Envkon. Scl. Technol. 1004, 28, 142-145
17a(H),21@(H)-Hopane as a Conserved Internal Marker for Estimatlng the Biodegradation of Crude Oil Roger C. Prlnce,'lt David L. Elmendorf,tl* James R. Lute,? Chang S. Hsu,~Copper E. Haith,? James D. Senius,t Gary J. Dechert,? Gregory S. Douglas,$ and Eric L. ButierS9II Exxon Research and Engineering Company. Annandale. New Jersey 0880 1, and Battelle Ocean Sciences,
Duxbury, Massachusetts 02332
Hopanes are common constituents of crude oils, and they are very resistant to biodegradation. They can therefore serve as conserved internal standards for assessing the biodegradation of the more degradable compounds in the oil. Here we address two important questions that attend such use. The first is whether the "internal standard" is being created during the biodegradation process itself, for this could result in an overestimate of the extent of biodegradation. The second is whether the internal standard is indeed relatively resistant to biodegradation on time scales of relevance to the biodegradation process under study; for if it was not, this could result in an underestimate of the extent of biodegradation. We find that 17a(H),21/3(H)-hopane is neither generated nor biodegraded during the biodegradation of crude oil fractions on time scales relevant to estimating the cleansing of oil spills, and so it has the appropriate characteristics to serve as an internal standard for studying the biodenrdation of crude oil in the environment.
17P(H), 21P(H)-hopane
Introduction The majority of the components of crude oil are biodegradable (1-41, but quantifying biodegradation in the field has proven to be a challenge. A well-known approach is to follow the degradation of a degradable component by reference to a relatively slowly degradable one, and the ratios of heptadecane:pristane and octadecane:phytane have often been used (see refs 1-7). Pristane and phytane are, however, rather biodegradable; so these ratios are only valuable in the early stages of biodegradation. In order to quantify biodegradation more rigorously, even after pristane and phytane have been substantially degraded, we have used 17a(H),2lp(H)-hopane as the conserved internal standard (5-7) (Figure 1). Hopanes are very resistant to biodegradation (8),and, indeed, may be the most abundant chemically defined organic species on earth (9). Hopanes are the molecular fossils of bacterial hopanoids, which themselves serve the membrane fluidizing role of sterols in eukaryotic membranes (IO). Stable isotope studies suggest that, at least in some oils, hopanes were originally synthesized by cyanobacteria (11). Since hopanes were originally synthesized by bacteria and are degradable, albeit slowly, it is important to determine whether such phenomena are important in the microbial degradation of spilled oil. Here we show that 17a(H),218(H)-hopaneis neither generated nor degraded ~
~~
t Esxon Research and Engineering Company. t Present address: Department of Biology, Universityof
25-norhopane Figure 1. Structuresof 17a(h),21@(Wopane, 17p(kfJ,2lj3(Wopane, and 25-norhopane.
in laboratory experiments that mimic biodegradation in the field, so hopane does have the appropriate properties to serve as a conserved internal marker.
Materials and Methods Two different fractions of Alaska North Slope crude oil were used in the experiments reported here. One is the fraction remaining when whole oil is distilled until the temperature reaches 272 "C; this material has lost 30% of its initial weight and is not subject to significant further evaporation. The other is the fraction that distills between 196 and 344 OC; it represents approximately 23% of the whole oil. Both oils are rich in alkanes, as shown in Figures 2 and 3, but while the former contains abundant hopanes, there are none detectable in the 196-344 cut because hopanes volatilize only at temperatures in excess of 344 OC.
Central
Oklahoma, Edmond, OK 73034. I Battelle Ocean Sciences. 11 Presentaddress: ENSR Cousultingand Engineering,Acton, MA 01720.
Biodegradation experiments were performed in Bushnell-Haas medium (12)supplemented with 10pg/L biotin, 50 pg/L p-aminobenzoic acid and vitamin BmlOO pg/L thiamine, 3% NaC1, Wolfe's minerals (13),and 1%oil.
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Figure 2. Gas chromatography-flame ionization detector chromatograms of 196-344 oil after extraction from flasks incubated at 15 O C for 14 days without (A) and with (B) microbial inoculum from Prince William Sound sediments. The large peak at about 33.5 min is the internal standard Sa-androstane added prior to the inJectioninto the instrument.
