enrichment Cultures Developed from Hydrocarbon Contaminated and Noncontaminated Soils contamination enrichment culture

location

source

concentrationa

crude oil

Light Crude (LC) James Island (JI) Washington Street (WS) End of Trestle (ET) Trestle Clay (TC)

Fairhope, AL James Island, SC Charleston, SC Charleston, SC Charleston, SC

creosote transformer oil natural gas natural gas natural gas

84.08 ( 12.15 3.23 ( 0.27 1.91 ( 0.07 7.50 ( 0.93 5.00 ( 0.44

Bonny Light crude ANS 521 ANS 521 ANS 521 ANS 521

a

mg of methylene chloride extractable material/g of dry soil.

TABLE 2. Crude Oil Characteristics Bonny Light Crude Alaskan North Slope 521 Venezuelan Oil

API gravitya

saturatesb

aromaticsb

polarsb

asphaltenesb

oleanane

35.3 27.8 11.8

56 48 24

31 37 39

11 13 28

2 2 9

+ +

a American Petroleum Institute Gravity (values are for nonweathered oils). (weight before fractionation) expressed in %.

oil 521 (ANS 521), or Venezuelan crude oil (VC) (Table 2). These cultures were maintained as described above. Subsequent studies examined the effect of time, oil concentration, and incubation temperature on the degradation of BLC by the LC culture. A time course experiment was conducted with BLC and the LC culture to determine the sequence of biomarker degradation and changes in the microbial community structure. Three inoculated and uninoculated (control) flasks were extracted for crude oil residues at days 0, 1, 3, 5, 7, 14, 21, 28, 35, and 64. Three additional flasks were used for microbial community analysis. The impact of crude oil concentration on biomarker degradation was determined by transferring the LC culture into sets of six flasks containing 1, 2, 4, 6, 8, or 10 mg/mL of BLC oil as the sole carbon source. Triplicate uninoculated controls were prepared for each oil concentration. Temperature effects on biomarker degradation by the LC culture were assessed in 50 mL glass test tubes with Teflonlined screw caps containing 9.6 mL of BMTM and Bonny Light crude oil. For each time point, six inoculated tubes (0.4 mL of LC culture inoculum) and three uninoculated controls were incubated at 4 °C, 15 °C, 30 °C, and 37 °C. After 30, 60, and 90 days of incubation, triplicate samples and uninoculated controls were extracted for crude oil analysis. Experiments were terminated upon detection of C30 17R(H),21β(H)hopane degradation or at 90 days. Crude Oil Extraction and Analysis. Crude oil extraction and gas chromatographic analysis of residues were described previously (22, 25). Hopanes were monitored at m/z ) 191, norhopanes at m/z ) 177, and steranes at m/z ) 217. All numerical values of individual compounds for calculation of ratios were obtained from mass chromatogram peak areas or quantitative analysis (22). Microbial Community Analysis. For heterotrophic plate counts, 30 day cultures (0.1 mL) were serially diluted and plated on Luria Broth (LB) agar plates (Difco, Detroit, MI). Morphologically distinct colonies were selected, streaked to purity, and frozen in 50% glycerol at -70 °C. The remaining culture from each flask was centrifuged at 5000 rpm for 5 min at 4 °C. The cell pellet was resuspended in BMTM and transferred into 1.5 mL microcentrifuge tubes for DNA extraction. Total genomic DNA from each culture as well as from morphologically distinct isolates was extracted from cell pellets (26). A 323 bp fragment of the V9 region of the 16S rRNA gene was amplified using a domain Bacteria-specific forward primer and a universal reverse primer (27). PCR4586

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b

(Weight of each fraction separated by column chromatography)/

DGGE analysis of amplified 16S rDNA sequences was performed on a Techne GeneMate thermal cycler (IFC BioExpress, Kaysville, UT) and separated on a DCode Universal Mutation Detection system (Bio-Rad Laboratories, Hercules, CA) using a 40 to 60% urea (7M) and deionized formamide (40%) gradient (28). The gels were stained with SYBR Green I (Molecular Probes, Eugene, OR) and analyzed using a laser fluoroimager (model 595, Molecular Dynamics, Sunnyvale, CA) with ImageQuant software. DGGE bands from the LC culture were excised with a sterile razor blade and eluted overnight into 50 µL of sterile deionized water. The eluted DNA (1 µL) was reamplified by PCR as already described and purified using a Wizard PCR Preps DNA purification kit (Promega, Madison, WI). Purified DNA was sequenced at the Biotechnology Resource Laboratory (Medical University of South Carolina, Charleston, SC) using an ABI 377 DNA Sequencer. Sequences were analyzed using the Sequence Match and Chimera check programs available from the Ribosomal Database Project II (29) and aligned by hand using BioEdit Sequence Alignment Editor (30). A phylogenetic tree based on this alignment was generated using PHYLIP software using maximum distance for the data set. The optimum tree was bootstrapped 100 times to establish maximum support for the phylogenetic branching indicated.

Results and Discussion Biodegradation of Hopanes and Homohopanes. Cultures enriched using hydrocarbon-impacted soils degraded the following biomarker classes: n-alkanes, branched alkanes (pristane and phytane), and the hopanes. GC-MS-SIM analysis (m/z ) 191) of BLC degraded by the LC, JI, WS, TC, and ET cultures showed similar biomarker biodegradation patterns, with depletion of the C30 17R(H), 21β(H)-hopane (relative to the C30 18R-oleanane peak) after 1 month (Figure 1A,D). This increased the oleanane index in all cultures when compared to uninoculated controls (Table 3). Depletion of the C31-C34 homohopanes was also observed, with preferential degradation of the lower molecular weight homologues (C31 > C32 > C33 > C34 > C35) (Figure 1A,D). Homohopane degradation favored the R epimer over the S epimer. In degraded crude oil residues, the homohopane index increased in all five cultures when compared to the uninoculated controls (Table 3). The m/z ) 191 chromatograms (Figure 1A,D) also demonstrated conservation of the C35 homohopane. These observations were further supported by decreased ratios of C31-C34/C35 homohopanes and C31-

FIGURE 1. GC-MS-SIM of the m/z ) 191 ion for (A) BLC, (B) ANS 521 and (C) VC uninoculated controls, and (D) BLC, (E) ANS 521, and (F) VC after growth with the LC culture.

TABLE 3. Changes in Bonny Light Crude Biomarker Ratios after Biodegradation by Enrichment Cultures enrichment cultures

oleanane indexa

homohopane Indexb

C31-C34/C35 homohopanesc

22R/22S d

Ts/Tme

C28TT/C27Df

uninoculated control Light Crude (LC) James Island (JI) Washington Street(WS) End of Trestle (ET) Trestle Clay (TC)

0.88 ( 0.02 5.64 ( 1.21 3.36 ( 0.72 2.60 ( 1.88 3.04 ( 0.24 2.15 ( 0.34

5.79 ( 0.24 27.91 ( 2.17 28.80 ( 3.97 23.18 ( 14.79 24.12 ( 0.41 18.27 ( 0.80

16.29 ( 0.72 2.60 ( 0.27 2.51 ( 0.48 4.42 ( 3.46 3.15 ( 0.71 4.48 ( 0.24

71.55 ( 1.57 53.93 ( 3.53 58.70 ( 0.32 69.43 ( 3.91 66.07 ( 2.62 65.14 ( 5.25

0.67 ( 0.02 1.31 ( 0.09 0.53 ( 0.09 0.58 ( 0.20 0.53 ( 0.04 0.56 ( 0.11

3.04 ( 0.28 1.26 ( 0.11 0.95 ( 0.05 1.71 ( 0.89 0.95 ( 0.35 1.16 ( 0.19

a C b (C c 30 18R-oleanane/C30 17R,21β-hopane. 35 (22R+22S)/C31-C35 homohopanes (22R+22S)) × 100. C31-C34 homohopanes (22R+22S)/C35 homohopanes (22R+22S). d (C31-C35 22R epimers/C31-C35 22S epimers) × 100. e C27 18R(H)-22,29,30-trisnorneohopane (Ts)/C27 17R(H)-22,29,30trisnorhopane (Tm). f C28 tricyclic terpanes (TT) (22R+22S)/C27 13β,17R-diasterane (D) (20S).