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iments with 196-344 oil. Microbial inocula came from oiled beach samples collected from Prince William Sound, AK, approximately 1year after the spill from the Exxon Vuldez. The sediment samples were shaken in saline Bushnell-Haas medium for 20 min at 160 rpm, and the medium was then decanted and used as a 2.5-10% inoculum. Care was taken not to add any oil from the sediment to the incubations. For 196-344 oil experiments, uninoculated flasks served as controls in case evaporation during incubation and extraction played a significant role in removing components of this oil. Bacterial numbers were assessed by most probable number techniques using the "sheen-screen"(14)for assessing oil-degrading bacteria and by a modification of this technique using Difco Marine Broth 2216 (Difco Laboratories, Detroit, MI) for heterotrophic organisms. In case the biodegradation of the hopanes in the 272+ oil was limited by the accumulation of water-soluble inhibitory products, the flasks were extracted with methylene chloride approximately ever 14 days, and the water phase was discarded. The oil solubilized in the methylene chloride was then returned to the flask, the methylene chloride was removed by evaporation, and the flasks were reinoculated with mediaand bacteria. Furthermore, Inipol EAP22, an oleophilic fertilizer (15), was added to 0.1% of the culture volume after the second and the following eight extractions. Inipol EAP22 was the oleophilic fertilizer used in the successful bioremediation program in Prince William Sound and the Gulf of Alaska (6, 7). At the end of the experiment (168 days for the 272+ experiment, 14 days for the 196-344 experiment), each flask was repetitively extracted with methylene chloride to remove all the oil. Water was removed from the methylene chloride extract with sodium sulfate, and the extract was filtered through an alumina column to remove polar material (5, 16, 17). I t was then analyzed by gas chromatography with detection by flame ionization (GC/ FID) and mass spectrometry (GCMS) (5, 16, 17). The initial oils for each experiment were analyzed at the same time as the biodegraded oils to minimize instrumental variations. A five-point calibration curve was generated using the commercially available 17@(H),2lO(H)-hopane(Chiron Laboratories, College Station, TX). This isomer is not present in Alaska North Slope crude oil, and it was added to each oil, just prior to GClMS analysis, to provide an internal standard. In addition to monitoring the principal fragment ion for 17a(H),2l@(H)-hopane(mlz 191), the principal reputed biodegradation product [25-norhopane, mlz 171 (see ref 811 was also monitored to detect degradation (17). Over a 6-month period, the standard deviation of 37 measurements of the amount of hopane in a reference oil was 1 2 % , but this variation was principally evident in differencesbetween different instruments, and over time; it was substantially smaller, typically 2-3%, in replicate samples run before and after a batch of experimental samples. Using hopane analysis, we calculate that the depletion of an artificially weathered (distilled) oil, with a gravimetrically measured depletion of 30.3%,was 30.1% .
Figure 3. Gas chromatography-flame ionization detector chromatograms of 272+ oil before (A) and after (B) 168 days of biodegradation as described in the text. The two peaks at approximately 28 and 30 min are the internal standards o-terphenyi and 5a-androstane added prior to injection into the instrument.