C35R/C31-C35S epimers (Table 3). Conservation of C35 homohopanes and degradation of the 22R over the 22S epimers was more pronounced in the LC and JI cultures where degradation was more extensive. The pattern of hopane degradation demonstrated by the LC, JI, WS, ET, and TC cultures is similar to that reported in some heavily biodegraded oils (1, 5) and in severely degraded tar sands from the Pt. Arena formation in Monterey (31). It has been suggested that this pattern of hopane biodegradation results from aerobic events during the history of reservoirs (12), and the same pattern has been reported after examination of waste petroleum sludge from a refinery landfarm (15). However, the data presented here are the first laboratory confirmation that aerobic biodegradation of crude oil can result in this specific pattern of biomarker degradation. Moldowan and colleagues (2) proposed two mechanisms of homohopane degradation. In one proposed mechanism, bacteria attack the homohopane molecule by oxidizing the side chain, thus favoring the higher molecular weight homologues (C35 > C34 > C33 > C32 > C31). This pattern has been observed in reservoir oils (32), in crude oil-contaminated mangrove soil (33), in laboratory studies using only crude oil as the microbial inoculum and carbon source (19), and with pure cultures of Nocardia grown on mineral media supplemented with saturate components of West Rozel oil, yeast extract, and glycerol (34). In the second proposed mechanism, bacteria attack constituents of the cyclic core, resulting in preferential degradation of the lower molecular weight homohopanes. Pure cultures of cholesterol induced Arthrobacter simplex transformed both synthesized tritium-labeled hopane and bacteriohopane, resulting in the formation of C30 hop-17,21-ene (23). Biodegradation of hopanes in reservoir oils has been associated with a proposed C-10 demethylation of the

hopane A/B rings that generates the corresponding 25norhopanes (1, 12, 35, 36). Peters et al. (12) contend that the order of homohopane degradation via C-10 demethylation results from the combined effects of molecular size and conformation, suggesting that as the molecular volumes (and weights) of homohopanes increase, the 22S epimers adopt a more compact scorpion configuration, with the side chain folding over and protecting the C-10 methyl group. This is in contrast to the side chain of the 22R epimers that have a rail shape conformation extending away from the molecule, leaving the C-10 position open to attack. In the present study, 25-norhopanes were not detected when monitored daily for 2 weeks during the period of hopane degradation (data not shown), suggesting an alternative mechanism of hopane degradation that may not involve C-10 demethylation. Concurrent degradation of norhopanes and hopanes by the LC culture has been observed, suggesting a similar mechanism of aerobic degradation for these compounds (22). Biodegradation of hopanes without formation of 25-norhopanes has been reported in naturally biodegraded crude oils (1, 32, 36). However, in these cases depletion of hopanes was preceded by the degradation of the steranes. The steranes and diasteranes (m/z ) 217) remained unaltered after incubation with all enrichment cultures examined (data not shown). Two separate hopane biodegradation pathways have been proposed: (1) demethylation of hopanes to 25norhopanes prior to sterane degradation or (2) degradation of hopanes without 25-norhopane formation preceded by sterane degradation (37). The data presented here demonstrate C30-C34 hopane degradation with no evidence of sterane degradation or 25-norhopane formation, suggesting an alternative pathway to the two proposed. Biodegradation of Other Biomarkers. As stated above, sterane and diasterane patterns remained unaltered for BLC, VOL. 36, NO. 21, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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ANS 521, and VC (data not shown). Further analysis of the m/z ) 191 chromatograms (Figure 1A,D) shows C28 tricyclic terpane degradation, demonstrated by a 69% decrease in its ratio to the 20S epimer of the C27 diasterane when compared to the uninoculated controls (Table 3). Biodegradation of the tricyclic terpanes in ANS 521 and VC is more evident due to the higher abundance of these compounds. There was no preferential C28 tricyclic terpane epimer degradation, and the C29 and C30 tricyclic terpanes were not degraded. Tricyclic terpanes are considered highly resistant to biodegradation (1) and may remain unaltered in heavily biodegraded oils, even after the hopanes have been removed (35, 36). Goodwin et al. (19) observed no alteration of the tricyclic terpanes in laboratory studies. However, biodegradation of these compounds has been previously reported in some heavily degraded petroleums, with preferential degradation of the lower molecular weight homologues (4). Degradation of the trisnorhopanes decreased the Ts (C27 18R-22,29,30-trisnorneohopane) and Tm (C27 17R-22,29,30trisnorhopane) peaks relative to C30 18R-oleanane (Figure 1A,D). However, while the trisnorhopanes were degraded by each of the five cultures, the LC culture was the only enrichment culture where preferential degradation of Tm was confirmed by an increase in the Ts/Tm ratio (Table 3). The ratio of Ts/(Ts+Tm) has been used as a maturity index for source-related oils because Tm is less thermally stable than Ts (1). Preferential degradation of Tm over Ts was consistent for the LC culture in the three oils tested, and, to the best of our knowledge, this pattern of trisnorhopane degradation has not been reported previously. Microbial Characterization of Enrichment Cultures. Only enrichment cultures initiated using soils with a history of hydrocarbon contamination degraded C30 17R(H),21β(H)hopane. In concurrent studies, cultures enriched from nonhydrocarbon impacted soils were unable to transform C30 17R(H),21β(H)-hopane or other biomarkers examined (data not shown). Previous studies have established a correlation between preexposure to hydrocarbons and hydrocarbon degradation efficiency of microorganisms (38-44). Heterotrophic plate counts of the enrichment cultures resulted in an average increase of 2 orders of magnitude from time zero. The number of microorganisms isolated from the cultures varied from 7 (ET culture) to 12 (JI culture), and most of the isolated microorganisms were Gram-negative bacilli (data now shown). Among cultures capable of hopane degradation, those enriched from soils with higher hydrocarbon concentrations had the highest microbial diversity and most hopane degradation (LC, JI). Microbial community structure analysis of the enrichment cultures was first initiated to determine the similarities and differences in community structure in cultures capable of hopane degradation. DGGE analysis of 16S rRNA gene fragments PCR-amplified from total nucleic acid extracts revealed distinct community profiles for each of the five enrichment cultures (Figure 2). The DGGE profiles for the JI, WS, ET, and TC cultures showed 9, 8, 8, and 7 visible bands, respectively. Morphologically distinct isolates of the same species often result in comigration of DGGE bands, which account for the differences between the number of bands and isolates recovered from the same culture. The profile for the LC culture, however, showed 15 visible bands (Figure 3). DNA eluted from excised DGGE bands from the LC culture profiles was sequenced and compared to the sequences of the same 16S rRNA gene region obtained from the LC isolates (Figure 4). Six out of the 15 bands were shown to represent organisms isolated from the LC culture. Heteroduplex sequences, as confirmed by DGGE, were not considered further (bands LCa, LCb, and LCe). Effect of Crude Oil Composition on Biomarker Degradation. Crude oil composition had no effect on the pattern 4588

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FIGURE 2. DGGE analysis of the hopane degrading enrichment cultures after 30 days. A denaturing gradient of 40 to 60% was used to separate the products of the 16S rDNA amplification. This figure shows the region of the gel approximately corresponding to a gradient of 45 to 55%, where all the visible bands were found. All lanes are representative of triplicate samples. of biomarker degradation as demonstrated by representative m/z ) 191 chromatograms of the LC culture grown on BLC, ANS 521, and VC (Figure 1). Gravimetric analysis of the BLC, ANS 521, and VC crude oils after 30 days incubation with the LC culture revealed losses of 58.2% ((1.9 standard deviation, 21.4% ((2.3), and 11.0% ((6.3), respectively. The observed pattern is similar to that described for BLC, with degradation of C30-34 17R(H), 21β(H)-hopanes (R over the S epimers) and preservation of C35 homohopanes. These observations were confirmed by similar changes in the biomarker ratios (Table 4). The oleanane index increased 5-fold in BLC and 10-fold in VC. Due to the absence of C30 18R oleanane in ANS 521, the ratio of C29-C33 17R(H), 21β(H)-hopanes (R+S) to regular steranes was used to monitor hopane degradation. This ratio increased 10-fold on BLC, while smaller increases were seen on the VC and ANS 521. The homohopane index increased from 5.8% to 27.9% for BLC, 15.5% to 41.4% for the VC, and 12.1% to 17.3% on ANS 521. When comparing the biodegradation of n-alkanes between two oils with similar saturate content, Westlake et al. (45) observed faster degradation in the oil containing the lowest concentration of polar compounds. When the biodegradability of several crude and fuel oils was tested, lighter oils were prone to more extensive degradation (46). The study of the bioremediation potential of a suite of 17 oils ranging in API gravity from 14° to 45° indicated that API gravity could be used as a reliable index for biodegradation potential (47). Oils with >30° API gravity were considered easily degradable, and oils with ANS 521 > VC), but no significant difference was observed in the pattern of biomarker degradation among the three oils tested. Sequence of Biomarker Degradation. The LC culture was monitored during growth on BLC over a period of 64 days