Results
For the experiments with 272+ oil, three 4-L flasks containing 1.2 L of medium were shaken at approximately 160 rpm at 15 "C in a water bath. Three l-L flasks containing 100 mL of medium were used for the exper-
The inocula from contaminated beach sediment from Prince William Sound, AK, grew well on both oils. Typical inocula contained on the order of lo6 heterotrophic and 5 X lo4 oil-degrading bacteria per milliliter, and these Environ. Sci. Technol.. Vol. 28, No. 1, 1994
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were added as 2.5-10% by volume. After 14-21 days, the microbial populations had increased to typically lo8 heterotrophic and lo6oil-degrading bacteria per milliliter. Biodegradation of the oils were readily apparent in the GC/FID results (Figures 2 and 3). The linear alkanes were substantially depleted after a single 2-week incubation (Figure 2) and were completely absent by day 168 (Figure 3). No 17a(H),21O(H)-hopanewas detected in the 196344 oil, either before or after microbial growth on this substrate (detectionlimit approximately 2-5 mg/kg of oil). In contrast, the batch of 272+ oil used in these experiments contained 245 mg of this compound per kilogram of oil, and there was no apparent change in the m/z = 191 mass spectrum of the oil before and after biodegradation over 168 days (Figure 4). In order to carry out a mass balance on this compound, we must make one of two assumptions about the nature of the oil lost at the various transfers during the experiment, since hopane concentrations were determined only at the beginning and the end of the experiment. A total of 12 g of oil was initially added to each of three flasks, 1.7-2.2 g was lost in the various transfers, and between 7.3 and 8.1 g was recovered on day 168. The two alternatives are that the lost oil was like either the initial (Le., we began with 9.8-10.3 g) or the final (Le,, we effectively ended with 9.0-9.8 g) oil. Averaging the estimates from these two alternatives for the triplicate experiments yielded an average recovery of hopane of 104.5 f 6% (95% confidence limits). 144
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Discussion This paper reports the results of two experiments to assess the validity of using 17a(H),21/3(H)-hopaneas a conserved internal marker of Alaska North Slope crude oil in order to assess biodegradation in the field. We chose this isomer because it is the most abundant hopane in this and many oils (8) and because integrating the area of a single peak in a mass spectrum (rather than, for example, summing all the hopanes in the oil) minimizes problems with baseline drift. The 196-344 oil provided a hopanefree substrate to determine whether biomass grown using this as sole carbon source produced any components that would interfere with the detection of the 17a(H),21/3(H)hopane in crude oil. The 272+ oil provided a carbon source rich in 17a(H),2lp(H)-hopane to see whether this was degraded on time scales relevant to the cleanup of the spill from the Exxon Vuldez. The data indicate that no significant amounts of 17a(H),2lp(H)-hopane are introduced or removed by microbial growth on Alaska North Slope crude oil. Bacterial grown on the 196-344 oil substantially degraded the alkanes and simple aromatics (Figure 2), with the generation of a substantial amount of biomass, but no 17a(H),2lp(H)-hopane was detected in either the uninoculated or inoculated flasks. This is not surprising, since the 196-344 oil contained no hopanes, and there are no hopanes in biomass. The poly01 hopanoids in biomass (8-10) are sufficiently polar that they would be excluded from the gas chromatography column by the alumina column step. Furthermore, the biological configuration is 17/3(H),21/3(H)-hopane,which is readily distinguished by GC/MS from the 17a(H),21/3(H)isomer (5,8, 16). Bacterial growth on the 272+ oil also produced a substantial degradation of the alkanes and simple aromatics. In these experiments,the oils were extracted from the medium a total of 10 times and reinoculated with medium and oil-degrading bacteria. The biodegradation in these samples was substantial. Estimates based on gravimetric recovery, which unfortunately includes methylene chloride soluble biomass, indicate that at least 27 % of the 30 % -depleted oil (equivalent to 49 % disappearance of whole oil) had been degraded by the end of the experiment, while 48% of the 30%-depleted oil had been lost based on the recovery of the GC-detectable material (equivalent to the disappearance of 64% of whole oil). Such estimates are in excellent agreement with earlier work (1-4). Furthermore, Inipol EAP22 was added to 0.1 % of the flask volume after the second and the followingeight extractions to mimic the bioremediation effort in Prince William Sound and to provide a readily degradable carbon source in case hopane degradation occurred only fortuitously by co-oxidation (18). Nevertheless, the hopane recovery at the end of the experiment was 104.5 f 6 % of that initially added to the flasks, indicating no degradation after 168 days. Furthermore, there was no significant change in the mlz = 191 mass spectrometry of the oil after biodegradation (Figure 4), providing further corroboration that there had been no degradation, and monitoring the mlz = 177 spectrum for demethylated hopanes (8) also indicated no degradation (data not shown). Hopanes are biodegradable, both in the natural environment (see ref 8) and in the laboratory (19,201.Under geological conditions, the process seems rather slow (81, but Parker and Acey (20)report 30% degradation in 5 weeks when hopanes are provided to Mycobacterium fortuitum growing on pristane. No degradation occurred
in the experiments reported here, and it seems likely that little biodegradation had occurred in the field samples collected as part of the U.S. EPA/Exxon/Alaska Department of Environmental Conservation Bioremediation Monitoring program carried out in Prince William Sound, AK, in 1990 (6, 7). If hopane has been biodegraded in these samples, our estimates of the extent of biodegradation (6, 7) would be underestimates.