FIGURE 3. DGGE analysis of the LC culture during the time course experiment. A denaturing gradient of 40 to 60% was used to separate the products of the DNA amplification. This figure shows the region of the gel approximately corresponding to a gradient of 45 to 55%, where all the visible bands were found. Each lane, labeled as time 0 and days (D) 1 to 64, is representative of triplicate samples taken at each time point. The bands excised from DGGE gels appear labeled in lower case letters. Bands that migrated to the same position as the LC isolates are also labeled with the isolate number. to determine the sequence of biomarker degradation (Figure 5). The n-alkanes and branched alkanes (pristane and phytane) were degraded by days 3 and 14, respectively. Biodegradation of C30 17R(H),21β(H)-hopane (relative to C30 18R-oleanane) was observed by day 21, with a 76% increase in the oleanane index compared to day 0. By day 64, there was an 86% increase in the oleanane index. GC-MS-SIM analysis (m/z ) 191) at each time point confirmed the loss of C30 17R(H),21β(H)-hopane observed by GC-FID (data not shown). Microbial community structure was analyzed at each time point, to both examine how crude oil compositional changes due to degradation influence the microbial community and to determine if there was community structure changes that could be correlated specifically with hopane degradation. Analysis of the microbial community profile by DGGE demonstrated a stable community structure over the 64-day sampling period, although subtle differences in band intensity and detection were observed (Figure 3). Several bands were more prominent at day 21 (LC7, LC4), when substantial hopane degradation was observed by GC-FID. However, the microorganisms isolated from the LC culture, including LC7 and LC4, were unable to degrade hopane when grown in pure culture on BLC (data not shown). The rapid depletion of the n-alkanes within the first week of incubation has been reported with pure (48, 49) and mixed cultures (8). Biodegradation of complex hydrocarbons is often thought to be the product of cometabolic events that require the presence of a cosubstrate (50, 51), and studies have demonstrated cometabolism of petroleum hydrocarbons, including the n-alkanes (52), cycloalkanes (53), and cyclosterols (54). Biodegradation of hopanes has been successful under laboratory conditions in the presence of pristane (55). However, in our study, hopane degradation occurred after the n-alkanes, pristane, and phytane had been depleted, suggesting that if a cometabolic event was involved, these biomarkers were not direct cosubstrates for the reaction. Effect of Oil Concentration and Temperature on Hopane Degradation. The effect of BLC concentration (1 to 10 mg/ mL) on biomarker degradation was examined using the LC culture. On BLC, the LC culture increased in total heterotrophic plate counts from 1.12 × 107 to 108 cfu/mL in all concentrations examined. Analysis of the DGGE patterns of the LC culture after growth with increasing oil concentrations

revealed no distinct changes in the community profiles (data not shown). Gravimetric analysis showed a decrease in the average percent degradation from 33.7% (1 mg/mL) to 16.7% (10 mg/mL) as oil concentration increased. Hopane degradation was statistically significant only at oil concentrations of 1 and 4 mg/mL (data not shown). The high standard deviation observed among the 2 mg/mL samples resulted from the lack of activity in one of the triplicate flasks, but biodegradation of hopanes at this concentration has been consistently observed in our experiments and is reported elsewhere in this study (Table 4). Hopane biodegradation was more efficient at low oil concentrations with 64% average degradation at 1 mg/mL and 40% and 47% degradation at 2 and 4 mg/mL, respectively. Hopane biodegradation declined to 23% at 6 mg/mL and was hindered at concentrations of 8 and 10 mg/mL, with only 8% and 11% degradation, respectively. Increased oil concentration resulted in a reduction of hopane degradation, but no change in the microbial profile and heterotrophic plate counts. These data suggest that while microorganisms present in the LC culture were able to survive at high oil concentrations, the population dynamics that favor hopane biodegradation were altered. A crude oil-degrading mixed culture consisting of 8 different isolates grown at high oil/water concentrations (50%) revealed that while total counts for the culture remained unchanged, 90% of the surviving bacteria represented one species, Pseudomonas aeruginosa (56). The same mixed cultures as well as pure cultures of the oil-resistant P. aeruginosa showed decreased biodegradation activity when grown at oil concentrations of 1% after induction at 50% oil:water concentrations. Biodegradation of C30 17R(H), 21β(H)-hopane was temperature dependent and only observed at 30 °C (data not shown). Biomarker degradation at 15 °C and 37 °C was limited to n-alkanes and branched alkanes, and no degradation was observed at 4 °C by GC-FID. Generally, the extent of crude oil biodegradation decreases with temperature (45, 57) due to slower microbial generation times, lack of volatilization of toxic low molecular weight compounds, and/or a decrease in enzymatic activity rates (58, 59). Prince et al. (18) did not observe degradation of C30 17R(H),21β(H)-hopane in ANS 521 at 15 °C after 168 days of incubation, although there was degradation of the saturate and aromatic fractions. Since VOL. 36, NO. 21, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Dendogram expressing the relationship between 16rDNA fragment sequences obtained from the LC isolates and the excised DGGE bands to partial reference sequences obtained from the RDP database. Sequences were analyzed using maximum distance with the Fitch-Margoliash method and bootstrap support using the PHYLIP programs. Bootstrap numbers higher than 50% are given at nodes and refer to the clusters to the right of each number.

TABLE 4. Biomarker Ratios after Biodegradation of Various Oils by the LC Culturee oleanane indexa

homohopane indexb

reg. steranes/hopanesc

C28TT/C27Dd

Bonny Light Crude uninoculated control sample

0.88 ( 0.02 5.64 ( 1.21

5.79 ( 0.24 27.91 ( 2.17

0.18 ( 0.00 1.90 ( 0.55

3.04 ( 0.28 1.26 ( 0.11

Alaskan North Slope 521 uninoculated control sample

NA NA

12.09 ( 0.88 17.33 ( 5.21

0.49 ( 0.02 0.87 ( 0.18

1.26 ( 0.09 0.58 ( 0.26

Venezuelan Oil uninoculated control sample

0.12 ( 0.01 1.50 ( 0.78

15.45 ( 0.32 41.42 ( 7.13

1.22 ( 0.03 7.75 ( 2.94

4.01 ( 0.39 0.73 ( 0.52

crude oil

a C 18R-oleanane/C 17R,21β-hopane. b (C /C -C homohopanes) × 100. c C , C , C RRR (20R + 20S) and Rββ (20R + 20S)/C -C 17R,21β30 30 35 31 35 27 28 29 29 33 hopanes (22R + 22S). d C28 tricyclic terpanes (22R+22S)/C27 13β,17R-diasterane (20S). e NA ) not applicable, C30 18R-oleanane is not present in ANS 521.

the LC culture was enriched and maintained at 30 °C using a microbial community enriched from a temperate soil (Fairhope, AL), more efficient crude oil biodegradation and depletion of C30 17R(H),21β(H)-hopane might be expected at this temperature. All examples of hopane degradation 4590

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reported in laboratory studies have been observed at 30 °C (19, 21, 22, 34, 55). A number of factors (i.e., microorganisms present, temperature, aeration, availability of nutrients and cosubstrates, salinity) influence crude oil biodegradation and should be

mentation Award for Science and Engineering Research Training, and the donors of the Petroleum Research Fund, administered by the American Chemical Society.

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FIGURE 5. GC-FID traces of BLC oil residues extracted after 0, 3, 14, 21, and 64 days of incubation with the LC culture. All chromatograms are representative of triplicate samples and are normalized to the weight of the oil extracted from each culture flask. The inserts represent amplified views of the portions corresponding to the elution of C30 18r-oleanane (O) and C30 17r(H),21β(H)-hopane (H). considered when interpreting the differences observed in degradation patterns among crude oils (6, 60, 61). For example, growth in rich media has been shown to limit the range of hydrocarbons degraded by bacteria (62). The use of pure cultures in biomarker degradation studies (34, 55) also limits the range of enzymatic activities and the possibility of degradation driven by interspecies cometabolic interactions. The data presented in this study demonstrate that C30 17R(H),21β(H)-hopane and other crude oil biomarkers are transformed by microbial cultures enriched from sites with varied sources of hydrocarbon contamination. Although the use of C30 17R(H),21β(H)-hopane to normalize crude oil biodegradation data is still a viable approach in many environments, care should be taken in the interpretation of hopane-normalized data. In assessing extensive degradation of crude oil, more accurate results could be obtained by applying a ranking scheme that also takes into account additional biomarkers (steranes, diasteranes, homohopanes, aromatics) (5). A better understanding of the transformation products of C30 17R(H),21β(H)-hopane may provide additional insights into the assessments of heavily degraded crude oils.

Acknowledgments The authors would like to thank Louise Weston for technical support and Dr. Ken Peters (ExxonMobil) for helpful discussions and for providing the Venezuelan crude oil. This work was supported by the U.S. Department of Energy grant DE-FG01-912EW506 to the Medical University of South Carolina, the Air Force Office of Scientific Research Aug-

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Received for review June 18, 2002. Revised manuscript received August 27, 2002. Accepted August 28, 2002. ES025894X