Literature Cited (1)Bertrand, J. C.; Rambeloarisoa, E.; Rontani, J. F.; Giusti, G.; Mattei, G. Biotechnol. Lett. 1983, 5, 567. (2) Oudot, J. Mar. Environ. Res. 1984, 13, 277. (3)Berwick,P.; Stafford, D. A. Process Biochem. 1985,20,175. (4) Kennicutt, M. C., 11. Oil Chem. Pollut. 1988,4, 89. (5) Butler, E. L.; Douglas, G. S.; Steinhauer, W. S.; Prince, R. C.; Aczel, T.; HSU,C. S.; Bronson, M. T.; Clark, J. R.; Lindstrom, J. E. In On-site Reclamation. Processes for xenobiotic and hydrocarbon treatment; Hinchee, R. E.; Olfenbuttel, R. F., Eds.; Butterworth-Heinemann: Boston, 1991;pp 515-521. (6) Bragg, J. R.; Prince, R. C.; Harner, E. J.; Atlas, R. M. In Proceedings of the 1993Znternational Oil Spill Conference; American Petroleum Institute: Washington, DC, 1993;pp 435-447. (7) Prince, R.C.; Clark, J. R.; Lindstrom, J. E.; Butler, E. L.; Brown, E. J.; Winter, G.; Grossman, M. J.; Parrish, P. R.; Bare, R. E.; Braddock, J. F.; Steinhauer, W. G.; Douglas, G. S.; Kennedy, J. M.; Barter, P. J.; Bragg, J. R.; Harner, E. J.; Atlas, R. M. In Hydrocarbon Bioremediation; Hinchee,
R. E., Alleman, B. C., Hoeppel, R. E., Miller, R. N., Eds.; Lewis Publishers: Ann Arbor, MI, 1994; pp 107-124.
(8) Peters, K. E.; Moldowan, J. M. The Biomarker Guide: Interpreting molecular fossils in petroleum and ancient sediments; Prentice Hall: Englewood Cliffs, NJ, 1993. (9) Ourisson, G.; Albrecht, P. Acc. Chem. Res. 1992, 25, 398. (10) Prince, R. C. Trends Biochem. Sci. 1987, 12, 455-6. (11) Schoell,M.; McCaffrey,M. A.; Fago, F. J.; Moldowan, J. M. Geochim. Cosmochim. Acta 1992,56, 1391. (12) Bushnell, L. D.; Haas, H. F. J. Eacteriol. 1941, 41, 653. (13) Wolin,E. A.; Wolin, M. J.; Wolfe,R. S. J.Biol. Chem. 1963, 238, 2882. (14) Brown, E. J.; Braddock, J. F. Appl. Environ. Microbiol. 1990,56, 232-237. (15)Ladousse, A.; Tramier, B. In Proceedings of the 1991 International Oil Spill Conference; American Petroleum Institute: Washington, DC, 1991;pp 577-81. (16) Douglas, G. S.;McCarthy, K. J.; Dahlen, D. T.; Seavey,J.
A.; Steinhauer, W. G.; Prince, R. C.; Elmendorf, D. L. J. Soil Contam. 1992, 1 , 197-216. (17) Douglas,G. S.;Prince, R. C.; Butler, E. L.; Steinhauer, W. G. In Hydrocarbon Bioremediation; Hinchee, R. E.,
Alleman, B. C., Hoeppel, R. E., Miller, R. N., Eds.; Lewis Publishers: Ann Arbor, MI, 1994;pp 219-236. (18)Perry, J. J. Microbiol. Rev. 1979,43, 59-72. (19) Goodwin,N. S.;Park, P. J. D.; Rawlinson,A. P. In Advances in Organic Geochemistry 1981; Bjoray, M., Ed.; Wiley: Chichester, U.K., 1983,pp 650-658. (20) Parker, S. L.; Acey, R. A. J. Cell Biochem. 1993, Suppl. 17C, 196. Received for review May 27,1993. Revised manuscript received September 10,1993. Accepted September 17, 1993." Abstract published in Advance ACS Abstracts, November 1,
1993.
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